Targeted and epigenetic therapies for acute myeloid leukemia treatment.

Introduction

Leukemia is a type of blood cancer originating in the bone marrow that produces abnormal, dysfunctional blood cells. Common symptoms include hemorrhage, ecchymosis, ostealgia, fatigue, fever, and cachexia; blood tests and a bone marrow biopsy can help diagnose the disease [ 1 ]. Leukemia is categorized as acute or chronic regarding the frequency of cell proliferation and as lymphoid or myeloid, depending on the origin of affected blood cells. In acute leukemia, blasts are typically 1–5% of bone marrow cells exceeding 20% of the total cell count, leading to a rapid onset of symptoms and disease progression [ 2 ]. Acute Myeloid Leukemia (AML) is caused by the aberrant proliferation and differentiation of myeloid cells in the bone marrow [ 3 ]. The malignancy is developed when the myeloid lineage is disrupted by cell development, leading to an accumulation of blasts or immature cells in the bone marrow. The clonal proliferation of myeloid progenitors (blasts) in peripheral blood and bone marrow is a hallmark of AML [ 4 ].

The primary treatment for AML is chemotherapy, which contains both induction and consolidation stages. While consolidation removes undetectable remaining disease to reach remission, induction lowers the number of leukemic cells. Another strategy is stem cell transplantation, which uses graft cells to elicit an immune response against leukemic cells [ 5 ]. The current studies focus on intracellular mechanisms and resistance to apoptotic signals in leukemia cells to target AML blasts [ 6 ].

Normal bone marrow cells can develop leukemic due to changes in their DNA, disrupting the usual regulation of cell growth and cell death. Thus, unregulated bone marrow cell division results in immature cells that evolve into leukemic white blood cells, known as myeloblasts. These cells may damage healthy cells and cause them not to function correctly [ 7 , 8 ].

Numerous genetic alterations are involved in the pathogenesis of AML, several of which represent potential novel therapeutic targets [ 9 ]. Among these, FLT3 mutations are some of the most frequently observed, occurring in approximately 30–35% of newly diagnosed AML cases. These mutations include internal tandem duplications (FLT3-ITD) and point mutations of the tyrosine kinase domain (FLT3-TKD), both of which are associated with poor prognosis and are targeted by inhibitors such as midostaurin and gilteritinib [ 10 , 11 ]. NPM1 mutations, found in approximately 30% of AML patients—particularly in cases with normal karyotypes—are associated with favorable prognosis in the absence of FLT3-ITD and serve as valuable markers for minimal residual disease (MRD) detection [ 12 ]. Mutations in isocitrate dehydrogenase (IDH) genes, including IDH1 and IDH2, present in 6–13% of cases, result in the production of 2-hydroxyglutarate, causing epigenetic dysregulation and impaired differentiation. Targeted therapies such as ivocidinib and enasitinib inhibit these mutations, allowing myeloid cells to mature properly by reducing 2-hydroxyglutarate levels [ 13 , 14 ]. TP53 mutations, though less prevalent, are associated with adverse outcomes and are being studied in the context of novel agents such as APR-246 and MDM2 inhibitors [ 15 ]. KIT mutations are particularly relevant in nuclear factor-binding leukemias and potentially affect response to therapy [ 16 ]. These molecular alterations provide important insights into the pathogenesis of AML and have facilitated the development of precision medicine strategies.

Gene therapy is a promising approach in the treatment of AML, especially for relapsed or refractory cases. While gene-based strategies—such as lentiviral transfer of T cells expressing leukemia-specific T-cell receptors (e.g., HA-1 via StrepTamer technology)—are under investigation following allogeneic stem cell transplantation, current clinical efforts are largely focused on exploiting targeted and epigenetic therapies [ 17 ]. The clinical success of agents such as FLT3 and IDH1/2 inhibitors, as well as hypomethylating agents (HMAs) (e.g., azacitidine and decitabine), highlights the therapeutic potential of targeting molecular abnormalities [ 13 , 14 , 18 ]. Despite these advances, major challenges remain, including the genetic and epigenetic heterogeneity of AML [ 19 ], the emergence of drug resistance [ 20 ], and the limited durability of response in advanced stages of the disease [ 21 ].

This article explores targeted and epigenetic therapies for AML. It reviews how the understanding of AML pathogenesis, driven by genetic and epigenetic alterations, has led to precision medicine strategies. The article discusses specific targeted agents like IDH, FLT3, BCL2, and Menin inhibitors that address molecular abnormalities in AML. It also covers epigenetic therapies targeting reversible modifications such as DNA methylation and histone changes, including DNMT inhibitors, HDAC inhibitors, and others. Finally, gene therapy is presented as a promising approach, highlighting advancements in gene editing and targeted strategies, and future directions like combination and personalized therapies.

