Small molecules in targeted cancer therapy: advances, challenges, and future perspectives.

Introduction

Drug treatment together with surgical operation, radiotherapy and biotherapy constitute the main approaches to cancer treatment. For a long time, chemotherapy, which is a method of killing tumor cells and/or inhibiting the growth and proliferation of tumor cells by chemical drugs, was the only approach to cancer drug therapy. The biggest characteristic of chemotherapy is the inability to distinguish between cancer cells and normal cells, resulting in significant toxicity and side effects. Over the past two decades, there has been a tremendous shift in cancer treatment, from broad-spectrum cytotoxic drugs to targeted drugs. 1 Compared with traditional chemotherapy drugs, targeted drugs can specifically target cancer cells but spare normal cells, hence having high potency and low toxicity. Encouraged by the approval of the first small-molecule tyrosine kinase inhibitor (TKI) imatinib for clinical use by the US Food and Drug Administration (FDA) in 2001, 2 targeted drugs have rapidly developed and entered a golden period of development. In the past 20 years, there has been a significant increase in FDA-approved targeted drugs for cancer treatment.

Targeted drugs can be roughly classified into two categories: small molecules and macromolecules (e.g., monoclonal antibodies, polypeptides, antibody–drug conjugates, and nucleic acids). 3 , 4 Compared with macromolecule drugs, small-molecule targeted drugs have advantages in some aspects such as the pharmacokinetic (PK) properties, costs, patient compliance, and drug storage and transportation (Supplementary Table S1 ). Despite challenged by macromolecule drugs represented by monoclonal antibodies in recent years, small-molecule targeted drugs still gain great development. To date, there are a total of 89 anti-cancer small molecules approved in the United States and China. Figure 1 summarizes the small-molecule anti-cancer drugs approved by the US FDA and National Medical Products Administration (NMPA) of China since 2001. The targets of these drugs cover a large scope including kinases, epigenetic regulatory proteins, DNA damage repair enzymes, and proteasomes. It is undeniable that small-molecule targeted anti-cancer drugs still face many challenges such as low response rate and drug resistance. Fig. 1 Timeline for the approval of small-molecule targeted anti-cancer drugs

To better promote the development of small-molecule targeted anti-cancer drugs, we will conduct a comprehensive review for them. In order to facilitate the description, protein targets of the approved agents will be taken as a clue. For each target, marketed small-molecule drugs and important drug candidates in clinical trials will be presented. Finally, an analysis of the current challenges in the field and a future perspective will also be given.

Kinase inhibitors

Protein kinase is a kind of enzyme that catalyzes the transfer of γ-phosphate group from ATP to protein residues containing hydroxyl groups. It has an important role in cell growth, proliferation, and differentiation (Fig. 2 ). 5 The human kinome comprises ~535 protein kinases. 6 According to the substrate residues, protein kinases can be classified as tyrosine kinases (including both receptor and non-receptor tyrosine kinases), serine/threonine kinases, and tyrosine kinase-like enzymes. Dysregulation of protein kinases is linked to various diseases, particularly cancer. Protein kinases are the most widely studied tumor therapeutic targets. Currently, a large number of protein kinase inhibitors have been reported. These kinase inhibitors can be classified into different categories by using many ways. Here we adopted an integrated classification system proposed by Roskoski, which is one of the most widely used methods. 7 According to this classification system, protein kinase inhibitors are classified into six types (Type-I–VI). Type-I inhibitors bind to the active conformation of the kinase (DFG-Asp in, αC-helix in). Type-I½ inhibitors bind to a DFG-Asp in inactive kinase conformation with αC-helix out, while type-II inhibitors bind to a DFG-Asp out inactive conformation. These types of inhibitors occupy part of the adenine binding pocket and form hydrogen bonds with the hinge region connecting the small and large lobes of the enzyme. Among them, type-I½ and type-II antagonists can be further divided into A and B subtypes. Type A inhibitors extend past the Sh2 gatekeeper residue into the back cleft, while type B inhibitors fail to extend into the back cleft. The possible importance of this difference is that type A inhibitors have longer residence times compared with type B inhibitors when binding to their targets. Type III and type IV kinase inhibitors are allosteric in nature. Type III inhibitors restrain kinase activity by binding to an allosteric site, which is in the cleft between the small and large kinase lobes adjacent to the ATP-binding pocket. Contrariwise, type IV inhibitors bind outside of the cleft. Moreover, the bivalent molecules that span two distinct regions of the kinase domain are type V inhibitors. Type-I–V inhibitors are all reversible. In contrast, compounds that bind covalently with the kinase active site are called type VI inhibitors (irreversible kinase inhibitors). Fig. 2 Activation of different protein kinase-dependent pathways. The set of RTKs influences a small number of intermediaries, such as phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinases (MAPK), thereby activating the complex signaling networks that are related to cell proliferation, differentiation, adhesion, apoptosis, and migration. The aggregation, activation, and depolymerization of the periodic CDK-cyclin complex are critical events driving cell cycle turnover. Figure created with BioRender.com

Receptor tyrosine kinase inhibitors

ALK inhibitors

Anaplastic lymphoma kinase (ALK) encoded by the ALK gene is a single transmembrane tyrosine kinase of the insulin receptor family. 8 ALK can activate multiple downstream signaling pathways and has an important role in the development of the nervous system. 9 Constitutive activation of ALK through point mutations or chromosomal rearrangements has been identified in multiple human cancers such as anaplastic large cell lymphoma, diffuse large B-cell lymphoma (DLBCL), 10 inflammatory myofibroblastic tumor, 11 and non-small cell lung cancer (NSCLC). 12 Fusion of echinoderm microtubule-associated protein-like 4 with ALK ( EML4-ALK ) in NSCLC was identified in 2007 by Soda et al. 12 this rearrangement of the ALK gene has been detected in ~3–7% of patients with NSCLC. EML4-ALK gene fusion is initiated by inversion in the short arm of chromosome 2, which juxtaposes the N-terminal of the EML4 promoter and the kinase domain of the ALK gene, ultimately leading to ligand-independent constitutive activation of ALK and promoting cancer cell proliferation and survival. Several other ALK gene fusions, such as NPM-ALK , ATIC-ALK , and RANBP2-ALK , have also been discovered; 13 – 15 these rearrangements define a specific subgroup of cancerous patients that can be treated with selective ALK inhibitors. 16

Crizotinib approved in 2011 is a first-generation ALK inhibitor targeting multiple tyrosine kinases including ALK, cellular-mesenchymal-epithelial transition factor (c-Met), and proto-oncogene tyrosine-protein kinase reactive oxygen species (ROS) (Table 1 ). 17 Two randomized phase III trials ( NCT00932893 , NCT01154140 ) established the superiority of crizotinib over chemotherapy in patients with advanced ALK -rearranged NSCLC, and it is now the standard drug therapy for metastatic ALK -positive NSCLC. 18 , 19 Unfortunately, most patients develop resistant mutations to crizotinib within 12 months, especially L1196M and G1269A mutations, which can lead to relapse. 20 The central nervous system (CNS) is the most common relapse site in patients with NSCLC treated with crizotinib, probably because of its poor blood–brain barrier (BBB) permeability. 21 The second-generation ALK inhibitors ceritinib, 22 alectinib, 23 and brigatinib 24 were subsequently developed for the treatment of crizotinib-resistant ALK -positive NSCLC, all of which are multikinase inhibitors (Table 1 ). Ceritinib is more potent than crizotinib and has doubled progression-free survival (PFS) compared with chemotherapy in clinical studies. 22 Alectinib has advantages over both crizotinib and ceritinib, and has shown inhibitory activity against several crizotinib or ceritinib-resistant ALK mutations such as L1196M, G1269A, C1156Y, and F1174L. 25 This agent is not a substrate of P-glycoprotein and can cross the BBB and effectively prevent the progression of CNS metastases. It was approved for NSCLC treatment in 2015 and recommended as first-line therapy for patients with ALK fusion-positive NSCLC in 2017. Moreover, brigatinib was granted accelerated approval by the FDA in 2017 as second-line therapy for patients with ALK -positive metastatic NSCLC, based on the considerable systemic and intracranial responses in clinical trials. 26 Similar to the experience with crizotinib, novel resistance mechanisms were observed in patients who relapsed after treatment with second-generation ALK inhibitors. Secondary ALK kinase domain mutations, such as the G1202R, V1180L, and I1171T mutants, are the most common resistance mechanisms. 27 Lorlatinib is an oral ATP-competitive brain penetrant inhibitor of ALK/ROS1 approved in 2018. 28 , 29 As a third-generation ALK inhibitor, all recognized ALK mutations (except L1198F mutation) can be targeted by lorlatinib. 29 Interestingly, lorlatinib is structurally distinct from most second-generation ALK inhibitors but has the same structural basis as crizotinib. However, patients harboring the L1198F mutation, which confers resistance to lorlatinib, have reported re-sensitivity to crizotinib. 30 This result indicates that retreatment under molecular guidance should be considered as a clinically meaningful approach for ALK -positive NSCLC. Table 1 Properties of approved small-molecule inhibitors of receptor tyrosine kinases Chemical structure Name Targets Approved indications (year) Corporation Crizotinib (Xalkori) ALK/ROS/c-Met NSCLC (2011) Pfizer Ceritinib (Zykadia) ALK/ROS NSCLC (2014) Novartis Alectinib (Alecensa) ALK NSCLC (2015) Roche/Chugai Brigatinib (Alunbrig) ALK/ROS/ IGF1R/EGFR/FLT3 NSCLC (2017) Ariad Lorlatinib (Lorbrena) ALK NSCLC (2018) Pfizer Capmatinib (Tabrecta) c-Met NSCLC (2020) Novartis Tepotinib (Tepmetko) c-Met NSCLC (2020) Merck Gefitinib (Iressa) EGFR NSCLC (2003) AstraZeneca Erlotinib (Tarceva) EGFR NSCLC (2004) Pancreatic cancer (2005) Roche/Astellas Lapatinib (Tykerb) EGFR/HER2 Breast cancer (2007) Novartis Icotinib (Conmana) EGFR NSCLC (2011) Betta Afatinib (Gilotrif) EGFR/HER2/HER4 NSCLC (2013) Boehringer Ingelheim Osimertinib (Tagrisso) EGFR NSCLC (2015) AstraZeneca Neratinib (Nerlynx) HER2/HER4/EGFR Breast cancer (2017) Puma Biotech Dacomitinib (Vizimpro) EGFR NSCLC (2018) Pfizer Almonertinib (Ameile) EGFR NSCLC (2020) Hansoh Tucatinib (Tukysa) HER2 Breast cancer (2020) Seattle Genetics Midostaurin (Rydapt) FLT3/c-Kit AML (2017) Novartis Gilteritinib (Xospata) FLT3/AXL AML (2018) Kotobuki/Astellas Quizartinib (Vanflyta) FLT3 AML (2019) Daiichi Sankyo Pexidartinib (Turalio) CSF1R/c-Kit/FLT3 TGCT (2019) Daiichi Sankyo Sorafenib (Nexavar) c-Kit/FLT3/RET/PTC/ VEGFR-1/2/3/PDGFR-β RCC (2005) HCC (2007) DTC (2013) Thyroid cancer (2014) Bayer Sunitinib (Sutent) PDGFR-α/β/VEGFR-1/2/3/CSF1R/c-Kit/RET/FLT3 RCC (2006) GIST (2006) Pancreas neuroendocrine tumor (2011) Pfizer Pazopanib (Votrient) PDGFR-β/VEGFR-1/2/3/ FGFR-1/3/c-Kit/Itk/Lck/c-GSK RCC (2009) STS (2012) Novartis Vandetanib (Caprelsa) EGFR/VEGFR/RET/BRK/TIE2/EPH MTC (2011) Genzyme Axitinib (Inlyta) VEGFR-1/2/3 RCC (2012) Pfizer Cabozantinib (Cometriq) VEGFR-1/2/3/TYRO3/ROS/TIE2/c-Met/HGFR/c-Kit/TRK2/RET MTC (2013) RCC (2016) HCC (2019) Exelixis Regorafenib (Stivarga) VEGFR-1/2/3/PDGFR-α/β/FGFR-1/2/RAF/RET/c-Kit CRC (2012) GIST (2013) HCC (2017) Bayer Apatinib (Aitan) VEGFR-2/Src/c-Kit Gastric cancer (2014) Hengrui Medicine Lenvatinib (Lenvima) PDGFR-α/VEGFR-1/2/3/FGFR-1/2/3/4/c-Kit/RET DTC (2015) Thyroid cancer (2015) RCC (2016) HCC (2018) Endometrial carcinoma (2019) Eisai Tivozanib (Fotivda) PDGFR-α/VEGFR-1/2/3/FGFR-1/2/3/4/c-Kit/RET RCC (2017) Eusa Fruquintinib (Elunate) VEGFR-1/2/3 CRC (2018) Chi-Med/Lilly Nintedanib (Ofev) VEGFR-1/2/3/PDGFR-α/β/ MDR1/BCRP/FGFR-1/3 NSCLC (2015) Boehringer Ingelheim Anlotinib (Focus V) VEGFR-2/3/PDGFR-β/FGFRs NSCLC (2018) STS (2019) SCLC (2020) Chia Tai Tianqing Erdafitinib (Balversa) FGFR-1/2/3/4 Urothelial carcinoma (2019) Janssen Pemigatinib (Pemazyre) FGFR-1/2/3/4 Cholangiocarcinoma (2020) Incyte Avapritinib (Ayvakit) c-Kit/PDGFR-α GIST (2020) Blueprint Ripretinib (Qinlock) c-Kit/PDGFR-α GIST (2020) Deciphera Selpercatinib (Retevmo) RET NSCLC (2020) MTC (2020) Thyroid cancer (2020) Loxo Pralsetinib (Gavreto) RET NSCLC (2020) MTC (2020) Thyroid cancer (2020) Blueprint Medicines Larotrectinib (Vitrakvi) TRKA/B/C Solid tumors with NTRK fusion (2018) Bayer Entrectinib (Rozlytrek) TRKA/B/C/ROS1/ALK Solid tumors with NTRK fusion (2019) Roche

Currently, there are still some ALK inhibitors under clinical investigation, such as the pan-TKIs entrectinib, 31 belizatinib, 32 and repotrectinib, 33 which target oncogenic rearrangements in ALK, ROS, and tropomyosin receptor kinase (TRK). 34 Among them, entrectinib was approved for the treatment of TRK fusion solid tumors in 2019. The clinical use of these drugs as ALK inhibitors is still under evaluation. 31 The aminopyrazine derivative ensartinib is a newly developed second-generation ALK inhibitor. It exhibits efficacy in crizotinib-naive and crizotinib-resistant patients with ALK -positive, locally advanced or metastatic NSCLC, as well as patients with brain metastases. 34 A phase III study is ongoing to compare ensartinib with crizotinib for the first-line treatment of ALK -positive NSCLC ( NCT02767804 ). Moreover, CEP-37440 is an orally administered inhibitor of ALK and focal adhesion kinase. A phase I trial ( NCT01922752 ) of this agent has been performed in patients with solid tumors, but no preliminary data are available. 35

Drug-resistant mutations are major obstacles that limit the clinical efficacy of ALK inhibitors. To date, ALK inhibitors have been developed to the third generation. Rational sequential therapy (using first-, second-, and third-generation ALK inhibitors for NSCLC therapy sequentially according to ALK gene mutations) can effectively overcome drug resistance and improve the survival of applicable patients. Of note is that retreatment with crizotinib can benefit patients harboring the ALK L1198F mutation, which is resistant to the latest generation of ALK inhibitors. 36 For resistance arising from bypass activation, combining ALK inhibitors with other targeted therapies such as a mitogen-activated protein kinase (MAPK) inhibitor, 37 , 38 cyclin-dependent kinase (CDK) inhibitor (ceritinib with ribociclib, NCT02292550 ), mammalian target of rapamycin (mTOR) inhibitor (ceritinib with everolimus, NCT02321501 ), and heat-shock protein 90 inhibitor has been assessed in a number of trials. 39 Since the expression of programmed death-ligand 1 is reportedly associated with EML4-ALK , combined treatments of ALK and immune checkpoint inhibitors have also been evaluated in ALK -positive NSCLC. 38 In addition to kinase inhibitors, degrading carcinogenic proteins using proteolysis targeting chimera (PROTAC) technology is an effective anti-cancer strategy. The PROTACs MS4077 and MS4078 designed by Zhang et al. have shown great potency in reducing ALK fusion protein in preclinical studies, suggesting a new approach to drug discovery targeting ALK. 40

c-Met inhibitors

Cellular-mesenchymal-epithelial transition factor (c-Met), also known as hepatocyte growth factor receptor (HGFR), is encoded by the MET proto-oncogene located on chromosome 7q21-31. 41 , 42 Under normal physiology, the binding of c-Met to its sole ligand HGF initiates the activation of the HGF/c-Met signaling pathway, which further activates several downstream signals including the PI3K/AKT, MAPK, STAT, and NF-κB pathways, and has a central role in a variety of cytoplasmic and nuclear processes, such as cell proliferation, survival, invasion, motility, scattering, angiogenesis, and epidermal–mesenchymal transition. 42 – 44 These normal regulatory functions mainly occur during embryonic development, wound healing, and post-injury tissue regeneration. However, aberrant activation of c-Met signaling caused by MET amplification, mutation, inadequate degradation, transcriptional deregulation, or aberrant HGF autocrine or paracrine has been implicated in the development of various solid tumors. 44 , 45 c-Met overexpression has also been reported to be related to poor prognosis and resistance to cytotoxic and molecular targeted therapy, especially for patients treated with EGFR inhibitors, in which MET amplification accounts for ~20% of resistant cases. 46 Activating MET mutations usually occur in the semaphoring domain (e.g., E168D) and juxtamembrane domain (e.g., T1010I, P1009S, skipping mutation) of exons 14, 18, and 19. Of these, c-Met exon 14 skipping mutations promote its oncogenic activity by suppressing c-Met receptor degradation. 43 , 45 , 47 These mutations are rare in patients with primary tumors but common in advanced cancers with metastases, especially in lung adenocarcinoma, brain gliomas, and renal cell carcinoma (RCC). 47 , 48

During the last decade, great progress has been made in antitumor therapy targeting the HGF/c-Met signaling pathway. The early developed c-Met inhibitors were multikinase inhibitors. As early as 2011 and 2012, two multitarget c-Met inhibitors, crizotinib and cabozantinib, were approved for the treatment of NSCLC and medullary thyroid cancer (MTC) as well as RCC, respectively. 49 , 50 However, the indications are not based on their ability to target c-Met but are due to the inhibitory effect of crizotinib on the ALK fusion protein and the multikinase inhibitory activity of cabozantinib. The development of selective c-Met inhibitors has progressed rapidly in recent years, and two highly selective c-Met inhibitors, capmatinib and tepotinib, were approved in the first half of 2020 (Table 1 ). Capmatinib (INCB28060) is an oral competitive c-Met inhibitor with ≥10,000-fold selectivity for c-Met compared with other kinases and potently inhibits c-Met activity at picomolar concentrations. 51 In the GEOMETRY mono-1 trial ( NCT02414139 ) conducted in patients with MET exon 14 skipping mutations, capmatinib exhibited a high objective response rate (ORR) and relatively durable responses in both previously treated and newly diagnosed patients, including those with brain metastases. 52 Combination therapy of capmatinib and gefitinib was also evaluated in a phase II trial in NSCLC patients with disease progression after gefitinib treatment ( NCT01610336 ). A disease control rate of 80% was achieved in 65 subjects, and more responses were observed in patients with MET amplification. 53 Similar results were reported in the combination therapy of capmatinib with other EGFR inhibitors, such as erlotinib. 54 Due to its significant efficacy compared to existing therapies, capmatinib granted a breakthrough therapy designation by the FDA for NSCLC patients harboring MET exon 14 skipping mutations in 2019 and was approved for this indication on May 6, 2020. Tepotinib (EMD1214063), developed by Merck, has more than 1000-fold selectivity for c-Met. 55 Clinical trials of tepotinib ( NCT04647838 and NCT03940703 ) also showed significant effectiveness in the treatment of cancer patients harboring MET mutations and in combination therapy with EGFR TKIs. 56 It has been approved by the Ministry of Health, Labour and Welfare (MHLW) of Japan for the treatment of unresectable, advanced, or recurrent NSCLC in patients with skipping mutations in MET exon 14. 57

There are also many small-molecule c-Met inhibitors at different stages of clinical trials. Representative multikinase c-Met inhibitors include foretinib (XL880/GSK1363089), glesatinib (MGCD265), BMS-777607, and S49076 , which target c-Met/RON/VEGFR-2/KIT/TIE2/PDGFR, c-Met/TIE2/RON/VEGFR-1/2/3, c-Met/RON/AXL, and c-Met/AXL/MER/FGFR, respectively. 58 – 61 Several clinical trials were carried out to test their efficacy for cancer therapy, but some of the results have not been disclosed. In a phase I trial for NSCLC patients who progressed after chemotherapy ( NCT01068587 ), combined foretinib with erlotinib could achieve a response rate of 17.8% in the evaluated patients, and the clinical response was closely associated with baseline c-Met expression. 58 In a phase I trial conducted in patients with advanced solid tumors (ISRCTN00759419), S49076 was administered orally once daily or twice daily in continuous 21-day cycles at escalating doses, followed by an expansion phase at the RP2D. The results showed that 83 patients (81.4%) had side effects, and 93% of them were grade 1–2. Nine patients had more than 6 months of stable disease, and the overall clinical benefit rate was 23%. 61 In addition, several c-Met-specific inhibitors are also undergoing clinical research. Tivantinib is a non-ATP-competitive inhibitor of c-Met. In phase II randomized open-label study conducted in previously treated locally advanced or metastatic NSCLC patients ( NCT00777309 ), the combination of tivantinib and erlotinib showed an ORR of 10% vs. 7% of the control arm and a median PFS of 3.8 vs. 2.3 months in the control arm. 62 Volitinib (savolitinib) selectively inhibits c-Met activity in an ATP-dependent manner. The tolerability and safety of volitinib as monotherapy or in combination with gefitinib in NSCLC patients with mutant or wild-type EGFR have been studied in several phase I trials ( NCT01773018 , NCT02374645 ). 63 It was also assessed in combination with osimertinib for patients with resistant NSCLC harboring the T790M mutation in a phase II clinical study ( NCT02143466 ). The preliminary results showed that the combination of volitinib plus osimertinib had reliable safety and effectiveness. SAR125844 derived from triazolopyridazine is an effective specific c-Met inhibitor with an IC 50 value of 4 nM. 64 A phase I trial ( NCT01657214 ) of SAR125844 displayed encouraging anti-NSCLC activity in patients with MET amplification, and the drug was well tolerated. 65 More than a dozen c-Met inhibitors are currently under clinical assessment in China for the treatment of various malignancies either alone or in combination, such as bozitinib, ningetinib, glumetinib, and kanitinib. Some of them are undergoing phase II/III clinical trials and have great potential for approval in the near future. 66

In addition to selective and non-selective small-molecule c-Met inhibitors, monoclonal antibodies against HGF ligand and c-Met are also effective strategies targeting the HGF/c-Met axis. One of the major challenges for the clinical use of these c-Met inhibitors is to distinguish the applicable population that is most likely to derive benefits from HGF/c-Met targeted therapy. It has been reported that c-Met expression detected by immunohistochemistry cannot accurately define the potential benefit patients. 67 Biomarkers or biomarker combinations, such as MET mutations, MET gene amplification, and HGF expression, should be evaluated in cohorts receiving anti-c-Met targeted therapy to identify potential predictors of efficacy for specific c-Met inhibitors. 67 , 68 For example, the MET exon 14 skipping mutation is a biomarker predicting the response to capmatinib and tepotinib. Drug resistance is another issue that needs to be considered in the development and clinical use of c-Met inhibitors. Multiple mechanisms have been identified to contribute to resistance to HGF/c-Met targeted therapy, including amplification of MET or KRAS , MET secondary mutations, induction of HGF secretion, and increased bypass activation. 67 , 69 , 70 The clinical response to currently approved c-Met inhibitors is largely restricted to patients with MET amplification or exon 14 deletion. NSCLC patients who are resistant to EGFR inhibitors mediated by c-Met overexpression can also respond to c-Met inhibitors. However, inhibition of either c-Met or its ligand alone has not been proven to be potent in unselected cancer patients. Rationally designed combination strategies, such as combining c-Met inhibitors with HGF neutralizing antibodies, and cooperatively targeting upstream, downstream or parallel signaling, can not only improve the clinical benefits but also overcome drug resistance to c-Met inhibitors. Such strategies may also benefit a wide range of patients who lack MET gene abnormalities.

EGFR inhibitors

The epidermal growth factor receptor (EGFR) is a transmembrane protein implicated in a wide range of biological processes. Members of this family also include ERBB2/HER2, ERBB3/HER3, and ERBB4/HER4, which are structurally similar and consist of an extramembrane ligand-binding region, a single-stranded transmembrane region, and a highly conserved intra EGFR membrane tyrosine kinase region. 71 , 72 When the EGFR extracellular domain binds to its ligand, such as EGF and TGF-α, EGFR dimerizes and autophosphorylates, thereby activating downstream intracellular signaling cascades, which are closely related to cell proliferation, survival, and apoptosis. 72 Abnormal activation of EGFR mutations is an important contributor to the tumorigenesis of multiple cancer types, especially lung cancer, breast cancer, and pancreatic cancer. 73 – 75

As shown in Table 1 , several EGFR TKIs are clinically available. The first generation of EGFR TKIs, such as gefitinib, erlotinib, and icotinib, are reversible inhibitors with a quinazoline structure. These drugs are highly effective in NSCLC patients harboring EGFR-activating mutations (exon 19 deletion and exon 21 L858R). 74 , 76 , 77 They demonstrated a significant PFS benefit over platinum doublet chemotherapy in the clinic. In addition, erlotinib has also been used in combination with gemcitabine for the clinical treatment of pancreatic cancer. 78 The EGFR L858R/T790M dual mutation is the major cause of treatment failure (>50%) after taking the first generation of EGFR inhibitors. 79 The second-generation irreversible EGFR-TKIs afatinib and dacomitinib are designed to conquer the T790M mutation. 80 , 81 They can covalently bind to the ATP-binding pocket of EGFR and show stronger pharmacological activity than gefitinib. However, they also strongly inhibit wild-type EGFR and cause severe rash and diarrhea, thereby limiting their clinical doses. Therefore, these agents are only used for NSCLC patients harboring EGFR-sensitive mutations but could not benefit sufferers harboring the T790M mutant. 79 , 80 , 82 Novel pyrimidine-based third-generation EGFR TKIs have inhibitory effects on EGFR-activating mutations and the T790M mutation specifically but show weak inhibitory activity on wild-type EGFR. Osimertinib is the first approved third-generation EGFR inhibitor and can achieve a PFS of over 10 months in patients harboring the EGFR T790M mutation. 83 Almonertinib, developed by Hansoh Pharma, is an analog of osimertinib. This drug also showed significant anti-cancer effects in resistant patients with NSCLC in clinical trials, and has been approved for NSCLC therapy by the NMPA recently. 84 The success of osimertinib and almonertinib in overcoming acquired resistance is mainly attributed to their high potency and selectivity against the EGFR T790M mutation.

Lapatinib and neratinib, which are clinically available for patients with breast cancer, are dual-target inhibitors that inhibit the activities of both EGFR and HER2 (Table 1 ). Among them, lapatinib is a reversible TKI and is mainly used in combination with capecitabine for the treatment of advanced or metastatic breast cancers that show HER2 overexpression and have previously received treatment by anthracycline, paclitaxel, or Herceptin. 85 Neratinib is an irreversible inhibitor mainly used in breast cancer patients who have completed standard Herceptin-assisted treatment and are currently without but at high risk of progression. 86 Besides, tucatinib (irbinitinib) is a potent and selective HER2 inhibitor with an IC 50 of 8 nM. This is a newly approved HER2 inhibitor, and is also used for the treatment of patients with advanced unresectable or metastatic HER2-positive breast cancer. 87

In addition, many other EGFR inhibitors are undergoing clinical trials. Typically, olmutinib is an irreversible anilino-thienopyrimidine inhibitor of EGFR that shows high inhibitory activity against the L858R/T790M dual mutation or exon 19 deletion. 88 Phase I and phase II trials ( NCT01588145 , NCT02444819 , and NCT02485652 ) have been conducted to evaluate the efficacy and safety of olmutinib alone or in combination with drugs such as afatinib, bevacizumab, or pembrolizumab on NSCLC patients. 89 So far this drug is only clinically available in South Korea, and has not been approved in other countries due to the potential serious side effects, such as Stevens-Johnson syndrome. 88 , 90 Avitinib is an irreversible pyrrolopyrimidine derivative that is evaluated clinically for the treatment of T790M mutant NSCLC ( NCT03574402 ). 91 Its inhibitory activity was 300 times higher on the T790M mutant than on wild-type EGFR. Pelitinib is an irreversible fluroanilino-quinoline EGFR inhibitor. This agent has been assessed in phase II clinical trials ( NCT00072748 , NCT00072748 ) for patients with NSCLC or colorectal carcinoma. 92 Moreover, the third-generation EGFR inhibitor furmonertinib (alflutinib) developed by Allist Pharmaceuticals is being evaluated for the treatment of NSCLC in several clinical trials ( NCT02973763 , NCT03452592 , and NCT03787992 ).

Acquired resistance to EGFR TKIs develops after 9–14 months of treatment. The main causes of drug resistance include EGFR secondary mutation, activation of alternative pathways, and phenotypic transformation, especially the former. The acquired EGFR C797S mutation mediates resistance to third-generation EGFR inhibitors in ∼40% of osimertinib-treated NSCLC patients. 93 Both cis and trans mutations of C797S and T790M were observed in resistant cases. If EGFR C797S and T790M mutations occur in trans, the combination of first- and third-generation EGFR TKIs has been reported to be an effective treatment strategy. 94 If they occur in cis, the patients are resistant to all approved EGFR TKIs; this is also a focus for research on fourth-generation EGFR TKIs. 94 Jia et al. reported an allosteric inhibitor EAI045 that targets both C797 and T790M mutations but spares wild-type EGFR. A combination of EAI045 and cetuximab is effective in mouse models of lung cancer driven by EGFR (L858R/T790M) and by EGFR (L858R/T790M/C797S) mutants. 95 Moreover, Shen et al. designed and synthesized a series of 5-methylpyrimidopyridone derivatives as EGFR (L858R/T790M/C797S) inhibitors. 96 , 97 The representative compound 8r-B inhibited EGFR (L858R/T790M/C797S) mutant with an IC 50 of 27.5 nM. 97 Further PK-oriented optimization of 8r-B is ongoing.

FLT3 inhibitors

Fms-like tyrosine kinase 3 (FLT3), which is widely expressed in hematopoietic stem and progenitor cells, is a transmembrane protein encoded by the proto-oncogene FLT3 . It belongs to the type III RTK family, which also includes PDGFR, FMS, and KIT. All of them consist of an extracellular ligand-binding domain, a single transmembrane hydrophobic alpha helix region, and an intracellular kinase domain. FLT3 is activated by binding to the ligands, which results in its dimerization and conformational changes. Subsequent autophosphorylation of FLT3 triggers signal transduction, activating intracellular signaling cascades such as PI3K/AKT/mTOR, RAS/RAF/MAPK, and JAK/STAT, 98 , 99 which are closely related to cell proliferation, differentiation, survival, and apoptosis. FLT3 is widely overexpressed in patients with acute myeloid leukemia (AML), and its mutations lead to the constitutive activation of downstream signals. 100 , 101 Internal tandem duplication (ITD) mutations in FLT3 (FLT3-ITD) are detected in ~25% of AML patients, and point mutations in the tyrosine kinase domain (TKD) are observed in 7–10% of patients. 102 These mutations have been identified to be involved in the occurrence of leukemia. Due to the established pathogenetic and prognostic roles of FLT3-ITD and FLT3-TKD in AML, several FLT3 inhibitors have been developed for AML therapy.

The first-generation FLT3 inhibitors, including sorafenib, sunitinib, midostaurin, tandutinib, and lestaurtinib, are multikinase inhibitors. 103 – 105 They are not specific for FLT3 and have inhibitory activity against various other RTKs, such as PDGFR, KIT, VEGFR, RAF, or JAK2. The clinical efficacy of most of these inhibitors as monotherapy for AML was unimpressive, and their off-target inhibition also increased adverse events. 106 Therefore, clinical studies on first-generation FLT3 inhibitors for AML monotherapy were discontinued except for midostaurin. A randomized phase III trial (RATIFY study, NCT00651261 ) showed that the addition of midostaurin to cytarabine chemotherapy significantly improved overall survival (OS) for FLT3-mutated AML patients. 104 Based on the beneficial results of the RATIFY study, midostaurin was approved by the US FDA for combination therapy with standard chemotherapy in 2017 (Table 1 ). Distinguishingly, pexidartinib (Turalio) is also an orally bioavailable multitarget inhibitor, with IC 50 values of 9, 12, and 17 nM against FLT3-ITD, c-Kit, and colony-stimulating factor 1 receptor (CSF1R), respectively. However, it is not used clinically for AML therapy but approved for the treatment of adult patients with tenosynovial giant cell tumors (TGCTs). This indication is based on its inhibitory effect on CSF1R, which is frequently overexpressed in TGCTs. 107 Second-generation FLT3 inhibitors developed by rational drug design are more potent and specific and have less toxicity related to off-target effects. Gilteritinib is the first approved second-generation FLT3 inhibitor and is also the first effective FLT3 inhibitor for AML monotherapy (Table 1 ). 108 A randomized open-label phase III trial (ADMIRAL study, NCT02421939 ) showed that the median OS was significantly longer in the gilteritinib monotherapy group (9.3 months) than in conventional chemotherapy-treated patients (5.6 months) ( p < 0.001). It was approved for the treatment of relapsed or refractory AML patients with FLT3 mutations in 2018. Quizartinib was screened by the KinomeScan technique to improve the affinity and specificity to FLT3 kinase, and it showed strong activity and selectivity against FLT3-ITD but not TKD. 109 The clinical efficacy of quizartinib was also superior to conventional chemotherapy ( NCT00989261 ); therefore, it was approved for relapsed or refractory AML patients with FLT3-ITD mutations in 2019. 110

Currently, many promising FLT3 inhibitors are still under clinical evaluation. Crenolanib, originally developed as an inhibitor of PDGFR, is also a second-generation FLT3 inhibitor with inhibitory activity against both FLT3-ITD and FLT3 D835 mutations. 111 Crenolanib development focused on assessing the combination effects of this drug with conventional chemotherapy in terms of first-line and relapse treatment. Several clinical trials are underway to evaluate the clinical efficacy of crenolanib, including a randomized phase III trial evaluating the potency of crenolanib in combination with induction chemotherapy for relapsed or refractory FLT3-mutated AML patients ( NCT02298166 ) and a multicentre phase III trial comparing the effects of crenolanib with midostaurin during induction chemotherapy and consolidation therapy for newly diagnosed FLT3-mutated AML patients ( NCT03258931 ). SKLB-1028 is a multitarget inhibitor with FLT3 inhibitory activity. 112 A phase I trial ( NCT02859948 ) was conducted to evaluate the safety, tolerability and pharmacokinetic characteristics of SKLB-1028 in FLT3 mutant AML subjects. 113 Moreover, it has been reported that SKLB-1028 also has inhibitory activity on FLT3 secondary mutations such as FLT3-D835Y and FLT3-F691L; therefore, it is considered a potential therapeutic drug for resistant AML patients harboring the corresponding mutations. 114 The Bcr-Abl1 inhibitor ponatinib is currently approved for chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) therapy, and it is also a FLT3 inhibitor with potent inhibition of FLT3-ITD. 115 Phase I/II studies are ongoing to evaluate the efficacy and safety of ponatinib in combination with cytarabine for AML patients with the FLT3-ITD mutation ( NCT02428543 ) and its monotherapy or combination with azacitidine for untreated AML patients who are unfit for conventional chemotherapy ( NCT02829840 ). In addition, the anti-AML activity and safety of several other multikinase inhibitors with FLT3 inhibitory activity, such as AT-9283, ENMD-981693, 4SC-203, cabozantinib, and CR-4, are also being evaluated in the clinic ( NCT01054937 , NCT01961765 ). 116

Primary and secondary resistance is a challenging issue for TKI treatment, including FLT3 inhibitors, which results in the limited and transient efficacy of FLT3 inhibitors. Primary resistance to FLT3 inhibitors involves insensitive FLT3 mutations, expression of FGF2 or CYP3A4 in the bone marrow microenvironment, upregulation of anti-apoptotic proteins MCL-1, BCL-XL, or BCL-2, and other activated signals. Acquired resistance includes TKD secondary mutations at the activating loop residues (e.g., D835, D839, I836, and Y842) or the gatekeeper site F691, 117 , 118 autocrine FLT3 signaling, and activation of alternative pathways. The understanding of the molecular mechanisms associated with resistance to FLT3 inhibitors lays a foundation for establishing strategies to overcome and reduce resistance. Many combination strategies have been evaluated to improve the treatment outcome, such as the combination with epigenetic therapy (e.g., HDAC inhibitors), proteasome inhibitors, and inhibitors targeting independent signaling pathways or downstream pathways of FLT3-ITD (e.g., STAT5 inhibitor, CDK4/6 inhibitors, PI3K/mTOR inhibitors). 119 – 123 Another strategy is to develop irreversible FLT3 inhibitors. FF-10101 is a covalent-binding FLT3 inhibitor and can maintain the ability to bind FLT3 in either an active or inactive conformation. 124 The irreversible binding of FF-10101 provides potent inhibitory effects on multiple secondary mutations, such as F691L gatekeeper mutation. A phase I/IIa study ( NCT03194685 ) was conducted to assess its safety, tolerability, pharmacokinetics, and efficacy in subjects with relapsed or refractory AML. Meanwhile, a variety of novel FLT3 inhibitors, including 7 h, 125 PLX3379, 126 and MZH29, 127 are in the preclinical research and development stage. They are potential next-generation FLT3 inhibitors to conquer secondary mutation-mediated resistance.

