The epigenetic revolution in hematology: from benchside breakthroughs to clinical transformations.

Introduction

The field of hematological malignancies has undergone a major shift in recent years, as studies have provided insight into the complex relationship between genetic mutations and epigenetic changes that influence the development and progression of these diseases [ 1 ]. Cancer research has traditionally focused on genetic changes, but it is now recognized that epigenetic changes, which affect gene expression without changing the underlying DNA sequence, play a key role in tumorigenesis [ 2 ]. These changes include chromatin remodeling, histone alteration and DNA methylation, all of which have major implications for the future and function of hematopoietic cells [ 3 ]. In addition to being essential for healthy hematopoietic development, epigenetic processes also play a role in abnormal gene expression patterns observed in hematological malignancies [ 4 ]. Dysregulation of epigenetic modulators-proteins that produce, maintain, or remove epigenetic markers-is involved in a variety of hematological disorders [ 5 ]. For example, leukemia cells often contain mutations in the genes that produce histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), which suppress tumor suppressor genes and activate oncogenes [ 6 ]. Because of the dynamic nature of this epigenetic landscape, it is possible to perform therapeutic interventions to reverse these changes [ 7 ]. Targeted epigenetic therapy has emerged as a recent development; it modifies the epigenetic framework to restore normal patterns of gene expression [ 8 ]. DNA methyltransferase inhibitors such as azacitidine and decitabine have emerged as key agents in AML and myelodysplastic syndromes (MDS), especially in patients who are not candidates for more intensive therapies such as bone marrow transplantation (BMT) [ 9 , 10 ]. Similarly, HDAC inhibitors have shown promise in altering gene expression and inducing apoptosis in cancer cells [ 9 , 11 ]. Other innovative therapies, including Enhancer of zeste homologue 2 (EZH2) inhibitors and Bromodomain and extra-terminal proteins (BET) inhibitors, are also being studied for their potential to target specific epigenetic alterations associated with hematological malignancies [ 12 ]. Even with this development, a number of challenges still need to be overcome before epigenetic therapy can be used in clinical practice. Challenges such as drug resistance, side effects and the need for individualized treatment approaches complicate the therapeutic landscape [ 13 , 14 ]. For example, although some patients respond well to azanucleosides, their effectiveness in hypo-proliferative leukemias is limited and long treatment durations may lead to serious side effects and costly costs. In addition, the variety of hematological malignancies, characterized by a range of genetic and epigenetic abnormalities, is an important obstacle to achieving reliable results from the treatment [ 5 ]. The objective of this review is to provide a comprehensive overview of the role of epigenetics in hematological malignancies, detailing the mechanisms underlying the epigenetic changes, the current state of epigenetic therapy and the challenges to be addressed in order to improve patient outcomes. By bridging the gap between the discoveries of the bench and the clinical applications, we hope to reveal the transformative potential of epigenetics in hematology.

Beyond the genome: epigenetic echoes in hematologic malignancies

DNA methylation: key players in hematological malignancies

DNA methylation changes are one of the most important epigenetic mechanisms that play a key role in regulating gene expression and regulating cellular activities. These changes are involved in various processes such as cell differentiation, proliferation and cell death. DNA methylation is actually a process in which methyl groups (-CH3) are added to cytosine bases in specific regions of DNA, and this process can be affected by various proteins [ 15 ]. These proteins are generally divided into three main categories: writers, readers and cleaners (Fig. 1 ).

Writer proteins are responsible for adding methyl groups to DNA. The most important of them are DNMTs. These enzymes cause changes in DNA structure and regulate gene activity by adding methyl groups to cytosines in DNA. These enzymes include DNMT1, DNMT3A, and DNMT3B. In hematological malignancies, increased activity of writer proteins can lead to increased methylation in the promoter regions of tumor suppressor genes, leading to their silencing [ 16 ]. For example, in acute myeloid leukemia (AML), increased methylation of tumor suppressor genes such as CDKN2A (which plays an important role in cell cycle regulation) has been observed. These changes disrupt the control of cell growth and cancer progression [ 17 ].

Reader proteins are responsible for recognizing and interpreting epigenetic changes in DNA. These proteins bind to methylated regions in DNA and thus regulate specific cellular processes such as cell differentiation and proliferation. One important group of reader proteins is the Methyl-CpG Binding Domain (MBD) family, which includes proteins such as MBD1, MBD2, and MECP2 [ 18 ]. In chronic myeloid leukemia (CML), changes in reader proteins can disrupt the activity of genes involved in cell cycle regulation and the immune system. For example, the protein MECP2, which binds to methylated regions of DNA, has been identified in some cases as a key factor in regulating the expression of genes associated with immune cells and tumor growth. In CML, changes in the reading of these regions can lead to impaired immune responses and disease progression [ 19 ].

Eraser proteins are responsible for removing methyl groups from DNA, causing reversible changes in methylation patterns. The most important of these are the proteins of the TET (Ten-Eleven Translocation) family, which are responsible for the conversion of methylated cytosine to hydroxymethylcytosine. In some hematological cancers, alterations in the function of scavenger proteins can lead to disease [ 20 ]. For example, in AML, a decrease in the activity of the TET2 protein, which is responsible for the clearance of DNA methylation, is observed. This change increases the stability of methylation changes in specific genes, which ultimately leads to impaired gene regulation and the development of cancer pathologies [ 21 , 22 ]. In non-Hodgkin lymphomas, a decrease in TET2 activity can also contribute to disease progression [ 23 ].