Targeted therapy in AML

Cancer has become a crucial public health challenge, with over 52,900 individuals diagnosed and more than 27,000 people losing their lives to the disease daily [ 22 ]. Cancer treatment has changed dramatically over the last ten years [ 23 ]. While traditional chemotherapy remains the primary option for treating various cancers, targeted therapies have become an integral part of treatment plans for a wide range of cancers, such as breast, colorectal, lung, and pancreatic cancers, as well as lymphoma, leukemia, and multiple myeloma. Since 2000, the U.S. Food and Drug Administration (FDA) has approved 15 targeted therapies, compared to five traditional chemotherapy drugs [ 24 ]. These therapies have expanded the concept of personalized cancer treatment, as they can be effective for patients whose cancers have specific molecular targets but may not work without them [ 24 ]. Targeted therapies focus on directing drugs toward genes or proteins unique to cancer cells or the tissue environment promoting cancer growth. Their success depends on delivering the treatment directly to the cancer site while minimizing damage to normal tissues. Targeted therapy is often combined with chemotherapy and other treatments. It includes the development of drugs that block cancer cell proliferation, regulate the cell cycle, or trigger apoptosis or autophagy, as well as delivering toxic substances specifically to cancer cells to destroy them. Targeted therapy can involve monoclonal antibodies or oral small-molecule drugs [ 25 ].

Targeted therapy, which focuses on specific molecules, is essential for improving outcomes against cancer cell survival [ 26 ]. Most targeted therapies use monoclonal antibodies or small-molecule medications [ 27 ]. According to cytogenetic and molecular approaches, the identification of MRD through flow cytometry, next-generation sequencing (NGS), and the integration of gene mutation-targeted therapies, recent advancements in AML treatment have rapidly developed. Alongside precise clinical diagnosis and detailed risk assessment, new pharmacological therapies (addressing gene mutations) have demonstrated significant advances for AML patients [ 28 , 29 ]. Recent advancements in AML treatment have been driven by the approval of targeted therapies, including midostaurin, enasidenib, and gemtuzumab ozogamicin (GO), which have significantly improved patient outcomes. These therapies focus on specific mutations prevalent in AML, and have revolutionized treatment strategies.

IDH inhibitors

Mutations in IDH1 and IDH2, occur in approximately 15–20% of AML patients and result in the neomorphic production of the oncometabolite 2-hydroxyglutarate (2-HG), which impairs cellular differentiation through epigenetic dysregulation [ 30 , 31 ]. Under normal physiological conditions, the isoenzymes IDH1 and IDH2 help regulate cellular metabolism within distinct cellular compartments—IDH1 functions in the cytoplasm and peroxisomes, whereas IDH2 is catalytically active in the mitochondria [ 32 ] (Fig. 1 ).

The development of IDH inhibitors, such as ivosidenib (IDH1 inhibitor) and enasidenib (IDH2 inhibitor), represents a major advancement in targeted therapy for AML. These agents work by inhibiting mutant IDH enzymes, reducing 2-HG levels, and restoring normal differentiation of leukemic cells [ 13 , 14 ]. The exploration of another IDH inhibitor, Olutasidenib, for AML and Myelodysplastic syndromes (MDS). Treatment has emerged [ 33 ]. Additionally, Glasdegib has undergone clinical trials in combination with standard chemotherapy for AML or high-risk MDS patients [ 34 ].

Clinical trials have demonstrated that IDH inhibitors, particularly in patients with relapsed or refractory AML harboring IDH mutations, lead to durable remissions with a favorable safety profile [ 35 ].

FLT3 inhibitors

FLT3 inhibitors are tyrosine kinase inhibitors (TKIs) that can be classified into first- and second-generation inhibitors based on their ability to inhibit FLT3 and associated downstream signaling pathways [ 36 , 37 ]. First-generation inhibitors include midostaurin and sorafenib, which can bind to both the active and inactive conformations of the FLT3 receptor as they target the FLT3 gatekeeper domain or ATP-binding pocket. These inhibitors have demonstrated improved overall survival in AML with FLT3 mutations, in combination with chemotherapy [ 38 ]. Second-generation inhibitors bind to the inactive form of the receptor adjacent to the ATP-binding domain located in the hydrophobic region. These inhibitors include quizartinib and gilteritinib, which are considered to offer better selectivity and efficacy [ 39 ]. In addition, gilteritinib, as a monotherapy for relapsed/refractory AML with FLT3 mutations, was confirmed to significantly improve overall survival compared with standard chemotherapy [ 18 ].

Despite these advances, the emergence of resistance to FLT3 inhibitors, often due to secondary TKD mutations or activation of alternative signaling pathways, has prompted investigations into sequential or dual inhibition strategies. These innovations are redefining the current standard of care and expanding the treatment options for patients with FLT3-mutated AML [ 40 ]. Recent studies have identified novel agents aimed at overcoming resistance mechanisms [ 41 ]. For example, fortitinib has been shown to inhibit FLT3 and overcome secondary mutations that confer resistance to quizartinib and gilteritinib [ 42 ]. Furthermore, the development of FLT3-PROTAC degraders, such as A20, offers a promising approach by inducing degradation of FLT3-ITD proteins and thereby overcoming resistance [ 43 ]. Also, the use of FLT3 inhibitors as maintenance therapy after allogeneic hematopoietic stem cell transplantation (allo-HCT) has been associated with improved overall survival and relapse-free survival in patients with FLT3-mutated AML [ 44 ]. Figure 2 illustrates the proposed intrinsic and extrinsic pathways and mechanisms that contribute to resistance against FLT3 inhibitors.