VEGFR/FGFR/PDGFR inhibitors

Angiogenesis is a complex process through which new blood vessels form from pre-existing vessels. 128 In physiological circumstances, angiogenesis is strictly regulated by various endogenous pro-angiogenic and anti-angiogenic factors. 129 Aberrant angiogenesis exists in a wide range of diseases including arthritis, retinopathies, atherosclerosis, endometriosis, and cancer. 130 – 132 In 1971, Judah Folkman raised the hypothesis that solid tumors cause new blood vessel growth (angiogenesis) in the tumor microenvironment by secreting pro-angiogenic factors, initiating the research between angiogenesis and cancer. 133 Angiogenesis is critical for the development and subsequent growth of human solid tumors; otherwise, tumor size will not exceed 1–2 mm. 134 Tumors require new blood capillaries to provide nutrient and oxygen, remove metabolic waste, and facilitate the formation of metastases. 135 , 136 As an increasing number of tumor angiogenesis-related genes, transcription factors, signaling pathways, and their mechanisms of action have been revealed, anti-angiogenesis has become an attractive strategy for cancer therapy. 137 , 138 Well-known pro-angiogenic factors mediating the angiogenic switch include vascular endothelial growth factor (VEGF), 139 basic fibroblast growth factor (bFGF), 140 platelet-derived growth factor (PDGF), 141 transforming growth factor (TGF), 142 insulin-like growth factor, epidermal growth factor (EGF), 143 and angiopoietin. 144 In the past few years, efforts to develop anti-angiogenic treatments have mainly focused on inhibiting the activities of their receptors such as VEGF receptors (VEGFR-1-3), FGF receptors (FGFR1–4), PDGF receptors (PDGFRα and PDGFRβ), and TGF-β receptors (TGF-βRI, TGF-βRII, and TGF-βRIII). 131 , 145 , 146

Currently, more than 10 anti-angiogenic TKIs have been approved by the FDA and NMPA of China for the treatment of multiple solid malignancies, and most of them are multikinase inhibitors (Table 1 ). Sorafenib can inhibit a number of receptor tyrosine kinases (RTKs) including VEGFR-1/2/3, c-Kit, FLT3, RET, PDGFRβ, and RAF, and is the first approved anti-angiogenic inhibitor. 147 It was initially approved for the treatment of advanced RCC in 2005. Subsequently, the FDA-approved sorafenib for the treatment of advanced hepatocellular carcinoma (HCC) in 2007 based on encouraging results from the SHARP trial, and for differentiated thyroid carcinoma (DTC) in 2013 based on beneficial results from the DECISION trial. 148 , 149 Sorafenib is also the first small-molecule targeted drug to be approved for these three cancer indications. The multikinase inhibitor lenvatinib approved in 2015 has the same clinical indications as sorafenib, and they are currently the only two targeted agents used clinically for the first-line treatment of HCC. 148 , 150 – 152 Other approved anti-angiogenic inhibitors for the first- or second-line treatment of RCC or DTC include sunitinib 153 (2006), pazopanib 154 (2009), axitinib 155 (2012), cabozantinib 156 (2016), and tivozanib 157 (2017). Among them, sunitinib, an indol-2-one multikinase inhibitor targeting VEGFR-1/2/3, PDGFRα/β, c-Kit, CSF1R, RET, and FLT3, is the second approved anti-angiogenic TKI, and was simultaneously approved for two distinct indications including RCC and imatinib-resistant gastrointestinal stromal tumor (GIST). 158 , 159 The anilinoquinazoline derivative vandetanib inhibits the activities of EGFR, VEGFR-2/3, RET, BRK, TIE2, and EPH. It is the first drug to be approved for the treatment of adult patients with metastatic MTC by the FDA. 160 However, this indication is most likely attributed to its inhibitory effect on RET, a tyrosine kinase hyperactivated by mutations in MTC. 161 Another anti-angiogenic inhibitor used for the clinical treatment of MTC is cabozantinib, which also has high RET inhibitory activity. 162 Relatedly, the FDA-approved two highly specific RET inhibitors (selpercatinib and pralsetinib) in 2020. Both of them show a wide range of therapeutic effects on RET-driven ( RET mutation or RET fusion-positive) malignancies in clinical trials and have been approved for the treatment of advanced or metastatic RET -mutant MTC, RET fusion-positive NSCLC, and radioactive iodine-refractory thyroid cancer (Table 1 ). 163 , 164 Regorafenib developed by Bayer is a fluoro-derivative of sorafenib with activity against multiple kinases including VEGFR-1/2/3, PDGFRα/β, FGFR1/2, BRAF, c-Kit, and RET. 165 It has shown clinical effectiveness for patients with metastatic colorectal cancer (mCRC), who progress after prior standard treatment ( NCT01103323 ), and received FDA approval in 2012. 166 Afterward, the FDA expanded its indication to advanced GIST in 2013 based on the results of GRID clinical trial ( NCT01271712 ). In this phase III study, although no difference was observed in the OS between regorafenib and placebo groups (hazard ratio = 0.77, p = 0.199), the PFS was significantly improved to 4.8 months in the treatment group, and the placebo arm was just 0.9 months. 167 The indolinone derivative nintedanib targets VEGFR-1/2/3, FGFR1/2, and PDGFRα/β. 168 It was initially used clinically for the treatment of idiopathic pulmonary fibrosis 169 and the combination therapy of nintedanib and docetaxel was approved as a second-line treatment for patients with NSCLC in the same year by the European Medicines Agency (EMA) but not the FDA. 170 In the past decade, Chinese researchers have also made great progress in developing anti-angiogenic drugs, and several have been approved by the NMPA of China. Apatinib developed by Hengrui Medicine inhibits the activities of VEGFR-2, c-Src, and c-Kit simultaneously and was approved for the treatment of advanced gastric cancer in October 2014. 171 The multitarget inhibitor anlotinib is developed by Chiatai Tianqing. 172 This anti-angiogenic agent has been used for the treatment of several malignant tumors including NSCLC, soft tissue sarcoma (STS), and small cell lung cancer (SCLC). 173 These two drugs have been identified as orphan drugs by the FDA, but have not been launched in the United States. Fruquintinib developed by Hutchison Whampoa Limited is a potent small-molecule inhibitor of VEGFR-1/2/3. 174 The FRESCO trial, a randomized double-blind phase III study ( NCT02314819 ), laid the foundation for the approval of fruquintinib in patients with mCRC in 2018. 175 Its effectiveness and safety are being explored in a phase I trial conducted in a non-Chinese population in the United States. 176

In addition to anti-angiogenic effects, several selective FGFR or PDGFR inhibitors have also been approved recently, mainly functioning as therapeutic agents for FGFR or PDGFR-driven malignancies (Table 1 ). Erdafitinib is an orally potent pan-FGFR inhibitor with IC 50 values of 1.2, 2.5, 3.0, and 5.7 nM against FGFR1, 2, 3, and 4, respectively. 177 As the first approved FGFR-selective inhibitor, it has been used for the second-line treatment of locally advanced or metastatic urothelial carcinoma. Meanwhile, it is undergoing clinical development as a treatment for other malignancies including NSCLC, gastric cancer, prostate cancer, cholangiocarcinoma, esophageal cancer, and lymphoma. 178 Pemigatinib, also known as INCB054828, is an orally potent FGFR-selective inhibitor with IC 50 values in the nanomolar range against FGFR1-3. Based on the results of FIGHT-202 ( NCT02924376 ), a phase II, open-label, single-arm, multicenter study to evaluate the efficacy and safety of pemigatinib in cholangiocarcinoma subjects, pemigatinib received accelerated approval in April 2020 for the treatment of patients with previously treated, advanced/metastatic, or surgically unresectable cholangiocarcinoma harboring FGFR2 fusions or other rearrangements. 179 This is also the first approved targeted treatment for cholangiocarcinoma. 180 Pemigatinib is being evaluated for clinical use in several other FGFR-driven cancers and received orphan designation in August 2019 by the FDA for the treatment of myeloid/lymphoid neoplasms with eosinophilia and rearrangement of FGFR1 or PDGFRα/β. 179 Avapritinib, developed by Blueprint Medicines, is an effective and selective inhibitor of PDGFRα and KIT activation loop mutants, which has recently been approved in the United States for adults with unresectable or metastatic GIST harboring PDGFRα exon 18 mutations. 181 GIST can be classified according to different molecular subtypes, and KIT or PDGFRα mutated GISTs are important subgroups that commonly arise in the stomach. Five TKIs of PDGFRs or KIT (imatinib, sunitinib, regorafenib, avapritinib, and ripretinib) are currently used clinically for GIST therapy. 182 As target-specific inhibitors, avapritinib and ripretinib have shown extensive inhibitory effects on KIT or PDGFRα mutant GIST, and are potent for patients harboring primary or secondary resistant mutations, including the PDGFRα D842V mutant. 183 – 185

Efforts are being made to expand many of the above-mentioned approved VEGFR, FGFR, or PDGFR inhibitors to other cancer indications. Meanwhile, a large number of novel inhibitors are also being developed, many of which have entered clinical trials. Several representative drug candidates with the potential to receive approval in the near future are presented here. Cediranib developed by AstraZeneca is a potent multikinase inhibitor targeting VEGFR-1/2/3, PDGFRα/β, and c-Kit. 186 However, AstraZeneca discontinued the development of cediranib for the treatment of mCRC, NSCLC, and recurrent glioblastoma due to the mediocre results from phase III clinical trials ( NCT00399035 , NCT00795340 , NCT00777153 ) in these indications. At present, it shows new hope in the combination therapy of ovarian cancer, and two phase III trials ( NCT03278717 and NCT02446600 ) are ongoing to compare the efficacy of cediranib plus olaparib with olaparib alone or in patients with ovarian cancer. 187 Dovitinib, also named CHIR-258, is a multitargeted anti-angiogenic inhibitor with IC 50 s of 10/13/8, 2, 1, 8/9, and 27/210 nM for VEGFR-1/2/3, c-Kit, FLT3, FGFR1/3, and PDGFRα/β, respectively. 188 In an open-label, randomized, phase III study to compare the safety and efficacy of dovitinib vs. sorafenib in patients with metastatic RCC ( NCT01223027 ), there was no difference in PFS between these two drugs in third-line treatment. 189 It received orphan drug designation for the treatment of adenoid cystic carcinoma in 2019. Motesanib is an orally administered multikinase inhibitor of VEGFR-1/2/3, PDGFRα/β, c-Kit, and RET, and was considered a potent anti-NSCLC drug in Asian patients based on the MONET1 trial. 190 However, the results of a later phase III trial ( NCT02629848 ) evaluating the efficacy of motesanib plus paclitaxel and carboplatin were disappointing. Motesanib plus paclitaxel/carboplatin did not significantly improve PFS vs. placebo plus paclitaxel/carboplatin (median PFS: 6.1 verse 5.6 months) in East Asian patients with stage IV/recurrent non-squamous NSCLC. 191 Nevertheless, it showed marked anti-cancer effects in patients with advanced thyroid cancer in two phase II studies ( NCT00121628 , NCT02084732 ). 192 , 193 Recently, a phase II study ( NCT00121628 ) assessing the efficacy of motesanib in low-grade neuroendocrine tumors (NETs) also achieved satisfactory treatment results with a 4-month PFS of 78.5%, and the median PFS of all patients was 8.7 months. 194 Sulfatinib is a potent inhibitor against VEGFR-1/2/3, FGFR1, and CSF1R with IC 50 values in the range of 1–24 nM. 195 An encouraging antitumor activity and acceptable safety profile were observed in a phase I trial ( NCT02133157 ), particularly in NETs. 196 It is currently being evaluated in advanced NETs in two phase III studies ( NCT02589821 and NCT02588170 ). 197 Crenolanib is an orally bioavailable TKI of PDGFRα/β and FLT3. 198 A multicenter, randomized, double-blinded, phase III trial ( NCT02847429 ) was conducted to assess the efficacy of oral crenolanib vs. oral placebo in combination with best supportive care in subjects with advanced or metastatic GIST with PDGFRα D842V mutation. 199 In addition, due to its high inhibitory activity on both FLT3-ITD and FLT3-TKD mutant subtypes, which are important therapeutic targets for AML, crenolanib is also evaluated in a phase III randomized multicenter study conducted in AML subjects with FLT3 mutation ( NCT03258931 ). 200 Lucitanib, developed by Shanghai HaiHe Biopharma, potently and selectively inhibits VEGFR-1, VEGFR-2, VEGFR3, FGFR1, and FGFR2 with IC 50 values of 7, 25, 10, 17.5, and 82.5 nM, respectively. 201 A multicenter phase III study is being conducted to evaluate the efficacy of lucitanib in combination with carboplatin plus etoposide in untreated participants with extensive-stage SCLC ( NCT04254471 ). Brivanib, in particular, is an orally active L-alanine ester prodrug that inhibits VEGFR-2 with an IC 50 of 25 nM, and has moderate potency against VEGFR-1 and FGFR1. 202 It is currently being evaluated in a phase III clinical trial on subjects with advanced HCC, who have failed or are intolerant to sorafenib ( NCT00858871 ). 203 , 204 Meanwhile, multiple noncovalent FGFR-selective inhibitors (pan-FGFR inhibitors) have also advanced to phase III trials, including the well-known AZD4547 205 and infigratinib, 206 both of which are typical type-I inhibitors. The former is being assessed for the treatment of patients with stage IV SCLC in phase II/III trial ( NCT02965378 ), and the latter was granted orphan drug designation by the FDA for the treatment of cholangiocarcinoma in 2019. 207 In addition, targeting TGF-β signaling is also a potential strategy for anti-angiogenic therapy. 208 However, no TGF-βR inhibitor has been approved for clinical use, and several drug candidates are undergoing evaluation in clinical trials including galunisertib, vactosertib, LY-3200882, PF-06952229, YL-13027, and GFH018. 209 – 211 Galunisertib, a selective TGF-βR type-I (TGF-βRI) kinase inhibitor with an IC 50 of 56 nM, is the most advanced drug candidate among them. 209 , 212 It has been evaluated clinically in several solid tumors including HCC, glioma, and glioblastoma multiforme, and achieved remarkable effect in the treatment of HCC. The median OS of galunisertib monotherapy for HCC can reach 16.8 months. 213 , 214

The concept of “starving tumors to death” by inhibiting tumor angiogenesis has promoted the development of anti-angiogenic therapy. 215 However, many anti-angiogenesis drugs produced only modest survival benefits for cancer patients in clinical trials. 216 , 217 One of the reasons can be explained by vascular normalization; this theory emphasizes that anti-angiogenesis agents mainly selectively block the formation of immature blood vessels rather than the mature and functional vasculatures. Therefore, anti-angiogenesis treatment alone is generally ineffective unless combined with chemotherapy. 218 , 219 On the other hand, tumor angiogenesis is regulated by multiple signaling pathways, and many interconnected pathways can compensate for the effect of single inhibition of one of these signals, such as the VEGFR pathway. This is why the approved small-molecule anti-angiogenesis TKIs are mostly multitarget inhibitors and only several selective inhibitors are used clinically for cancer patients with specific mutations. This also indicates the importance of combination therapy in the clinical use of anti-angiogenesis agents. 220 , 221 Accumulating evidences have demonstrated that anti-angiogenic therapy can not only inhibit the formation of neo-vascular, but also regulate the immune microenvironment, which provides a theoretic basis for the combination of anti-angiogenesis agents with immunotherapy. 221 Hundreds of clinical studies have been designed to evaluate such combination strategies. Encouragingly, the combination of axitinib with PD-1 antibody pembrolizumab has been approved for the treatment of patients with advanced RCC in 2019. 222 Another challenge of anti-angiogenic therapy is the lack of more personalized use of existing anti-angiogenesis agents. This is also a significant feature that distinguishes most anti-angiogenesis TKIs from other molecular targeted therapies; the former is given to unselected patients within approved indications, while the latter are used in proper patients selected by robust biomarkers, which markedly improve their clinical benefits. Most efforts have been made to identify molecular biomarkers for anti-angiogenesis agents, such as expression of VEGF and FGF in blood and tumors, 223 tumor perfusion status, 224 and other angiogenic factors, but none of them have yet been validated for routine clinical use. Recently, several studies have suggested that anti-angiogenesis-related side effects, such as hypothyroidism, high blood pressure, or hand-foot syndrome, may be associated with the antitumor efficacy of angiogenesis inhibitors. 223 , 224 As the lack of reliable predictive biomarkers in the clinic, these side effects may contribute to clinical decision, but further clinical verification is still needed.

TRK inhibitors

The tropomyosin receptor kinase (TRK) family is composed of three members, TRKA, TRKB, and TRKC, which are encoded by the neurotrophic tyrosine receptor kinase (NTRK) genes NTRK1 , NTRK2 , and NTRK3 , respectively. 225 To activate TRK receptors, neurotrophins (TRK ligands) bind to the extracellular domain of the receptors, stimulating homodimerization and autophosphorylation of TRK proteins, thereby activating downstream signaling pathways, such as RAS/MAPK/ERK, PI3K/AKT, and PLCγ. NTRK gene rearrangements containing a kinase domain of one of the three TRKs and a dimerization domain of another gene generate fusion proteins and result in aberrant activation of TRKs, which have been identified as oncogenic drivers of various cancers. 226 – 228 Therefore, TRKs are emerging as important targets for cancer therapy. The rearrangements of NTRK genes occur in only 1% of all malignancies; they have been widely detected at low frequencies in some common cancers, such as lung cancer, thyroid carcinoma, glioblastoma, and colorectal cancer. However, in several rare pediatric and adult cancer types, including infantile fibrosarcoma, secretory breast carcinoma, and salivary gland secretory carcinoma, NTRK gene rearrangements are common.

The discovery of TRK inhibitors renewed interest in NTRK gene rearrangements as oncogenes. Currently, two first-generation TRK inhibitors are available for clinical cancer treatment (Table 1 ). Larotrectinib is the first approved selective oral pan-TRK inhibitor with high potency against TRKA, TRKB, and TRKC. 229 Entrectinib (RXDX-101/NMS-E628) is a potent multikinase inhibitor targeting TRKA/B/C, ROS1, and ALK. 230 Both agents received the FDA breakthrough therapy identification; this breakthrough designation highlights the efficacy of TRK inhibitors in various cancers that have the same mutation, regardless of cancer type and patient age. Based on the tumor-agnostic efficacy of the “basket trail” conducted in diverse NTRK fusion-positive cancers, larotrectinib and entrectinib granted FDA approval for the treatment of adult and pediatric patients with TRK fusion solid tumors. In clinical use, NTRK gene fusions should be diagnosed to select patients for targeted TRK therapy. Remarkably, both larotrectinib and entrectinib displayed activity against CNS tumors with NTRK fusions, indicating the ability for BBB penetration. 231 , 232 When referring to the adverse events of first-generation TRK inhibitors, it should be noted that both agents have favorable overall safety profiles compared to other small-molecule TKIs. These drugs are generally well-tolerated in patients, with low incidences of dose reductions, discontinuations, and grade 3–4 adverse events. 225

Recently, there have been several small-molecule TRK inhibitors in different stages of clinical research, some of which target multiple kinases, such as cabozantinib (targeting c-Met, RET, VEGFR-2, ROS1, ALK, and TRK), 233 merestinib (targeting c-Met, TEK, ROS1, and TRK), 234 belizatinib (targeting ALK and TRK), 235 sitravatinib (targeting c-Met, RET, AXL, and TRK), 236 altiratinib (targeting c-Met, TIE2, VEGFR-2, FLT3, and TRK), 237 and DS-6051b (targeting ROS and TRK). 238 These inhibitors displayed varying degrees of inhibitory activity against TRK. Some of them have been approved for indications other than TRK fusion tumors; for example, cabozantinib was approved as an anti-angiogenic inhibitor for the treatment of patients with advanced RCC in 2016, and data are limited on its efficacy against NTRK fusions. However, with the increasing interest in TRK as a cancer therapy target, an increasing number of clinical trials have been performed to evaluate the effects of these inhibitors in patients with TRK fusion-positive tumors. In addition, a phase I study was carried out to assess the safety, PKs, and PDs of the selective TRK inhibitor PLX7486 as a single agent in patients with any histological solid tumors with activating NTRK point or NTRK fusion mutations ( NCT01804530 ). 239 However, the results were not disclosed.

Acquired resistance to TKI treatment can be mediated by on-target mutations or off-target (bypass activation) mechanisms. Until now, on-target mutations in the kinase domain of NTRK fusion have been the only resistance mechanism of first-generation TRK inhibitors, which can result in amino acid substitutions of the solvent front, activation loop xDFG motif, and gatekeeper residues in the kinase domain of TRK fusion proteins, interfering with TRK inhibitor binding. 225 The first resistance case to TRK inhibition was discovered in a colorectal cancer patient treated with entrectinib, and two acquired resistance mutations, TRKA G595R and TRKA G667C, were detected in the plasma cfDNA of this patient. 240 Several other resistant mutations were subsequently identified in patients resistant to larotrectinib and entrectinib, including the acquired TRKC G623R substitution, A608D mutation and gatekeeper F589L substitution in TRKA, and the substitutions involving the xDFG site of TRKA (G667S) and TRKC (G696A). 229 , 241 Fortunately, next-generation TRK inhibitors are currently in development to overcome acquired resistance to larotrectinib and entrectinib. In particular, selitrectinib (LOXO-195), repotrectinib (TPX-0005), and ONO-5390556 have demonstrated nanomolar inhibitory activity against the TRK mutants mentioned above. 242 Among them, the safety and efficacy of selitrectinib and repotrectinib are currently under assessment in phase I/II trials ( NCT04275960 , NCT04094610 ).

Non-receptor tyrosine kinase inhibitors

Bcr-Abl1 inhibitors

c-Abl is encoded by the abelson murine leukemia 1 ( ABL1 ) gene on chromosome 9 and belongs to the Abl family of non-receptor tyrosine kinase; it has been implicated in a range of cellular processes including the regulation of cell differentiation, cell cycle, and survival. Philadelphia (Ph) chromosome translocation results in the molecular juxtaposition of ABL1 and the breakpoint cluster region ( BCR ) of chromosome 22, forming an aberrant BCR-ABL fusion gene on chromosome 22. 243 This gene encodes a 210 kDa oncoprotein (p210 Bcr-Abl1) that is capable of autophosphorylation and constitutively activates the downstream pathway, thereby driving the uncontrolled proliferation of leukemia cells in almost all cases of CML and ~20% of patients with ALL. 244 – 246 The BCR-ABL fusion gene was identified as a specific biomarker for diagnosis and prediction of response to treatment, while Bcr-Abl1 fusion tyrosine kinase is considered to be a susceptible target for certain leukemias.

As indicated in Table 2 , imatinib is the first approved Bcr-Abl1 inhibitor as well as the first approved small-molecule TKI, which launches a new era of tumor-targeted therapy. This agent has shown striking activity in patients with chronic phase CML (CML-CP) and Ph + ALL. 247 A 5-year follow-up study conducted in patients with CML-CP receiving interferon or imatinib treatment showed that the OS and PFS of patients taking imatinib could reach 89% and 93%, respectively. 248 The introduction of imatinib for the treatment of CML patients with Ph chromosome translocation provides a proof-of-principle for using aberrant kinases as therapeutic targets. Currently, this drug represents the gold therapeutic standard in patients with CML in the clinical setting. Although treatment with imatinib has achieved exciting results, drug resistance caused by point mutations in the kinase domain of BCR-ABL has frequently emerged such as G250E, Q252H, Y253H/F, and E255K/V mutations located in the P loop region, T315I mutation in the ATP-binding region, and H395P/R mutation in the activation region. 248 – 251 Point mutations decrease the affinity of imatinib to the Bcr-Abl1 kinase domain, resulting in reduced imatinib inhibitory activity. 249 , 252 The increasing recognition of imatinib resistance stimulates the development of second-generation Bcr-Abl1 inhibitors including dasatinib, nilotinib, bosutinib, and radotinib, which were approved in 2006, 2007, 2012, and 2012, respectively. 253 – 256 Both dasatinib and bosutinib are oral dual Src/Abl1 kinase inhibitors, and the former is ~300-fold more potent than imatinib. Nilotinib, an aniline pyrimidine derivative developed from imatinib by crystallographic analysis and structural modification, has better lipophilicity and solubility and ~30-fold higher potency. Radotinib is the structural analog of nilotinib and is used as a second-line treatment in the clinic. These inhibitors can suppress most clinically relevant BCR-ABL mutants, except T315I gatekeeper mutation, which occurs in up to 20% of patients with resistant CML. 256 , 257 Ponatinib is a third-generation Bcr-Abl1 inhibitor with activity against T315I mutation. 258 The binding pattern of ponatinib is similar to imatinib, except that the carbon-carbon triple bond extending from the purine of ponatinib enforces compatibility with T315I residue. It is currently approved for the treatment of patients with CML or ALL that are either resistant or unable to tolerate other Bcr-Abl1 inhibitors. In addition, due to the multitarget properties of imatinib, dasatinib, and nilotinib, they were also evaluated clinically for the treatment of some solid tumors. Among them, imatinib was approved for GIST therapy in 2003 (Table 2 ). Table 2 Properties of approved small-molecule inhibitors of non-receptor tyrosine kinases Chemical structure Name Targets Approved indications (year) Corporation Imatinib (Gleevec) Bcr-Abl/PDGFR-β/c-Kit CML (2001) GIST (2003) ALL (2006) Novartis Dasatinib (Spraysel) Bcr-Abl/Src/c-Kit/LCK/PDGFR-β CML (2006) ALL (2006) Bristol-Myers Squibb Nilotinib (Tasigna) Bcr-Abl/DDR1/2 CML (2007) Novartis Bosutinib (Bosulif) Abl1/Src CML (2012) Pfizer Radotinib (Supect) Bcr-Abl CML (2012) IL-Yang Ponatinib (Iclusig) Bcr-Abl /PDGFR-α/VEGFR-2/FGFR-1/Src/FLT3/c-Kit CML (2013) ALL (2013) Incyte/Takeda Ibrutinib (Imbruvica) BTK MCL (2013) CLL (2014) WM (2015) SLL (2016) MZL (2017) AbbVie/ Johnson & Johnson Acalabrutinib (Calquence) BTK MCL (2017) AstraZeneca Zanubrutinib (Brukinsa) BTK MCL (2019) BeiGene Ruxolitinib (Jakafi) JAK1/2 Myelofibrosis (2011) Incyte/Novartis Fedratinib (Inrebic) JAK2 Myelofibrosis (2019) Impact

Research on the development of novel Bcr-Abl1 inhibitors against drug-resistant mutations is ongoing. To date, up to 13 inhibitors have entered clinical trials. Typically, asciminib (ABL001) is a potent and selective allosteric Abl1 inhibitor, which binds to the myristoyl pocket of Abl1 and induces the formation of an inactive kinase conformation. 259 Phase I clinical trials are being conducted to evaluate the efficacy and safety of this drug alone or in combination with dasatinib and prednisone in patients with CML or BCR-ABL -positive B-cell ALL ( NCT02081378 , NCT03595917 ). Meanwhile, it is also evaluated in phase III clinical trial to compare its efficacy with bosutinib in patients with CML-CP ( NCT03106779 ). Rebastinib (DCC-2036) is a potent conformational control inhibitor, designed to conquer Bcr-Abl1 resistant mutations, mainly T315I. 260 It induces the kinase to a catalytically inactive state, regardless of gatekeeper mutations. Clinically, a phase I dose-finding study of rebastinib in patients with relapsed CML has been completed ( NCT00827138 ); however, the clinical benefit was considered insufficient to support its continued use in leukemia treatment since the advent of ponatinib. Bafetinib (INNO-406) was developed to expand the susceptibility spectrum of mutations to TKIs and increase the selectivity to Bcr-Abl1 to reduce adverse reactions. 261 In a phase I trial ( NCT00352677 ), bafetinib as second-line treatment can achieve complete cytogenetic response in 19% of patients with CML and Ph + ALL that is resistant or intolerant to imatinib, indicating its potential clinical efficacy.

Outcomes for patients with CML have been greatly improved since the clinical application of Bcr-Abl1 TKIs. However, despite the high initial response rate of these inhibitors, drug resistance and adverse events are two main problems influencing the achievement of the best response and quality of life for patients. Sequential therapy with Bcr-Abl1 inhibitors leads to the continuous acquisition of novel mutations, especially compound mutants, which refers to the accumulation of more than one mutation in the same allele. Ponatinib is the latest generation of Bcr-Abl1 inhibitor, and patients treated with this drug have developed new resistance mutations, such as T315M single-point mutation and complex mutations T315I/E255V and E255V/Y253H. 262 – 264 Overcoming these resistance mutations requires the development of next-generation inhibitors and combination therapy strategies. Meanwhile, it has also been reported that some mutations are sensitive to the second-generation inhibitors, such as T315A to nilotinib, E255K/V and Y253H to dasatinib and bosutinib, and patients harboring such mutations can be retreated with these drugs. The clinical use of ponatinib is associated with cardiovascular events, and concerns about arterial thrombosis may limit its treatment in some patients with T315I mutations. For these patients, omacetaxine mepesuccinate approved by the FDA in 2012 is a proper treatment option. 265 , 266 It has shown encouraging therapeutic results in patients harboring T315I mutation and is tolerable without cardiovascular toxicity. Moreover, some patients with GIST and systemic mastocytosis can benefit from the treatment of imatinib, dasatinib, or nilotinib due to the broad-spectrum selectivity of them against c-Kit, PDGFR, or Src. 267 , 268 Mutations of these kinases can also drive the selection of appropriate inhibitors. An in vitro study showed that imatinib-resistant mutations PDGFRα D842V and c-Kit D816V that commonly occur in GISTs and mastocytosis, respectively, could be strongly inhibited by dasatinib. 269

BTK inhibitors

The B-cell receptor (BCR) pathway has a key role in the progression of a variety of B-cell malignancies. 270 Abnormal activation of BCR signaling has been identified in multiple heterogeneous hematologic malignancies, including B-cell non-Hodgkin’s lymphoma (NHL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma, Waldenstrom’s macroglobulinemia (WM), and DLBCL. 271 Bruton’s agammaglobulinemia tyrosine kinase (BTK), a crucial component of the BCR pathway, belongs to the non-receptor tyrosine kinase of the TEC family, which contains four other members: tyrosine kinase expressed in hepatocellular carcinoma (TEC), interleukin-2-inducible T-cell kinase (ITK), resting lymphocyte kinase (RLK/TXK), and bone marrow expressed kinase (BMX). 272 BTK is abundantly expressed in B-cell leukemias and lymphomas and functions as a vital regulator of cell proliferation and survival in various B-cell malignancies. 273 Inhibiting BTK is considered an effective therapeutic strategy for some hematologic malignancies. 274

Ibrutinib is the first-generation BTK inhibitor and has been proven to be superior to standard chemotherapy in multiple studies including older patients with significant comorbidity. It is an irreversible small-molecule inhibitor that covalently binds to Cys-481 within the ATP-binding pocket of BTK. Based on the high response rates and durable responses of its monotherapy 275 or in combination with anti-CD20 antibody, 276 ibrutinib has been approved by the FDA for the treatment of MCL, CLL, WM, SLL, and MZL between 2013 and 2017 (Table 2 ). 277 – 279 The clinical efficacy of ibrutinib in the treatment of DLBCL, refractory/recurrent primary central nervous system lymphoma, and secondary central nervous system lymphoma is still undergoing evaluation to expand its indications. 280 Despite the clinical achievement of ibrutinib, side effects including arthralgia, atrial fibrillation, pneumonitis and rash have also been reported and limit its clinical use. Most of the toxicity of ibrutinib is due to its off-target activities against four other TEC family kinases, EGFR, HER2, and Janus kinase 3 (JAK3). 281 Particularly, in combination therapy with the CD20 antibody rituximab, off-target inhibition of ITK by ibrutinib led to an antagonistic effect on antibody-dependent cell-mediated cytotoxicity, influencing the combined effects. 282 The off-target activity of ibrutinib triggered the development of more selective second-generation BTK inhibitors. Acalabrutinib 283 and zanubrutinib 284 are currently approved second-generation BTK inhibitors (Table 2 ). Similar to ibrutinib, they are irreversible inhibitors and form covalent bonds with the Cys-481 residue of the BTK active site. 284 And their selectivity is significantly improved. Acalabrutinib inhibited BTK with an IC 50 of 3 nM and had less off-target activity on EGFR, ITK, or TEC; 285 zanubrutinib had similar inhibitory activity to ibrutinib against BTK, but its IC 50 s on TEC, ITK, EGFR, HER2, and JAK3 were 2–70 times higher than those of ibrutinib. 286 Currently, the FDA has approved them for the treatment of adult MCL patients who have received at least one prior therapy. 287 Both of them are still evaluated in the clinic for the treatment of some other malignancies, such as NHL, 288 multiple myeloma (MM), 289 and ovarian cancer. 290

Several promising BTK irreversible inhibitors are under clinical evaluation. 291 Tirabrutinib (ONO-4059) is a highly selective covalent inhibitor of BTK with an IC 50 of 2.2 nM. In a phase I trial conducted in patients with B-cell malignancies ( NCT02457559 ), tirabrutinib showed significant potency on patients in the CLL group. Ninety-six percent of CLL patients responded to tirabrutinib, and all of the evaluated CLL patients harboring del 17p or TP53 mutations without del 17p responded. 292 Moreover, a phase II trial ( NCT02968563 ) is underway to assess the efficacy and safety of tirabrutinib in combination with the PI3K inhibitor idelalisib and the anti-CD20 antibody obinutuzumab. 293 Spebrutinib (CC-292/AVL-292) is also a second-generation BTK inhibitor and inhibits BTK activity with an IC 50 of 0.5 nM. 294 The results of phase I studies ( NCT01692184 , NCT01732861 , and NCT01351935 ) showed that spebrutinib was safe and well-tolerated following once-daily administration in patients with relapsed or refractory CLL/SLL, WM, and NHL. 295 Despite its high in vitro activity, spebrutinib exhibited inferior clinical efficacy compared with the approved BTK inhibitors. The reasons for the suboptimal effect are not fully understood, but the highly variable PK and pharmacodynamics (PD) seem to limit spebrutinib to continuously reach the in vivo targets. 296 In addition, due to the critical role of BTK in the development and function of B cells, BTK has also been confirmed as a potential therapeutic target for autoimmune disorders. Several BTK inhibitors including evobrutinib, 297 spebrutinib, 294 branebrutinib, 298 and HM71224 299 have been assessed in clinical trials for the treatment of autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythaematosus, and remitting multiple sclerosis.

Both the first and second generations of BTK inhibitors covalently bind to the sulfhydryl group of Cys-481 in the active site of BTK. Cys-481 is reported to be the frequently mutated residue of BTK in cases of resistance to irreversible BTK inhibitors. Several mutants, including C481S, C481R, C481F, C481Y, and C481T, have been identified in resistant patients, especially C481S, which results in the vast majority of drug resistance. 300 Moreover, PLCγ2 mutations have also been implicated in BTK inhibitor resistance. As a downstream protein of BTK, PLCγ2 mutations drive continued signaling regardless of BTK activity. 301 Developing noncovalent BTK inhibitors is one of the available strategies to overcome resistance caused by Cys-481 mutations. Vecabrutinib, 302 LOXO-305, 303 fenebrutinib 304 and ARQ-531 305 are all noncovalent reversible inhibitors of BTK undergoing clinical evaluation. These agents inhibit BTK activity without forming covalent bonds with the Cys-481 residue; therefore, they have the same inhibitory effects on either wild-type BTK or its mutants. Another strategy is to target other components of the BCR pathway, such as inhibition of the upstream signaling pathways LYN or SYK, which can restrain BTK phosphorylation and conquer resistance caused by PLCγ2 mutations. In addition, the combined treatment of BTK inhibitors and other targeted agents is also considered a potential strategy for patients resistant to BTK inhibitor monotherapy. 306

JAK inhibitors

Janus kinases (JAKs) belong to the family of non-receptor tyrosine kinases and are composed of four isoforms, JAK1, JAK2, JAK3, and TYK2, with up to 70% homology. 307 , 308 JAK1, JAK2, and TYK2 are widely distributed in various tissues and cells, while JAK3 is only expressed in the bone marrow and lymphatic-derived cells. 309 JAKs are able to transfer extracellular signals to the nucleus and mediate DNA transcription and protein expression. 310 Receptor-coupled JAKs can be activated when inflammatory cytokines such as interleukin and interferon bind to cytokine receptors. 311 Then JAKs catalyze the phosphorylation of receptor tyrosine residues and recruit and phosphorylate downstream signal transducer and activator of transcription (STAT) proteins. Activated STAT proteins promote their translocation to the nucleus and regulation of target-gene transcription and expression. Distinct cytoplasmic domains of cytokine receptors activate different JAKs and STATs. 312 The JAK/STAT pathway runs downstream of more than 50 cytokines and growth factors and is considered to be the central communication node for the immune system. JA-7 Given the important role of the JAK/STAT pathway in cytokine signal transduction, targeting JAK/STAT is considered a promising strategy for the treatment of multiple autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. Additionally, STAT signals (e.g., STAT3, STAT5, or STAT6) have been found to be frequently activated in malignant tumors, especially hematopoietic cancers and are involved in cell proliferation, survival, invasion, or inflammation. 313 As the critical upstream protein of STAT signals, JAKs are also potential targets for cancer treatment. The application of JAK inhibitors in cancer is mainly focused on hematologic malignancies. 314

Thus far, four JAK inhibitors have been approved for clinical use. As shown in Table 2 , ruxolitinib is the first launched JAK inhibitor and selectively targets JAK1 and JAK2 with moderate activity against TYK2. It was approved for the treatment of myelofibrosis (a myeloproliferative neoplasm), polycythemia vera, and bone marrow cancer in 2011 by the FDA. 315 The recently approved fedratinib is a selective JAK2 inhibitor and is used for myelofibrosis treatment in the clinic. 316 Meanwhile, it has been reported that fedratinib also showed efficacy in the treatment of NSCLC in preclinical studies. It can reverse the resistance of NSCLC cells to erlotinib and abrogate PD-L1 expression, which mediates immune checkpoint blockade therapy in NSCLC. 317 Another two approved JAK inhibitors are tofacitinib and baricitinib. 318 , 319 They are all used clinically for the treatment of autoimmune diseases. 313

Recently, the role of several new JAK inhibitors in cancer treatment is undergoing clinical evaluation. WP1066 is a novel JAK2 and STAT3 inhibitor with little activity against JAK1 and JAK3. A phase I trial of WP1066 was performed to evaluate its effects in the treatment of melanoma and glioblastoma ( NCT01904123 ). 320 Gandotinib (LY2784544) is an orally potent inhibitor of JAK1 and JAK2. 274 Clinical studies have demonstrated that gandotinib has adequate efficacy, safety, and tolerability profiles in patients with myeloproliferative neoplasms ( NCT01594723 ). Lestaurtinib (CEP701) is a multikinase inhibitor targeting JAK2, FLT3, and neurotropin receptor TrkA. The use of lestaurtinib to treat multiple cancers, including AML, Hodgkin lymphoma, neuroblastoma, prostate cancer, and myeloproliferative disorders, has been reported in experimental or clinical studies. 321 A phase II study ( NCT00668421 ) exhibited moderate efficacy and moderate but frequent gastrointestinal toxicity of lestaurtinib in myelofibrosis patients. 322 Further studies are still needed to assess its clinical benefits. INCB039110 is an effective JAK1 inhibitor with >20-fold selectivity over JAK2 and >100-fold over JAK3 and TYK2. 323 In an open-label phase II trial ( NCT01633372 ), myelofibrosis-related symptoms were obviously ameliorated after INCB039110 treatment. Moreover, pacritinib (SB1518) is a dual JAK2 and FLT3 inhibitor that can inhibit JAK2 and JAK2 V617F mutations as well as FLT3 and FLT3-D835Y mutations. It has been clinically tested in myelofibrosis and AML patients ( NCT03645824 , NCT02532010 ), and has shown efficacy for myelofibrosis therapy. 324

JAK inhibitors are effective for the management of immune‐mediated diseases (rheumatoid arthritis, ulcerative colitis, and psoriatic arthritis), 313 myelofibrosis, 325 and polycythaemia vera; these diseases generally respond well to JAK inhibitors. A large number of clinical trials related to JAK inhibitors are still in progress. 326 With further understanding of the clinical potential of JAK inhibitors, their indications will also be expanded. 327 For example, the combination of JAK inhibitors with PD-L1 monoclonal antibodies or inhibitors of relevant kinases such as STAT inhibitors in cancer treatment has a certain theoretical basis and data support. 328 In addition, side effects should be considered in the clinical use of JAK inhibitors. The adverse reactions related to JAK inhibitors include infection, neutropenia, venous thromboembolism, anemia, hypercholesterolemia, and even malignancy. Susceptibility to various infections is the main side effect after treatment with these inhibitors. Therefore, reliable prevention and monitoring strategies are needed during the medication process. 329 , 330

Serine/theonine kinase inhibitors

BRAF/MEK/ERK inhibitors

Rat sarcoma virus (RAS)-rapidly accelerated fibrosarcoma (RAF)-mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) signaling is a well-established pathway that controls cell growth, proliferation, and survival in normal cells and cancer cells. 331 , 332 Core components of RAS-RAF-MAPK-ERK signaling include the small GTPase RAS, the serine/threonine kinase RAF, the protein kinases MEK1/2 and ERK1/2. RAF, including ARAF, BRAF, and CRAF, is the direct downstream of RAS, which serves as a transducer of extracellular stimuli. 332 RAF activates the dual-specificity protein kinase MEK1/2, which subsequently phosphorylates ERK1/2. Activated ERK phosphorylates multiple substrates in the cytosol and nucleus, and then promote cell proliferation and survival. 332 Dysregulation of RAS/RAF/MEK/ERK signaling can be observed in a large number of cancers, which is most commonly due to mutations in RAS or BRAF. 332 , 333 BRAF mutations (mostly BRAF V600E) have been identified in ~40–50% of melanomas and 6% of other malignancies, which results in several-fold hyperactivation of BRAF and continuous activation of downstream MEK and ERK. 333 , 334 Due to the picomolar affinities of RAS to GTP, RAS was taken as an undruggable target until the appearance of irreversible small-molecule inhibitors of KRAS G12C in recent years. 335 Therefore, BRAF and the downstream kinases MEK1/2 and ERK are considered attractive therapeutic targets for malignant tumors, especially melanoma.