BCL2 inhibitors

BCL2 inhibitors, a type of BH3 mimetic, target anti-apoptotic BCL2 family proteins to induce apoptosis in cancer cells. These inhibitors promote cell death by binding to anti-apoptotic proteins like BCL2, freeing pro-apoptotic proteins such as BAK and BAX1. Venetoclax (ABT-199) is a selective BCL2 inhibitor that was the first FDA-approved drug in this class [ 45 ] (Fig. 3 ). Unlike earlier inhibitors, venetoclax selectively binds to BCL2 with significantly lower avidity for BCL-XL, which helps mitigate dose-limiting thrombocytopenia [ 46 ].

Venetoclax has demonstrated potent antitumor activity, particularly in hematological malignancies like chronic lymphocytic leukemia (CLL) and AML. It is incorporated into treatment regimens for these diseases [ 47 ]. For example, venetoclax combined with azacitidine or decitabine has shown efficacy in previously untreated elderly patients with AML [ 48 ].

It has also been studied in combination with intensive chemotherapy like FLAG-IDA in newly diagnosed and relapsed/refractory AML. Combinations of venetoclax with obinutuzumab are used for previously untreated CLL. Venetoclax has also shown promise in combination therapies for multiple myeloma and non-Hodgkin lymphoma [ 47 ].

Despite its success, resistance to venetoclax can occur. Combination strategies are being explored to overcome resistance and enhance efficacy. In AML, combinations of venetoclax with FLT3 inhibitors like midostaurin or gilteritinib have shown synergistic effects, particularly in FLT3-mutated subtypes. Additionally, concurrent targeting of other anti-apoptotic proteins like MCL-1 or BCL-XL, often alongside venetoclax, is being investigated in preclinical models and clinical trials to overcome resistance mechanisms [ 47 ]. Deficiency in BAX can lead to reduced sensitivity to combined BCL2 and MCL-1 inhibition [ 49 ]. Combinations of venetoclax with PARP inhibitors also show promise, especially in AML with KDM6A deficiency [ 48 ].

Menin inhibitors

Menin inhibitors represent a targeted therapeutic approach in AML, particularly for subtypes driven by KMT2A rearrangements (KMT2Ar) or NPM1 mutations (NPM1mt) [ 50 ]. Menin is a nuclear scaffold protein encoded by the MEN1 gene, known for its paradoxical oncogenic role in these specific leukemias despite being a tumor suppressor in other contexts [ 51 , 52 ]. In KMT2Ar leukemia, menin is essential for the oncogenic activity of KMT2A fusion proteins, mediating the aberrant expression of critical leukemogenic genes such as HOXA9 and MEIS1 [ 53 , 54 ] Similarly, menin contributes to maintaining the leukemic state in NPM1mt AML, which shares transcriptional similarities with KMT2Ar leukemia, including elevated HOXA and MEIS1 expression [ 55 ].

Menin inhibitors are small molecules designed to block the interaction between menin and KMT2A fusion proteins by binding to the KMT2A binding pocket on menin. This disruption prevents the activation of the aberrant transcriptional program responsible for leukemia cell proliferation and survival [ 56 ].

Preclinical studies in cell lines and patient-derived xenograft (PDX) models of both KMT2Ar and NPM1mt AML have demonstrated that menin inhibition leads to differentiation and reduced viability of leukemia cells [ 57 ]. Several menin inhibitors are undergoing clinical evaluation [ 50 ]. For example, revumenib (SNDX-5613) and ziftomenib have shown promising clinical activity in relapsed/refractory KMT2Ar and NPM1mt acute leukemias [ 57 , 58 ]. However, acquired resistance to menin inhibitors can emerge, often involving mutations in the MEN1 gene itself, such as M327I and T349M, which can impair the inhibitor’s binding [ 59 ].

Strategies combining menin inhibitors with other agents, including BCL2 inhibitors (like venetoclax), are being explored in preclinical studies and clinical trials to overcome resistance and enhance efficacy [ 60 , 61 ].

Epigenetics in AML

Epigenetics primarily refers to alterations in chromatin structure, including DNA methylation, histone modifications, and changes in higher-order chromatin structures that ultimately influence gene expression [ 62 ]. These epigenetic regulators are broadly categorized as “writers” (e.g., DNA and histone methyltransferases), “erasers” (e.g., histone deacetylases), or “readers” (e.g., bromodomain-containing proteins) [ 63 ]. The substantial involvement of epigenetics in cancer progression has been extensively documented over several decades, spanning various solid tumors and hematologic malignancies [ 64 ]. Around 70% of recurring mutations in AML patients target diverse gene expression regulators, including epigenetic proteins, transcription factors (TFs), and splicing machinery components [ 65 , 66 ]. This high prevalence of mutations, combined with the relatively low mutational burden in AML cases, strongly indicates the profound involvement of epigenome dysregulation in the development and progression of this specific form of cancer [ 19 , 67 ]. According to the pivotal role of epigenetic proteins in managing the balance of cellular self-renewal and differentiation, it is unsurprising that a significant portion of mutations detected in AML genomes occur within these epigenetic regulatory elements. This finding is of tremendous interest to researchers, healthcare providers, and the pharmaceutical industry in studying, characterizing, and testing small molecules that use the adaptable nature of the epigenome to disrupt malignant transcriptional pathways [ 68 ].

In AML, specific mutations impacting epigenetic mechanisms, such as histone modification (e.g., Enhancer of Zeste Homologue 2 - EZH2, and ASXL1), DNA methylation regulation (e.g., DNMT3A, TET2), and enzymes catalyzing metabolism (e.g., IDH1/2), have emerged as crucial contributors to disease pathogenesis, with prognostic implications [ 19 , 67 ]. DNA methylation and hydroxymethylation pathways have been directly related to malignant transformation by inactivating tumor suppressor genes [ 63 , 69 ]. Mutations in genes involved in DNA methylation (e.g., DNMT3A) and demethylation (e.g., TET2) are detected in approximately 22% and 23% of AML patients, respectively [ 70 , 71 ].

In addition to DNA methylation, the dynamic process of histone acetylation, regulated by histone lysine acetyltransferases and histone deacetylases (HDACs), significantly affects gene transcription by modifying chromatin accessibility [ 72 ]. Mutations in other genes affecting histone-modifying enzymes, such as EZH2 and ASXL1, are identified in up to 30% of AML patients [ 73 , 74 ].

In addition to DNA methylation and histone modifications, RNA modifications have recently emerged as an additional layer of epigenetic regulation involved in the pathogenesis of AML [ 75 ]. Among these modifications, N6-methyladenosine (m6A) is the most common internal modification in eukaryotic mRNA and plays a crucial role in RNA metabolism, including splicing, stability, nuclear export, and translation [ 76 – 78 ]. These modifications are regulated by a dynamic interplay of m6A “writers” (e.g., METTL3, METTL14), “erasers” (e.g., FTO, ALKBH5), and “readers” (e.g., YTHDF1–3) [ 79 – 81 ]. Dysregulation of m6A pathways has been shown to affect leukemogenesis by modulating the expression and function of key oncogenes and tumor suppressors. For example, overexpression of METTL3 increases the translation of transcripts such as MYC and BCL2, contributing to AML cell proliferation and impaired differentiation. Conversely, inhibition of FTO, an m6A demethylase, suppresses AML cell growth and increases sensitivity to chemotherapeutic agents [ 79 , 80 ].

Epigenetic therapies for AML treatment

Epigenetic therapy in AML targets reversible epigenetic modifications such as DNA methylation and histone changes. DNMT inhibitors like 5-azacytidine (5-AZA) and decitabine have been used for over a decade to treat AML patients who are not candidates for intensive chemotherapy, as well as those with MDS [ 82 , 83 ]. Histone deacetylase (HDAC) inhibitors enhance histone acetylation, promoting gene expression in cell differentiation, cell cycle regulation, and apoptosis [ 84 ].

HDAC and BET inhibitors are currently in trials, showing promise for targeting AML-specific epigenetic alterations. Recent studies have also explored combining DNMT and HDAC inhibitors, demonstrating potential synergistic effects [ 74 ]. While monotherapy with DNMT and HDAC inhibitors has shown limited efficacy, combination therapies targeting multiple levels of AML pathogenesis, including combinations with conventional chemotherapy, immunotherapy, or other epigenetic therapies, are showing promising early results [ 85 ]. Despite these advancements, challenges remain, including off-target effects and the need for personalized therapies. Future research will focus on refining these treatments and identifying biomarkers to optimize patient responses [ 86 ].

LSD1 inhibitors, such as tranylcypromine and ORY-1001, inhibit the demethylation of histone H3K4 and H3K9, promoting myeloid differentiation and apoptosis, particularly in AML with MLL rearrangements. Clinical trials have shown promising results, with phase 1 studies reporting a 36% response rate in MLL-translocated AML when combined with all-trans-retinoic acid (ATRA), though challenges like toxicity and limited single-agent efficacy persist [ 87 ].

IDH inhibitors, such as ivosidenib and enasidenib, target mutant IDH1 or IDH2 enzymes, reducing oncometabolite 2-hydroxyglutarate levels to restore normal DNA demethylation. These agents are approved for IDH-mutant AML, achieving approximately 30% response rates in relapsed/refractory cases, and are under investigation for MDS, with ongoing trials exploring combinations to enhance efficacy [ 14 ].

DOT1L inhibitors, like pinometostat, target H3K79 methylation to disrupt transcription elongation in MLL-rearranged AML, a subtype with poor prognosis. Preclinical studies demonstrate robust anti-leukemic effects, but phase 1 trials indicate limited single-agent activity, prompting research into combinations with HMAs or chemotherapy to overcome resistance [ 88 ]. Table 1 summarizes the key epigenetic therapies for AML, highlighting their mechanisms of action and potential therapeutic benefits.

Overview of AML treatments

The bone marrow microenvironment emerges as a crucial player in AML pathogenesis and progression. Studies show that the interactions between AML cells and surrounding stromal cells, extracellular matrix, and immune cells contribute to the survival and drug resistance of AML cells. As a result, strategies aimed at disrupting these interactions may offer a viable approach to enhance the effectiveness of AML therapies [ 89 ].

Historically, the lack of suitable preclinical models has impeded the development of new drugs for AML precisely due to the challenge of replicating the disease’s complexity and heterogeneity. However, recent advances in PDX models, 3D organoid cultures, and genome editing technologies have mostly overcome these limitations. These evolving models offer a more physiologically relevant platform for drug discovery and development, potentially leading to more effective and targeted therapies for AML patients [ 90 ].