Small-molecule BRAF inhibitors can be classified into two types. Type-I inhibitors stabilize the kinase in its active (DFG-in) conformation by occupying the ATP-binding pocket. 336 The FDA has approved three type-I competitive BRAF inhibitors for the treatment of non-resectable BRAF V600E/K/D mutant melanoma as single agents or combined with MEK inhibitors including vemurafenib in 2011, 337 dabrafenib in 2013, 338 and encorafenib in 2018 (Table 3 ). 339 Type-II BRAF inhibitors bind to the adjacent hydrophobic sites of ATP-binding pocket and stabilize the kinase in its inactive (DFG-out) conformation. 336 Sorafenib, a representative type-II pan-RAF inhibitor, is a multikinase inhibitor. This agent was initially developed as a RAF kinase inhibitor; however, it also showed inhibitory activity against VEGFR-1/2/3, PDGFR, FLT1, KIT, and RET. It is currently approved by FDA for the treatment of unresectable HCC, advanced RCC, and thyroid carcinoma refractory to radioactive iodine, but not for melanoma due to the unsatisfactory results of clinical trials. 340 Table 3 Properties of approved small-molecule inhibitors of serine/theonine kinases Chemical structure Name Targets Approved indications (year) Corporation Vemurafenib (Zelboraf) BRAF Melanoma (2011) Roche/Genentech Dabrafenib (Tafinlar) BRAF/CRAF Melanoma (2013) NSCLC (2017) ATC (2018) Novartis/GlaxoSmithkline Encorafenib (Braftovi) BRAF Melanoma (2018) Array Trametinib (Mekinist) MEK1/2 Melanoma (2013) NSCLC (2017) ATC (2018) Novartis/GlaxoSmithkline Cobimetinib (Cotellic) MEK1/2 Melanoma (2015) Roche/Genentech/ Exelixis Binimetinib (Balimek) MEK1/2 Melanoma (2018) Array Selumetinib (Koselugo) MEK1/2 Neurofibromatosis type 1 (2020) Plexiform neurofibromas (2020) AstraZeneca/Merck Palbociclib (Aiboxin) CDK4/6 Breast cancer (2015) Pfizer Ribociclib (Kisqali) CDK4/6 Breast cancer (2017) Novartis Abemaciclib (Verzenio) CDK4/6 Breast cancer (2017) Lilly Idelalisib (Zydelig) PI3Kδ CLL (2014) Follicular lymphoma (2014) Gilead Copanlisib (Aliqopa) Pan-PI3K Follicular lymphoma (2017) Bayer Duvelisib (Copiktra) PI3Kγ/δ CLL (2018) SLL (2018) Follicular lymphoma (2018) Verastem Alpelisib (Piqray) PI3Kα Breast cancer (2019) Novartis Temsirolimus (Torisel) mTOR RCC (2007) Pfizer Everolimus (Afinitor) mTOR RCC (2009) Pancreatic cancer (2011) Breast cancer (2012) Novartis Sirolimus (Rapamune) mTOR LAM (2015) Pfizer

The discovery and use of BRAF inhibitors opened up an avenue for the development of inhibitors of MEK1/2 and ERK, downstream targets of RAS/RAF/MEK/ERK signaling. Three MEK1/2 inhibitors have been approved in combination with BRAF inhibitors for the treatment of unresectable or metastatic melanoma harboring BRAF V600E/K mutation including reversible allosteric inhibitors trametinib and binimetinib as well as cobimetinib, a highly selective inhibitor that restrains the catalytic activity of MEK1/2 (Table 3 ); trametinib is also administered as a monotherapy. 341 The FDA-approved combination regimens of BRAF and MEK inhibitors (vemurafenib plus cobimetinib, dabrafenib plus trametinib, and encorafenib plus binimetinib) can achieve a longer PFS in patients with melanoma compared with BRAF inhibitor monotherapy in clinical trials, and encorafenib-binimetinib combination had the best toxicity profile. 342 The combination of dabrafenib and trametinib has also been approved for the treatment of NSCLC or anaplastic thyroid cancer (ATC) patients harboring BRAF V600E mutation. Moreover, the MEK inhibitor binimetinib (as monotherapy or in combination therapy) has been reported to be effective for patients with NRAS mutant melanoma. 343 Recently, the FDA-approved a novel MEK1/2 inhibitor named selumetinib. It is a highly selective allosteric inhibitor of MEK1/2 and was initially assessed in patients with metastatic uveal melanoma. It has been granted orphan drug status as adjuvant treatment for thyroid cancer and as treatment for neurofibromatosis type 1. Based on the results of the phase II SPRINT trial, selumetinib was approved for pediatric patients with neurofibromatosis type 1 and symptomatic, inoperable plexiform neurofibromas in April 2020 (Table 3 ). 344 Currently, no ERK inhibitor has been approved for clinical use.

The success of RAS/MAPK signaling inhibitors in the treatment of melanoma has promoted the further development of such inhibitors, and a variety of novel inhibitors have entered clinical trials. 345 , 346 Typically, pimasertib (AS703026) is a highly selective ATP non-competitive allosteric inhibitor of MEK1/2. A phase II trial comparing the combination of pimasertib with SAR245409 (PI3K inhibitor) to pimasertib alone in patients with recurrent unresectable borderline or low-grade ovarian cancer has been completed ( NCT01936363 ). Response to pimasertib alone (ORR: 12%) was as effective as the combination, suggesting that MEK inhibition could be used as an alternative treatment method to cytotoxic chemotherapy in this population. 347 RO5126766 is a dual RAF/MEK allosteric inhibitor with a novel structure based on coumarin skeleton. A phase I clinical trial ( NCT02407509 ) is conducted to assess RO5126766 alone or together with everolimus on patients with solid tumors or MM harboring BRAF, NRAS, and/or KRAS mutation; 348 the completion date of this trial is targeted for June 2020. The pyrazole amino-pyrimidine derivative ravoxertinib (GDC-0994) is a selective ERK1/2 inhibitor with an IC 50 in the subnanomolar range. Preclinical studies have demonstrated that ravoxertinib had good PD and PK properties. Interestingly, it also showed strong antitumor activity in nude mice harboring KRAS-mutant xenografts. 349 The efficacy and safety of ravoxertinib in combination with cobimetinib in the treatment of locally advanced or metastatic solid tumors were evaluated in a phase I study ( NCT02457793 ), but the data are not yet available.

The clinical application of BRAF and MEK inhibitors has changed the prospect of melanoma treatment, especially the combination therapy with both agents. Treatment with BRAF inhibitors is often accompanied by paradoxical MAP kinase reactivation that limits the clinical response. 350 The combination with MEK inhibitors alleviates this situation to some extent, thereby significantly improving the response rate and survival of patients with melanoma. However, the efficacy of this therapy is limited by acquired resistance caused by gene mutation and bypass activation. 351 Some novel combinations of targeted therapy are expected to improve efficacy and overcome drug resistance, such as the combination with ERK inhibitors, CDK4/6 inhibitors, or inhibitors of the PI3K/AKT/mTOR pathway. 352 In addition, resistance to BRAF and MEK inhibitors is driven partly by immune-mediated mechanisms; therefore, combined targeted therapy and immunotherapy have become the focus in clinical melanoma treatment in recent years. 351 An array of such trials is under assessment for toxicity, efficacy, and treatment sequence to support the continuous progress of individualized treatment of melanoma. 351 , 352

CDK inhibitors

Cell cycle abnormalities result in uncontrolled cell proliferation and have been considered one of the important hallmarks of cancer. 353 Cyclin-dependent kinases (CDKs) are critical enzymes regulating cell cycle progression and require cyclin proteins for activation and downstream phosphorylation. 354 To date, at least 20 CDKs and 29 cyclins have been identified in humans. 355 Among them, CDK4 and CDK6 are necessary for regulating growth signaling and driving the transition of the cell cycle from G1 to S phase. During the process of regulation, CDK4 and CDK6 are activated by D-type cyclins, which induce the phosphorylation of tumor suppressor retinoblastoma protein 1 (RB1) early in the G1 phase. This leads to the inactivation of RB and release of E2F transcription factors, thereby promoting the transcription of target genes related to cell cycle progression. 356 – 358 Given the critical role of the CDK4/6-RB1 axis in mediating cellular proliferation and tumorigenesis, inhibition of CDK4/6 is a promising strategy for cancer therapy that can induce cell cycle arrest in G1 phase and result in decreased cell viability. 359 Notably, CDK4 and CDK6 are functionally identical in their biological effects; therefore, dual inhibition of CDK4 and CDK6 is essential because of the compensatory effects. Cyclin D, the catalyst for CDK4/6, is a major transcriptional target of the estrogen receptor (ER). Estrogen binding to the ER initiates cyclin D transcription, followed by activation of the CDK4/6-RB1 pathway. 360 – 362 Hence, dysregulation of the CDK4/6-RB1 pathway is a significant feature of hormone receptor (HR)-positive breast cancers. 363

In the past three decades, great progress has been made in the development of CDK inhibitors. 364 However, many early developed non-selective and pan-CDK inhibitors (e.g., flvopiridol, seliciclib, UCN-01) have been discontinued due to their limited clinical efficacy or serious side effects. To date, three CDK inhibitors are clinically available: palbociclib, 365 ribociclib, 366 and abemaciclib (Table 3 ). 367 All of them have similar chemical structures and are orally selective reversible inhibitors specifically targeting CDK4/6. They are approved for the treatment of metastatic HR-positive, HER2-negative breast cancer in combination with specific endocrine therapies. 368 In contrast, palbociclib and ribociclib are used in combination with non-steroidal aromatase inhibitors (e.g., letrozole) in postmenopausal women with HR-positive HER2-negative metastatic breast cancer, 369 and palbociclib is also combined with fulvestrant for breast cancer patients with disease progression following endocrine therapy. 370 Abemaciclib shares an indication with palbociclib for use in combination with fulvestrant in HR-positive HER2-negative breast cancer progressing after endocrine therapy. 371 In particular, it is also the only CDK4/6 inhibitor approved as monotherapy for HR-positive HER2-negative metastatic breast cancer pretreated with endocrine therapy and chemotherapy. 372 Despite being approved only for specific breast cancer, the three approved CDK4/6 inhibitors have also been assessed in other solid tumors and hematologic malignancies, such as lung cancer, prostate cancer, melanoma, glioblastoma, and myelofibrosis, and have shown promising results in preclinical studies or clinical trials of some malignancies. 373 , 374 Nevertheless, further clinical research is needed to confirm their efficacies.

Additionally, several newly developed CDK4/6 inhibitors have entered clinical trials. Trilaciclib (G1T28) inhibits CDK4 and CDK6 with IC 50 values of 1 and 4 nM, respectively. It has been evaluated clinically for the prevention of chemotherapy-induced myelosuppression in triple-negative breast cancer (TNBC) and SCLC. 375 The pyridopyrimidine derivative PF-06873600 is potent against CDK2/4/6 and can overcome palbociclib resistance in preclinical studies. Phase II trials of PF-06873600 in the treatment of metastatic breast cancer and other gynecological cancers are ongoing ( NCT03519178 ). SHR-6390 conquered resistance to tamoxifen or trastuzumab, and its combination with endocrine therapy had significant synergistic effects in breast cancer. 376 BPI-16350, FCN-437, and XZP-3287 can effectively penetrate the blood–brain barrier. These CDK4/6 inhibitors are currently being studied in phase I/II trials ( NCT03791112 , NCT04488107 , and NCT04539496 ). 364 A variety of reversible ATP-competitive inhibitors of other CDK isoforms have been evaluated in the clinic in combination with standard-of-care agents or as monotherapy, such as the CDK2 inhibitors inditinib (AGM-130) 377 and FN-1501, 378 the selective CDK7 inhibitor ICEC0942, 379 the CDK9 inhibitors BAY-1251152, 364 and CYC-065, a second-generation inhibitor of CDK2/5/9. Meanwhile, with the development of modern biotechnology, non-classical CDK inhibitors (allosteric inhibitors, covalent inhibitors, and PROTACS) have laid the foundation for the discovery of a novel generation of selective CDK inhibitors. Notably, SY-1365 is an ATP-competitive covalent inhibitor of CDK7 and is the first irreversible CDK inhibitor entering the clinical study. 380 However, the development of this drug was discontinued in September 2019 by Syros Pharmaceuticals due to poor clinical data, which showed that sustaining CDK7 target coverage levels to enhance clinical activity would require more frequent doses or a higher dose, and this may lead to an overly burdensome dosing schedule for patients. 364

Attention should be paid to the adverse events related to the approved CDK4/6 inhibitors. Hematological toxicities such as neutropaenia are often observed in the clinical use of palbociclib and ribociclib, 359 while gastrointestinal toxicities, especially diarrhea, are common in abemaciclib treatment. 368 Most adverse reactions are normally easily controlled by standard supportive care and dose adjustments. In September 2019, the FDA issued a drug safety newsletter and warned that palbociclib, ribociclib, and abemaciclib used to treat advanced breast cancer could trigger rare but serious pneumonia called interstitial lung disease. 381 This risk warning information must be added to the drug labels and patient instructions of all approved CDK4/6 inhibitors. 382 Moreover, understanding the mechanisms of resistance to CDK4/6 inhibitors is another issue that needs to be considered. RB1 is the phosphorylation target of CDK4/6; therefore, loss of RB1 expression is one of the potential mechanisms leading to CDK4/6 inhibitor resistance. RB1 mutations have been detected in the ctDNA of breast cancer patients resistant to CDK4/6 inhibitors, with an estimated frequency of 5%. 383 Ectopic overexpression of cyclin E1 (CCNE1) and cyclin E2 (CCNE2) can also result in resistance to antioestrogen and palbociclib monotherapy through activation of CDK2. 384 In addition to cell cycle alterations, overexpression or mutation in upstream proteins, including AKT1, KRAS/HRAS/NRAS, ERBB2, and FGFR2, have also been observed as potential resistance mechanisms. Given these findings, exploring rational combination treatment strategies may be efficacious to conquer CDK inhibitor resistance. 385

PI3K/AKT/mTOR inhibitors

The phosphatidylinositol 3-kinase (PI3K)/V-AKT murine thymoma viral oncogene homolog (AKT)/mammalian target of rapamycin (mTOR) signaling pathway has an important role in cell growth, proliferation, survival, apoptosis, and motility and is frequently activated in human cancer. 353 PI3Ks are a family of lipid kinases that catalyze the phosphorylation of phosphatidylinositol D3. According to the structural characteristics of the subunits and substrates, PI3Ks can be divided into three classes. Of these, class I PI3K is the major isoform implicated in cancer and can be further subdivided into class IA and class IB, which are activated by RTKs and GPCRs, respectively. Class IA consists of PI3Kα, PI3Kβ, and PI3Kδ encoded by PIK3CA , PIK3CB , and PIK3CD genes, respectively. Class IB comprises only the PI3Kγ subtype encoded by PIK3CG. 386 , 387 PI3K/AKT/mTOR pathway is activated by various mechanisms in oncogenesis and progression. PIK3CA gene is frequently dysregulated in multiple cancers, both by point mutations and amplification. The tumor suppressor PTEN negatively regulates the PI3K pathway by dephosphorylating PIP3 to PIP2. It is also mutated frequently and results in loss of function in human cancers, which upregulates PIP3 levels and leads to constitutive activation of AKT and downstream components. Moreover, as the immediate downstream effector of PI3K, amplification and activating mutations of AKT are often observed in solid tumors. 388 Hyperactivation of PI3K/AKT/mTOR signaling is not only common in a variety of tumors but also closely related to drug resistance (e.g., resistance mechanism of EGFR inhibitors); thus, this pathway has become an attractive target for developing antitumor targeted drugs. Many small-molecule inhibitors of PI3K, AKT, and mTOR have been developed in the past few years. However, only several PI3K and mTOR inhibitors have been approved for cancer treatment.

The early developed PI3K inhibitors are mostly pan-PI3K inhibitors that are capable of binding to all class I PI3Ks, such as wortmannin and LY294002. However, due to their poor PK properties, they are not ultimately approved for clinical use. The second-generation isoform-selective PI3K inhibitors are highly selective for the four isoforms of the class I PI3K catalytic subunit p110 (α, β, γ, and δ). As indicated in Table 3 , idelalisib is the first approved selective PI3Kδ inhibitor based on its efficacy in the treatment of relapsed or refractory CLL patients. It is also recommended for the treatment of lymphocytic lymphoma patients who have received at least two prior systemic therapies or patients with relapsed follicular B-cell NHL. 389 Copanlisib, approved in September 2017, is a pan-PI3K inhibitor with IC 50 values of 0.5, 3.7, 6.4, and 0.7 nM against class I PI3K-α, β, γ, and δ isoforms, respectively. 390 It is used clinically for the treatment of adult patients with relapsed follicular lymphoma who have received at least two prior systemic therapies. Duvelisib is an oral dual PI3Kγ and PI3Kδ inhibitor with IC 50 values of 27 and 2.5 nM, respectively. 391 It prevents the activation of PI3K-γ and δ isoforms by competitively and reversibly binding to the ATP-binding pocket of the p110 subunit. 392 The FDA has approved duvelisib for the treatment of adult patients with relapsed or refractory CLL, SLL, and follicular lymphoma after at least two prior therapies. The newly approved alpelisib is a selective inhibitor targeting the α-isoform of class I PI3K, with an in vitro IC 50 of 4.6 nM. It is indicated in combination with fulvestrant for the treatment of postmenopausal women and men with HR-positive, HER2-negative, PIK3CA -mutated, advanced, or metastatic breast cancer as detected by an FDA-approved test following progression on or after an endocrine-based regimen. 393 This is also the first PI3K inhibitor approved for clinical treatment of breast cancer. In addition, there are also a variety of PI3K inhibitors undergoing clinical evaluation, such as the pan-class I PI3K inhibitors XL147 and ZSTK474, PI3Kγ inhibitor IPI-549, and PI3Kα inhibitor serabelisib. 394 – 397 Some of them have progressed to phase II or III trials and have good prospects for approval. In addition, the promising efficacy of PI3K inhibitors raised the question of whether the combined inhibition of multiple pathway components could further improve the efficacy without excessive side effects. Developing dual PI3K/mTOR inhibitors is a predominant strategy due to the high homology between the ATP-binding domain of p110 and the catalytic site of mTOR. Several dual PI3K/mTOR inhibitors have entered clinical trials, such as bimiralisib, dactolisib (BEZ235), GDC-0084 (RG7666), and gedatolisib (PKI-587/PF-05212384). 398 – 400 Current clinical trials focus on combining them with a range of other antitumor agents. Notably, GDC-0084 is orally administered and can penetrate the BBB. It is currently specifically developed for patients with glioma or brain metastases from solid tumors.

Compared with PI3K and mTOR inhibitors, the development of AKT inhibitors is relatively slow. The obtained results from numerous preclinical studies revealed that AKT inhibitors are potent for tumors with PIK3CA mutations or loss of PTEN function. 386 However, most of these agents displayed limited clinical effects in patient settings, especially as monotherapy. Only several AKT inhibitors, including ATP-competitive inhibitors and allosteric inhibitors, have progressed to clinical trials. The ATP-competitive inhibitors ipatasertib, capivasertib, afuresertib, and uprosertib are all highly selective pan-AKT inhibitors and can prevent AKT activation and enhance the antitumor activity of chemotherapeutic drugs. 401 , 402 A phase III trial ( NCT03072238 ) of ipatasertib is being conducted to evaluate the efficacy of its combination with abiraterone and prednisone/prednisolone in adult male patients with metastatic castration-resistant prostate cancer (CRPC). Three other phase III trials related to ipatasertib combination are actively recruiting patients with HR-positive and HER2-negative locally advanced unresectable or metastatic breast cancer ( NCT04060862 , NCT04177108 , and NCT03337724 ). The efficacy of capivasertib (AZD5363) combined with paclitaxel as a first-line treatment for locally advanced or metastatic TNBC patients is under evaluation in a phase III trial ( NCT03997123 ). 403 Afuresertib (GSK2110183) monotherapy showed satisfactory safety and clinical activity against hematological malignancies, including MM. 404 Uprosertib (GSK2141795) was used for relapsed or refractory MM either alone or in combination with trametinib in phase I and II clinical trials ( NCT01989598 , NCT01979523 ). 405 In addition, allosteric AKT inhibitors are represented by MK-2206. Several clinical trials are ongoing to test MK-2206 monotherapy or in combination with targeted therapy or chemotherapy for metastatic breast cancer and in combination with lapatinib and trastuzumab for HER2-positive breast cancer ( NCT01277757 , NCT01245205 , and NCT01235897 ). Notably, there is currently an AKT inhibitor approved for marketing, named miltefosine. However, it is used clinically for the treatment of visceral and cutaneous leishmaniasis but not for malignancies.

mTOR inhibitors can be divided into two categories: rapamycin analogs (rapalogs) and ATP-competitive inhibitors. The former is the first-generation mTOR inhibitor, which forms a complex with FK506 binding protein 12 (FKBP12) and inhibits the activity of only mTORC1 but not mTORC2. 406 They are also the first compounds developed to target the PI3K/mTOR pathway. Currently, multiple rapalogs, such as sirolimus (rapamycin), temsirolimus, and everolimus, have been approved for the treatment of various cancers, including RCC, breast cancer, and pancreatic cancer (Table 3 ). Of these, rapamycin was initially approved as an immunosuppressant for transplant patients, and its indications were subsequently extended to lymphangioleiomyomatosis (LAM). 407 Although patients can benefit from such medications, drug resistance caused by mTORC2-mediated negative feedback limits their clinical use. Second-generation mTOR inhibitors are designed as ATP-competitive inhibitors, which can simultaneously suppress the activity of both mTORC1 and mTORC2. At present, no such inhibitors have received approval, but some of them are under clinical evaluation, such as sapanisertib (TAK-228), vistausertib (AZD2014), CC-223, BI860585 , DS-3078a, ME-344, GDC-0349, OSI-027, and P529. 406 Representatively, sapanisertib (TAK-228), formerly known as INK128 or MLN0128, is an orally effective mTOR inhibitor with an IC 50 of 1 nM. 408 It is being evaluated for the treatment of multiple cancers in several phase II trials ( NCT02484430 , NCT03047213 , and NCT02893930 ). Vistusertib is a selective mTOR inhibitor with an IC 50 of 2.8 nM. In preclinical studies, vistusertib exhibited a broad spectrum of antitumor activity. 409 It is undergoing evaluation in a phase II clinical trial for the treatment of recurrent grade II–III meningiomas ( NCT03071874 ).

Isoform-specific inhibitors have fewer off-target effects and side effects than pan-PI3K, pan-AKT, and dual PI3K/mTOR inhibitors. However, due to their limited therapeutic efficacy caused by compensation effects or various mutations of the PI3K/AKT/mTOR pathway, most clinical trials still tend to use pan-inhibitors. Therefore, clarifying the mutant types via molecular pathological diagnosis in advance can improve the clinical application of specific inhibitors. On the other hand, the clinical efficacy using only PI3K/AKT/mTOR inhibitors were shown to have modest effects for patients in actual treatment. More importantly, PI3K/AKT/mTOR cascade exchanges crosstalk with many other signaling pathways, such as Wnt and MAPK signaling. Hence, using such inhibitors is prone to negative feedback regulation, resulting in resistance. These problems highlight the limitations of PI3K/AKT/mTOR inhibitors as monotherapy in malignancies. Currently, efforts have focused on combination therapy, including inhibition of parallel pathways as well as targeted therapy combined with cytotoxic drugs, 388 and the results of most clinical trials are promising. Moreover, treatment with PI3K pathway inhibitors could also induce adverse effects, even though isoform-specific inhibitors are no exception. For example, the PI3Kδ inhibitor idelalisib was shipped with four black-frame warnings, suggesting fatal and severe liver toxicity, diarrhea and colitis, pneumonia, and intestinal perforation. 410 Other toxicities reported in clinical trials of the PI3K/AKT/mTOR inhibitors include immunosuppression, hypoglycemic, cardiac toxicity, neuropsychiatric effects, cutaneous reactions, nausea, mouth ulcers, constipation, etc. A better understanding of the mechanism of side effects caused by such drugs and their management might advance novel PI3K/AKT/mTOR inhibitors from clinical trials to the bedside, with new treatment options for patients.

We in the above summarize the anti-cancer kinase inhibitors approved by the US FDA and NMPA of China. To date, a total of 70 kinase inhibitors have been approved to use in the clinic, which accounts for almost 80% of all approved small-molecule targeted anti-cancer drugs (89 in total). Despite this great achievement, these kinase inhibitors just act on a small number of kinase targets (<30 kinases). Many kinases that are thought of as potential anti-cancer targets still lack inhibitors or at least potent and selective inhibitors. Therefore, drug discovery targeting kinases still has a lot of room for development. Another hot topic of debate regarding kinase inhibitors is whether single-target drugs or multitarget drugs have more advantages. Target-specific kinase inhibitors generally have low toxicity but are easy to develop resistance. While multikinase inhibitors often have high side effects, but have advantages in anti-cancer efficacy and overcoming drug resistance.

Epigenetic inhibitors

Epigenetics is a branch of genetics that studies the heritable changes of gene expression without changing the nucleotide sequence of genes. It is strictly regulated by a variety of chemical modifying enzymes and recognition proteins, which are often called “writers”, “erasers”, and “readers”. 411 , 412 The writers refer to enzymes that transfer chemical groups to DNA or histones, which include DNA methyltransferases (DNMTs), histone acetyltransferases (HATs), and histone lysine methyltransferases (KMTs). The erasers remove post-translational modifications, and include histone deacetylases (HDACs) and histone lysine demethylases (KDMs). The readers are proteins that can recognize the modified histones or DNA, such as methyl-binding domain proteins, and bromodomain and extra-terminal (BET) family proteins (Fig. 3 ). Abnormal epigenetic regulation is also closely related to various diseases including tumor, immune diseases, and many rare diseases. Though numerous epigenetic regulatory proteins have been identified as potential disease targets, only fewer epigenetic drugs are approved for clinical use at present. Fig. 3 Commonly altered epigenetic regulatory proteins implicated in cancer. Gene silencing in mammalian cells is usually caused by methylation of DNA CpG islands as well as hypermethylation or hypoacetylation of histones. The writers (DNMTs, HATs, and HMTs) refer to enzymes that transfer chemical groups to DNA or histones; the erasers (HDACs and KDMs) are enzymes responsible for removing chemical groups from histones; the proteins (MBDs and BET family proteins) that can recognize the methyl-CpGs and modified histones are readers. Mutated IDH1/2 catalyzes the reduction of α-KG to 2-HG, which inhibits the activity of TET and lysine demethylases, resulting in DNA hypermethylation and increased histone lysine methylation. Figure created with BioRender.com

EZH2 Inhibitors

Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, functions as a catalytic subunit of the polycomb repressor complex 2 (PRC2), which also comprises other members including embryonic ectoderm development (EED), suppressor of zeste 12 (SUZ12), and histone-binding proteins RbAp46/48. 413 PRC2 is one of the two core complexes of polycomb group proteins (PcGs), and is responsible for transferring methyl groups from S-adenosyl-L-methionine (SAM) to lysine 27 on histone H3 (H3K27) through its C-terminal SET domain, resulting in chromatin compaction and transcriptional silencing of target genes. As the central component of PRC2, EZH2 is involved in numerous epigenetic modifications that are associated with cell proliferation, differentiation, survival, adhesion, and DNA damage repair. 414 Dysfunction of EZH2 is closely related to tumorigenesis and progression. Accumulating evidence has confirmed that EZH2 is frequently mutated and abnormally overexpressed in various malignant tumors including prostate cancer, 415 , 416 ovarian cancer, 417 endometrial carcinoma, 418 breast cancer, 419 melanoma as well as hematological malignancies, 420 such as NHL, B-cell lymphoma, and T-cell ALL. 421 – 424 It promotes tumorigenesis mainly through three mechanisms: PRC2-dependent H3K27 methylation, PRC2-dependent non-histone protein methylation, and PRC2-independent coactivator of transcriptional factors. Given the evidence for EZH2 enzymatic gain of function being a cancer driver, inhibition of EZH2 has been thought of as a novel and promising approach for cancer therapy. 413

The first reported small-molecule EZH2 inhibitor is 3-deazaneplanocin A (DZNep), a cyclopentanyl analog of 3-deazaadenosine. This compound can potently inhibit the S -adenosyl- l -homocysteine (SAH) hydrolase activity and induce the increase of cellular 5-adensylhomocystein levels, thus suppressing the activity of global S -adenosyl- l -methionine (SAM)-dependent histone lysine methyltransferase, including EZH2-mediated histone methylation. Therefore, DZNep is a non-specific EZH2 inhibitor. 425 Although treatment with DZNep showed significant antitumor activity in various preclinical studies, the poor PK profile and safety encouraged further development of more potent and selective EZH2 inhibitors. Since 2012, multiple SAM-competitive EZH2 inhibitors have been reported, including EI1, EPZ005687, GSK126, GSK343, UNC1999, tazemetostat (EPZ6438), SHR2554, CPI-1205, DS-3201, PF-06821497, and HH2853 ( NCT04390737 ). 413 These compounds display better selectivity for EZH2 or EZH1 compared with other methyltransferases, and most of them bind to both wild-type and mutant EZH2, particularly Y641 and Y646 mutations. 426 , 427 Currently, several of these inhibitors (e.g., tazemetostat, SHR2554, CPI-1205, DS-3201, PF-06821497, and HH2853) have entered clinical trials to evaluate their clinical efficacy and safety in a variety of solid tumors or hematological malignancies. Among them, tazemetostat developed by Epizyme and Eisai is an oral competitive inhibitor of the SAM pocket of the EZH2 SET domain, and received accelerated approval in January 2020 in the USA for the treatment of adults and adolescents aged ≥16 years with locally advanced or metastatic epithelioid sarcoma not eligible for complete resection (Table 4 ). Tazemetostat is the first approved EZH2 inhibitor and also the first therapy specifically for the treatment of epithelioid sarcoma. Table 4 Properties of approved small-molecule inhibitors of epigenetic targets Chemical structure Name Targets Approved indications (year) Corporation Tazemetostat (Tazverik) EZH2 Epithelioid sarcoma (2020) Epizyme/Eisai Vorinostat (Zolinza) HDAC1/2/3/6 CTCL (2006) Merck Romidepsin (Istodax) HDACs CTCL (2010) PTCL (2011) Celgene Belinostat (Beleodaq) HDAC1/2 PTCL (2014) Onxeo/Spectrum Tucidinostat (Epidaza) HDAC1/2/3/10 PTCL (2015) Chipscreen Biosciences Panobinostat (Farydak) HDACs MM (2015) Secura Bio Enasidenib (Idhifa) IDH2 AML (2017) Agios/Celgene Ivosidenib (Tibsovo) IDH1 AML (2018) Agios

In addition to the highly selective SAM-competitive inhibitors, EZH2 can also be inhibited by disrupting its interaction with other PRC2 subunits. EED226 (MAK683) is a potent and selective PRC2 allosteric antagonist, which directly binds to the H3K27me3 pocket of EED and induces a conformational change, therefore disrupting EZH2-EED interaction and resulting in loss of PRC2 activity. 428 EED226 shows similar activity to SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 could also potently inhibit the activity of PRC2 containing a mutant EZH2 protein resistant to SAM-competitive inhibitors. 428 A phase I trial ( NCT02900651 ) is ongoing to evaluate the safety and efficacy of EED226 in patients with advanced malignancies, such as DLBCL, nasopharyngeal carcinoma (NPC) or other advanced solid tumors for whom no further effective standard treatment is available. Besides, some other small-molecule inhibitors, such as A-395, 429 BR-001, and UNC6852, were also reported as EED inhibitors that inhibit the interaction between EZH2 and EED and destabilize PRC2 complex, 430 , 431 thereby exerting antitumor activity. Moreover, Wang et al. developed a unique strategy to target EZH2 by protein degradation. 432 They identified a gambogenic acid (GNA) derivative GNA002 that could specifically and covalently bind to Cys668 within the EZH2-SET domain and trigger EZH2 degradation through COOH terminus of Hsp70-interacting protein (CHIP)-mediated ubiquitination. In this study, GNA002 significantly suppressed H3K27Me3 and effectively reactivated PRC2-silenced tumor suppressor genes, and could inhibit tumor growth in an EZH2-dependent manner. 432

The efficacy of EZH2 inhibitors is limited by both primary and acquired resistance, which is the main challenge for the clinical use of EZH2 inhibitors. Primary resistance is often due to EZH2 resistant mutations, differences in the tumor microenvironment, heterogeneity of tumor tissue structure (influencing PKs and drug delivery), or activation of prosurvival pathways as reported in DLBCL and SWI/SNF mutant cancer cells. 433 , 434 Huang et al. profiled global post-translational histone modification changes across a large panel of cancer cell lines with various sensitivities to EZH2 inhibitors, and found that oncogenic transcriptional reprogramming mediated by MLL1’s interaction with the p300/CBP complex directed H3K27me loss to reciprocal H3K27ac gain and restricted response to EZH2 inhibition. 435 This resistance could be effectively reversed by concurrent inhibition of H3K27me and H3K27ac with a combination of EZH2 and BRD4 inhibitors. Acquired resistance is usually because of multiple secondary mutations of EZH2, such as Y641F, C663Y, E720G, Y726F, Y111L, and Y661D. 433 , 434 , 436 , 437 Combination therapy is thought as the most efficient strategy to improve the limited effectiveness of EZH2 inhibitors. 438 For example, the approved drug tazemetostat is currently assessed clinically in several combined treatments for a variety of malignant tumors. Exploring various effective combination strategies, including combination with chemotherapy, immunotherapy, kinase targeted therapy, and metabolic regulators, is a promising direction in the near future. 414 , 439 , 440

HDAC inhibitors

Histone deacetylases (HDACs) are important epigenetic regulators that remove the acetyl groups from the N-acetylated lysine residues of histones and various non-histone substrates. To date, 18 HDACs have been identified in mammals and are characterized into 4 subfamilies: class I HDACs (HDACs 1, 2, 3, 8), class II HDACs (class IIa: HDACs 4, 5, 7, 9; class IIb: HDACs 6, 10), class III HDACs (Sirt1-7) and class IV HDAC (HDAC11). 441 , 442 Class I, II, and IV HDACs are Zn 2+ -dependent histone deacetylases and can be restrained by inhibitors (such as TSA and SAHA) that occupy the catalytic core of the zinc-binding site. Class III HDACs require nicotinamide adenine dinucleotide (NAD + ) for their activity. 441 – 443 Aberrant upregulation of HDACs has been reported in various types of cancers. Such changes alter the transcription of oncogenes and tumor suppressor genes, which are closely associated with cell proliferation, apoptosis, differentiation, migration, and cancer angiogenesis. 443 , 444 Inhibition of HDACs has proven to be an effective strategy for cancer therapy. To date, a variety of HDAC inhibitors have been approved by the FDA (Table 4 ) or are undergoing clinical trials.

Based on chemical structural differences, HDAC inhibitors can be divided into four categories: hydroxamates, cyclic tetrapeptides, benzamides, and aliphatic acids. 445 Hydroxamide HDAC inhibitors inhibit HDAC activity through coordination with Zn 2+ . TSA was the first natural hydroxamate HDAC inhibitor. Its structural analog vorinostat (SAHA) is a pan-inhibitor of classical classes of HDACs (I, II, and IV) and was approved in October 2006 for the treatment of cutaneous T-cell lymphoma (CTCL) (Table 4 ). 446 Belinostat and panobinostat are two other hydroxamate pan-HDAC inhibitors that are approved for the treatment of relapsed or refractory peripheral T-cell lymphomas (PTCL) and drug-resistant MM, respectively. 447 , 448 Both of them are derivatives of M-carboxycinnamic acid bishydroxamate. Cyclic tetrapeptides are a class of HDAC inhibitors with complex structures. Romidepsin isolated from Chromobacterium violaceum is the only such inhibitor granted US FDA approval. Its indications were initially only CTCL and expanded to PTCL in November 2011, and the objective response rate was 34% in CTCL patients and 25% in patients with PTCL. 449 Moreover, tucidinostat (chidamide), containing pyridine and N -(2-amino-5-fluorophenyl)-benzamide groups, is a benzamide inhibitor of HDAC1/2/3/10. It was developed wholly in China and approved for the treatment of refractory or relapsed PTCL in 2015 by the NMPA. 450

Aliphatic acids, such as valproic acid and phenylbutyrate, show relatively weak inhibitory activity against class I and class II HDACs, and both of these aliphatic acid HDAC inhibitors have already been approved for some non-oncological uses in the clinic and are recently under clinical evaluation for cancer therapy. 443 Sirtuin (class III HDAC) inhibitors include the allosteric non-competitive pan-inhibitor nicotinamide and Sirt-specific inhibitors such as EX-527, sirtinol, and cambinol. 451 Although Sirt inhibitors have been reported to be useful for cancer treatment, so far no such inhibitors have been approved for clinical use. Moreover, more than 10 other HDAC inhibitors have undergone or are undergoing clinical trials as monotherapy or in combination therapy in patients with hematologic malignancies or solid tumors. 452 , 453 For example, the benzamide inhibitor entinostat is currently assessed in phase III trials ( NCT02115282 and NCT03538171 ) for the clinical benefit in patients with HR-positive, HER2-negative, locally advanced, or metastatic breast cancer. A phase I dose-escalation multicentre trial ( NCT00741234 ) demonstrated that the hydroxamate HDAC inhibitor pracinostat was safe, with modest single-agent activity in patients with advanced hematological malignancies. 454

Currently, the clinical activity of HDAC inhibitors as monotherapy is largely restricted to hematological malignancies, including lymphomas, leukemia, and MM. In solid tumors, HDAC inhibitors just showed limited single-agent activity, which may be attributed to the non-specific blocking of angiogenesis and inflammation as well as the poor PK properties of some agents. 443 , 455 Inhibition of tumor angiogenesis hinders drug delivery in solid tumors. The anti-inflammatory effect may induce apoptosis of tumor-fighting immune cells. Another obstacle limiting the clinical use of HDAC inhibitors is their side effects. The common side effects associated with vorinostat, belinostat, and romidepsin were nausea, anorexia, fatigue, and vomiting, which are mostly manageable, but some agents may cause more serious toxicities. 455 Currently, major efforts to overcome these obstacles are focused on the development of small-molecule isoforms or class-selective HDAC inhibitors. Selective inhibitors can also be used as chemical probes to explore the epigenetic effects and biological processes induced by HDACs in cancer cells. Additionally, HDAC inhibitors are used in combination with other antitumor drugs to optimize their efficacy and conquer drug toxicity and resistance. Many combination therapy regimens have been used in clinical oncology treatment. 452 , 456 , 457 Furthermore, some novel combination strategies, such as combining HDAC inhibitors with epigenetic-targeted drugs, proteasome inhibitors, or immunotherapy, have also exhibited good therapeutic effects in preclinical studies. 453 , 458 – 460

IDH1/2 inhibitors

Isocitrate dehydrogenases (IDHs) include three subtypes (IDH1, IDH2, and IDH3) and are key enzymes that catalyze the conversion of isocitrate to α-ketoglutarate (α-KG) using nicotinamide adenine dinucleotide phosphate (NADP + ) or NAD + as a cofactor in the tricarboxylic acid (TCA) cycle. 461 NADP-dependent IDH1 and IDH2 are homodimeric isoenzymes with 70% sequence similarity and almost the same protein structure, whereas IDH3 is a NAD-dependent enzyme that has a unique sequence. 462 , 463 IDH1/2 are mutated in several types of tumors, including AML, glioma, myelodysplastic syndrome (MDS), myeloproliferative neoplasms, chondrosarcoma, and cholangiocarcinoma. 463 – 467 Mutations in IDH3 are rarely reported in cancer. IDH1 R132 H/C, IDH2 R140Q, and R172K are the main identified mutants of IDH1/2, and mutant IDH1/2 (mutIDH1/2) loses its normal catalytic function and instead catalyzes the reduction of α-KG to the oncometabolite product 2-hydroxyglutarate (2-HG). 468 , 469 The accumulation of 2-HG competitively inhibits the α-KG-dependent dioxygenases involved in epigenetic remodeling and DNA repair, such as JMJD-containing histone lysine demethylases (KDMs) and the ten-eleven translocation (TET) family of 5-methylcytosine hydroxylases (DNA demethylases) (Fig. 3 ), thereby promoting oncogenesis through transcriptional dysregulation and impairing normal cellular differentiation. 470 Thus, targeted inhibition of mutated IDH1/2 can reduce the serum 2-HG level and may be therapeutically beneficial for malignancies with IDH1 or IDH2 mutations. 471 , 472

Two IDH1/2 inhibitors have been approved for AML therapy (Table 4 ). Enasidenib (AG-221) is an oral selective inhibitor of mutIDH2 with IC 50 values of 4.0 nM on IDH2 R140Q and 340 nM on wild-type IDH2. 471 The precursor form of enasidenib was initially screened by Agios Pharmaceuticals to find potent inhibitors of the IDH R140Q mutant, the most common form of mutIDH2 in AML, and then licensed to Celgene for further development. 472 , 473 Enasidenib binds to IDH2 R140Q at an allosteric site within the heterodimer interface of the enzyme, which forces mutIDH2 to form an open conformation with no catalytic activity. 474 , 475 It also showed inhibitory activity on IDH2 R172K, and its clinical efficacy was stronger in patients harboring the IDH2 R172K mutation than in patients with the IDH1 R140Q mutation (ORR: 53.3% vs. 35.4%; CR: 24.4% vs. 17.7%). 476 In 2017, enasidenib received FDA approval for the treatment of IDH2-mutated relapsed/refractory AML. 473 Ivosidenib (AG-120) is a reversible allosteric mutIDH1 inhibitor optimized from AGI-5198; the development of the latter has been discontinued due to its poor PKs. 477 Ivosidenib was also developed by Agios and Celgene Pharmaceuticals and prevents the formation of catalytically active sites by binding with the cofactor (magnesium ion) of IDH1. 478 It is highly selective for IDH1 R132 mutants but has little inhibitory activity against wild-type or mutant IDH2. 479 The successes of preclinical studies and phase I trials earned ivosidenib orphan drug designation for glioma treatment, and it was first approved by the FDA on 20 July 2018 for adult relapsed/refractory AML patients with mutIDH1. 480 In addition to AML indications, several clinical trials evaluating the efficacy and safety of enasidenib and ivosidenib in other tumors, such as glioma, T-cell lymphoma, MDS, or cholangiocarcinoma, are ongoing. 481 , 482 Some of these studies may support the future expansion of these two drugs.

In the past few years, a variety of other IDH inhibitors have also been developed, and several of them have entered clinical trials. 483 Typically, vorasidenib (AG-881) is an orally available pan-IDH inhibitor with IC 50 ranges of 0.04–130 nM against various IDH1 R132, IDH2 R140, and R172 mutations. It could easily penetrate the BBB in preclinical studies, indicating the potential for glioma therapy. The drug is undergoing clinical investigation in hematologic malignancies and solid tumors including glioma. BAY-1436032 developed by Bayer is a specific allosteric inhibitor of mutIDH1, which is potent against all reported IDH R132 mutants. It could also efficaciously pass through the BBB and exert antitumor effects in glioma and AML animal models with IDH R132 mutations. 484 Two open-label phase I trials of BAY-1436032 are ongoing to evaluate its PDs, safety, tolerability, and preliminary efficacy in patients with AML ( NCT03127735 ) and solid tumors ( NCT02746081 ), but the initial results have not been announced. Clinical trials of other mutIDH1 inhibitors (e.g., FT-2102, DS-1001b, and IDH305) in hematological malignancies or glioma are also underway. 485 , 486 Notably, the development of IDH305 has been halted due to its dose-limiting adverse events observed in clinical studies, such as transaminitis and hyperbilirubinaemia. 487

Although two IDH inhibitors have been approved for AML therapy and showed efficacy and safety in clinical trials of other solid tumors, including glioma, some issues still need to be resolved. Acquired resistance caused by mutIDH isoform switching was detected after treatment with specific IDH inhibitors, such as the IDH2 R140Q mutation after ivosidenib treatment and IDH1 R132C or R132H mutations after enasidenib treatment. 488 Moreover, IDH2 secondary mutations occurring at the glutamine 316 site (Q316E mutation) or isoleucine 319 site (I319M mutation) after enasidenib treatment will change the conformation of the IDH2 trans-dimer interface and result in resistance to enasidenib. 489 The pan-IDH inhibitor AG-881 has been reported to be effective for some mutIDH isoform switching-induced resistance. 490 Nevertheless, there is still a demand to develop novel pan- or specific IDH inhibitors, especially mutIDH2 inhibitors, which were all previously optimized from the scaffolds of enasidenib, to conquer special-site mutations. In addition, due to the complex global epigenetic alterations in oncogenesis, IDH inhibitor monotherapy may be insufficient to treat IDH-mutated malignancies, in particular in glioma; this is also observed in clinical trials. Therefore, reasonable combination therapy with other antitumor agents, especially histone-modifying drugs or BET inhibitors, may be therapeutically beneficial for IDH-mutated patients. Interestingly, Fujiwara H et al. reported that AML patients with IDH mutations had a 14-fold higher response rate to BET inhibitors than common patients. 491 The combination strategy of IDH inhibitors with the DNMT inhibitor azacitidine in AML treatment has also been evaluated in several clinical trials, and the preliminary results are encouraging. 492 , 493

In addition to EZH2, HDAC, and IDH inhibitors, targeting DNMT is also a potential strategy for cancer therapy. Two DNMT inhibitors (azacytidine and decitabine) cxvdv have been approved for the treatment of hematologic malignancies, particularly AML and MDS. 494 – 496 These cytosine analogs can incorporate into the DNA or RNA backbone to replace C-5 of cytosine with N-5 and interfere with the methylation as well as induce DNMTs degradation. Moreover, agents for other epigenetic targets are also in development. DOT1L methyltransferase inhibitor (e.g., pinometostat), BET inhibitors (e.g., birabresib, molibresib, ZEN003694, PLX51107 ), and lysine-specific demethylase 1 (LSD1) inhibitors (e.g., iadademstat, INCB059872, CC-90011) have progressed to different stages of clinical trials. 412 , 497 , 498 Despite the availability of more and more epigenetic agents, there are still many problems for epigenetic therapies. Epigenetic alterations are widely distributed across normal and cancer cells. Even in the cancer cells, these changes may occur early as foundational mutations or arise late and drive clonal subgroups. Furthermore, the sensitivity to certain epigenetic regulation is discrepant in different cancers. These factors are the root causes of drug resistance and side effects. Deeper understanding of the mechanisms and difference of epigenetic alterations in different cancers may contribute to further development and optimization of epigenetic therapies. Normalization of the epigenome can sensitize cancer chemotherapy, molecular targeted therapy, and immunotherapy. Combination therapies are evaluated clinically to broaden the response rates among patients with hematologic malignancies and to expand the indication to solid tumors. However, the current results are not satisfactory. Thus far, only the combination of panobinostat, dexamethasone, and bortezomib has received FDA approval. 499 The hope is still the deeper epigenetic insights, which may eventually guide the development of epigenetic agents and the relevant combinations.

BCL-2 inhibitors

The B-cell lymphoma 2 (BCL-2) family of proteins consists of more than 20 members that regulate the intrinsic apoptosis pathway, and fall into three subfamilies (anti-apoptotic proteins, pro-apoptotic proteins, and the cell death mediators) based on their structure and function. 500 Anti-apoptotic proteins such as BCL-2 and the closely related proteins BCL-XL, BCL-W, MCL-1, and A1/BFL-1 have four tandem BCL-2 homology (BH) domains and promote cell survival. 500 , 501 While the multi-region pro-apoptotic effector proteins (BAX and BAK) and BCL-2 homology 3 (BH3)-only pro-apoptotic proteins (BIM, PUMA, BAD, or NOXA) promote cell apoptosis upon diverse cellular stresses by the alternation of mitochondrial outer membrane permeabilization and release of cytochrome C from the mitochondria (Fig. 4 ). 501 , 502 The interaction between the anti-apoptotic and pro-apoptotic BCL-2 family of proteins regulates the apoptotic state of cells. Dysregulation of the apoptosis pathway is common in malignant tumors, especially in hematologic malignancies. 503 – 505 BCL-2 was first discovered as an inhibitor of cellular apoptosis, and the in-depth understanding of this target promotes the development and application of BCL-2 inhibitors in cancer treatment. 506 , 507 Fig. 4 Schematic illustration of extrinsic and intrinsic pathways of apoptosis. In healthy cells, anti-apoptotic BCL-2 proteins (BCL-2, BCL-XL, BCL-W, MCL-1, and A1/BFL-1) bind to and inhibit activators (BH3-only proteins) and effectors (BAX and BAK). Treatment with BCL-2 inhibitors releases the inhibitory effects of anti-apoptotic BCL-2 proteins on activators and effectors. The subsequent activation and oligomerization of the pro-apoptotic proteins BAK and BAX result in the formation of mitochondrial outer membrane permeabilization (MOMP) and the release of cytochrome C as well as a second mitochondria-derived activator of caspase (SMAC) from the mitochondria. Cytochrome C can form a complex with procaspase 9 and apoptosis protease-activating factor 1 (APAF1), thereby activating caspase 9. Caspase 9 then activates procaspase 3 and procaspase 7, resulting in cell apoptosis. Figure created with BioRender.com

Several early efforts have been made to target the anti-apoptotic BCL-2 family members including antisense oligonucleotide drug oblimersen, the natural product gossypol, and its synthetic derivatives. 508 A major breakthrough in targeting BCL-2 is the development of small-molecule BH3 mimetics ABT-737 and ABT-263 (navitoclax). 509 , 510 These agents can mimic the physiologic inhibitors of anti-apoptotic BCL-2 and its relatives, thereby promoting apoptosis. ABT-737 is the first synthetic BH3 mimetic discovered by structure-activity relationship and nuclear magnetic resonance screening. It binds to BCL-2, BCL-XL, and BCL-W with high affinity, and exhibits 500-fold weaker affinity for MCL1 and BFL-1/A1. 509 However, ABT-737 has poor bioavailability and requires continuous parenteral administration, which hindered its clinical development. Navitoclax is a structurally related molecule of ABT-737 with high oral bioavailability (~50% in dogs) and entered clinical trials in 2006. 511 Although the efficacy of navitoclax was observed in preclinical and clinical studies, dose-dependent thrombocytopenia caused by BCL-XL suppression is the major obstacle restricting the clinical application of navitoclax. 510 Initial efforts on the BH3 mimetics ABT-737 and navitoclax facilitated the successful development of ABT-199 (venetoclax), a highly selective BH3 mimetic with greater affinity for BCL-2 but a much lower affinity for BCL-XL and BCL-W. 512 In a single-arm phase II trial of venetoclax monotherapy, 79.4% of patients with CLL with the 17p deletion achieved an objective response over a median of 12 months among 107 enrolled patients, and complete remission was achieved in a median of 8.2 months ( NCT01889186 ). 513 Due to its remarkable efficacy and safety, the FDA-approved venetoclax for the treatment of CLL patients with 17p deletion in April 2016 (Table 5 ). This indication was subsequently expanded in June 2018 to include patients with CLL or SLL, with or without 17p deletion, who have received at least one prior therapy. Moreover, in November 2018, venetoclax was approved in combination with standard chemotherapy agents, including azacitidine, decitabine, and low-dose cytarabine, for the treatment of newly diagnosed AML in adults who are 75 years of age or older, or who have comorbidities that preclude the use of intensive induction chemotherapy; this approval was based on the impressive results of two clinical studies conducted in this population. 514 , 515 As a BCL-2-specific inhibitor, venetoclax is also under assessment for the treatment of many other malignancies in the clinic, including solid tumors, and most of the clinical trials have tested its role in combination therapies. 516 Table 5 Properties of approved small-molecule inhibitors of BCL-2, hedgehog pathway, proteasome, and PARP Chemical structure Name Targets Approved indications (year) Corporation Venetoclax (Venclyxto) BCL-2 CLL (2016) SLL (2018) AML (2018) Abbvie/Genentech Vismodegib (Erivedge) SMO BCC (2012) Genentech Sonidegib (Odomzo) SMO BCC (2015) Novartis Glasdegib (Daurismo) SMO AML (2018) Pfizer Bortezomib (Velcade) Proteasome MM (2003) MCL (2006) Millennium Carfilzomib (Kyprolis) Proteasome MM (2012) Onyx Ixazomib (Ninlaro) Proteasome MM (2015) Takeda Olaparib (Lynparza) PARP1/2/3 Ovarian cancer (2014) FTC (2015) Breast cancer (2018) Pancreatic adenocarcinoma (2019) Prostate cancer (2020) AstraZeneca Rucaparib (Rubraca) PARP1/2/3 Ovarian cancer (2016) FTC (2016) PPC (2018) Prostate cancer (2020) Clovis Niraparib (Zejula) PARP1/2 Ovarian cancer (2017) FTC (2017) PPC (2017) Tesaro/Takeda/Janssen Talazoparib (Talzenna) PARP1/2 Breast cancer (2018) Pfizer

S55746 (also known as BCL201 or Servier-1) is another orally bioavailable small-molecule BCL-2 inhibitor being studied in clinical trials. It is a BH3 mimetic derived from tetrahydroisoquinoline amide-substituted phenyl pyrazoles, and shows high affinity and selectivity for BCL-2 with a Ki value of 1.3 nM. 517 The results of a phase I study ( NCT02920697 ) indicated the efficacy and safety of S55746 in patients with B-cell NHL. In addition to the BCL-2 inhibitor, several inhibitors targeting MCL-1 or BCL-XL are also being evaluated in clinical trials. AMG-176 was the first selective MCL-1 inhibitor to be evaluated in the clinic, and is currently being assessed in a phase I study for efficacy, tolerability, and PK properties in patients with relapsed/refractory MM or AML ( NCT02675452 ). 518 S64315 (MIK665), another MCL-1 inhibitor derived from S63845 , can induce apoptosis in diverse hematological malignancies in a Bax/Bak-dependent manner. 519 It is undergoing evaluation in several phase I clinical trials in patients with relapsed/refractory AML or MDS ( NCT02979366 ), and relapsed/refractory MM or lymphoma ( NCT02992483 ). Two other MCL-1 inhibitors AZD-5991 520 and AMG-176 521 have also entered clinical trials to evaluate their safety, PKs, and antitumor response in patients with hematological malignancies ( NCT03218683 and NCT02675452 ). Neither efficacy nor safety data of MCL-1 inhibitors are currently available. In addition, since BCL-XL overexpression is closely related to the oncogenesis of various solid tumors, targeting BCL-XL is a promising therapeutic strategy for some malignancies, although thrombocytopenia induced by BCL-XL inhibition remains a concern. Multiple BCL-XL inhibitors such as WEHI-539 and A-1331852 have been developed, and exhibit significant monotherapy activity in preclinical studies carried out in solid tumors. 522 As the time of this review, none of the selective BCL-XL inhibitors have entered clinical trials.

The success of BCL-2 family inhibitors in the treatment of hematological malignancies has broken new ground in small-molecule targeted therapy by confirming that protein–protein interactions can also be efficiently and specifically targeted. Despite favorable outcomes, challenges for these inhibitors remain, including identifying reliable biomarkers of response and elucidating resistant mechanisms to BCL-2 inhibition. A study conducted using samples from patients with venetoclax-treated CLL found that neither a cell sensitivity test in vitro nor TP53 status was a predictor of the antitumor response to venetoclax. 523 BH3 profiling, an in vitro assay to determine the dependence of tumor on individual anti-apoptotic protein, was reported as a potential solution to this problem. 523 However, due to the diversity of factors influencing the efficacy of BCL-2 inhibitors, the reliability of this finding still requires validation by systematic clinical evaluation. As has been demonstrated repeatedly, cancer cells eventually become resistant to targeted therapies, and BCL-2 apoptotic pathway-dependent malignancies are no exception. The resistance mechanisms involve upregulation of alternative anti-apoptotic BCL-2 family members such as BCL-XL and MCL-1, mutation or phosphorylation of BCL-2, and loss-of-function mutations of pro-apoptotic proteins BAX and BAK. 524 At present, combination therapy either with diverse anti-apoptotic BCL-2 family inhibitors or with BCL-2 family inhibitors and conventional chemotherapy is the main strategy to overcome resistance. There are considerable clinical studies focusing on these combined effects. 524 , 525 How to choose synergistic drugs and avoid the possible side effects are new challenges for researchers. As data from multiple phase I trials of combination therapy become available, close and critical assessments are needed.

Hedgehog pathway inhibitors

The hedgehog (HH) signaling pathway is highly conserved and has an important role in embryonic development and tissue regeneration. The HH pathway can be divided into canonical and noncanonical pathways. Activation of the canonical HH pathway is initiated by the release of HH ligands (Desert-DHH, Indian-IHH, and Sonic-SHH), and these ligands can bind and suppress the 12-pass transmembrane receptor Patched-1 (PTCH1). 526 In the absence of HH ligands, PTCH1 constitutively inhibits HH signaling by suppressing the transmembrane transducer Smoothened (SMO). Upon ligand binding, PTCH1 is internalized from the cell membrane and degraded, which results in the release of SMO to the primary cilium and phosphorylation at the cytoplasmic end. Active SMO promotes the activation of the glioma-associated oncogene (GLI) transcription factors GLI1, GLI2, and GLI3 and then induces the expression of target genes related to cell proliferation, survival, and differentiation (Fig. 5 ). 526 HH signaling can be abnormally activated through various mechanisms, such as HH ligand upregulation and PTCH1 or SMO mutations. Accumulating evidence has suggested that aberrant activation of the HH pathway is closely related to the oncogenesis and progression of a variety of tumors, including solid carcinomas and hematological tumors, as well as self-renewal of cancer stem cells (CSCs). 527 , 528 Therefore, the HH pathway has emerged as an attractive target for cancer therapy. Fig. 5 Canonical SMO-dependent hedgehog (HH) signaling pathway. Unliganded PTCH1 prevents the ciliary translocation of SMO effector protein. GLI2 and GLI3 proteins are sequestered in the cytoplasm by SUFU and phosphorylated by protein kinases, thereby preventing HH target-gene transcription. HH ligands binding triggers endocytic internalization of PTCH1, which results in the accumulation and activation of SMO. Active SMO relieves SUFU-mediated inhibition of GLI2 and GLI3. Activator forms of GLI (GLI1 A /GLI2 A /GLI3 A ) translocate into the nucleus and initiate the transcription of target genes. Figure created with BioRender.com

To date, three HH pathway inhibitors have been approved by the FDA for clinical oncology treatment (Table 5 ). Among them, both vismodegib and sonidegib are oral SMO inhibitors and are used for the treatment of locally advanced, unresectable, or metastatic basal cell carcinoma (mBCC). 529 , 530 The results of an open-label trial ( NCT01327053 ) for vismodegib showed a response rate of 68.5% in 1119 locally advanced BCCs (laBCCs) and 36.9% in 96 mBCC patients. The median PFS in laBCC and mBCC was 13.1 and 23.2 months, respectively. 531 Moreover, in a randomized trial of sonidegib for 66 laBCCs and 13 mBCCs ( NCT00833417 ), the 200 mg/day treatment group had response rates of 57.6% and 7.7%, respectively. The disease control rate, including stable disease, was 91.9% in laBCC and 92.3% in mBCC. 532 In addition to BCC, many clinical trials have been performed to evaluate the efficacy of these two agents in the treatment of various tumors, including rare tumors. 533 – 536 Glasdegib is also a selective HH pathway inhibitor that binds to SMO. In 2018, it was approved by the FDA in combination with low-dose cytarabine chemotherapy for newly diagnosed AML patients who were older than 75 years or unable to receive intensive chemotherapy due to chronic health problems and diseases. 537 This is also the first HH pathway inhibitor approved to treat AML.

Several other SMO inhibitors have also entered clinical trials to evaluate their therapeutic effects on BCC, pancreatic cancer, colon cancer, and breast cancer. 538 However, clinical studies of some drugs, such as saridegib (a cyclopamine analog), TAK-441, IPI-926, and CUR-61414, have been terminated due to detrimental effects and lack of response. 539 It is worth mentioning that the FDA-approved antifungal drug itraconazole was found to have an inhibitory effect against the HH pathway by antagonizing SMO. It has been reported that the clinical benefit of high-dose itraconazole in prostate cancer was mainly attributed to HH signaling inhibition rather than an anti-androgen effect. 540 Moreover, vitamin D3 could also bind SMO with high affinity and is currently in phase I or phase III clinical trials as a neoadjuvant for the treatment of BCC, pancreatic cancer, CLL, and NHL. 539

Inhibition of GLI-mediated transcription is an alternative strategy for developing HH signaling inhibitors, and this strategy has the potential to overcome acquired resistance of the approved SMO inhibitors. Currently, there are many reports on GLI inhibitors, but most of them are in the preclinical stage. Arsenic trioxide (ATO), a well-known agent approved by the FDA for acute promyelocytic leukemia treatment, is also a GLI inhibitor. Mechanistically, ATO blocks GLI2 accumulation and thus inhibits the transcriptional activation of GLI target genes. In a preclinical study, treatment with ATO or its combination with itraconazole effectively inhibited the growth of medulloblastoma harboring the SMO D477G mutation (or SMO D473H in humans) that is resistant to SMO inhibitors. 541 In a clinical study conducted in patients with refractory mBCC, combined treatment with ATO and itraconazole induced stable disease in 3 of 4 patients. 542 The hexahydropyrimidine derivatives GANT58 and GANT61 screened by constructed cells are selective GLI inhibitors. 543 They could interfere with GLI1/2-mediated transcriptional activity by blocking their DNA binding and restraining the growth of multiple tumors in a GLI-dependent manner. However, there has been no clinical study of these drugs due to their restricted pharmacological applicability.

Although the approved HH pathway inhibitors (SMO inhibitors) have shown significant antineoplastic activity in a variety of tumors, especially BCC, the diseases relapse and progress within several months due to acquired resistance. The resistance mechanisms involve SMO mutations, such as SMO D473H, activation of HH signaling downstream of SMO, and activation of compensatory signaling pathways. 544 As mentioned above, GLI inhibitors have the potential to conquer resistance to SMO inhibitors. Moreover, novel therapeutics targeting HH signaling has also been reported. LEQ-506 is an SMO inhibitor undergoing phase I clinical studies in some advanced solid tumors ( NCT01106508 ). It could suppress the proliferation of medulloblastoma cells harboring the SMO D473H mutation, which is derived from a patient who relapsed after an initial response to vismodegib treatment. 545 The SMO inhibitor taladegib can bind to and inhibit SMO D473H activity. A phase I trial ( NCT01919398 ) assessed the efficacy of taladegib in several advanced solid tumors and suggested that it is effective only in BCC with an estimated ORR of 46.8%. 546 This clinical trial also indicated that taladegib could benefit patients who were resistant to SMO inhibitors. As the compensatory upregulation of RAS/MAPK and PI3K-mTOR signaling pathways contributes to the mechanism of SMO-independent GLI regulation and resistance to SMO inhibitors, the combined use of HH signaling inhibitors and PI3K or MAPK pathway inhibitors may be a potential strategy to overcome the current resistance to SMO inhibitors. 528 , 547

Proteasome inhibitors

Proteasomes are large multicatalytic enzyme complexes that are expressed in the nucleus and cytoplasm of all eukaryotic cells and are responsible for more than 80% protein degradation in human cells. 548 The ubiquitin–proteasome system (UPS) has an important role in maintaining cellular protein homeostasis and regulating numerous biological processes, such as cell survival, signal transduction, DNA repair, and antigen presentation. 549 Most misfolded, unassembled, or damaged proteins that could otherwise form potentially toxic aggregates are degraded via UPS, in which proteins are tagged by ubiquitin and then recognized and degraded into small peptides by the proteasome complex (Fig. 6 ). Structurally, all proteasomes contain a common core, referred to as the 20S proteasome. The 20S core consists of a cylinder made of four stacked rings: 2 identical outer α-rings and 2 identical inner β-rings, each containing 7 distinct but related subunits. 548 , 550 The specificity of the 20S proteasome for substrate action depends on the peptide bond on the N2 terminal threonine residue of the β1, β2, β5 subunits. Dysfunction of the UPS is related to multiple human diseases, such as cancers, autoimmune diseases, and genetic diseases; 550 , 551 thus, much work has been conducted by targeting the UPS as a potential treatment strategy. Fig. 6 Proteasome inhibition acts through multiple mechanisms to induce cell apoptosis. Proteasome inhibition leads to NF-κB deactivation, thereby downregulating multiple pro-neoplastic pathways associated with cell proliferation, invasion, metastasis, and angiogenesis. Inhibition of proteasome activates the JNK signaling pathway and results in programmed cell death via caspase 3 and 7. Additionally, proteasome inhibition can indirectly cause apoptosis by preventing the degradation of pro-apoptotic family proteins such as BAX, BID, BIK, and BIM as well as NOXA. Inhibition of proteasome prevents the degradation of ubiquitinated proteins, which can increase endoplasmic reticulum (ER) stress and activate the UPR, cell cycle arrest, and subsequent apoptosis. Figure created with BioRender.com

Multiple myeloma (MM) cells produce excessive paraproteins, and their growth is dependent on proteasome-regulated signaling pathways. Therefore, MM cells are particularly susceptible to proteasome inhibition, and proteasome inhibitors (PIs) have become the backbone of MM clinical therapy. 552 Bortezomib is the first approved PI for the treatment of relapsed or refractory MM (Table 5 ). It is a peptide boronic acid and reversibly acts on the β5 catalytic subunit of the proteasome. 553 The clinical application of bortezomib significantly improves the long-term outcomes for MM patients. Moreover, Bortezomib has also been approved for the treatment of MCL. Currently, there are more than 200 clinical trials related to bortezomib that focus on its combination with other agents, efficacy in other cancers, and even other noncancer applications such as graft-versus-host disease. 554 However, bortezomib treatment has several limitations including primary resistance in MM and MCL patients, relapse in many initially-responding patients, and induction of dose-limiting peripheral neuropathy (PN). 554 , 555 To conquer these limitations, the second-generation PIs have been developed, and many of them are derived from synthetic and natural products. 552 , 556 Carfilzomib approved by the FDA in 2012 is a second-generation PI and is derived from the natural product epoxomicin (Table 5 ). 557 Unlike bortezomib, carfilzomib is an irreversible inhibitor that contains an epoxyketone warhead, which could covalently bind to the N-terminal threonine-containing active sites of the 20S proteasome. 558 The irreversible nature of carfilzomib contributes to its efficacy even in MM patients relapsed from or refractory to bortezomib. In addition, the carfilzomib-containing regimens exhibit significantly reduced peripheral neurotoxicity, while cardiovascular events were observed in MM patients treated with carfilzomib. 557 Another second-generation PI ixazomib was approved in 2015 in combination with lenalidomide and dexamethasone for MM patients who have received at least one prior therapy (Table 5 ). 559 Ixazomib is an N-capped dipeptidyl leucine boronic acid that reversibly inhibits the CT-L proteolytic (β5) site of the 20S proteasome. 560 Ixazomib shares the same pharmacophore boronic acid residue with bortezomib, however, the elimination half-life of the former is much shorter than that of the latter (18 vs. 110 min), which may contribute to the improved safety profiles of xazomib over bortezomib. 559 , 560 Patients resistant to bortezomib can still benefit from ixazomib treatment. Moreover, it is worth mentioning that both bortezomib and carfilzomib require parenteral (intravenous or subcutaneous) administration, whereas ixazomib is the first oral PI and is a prodrug. 561 Ixazomib is rapidly hydrolyzed to the active form (MLN2238) under physiological conditions.

Following the clinical success of the PIs, a number of novel PIs have been developed in recent years but only three of them are currently being evaluated in clinical trials. Marizomib (Salinosporamide A) is a naturally occurring β-lactone-γ-lactam bicyclic compound isolated from Salinispora bacteria. It can bind to 3 major catalytic sites on β5, β1, and β2 of proteasome irreversibly and inhibit proteasome activity at nanomolar concentrations. 562 The efficacy of marizomib in relapse or refractory MM and lymphoma is evaluated in phase I/II trials ( NCT00461045 , NCT00396864 ). Moreover, marizomib is reported to have CNS adverse events, which suggests that it can penetrate the BBB. 563 Therefore, it is currently under evaluation in a phase III clinical trial for malignant glioblastoma treatment ( NCT03345095 ). Oprozomib is a structural analog of carfilzomib, with a peptide-like backbone and an epoxyketone warhead. 564 The development of oprozomib aims to find an oral PI by chemical modification of carfilzomib. Currently, it is assessed in clinical trials for the treatment of refractory MM or relapsed MM after receiving bortezomib- or carfilzomib-based therapies ( NCT02939183 ). Another reversible PI under clinical assessment is delanzomib, an orally bioavailable structural homolog of bortezomib with a peptide-like backbone and a boronate warhead. 565 It is assessed in several phase I/II clinical trials for MM, lymphoma, and solid tumors. 566 , 567 Compared with BTZ, delanzomib showed slightly reduced efficacy against MM and some solid tumor cells, but had higher selectivity to cancer cells over normal cells, which means better drug safety. A phase I study of delanzomib also exhibits that it has a favorable safety profile with limited PN. 566

While emerging as an important treatment strategy for MM, PIs showed weak efficacy against solid cancers. The lack of clinical benefits of PIs in treating solid cancers was speculated to be attributed to their short elimination time and poor distribution to the proteasome target located in solid cancers. 554 , 568 Moreover, drug resistance has also been a major hurdle for PIs. As mentioned above, the next-generation PIs with improved PK/PD characteristics may promote the clinical efficacy of MM patients as well as those who are resistant to existing PI treatment, and extend treatment benefits to solid cancers. Meanwhile, combined proteasome inhibition with other active agents, such as BCL-2 inhibitor, HDAC inhibitor, immunomodulatory agents, and some other inhibitors of signaling pathways related to resistant mechanisms, is also a strategy to conquer resistance to PIs. 569 These combination therapies are being assessed in many preclinical and clinical studies.

PARP inhibitors

Genomic instability is one of the typical characteristics of tumor cells. To maintain genomic integrity, tumor cells have multiple mechanisms to repair DNA lesions, such as the repair pathways of DNA double-strand breaks (DSBs) and single-strand breaks (SSBs). Among them, the former includes homologous recombination and non-homologous end joining (NHEJ), while the latter includes base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). 570 Poly (ADP-ribose) polymerases (PARPs) are a group of multifunctional post-translational modification enzymes that engage in a diverse set of cellular processes, including DNA repair, transcription, mitosis, and cell cycle regulation. 571 To date, 18 members have been identified in PARP family proteins, among which PARP1 is the best-studied PARP member and has an important role in the repair of DNA SSBs. Once DNA SSBs occur, PARP1 binds to damaged DNA through N-terminal zinc finger domains, allowing its cofactor β-nicotinamide adenine dinucleotide (β-NAD) to bind to the active site of the enzyme and activating the catalytic function of the ADP-ribosyltransferase catalytic domain. PARP1 then catalyzes the transfer of PAR chains to the target proteins (PARylation) in the vicinity of the DNA breaks, which promotes chromatin remodeling and the recruitment of a series of DNA repair effectors and completes the DNA repair process (Fig. 7 ). 570 , 571 PARP1 autoPARylation eventually causes its dissociation from DNA damage and restores its autoinhibitory status. Inhibition of another DNA repair pathway in cancer cells with defective DNA repair mechanisms may create a “synergistic lethal” effect; this theory was first proposed by Theodosius Dobzhansky et al. 572 Breast cancer susceptibility genes BRCA1 and BRCA2 are two key tumor suppressors that repair DNA DSBs. Mutations in BRCA1 and BRCA2 are susceptible to breast and ovarian cancers, and DSBs are not easily repaired in BRCA -mutant tumor cells. 573 Therefore, PARP inhibition in BRCA- mutant cancers can induce synthetic lethality due to the simultaneous blockade of both DSB and SSB repair pathways (Fig. 7 ). Fig. 7 Molecular process of DNA damage repair related to PARP and the mechanism of action of PARP inhibitors. Endogenous single-strand breaks (SSB) are repaired mostly by PARP-dependent base excision repair (BER) pathway. PARP inhibitors suppress the repair of SSB and the unrepaired SSB can be converted to double-strand breaks (DSB) that are toxic to cells. Homologous recombination (HR) is the major pathway to repair DSB. However, the DSB in BRCA1/2 mutant cells cannot be repaired through HR, thus resulting in genomic instability and cell death. Figure created with BioRender.com

Nicotinamide, the cofactor of PARPs, which competes with NAD for the catalytic pocket of PARPs, is the first identified PARP inhibitor. 574 Currently, four PARP inhibitors with nicotinamide pharmacophores (olaparib, rucaparib, niraparib, and talazoparib) have been approved by the FDA or the EMA (Table 5 ). 575 – 578 All PARP inhibitors have the ability to inhibit the catalytic activity of PARPs. However, this mechanism cannot fully explain the antitumor activity of PARP inhibitors. They can also trap PARP in a non-effective state at chromatin, and such binding to the PARP–chromatin complex will produce more effective cytotoxicity. 579 Based on in-depth research on the mechanisms of action of PARP inhibitors and the results of clinical trials, indications of PARP inhibitors have been continuously updated since the first inhibitor olaparib was approved in 2014. Olaparib was originally approved for patients with deleterious or suspected deleterious germline BRCA -mutant advanced ovarian cancer who had undergone three or more prior lines of chemotherapy, followed by rucaparib in 2016, and niraparib in 2017. 575 , 576 Later, in 2017 and 2018, olaparib, niraparib, and rucaparib were approved as maintenance therapies in recurrent platinum-sensitive cancers, including epithelial ovarian cancer, fallopian tube cancer (FTC), or primary peritoneal cancer (PPC), regardless of BRCA status. 575 – 577 Furthermore, olaparib became a first-line maintenance treatment in germline or somatic BRCA -mutated cancer patients who responded to platinum-based chemotherapy according to the data of clinical trial ( NCT01874353 ). 580 In 2018, the FDA successively approved olaparib and talazoparib for BRCA -mutated HER2-negative locally advanced or metastatic breast cancer. In addition, niraparib expanded the indications for the treatment of advanced ovarian cancer, FTC, and PPC associated with homologous recombination deficiency positive status in 2019. 581

At present, a large number of clinical trials related to olaparib, rucaparib, niraparib, or talazoparib alone or in combination are still underway to identify responding patients beyond the ovarian or breast cancer population. Additionally, several other PARP inhibitors, such as pamiparib, veliparib, INO-1001, E7449, iniparib, AZD2461, amelparib, IMP4297, RBN-2397, and fluzoparib, have also entered clinical trials and are in different stages. 582 , 583 Among them, veliparib (ABT-888) is an orally bioavailable PARP inhibitor developed by Abbvie that can cross the blood–brain barrier. 584 Its clinical efficacy has been evaluated in multiple solid tumors. A phase III trial of veliparib with first-line chemotherapy showed an increased PFS in high-grade serous ovarian carcinoma compared with paclitaxel and carboplatin alone ( NCT02470585 ). However, the clinical outcomes could not be significantly improved by veliparib as monotherapy in patients with ovarian or pancreatic cancer as well as by its combination with carboplatin/paclitaxel in TNBC. 585 To date, veliparib has not been approved by any agency.