In recent years, advances in understanding the genomic and molecular aspects of AML have significantly broadened treatment options [ 91 ]. Among the auspicious therapeutic interventions, the combination of venetoclax, a selective BCL-2 inhibitor, with HMAs has exhibited efficacy in older individuals diagnosed with AML who are not candidates for intensive chemotherapy, thereby enhancing overall survival in contrast to the use of HMA alone [ 92 , 93 ]. Advancements in genomic sequencing have identified new genetic mutations and molecular pathways involved in AML pathogenesis. These findings contribute to the development of more personalized and targeted therapies, ultimately improving patient outcomes [ 94 ].

Agents targeting oncogenic mutations—such as FLT3 and IDH inhibitors—are among the AML-targeted therapies. FLT3 is a cytokine receptor primarily expressed in hematopoietic tissues, while IDH catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate [ 95 ]. Recent developments, such as FLT3 inhibitors (e.g., midostaurin and gilteritinib), have notably improved survival in FLT3-mutated patients, particularly when combined with chemotherapy. IDH inhibitors, including enasidenib and ivosidenib, have shown promising results in relapsed or refractory AML. Additionally, combining HMAs with venetoclax has significantly improved outcomes in older, less fit patients, a group traditionally associated with poor prognoses [ 96 ].

In combination with chemotherapy, Midostaurin has improved survival rates in FLT3-mutated patients, while enasidenib has shown efficacy in patients with IDH2 mutations, promoting differentiation of leukemic blasts and increasing remission rates [ 95 ].

Small molecular inhibitors, enasidenib and ivosidenib, show potential effects in treating AML patients, harboring IDH2 and IDH1 mutations, respectively, which are present in about 20% of AML cases. Clinical trials indicate that both inhibitors can induce complete remission and enhance overall survival in AML patients with IDH2 and IDH1 mutations [ 14 , 97 ]. Gilteritinib, a selective FLT3 inhibitor, targeting the mutated receptor tyrosine kinase in around 30% of AML patients and displayed promising results in clinical trials, leading to complete remission and improved overall survival in AML patients with FLT3 mutations [ 18 , 98 ]. Furthermore, clinical trials suggest that combining azacitidine with venetoclax and/or gilteritinib can benefit patients with newly diagnosed or relapsed/refractory AML [ 99 ].

The FDA approved Midostaurin, for AML patients harbouring FLT3 mutations, as the first approval of a tyrosine kinase inhibitor (TKI) for AML and the inaugural drug for a mutation-specific non-acute promyelocytic leukemia (APL) subtype [ 38 ]. Subsequently, many gene mutation-targeted therapies for AML emerged, including Enasidenib, an inhibitor of IDH2, designed for relapsed/refractory (R/R) AML with IDH2 mutations [ 14 , 97 ].

Advancements in understanding FLT3 gene mutations have caused the development of TKI agents targeting various points within the ATP binding site of the FLT3 RTK. Sunitinib, Lestaurtinib, Midostaurin, Crenolanib, and Gilteritinib are type 1 inhibitors [ 100 ] that bind to the RTK ATP-binding site in active and inactive conformations. In contrast, Type 2 inhibitors, including Sorafenib, Quizartinib, and Ponatinib, bind to the hydrophobic region adjacent to the ATP-binding domain, inhibiting receptor activation while the RTK is inactive [ 101 ].

Ivosidenib, an IDH1 inhibitor, has received FDA approval for safety and efficacy in patients with IDH1-mutated R/R AML [ 14 ]. Enasidenib, a pioneer mutant IDH2 inhibitor, has also demonstrated promising safety and efficacy [ 97 ], exploring another IDH inhibitor, Olutasidenib, for AML and MDS [ 33 ].

In AML gene therapy, a compelling opportunity lies in overcoming drug resistance and enhancing long-term patient outcomes [ 102 ]. Targeting specific genetic mutations or molecular pathways implicated in AML pathogenesis through gene therapies may yield more enduring responses than traditional chemotherapy or targeted approaches [ 103 ]. Moreover, there is a growing prospect of deploying gene therapies at earlier stages in the course of AML, including in patients with measurable residual disease (MRD) or high-risk precancerous conditions. Consequently, early implementation of gene therapy shows the potential to halt disease advancement and increase overall survival rates [ 104 ].

The complexity of AML presents challenges in identifying patients who will respond to treatment. However, advancements in genomic sequencing and gene expression analysis have helped classify AML into distinct genetic and molecular subtypes, paving the way for more precise and personalized gene therapies [ 67 ]. Ensuring safety is a critical aspect of gene therapy, as certain strategies may lead to off-target effects or unintended immune responses. To minimize these risks, researchers utilize precise gene-editing technologies such as CRISPR/Cas9 and implement monitoring techniques to assess the viability and biodistribution of gene-modified cells [ 105 , 106 ].

The foundational concept of gene therapy with hematopoietic stem cells lies in transferring functional genes to the patient’s stem cells to correct genetic defects or enhance immune function [ 107 ]. Current research in gene therapy for hematopoietic stem cells targets frequently mutated genes in AML, such as IDH1/2, DNMT3A, and TET2, evaluating safety and efficacy in early-phase clinical trials [ 108 , 109 ]. CRISPR/Cas9 gene editing application in AML is also undergoing assessment for safety and effectiveness in multiple early-phase clinical trials [ 110 , 111 ]. In one such trial, a CRISPR/Cas9 gene editing strategy targeted a common AML mutation, FLT3-ITD, demonstrating effective mutation abrogation and impaired AML cell growth in vitro and in vivo [ 112 , 113 ].

Gene therapy for AML

Recent advancements in gene therapy for AML involve several strategies, often leveraging gene editing technologies and exploring new targets or delivery methods [ 114 , 115 ]. One significant approach aims to protect healthy hematopoietic cells from targeted therapies by genetically modifying them. For instance, researchers are developing methods to edit HSPCs to remove or modify the expression of the antigen targeted by immunotherapy [ 116 ].

This strategy allows for the administration of targeted therapies post-transplant at optimal doses without causing severe toxicity to the newly engrafted hematopoietic system, thereby potentially increasing the therapeutic index. A prime example of this is the development of tremtelectogene empogeditemcel (trem-cel), formerly known as VOR33. Trem-cel is an allogeneic hematopoietic stem and progenitor cell transplant product generated from healthy donor hematopoietic stem and progenitor cells (HSPCs) where the myeloid protein CD33 has been genetically ablated using CRISPR/Cas9 technology [ 117 ].

CD33 is a frequently targeted antigen in AML, expressed on over 80% of AML blasts, and existing therapies like GO target it. However, GO’s use is limited by on-target, off-tumor hematopoietic toxicity [ 118 ]. Trem-cel is designed to be resistant to CD33-targeted drug cytotoxicity, allowing for subsequent CD33-directed maintenance therapy post-transplant with minimal toxicity to the transplanted cells. Preclinical studies supported the concept with efficient CD33 ablation in HSPCs without impacting myeloid cell differentiation or function, and edited cells showed resistance to GO14. These promising preclinical results supported the initiation of the first-in-human, multicenter clinical trial evaluating the safety and efficacy of trem-cel in patients with AML ( NCT04849910 ) [ 117 ]. Initial results from this trial show rapid neutrophil engraftment, high CD33 editing efficiency in myeloid cells, and minimal effect on counts with GO administration, suggesting the edited donor cells are protected [ 118 ].

Another antigen targeted in AML is CD123, which is overexpressed on AML blasts and leukemic stem cells [ 119 ]. Similar to CD33, CD123 is also present on healthy HSPCs [ 120 ]. To mitigate the on-target toxicity to healthy cells, researchers are exploring epitope editing of CD123 on HSPCs. This approach uses base editors (BEs) and prime editors (PEs) to precisely modify the CD123 epitope on HSPCs, aiming to prevent CAR-T cell recognition and killing while preserving the normal function of the HSPCs. Optimized prime editing techniques have shown high efficiency in introducing these modifications in HSPCs in preclinical studies. Infused edited HSPCs were resistant to CD123-CAR-T lysis in humanized mouse models, demonstrating a proof-of-concept for shielding healthy cells and potentially enabling CD123-targeted therapy for relapsed AML [ 116 ].

Beyond protecting healthy cells, gene therapy approaches also focus on improving the efficacy and safety of the immune effector cells themselves, such as CAR-T cells. Gene editing, notably with CRISPR/Cas9 technology, is used to modify CAR-T cells to address limitations like T cell exhaustion or antigen escape. Strategies include optimizing costimulatory domains or suppressing inhibitory signals within the CAR construct or the T cell [ 121 – 124 ].

Furthermore, targeting alternative antigens with different expression profiles is being investigated [ 115 ]. CD7 is expressed on AML cells and T cells but is underexpressed or absent on myeloid cells, potentially posing less risk of severe myelosuppression compared to targeting CD33 or CD123. A strategy involving “naturally selected” CD7 CAR-T cells is being explored, where CD7 expression on the CAR-T cells themselves is naturally lost during the manufacturing process, potentially avoiding the need for complex gene editing to prevent fratricide [ 125 ]. Preclinical studies have demonstrated the powerful cytotoxic effects of naturally selected CD7 CAR-T cells against CD7 + AML cells, and clinical trials are underway to assess their performance in R/R AML patients. A phase I clinical study ( NCT04938115 ) is investigating naturally selected CD7 CAR-T therapy in patients with CD7-positive R/R AML [ 126 ].

Another target being explored is CD19, which is aberrantly expressed in certain AML subtypes, such as relapsed t(8;21) AML. While typically associated with B-cell malignancies, CD19-directed CAR T-cell therapy has shown high safety and efficacy, achieving high response rates, in a clinical trial for CD19-positive relapsed t(8;21) AML patients ( NCT03896854 ) [ 127 ].

For patients who experience relapse after allogeneic hematopoietic stem cell transplantation (alloHCT), donor-derived CAR-T cells are being developed. One such approach involves VCAR33, a CD33-directed CAR T-cell product manufactured from the lymphocytes of the patient’s original allogeneic stem cell donor. This strategy aims to address challenges related to T-cell dysfunction and manufacturing difficulties in AML patients by utilizing healthy donor T cells. A Phase 1/2 clinical trial is currently evaluating the safety and efficacy of VCAR33 in patients who have relapsed following alloHCT [ 128 ].

Beyond CAR-T, T cell receptor (TCR)-engineered T cell therapy is also being developed for AML, targeting antigens presented by HLA molecules. For example, Wilms tumor 1 (WT1) is a target for TCR therapy. Gene editing using CRISPR/Cas9 is being applied to develop WT1-specific TCR-engineered T cells from cord blood, specifically editing out the endogenous TCR chains to improve the expression and specificity of the introduced therapeutic TCR and avoid mispairing. Preclinical studies indicate this approach can generate potent T cells targeting AML blasts without harming healthy cells [ 129 ].