The efficacy of PARP inhibitors in cancer treatment is not restricted to BRCA1/2 mutant sufferers. In the treatment of ovarian cancers, 40–70% of BRCA1/2 mutated patients failed to respond to PARP inhibitors. 586 Platinum sensitivity is also reported as a prospective indicator for predicting the response to these inhibitors. 587 Nevertheless, these two predictors still cannot completely cover the tumor types suitable for PARP inhibitor treatment. This is one of the problems that need further research in PARP inhibition therapy. In addition, with the widespread use of PARP inhibitors in the clinic, the issue of acquired resistance has emerged frequently. Mechanisms of resistance are multifactorial, mainly involving the restoration of HR function caused by secondary mutations of homologous recombination repair genes ( BRCA1/2 , RAD51C/D , PALB2 ) and defects in NHEJ as well as loss of 53BP1, stabilization of replication forks, PARP1 mutations, and drug efflux. 588 Combined therapy is increasingly explored as a means to enhance the efficacy and overcome drug resistance to PARP inhibitors. Many studies have shown that simultaneously targeting PARPs and cell cycle checkpoints (ATR, CHK1, and WEE1) can overcome the resistance caused by restored replication fork stabilization. 589 , 590 A phase II study of olaparib plus ATR/WEE1 inhibitor vs. olaparib monotherapy is currently underway in TNBC patients ( NCT04191135 ). 591 Moreover, it should be noted that combination therapy may also increase the risk of side effects. Severe myelosuppression was observed in several phase I trials of PARP inhibitors combined with chemotherapy drugs. 592 It is still of great importance to better understand the predictors and resistance mechanisms of PARP inhibitors and ultimately improve diagnosis, treatment strategy, and outcome.

Concluding remarks: challenges and future perspectives

With the evolution of modern molecular biology and the application of some advanced technologies such as computer-aided drug design, structure biology, and combinatorial chemistry, small-molecule targeted anti-cancer drugs have entered a rapid development stage. To date, 89 small-molecule targeted drugs have been approved by the FDA and/or NMPA to treat various cancers (Fig. 1 ). Thousands of targeted agents are undergoing clinical trials for cancer treatment (Supplementary Fig. S1 ). Among them, a large number of promising agents have advanced to phase III trials (Supplementary Tables S2 – S5 ). According to the prediction of the Business Research Company, the global anti-cancer drug market size will reach 200 billion dollars in 2021, among which targeted drugs are the “main force”. Despite the significant progress achieved, there are still some challenges that small-molecule targeted anti-cancer drugs face.

The first major challenge is drug resistance. Almost all targeted anti-cancer drugs come across resistance after a period of time of clinical use. Drug resistance has been linked to many mechanisms, including gene mutation, amplification, CSCs, efflux transporters, apoptosis dysregulation, and autophagy, etc (Fig. 8 ). 593 – 599 Gene mutation is the main reason leading to anti-cancer drug resistance. There are two different views regarding drug-resistant gene mutations. One is that the gene mutations are induced by drugs. The other one is that the drug-resistant mutations have already existed. In the early stage of treatment, cancer cells with drug-sensitive mutations dominate and suppress the proliferation of cells containing drug-resistant mutations. After cells with sensitive mutations are killed, the resistant mutant cells become the mainstream and show resistance. Amplification of other genes is another common reason for anti-cancer drug resistance. For example, MET amplification accounts for about 20% of EGFR inhibitor-resistant cases. 46 CSCs are also thought to be an important reason for drug resistance and recurrence. 595 , 596 The CSC theory proposes that the different cells within a tumor, as well as metastasis deriving from it, originate from a single subpopulation of cells with self-renewal and differentiation capacities, which are similar to stem cells. Overexpression of efflux transporters, such as multidrug resistance transporter proteins, especially P-glycoprotein, which renders the resistance to chemotherapeutic drugs, also has a role in the targeted anti-cancer drug resistance. 597 , 598 In addition to these causes, apoptosis dysregulation and autophagy also could be responsible for anti-cancer drug resistance. 593 , 599 Fig. 8 Mechanisms and insights in drug resistance of small-molecule targeted anti-cancer agents. Figure created with BioRender.com

Low efficiency is another major challenge for targeted anti-cancer drugs. As mentioned many times before, targeted anti-cancer drugs are effective only in a limited number of patients. For example, less than 20% of patients with NSCLC are sensitive to EGFR inhibitors (e.g., gefitinib and erlotinib). Patients that are sensitive to these EGFR inhibitors are found to harbor EGFR-activating mutations (for example, exon 19 deletion or exon 21 L858R point mutations). 74 TRK inhibitors larotrectinib and entrectinib have been approved for the treatment of patients with NTRK gene rearrangements, regardless of cancer type and patient age. These rearrangements can be found at high frequencies (up to 90%) in certain types of cancer, such as infantile fibrosarcoma (a rare disease), but the incidence is only 1% of all malignancies. 225 These highlight the importance of the identification of predictive biomarkers for response to targeted anti-cancer drugs.

Currently, in order to deal with the major challenges of targeted anti-cancer drugs, many strategies have been applied, such as new generation anti-cancer drugs against drug resistance mutations, multitarget drugs, combination therapy, and drugs targeting CSCs. 594 , 600 , 601 In addition to these, several new research trends in this area deserve attention.

The first one is the drug discovery against new type cancer targets. For example, in the past years, some new epigenetic regulatory proteins have garnered increasing attention, such as RNA m6A methylation-related proteins (METTL3/14, FTO, ALKBH5, WTAP, and YTHDFs). 602 , 603 microRNAs (miRNAs) are another new type of cancer targets, which are frequently dysregulated in cancers and may serve as promising targets for cancer therapy. Currently, some effects have been paid to discover small-molecule inhibitors against miRNAs, such as miR-21 inhibitor AC1MMYR2 (also known as NSC211332), Lin28-let-7 inhibitors 6-hydroxy-DL-DOPA, SB/ZW/0065, and KCB3602. 604

Furthermore, some proteins that are previously thought undruggable may also be attractive anti-cancer targets. A typical example is KRAS , a most frequently mutated isoform of RAS proto-oncogene, which has a predominant role in driving the initiation and progression of cancers. 605 , 606 Decades of efforts failed to target KRAS with small molecules due to the high binding affinity of the intrinsic ligand GTP to KRAS, the relatively small size and smooth surface of KRAS, and the high flexibility of KRAS switch regions. 607 – 609 However, this dilemma has begun to change recently. A variety of novel small molecules that directly target KRAS are being developed, including covalent allosteric inhibitors for KRAS G12C mutant, protein–protein interaction inhibitors that bind in the switch I/II pocket or the A59 site, and GTP-competitive inhibitors targeting the nucleotide-binding site. 609 , 610 To date, four KRAS G12C covalent inhibitors (AMG510, JNJ-74699157, MRTX849, and GDC-6036) with similar allosteric mechanisms have advanced to clinical trials. 611 , 612 In addition to KRAS, other types of such previously undruggable cancer targets include MYC, 613 phosphatases, 614 and protein–protein interactions, 615 , 616 etc.

The second one is the combination of small-molecule targeted drugs with immunotherapy such as PD-1 antibody. Lenvatinib plus pembrolizumab, a PD-1 antibody, was designated as a breakthrough therapy in 2018 by the FDA for patients with advanced or metastatic RCC. The ORR was 63.3% for all RCC patients receiving this combination therapy, and 83.3% in the first-line treatment setting. 617 Inspiringly, the combination of pembrolizumab with another small-molecule anti-angiogenesis agent axitinib has been approved for the treatment of patients with advanced RCC. 618 , 619 This is also the first combination therapy of PD-1 antibody plus targeted drugs approved by the FDA for the first-line treatment of advanced RCC.

The third one is antibody-drug conjugate (ADC) drugs. Though the first ADC drug (Mylotarg, Pfizer) suffered setback in 2010 due to the limitations of coupling technology, targeting, and effectiveness, people did not lose confidence in ADC drugs. 620 , 621 With the improvement of antibody-conjugate technology, ten ADC drugs such as polatuzumab vedotin-piiq (Polivy), enfortumab vedotin-ejfv (Padcev), fam-trastuzumab deruxtecan-nxki (Enhertu), sacituzumab govitecan-hziy (Trodelvy), and belantamab mafodotin-blmf (Blenrep) have been approved by the FDA in the past decade. 622 – 624 ADC drugs will be an important development direction in the future.

The fourth one is PROTAC, which employs small molecules that recruit target proteins for ubiquitination and removal by the proteasome. 625 PROTAC technology is different from the traditional “target occupying” inhibitor therapy. It reduces the activity of target proteins by catalyzing the degradation of target proteins. Due to the slow biosynthesis of target proteins, this method can greatly slow down the recovery of target protein activity. PROTAC is a rapidly emerging alternative therapeutic strategy with the potential to address many of the challenges currently faced in modern drug development programs. Currently, two drugs (ARV-110 and ARV-471) designed by PROTAC technology have entered clinical trials. 625 Both of them are developed by Arvinas and approved by the FDA in 2019 for phase I clinical trials. Among them, ARV-110 specifically binds to androgen receptor (AR) and mediates AR degradation, and is used for the treatment of patients with metastatic CRPC. 626 ARV-471 is an ER protein degrading agent. In preclinical studies, ARV-471 led to almost completely ER degradation in tumor cells and exhibited strong growth suppression in multiple ER-driven xenograft models. 627 It is now evaluated clinically for the treatment of patients with locally advanced or metastatic ER + /HER2 - breast cancer.

The fifth one is synthetic lethality, which means that loss of function of either of the two genes individually has little effect on cancer cell viability, but the inactivation of both genes simultaneously by gene mutation/deletion or pharmacological inhibition leads to cell death. 628 Normal cells are usually spared the effect of synthetic lethality due to the lack of the fixed genetic alteration. The concept of synthetic lethality has been used in the development of targeted therapy for many years. The most popular examples are the synthetic lethal interaction of combined BCL-XL and MEK inhibition in KRAS-mutant cancer models as well as the successful clinical application of PARP inhibitors in BRCA-mutant ovarian cancer. 574 , 629 Although it is not a novel idea, synthetic lethality provides an alternative treatment strategy for some known genetic drivers of cancer that cannot be directly targeted owing to their molecular structure (undruggable oncogenes) or because they result in a functional loss (tumor suppressor genes). Therefore, synthetic lethality has promising potential to drive the discovery of new anti-cancer targets and subsequently the development of effective medicines or combination strategies that are still needed for most cancers.

In summary, small-molecule targeted drugs will continue to be the mainstream in cancer treatment because of their unique advantages, despite the competition from macromolecule drugs. With an in-depth understanding of tumor pathology and the evolution of new drug research and development technology, we believe that more new small-molecule anti-cancer drugs that target novel genes or the mechanism of action will be developed in the near future. It is also expected that some new areas such as the combination of small-molecule targeted drugs with tumor immunotherapy, ADC, and PROTAC will gain significant development in the next decade.

Supplementary information

Supplemental Material File #1

These authors contributed equally: Lei Zhong, Yueshan Li, Liang Xiong, Wenjing Wang

Supplementary information

The online version contains supplementary material available at 10.1038/s41392-021-00572-w.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81930125, 81702286, 81773633, and 21772130), the Research Project of Science and Technology Department of Sichuan Province (2021YJ0134, 2019YFS0514, and 2020YFS0006), the 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (ZYGD18001), and partially by the fast-track grants from West China Hospital, Sichuan University (HX-2019-nCoV-053), and the Open Project of Radiation Oncology Key Laboratory of Sichuan Province (2020FSZLX04).

Competing interests

Shengyong Yang is the editorial board member of Signal Transduction and Targeted Therapy, but he has not been involved in the process of manuscript handling. The remaining authors declare no competing interests.

References

1. Bedard PL Hyman DM Davids MS Siu LL Small molecules, big impact: 20 years of targeted therapy in oncology Lancet 2020 395 1078 1088 10.1016/S0140-6736(20)30164-1 32222192

2. Savage DG Antman KH Imatinib mesylate–a new oral targeted therapy N. Engl. J. Med. 2002 346 683 693 10.1056/NEJMra013339 11870247

3. Wilkes GM Targeted therapy: attacking cancer with molecular and immunological targeted agents Asia Pac. J. Oncol. Nurs. 2018 5 137 155 10.4103/apjon.apjon_79_17 29607374 PMC5863423

4. Lee YT Tan YJ Oon CE Molecular targeted therapy: treating cancer with specificity Eur. J. Pharm. 2018 834 188 196 10.1016/j.ejphar.2018.07.034 30031797

5. Ardito F The crucial role of protein phosphorylation in cell signaling and its use as targeted therapy (review) Int J. Mol. Med. 2017 40 271 280 10.3892/ijmm.2017.3036 28656226 PMC5500920

6. Wilson LJ New perspectives, opportunities, and challenges in exploring the human protein kinome Cancer Res. 2018 78 15 29 10.1158/0008-5472.CAN-17-2291 29254998

7. Roskoski R Jr. Classification of small molecule protein kinase inhibitors based upon the structures of their drug-enzyme complexes Pharm. Res. 2016 103 26 48 10.1016/j.phrs.2015.10.021 26529477

8. Morris SW Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin’s lymphoma Science 1995 267 316 317 10.1126/science.267.5196.316-b 7824924

9. Iwahara T Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system Oncogene 1997 14 439 449 10.1038/sj.onc.1200849 9053841

10. McManus DT ALK-positive diffuse large B-cell lymphoma of the stomach associated with a clathrin-ALK rearrangement Hum. Pathol. 2004 35 1285 1288 10.1016/j.humpath.2004.06.001 15492998

11. Cessna MH Expression of ALK1 and p80 in inflammatory myofibroblastic tumor and its mesenchymal mimics: a study of 135 cases Mod. Pathol. 2002 15 931 938 10.1097/01.MP.0000026615.04130.1F 12218210

12. Soda M Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer Nature 2007 448 561 566 10.1038/nature05945 17625570

13. Waggott W Detection of NPM-ALK DNA rearrangement in CD30 positive anaplastic large cell lymphoma Br. J. Haematol. 1995 89 905 907 10.1111/j.1365-2141.1995.tb08434.x 7772531

14. Trinei M A new variant anaplastic lymphoma kinase (ALK)-fusion protein (ATIC-ALK) in a case of ALK-positive anaplastic large cell lymphoma Cancer Res. 2000 60 793 798 10706082

15. Ma Z Fusion of ALK to the Ran-binding protein 2 (RANBP2) gene in inflammatory myofibroblastic tumor Genes Chromosomes Cancer 2003 37 98 105 10.1002/gcc.10177 12661011

16. Kong X Drug discovery targeting anaplastic lymphoma kinase (ALK) J. Med. Chem. 2019 62 10927 10954 10.1021/acs.jmedchem.9b00446 31419130

17. Cui JJ Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK) J. Med. Chem. 2011 54 6342 6363 10.1021/jm2007613 21812414

18. Shaw AT Crizotinib versus chemotherapy in advanced ALK-positive lung cancer N. Engl. J. Med. 2013 368 2385 2394 10.1056/NEJMoa1214886 23724913

19. Solomon BJ First-line crizotinib versus chemotherapy in ALK-positive lung cancer N. Engl. J. Med. 2014 371 2167 2177 10.1056/NEJMoa1408440 25470694

20. Sasaki T A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors Cancer Res. 2011 71 6051 6060 10.1158/0008-5472.CAN-11-1340 21791641 PMC3278914

21. Costa DB Clinical experience with crizotinib in patients with advanced ALK-rearranged non-small-cell lung cancer and brain metastases J. Clin. Oncol. 2015 33 1881 1888 10.1200/JCO.2014.59.0539 25624436 PMC4451171

22. Marsilje TH Synthesis, structure-activity relationships, and in vivo efficacy of the novel potent and selective anaplastic lymphoma kinase (ALK) inhibitor 5-chloro-N2-(2-isopropoxy-5-methyl-4-(piperidin-4-yl)phenyl)-N4-(2-(isopropylsulf onyl)phenyl)pyrimidine-2,4-diamine (LDK378) currently in phase 1 and phase 2 clinical trials J. Med. Chem. 2013 56 5675 5690 10.1021/jm400402q 23742252

23. Kinoshita K 9-substituted 6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazoles as highly selective and potent anaplastic lymphoma kinase inhibitors J. Med. Chem. 2011 54 6286 6294 10.1021/jm200652u 21823617

24. Huang WS Discovery of brigatinib (AP26113), a phosphine oxide-containing, potent, orally active inhibitor of anaplastic lymphoma kinase J. Med. Chem. 2016 59 4948 4964 10.1021/acs.jmedchem.6b00306 27144831

25. Friboulet L The ALK inhibitor ceritinib overcomes crizotinib resistance in non-small cell lung cancer Cancer Discov. 2014 4 662 673 10.1158/2159-8290.CD-13-0846 24675041 PMC4068971

26. Markham A Brigatinib: first global approval Drugs 2017 77 1131 1135 10.1007/s40265-017-0776-3 28597393

27. Qian M Zhu B Wang X Liebman M Drug resistance in ALK-positiveNon-small cell lungcancer patients Semin. Cell Dev. Biol. 2017 64 150 157 10.1016/j.semcdb.2016.09.016 27693505

28. Basit S Ashraf Z Lee K Latif M First macrocyclic 3(rd)-generation ALK inhibitor for treatment of ALK/ROS1 cancer: clinical and designing strategy update of lorlatinib Eur. J. Med. Chem. 2017 134 348 356 10.1016/j.ejmech.2017.04.032 28431340

29. Syed YY Lorlatinib: first global approval Drugs 2019 79 93 98 10.1007/s40265-018-1041-0 30604291

30. Guan J The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN Dis. Model Mech. 2016 9 941 952 27483357 10.1242/dmm.024448 PMC5047689

31. Drilon A Safety and antitumor activity of the multitargeted pan-TRK, ROS1, and ALK inhibitor entrectinib: combined results from two phase I trials (ALKA-372-001 and STARTRK-1) Cancer Discov. 2017 7 400 409 10.1158/2159-8290.CD-16-1237 28183697 PMC5380583

32. Sachdev J 506 Phase (Ph) 1/2a study of TSR-011, a potent inhibitor of ALK and TRK, in advanced solid tumors including crizotinib-resistant ALK positive non-small cell lung cancer Eur. J. Cancer 2014 50 165 10.1016/S0959-8049(14)70632-X

33. Drilon A Repotrectinib (TPX-0005) Is a next-generation ROS1/TRK/ALK inhibitor that potently inhibits ROS1/TRK/ALK solvent-front mutations Cancer Discov. 2018 8 1227 1236 10.1158/2159-8290.CD-18-0484 30093503

34. Menichincheri M Discovery of entrectinib: a new 3-aminoindazole as a potent anaplastic lymphoma kinase (ALK), c-ros oncogene 1 kinase (ROS1), and pan-tropomyosin receptor kinases (Pan-TRKs) inhibitor J. Med. Chem. 2016 59 3392 3408 10.1021/acs.jmedchem.6b00064 27003761

35. Infante JR Safety, pharmacokinetic, and pharmacodynamic phase I dose-escalation trial of PF-00562271, an inhibitor of focal adhesion kinase, in advanced solid tumors J. Clin. Oncol. 2012 30 1527 1533 10.1200/JCO.2011.38.9346 22454420

36. Shaw AT Resensitization to crizotinib by the lorlatinib ALK resistance mutation L1198F N. Engl. J. Med. 2016 374 54 61 10.1056/NEJMoa1508887 26698910 PMC4773904

37. Chen Z Inhibition of ALK, PI3K/MEK, and HSP90 in murine lung adenocarcinoma induced by EML4-ALK fusion oncogene Cancer Res. 2010 70 9827 9836 10.1158/0008-5472.CAN-10-1671 20952506 PMC3043107

38. Roskoski R Jr. Anaplastic lymphoma kinase (ALK) inhibitors in the treatment of ALK-driven lung cancers Pharm. Res. 2017 117 343 356 10.1016/j.phrs.2017.01.007 28077299

39. Courtin A Emergence of resistance to tyrosine kinase inhibitors in non-small-cell lung cancer can be delayed by an upfront combination with the HSP90 inhibitor onalespib Br. J. Cancer 2016 115 1069 1077 10.1038/bjc.2016.294 27673365 PMC5117788

40. Zhang C Proteolysis targeting chimeras (PROTACs) of anaplastic lymphoma kinase (ALK) Eur. J. Med. Chem. 2018 151 304 314 10.1016/j.ejmech.2018.03.071 29627725 PMC5924614

41. Park M Mechanism of met oncogene activation Cell 1986 45 895 904 10.1016/0092-8674(86)90564-7 2423252

42. Baldanzi G Graziani A Physiological signaling and structure of the HGF receptor MET Biomedicines 2014 3 1 31 10.3390/biomedicines3010001 28536396 PMC5344233

43. Holmes O Insights into the structure/function of hepatocyte growth factor/scatter factor from studies with individual domains J. Mol. Biol. 2007 367 395 408 10.1016/j.jmb.2006.12.061 17258232

44. Organ SL Tsao MS An overview of the c-MET signaling pathway Ther. Adv. Med. Oncol. 2011 3 S7 s19 10.1177/1758834011422556 22128289 PMC3225017

45. Blumenschein GR Jr. Mills GB Gonzalez-Angulo AM Targeting the hepatocyte growth factor-cMET axis in cancer therapy J. Clin. Oncol. 2012 30 3287 3296 10.1200/JCO.2011.40.3774 22869872 PMC3434988

46. Turke AB Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC Cancer Cell 2010 17 77 88 10.1016/j.ccr.2009.11.022 20129249 PMC2980857

47. Frampton GM Activation of MET via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors Cancer Discov. 2015 5 850 859 10.1158/2159-8290.CD-15-0285 25971938

48. Garajova I Giovannetti E Biasco G Peters GJ c-Met as a Target for Personalized Therapy Transl. Oncogenomics 2015 7 13 31 26628860 10.4137/TOG.S30534 PMC4659440

49. Heigener DF Reck M Crizotinib Recent Results Cancer Res. 2018 211 57 65 10.1007/978-3-319-91442-8_4 30069759

50. Choueiri TK Cabozantinib versus everolimus in advanced renal cell carcinoma (METEOR): final results from a randomised, open-label, phase 3 trial Lancet Oncol. 2016 17 917 927 10.1016/S1470-2045(16)30107-3 27279544

51. Liu X A novel kinase inhibitor, INCB28060, blocks c-MET-dependent signaling, neoplastic activities, and cross-talk with EGFR and HER-3 Clin. Cancer Res. 2011 17 7127 7138 10.1158/1078-0432.CCR-11-1157 21918175

52. Wolf J Capmatinib in MET exon 14-mutated or MET-amplified non-small-cell lung cancer N. Engl. J. Med. 2020 383 944 957 10.1056/NEJMoa2002787 32877583

53. Wu YL Phase Ib/II study of capmatinib (INC280) plus gefitinib after failure of epidermal growth factor receptor (EGFR) inhibitor therapy in patients with EGFR-mutated, MET factor-dysregulated non-small-cell lung cancer J. Clin. Oncol. 2018 36 3101 3109 10.1200/JCO.2018.77.7326 30156984

54. Lara MS Preclinical evaluation of MET inhibitor INC-280 with or without the epidermal growth factor receptor inhibitor erlotinib in non-small-cell lung cancer Clin. Lung Cancer 2017 18 281 285 10.1016/j.cllc.2016.11.006 28038979 PMC5837026

55. Bladt F EMD 1214063 and EMD 1204831 constitute a new class of potent and highly selective c-Met inhibitors Clin. Cancer Res. 2013 19 2941 2951 10.1158/1078-0432.CCR-12-3247 23553846

56. Friese-Hamim M The selective c-Met inhibitor tepotinib can overcome epidermal growth factor receptor inhibitor resistance mediated by aberrant c-Met activation in NSCLC models Am. J. Cancer Res. 2017 7 962 972 28469968 PMC5411803

57. Markham A Tepotinib: first approval Drugs 2020 80 829 833 10.1007/s40265-020-01317-9 32361823

58. Leighl NB A phase I study of foretinib plus erlotinib in patients with previously treated advanced non-small cell lung cancer: Canadian cancer trials group IND.196 Oncotarget 2017 8 69651 69662 10.18632/oncotarget.18753 29050231 PMC5642506

59. Engstrom LD Glesatinib exhibits antitumor activity in lung cancer models and patients harboring MET exon 14 mutations and overcomes mutation-mediated resistance to type I MET inhibitors in nonclinical models Clin. Cancer Res. 2017 23 6661 6672 10.1158/1078-0432.CCR-17-1192 28765324

60. Miranda, O., Farooqui, M. & Siegfried, J. M. Status of agents targeting the HGF/c-met axis in lung cancer. Cancers 10 , 280 (2018). 10.3390/cancers10090280 PMC6162713 30134579

61. Rodon J First-in-human phase I study of oral S49076, a unique MET/AXL/FGFR inhibitor, in advanced solid tumours Eur. J. Cancer 2017 81 142 150 10.1016/j.ejca.2017.05.007 28624695

62. Sequist LV Randomized phase II study of erlotinib plus tivantinib versus erlotinib plus placebo in previously treated non-small-cell lung cancer J. Clin. Oncol. 2011 29 3307 3315 10.1200/JCO.2010.34.0570 21768463

63. Yang J-J Preliminary results of a phase Ib trial of savolitinib combined with gefitinib in EGFR-mutant lung cancer J. Clin. Oncol. 2016 34 e20559 e20559 10.1200/JCO.2016.34.15_suppl.e20559

64. Egile C The selective intravenous inhibitor of the MET tyrosine kinase SAR125844 inhibits tumor growth in MET-amplified cancer Mol. Cancer Ther. 2015 14 384 394 10.1158/1535-7163.MCT-14-0428 25504634

65. Shitara K Phase I dose-escalation study of the c-Met tyrosine kinase inhibitor SAR125844 in Asian patients with advanced solid tumors, including patients with MET-amplified gastric cancer Oncotarget 2017 8 79546 79555 10.18632/oncotarget.18554 29108334 PMC5668067

66. Parikh PK Ghate MD Recent advances in the discovery of small molecule c-Met Kinase inhibitors Eur. J. Med. Chem. 2018 143 1103 1138 10.1016/j.ejmech.2017.08.044 29157685

67. Bradley CA Targeting c-MET in gastrointestinal tumours: rationale, opportunities and challenges Nat. Rev. Clin. Oncol. 2017 14 562 576 10.1038/nrclinonc.2017.40 28374784

68. Salgia R MET in lung cancer: biomarker selection based on scientific rationale Mol. Cancer Ther. 2017 16 555 565 10.1158/1535-7163.MCT-16-0472 28373408

69. Qi J Multiple mutations and bypass mechanisms can contribute to development of acquired resistance to MET inhibitors Cancer Res. 2011 71 1081 1091 10.1158/0008-5472.CAN-10-1623 21266357 PMC3208270

70. Cepero V MET and KRAS gene amplification mediates acquired resistance to MET tyrosine kinase inhibitors Cancer Res. 2010 70 7580 7590 10.1158/0008-5472.CAN-10-0436 20841479

71. Threadgill DW Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype Science 1995 269 230 234 10.1126/science.7618084 7618084

72. Blobel CP ADAMs: key components in EGFR signalling and development Nat. Rev. Mol. Cell Biol. 2005 6 32 43 10.1038/nrm1548 15688065

73. Salomon DS Brandt R Ciardiello F Normanno N Epidermal growth factor-related peptides and their receptors in human malignancies Crit. Rev. Oncol./Hematol. 1995 19 183 232 10.1016/1040-8428(94)00144-I 7612182

74. Metro G Epidermal growth factor receptor (EGFR) targeted therapies in non-small cell lung cancer (NSCLC) Rev. Recent Clin. trials 2006 1 1 13 10.2174/157488706775246157 18393776

75. Roskoski R Jr. The ErbB/HER family of protein-tyrosine kinases and cancer Pharm. Res. 2014 79 34 74 10.1016/j.phrs.2013.11.002 24269963

76. Gu A Efficacy and safety evaluation of icotinib in patients with advanced non-small cell lung cancer Chin. J. Cancer Res. 2013 25 90 94 23372346 10.3978/j.issn.1000-9604.2012.12.07 PMC3555305

77. Lynch TJ Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib N. Engl. J. Med. 2004 350 2129 2139 10.1056/NEJMoa040938 15118073

78. Kulke MH Capecitabine plus erlotinib in gemcitabine-refractory advanced pancreatic cancer J. Clin. Oncol. 2007 25 4787 4792 10.1200/JCO.2007.11.8521 17947726

79. Maione P Overcoming resistance to targeted therapies in NSCLC: current approaches and clinical application Ther. Adv. Med. Oncol. 2015 7 263 273 10.1177/1758834015595048 26327924 PMC4543855

80. Miller VA Afatinib versus placebo for patients with advanced, metastatic non-small-cell lung cancer after failure of erlotinib, gefitinib, or both, and one or two lines of chemotherapy (LUX-Lung 1): a phase 2b/3 randomised trial Lancet Oncol. 2012 13 528 538 10.1016/S1470-2045(12)70087-6 22452896

81. Lavacchi D Mazzoni F Giaccone G Clinical evaluation of dacomitinib for the treatment of metastatic non-small cell lung cancer (NSCLC): current perspectives Drug Des. Dev. Ther. 2019 13 3187 3198 10.2147/DDDT.S194231 PMC6735534 31564835

82. Schrank, Z. et al. Current molecular-targeted therapies in NSCLC and their mechanism of resistance. Cancers 10 , 224 (2018). 10.3390/cancers10070224 PMC6071023 29973561

83. Yver A Osimertinib (AZD9291)-a science-driven, collaborative approach to rapid drug design and development Ann. Oncol. 2016 27 1165 1170 10.1093/annonc/mdw129 26961148

84. Yang, J. C. et al. Safety, efficacy, and pharmacokinetics of almonertinib (HS-10296) in pretreated patients with EGFR-mutated advanced NSCLC: a multicenter, open-label, phase 1 trial. J. Thorac. Oncol . 15 , 1907–1918 (2020). 10.1016/j.jtho.2020.09.001 32916310

85. Paul B Trovato JA Thompson J Lapatinib: a dual tyrosine kinase inhibitor for metastatic breast cancer Am. J. Health Syst. Pharm. 2008 65 1703 1710 10.2146/ajhp070646 18768996

86. Deeks ED Neratinib: first global approval Drugs 2017 77 1695 1704 10.1007/s40265-017-0811-4 28884417

87. Murthy RK Tucatinib, trastuzumab, and capecitabine for HER2-positive metastatic breast cancer N. Engl. J. Med. 2020 382 597 609 10.1056/NEJMoa1914609 31825569

88. Kim ES Olmutinib: first global approval Drugs 2016 76 1153 1157 10.1007/s40265-016-0606-z 27357069

89. Roskoski R Jr. Small molecule inhibitors targeting the EGFR/ErbB family of protein-tyrosine kinases in human cancers Pharm. Res. 2019 139 395 411 10.1016/j.phrs.2018.11.014 30500458

90. Kim DW Safety, tolerability, and anti-tumor activity of olmutinib in non-small cell lung cancer with T790M mutation: a single arm, open label, phase 1/2 trial Lung Cancer 2019 135 66 72 10.1016/j.lungcan.2019.07.007 31447004

91. Ma Y First-in-human phase I study of AC0010, a mutant-selective EGFR inhibitor in non-small cell lung cancer: safety, efficacy, and potential mechanism of resistance J. Thorac. Oncol. 2018 13 968 977 10.1016/j.jtho.2018.03.025 29626621

92. Erlichman C Phase I study of EKB-569, an irreversible inhibitor of the epidermal growth factor receptor, in patients with advanced solid tumors J. Clin. Oncol. 2006 24 2252 2260 10.1200/JCO.2005.01.8960 16710023

93. Thress KS Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M Nat. Med. 2015 21 560 562 10.1038/nm.3854 25939061 PMC4771182

94. Zhou Z Durable clinical response of lung adenocarcinoma harboring EGFR 19Del/T790M/in trans-C797S to combination therapy of first- and third-generation EGFR tyrosine kinase inhibitors J. Thorac. Oncol. 2019 14 e157 e159 10.1016/j.jtho.2019.04.020 31075545

95. Jia Y Overcoming EGFR(T790M) and EGFR(C797S) resistance with mutant-selective allosteric inhibitors Nature 2016 534 129 132 10.1038/nature17960 27251290 PMC4929832

96. Lu X Discovery of JND3229 as a new EGFR(C797S) mutant inhibitor with in vivo monodrug efficacy ACS Med. Chem. Lett. 2018 9 1123 1127 10.1021/acsmedchemlett.8b00373 30429956 PMC6231186

97. Shen, J. et al. Structure-based design of 5-methylpyrimidopyridone derivatives as new wild-type sparing inhibitors of the epidermal growth factor receptor triple mutant (EGFR(L858R/T790M/C797S). J. Med. Chem. 62 , 7302–7308 (2019). 10.1021/acs.jmedchem.9b00576 31298540

98. Gilliland DG Griffin JD The roles of FLT3 in hematopoiesis and leukemia Blood 2002 100 1532 1542 10.1182/blood-2002-02-0492 12176867

99. Kiyoi H Mechanism of constitutive activation of FLT3 with internal tandem duplication in the juxtamembrane domain Oncogene 2002 21 2555 2563 10.1038/sj.onc.1205332 11971190

100. Quentmeier H Reinhardt J Zaborski M Drexler HG FLT3 mutations in acute myeloid leukemia cell lines Leukemia 2003 17 120 124 10.1038/sj.leu.2402740 12529668

101. Tallman MS Gilliland DG Rowe JM Drug therapy for acute myeloid leukemia Blood 2005 106 1154 1163 10.1182/blood-2005-01-0178 15870183

102. Daver N Schlenk RF Russell NH Levis MJ Targeting FLT3 mutations in AML: review of current knowledge and evidence Leukemia 2019 33 299 312 10.1038/s41375-018-0357-9 30651634 PMC6365380

103. Auclair D Antitumor activity of sorafenib in FLT3-driven leukemic cells Leukemia 2007 21 439 445 10.1038/sj.leu.2404508 17205056

104. Stone RM Midostaurin plus chemotherapy for acute myeloid leukemia with a FLT3 mutation N. Engl. J. Med. 2017 377 454 464 10.1056/NEJMoa1614359 28644114 PMC5754190

105. Garcia JS Stone RM The development of FLT3 inhibitors in acute myeloid leukemia Hematol. Oncol. Clin. North Am. 2017 31 663 680 10.1016/j.hoc.2017.03.002 28673394

106. Knapper S The clinical development of FLT3 inhibitors in acute myeloid leukemia Expert Opin. Investig. Drugs 2011 20 1377 1395 10.1517/13543784.2011.611802 21895538

107. Baldi GG Gronchi A Stacchiotti S Pexidartinib for the treatment of adult symptomatic patients with tenosynovial giant cell tumors Expert Rev. Clin. Pharm. 2020 13 571 576 10.1080/17512433.2020.1771179 32478598

108. Mori M Gilteritinib, a FLT3/AXL inhibitor, shows antileukemic activity in mouse models of FLT3 mutated acute myeloid leukemia Invest. N. Drugs 2017 35 556 565 10.1007/s10637-017-0470-z PMC5613053 28516360

109. Zarrinkar PP AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML) Blood 2009 114 2984 2992 10.1182/blood-2009-05-222034 19654408 PMC2756206

110. Cortes J Quizartinib, an FLT3 inhibitor, as monotherapy in patients with relapsed or refractory acute myeloid leukaemia: an open-label, multicentre, single-arm, phase 2 trial Lancet Oncol. 2018 19 889 903 10.1016/S1470-2045(18)30240-7 29859851 PMC8152787

111. Galanis A Crenolanib is a potent inhibitor of FLT3 with activity against resistance-conferring point mutants Blood 2014 123 94 100 10.1182/blood-2013-10-529313 24227820 PMC3879910

112. Cao ZX SKLB1028, a novel oral multikinase inhibitor of EGFR, FLT3 and Abl, displays exceptional activity in models of FLT3-driven AML and considerable potency in models of CML harboring Abl mutants Leukemia 2012 26 1892 1895 10.1038/leu.2012.67 22402607

113. Sutamtewagul G Vigil CE Clinical use of FLT3 inhibitors in acute myeloid leukemia Onco Targets Ther. 2018 11 7041 7052 10.2147/OTT.S171640 30410361 PMC6198878

114. Lim SH Dubielecka PM Raghunathan VM Molecular targeting in acute myeloid leukemia J. Transl. Med. 2017 15 183 10.1186/s12967-017-1281-x 28851395 PMC5576374

115. Miller GD Bruno BJ Lim CS Resistant mutations in CML and Ph(+)ALL - role of ponatinib Biologics 2014 8 243 254 25349473 10.2147/BTT.S50734 PMC4208348

116. Elshoury A Przespolewski A Baron J Wang ES Advancing treatment of acute myeloid leukemia: the future of FLT3 inhibitors Expert Rev. Anticancer Ther. 2019 19 273 286 10.1080/14737140.2019.1573679 30681373

117. Smith CC Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia Nature 2012 485 260 263 10.1038/nature11016 22504184 PMC3390926

118. Sung L Predictors and short-term outcomes of hyperleukocytosis in children with acute myeloid leukemia: a report from the Children’s Oncology Group Haematologica 2012 97 1770 1773 10.3324/haematol.2012.065490 22801969 PMC3487455

119. Lin WH Evaluation of the antitumor effects of BPR1J-340, a potent and selective FLT3 inhibitor, alone or in combination with an HDAC inhibitor, vorinostat, in AML cancer PLoS ONE 2014 9 e83160 10.1371/journal.pone.0083160 24416160 PMC3885398

120. Larrosa-Garcia M Baer MR FLT3 inhibitors in acute myeloid leukemia: current status and future directions Mol. Cancer Ther. 2017 16 991 1001 10.1158/1535-7163.MCT-16-0876 28576946 PMC5600895

121. Cao T The FLT3-ITD mutation and the expression of its downstream signaling intermediates STAT5 and Pim-1 are positively correlated with CXCR4 expression in patients with acute myeloid leukemia Sci. Rep. 2019 9 12209 10.1038/s41598-019-48687-z 31434952 PMC6704161

122. Uras IZ Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6 Blood 2016 127 2890 2902 10.1182/blood-2015-11-683581 27099147 PMC4920675

123. Sandhofer N Dual PI3K/mTOR inhibition shows antileukemic activity in MLL-rearranged acute myeloid leukemia Leukemia 2015 29 828 838 10.1038/leu.2014.305 25322685

124. Yamaura T A novel irreversible FLT3 inhibitor, FF-10101, shows excellent efficacy against AML cells with FLT3 mutations Blood 2018 131 426 438 10.1182/blood-2017-05-786657 29187377

125. Chen CT Identification of a potent 5-phenyl-thiazol-2-ylamine-based inhibitor of FLT3 with activity against drug resistance-conferring point mutations Eur. J. Med. Chem. 2015 100 151 161 10.1016/j.ejmech.2015.05.008 26081023

126. Smith CC Characterizing and overriding the structural mechanism of the quizartinib-resistant FLT3 “Gatekeeper” F691L mutation with PLX3397 Cancer Discov. 2015 5 668 679 10.1158/2159-8290.CD-15-0060 25847190 PMC4522415

127. Xu B MZH29 is a novel potent inhibitor that overcomes drug resistance FLT3 mutations in acute myeloid leukemia Leukemia 2017 31 913 921 10.1038/leu.2016.297 27773927

128. Park I Angiogenesis and microsatellite alterations in oral cavity and oropharynx cancer Otolaryngol. Head Neck Surg. 2003 129 P161 10.1016/S0194-5998(03)00974-4

129. Risau W Mechanisms of angiogenesis Nature 1997 386 671 674 10.1038/386671a0 9109485

130. Klagsbrun M Moses MA Molecular angiogenesis Chem. Biol. 1999 6 R217 R224 10.1016/S1074-5521(99)80081-7 10421764

131. Yadav L Tumour angiogenesis and angiogenic inhibitors: a review J. Clin. Diagn. Res. 2015 9 Xe01 xe05 26266204 10.7860/JCDR/2015/12016.6135 PMC4525594

132. Yang WH Xu J Mu JB Xie J Revision of the concept of anti-angiogenesis and its applications in tumor treatment Chronic Dis. Transl. Med. 2017 3 33 40 29063054 10.1016/j.cdtm.2017.01.002 PMC5627689

133. Folkman J Tumor angiogenesis - therapeutic implications N. Engl. J. Med. 1971 285 1182 10.1056/NEJM197108122850711 4938153

134. Potente M Gerhardt H Carmeliet P Basic and therapeutic aspects of angiogenesis Cell 2011 146 873 887 10.1016/j.cell.2011.08.039 21925313

135. Hanahan D Folkman J Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis Cell 1996 86 353 364 10.1016/S0092-8674(00)80108-7 8756718

136. Kerbel RS Tumor angiogenesis: past, present and the near future Carcinogenesis 2000 21 505 515 10.1093/carcin/21.3.505 10688871

137. Fagiani E Christofori G Angiopoietins in angiogenesis Cancer Lett. 2013 328 18 26 10.1016/j.canlet.2012.08.018 22922303

138. Zheng X The regulation of cytokine signaling by retinal determination gene network pathway in cancer Onco Targets Ther. 2018 11 6479 6487 10.2147/OTT.S176113 30323623 PMC6177397

139. Leung DW Vascular endothelial growth factor is a secreted angiogenic mitogen Science 1989 246 1306 1309 10.1126/science.2479986 2479986

140. Itoh N Ornitz DM Evolution of the Fgf and Fgfr gene families Trends Genet. 2004 20 563 569 10.1016/j.tig.2004.08.007 15475116

141. Chen PH Chen X He X Platelet-derived growth factors and their receptors: structural and functional perspectives Biochim. Biophys. Acta 2013 1834 2176 2186 10.1016/j.bbapap.2012.10.015 23137658 PMC3612563

142. Shi Y Massagué J Mechanisms of TGF-β signaling from cell membrane to the nucleus Cell 2003 113 685 700 10.1016/S0092-8674(03)00432-X 12809600

143. Rak J Yu JL Klement G Kerbel RS Oncogenes and angiogenesis: signaling three-dimensional tumor growth J. Investig. Dermatol Symp. Proc. 2000 5 24 33 10.1046/j.1087-0024.2000.00012.x 11147671

144. Ferrara N Davis-Smyth T The biology of vascular endothelial growth factor Endocr. Rev. 1997 18 4 25 10.1210/edrv.18.1.0287 9034784

145. Eskens FA Verweij J The clinical toxicity profile of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR) targeting angiogenesis inhibitors; a review Eur. J. Cancer 2006 42 3127 3139 10.1016/j.ejca.2006.09.015 17098419

146. Kerbel R Folkman J Clinical translation of angiogenesis inhibitors Nat. Rev. Cancer 2002 2 727 739 10.1038/nrc905 12360276

147. Wilhelm SM Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling Mol. Cancer Ther. 2008 7 3129 3140 10.1158/1535-7163.MCT-08-0013 18852116 PMC12261297

148. Llovet JM Sorafenib in advanced hepatocellular carcinoma N. Engl. J. Med. 2008 359 378 390 10.1056/NEJMoa0708857 18650514

149. Wilhelm S Discovery and development of sorafenib: a multikinase inhibitor for treating cancer Nat. Rev. Drug Discov. 2006 5 835 844 10.1038/nrd2130 17016424

150. Woo HY Heo J Sorafenib in liver cancer Expert Opin. Pharmacother. 2012 13 1059 1067 10.1517/14656566.2012.679930 22519770

151. Escudier B Sorafenib in advanced clear-cell renal-cell carcinoma N. Engl. J. Med. 2007 356 125 134 10.1056/NEJMoa060655 17215530

152. Brose MS Sorafenib in radioactive iodine-refractory, locally advanced or metastatic differentiated thyroid cancer: a randomised, double-blind, phase 3 trial Lancet 2014 384 319 328 10.1016/S0140-6736(14)60421-9 24768112 PMC4366116

153. Motzer RJ Escudier B Gannon A Figlin RA Sunitinib: ten years of successful clinical use and study in advanced renal cell carcinoma Oncologist 2017 22 41 52 10.1634/theoncologist.2016-0197 27807302 PMC5313263

154. van der Graaf WTA Pazopanib for metastatic soft-tissue sarcoma (PALETTE): a randomised, double-blind, placebo-controlled phase 3 trial Lancet 2012 379 1879 1886 10.1016/S0140-6736(12)60651-5 22595799

155. Keating GM Axitinib: a review in advanced renal cell carcinoma Drugs 2015 75 1903 1913 10.1007/s40265-015-0483-x 26487541

156. Elisei R Cabozantinib in progressive medullary thyroid cancer J. Clin. Oncol. 2013 31 3639 3646 10.1200/JCO.2012.48.4659 24002501 PMC4164813

157. Escudier B The role of tivozanib in advanced renal cell carcinoma therapy Expert Rev. Anticancer Ther. 2018 18 1113 1124 10.1080/14737140.2018.1508348 30084668

158. Schenone S Brullo C Botta M Small molecules ATP-competitive inhibitors of FLT3: a chemical overview Curr. Med. Chem. 2008 15 3113 3132 10.2174/092986708786848613 19075657

159. Roskoski R Jr. Sunitinib: a VEGF and PDGF receptor protein kinase and angiogenesis inhibitor Biochem. Biophys. Res. Commun. 2007 356 323 328 10.1016/j.bbrc.2007.02.156 17367763

160. Morabito A Vandetanib: an overview of its clinical development in NSCLC and other tumors Drugs Today 2010 46 683 698 10.1358/dot.2010.46.9.1516989 20967300

161. Yoh K Vandetanib in patients with previously treated RET-rearranged advanced non-small-cell lung cancer (LURET): an open-label, multicentre phase 2 trial Lancet Respir. Med. 2017 5 42 50 10.1016/S2213-2600(16)30322-8 27825616

162. Zhang Y XL-184, a MET, VEGFR-2 and RET kinase inhibitor for the treatment of thyroid cancer, glioblastoma multiforme and NSCLC IDrugs Investig. Drugs J. 2010 13 112 121 PMC3268517 20127563

163. Markham A Selpercatinib: first approval Drugs 2020 80 1119 1124 10.1007/s40265-020-01343-7 32557397 PMC7716849

164. Markham A Pralsetinib: first approval Drugs 2020 80 1865 1870 10.1007/s40265-020-01427-4 33136236

165. Wilhelm SM Regorafenib (BAY 73-4506): a new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with potent preclinical antitumor activity Int J. Cancer 2011 129 245 255 10.1002/ijc.25864 21170960

166. Strumberg D Regorafenib (BAY 73-4506) in advanced colorectal cancer: a phase I study Br. J. Cancer 2012 106 1722 1727 10.1038/bjc.2012.153 22568966 PMC3364125

167. Demetri GD Efficacy and safety of regorafenib for advanced gastrointestinal stromal tumours after failure of imatinib and sunitinib (GRID): an international, multicentre, randomised, placebo-controlled, phase 3 trial Lancet 2013 381 295 302 10.1016/S0140-6736(12)61857-1 23177515 PMC3819942

168. Dhillon S Nintedanib: a review of its use as second-line treatment in adults with advanced non-small cell lung cancer of adenocarcinoma histology Target Oncol. 2015 10 303 310 10.1007/s11523-015-0367-8 25894578

169. Keating GM Nintedanib: a review of its use in patients with idiopathic pulmonary fibrosis Drugs 2015 75 1131 1140 10.1007/s40265-015-0418-6 26063212

170. Alshangiti A Chandhoke G Ellis PM Antiangiogenic therapies in non-small-cell lung cancer Curr. Oncol. 2018 25 S45 S58 10.3747/co.25.3747 29910647 PMC6001774

171. Aoyama T Yoshikawa T Targeted therapy: apatinib - new third-line option for refractory gastric or GEJ cancer Nat. Rev. Clin. Oncol. 2016 13 268 270 10.1038/nrclinonc.2016.53 27071350

172. Syed YY Anlotinib: first global approval Drugs 2018 78 1057 1062 10.1007/s40265-018-0939-x 29943374

173. Han B Anlotinib as a third-line therapy in patients with refractory advanced non-small-cell lung cancer: a multicentre, randomised phase II trial (ALTER0302) Br. J. Cancer 2018 118 654 661 10.1038/bjc.2017.478 29438373 PMC5846072

174. Shirley M Fruquintinib: first global approval Drugs 2018 78 1757 1761 10.1007/s40265-018-0998-z 30357594

175. Li J Effect of fruquintinib vs placebo on overall survival in patients with previously treated metastatic colorectal cancer: The FRESCO Randomized Clinical Trial JAMA 2018 319 2486 2496 10.1001/jama.2018.7855 29946728 PMC6583690

176. Chen Z Jiang L The clinical application of fruquintinib on colorectal cancer Expert Rev. Clin. Pharm. 2019 12 713 721 10.1080/17512433.2019.1630272 31177854

177. Perera TPS Discovery and pharmacological characterization of JNJ-42756493 (Erdafitinib), a functionally selective small-molecule FGFR family inhibitor Mol. Cancer Ther. 2017 16 1010 1020 10.1158/1535-7163.MCT-16-0589 28341788

178. Zhang Y Constitutive activating mutation of the FGFR3b in oral squamous cell carcinomas Int J. Cancer 2005 117 166 168 10.1002/ijc.21145 15880580

179. Hoy SM Pemigatinib: first approval Drugs 2020 80 923 929 10.1007/s40265-020-01330-y 32472305

180. Okamoto I Comparison of carboplatin plus pemetrexed followed by maintenance pemetrexed with docetaxel monotherapy in elderly patients with advanced nonsquamous non-small cell lung cancer: A Phase 3 Randomized Clinical Trial JAMA Oncol. 2020 6 e196828 10.1001/jamaoncol.2019.6828 32163097 PMC7068674

181. Dhillon S Avapritinib: first approval Drugs 2020 80 433 439 10.1007/s40265-020-01275-2 32100250

182. Kasireddy V von Mehren M Emerging drugs for the treatment of gastrointestinal stromal tumour Expert Opin. Emerg. Drugs 2017 22 317 329 10.1080/14728214.2017.1411479 29183145

183. Heinrich MC Avapritinib in advanced PDGFRA D842V-mutant gastrointestinal stromal tumour (NAVIGATOR): a multicentre, open-label, phase 1 trial Lancet Oncol. 2020 21 935 946 10.1016/S1470-2045(20)30269-2 32615108

184. Saleh, N. Avapritinib approved for GIST subgroup. Cancer Discov . 10 , 334 (2020). 10.1158/2159-8290.CD-NB2020-002 31974168

185. Dhillon S Ripretinib: first approval Drugs 2020 80 1133 1138 10.1007/s40265-020-01348-2 32578014 PMC7595980

186. Batchelor TT Phase II study of cediranib, an oral pan-vascular endothelial growth factor receptor tyrosine kinase inhibitor, in patients with recurrent glioblastoma J. Clin. Oncol. 2010 28 2817 2823 10.1200/JCO.2009.26.3988 20458050 PMC2903316

187. Liu JF Combination cediranib and olaparib versus olaparib alone for women with recurrent platinum-sensitive ovarian cancer: a randomised phase 2 study Lancet Oncol. 2014 15 1207 1214 10.1016/S1470-2045(14)70391-2 25218906 PMC4294183

188. Renhowe PA Design, structure-activity relationships and in vivo characterization of 4-amino-3-benzimidazol-2-ylhydroquinolin-2-ones: a novel class of receptor tyrosine kinase inhibitors J. Med. Chem. 2009 52 278 292 10.1021/jm800790t 19113866

189. Kim KB Phase I/II and pharmacodynamic study of dovitinib (TKI258), an inhibitor of fibroblast growth factor receptors and VEGF receptors, in patients with advanced melanoma Clin. Cancer Res. 2011 17 7451 7461 10.1158/1078-0432.CCR-11-1747 21976540

190. Raghav KP Blumenschein GR Motesanib and advanced NSCLC: experiences and expectations Expert Opin. Investig. Drugs 2011 20 859 869 10.1517/13543784.2011.579103 21534718

191. Kubota K Phase III, randomized, placebo-controlled, double-blind trial of motesanib (AMG-706) in combination with paclitaxel and carboplatin in East Asian patients with advanced nonsquamous non-small-cell lung cancer J. Clin. Oncol. 2017 35 3662 3670 10.1200/JCO.2017.72.7297 28902534

192. Schlumberger MJ Phase II study of safety and efficacy of motesanib in patients with progressive or symptomatic, advanced or metastatic medullary thyroid cancer J. Clin. Oncol. 2009 27 3794 3801 10.1200/JCO.2008.18.7815 19564535

193. Bass MB Biomarkers as predictors of response to treatment with motesanib in patients with progressive advanced thyroid cancer J. Clin. Endocrinol. Metab. 2010 95 5018 5027 10.1210/jc.2010-0947 20739388

194. Sherman SI Motesanib diphosphate in progressive differentiated thyroid cancer N. Engl. J. Med. 2008 359 31 42 10.1056/NEJMoa075853 18596272

195. Xu JM Sulfatinib, a novel kinase inhibitor, in patients with advanced solid tumors: results from a phase I study Oncotarget 2017 8 42076 42086 10.18632/oncotarget.14942 28159938 PMC5522050

196. Xu J Surufatinib in advanced well-differentiated neuroendocrine tumors: A Multicenter, Single-arm, Open-label, Phase Ib/II Trial Clin. Cancer Res. 2019 25 3486 3494 30833272 10.1158/1078-0432.CCR-18-2994

197. Xu, J. et al. Surufatinib in advanced extrapancreatic neuroendocrine tumours (SANET-ep): a randomised, double-blind, placebo-controlled, phase 3 study. Lancet Oncol. 21 , 1500–1512 (2020). 10.1016/S1470-2045(20)30496-4 32966811

198. Heinrich MC Crenolanib inhibits the drug-resistant PDGFRA D842V mutation associated with imatinib-resistant gastrointestinal stromal tumors Clin. Cancer Res. 2012 18 4375 4384 10.1158/1078-0432.CCR-12-0625 22745105

199. Indio, V. et al. Integrated molecular characterization of gastrointestinal stromal tumors (GIST) harboring the rare D842V mutation in PDGFRA gene. Int. J. Mol. Sci . 19 , 732 (2018). 10.3390/ijms19030732 PMC5877593 29510530

200. Jetani H CAR T-cells targeting FLT3 have potent activity against FLT3(-)ITD(+) AML and act synergistically with the FLT3-inhibitor crenolanib Leukemia 2018 32 1168 1179 10.1038/s41375-018-0009-0 29472720

201. Guffanti F In vitro and in vivo activity of lucitanib in FGFR1/2 amplified or mutated cancer models Neoplasia 2017 19 35 42 10.1016/j.neo.2016.11.008 27988457 PMC5167242

202. Cai ZW Discovery of brivanib alaninate ((S)-((R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate), a novel prodrug of dual vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 kinase inhibitor (BMS-540215) J. Med. Chem. 2008 51 1976 1980 10.1021/jm7013309 18288793

203. Johnson PJ Brivanib versus sorafenib as first-line therapy in patients with unresectable, advanced hepatocellular carcinoma: results from the randomized phase III BRISK-FL study J. Clin. Oncol. 2013 31 3517 3524 10.1200/JCO.2012.48.4410 23980084

204. Kudo M Brivanib as adjuvant therapy to transarterial chemoembolization in patients with hepatocellular carcinoma: a randomized phase III trial Hepatology 2014 60 1697 1707 10.1002/hep.27290 24996197

205. Tucker JA Structural insights into FGFR kinase isoform selectivity: diverse binding modes of AZD4547 and ponatinib in complex with FGFR1 and FGFR4 Structure 2014 22 1764 1774 10.1016/j.str.2014.09.019 25465127

206. Felix NS Effects of the FGF receptor-1 inhibitor, infigratinib, with or without sildenafil, in experimental pulmonary arterial hypertension Br. J. Pharm. 2019 176 4462 4473 10.1111/bph.14807 PMC6932937 31351013

207. Makawita S Infigratinib in patients with advanced cholangiocarcinoma with FGFR2 gene fusions/translocations: the PROOF 301 trial Future Oncol. 2020 16 2375 2384 10.2217/fon-2020-0299 32580579

208. Lahn M Kloeker S Berry BS TGF-beta inhibitors for the treatment of cancer Expert Opin. Investig. Drugs 2005 14 629 643 10.1517/13543784.14.6.629 16004592

209. Herbertz S Clinical development of galunisertib (LY2157299 monohydrate), a small molecule inhibitor of transforming growth factor-beta signaling pathway Drug Des. Dev. Ther. 2015 9 4479 4499 10.2147/DDDT.S86621 PMC4539082 26309397

210. Jung SY Population pharmacokinetics of vactosertib, a new TGF-beta receptor type Iota inhibitor, in patients with advanced solid tumors Cancer Chemother. Pharm. 2020 85 173 183 10.1007/s00280-019-03979-z 31673825

211. Xu G Synthesis and biological evaluation of 4-(pyridin-4-oxy)-3-(3,3-difluorocyclobutyl)-pyrazole derivatives as novel potent transforming growth factor-beta type 1 receptor inhibitors Eur. J. Med. Chem. 2020 198 112354 10.1016/j.ejmech.2020.112354 32387837

212. Yingling JM Preclinical assessment of galunisertib (LY2157299 monohydrate), a first-in-class transforming growth factor-β receptor type I inhibitor Oncotarget 2018 9 6659 6677 10.18632/oncotarget.23795 29467918 PMC5805504

213. Santini V Phase II study of the ALK5 inhibitor galunisertib in very low-, low-, and intermediate-risk myelodysplastic syndromes Clin. Cancer Res. 2019 25 6976 6985 10.1158/1078-0432.CCR-19-1338 31481511

214. Holmgaard RB Targeting the TGFbeta pathway with galunisertib, a TGFbetaRI small molecule inhibitor, promotes anti-tumor immunity leading to durable, complete responses, as monotherapy and in combination with checkpoint blockade J. Immunother. Cancer 2018 6 47 10.1186/s40425-018-0356-4 29866156 PMC5987416

215. Teleanu, R. I., Chircov, C., Grumezescu, A. M. & Teleanu, D. M. Tumor angiogenesis and anti-angiogenic strategies for cancer treatment. J. Clin. Med . 9 , 84 (2019). 10.3390/jcm9010084 PMC7020037 31905724

216. Rajabi, M. & Mousa, S. A. The role of angiogenesis in cancer treatment. Biomedicines . 5 , 34 (2017). 10.3390/biomedicines5020034 PMC5489820 28635679

217. Russo M Giavazzi R Anti-angiogenesis for cancer: current status and prospects Thromb. Res. 2018 164 S3 S6 10.1016/j.thromres.2018.01.030 29703482

218. Mauceri HJ Combined effects of angiostatin and ionizing radiation in antitumour therapy Nature 1998 394 287 291 10.1038/28412 9685160

219. Hlatky L Hahnfeldt P Folkman J Clinical application of antiangiogenic therapy: microvessel density, what it does and doesn’t tell us J. Natl Cancer Inst. 2002 94 883 893 10.1093/jnci/94.12.883 12072542

220. Giuliano S Pagès G Mechanisms of resistance to anti-angiogenesis therapies Biochimie 2013 95 1110 1119 10.1016/j.biochi.2013.03.002 23507428

221. Posadas EM Limvorasak S Figlin RA Targeted therapies for renal cell carcinoma Nat. Rev. Nephrol. 2017 13 496 511 10.1038/nrneph.2017.82 28691713

222. Sheng X Axitinib in combination with toripalimab, a humanized immunoglobulin G(4) monoclonal antibody against programmed cell death-1, in patients with metastatic mucosal melanoma: An Open-Label Phase IB Trial J. Clin. Oncol. 2019 37 2987 2999 10.1200/JCO.19.00210 31403867 PMC6839911

223. Liu X Early presence of anti-angiogenesis-related adverse events as a potential biomarker of antitumor efficacy in metastatic gastric cancer patients treated with apatinib: a cohort study J. Hematol. Oncol. 2017 10 153 10.1186/s13045-017-0521-0 28870253 PMC5584332

224. Rini BI Diastolic blood pressure as a biomarker of axitinib efficacy in solid tumors Clin. Cancer Res. 2011 17 3841 3849 10.1158/1078-0432.CCR-10-2806 21531811

225. Cocco E Scaltriti M Drilon A NTRK fusion-positive cancers and TRK inhibitor therapy Nat. Rev. Clin. Oncol. 2018 15 731 747 10.1038/s41571-018-0113-0 30333516 PMC6419506

226. Scott-Solomon E Kuruvilla R Mechanisms of neurotrophin trafficking via Trk receptors Mol. Cell. Neurosci. 2018 91 25 33 10.1016/j.mcn.2018.03.013 29596897 PMC6128733

227. Nakagawara A Trk receptor tyrosine kinases: a bridge between cancer and neural development Cancer Lett. 2001 169 107 114 10.1016/S0304-3835(01)00530-4 11431098

228. Bertrand T The crystal structures of TrkA and TrkB suggest key regions for achieving selective inhibition J. Mol. Biol. 2012 423 439 453 10.1016/j.jmb.2012.08.002 22902478

229. Drilon A Efficacy of larotrectinib in TRK fusion-positive cancers in adults and children N. Engl. J. Med. 2018 378 731 739 10.1056/NEJMoa1714448 29466156 PMC5857389

230. Doebele RC Entrectinib in patients with advanced or metastatic NTRK fusion-positive solid tumours: integrated analysis of three phase 1-2 trials Lancet Oncol. 2020 21 271 282 10.1016/S1470-2045(19)30691-6 31838007 PMC7461630

231. Ardini E Entrectinib, a pan-TRK, ROS1, and ALK inhibitor with activity in multiple molecularly defined cancer indications Mol. Cancer Ther. 2016 15 628 639 10.1158/1535-7163.MCT-15-0758 26939704

232. Federman N McDermott R Larotrectinib, a highly selective tropomyosin receptor kinase (TRK) inhibitor for the treatment of TRK fusion cancer Expert Rev. Clin. Pharm. 2019 12 931 939 10.1080/17512433.2019.1661775 31469968

233. Grüllich C Cabozantinib: multi-kinase Inhibitor of MET, AXL, RET, and VEGFR2 Recent Results Cancer Res. 2018 211 67 75 10.1007/978-3-319-91442-8_5 30069760

234. Konicek BW Merestinib (LY2801653) inhibits neurotrophic receptor kinase (NTRK) and suppresses growth of NTRK fusion bearing tumors Oncotarget 2018 9 13796 13806 10.18632/oncotarget.24488 29568395 PMC5862616

235. Lin CC A phase 1, open-label, dose-escalation trial of oral TSR-011 in patients with advanced solid tumours and lymphomas Br. J. Cancer 2019 121 131 138 10.1038/s41416-019-0503-9 31217479 PMC6738096

236. Yang, Y. et al. Sitravatinib, a tyrosine kinase inhibitor, inhibits the transport function of ABCG2 and restores sensitivity to chemotherapy-resistant cancer cells in vitro. Front. Oncol . 10 , 700 (2020). 10.3389/fonc.2020.00700 PMC7236772 32477943

237. Piao Y Novel MET/TIE2/VEGFR2 inhibitor altiratinib inhibits tumor growth and invasiveness in bevacizumab-resistant glioblastoma mouse models Neuro-Oncol. 2016 18 1230 1241 10.1093/neuonc/now030 26965451 PMC4998992

238. Katayama R The new-generation selective ROS1/NTRK inhibitor DS-6051b overcomes crizotinib resistant ROS1-G2032R mutation in preclinical models Nat. Commun. 2019 10 3604 10.1038/s41467-019-11496-z 31399568 PMC6688997

239. Khotskaya YB Targeting TRK family proteins in cancer Pharmacol. Therap. 2017 173 58 66 10.1016/j.pharmthera.2017.02.006 28174090

240. Russo M Acquired resistance to the TRK inhibitor entrectinib in colorectal cancer Cancer Discov. 2016 6 36 44 10.1158/2159-8290.CD-15-0940 26546295

241. Drilon A A next-generation TRK kinase inhibitor overcomes acquired resistance to prior trk kinase inhibition in patients with TRK fusion-positive solid tumors Cancer Discov. 2017 7 963 972 10.1158/2159-8290.CD-17-0507 28578312 PMC5581710

242. Drilon A TRK inhibitors in TRK fusion-positive cancers Ann. Oncol. 2019 30 viii23 viii30 10.1093/annonc/mdz282 31738426 PMC6859818

243. Quintas-Cardama A Cortes J Molecular biology of bcr-abl1-positive chronic myeloid leukemia Blood 2009 113 1619 1630 10.1182/blood-2008-03-144790 18827185 PMC3952549

244. Redaelli A Clinical and epidemiologic burden of chronic myelogenous leukemia Expert Rev. Anticancer Ther. 2004 4 85 96 10.1586/14737140.4.1.85 14748660

245. Kurzrock R Kantarjian HM Druker BJ Talpaz M Philadelphia chromosome-positive leukemias: from basic mechanisms to molecular therapeutics Ann. Intern. Med. 2003 138 819 830 10.7326/0003-4819-138-10-200305200-00010 12755554

246. Klein F The BCR-ABL1 kinase bypasses selection for the expression of a pre-B cell receptor in pre-B acute lymphoblastic leukemia cells J. Exp. Med. 2004 199 673 685 10.1084/jem.20031637 14993251 PMC2213306

247. Deininger M Buchdunger E Druker BJ The development of imatinib as a therapeutic agent for chronic myeloid leukemia Blood 2005 105 2640 2653 10.1182/blood-2004-08-3097 15618470

248. Druker BJ Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia N. Engl. J. Med. 2006 355 2408 2417 10.1056/NEJMoa062867 17151364

249. Hochhaus A Molecular and chromosomal mechanisms of resistance to imatinib (STI571) therapy Leukemia 2002 16 2190 2196 10.1038/sj.leu.2402741 12399961

250. Hantschel O Grebien F Superti-Furga G The growing arsenal of ATP-competitive and allosteric inhibitors of BCR-ABL Cancer Res. 2012 72 4890 4895 10.1158/0008-5472.CAN-12-1276 23002203 PMC3517953

251. Azam M Latek RR Daley GQ Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL Cell 2003 112 831 843 10.1016/S0092-8674(03)00190-9 12654249

252. Jabbour E Cortes J Kantarjian H Treatment selection after imatinib resistance in chronic myeloid leukemia Target Oncol. 2009 4 3 10 10.1007/s11523-008-0100-y 19343297

253. Weisberg E Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl Cancer Cell 2005 7 129 141 10.1016/j.ccr.2005.01.007 15710326

254. Puttini M In vitro and in vivo activity of SKI-606, a novel Src-Abl inhibitor, against imatinib-resistant Bcr-Abl+ neoplastic cells Cancer Res. 2006 66 11314 11322 10.1158/0008-5472.CAN-06-1199 17114238

255. Sawyers CL Even better kinase inhibitors for chronic myeloid leukemia N. Engl. J. Med. 2010 362 2314 2315 10.1056/NEJMe1004430 20525994

256. O’Hare T In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant imatinib-resistant Abl kinase domain mutants Cancer Res. 2005 65 4500 4505 10.1158/0008-5472.CAN-05-0259 15930265

257. O’Hare T Targeting the BCR-ABL signaling pathway in therapy-resistant Philadelphia chromosome-positive leukemia Clin. Cancer Res. 2011 17 212 221 10.1158/1078-0432.CCR-09-3314 21098337

258. O’Hare T AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance Cancer Cell 2009 16 401 412 10.1016/j.ccr.2009.09.028 19878872 PMC2804470

259. Wylie AA The allosteric inhibitor ABL001 enables dual targeting of BCR-ABL1 Nature 2017 543 733 737 10.1038/nature21702 28329763

260. Eide CA The ABL switch control inhibitor DCC-2036 is active against the chronic myeloid leukemia mutant BCR-ABLT315I and exhibits a narrow resistance profile Cancer Res. 2011 71 3189 3195 10.1158/0008-5472.CAN-10-3224 21505103 PMC3206627

261. Yokota A INNO-406, a novel BCR-ABL/Lyn dual tyrosine kinase inhibitor, suppresses the growth of Ph+ leukemia cells in the central nervous system, and cyclosporine A augments its in vivo activity Blood 2007 109 306 314 10.1182/blood-2006-03-013250 16954504

262. Zabriskie MS BCR-ABL1 compound mutations combining key kinase domain positions confer clinical resistance to ponatinib in Ph chromosome-positive leukemia Cancer Cell 2014 26 428 442 10.1016/j.ccr.2014.07.006 25132497 PMC4160372

263. O’Hare T Zabriskie MS Eiring AM Deininger MW Pushing the limits of targeted therapy in chronic myeloid leukaemia Nat. Rev. Cancer 2012 12 513 526 10.1038/nrc3317 22825216

264. Gibbons DL Molecular dynamics reveal BCR-ABL1 polymutants as a unique mechanism of resistance to PAN-BCR-ABL1 kinase inhibitor therapy Proc. Natl Acad. Sci. USA 2014 111 3550 3555 10.1073/pnas.1321173111 24550512 PMC3948238

265. Alvandi F U.S. Food and Drug Administration approval summary: omacetaxine mepesuccinate as treatment for chronic myeloid leukemia Oncologist 2014 19 94 99 10.1634/theoncologist.2013-0077 24309980 PMC3903068

266. Khoury HJ Omacetaxine mepesuccinate in patients with advanced chronic myeloid leukemia with resistance or intolerance to tyrosine kinase inhibitors Leuk. Lymphoma 2015 56 120 127 10.3109/10428194.2014.889826 24650054

267. Ogbogu PU Hypereosinophilic syndrome: a multicenter, retrospective analysis of clinical characteristics and response to therapy J. Allergy Clin. Immunol. 2009 124 1319 1325.e1313 10.1016/j.jaci.2009.09.022 19910029 PMC2829669

268. Montemurro M Long-term outcome of dasatinib first-line treatment in gastrointestinal stromal tumor: a multicenter, 2-stage phase 2 trial (Swiss Group for Clinical Cancer Research 56/07) Cancer 2018 124 1449 1454 10.1002/cncr.31234 29315500

269. Rossari F Minutolo F Orciuolo E Past, present, and future of Bcr-Abl inhibitors: from chemical development to clinical efficacy J. Hematol. Oncol. 2018 11 84 10.1186/s13045-018-0624-2 29925402 PMC6011351

270. Tanaka S Baba Y B cell receptor signaling Adv. Exp. Med. Biol. 2020 1254 23 36 10.1007/978-981-15-3532-1_2 32323266

271. Hendriks RW Yuvaraj S Kil LP Targeting Bruton’s tyrosine kinase in B cell malignancies Nat. Rev. Cancer 2014 14 219 232 10.1038/nrc3702 24658273

272. Schwartzberg PL Finkelstein LD Readinger JA TEC-family kinases: regulators of T-helper-cell differentiation Nat. Rev. Immunol. 2005 5 284 295 10.1038/nri1591 15803148

273. Khan WN Defective B cell development and function in Btk-deficient mice Immunity 1995 3 283 299 10.1016/1074-7613(95)90114-0 7552994

274. Ma L Discovery and characterization of LY2784544, a small-molecule tyrosine kinase inhibitor of JAK2V617F Blood Cancer J. 2013 3 e109 10.1038/bcj.2013.6 23584399 PMC3641321

275. Davids MS Brown JR Ibrutinib: a first in class covalent inhibitor of Bruton’s tyrosine kinase Future Oncol. 2014 10 957 967 10.2217/fon.14.51 24941982 PMC4632638

276. Skarzynski M Interactions between ibrutinib and anti-CD20 antibodies: competing effects on the outcome of combination therapy Clin. Cancer Res. 2016 22 86 95 10.1158/1078-0432.CCR-15-1304 26283682 PMC4703510

277. Akinleye A Ibrutinib and novel BTK inhibitors in clinical development J. Hematol. Oncol. 2013 6 59 10.1186/1756-8722-6-59 23958373 PMC3751776

278. Wang ML Targeting BTK with ibrutinib in relapsed or refractory mantle-cell lymphoma N. Engl. J. Med. 2013 369 507 516 10.1056/NEJMoa1306220 23782157 PMC4513941

279. Byrd JC Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia N. Engl. J. Med. 2013 369 32 42 10.1056/NEJMoa1215637 23782158 PMC3772525

280. Noy A Targeting Bruton tyrosine kinase with ibrutinib in relapsed/refractory marginal zone lymphoma Blood 2017 129 2224 2232 10.1182/blood-2016-10-747345 28167659 PMC5399483

281. Mato AR Toxicities and outcomes of 616 ibrutinib-treated patients in the United States: a real-world analysis Haematologica 2018 103 874 879 10.3324/haematol.2017.182907 29419429 PMC5927982

282. Rajasekaran, N. et al. Three BTK-specific inhibitors, in contrast to ibrutinib, do not antagonize rituximab-dependent NK-cell mediated cytotoxicity. Blood . 124 , 3118 (2014).