Furthermore, combination strategies are being investigated to enhance the efficacy of CAR-T therapy in AML. Combining CAR-T cells with other agents, such as immune checkpoint inhibitors (ICIs) or TKIs, or epigenetic modulators like demethylating drugs, is being explored to potentially improve anti-tumor effects, overcome resistance mechanisms, or manage toxicities. Demethylating drugs, for instance, have been shown to increase antigen density in preclinical trials, which could potentially improve CAR therapy efficacy [ 115 ].

Future perspective

Enhancing survival rates among AML patients while mitigating treatment-induced toxicity stands as a primary concern for medical practitioners [ 130 ]. Amidst various challenges, gene therapy emerges as a potential future avenue for AML treatment. The therapeutic transgenes’ repertoire in targeting malignant cells spans genes encoding cytotoxic, pro-apoptotic, antiproliferative proteins alongside conventional suicide genes like the herpes simplex virus thymidine kinase gene. Alternatively, augmenting the immune system’s ability to eradicate cancerous cells could be facilitated by delivering genes encoding costimulatory molecules, such as IL-2, IL-7, and IL-12 [ 131 – 133 ], or immunomodulatory molecules like CD40, CD80 [ 134 , 135 ], and interferon β [ 136 ]. The use of RNA molecules in cancer treatment is also a therapeutic approach for this disease. microRNAs (miRNAs), a key subgroup of non-coding RNAs (ncRNAs), have attracted significant scientific interest. Emerging evidence suggests that these molecules hold great potential as promising biomarkers for cancer diagnosis [ 137 ]. Recently, researchers have shown that the interaction of Loc646329 as a sponge for miR-21 can target the RAS/MAPK pathway, resulting in the induction of apoptosis and cell cycle arrest [ 138 ].

Improved delivery methods and targeting strategies

The efficient delivery of vectors to specific cells or tissues remains a significant challenge in gene therapy and is pivotal for successful gene transfer. Viral vectors dominate therapeutic gene delivery despite advancements in non-viral techniques, notably for hematopoietic cells, encompassing electroporation, nucleofection, and particle bombardment [ 139 ]. Preliminary in vitro experiments suggest promising prospects for lentiviral, retroviral, or adenoviral vectors as delivery vehicles for leukemic cells [ 140 , 141 ]. However, concerns regarding unintended genomic integration and adverse immune responses with specific viral vectors impede their clinical applicability. In contrast, due to their low genomic integration frequency and minimal immune response, adeno-associated virus (AAV) vectors have emerged as efficacious tools for long-term gene expression across diverse cell types [ 142 – 144 ].

Strategies aimed at enhancing therapeutic vector binding to target cells encompass the use of bispecific conjugates and modifications of AAV for improved targeting. Nevertheless, ensuring the universal efficacy of these modifications across diverse vector capsids presents challenges. Addressing this challenge, researchers have developed random peptide-display libraries based on gene therapy vector capsids, specifically for AAV and retroviruses [ 145 , 146 ]. These innovative libraries enable the selection of peptide ligands that efficiently transduce specific cell types within the viral capsid context, facilitating the isolation of tailored vectors designed for targeted cell transduction [ 147 , 148 ].

Combination therapies with existing treatments

In the last decades, the vulnerability of malignant cells to concurrent disruption of multiple cooperative survival signaling pathways has gained attention [ 149 , 150 ]. Combining targeted agents to counteract the redundancy of survival pathways in neoplastic cells has gained traction across various tumor types [ 151 ]. Also, an increasingly popular combination method has been used with DNA methyltransferase inhibitors (DNMTIs) coupled with histone deacetylase inhibitors (HDACIs) [ 152 ]. This tandem synergistically triggers apoptosis and enhances the expression of microRNAs, subsequently down-regulating oncogenes [ 153 ].

In AML, DNMTIs have been paired with diverse targeted agents. For instance, a phase I trial combining the proteasome inhibitor bortezomib with decitabine showcased promising results by up-regulating miR-29b, leading to the inhibition of genes involved in myeloid leukemogenesis, resulting in notable remission rates [ 154 ].

Recent advancements in cancer epigenetics have identified potent and specific small-molecule BET (bromodomain and extra terminal) inhibitors [ 62 ]. The BRD4 antagonist JQ1, when combined with the HDACI panobinostat, demonstrated synergistic apoptosis induction in AML cells and enhanced survival rates in mouse xenograft models [ 155 ].

Development of personalized gene therapy

Advancements in genomic technologies, including global gene-expression analysis [ 156 ], NGS [ 157 , 158 ], and single nucleotide polymorphism (SNP) array analysis, have significantly facilitated the discovery of genetic alterations in AML [ 159 ]. These tools have enabled precise identification of somatic mutations, gene fusions, and chromosomal abnormalities driving leukemogenesis, forming the basis for molecularly targeted therapies. Recent years have witnessed a growing integration of genetic diagnostics into clinical practice. The current World Health Organization (WHO) classification system relies on underlying genetic defects, categorizing over half of AML cases and delineating distinct clinicopathologic entities [ 160 ]. Notably, cytogenetic and molecular genetic changes serve as robust prognostic markers. Furthermore, ongoing efforts focus on developing novel therapies targeting specific genetic and epigenetic alterations, exemplified by the use of TKIs and demethylating agents [ 161 ].

Experts increasingly envision that the integration of germline and somatic genomic data, along with epigenetic and transcriptional profiling, will form the molecular foundation for future treatment decision-making in AML. This integrated approach promises to offer the highest predictive potential for identifying actionable vulnerabilities and tailoring patient-specific therapeutic strategies [ 162 ]. Recent studies using CRISPR/Cas9 genomic screens have identified key AML survival genes, including epigenetic regulators such as KAT7 and YTHDC1. This research paves the way for the development of personalized therapies that target specific affinities of leukemia cells [ 163 ]. Meanwhile, the combination of CRISPR-based editing and CAR T-cell engineering has shown improved efficacy against resistant AML clones in preclinical models [ 164 ].

Single-cell RNA sequencing (scRNA-seq) has become a powerful tool to map intratumoral heterogeneity in AML and uncover rare subpopulations responsible for therapy resistance or relapse. A 2025 study identified novel cell states and surface markers that could serve as the basis for future gene therapy targets or drug delivery systems, pushing the boundaries of precision oncology [ 165 ]. Efforts are also ongoing to increase the safety of gene-editing therapies. A 2023 clinical trial study described a strategy involving engineered hematopoietic stem cells with deleted antigen targets (e.g., CD33) to avoid off-target toxicity of CAR T-cell therapies while preserving hematopoiesis. This approach allows for selective targeting of leukemia cells with minimal damage to normal tissues [ 166 ].

Conclusion

Acute Myeloid Leukemia (AML) remains a significant challenge in hematological oncology, characterized by its complex biology, genetic and epigenetic heterogeneity, and the frequent emergence of therapeutic resistance. While traditional chemotherapy and stem cell transplantation have long been the cornerstones of AML treatment, a deeper understanding of the molecular underpinnings of the disease has fundamentally shifted the therapeutic paradigm towards precision medicine.

This article has highlighted the critical role of targeted therapies that exploit specific molecular vulnerabilities in AML cells. Agents targeting frequently mutated genes such as FLT3 (e.g., midostaurin, gilteritinib) and IDH1/2 (e.g., ivosidenib, enasidenib) have demonstrated improved outcomes, particularly in patients harboring these alterations. Furthermore, therapies like BCL2 inhibitors (e.g., venetoclax) that restore apoptosis, and novel agents such as Menin inhibitors for specific subtypes like KMT2Ar or NPM1-mutant AML, represent targeted strategies that directly address critical survival pathways in leukemia cells.

Alongside targeted agents, epigenetic therapies play a crucial role by modulating reversible modifications that are dysregulated in AML. DNMT inhibitors (e.g., azacitidine, decitabine) have been established for their ability to restore proper DNA methylation patterns. HDAC inhibitors and other emerging agents targeting epigenetic regulators like LSD1 and DOT1L are also being explored to disrupt aberrant gene expression programs that maintain the leukemic state.

A particularly promising frontier lies in gene therapy, which offers potential strategies to overcome inherent challenges like drug resistance and treatment toxicity. Innovative approaches include the genetic modification of healthy hematopoietic stem cells (HSPCs) using tools like CRISPR/Cas9 to render them resistant to targeted immunotherapies (e.g., against CD33 or CD123), thereby protecting normal hematopoiesis while allowing for aggressive targeting of leukemia cells. Simultaneously, gene therapy is being leveraged to enhance the anti-leukemic activity of immune cells, such as through the development of improved CAR-T and TCR-engineered therapies targeting various antigens expressed on AML blasts.

Despite these significant advancements, challenges such as the emergence of resistance mechanisms and the inherent heterogeneity of AML necessitate continued innovation. The future of AML treatment will increasingly rely on synergistic combination therapies designed to simultaneously block multiple survival pathways and circumvent resistance. Critically, ongoing progress in genomic sequencing, single-cell analysis, and other molecular profiling technologies is paving the way for truly personalized treatment approaches, where therapeutic decisions are guided by the unique molecular landscape of each patient’s disease. By integrating these sophisticated molecular insights with the growing armamentarium of targeted, epigenetic, and gene therapies, the medical community aims to improve response durability, reduce treatment-related toxicity, and ultimately enhance the long-term survival and quality of life for patients with AML.

Acknowledgements

This study was supported by Amino Techno Gene Private Laboratory, Tehran, Iran.

Author contributions

M.S. conceived the original idea for this work, led the manuscript writing, and drafted the majority of the main content. M.G. wrote the clinical trial section, contributed to the targeted therapy section, and assisted with proofreading and language editing. M.M. drafted the introduction section. H.A. assisted M.G. with proofreading and language review. S.M. and A.F. coordinated the team, supported the corresponding author with organizational tasks, and helped M.S. verify the accuracy of the references. All authors reviewed and approved the final version of the manuscript.

Funding

No funding was received for this study.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Marzieh Shokoohi, Email: shokoohimarzieh@gmail.com.

Mobin Ghaderi, Email: moobinghaderi62@gmail.com.

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Associated Data

Data Availability Statement

No datasets were generated or analysed during the current study.