283. Wu J Zhang M Liu D Acalabrutinib (ACP-196): a selective second-generation BTK inhibitor J. Hematol. Oncol. 2016 9 21 10.1186/s13045-016-0250-9 26957112 PMC4784459

284. Wu J Liu C Tsui ST Liu D Second-generation inhibitors of Bruton tyrosine kinase J. Hematol. Oncol. 2016 9 80 10.1186/s13045-016-0313-y 27590878 PMC5010774

285. Herman SEM The Bruton tyrosine kinase (BTK) inhibitor acalabrutinib demonstrates potent on-target effects and efficacy in two mouse models of chronic lymphocytic leukemia Clin. Cancer Res. 2017 23 2831 2841 10.1158/1078-0432.CCR-16-0463 27903679 PMC5548968

286. Guo Y Discovery of zanubrutinib (BGB-3111), a novel, potent, and selective covalent inhibitor of Bruton’s tyrosine kinase J. Med. Chem. 2019 62 7923 7940 10.1021/acs.jmedchem.9b00687 31381333

287. Syed YY Zanubrutinib: first approval Drugs 2020 80 91 97 10.1007/s40265-019-01252-4 31933167

288. Blum KA B-cell receptor pathway modulators in NHL Hematol. Am. Soc. Hematol. Educ. Program. 2015 2015 82 91 10.1182/asheducation-2015.1.82 PMC4871119 26637705

289. Naymagon L Abdul-Hay M Novel agents in the treatment of multiple myeloma: a review about the future J. Hematol. Oncol. 2016 9 52 10.1186/s13045-016-0282-1 27363832 PMC4929712

290. Schwartz M Zhang Y Rosenblatt JD B cell regulation of the anti-tumor response and role in carcinogenesis J. Immunother. Cancer 2016 4 40 10.1186/s40425-016-0145-x 27437104 PMC4950763

291. Burger JA Bruton’s tyrosine kinase (BTK) inhibitors in clinical trials Curr. Hematol. Malig. Rep. 2014 9 44 49 10.1007/s11899-013-0188-8 24357428

292. Walter HS A phase 1 clinical trial of the selective BTK inhibitor ONO/GS-4059 in relapsed and refractory mature B-cell malignancies Blood 2016 127 411 419 10.1182/blood-2015-08-664086 26542378 PMC4731845

293. Danilov AV Phase Ib study of tirabrutinib in combination with idelalisib or entospletinib in previously treated chronic lymphocytic leukemia Clin. Cancer Res. 2020 26 2810 2818 10.1158/1078-0432.CCR-19-3504 32156743

294. Evans EK Inhibition of Btk with CC-292 provides early pharmacodynamic assessment of activity in mice and humans J. Pharm. Exp. Ther. 2013 346 219 228 10.1124/jpet.113.203489 23709115

295. Abdelhameed AS Attwa MW Al-Shaklia NS Kadi AA A highly sensitive LC-MS/MS method to determine novel Bruton’s tyrosine kinase inhibitor spebrutinib: application to metabolic stability evaluation R. Soc. Open Sci. 2019 6 190434 10.1098/rsos.190434 31312501 PMC6599791

296. Brown JR Phase I study of single-agent CC-292, a highly selective Bruton’s tyrosine kinase inhibitor, in relapsed/refractory chronic lymphocytic leukemia Haematologica 2016 101 e295 e298 10.3324/haematol.2015.140806 27151992 PMC5004476

297. Haselmayer P Efficacy and pharmacodynamic modeling of the BTK inhibitor evobrutinib in autoimmune disease models J. Immunol. 2019 202 2888 2906 10.4049/jimmunol.1800583 30988116 PMC6500888

298. Watterson SH Discovery of Branebrutinib (BMS-986195): a strategy for identifying a highly potent and selective covalent inhibitor providing rapid in vivo inactivation of Bruton’s tyrosine kinase (BTK) J. Med. Chem. 2019 62 3228 3250 10.1021/acs.jmedchem.9b00167 30893553

299. Park JK HM71224, a novel Bruton’s tyrosine kinase inhibitor, suppresses B cell and monocyte activation and ameliorates arthritis in a mouse model: a potential drug for rheumatoid arthritis Arthritis Res. Ther. 2016 18 91 10.1186/s13075-016-0988-z 27090981 PMC4835877

300. Zhang Z Targeting Bruton’s tyrosine kinase for the treatment of B cell associated malignancies and autoimmune diseases: preclinical and clinical developments of small molecule inhibitors Arch. Pharm. 2018 351 e1700369 10.1002/ardp.201700369 29741794

301. Kim HO Development of BTK inhibitors for the treatment of B-cell malignancies Arch. Pharm. Res. 2019 42 171 181 10.1007/s12272-019-01124-1 30706214

302. Thompson PA Burger JA Bruton’s tyrosine kinase inhibitors: first and second generation agents for patients with chronic lymphocytic leukemia (CLL) Expert Opin. Investig. Drugs 2018 27 31 42 10.1080/13543784.2018.1404027 29125406 PMC11784538

303. Brandhuber B LOXO-305, a next generation reversible BTK inhibitor, for overcoming acquired resistance to irreversible BTK inhibitors Clin. Lymphoma Myeloma Leuk. 2018 18 S216 10.1016/j.clml.2018.07.081

304. Erickson RI Bruton’s tyrosine kinase small molecule inhibitors induce a distinct pancreatic toxicity in rats J. Pharm. Exp. Ther. 2017 360 226 238 10.1124/jpet.116.236224 27821712

305. Eathiraj S Targeting ibrutinib-resistant BTK-C481S mutation with ARQ 531, a reversible non-covalent inhibitor of BTK Clin. Lymphoma Myeloma Leuk. 2016 16 S47 S48 10.1016/j.clml.2016.07.068

306. Woyach JA BTK(C481S)-mediated resistance to ibrutinib in chronic lymphocytic leukemia J. Clin. Oncol. 2017 35 1437 1443 10.1200/JCO.2016.70.2282 28418267 PMC5455463

307. Serra López-Matencio JM Morell Baladrón A Castañeda S JAK-STAT inhibitors for the treatment of immunomediated diseases Med. Clín. 2019 152 353 360 10.1016/j.medcli.2018.10.020 30527218

308. Villarino AV Kanno Y O’Shea JJ Mechanisms and consequences of Jak-STAT signaling in the immune system Nat. Immunol. 2017 18 374 384 10.1038/ni.3691 28323260 PMC11565648

309. Cacalano NA Autosomal SCID caused by a point mutation in the N-terminus of Jak3: mapping of the Jak3-receptor interaction domain EMBO J. 1999 18 1549 1558 10.1093/emboj/18.6.1549 10075926 PMC1171243

310. Banerjee S JAK-STAT signaling as a target for inflammatory and autoimmune diseases: current and future prospects Drugs 2017 77 521 546 10.1007/s40265-017-0701-9 28255960 PMC7102286

311. O’Shea JJ The JAK-STAT pathway: impact on human disease and therapeutic intervention Annu Rev. Med. 2015 66 311 328 10.1146/annurev-med-051113-024537 25587654 PMC5634336

312. Bryan MC Rajapaksa NS Kinase inhibitors for the treatment of immunological disorders: recent advances J. Med. Chem. 2018 61 9030 9058 10.1021/acs.jmedchem.8b00667 29870256

313. Schwartz DM JAK inhibition as a therapeutic strategy for immune and inflammatory diseases Nat. Rev. Drug Discov. 2017 16 843 862 10.1038/nrd.2017.201 29104284

314. Strand V Systematic review and meta-analysis of serious infections with tofacitinib and biologic disease-modifying antirheumatic drug treatment in rheumatoid arthritis clinical trials Arthritis Res. Ther. 2015 17 362 10.1186/s13075-015-0880-2 26669566 PMC4704538

315. Mesa RA Yasothan U Kirkpatrick P Ruxolitinib Nat. Rev. Drug Discov. 2012 11 103 104 10.1038/nrd3652 22293561

316. Jamieson C Effect of treatment with a JAK2-selective inhibitor, fedratinib, on bone marrow fibrosis in patients with myelofibrosis J. Transl. Med. 2015 13 294 10.1186/s12967-015-0644-4 26357842 PMC4566296

317. Zhang FQ JAK2 inhibitor TG101348 overcomes erlotinib-resistance in non-small cell lung carcinoma cells with mutated EGF receptor Oncotarget 2015 6 14329 14343 10.18632/oncotarget.3685 25869210 PMC4546470

318. Gremese E Ferraccioli G Tofacitinib for rheumatoid arthritis Lancet 2013 381 1812 10.1016/S0140-6736(13)61114-9 23706794

319. Sanchez GAM JAK1/2 inhibition with baricitinib in the treatment of autoinflammatory interferonopathies J. Clin. Invest. 2018 128 3041 3052 10.1172/JCI98814 29649002 PMC6026004

320. Verstovsek S WP1066, a novel JAK2 inhibitor, suppresses proliferation and induces apoptosis in erythroid human cells carrying the JAK2 V617F mutation Clin. Cancer Res. 2008 14 788 796 10.1158/1078-0432.CCR-07-0524 18245540

321. Hexner EO Lestaurtinib (CEP701) is a JAK2 inhibitor that suppresses JAK2/STAT5 signaling and the proliferation of primary erythroid cells from patients with myeloproliferative disorders Blood 2008 111 5663 5671 10.1182/blood-2007-04-083402 17984313 PMC2424161

322. Mascarenhas J Phase II trial of Lestaurtinib, a JAK2 inhibitor, in patients with myelofibrosis Leuk. Lymphoma 2019 60 1343 1345 10.1080/10428194.2018.1532509 30668266 PMC6530779

323. Mascarenhas JO Primary analysis of a phase II open-label trial of INCB039110, a selective JAK1 inhibitor, in patients with myelofibrosis Haematologica 2017 102 327 335 10.3324/haematol.2016.151126 27789678 PMC5286940

324. Yang EG Design and synthesis of Janus kinase 2 (JAK2) and histone deacetlyase (HDAC) bispecific inhibitors based on pacritinib and evidence of dual pathway inhibition in hematological cell lines J. Med. Chem. 2016 59 8233 8262 10.1021/acs.jmedchem.6b00157 27541357

325. Mesa RA Pacritinib versus best available therapy for the treatment of myelofibrosis irrespective of baseline cytopenias (PERSIST-1): an international, randomised, phase 3 trial Lancet Haematol. 2017 4 e225 e236 10.1016/S2352-3026(17)30027-3 28336242 PMC8209752

326. Harrington R Al Nokhatha SA Conway R JAK inhibitors in rheumatoid arthritis: an evidence-based review on the emerging clinical data J. Inflamm. Res. 2020 13 519 531 10.2147/JIR.S219586 32982367 PMC7500842

327. Xu P Janus kinases (JAKs): The efficient therapeutic targets for autoimmune diseases and myeloproliferative disorders Eur. J. Med. Chem. 2020 192 112155 10.1016/j.ejmech.2020.112155 32120325

328. Chan LC IL-6/JAK1 pathway drives PD-L1 Y112 phosphorylation to promote cancer immune evasion J. Clin. Invest. 2019 129 3324 3338 10.1172/JCI126022 31305264 PMC6668668

329. Dai Z Zeng W Christiano AM 1098 Efficacy of selective next-generation JAK inhibitors in the treatment of alopecia areata J. Investig. Dermatol. 2018 138 S186 10.1016/j.jid.2018.03.1111

330. Norman P Selective JAK inhibitors in development for rheumatoid arthritis Expert Opin. Investig. Drugs 2014 23 1067 1077 10.1517/13543784.2014.918604 24818516

331. Santarpia L Lippman SM El-Naggar AK Targeting the MAPK-RAS-RAF signaling pathway in cancer therapy Expert Opin. Ther. Targets 2012 16 103 119 10.1517/14728222.2011.645805 22239440 PMC3457779

332. Degirmenci, U., Wang, M. & Hu, J. Targeting aberrant RAS/RAF/MEK/ERK signaling for cancer therapy. Cells . 9 , 198 (2020). 10.3390/cells9010198 PMC7017232 31941155

333. Flaherty KT McArthur G BRAF, a target in melanoma: implications for solid tumor drug development Cancer 2010 116 4902 4913 10.1002/cncr.25261 20629085

334. Long GV Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma J. Clin. Oncol. 2011 29 1239 1246 10.1200/JCO.2010.32.4327 21343559

335. Ostrem JM K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions Nature 2013 503 548 551 10.1038/nature12796 24256730 PMC4274051

336. Liu T Wang Z Guo P Ding N Electrostatic mechanism of V600E mutation-induced B-Raf constitutive activation in colorectal cancer: molecular implications for the selectivity difference between type-I and type-II inhibitors Eur. Biophys. J. 2019 48 73 82 10.1007/s00249-018-1334-y 30218115

337. Tsai J Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity Proc. Natl Acad. Sci. USA 2008 105 3041 3046 10.1073/pnas.0711741105 18287029 PMC2268581

338. Hauschild A Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial Lancet 2012 380 358 365 10.1016/S0140-6736(12)60868-X 22735384

339. Li Z Encorafenib (LGX818), a potent BRAF inhibitor, induces senescence accompanied by autophagy in BRAFV600E melanoma cells Cancer Lett. 2016 370 332 344 10.1016/j.canlet.2015.11.015 26586345

340. Molnar E Pan-RAF and MEK vertical inhibition enhances therapeutic response in non-V600 BRAF mutant cells BMC Cancer 2018 18 542 10.1186/s12885-018-4455-x 29739364 PMC5941622

341. Saro S Diwakar D MEK inhibitors for the treatment of NRAS mutant melanoma Drug Des. Devel. Ther 2018 12 2553 2565 10.2147/DDDT.S131721 PMC6108333 30154648

342. Roskoski R Jr. Targeting oncogenic Raf protein-serine/threonine kinases in human cancers Pharm. Res. 2018 135 239 258 10.1016/j.phrs.2018.08.013 30118796

343. Khunger A Khunger M Velcheti V Dabrafenib in combination with trametinib in the treatment of patients with BRAF V600-positive advanced or metastatic non-small cell lung cancer: clinical evidence and experience Ther. Adv. Respir. Dis. 2018 12 1753466618767611 10.1177/1753466618767611 29595366 PMC5941661

344. Markham A Keam SJ Selumetinib: first approval Drugs 2020 80 931 937 10.1007/s40265-020-01331-x 32504375

345. Luebker SA Koepsell SA Diverse mechanisms of BRAF inhibitor resistance in melanoma identified in clinical and preclinical studies Front. Oncol. 2019 9 268 10.3389/fonc.2019.00268 31058079 PMC6478763

346. Al-Olabi L Mosaic RAS/MAPK variants cause sporadic vascular malformations which respond to targeted therapy J. Clin. Invest 2018 128 1496 1508 10.1172/JCI98589 29461977 PMC5873857

347. Arend RC EMR 20006-012: a phase II randomized double-blind placebo controlled trial comparing the combination of pimasertib (MEK inhibitor) with SAR245409 (PI3K inhibitor) to pimasertib alone in patients with previously treated unresectable borderline or low grade ovarian cancer Gynecol. Oncol. 2020 156 301 307 10.1016/j.ygyno.2019.12.002 31870556

348. Chenard-Poirier M Results from the biomarker-driven basket trial of RO5126766 (CH5127566), a potent RAF/MEK inhibitor, in RAS- or RAF-mutated malignancies including multiple myeloma J. Clin. Oncol. 2017 35 2506 2506 10.1200/JCO.2017.35.15_suppl.2506

349. Blake JF Discovery of (S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((1-methyl-1H-pyrazol-5-y l)amino)pyrimidin-4-yl)pyridin-2(1H)-one (GDC-0994), an Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibitor in Early Clinical Development J. Med. Chem. 2016 59 5650 5660 10.1021/acs.jmedchem.6b00389 27227380

350. Okimoto RA Preclinical efficacy of a RAF inhibitor that evades paradoxical MAPK pathway activation in protein kinase BRAF-mutant lung cancer Proc. Natl Acad. Sci. USA 2016 113 13456 13461 10.1073/pnas.1610456113 27834212 PMC5127364

351. Pelster MS Amaria RN Combined targeted therapy and immunotherapy in melanoma: a review of the impact on the tumor microenvironment and outcomes of early clinical trials Ther. Adv. Med. Oncol. 2019 11 1758835919830826 10.1177/1758835919830826 30815041 PMC6384439

352. Kakadia S Mechanisms of resistance to BRAF and MEK inhibitors and clinical update of US Food and Drug Administration-approved targeted therapy in advanced melanoma Onco Targets Ther. 2018 11 7095 7107 10.2147/OTT.S182721 30410366 PMC6200076

353. Hanahan D Weinberg RA Hallmarks of cancer: the next generation Cell 2011 144 646 674 10.1016/j.cell.2011.02.013 21376230

354. Weinberg RA The retinoblastoma protein and cell cycle control Cell 1995 81 323 330 10.1016/0092-8674(95)90385-2 7736585

355. Hunter T Pines J Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age Cell 1994 79 573 582 10.1016/0092-8674(94)90543-6 7954824

356. O’Leary B Finn RS Turner NC Treating cancer with selective CDK4/6 inhibitors Nat. Rev. Clin. Oncol. 2016 13 417 430 10.1038/nrclinonc.2016.26 27030077

357. Chen P Spectrum and degree of CDK drug interactions predicts clinical performance Mol. Cancer Ther. 2016 15 2273 2281 10.1158/1535-7163.MCT-16-0300 27496135

358. Anders L A systematic screen for CDK4/6 substrates links FOXM1 phosphorylation to senescence suppression in cancer cells Cancer Cell 2011 20 620 634 10.1016/j.ccr.2011.10.001 22094256 PMC3237683

359. Asghar U Witkiewicz AK Turner NC Knudsen ES The history and future of targeting cyclin-dependent kinases in cancer therapy Nat. Rev. Drug Discov. 2015 14 130 146 10.1038/nrd4504 25633797 PMC4480421

360. Choi YJ The requirement for cyclin D function in tumor maintenance Cancer Cell 2012 22 438 451 10.1016/j.ccr.2012.09.015 23079655 PMC3487466

361. Sawai CM Therapeutic targeting of the cyclin D3:CDK4/6 complex in T cell leukemia Cancer Cell 2012 22 452 465 10.1016/j.ccr.2012.09.016 23079656 PMC3493168

362. Ameratunga M Kipps E Okines AFC Lopez JS To cycle or fight-CDK4/6 inhibitors at the crossroads of anticancer immunity Clin. Cancer Res. 2019 25 21 28 10.1158/1078-0432.CCR-18-1999 30224338

363. Chen X Latest overview of the cyclin-dependent kinases 4/6 inhibitors in breast cancer: the past, the present and the future J. Cancer 2019 10 6608 6617 10.7150/jca.33079 31777590 PMC6856891

364. Sanchez-Martinez C Lallena MJ Sanfeliciano SG de Dios A Cyclin dependent kinase (CDK) inhibitors as anticancer drugs: recent advances (2015-2019) Bioorg. Med. Chem. Lett. 2019 29 126637 10.1016/j.bmcl.2019.126637 31477350

365. Laderian B Fojo T CDK4/6 Inhibition as a therapeutic strategy in breast cancer: palbociclib, ribociclib, and abemaciclib Semin. Oncol. 2017 44 395 403 10.1053/j.seminoncol.2018.03.006 29935901

366. Infante JR A phase I study of the cyclin-dependent kinase 4/6 inhibitor ribociclib (LEE011) in patients with advanced solid tumors and lymphomas Clin. Cancer Res. 2016 22 5696 5705 10.1158/1078-0432.CCR-16-1248 27542767 PMC5621377

367. Patnaik A Efficacy and safety of abemaciclib, an inhibitor of CDK4 and CDK6, for patients with breast cancer, non-small cell lung cancer, and other solid tumors Cancer Discov. 2016 6 740 753 10.1158/2159-8290.CD-16-0095 27217383

368. Spring LM Zangardi ML Moy B Bardia A Clinical management of potential toxicities and drug interactions related to cyclin-dependent kinase 4/6 inhibitors in breast cancer: practical considerations and recommendations Oncologist 2017 22 1039 1048 10.1634/theoncologist.2017-0142 28706010 PMC5599204

369. Finn RS The cyclin-dependent kinase 4/6 inhibitor palbociclib in combination with letrozole versus letrozole alone as first-line treatment of oestrogen receptor-positive, HER2-negative, advanced breast cancer (PALOMA-1/TRIO-18): a randomised phase 2 study Lancet Oncol. 2015 16 25 35 10.1016/S1470-2045(14)71159-3 25524798

370. Cristofanilli M Fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone-receptor-positive, HER2-negative metastatic breast cancer that progressed on previous endocrine therapy (PALOMA-3): final analysis of the multicentre, double-blind, phase 3 randomised controlled trial Lancet Oncol. 2016 17 425 439 10.1016/S1470-2045(15)00613-0 26947331

371. Sledge GW Jr. MONARCH 2: abemaciclib in combination with fulvestrant in women with HR+/HER2- advanced breast cancer who had progressed while receiving endocrine therapy J. Clin. Oncol. 2017 35 2875 2884 10.1200/JCO.2017.73.7585 28580882

372. Barroso-Sousa R Shapiro GI Tolaney SM Clinical development of the CDK4/6 inhibitors ribociclib and abemaciclib in breast cancer Breast Care 2016 11 167 173 10.1159/000447284 27493615 PMC4960359

373. Goldman JW Treatment Rationale and Study Design for the JUNIPER Study: a randomized phase III study of abemaciclib with best supportive care versus erlotinib with best supportive care in patients with stage IV non-small-cell lung cancer with a detectable KRAS mutation whose disease has progressed after platinum-based chemotherapy Clin. Lung Cancer 2016 17 80 84 10.1016/j.cllc.2015.08.003 26432508

374. Tripathy D Bardia A Sellers WR Ribociclib (LEE011): mechanism of action and clinical impact of this selective cyclin-dependent kinase 4/6 inhibitor in various solid tumors Clin. Cancer Res. 2017 23 3251 3262 10.1158/1078-0432.CCR-16-3157 28351928 PMC5727901

375. He, S. et al. Transient CDK4/6 inhibition protects hematopoietic stem cells from chemotherapy-induced exhaustion. Science Transl. Med. 9 , eaal3986 (2017). 10.1126/scitranslmed.aal3986 PMC5774632 28446688

376. Wang J Critical roles of conventional dendritic cells in promoting T cell-dependent hepatitis through regulating natural killer T cells Clin. Exp. Immunol. 2017 188 127 137 10.1111/cei.12907 27891589 PMC5343358

377. Kim WS 5’-OH-5-nitro-indirubin oxime (AGM130), an indirubin derivative, induces apoptosis of Imatinib-resistant chronic myeloid leukemia cells Leuk. Res. 2013 37 427 433 10.1016/j.leukres.2012.12.017 23337400

378. Wang Y Discovery of 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-methylpiperazin-1-yl)methyl)p henyl)-1H-pyrazole-3-carboxamide (FN-1501), an FLT3- and CDK-kinase inhibitor with potentially high efficiency against acute myelocytic leukemia J. Med Chem. 2018 61 1499 1518 10.1021/acs.jmedchem.7b01261 29357250

379. Hazel P Corrigendum: inhibitor selectivity for cyclin-dependent kinase7: a structural, thermodynamic, and modelling study ChemMedChem 2018 13 207 10.1002/cmdc.201700826 29372749

380. Hu S Discovery and characterization of SY-1365, a selective, covalent inhibitor of CDK7 Cancer Res. 2019 79 3479 3491 10.1158/0008-5472.CAN-19-0119 31064851

381. Sielecki TM Boylan JF Benfield PA Trainor GL Cyclin-dependent kinase inhibitors: useful targets in cell cycle regulation J. Med. Chem. 2000 43 1 18 10.1021/jm990256j 10633033

382. Condorelli R Polyclonal RB1 mutations and acquired resistance to CDK 4/6 inhibitors in patients with metastatic breast cancer Ann. Oncol. 2018 29 640 645 10.1093/annonc/mdx784 29236940

383. Herrera-Abreu MT Early adaptation and acquired resistance to CDK4/6 inhibition in estrogen receptor-positive breast cancer Cancer Res. 2016 76 2301 2313 10.1158/0008-5472.CAN-15-0728 27020857 PMC5426059

384. Caldon CE Cyclin E2 overexpression is associated with endocrine resistance but not insensitivity to CDK2 inhibition in human breast cancer cells Mol. Cancer Ther. 2012 11 1488 1499 10.1158/1535-7163.MCT-11-0963 22564725

385. Spring LM Cyclin-dependent kinase 4 and 6 inhibitors for hormone receptor-positive breast cancer: past, present, and future Lancet 2020 395 817 827 10.1016/S0140-6736(20)30165-3 32145796

386. Bilanges B Posor Y Vanhaesebroeck B PI3K isoforms in cell signalling and vesicle trafficking Nat. Rev. Mol. Cell Biol. 2019 20 515 534 10.1038/s41580-019-0129-z 31110302

387. Jean S Kiger AA Classes of phosphoinositide 3-kinases at a glance J. Cell Sci. 2014 127 923 928 10.1242/jcs.093773 24587488 PMC3937771

388. Fruman DA The PI3K pathway in human disease Cell 2017 170 605 635 10.1016/j.cell.2017.07.029 28802037 PMC5726441

389. Fresno Vara JA PI3K/Akt signalling pathway and cancer Cancer Treat. Rev. 2004 30 193 204 10.1016/j.ctrv.2003.07.007 15023437

390. Liu N BAY 80-6946 is a highly selective intravenous PI3K inhibitor with potent p110alpha and p110delta activities in tumor cell lines and xenograft models Mol. Cancer Ther. 2013 12 2319 2330 10.1158/1535-7163.MCT-12-0993-T 24170767

391. Vangapandu HV Jain N Gandhi V Duvelisib: a phosphoinositide-3 kinase delta/gamma inhibitor for chronic lymphocytic leukemia Expert Opin. Investig. Drugs 2017 26 625 632 10.1080/13543784.2017.1312338 28388280 PMC5584596

392. Vangapandu HV B-cell receptor signaling regulates metabolism in chronic lymphocytic leukemia Mol. Cancer Res. 2017 15 1692 1703 10.1158/1541-7786.MCR-17-0026 28835371 PMC5714317

393. James A Absorption, distribution, metabolism, and excretion of [(14)C]BYL719 (alpelisib) in healthy male volunteers Cancer Chemother. Pharm. 2015 76 751 760 10.1007/s00280-015-2842-4 26254025

394. Foster P The selective PI3K inhibitor XL147 (SAR245408) inhibits tumor growth and survival and potentiates the activity of chemotherapeutic agents in preclinical tumor models Mol. Cancer Ther. 2015 14 931 940 10.1158/1535-7163.MCT-14-0833 25637314

395. Yaguchi S Antitumor activity of ZSTK474, a new phosphatidylinositol 3-kinase inhibitor J. Natl Cancer Inst. 2006 98 545 556 10.1093/jnci/djj133 16622124

396. Evans CA Discovery of a selective phosphoinositide-3-kinase (PI3K)-gamma inhibitor (IPI-549) as an immuno-oncology clinical candidate ACS Med. Chem. Lett. 2016 7 862 867 10.1021/acsmedchemlett.6b00238 27660692 PMC5018865

397. Juric D A first-in-human, phase I, dose-escalation study of TAK-117, a selective PI3Kalpha isoform inhibitor, in patients with advanced solid malignancies Clin. Cancer Res. 2017 23 5015 5023 10.1158/1078-0432.CCR-16-2888 28490463 PMC6858996

398. Serra V NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations Cancer Res. 2008 68 8022 8030 10.1158/0008-5472.CAN-08-1385 18829560

399. Chang KY Novel phosphoinositide 3-kinase/mTOR dual inhibitor, NVP-BGT226, displays potent growth-inhibitory activity against human head and neck cancer cells in vitro and in vivo Clin. Cancer Res. 2011 17 7116 7126 10.1158/1078-0432.CCR-11-0796 21976531

400. Lin J Targeting activated Akt with GDC-0068, a novel selective Akt inhibitor that is efficacious in multiple tumor models Clin. Cancer Res. 2013 19 1760 1772 10.1158/1078-0432.CCR-12-3072 23287563

401. Alzahrani AS PI3K/Akt/mTOR inhibitors in cancer: At the bench and bedside Semin. Cancer Biol. 2019 59 125 132 10.1016/j.semcancer.2019.07.009 31323288

402. McKenna M McGarrigle S Pidgeon GP The next generation of PI3K-Akt-mTOR pathway inhibitors in breast cancer cohorts Biochim. Biophys. Rev. Cancer 2018 1870 185 197 10.1016/j.bbcan.2018.08.001 30318472

403. Schmid P Capivasertib plus paclitaxel versus placebo plus paclitaxel as first-line therapy for metastatic triple-negative breast cancer: The PAKT Trial J. Clin. Oncol. 2020 38 423 433 10.1200/JCO.19.00368 31841354

404. Spencer A The novel AKT inhibitor afuresertib shows favorable safety, pharmacokinetics, and clinical activity in multiple myeloma Blood 2014 124 2190 2195 10.1182/blood-2014-03-559963 25075128 PMC4229853

405. Tolcher AW Phase I dose-escalation trial of the oral AKT inhibitor uprosertib in combination with the oral MEK1/MEK2 inhibitor trametinib in patients with solid tumors Cancer Chemother. Pharm. 2020 85 673 683 10.1007/s00280-020-04038-8 32062691

406. Benjamin D Colombi M Moroni C Hall MN Rapamycin passes the torch: a new generation of mTOR inhibitors Nat. Rev. Drug Discov. 2011 10 868 880 10.1038/nrd3531 22037041

407. Li J Kim SG Blenis J Rapamycin: one drug, many effects Cell Metab. 2014 19 373 379 10.1016/j.cmet.2014.01.001 24508508 PMC3972801

408. Hsieh AC The translational landscape of mTOR signalling steers cancer initiation and metastasis Nature 2012 485 55 61 10.1038/nature10912 22367541 PMC3663483

409. Basu B First-in-human pharmacokinetic and pharmacodynamic study of the dual m-TORC 1/2 inhibitor AZD2014 Clin. Cancer Res. 2015 21 3412 3419 10.1158/1078-0432.CCR-14-2422 25805799 PMC4512239

410. Mosedale M Identification of candidate risk factor genes for human idelalisib toxicity using a collaborative cross approach Toxicol. Sci. 2019 172 265 278 10.1093/toxsci/kfz199 31501888 PMC6876541

411. Tarakhovsky A Tools and landscapes of epigenetics Nat. Immunol. 2010 11 565 568 10.1038/ni0710-565 20562839

412. Cheng Y Targeting epigenetic regulators for cancer therapy: mechanisms and advances in clinical trials Signal Transduct. Target Ther. 2019 4 62 10.1038/s41392-019-0095-0 31871779 PMC6915746

413. Duan R Du W Guo W EZH2: a novel target for cancer treatment J. Hematol. Oncol. 2020 13 104 10.1186/s13045-020-00937-8 32723346 PMC7385862

414. Yang YX Therapeutic potential of enhancer of zeste homolog 2 in autoimmune diseases Expert Opin. Ther. Targets 2019 23 1015 1030 10.1080/14728222.2019.1696309 31747802

415. Wu X Increased EZH2 expression in prostate cancer is associated with metastatic recurrence following external beam radiotherapy Prostate 2019 79 1079 1089 10.1002/pros.23817 31104332 PMC6563086

416. Bai Y Inhibition of enhancer of zeste homolog 2 (EZH2) overcomes enzalutamide resistance in castration-resistant prostate cancer J. Biol. Chem. 2019 294 9911 9923 10.1074/jbc.RA119.008152 31085587 PMC6597805

417. Jones BA Varambally S Arend RC Histone methyltransferase EZH2: A Therapeutic Target For Ovarian Cancer Mol. Cancer Ther. 2018 17 591 602 10.1158/1535-7163.MCT-17-0437 29726819 PMC5939583

418. Jia N Enhancer of zeste homolog 2 is involved in the proliferation of endometrial carcinoma Oncol. Lett. 2014 8 2049 2054 10.3892/ol.2014.2437 25295088 PMC4186594

419. Puppe J EZH2 is overexpressed in BRCA1-like breast tumors and predictive for sensitivity to high-dose platinum-based chemotherapy Clin. Cancer Res. 2019 25 4351 4362 10.1158/1078-0432.CCR-18-4024 31036541

420. Mahmoud F Role of EZH2 histone methyltrasferase in melanoma progression and metastasis Cancer Biol. Ther. 2016 17 579 591 10.1080/15384047.2016.1167291 27105109 PMC4990393

421. Sashida G Iwama A Multifaceted role of the polycomb-group gene EZH2 in hematological malignancies Int J. Hematol. 2017 105 23 30 10.1007/s12185-016-2124-x 27830540

422. Italiano A Tazemetostat, an EZH2 inhibitor, in relapsed or refractory B-cell non-Hodgkin lymphoma and advanced solid tumours: a first-in-human, open-label, phase 1 study Lancet Oncol. 2018 19 649 659 10.1016/S1470-2045(18)30145-1 29650362

423. Lue JK Amengual JE Emerging EZH2 inhibitors and their application in lymphoma Curr. Hematol. Malig. Rep. 2018 13 369 382 10.1007/s11899-018-0466-6 30112706

424. Danis E Ezh2 controls an early hematopoietic program and growth and survival signaling in early T cell precursor acute lymphoblastic leukemia Cell Rep. 2016 14 1953 1965 10.1016/j.celrep.2016.01.064 26904942 PMC4790111

425. Miranda TB DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation Mol. Cancer Ther. 2009 8 1579 1588 10.1158/1535-7163.MCT-09-0013 19509260 PMC3186068

426. Yap DB Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation Blood 2011 117 2451 2459 10.1182/blood-2010-11-321208 21190999 PMC3062411

427. Bodor C EZH2 mutations are frequent and represent an early event in follicular lymphoma Blood 2013 122 3165 3168 10.1182/blood-2013-04-496893 24052547 PMC3814734

428. Qi W An allosteric PRC2 inhibitor targeting the H3K27me3 binding pocket of EED Nat. Chem. Biol. 2017 13 381 388 10.1038/nchembio.2304 28135235

429. He Y The EED protein-protein interaction inhibitor A-395 inactivates the PRC2 complex Nat. Chem. Biol. 2017 13 389 395 10.1038/nchembio.2306 28135237

430. Dong H An allosteric PRC2 inhibitor targeting EED suppresses tumor progression by modulating the immune response Cancer Res. 2019 79 5587 5596 10.1158/0008-5472.CAN-19-0428 31395608

431. Potjewyd F Degradation of polycomb repressive complex 2 with an EED-targeted bivalent chemical degrader Cell Chem. Biol. 2020 27 47 56.e15 10.1016/j.chembiol.2019.11.006 31831267 PMC7004250

432. Wang X A covalently bound inhibitor triggers EZH2 degradation through CHIP-mediated ubiquitination EMBO J. 2017 36 1243 1260 10.15252/embj.201694058 28320739 PMC5412902

433. Bisserier M Wajapeyee N Mechanisms of resistance to EZH2 inhibitors in diffuse large B-cell lymphomas Blood 2018 131 2125 2137 10.1182/blood-2017-08-804344 29572378 PMC5946763

434. Wu S SWI/SNF catalytic subunits’ switch drives resistance to EZH2 inhibitors in ARID1A-mutated cells Nat. Commun. 2018 9 4116 10.1038/s41467-018-06656-6 30297712 PMC6175882

435. Huang X Targeting epigenetic crosstalk as a therapeutic strategy for EZH2-aberrant solid tumors Cell 2018 175 186 199 e119 10.1016/j.cell.2018.08.058 30220457

436. Gibaja V Development of secondary mutations in wild-type and mutant EZH2 alleles cooperates to confer resistance to EZH2 inhibitors Oncogene 2016 35 558 566 10.1038/onc.2015.114 25893294 PMC4744243

437. Tremblay-LeMay R Rastgoo N Pourabdollah M Chang H EZH2 as a therapeutic target for multiple myeloma and other haematological malignancies Biomark. Res. 2018 6 34 10.1186/s40364-018-0148-5 30555699 PMC6286605

438. Zhang Y Combination of EZH2 inhibitor and BET inhibitor for treatment of diffuse intrinsic pontine glioma Cell Biosci. 2017 7 56 10.1186/s13578-017-0184-0 29118968 PMC5663148

439. Lue JK Precision targeting with EZH2 and HDAC inhibitors in epigenetically dysregulated lymphomas Clin. Cancer Res. 2019 25 5271 5283 10.1158/1078-0432.CCR-18-3989 30979734 PMC6726529

440. Richart L Margueron R Drugging histone methyltransferases in cancer Curr. Opin. Chem. Biol. 2020 56 51 62 10.1016/j.cbpa.2019.11.009 31981999

441. Park SY Kim JS A short guide to histone deacetylases including recent progress on class II enzymes Exp. Mol. Med. 2020 52 204 212 10.1038/s12276-020-0382-4 32071378 PMC7062823

442. Delcuve GP Khan DH Davie JR Roles of histone deacetylases in epigenetic regulation: emerging paradigms from studies with inhibitors Clin. Epigenet. 2012 4 5 10.1186/1868-7083-4-5 PMC3320549 22414492

443. Li, Y. & Seto, E. HDACs and HDAC inhibitors in cancer development and therapy. Cold Spring Harb. Perspect. Med . 6 , a026831 (2016). 10.1101/cshperspect.a026831 PMC5046688 27599530

444. Eckschlager, T., Plch, J., Stiborova, M. & Hrabeta, J. Histone deacetylase inhibitors as anticancer drugs. Int. J. Mol. Sci . 18 , 1414 (2017). 10.3390/ijms18071414 PMC5535906 28671573

445. Dokmanovic M Clarke C Marks PA Histone deacetylase inhibitors: overview and perspectives Mol. Cancer Res. 2007 5 981 989 10.1158/1541-7786.MCR-07-0324 17951399

446. Mann BS Vorinostat for treatment of cutaneous manifestations of advanced primary cutaneous T-cell lymphoma Clin. Cancer Res. 2007 13 2318 2322 10.1158/1078-0432.CCR-06-2672 17438089

447. Lee HZ FDA approval: belinostat for the treatment of patients with relapsed or refractory peripheral T-cell lymphoma Clin. Cancer Res. 2015 21 2666 2670 10.1158/1078-0432.CCR-14-3119 25802282

448. Yee AJ Raje NS Panobinostat and multiple myeloma in 2018 Oncologist 2018 23 516 517 10.1634/theoncologist.2017-0644 29445026 PMC5947458

449. Piekarz RL Phase 2 trial of romidepsin in patients with peripheral T-cell lymphoma Blood 2011 117 5827 5834 10.1182/blood-2010-10-312603 21355097 PMC3112033

450. Xu Y Zhang P Liu Y Chidamide tablets: HDAC inhibition to treat lymphoma Drugs Today 2017 53 167 176 10.1358/dot.2017.53.3.2595452 28447074

451. Cea M Synergistic interactions between HDAC and sirtuin inhibitors in human leukemia cells PLoS ONE 2011 6 e22739 10.1371/journal.pone.0022739 21818379 PMC3144930

452. Suraweera A O’Byrne KJ Richard DJ Combination therapy with histone deacetylase inhibitors (HDACi) for the treatment of cancer: achieving the full therapeutic potential of HDACi Front. Oncol. 2018 8 92 10.3389/fonc.2018.00092 29651407 PMC5884928

453. Pan D Lu X New therapeutic avenue of epigenetic modulations in cancer Transl. Breast Cancer Res. 2020 1 2 2 10.21037/tbcr.2020.03.03

454. Abaza YM Phase 1 dose escalation multicenter trial of pracinostat alone and in combination with azacitidine in patients with advanced hematologic malignancies Cancer 2017 123 4851 4859 10.1002/cncr.30949 28841236 PMC5901648

455. Qin HT Li HQ Liu F Selective histone deacetylase small molecule inhibitors: recent progress and perspectives Expert Opin. Ther. Pat. 2017 27 621 636 10.1080/13543776.2017.1276565 28033734

456. Kyaw MTH The HDAC inhibitor, SAHA, combined with cisplatin synergistically induces apoptosis in alpha-fetoprotein-producing hepatoid adenocarcinoma cells Acta Histochem. Cytochem. 2019 52 1 8 10.1267/ahc.18044 30923410 PMC6434315

457. Vitkeviciene A HDAC and HMT inhibitors in combination with conventional therapy: a novel treatment option for acute promyelocytic leukemia J. Oncol. 2019 2019 6179573 10.1155/2019/6179573 31396278 PMC6668531

458. Gray J Cubitt CL Zhang S Chiappori A Combination of HDAC and topoisomerase inhibitors in small cell lung cancer Cancer Biol. Ther. 2012 13 614 622 10.4161/cbt.19848 22441819 PMC3408970

459. Zhang Y Combined HDAC and bromodomain protein inhibition reprograms tumor cell metabolism and elicits synthetic lethality in glioblastoma Clin. Cancer Res. 2018 24 3941 3954 10.1158/1078-0432.CCR-18-0260 29764852 PMC6095717

460. Banik, D., Moufarrij, S. & Villagra, A. Immunoepigenetics combination therapies: an overview of the role of HDACs in cancer immunotherapy. Int. J. Mol. Sci . 20 , 2241 (2019). 10.3390/ijms20092241 PMC6539010 31067680

461. Stoddard BL Dean A Koshland DE Jr. Structure of isocitrate dehydrogenase with isocitrate, nicotinamide adenine dinucleotide phosphate, and calcium at 2.5-A resolution: a pseudo-Michaelis ternary complex Biochemistry 1993 32 9310 9316 10.1021/bi00087a008 8369300

462. Xu X Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity J. Biol. Chem. 2004 279 33946 33957 10.1074/jbc.M404298200 15173171

463. Henderson NS Isozymes of isocitrate dehydrogenase: subunit structure and intracellular location J. Exp. Zool. 1965 158 263 273 10.1002/jez.1401580303 14327193

464. Yang H Ye D Guan KL Xiong Y IDH1 and IDH2 mutations in tumorigenesis: mechanistic insights and clinical perspectives Clin. Cancer Res. 2012 18 5562 5571 10.1158/1078-0432.CCR-12-1773 23071358 PMC3897211

465. Amary MF IDH1 and IDH2 mutations are frequent events in central chondrosarcoma and central and periosteal chondromas but not in other mesenchymal tumours J. Pathol. 2011 224 334 343 10.1002/path.2913 21598255

466. Ganguly BB Kadam NN Mutations of myelodysplastic syndromes (MDS): an update Mutat. Res Rev. Mutat. Res. 2016 769 47 62 10.1016/j.mrrev.2016.04.009 27543316

467. Farshidfar F Integrative genomic analysis of cholangiocarcinoma identifies distinct IDH-mutant molecular profiles Cell Rep. 2017 18 2780 2794 10.1016/j.celrep.2017.02.033 28297679 PMC5493145

468. Gross S Cancer-associated metabolite 2-hydroxyglutarate accumulates in acute myelogenous leukemia with isocitrate dehydrogenase 1 and 2 mutations J. Exp. Med. 2010 207 339 344 10.1084/jem.20092506 20142433 PMC2822606

469. Ward PS The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate Cancer Cell 2010 17 225 234 10.1016/j.ccr.2010.01.020 20171147 PMC2849316

470. Lu C IDH mutation impairs histone demethylation and results in a block to cell differentiation Nature 2012 483 474 478 10.1038/nature10860 22343901 PMC3478770

471. Prensner JR Chinnaiyan AM Metabolism unhinged: IDH mutations in cancer Nat. Med. 2011 17 291 293 10.1038/nm0311-291 21383741

472. Dang L Jin S Su SM IDH mutations in glioma and acute myeloid leukemia Trends Mol. Med. 2010 16 387 397 10.1016/j.molmed.2010.07.002 20692206

473. Kim ES Enasidenib: first global approval Drugs 2017 77 1705 1711 10.1007/s40265-017-0813-2 28879540

474. Stein EMEnasidenib a targeted inhibitor of mutant IDH2 proteins for treatment of relapsed or refractory acute myeloid leukemia Future Oncol. 2018 14 23 40 10.2217/fon-2017-0392 29243965

475. Yen K AG-221, a first-in-class therapy targeting acute myeloid leukemia harboring oncogenic IDH2 mutations Cancer Discov. 2017 7 478 493 10.1158/2159-8290.CD-16-1034 28193778

476. Galkin M Jonas BA Enasidenib in the treatment of relapsed/refractory acute myeloid leukemia: an evidence-based review of its place in therapy Core Evid. 2019 14 3 17 10.2147/CE.S172912 31118877 PMC6503332

477. Popovici-Muller J Discovery of AG-120 (Ivosidenib): a first-in-class mutant IDH1 inhibitor for the treatment of IDH1 mutant cancers ACS Med. Chem. Lett. 2018 9 300 305 10.1021/acsmedchemlett.7b00421 29670690 PMC5900343

478. DiNardo CD Durable remissions with ivosidenib in IDH1-mutated relapsed or refractory AML N. Engl. J. Med. 2018 378 2386 2398 10.1056/NEJMoa1716984 29860938

479. Norsworthy KJ FDA approval summary: ivosidenib for relapsed or refractory acute myeloid leukemia with an isocitrate dehydrogenase-1 mutation Clin. Cancer Res. 2019 25 3205 3209 10.1158/1078-0432.CCR-18-3749 30692099

480. Duggan S Caplacizumab: first global approval Drugs 2018 78 1639 1642 10.1007/s40265-018-0989-0 30298461 PMC6280848

481. Feliciano P PAK inhibitor in fragile X Nat. Genet. 2013 45 477 477

482. de Botton S Targeting isocitrate dehydrogenase (IDH)1 and IDH2 mutations clinical results in advanced hematologic malignancies Ann. Oncol. 2015 26 ii15 10.1093/annonc/mdv089.3

483. Ma T Inhibitors of mutant isocitrate dehydrogenases 1 and 2 (mIDH1/2): an update and perspective J. Med. Chem. 2018 61 8981 9003 10.1021/acs.jmedchem.8b00159 29847930

484. Chaturvedi A Pan-mutant-IDH1 inhibitor BAY1436032 is highly effective against human IDH1 mutant acute myeloid leukemia in vivo Leukemia 2017 31 2020 2028 10.1038/leu.2017.46 28232670 PMC5629366

485. Caravella JA Structure-based design and identification of FT-2102 (olutasidenib), a potent mutant-selective IDH1 inhibitor J. Med. Chem. 2020 63 1612 1623 10.1021/acs.jmedchem.9b01423 31971798

486. Cho YS Discovery and evaluation of clinical candidate IDH305, a brain penetrant mutant IDH1 inhibitor ACS Med. Chem. Lett. 2017 8 1116 1121 10.1021/acsmedchemlett.7b00342 29057061 PMC5641959

487. DiNardo CD Stein EM SOHO state of the art update and next questions: IDH therapeutic targeting in AML Clin. Lymphoma Myeloma Leuk. 2018 18 769 772 10.1016/j.clml.2018.10.007 30416011

488. Harding JJ Isoform switching as a mechanism of acquired resistance to mutant isocitrate dehydrogenase inhibition Cancer Discov. 2018 8 1540 1547 10.1158/2159-8290.CD-18-0877 30355724 PMC6699636

489. Intlekofer AM Acquired resistance to IDH inhibition through trans or cis dimer-interface mutations Nature 2018 559 125 129 10.1038/s41586-018-0251-7 29950729 PMC6121718

490. Medeiros BC Isocitrate dehydrogenase mutations in myeloid malignancies Leukemia 2017 31 272 281 10.1038/leu.2016.275 27721426 PMC5292675

491. Fujiwara H Isocitrate dehydrogenase 1 mutation sensitizes intrahepatic cholangiocarcinoma to the BET inhibitor JQ1 Cancer Sci. 2018 109 3602 3610 10.1111/cas.13784 30156013 PMC6215870

492. DiNardo CD Venetoclax combined with decitabine or azacitidine in treatment-naive, elderly patients with acute myeloid leukemia Blood 2019 133 7 17 10.1182/blood-2018-08-868752 30361262 PMC6318429

493. Chaturvedi, A. et al. Synergistic activity of IDH1 inhibitor BAY1436032 with azacitidine in IDH1 mutant acute myeloid leukemia. Haematologica 106 , 565–573 (2020). 10.3324/haematol.2019.236992 PMC7849562 32241846

494. Kantarjian H Decitabine improves patient outcomes in myelodysplastic syndromes: results of a phase III randomized study Cancer 2006 106 1794 1803 10.1002/cncr.21792 16532500

495. Silverman LR Randomized controlled trial of azacitidine in patients with the myelodysplastic syndrome: a study of the cancer and leukemia group B J. Clin. Oncol. 2002 20 2429 2440 10.1200/JCO.2002.04.117 12011120

496. Olino K Park T Ahuja N Exposing hidden targets: combining epigenetic and immunotherapy to overcome cancer resistance Semin. Cancer Biol. 2020 65 114 122 10.1016/j.semcancer.2020.01.001 31911188

497. Bates SE Epigenetic therapies for cancer N. Engl. J. Med. 2020 383 650 663 10.1056/NEJMra1805035 32786190

498. Ghasemi S Cancer’s epigenetic drugs: where are they in the cancer medicines? Pharmacogenomics J. 2020 20 367 379 10.1038/s41397-019-0138-5 31819161

499. Laubach JP Moreau P San-Miguel JF Richardson PG Panobinostat for the treatment of multiple myeloma Clin. Cancer Res. 2015 21 4767 4773 10.1158/1078-0432.CCR-15-0530 26362997

500. Knight T A delicate balance - The BCL-2 family and its role in apoptosis, oncogenesis, and cancer therapeutics Biochem. Pharm. 2019 162 250 261 10.1016/j.bcp.2019.01.015 30668936

501. Warren CFA Wong-Brown MW Bowden NA BCL-2 family isoforms in apoptosis and cancer Cell Death Dis. 2019 10 177 10.1038/s41419-019-1407-6 30792387 PMC6384907

502. Huang K BH3-only proteins target BCL-xL/MCL-1, not BAX/BAK, to initiate apoptosis Cell Res. 2019 29 942 952 10.1038/s41422-019-0231-y 31551537 PMC6888900

503. Fabregat I Dysregulation of apoptosis in hepatocellular carcinoma cells World J. Gastroenterol. 2009 15 513 520 10.3748/wjg.15.513 19195051 PMC2653340

504. Schattenberg JM Schuchmann M Galle PR Cell death and hepatocarcinogenesis: dysregulation of apoptosis signaling pathways J. Gastroenterol. Hepatol. 2011 26 213 219 10.1111/j.1440-1746.2010.06582.x 21199533

505. Perini GF BCL-2 as therapeutic target for hematological malignancies J. Hematol. Oncol. 2018 11 65 10.1186/s13045-018-0608-2 29747654 PMC5946445

506. Ashkenazi A Fairbrother WJ Leverson JD Souers AJ From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors Nat. Rev. Drug Discov. 2017 16 273 284 10.1038/nrd.2016.253 28209992

507. Zhu Y Identification of a novel senolytic agent, navitoclax, targeting the Bcl-2 family of anti-apoptotic factors Aging Cell 2016 15 428 435 10.1111/acel.12445 26711051 PMC4854923

508. Lampson BL Davids MS The development and current use of BCL-2 inhibitors for the treatment of chronic lymphocytic leukemia Curr. Hematol. Malig. Rep. 2017 12 11 19 10.1007/s11899-017-0359-0 28116634 PMC5862560

509. Anderson MA Huang D Roberts A Targeting BCL2 for the treatment of lymphoid malignancies Semin. Hematol. 2014 51 219 227 10.1053/j.seminhematol.2014.05.008 25048785

510. Valentin R Grabow S Davids MS The rise of apoptosis: targeting apoptosis in hematologic malignancies Blood 2018 132 1248 1264 10.1182/blood-2018-02-791350 30012635

511. Tse C ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor Cancer Res. 2008 68 3421 3428 10.1158/0008-5472.CAN-07-5836 18451170

512. Stilgenbauer S Venetoclax in relapsed or refractory chronic lymphocytic leukaemia with 17p deletion: a multicentre, open-label, phase 2 study Lancet Oncol. 2016 17 768 778 10.1016/S1470-2045(16)30019-5 27178240

513. Deeks ED Venetoclax: first global approval Drugs 2016 76 979 987 10.1007/s40265-016-0596-x 27260335

514. Guerra VA DiNardo C Konopleva M Venetoclax-based therapies for acute myeloid leukemia Best Pr. Res. Clin. Haematol. 2019 32 145 153 10.1016/j.beha.2019.05.008 PMC6581210 31203996

515. Caenepeel S AMG 176, a selective MCL1 inhibitor, is effective in hematologic cancer models alone and in combination with established therapies Cancer Discov. 2018 8 1582 1597 30254093 10.1158/2159-8290.CD-18-0387

516. Casara P S55746 is a novel orally active BCL-2 selective and potent inhibitor that impairs hematological tumor growth Oncotarget 2018 9 20075 20088 10.18632/oncotarget.24744 29732004 PMC5929447

517. McBride A The role of inhibition of apoptosis in acute leukemias and myelodysplastic syndrome Front. Oncol. 2019 9 192 10.3389/fonc.2019.00192 30972300 PMC6445951

518. Tron AE Discovery of Mcl-1-specific inhibitor AZD5991 and preclinical activity in multiple myeloma and acute myeloid leukemia Nat. Commun. 2018 9 5341 10.1038/s41467-018-07551-w 30559424 PMC6297231

519. Yalniz FF Wierda WG Targeting BCL2 in chronic lymphocytic leukemia and other hematologic malignancies Drugs 2019 79 1287 1304 10.1007/s40265-019-01163-4 31313099

520. Yang S The chemical biology of apoptosis: revisited after 17 years Eur. J. Med. Chem. 2019 177 63 75 10.1016/j.ejmech.2019.05.019 31129454

521. Aldoss I Venetoclax and hypomethylating agents in TP53-mutated acute myeloid leukaemia Br. J. Haematol. 2019 187 e45 e48 10.1111/bjh.16166 31441045 PMC6786919

522. Villalobos-Ortiz M BH3 profiling discriminates on-target small molecule BH3 mimetics from putative mimetics Cell Death Differ. 2020 27 999 1007 10.1038/s41418-019-0391-9 31332296 PMC7205860

523. Thomas S Targeting the Bcl-2 family for cancer therapy Expert Opin. Ther. Targets 2013 17 61 75 10.1517/14728222.2013.733001 23173842 PMC3955095

524. Tahir SK Potential mechanisms of resistance to venetoclax and strategies to circumvent it BMC Cancer 2017 17 399 10.1186/s12885-017-3383-5 28578655 PMC5457565

525. Lok SW A phase Ib dose-escalation and expansion study of the BCL2 inhibitor venetoclax combined with tamoxifen in ER and BCL2-positive metastatic breast cancer Cancer Discov. 2019 9 354 369 10.1158/2159-8290.CD-18-1151 30518523

526. Pak E Segal RA Hedgehog signal transduction: key players, oncogenic drivers, and cancer therapy Dev. Cell 2016 38 333 344 10.1016/j.devcel.2016.07.026 27554855 PMC5017307

527. Tukachinsky H Petrov K Watanabe M Salic A Mechanism of inhibition of the tumor suppressor Patched by Sonic Hedgehog Proc. Natl Acad. Sci. USA 2016 113 E5866 e5875 10.1073/pnas.1606719113 27647915 PMC5056095

528. Dlugosz A Agrawal S Kirkpatrick P Vismodegib Nat. Rev. Drug Discov. 2012 11 437 438 10.1038/nrd3753 22653209 PMC3383648

529. Jain S Song R Xie J Sonidegib: mechanism of action, pharmacology, and clinical utility for advanced basal cell carcinomas Onco Targets Ther. 2017 10 1645 1653 10.2147/OTT.S130910 28352196 PMC5360396

530. Basset-Seguin N Vismodegib in patients with advanced basal cell carcinoma: Primary analysis of STEVIE, an international, open-label trial Eur. J. Cancer 2017 86 334 348 10.1016/j.ejca.2017.08.022 29073584

531. Lear JT Long-term efficacy and safety of sonidegib in patients with locally advanced and metastatic basal cell carcinoma: 30-month analysis of the randomized phase 2 BOLT study J. Eur. Acad. Dermatol Venereol. 2018 32 372 381 10.1111/jdv.14542 28846163 PMC5873455

532. Sekulic A Efficacy and safety of vismodegib in advanced basal-cell carcinoma N. Engl. J. Med. 2012 366 2171 2179 10.1056/NEJMoa1113713 22670903 PMC5278761

533. LoRusso PM Phase I trial of hedgehog pathway inhibitor vismodegib (GDC-0449) in patients with refractory, locally advanced or metastatic solid tumors Clin. Cancer Res. 2011 17 2502 2511 10.1158/1078-0432.CCR-10-2745 21300762 PMC5244484

534. Stathis A Phase I trial of the oral smoothened inhibitor sonidegib in combination with paclitaxel in patients with advanced solid tumors Invest. N. Drugs 2017 35 766 772 10.1007/s10637-017-0454-z 28317088

535. Li Y Song Q Day BW Phase I and phase II sonidegib and vismodegib clinical trials for the treatment of paediatric and adult MB patients: a systemic review and meta-analysis Acta Neuropathol. Commun. 2019 7 123 10.1186/s40478-019-0773-8 31362788 PMC6668073

536. Jamieson C Martinelli G Papayannidis C Cortes JE Hedgehog pathway inhibitors: a new therapeutic class for the treatment of acute myeloid leukemia Blood Cancer Discov. 2020 1 134 145 10.1158/2643-3230.BCD-20-0007 PMC8447269 34661144

537. Xie, H., Paradise, B. D., Ma, W. W. & Fernandez-Zapico, M. E. Recent advances in the clinical targeting of Hedgehog/GLI signaling in cancer. Cells . 8 , 394 (2019). 10.3390/cells8050394 PMC6562674 31035664

538. Pietrobono, S. & Stecca, B. Targeting the oncoprotein smoothened by small molecules: focus on novel acylguanidine derivatives as potent smoothened inhibitors. Cells 7 , 272 (2018). 10.3390/cells7120272 PMC6316656 30558232

539. Suzman DL Antonarakis ES Clinical implications of hedgehog pathway signaling in prostate cancer Cancers 2015 7 1983 1993 10.3390/cancers7040871 26426053 PMC4695871

540. Cortes JE Gutzmer R Kieran MW Solomon JA Hedgehog signaling inhibitors in solid and hematological cancers Cancer Treat. Rev. 2019 76 41 50 10.1016/j.ctrv.2019.04.005 31125907

541. Girardi, D., Barrichello, A., Fernandes, G. & Pereira, A. Targeting the Hedgehog pathway in cancer: current evidence and future perspectives. Cells 8 , 153 (2019). 10.3390/cells8020153 PMC6406365 30759860

542. Bhateja, P., Cherian, M., Majumder, S. & Ramaswamy, B. The Hedgehog signaling pathway: a viable target in breast cancer? Cancers 11 , 1126 (2019). 10.3390/cancers11081126 PMC6721501 31394751

543. Kim J Arsenic antagonizes the Hedgehog pathway by preventing ciliary accumulation and reducing stability of the Gli2 transcriptional effector Proc. Natl Acad. Sci. USA 2010 107 13432 13437 10.1073/pnas.1006822107 20624968 PMC2922148

544. Liu, X. et al. Development of hedgehog pathway inhibitors by epigenetically targeting GLI through BET bromodomain for the treatment of medulloblastoma. Acta Pharm. Sin. B 11 , 488–504 (2020). 10.1016/j.apsb.2020.07.007 PMC7893122 33643826

545. Tu J Molecular modeling study on resistance of WT/D473H SMO to antagonists LDE-225 and LEQ-506 Pharm. Res. 2018 129 491 499 10.1016/j.phrs.2017.11.025 29175550

546. Ueno H A phase I and pharmacokinetic study of taladegib, a Smoothened inhibitor, in Japanese patients with advanced solid tumors Invest. N. Drugs 2018 36 647 656 10.1007/s10637-017-0544-y 29453627

547. Dong X Wang C Chen Z Zhao W Overcoming the resistance mechanisms of smoothened inhibitors Drug Discov. Today 2018 23 704 710 10.1016/j.drudis.2018.01.012 29326074

548. Adams J The proteasome: structure, function, and role in the cell Cancer Treat. Rev. 2003 29 3 9 10.1016/S0305-7372(03)00081-1 12738238

549. Burger AM Seth AK The ubiquitin-mediated protein degradation pathway in cancer: therapeutic implications Eur. J. Cancer 2004 40 2217 2229 10.1016/j.ejca.2004.07.006 15454246

550. Kodroń, A., Mussulini, B. H., Pilecka, I. & Chacińska, A. The ubiquitin-proteasome system and its crosstalk with mitochondria as therapeutic targets in medicine. Pharmacol Res. 163 , 105248 (2020). 10.1016/j.phrs.2020.105248 33065283

551. Adams J The proteasome: a suitable antineoplastic target Nat. Rev. Cancer 2004 4 349 360 10.1038/nrc1361 15122206

552. Gandolfi S The proteasome and proteasome inhibitors in multiple myeloma Cancer Metastasis Rev. 2017 36 561 584 10.1007/s10555-017-9707-8 29196868

553. Adams J Kauffman M Development of the proteasome inhibitor Velcade (Bortezomib) Cancer Investig. 2004 22 304 311 10.1081/CNV-120030218 15199612

554. Fricker LD Proteasome inhibitor drugs Annu. Rev. Pharmacol. Toxicol. 2020 60 457 476 10.1146/annurev-pharmtox-010919-023603 31479618

555. Dimopoulos MA Richardson PG Moreau P Anderson KC Current treatment landscape for relapsed and/or refractory multiple myeloma Nat. Rev. Clin. Oncol. 2015 12 42 54 10.1038/nrclinonc.2014.200 25421279

556. Dick LR Fleming PE Building on bortezomib: second-generation proteasome inhibitors as anti-cancer therapy Drug Discov. Today 2010 15 243 249 10.1016/j.drudis.2010.01.008 20116451

557. Herndon TM U.s. Food and Drug Administration approval: carfilzomib for the treatment of multiple myeloma Clin. Cancer Res. 2013 19 4559 4563 10.1158/1078-0432.CCR-13-0755 23775332

558. Demo SD Antitumor activity of PR-171, a novel irreversible inhibitor of the proteasome Cancer Res. 2007 67 6383 6391 10.1158/0008-5472.CAN-06-4086 17616698

559. Zanwar S Abeykoon JP Kapoor P Ixazomib: a novel drug for multiple myeloma Expert Rev. Hematol. 2018 11 761 771 10.1080/17474086.2018.1518129 30173621

560. Kupperman E Evaluation of the proteasome inhibitor MLN9708 in preclinical models of human cancer Cancer Res. 2010 70 1970 1980 10.1158/0008-5472.CAN-09-2766 20160034

561. Xie J Ixazomib - the first oral proteasome inhibitor Leuk. Lymphoma 2019 60 610 618 10.1080/10428194.2018.1523398 30614337

562. Groll M Huber R Potts BC Crystal structures of Salinosporamide A (NPI-0052) and B (NPI-0047) in complex with the 20S proteasome reveal important consequences of beta-lactone ring opening and a mechanism for irreversible binding J. Am. Chem. Soc. 2006 128 5136 5141 10.1021/ja058320b 16608349

563. Ma L Diao A Marizomib, a potent second generation proteasome inhibitor from natural origin Anti-Cancer Agents Med. Chem. 2015 15 298 306 10.2174/1871520614666141114202606 25403165

564. Zhou HJ Design and synthesis of an orally bioavailable and selective peptide epoxyketone proteasome inhibitor (PR-047) J. Med. Chem. 2009 52 3028 3038 10.1021/jm801329v 19348473

565. Piva R CEP-18770: a novel, orally active proteasome inhibitor with a tumor-selective pharmacologic profile competitive with bortezomib Blood 2008 111 2765 2775 10.1182/blood-2007-07-100651 18057228

566. Gallerani E A first in human phase I study of the proteasome inhibitor CEP-18770 in patients with advanced solid tumours and multiple myeloma Eur. J. Cancer 2013 49 290 296 10.1016/j.ejca.2012.09.009 23058787

567. Vogl DT Phase I/II study of the novel proteasome inhibitor delanzomib (CEP-18770) for relapsed and refractory multiple myeloma Leuk. Lymphoma 2017 58 1872 1879 10.1080/10428194.2016.1263842 28140719

568. Park JE Next-generation proteasome inhibitors for cancer therapy Transl. Res. 2018 198 1 16 10.1016/j.trsl.2018.03.002 29654740 PMC6151281

569. Dolloff NGEmerging Therapeutic strategies for overcoming proteasome inhibitor resistance Adv. Cancer Res. 2015 127 191 226 10.1016/bs.acr.2015.03.002 26093901

570. Caldecott KW Protein ADP-ribosylation and the cellular response to DNA strand breaks DNA Repair 2014 19 108 113 10.1016/j.dnarep.2014.03.021 24755000

571. Vyas S Chang P New PARP targets for cancer therapy Nat. Rev. Cancer 2014 14 502 509 10.1038/nrc3748 24898058 PMC4480224

572. Dobzhansky T Genetics of natural populations; recombination and variability in populations of Drosophila pseudoobscura Genetics 1946 31 269 290 10.1093/genetics/31.3.269 20985721 PMC1209328

573. King MC Marks JH Mandell JB New York Breast Cancer Study, Group. Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2 Science 2003 302 643 646 10.1126/science.1088759 14576434

574. Lord CJ Ashworth A PARP inhibitors: synthetic lethality in the clinic Science 2017 355 1152 1158 10.1126/science.aam7344 28302823 PMC6175050

575. Kim G FDA approval summary: olaparib monotherapy in patients with deleterious germline BRCA-mutated advanced ovarian cancer treated with three or more lines of chemotherapy Clin. Cancer Res. 2015 21 4257 4261 10.1158/1078-0432.CCR-15-0887 26187614

576. Dockery LE Gunderson CC Moore KN Rucaparib: the past, present, and future of a newly approved PARP inhibitor for ovarian cancer Onco Targets Ther. 2017 10 3029 3037 10.2147/OTT.S114714 28790837 PMC5488752

577. Essel KG Moore KN Niraparib for the treatment of ovarian cancer Expert Rev. Anticancer Ther. 2018 18 727 733 10.1080/14737140.2018.1490180 29911447

578. Hoy SM Talazoparib: first global approval Drugs 2018 78 1939 1946 10.1007/s40265-018-1026-z 30506138

579. Strom CE Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate Nucleic Acids Res. 2011 39 3166 3175 10.1093/nar/gkq1241 21183466 PMC3082910

580. Pujade-Lauraine E Olaparib tablets as maintenance therapy in patients with platinum-sensitive, relapsed ovarian cancer and a BRCA1/2 mutation (SOLO2/ENGOT-Ov21): a double-blind, randomised, placebo-controlled, phase 3 trial Lancet Oncol. 2017 18 1274 1284 10.1016/S1470-2045(17)30469-2 28754483

581. Konstantinopoulos, P. A. et al. Single-Arm phases 1 and 2 trial of niraparib in combination with pembrolizumab in patients with recurrent platinum-resistant ovarian carcinoma. JAMA Oncol . 5 , 1141–1149 (2019). 10.1001/jamaoncol.2019.1048 PMC6567832 31194228

582. Sachdev E PARP inhibition in cancer: an update on clinical development Target Oncol. 2019 14 657 679 10.1007/s11523-019-00680-2 31625002

583. Mateo J A decade of clinical development of PARP inhibitors in perspective Ann. Oncol. 2019 30 1437 1447 10.1093/annonc/mdz192 31218365 PMC6771225

584. Donawho CK ABT-888, an orally active poly(ADP-ribose) polymerase inhibitor that potentiates DNA-damaging agents in preclinical tumor models Clin. Cancer Res. 2007 13 2728 2737 10.1158/1078-0432.CCR-06-3039 17473206

585. Coleman RL Veliparib with first-line chemotherapy and as maintenance therapy in ovarian cancer N. Engl. J. Med. 2019 381 2403 2415 10.1056/NEJMoa1909707 31562800 PMC6941439

586. Moore K Maintenance olaparib in patients with newly diagnosed advanced ovarian cancer N. Engl. J. Med. 2018 379 2495 2505 10.1056/NEJMoa1810858 30345884

587. Jiang X PARP inhibitors in ovarian cancer: Sensitivity prediction and resistance mechanisms J. Cell Mol. Med. 2019 23 2303 2313 10.1111/jcmm.14133 30672100 PMC6433712

588. D’Andrea AD Mechanisms of PARP inhibitor sensitivity and resistance DNA Repair 2018 71 172 176 10.1016/j.dnarep.2018.08.021 30177437

589. Haynes B Murai J Lee JM Restored replication fork stabilization, a mechanism of PARP inhibitor resistance, can be overcome by cell cycle checkpoint inhibition Cancer Treat. Rev. 2018 71 1 7 10.1016/j.ctrv.2018.09.003 30269007 PMC7429716

590. Carrassa L Colombo I Damia G Bertoni F Targeting the DNA damage response for patients with lymphoma: Preclinical and clinical evidences Cancer Treat. Rev. 2020 90 102090 10.1016/j.ctrv.2020.102090 32892059

591. Burgess, B. T. et al. Olaparib combined with an ATR or Chk1 inhibitor as a treatment strategy for acquired olaparib-resistant BRCA1 mutant ovarian cells. Diagnostics 10 , 121 (2020). 10.3390/diagnostics10020121 PMC7168282 32098452

592. Awada A An open-label, dose-escalation study to evaluate the safety and pharmacokinetics of CEP-9722 (a PARP-1 and PARP-2 inhibitor) in combination with gemcitabine and cisplatin in patients with advanced solid tumors Anticancer Drugs 2016 27 342 348 10.1097/CAD.0000000000000336 26796987

593. Schram AM Chang MT Jonsson P Drilon A Fusions in solid tumours: diagnostic strategies, targeted therapy, and acquired resistance Nat. Rev. Clin. Oncol. 2017 14 735 748 10.1038/nrclinonc.2017.127 28857077 PMC10452928

594. Pottier, C. et al. Tyrosine kinase inhibitors in cancer: breakthrough and challenges of targeted therapy. Cancers 12 , 731 (2020). 10.3390/cancers12030731 PMC7140093 32244867

595. Gasch C Ffrench B O’Leary JJ Gallagher MF Catching moving targets: cancer stem cell hierarchies, therapy-resistance & considerations for clinical intervention Mol. Cancer 2017 16 43 10.1186/s12943-017-0601-3 28228161 PMC5322629

596. Najafi M Mortezaee K Majidpoor J Cancer stem cell (CSC) resistance drivers Life Sci. 2019 234 116781 10.1016/j.lfs.2019.116781 31430455

597. Erin N Grahovac J Brozovic A Efferth T Tumor microenvironment and epithelial mesenchymal transition as targets to overcome tumor multidrug resistance Drug Resist. Updat. 2020 53 100715 10.1016/j.drup.2020.100715 32679188

598. Bukowski K Kciuk M Kontek R Mechanisms of multidrug resistance in cancer chemotherapy Int. J. Mol. Sci. 2020 21 3233 10.3390/ijms21093233 32370233 PMC7247559

599. Mele L The role of autophagy in resistance to targeted therapies Cancer Treat. Rev. 2020 88 102043 10.1016/j.ctrv.2020.102043 32505806

600. Hussain S Cancer drug resistance: a fleet to conquer J. Cell Biochem. 2019 120 14213 14225 10.1002/jcb.28782 31037763

601. Boumahdi S de Sauvage FJ The great escape: tumour cell plasticity in resistance to targeted therapy Nat. Rev. Drug Discov. 2020 19 39 56 10.1038/s41573-019-0044-1 31601994

602. Shi H Wei J He C Where, when, and how: context-dependent functions of RNA methylation writers, readers, and erasers Mol. Cell 2019 74 640 650 10.1016/j.molcel.2019.04.025 31100245 PMC6527355

603. Meyer KD Jaffrey SR Rethinking m(6)A readers, writers, and erasers Annu Rev. Cell Dev. Biol. 2017 33 319 342 10.1146/annurev-cellbio-100616-060758 28759256 PMC5963928

604. Van Meter EN Onyango JA Teske KA A review of currently identified small molecule modulators of microRNA function Eur. J. Med. Chem. 2020 188 112008 10.1016/j.ejmech.2019.112008 31931338

605. Pylayeva-Gupta Y Grabocka E Bar-Sagi D RAS oncogenes: weaving a tumorigenic web Nat. Rev. Cancer 2011 11 761 774 10.1038/nrc3106 21993244 PMC3632399

606. Jancik S Drabek J Radzioch D Hajduch M Clinical relevance of KRAS in human cancers J. Biomed. Biotechnol. 2010 2010 150960 10.1155/2010/150960 20617134 PMC2896632

607. Cox AD Drugging the undruggable RAS: mission possible? Nat. Rev. Drug Discov. 2014 13 828 851 10.1038/nrd4389 25323927 PMC4355017

608. Cox AD Der CJ Philips MR Targeting RAS membrane association: back to the future for anti-RAS drug discovery? Clin. Cancer Res. 2015 21 1819 1827 10.1158/1078-0432.CCR-14-3214 25878363 PMC4400837

609. Papke B Der CJ Drugging RAS: know the enemy Science 2017 355 1158 1163 10.1126/science.aam7622 28302824

610. Chen H Small-molecule inhibitors directly targeting KRAS as anticancer therapeutics J. Med. Chem. 2020 63 14404 14424 10.1021/acs.jmedchem.0c01312 33225706

611. Nagasaka M KRAS G12C Game of Thrones, which direct KRAS inhibitor will claim the iron throne? Cancer Treat. Rev. 2020 84 101974 10.1016/j.ctrv.2020.101974 32014824 PMC7041424

612. Liu P Wang Y Li X Targeting the untargetable KRAS in cancer therapy Acta Pharm. Sin. B 2019 9 871 879 10.1016/j.apsb.2019.03.002 31649840 PMC6804475

613. Bayliss R Burgess SG Leen E Richards MW A moving target: structure and disorder in pursuit of Myc inhibitors Biochem. Soc. Trans. 2017 45 709 717 10.1042/BST20160328 28620032

614. Krzyzosiak A Target-based discovery of an inhibitor of the regulatory phosphatase PPP1R15B Cell 2018 174 1216 1228.e1219 10.1016/j.cell.2018.06.030 30057111 PMC6108835

615. Kieffer C Jourdan JP Jouanne M Voisin-Chiret AS Noncellular screening for the discovery of protein-protein interaction modulators Drug Discov. Today 2020 25 1592 1603 10.1016/j.drudis.2020.07.012 32682931

616. Mabonga L Kappo AP Protein-protein interaction modulators: advances, successes and remaining challenges Biophys. Rev. 2019 11 559 581 10.1007/s12551-019-00570-x 31301019 PMC6682198

617. Taylor MH Phase IB/II trial of lenvatinib plus pembrolizumab in patients with advanced renal cell carcinoma, endometrial cancer, and other selected advanced solid tumors J. Clin. Oncol. 2020 38 1154 1163 10.1200/JCO.19.01598 31961766 PMC7145588

618. Chau V Bilusic M Pembrolizumab in combination with axitinib as first-line treatment for patients with renal cell carcinoma (RCC): evidence to date Cancer Manag. Res. 2020 12 7321 7330 10.2147/CMAR.S216605 32884346 PMC7443011

619. Rini BI Pembrolizumab plus axitinib versus sunitinib for advanced renal-cell carcinoma N. Engl. J. Med. 2019 380 1116 1127 10.1056/NEJMoa1816714 30779529

620. Schapira L Simple rules can improve prognostic accuracy J. Clin. Oncol. 2011 29 347 349 10.1200/JCO.2010.32.9086 21189394

621. Beck A The next generation of antibody-drug conjugates comes of age Discov. Med. 2010 10 329 339 21034674

622. Thomas A Teicher BA Hassan R Antibody–drug conjugates for cancer therapy Lancet Oncol. 2016 17 e254 e262 10.1016/S1470-2045(16)30030-4 27299281 PMC6601617

623. Yaghoubi S Potential drugs used in the antibody-drug conjugate (ADC) architecture for cancer therapy J. Cell Physiol. 2020 235 31 64 10.1002/jcp.28967 31215038

624. Torre, B. G. & Albericio, F. An analysis of FDA drug approvals from the perspective of molecules. Molecules 26 , 627 (2021). 10.3390/molecules26030627 PMC7865374 33504104

625. An S Fu L Small-molecule PROTACs: an emerging and promising approach for the development of targeted therapy drugs EBioMedicine 2018 36 553 562 10.1016/j.ebiom.2018.09.005 30224312 PMC6197674

626. Wang Y Degradation of proteins by PROTACs and other strategies Acta Pharm. Sin. B 2020 10 207 238 10.1016/j.apsb.2019.08.001 32082969 PMC7016280

627. Lin X Xiang H Luo G Targeting estrogen receptor alpha for degradation with PROTACs: A promising approach to overcome endocrine resistance Eur. J. Med. Chem. 2020 206 112689 10.1016/j.ejmech.2020.112689 32829249

628. Huang A Garraway LA Ashworth A Weber B Synthetic lethality as an engine for cancer drug target discovery Nat. Rev. Drug Discov. 2020 19 23 38 10.1038/s41573-019-0046-z 31712683

629. Corcoran RB Synthetic lethal interaction of combined BCL-XL and MEK inhibition promotes tumor regressions in KRAS mutant cancer models Cancer Cell 2013 23 121 128 10.1016/j.ccr.2012.11.007 23245996 PMC3667614