In summary, writer, reader, and eraser proteins are simultaneously involved in the regulation of DNA methylation, and alterations in the function of any of these proteins can lead to the generation of epigenetic changes that contribute to the development of hematological malignancies. A more detailed understanding of these proteins and the related mechanisms could help develop targeted therapies and innovative diagnostic methods for these types of cancers [ 24 ].

Histone modifications behind hematological malignancies

Histone modifications are among the most important epigenetic mechanisms in regulating chromatin structure and gene expression. These modifications include acetylation, methylation, phosphorylation, ubiquitination, and other types of chemical modifications on histone tails. These modifications are controlled by three main groups of proteins: writers, readers, and erasers. Each of these groups plays a critical role in epigenetic balance, and disruption of their function can contribute to various pathologies, including hematological malignancies [ 25 ].

Writer proteins are responsible for adding chemical groups to histone tails. For example, histone methyltransferases (HMTs) such as EZH2 and histone acetyltransferases (HATs) such as CBP and p300 add methyl or acetyl groups to lysines or arginines on histones [ 26 ]. In acute lymphoblastic leukemia (ALL), increased expression of EZH2 leads to increased H3K27 methylation, which is associated with silencing of differentiation genes in lymphocytes [ 27 , 28 ]. Mutations in the CREBBP gene (which encodes one of the HATs) are also common in B-cell lymphomas (such as diffuse large B-cell lymphoma(DLBCL)), causing reduced acetylation and impaired expression of genes that regulate the immune response [ 29 ].

Reader proteins recognize and bind to specific modifications on histones to interpret epigenetic signals. The most important of these proteins is BRD4 (a member of the BET family), which recognizes histone acetylation. In blood cancers such as ALL and AML, BRD4 binds to acetylated regions and activates the expression of oncogenes such as MYC. BET inhibitor drugs (such as JQ1) have been able to inhibit the growth of leukemia cells in laboratory models by inhibiting the function of BRD4 [ 30 ].

Erasers are the group of proteins is responsible for removing epigenetic modifications from histones. Demethylases such as KDM6A (which removes H3K27 methylation) [ 31 ] and HDACs such as HDAC1 and HDAC2 are included in this group [ 32 ]. In myelodysplastic syndrome and AML, increased HDAC activity leads to the elimination of histone acetylation and chromatin compaction, which is associated with the silencing of tumor suppressor genes [ 33 ]. In addition, mutations in the KDM6A gene are associated with reduced H3K27me3 demethylation and have been reported in various types of lymphomas [ 34 ].

The coordinated function of writers, readers, and erasers in regulating the dynamics of histone modifications is essential for maintaining the health of blood cells. Dysfunction of any of these groups can cause persistent changes in gene expression and play a key role in the occurrence and progression of hematological malignancies. A detailed understanding of these mechanisms can provide a basis for the development of targeted epigenetic therapies. This classification is in fact a general functional framework in epigenetics that is applicable to most types of protein modifications. The following are the changes in phosphorylation, ubiquitination, and sumoylation [ 35 ].

Phosphorylation: kinases (e.g., MSK1, Aurora B, JAK2) that add a phosphate group to specific amino acids such as serine or threonine in histone tails are considered writer proteins. Proteins that recognize phosphorylation (reader), such as 14–3–3, which binds to phosphorylated histones and causes changes in gene expression [ 36 ], and phosphatases (e.g., PP1 and PP2A) that remove the phosphate group are known as Eraser proteins [ 37 ]. In hematology, phosphorylation of H3S10 by Aurora B is involved in cell division and is associated with abnormal cell division and aneuploidy in some leukemias [ 38 ] and lymphomas [ 39 ].

Ubiquitination: ubiquitin ligases (E3 ligases) such as RNF20 that attach ubiquitin to specific lysines are referred to as writer proteins, while proteins with ubiquitin-binding domains (UBDs) that recognize ubiquitin are referred to as reader proteins, and finally deubiquitinases (DUBs) such as USP22 that remove ubiquitin act as eraser proteins. In hematology, reduced activity of RNF220 causes chromatin dysregulation in leukemia, and USP22 is associated with poor prognosis in myeloid leukemias [ 40 ].

SUMOylation: SUMO E3 ligases such as PIAS1 that attach SUMO to proteins, proteins with a SIM (SUMO-interacting motif) that recognize SUMOylation, and SUMO-specific proteases such as SENPs that remove SUMO are referred to as writer, reader, and eraser proteins, respectively. In hematology, excessive sumoylation of some transcription factors can lead to the repression of tumor suppressor genes in AML. Increased PIAS1 activity has been observed in some leukemia subtypes [ 41 ].

Non-coding RNAs: epigenetic conductors of hematological malignancies

Non-coding RNAs, particularly microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), play key roles in the epigenetic regulation of gene expression and chromatin structure in hematological malignancies. These molecules can contribute to the malignant phenotype of blood cells by influencing translational regulatory pathways and directing chromatin remodeling complexes [ 42 ]. miRNAs usually repress translation or induce mRNA degradation by binding to the 3'UTR of target mRNA. In hematological malignancies, alterations in the expression of miRNAs such as miR-29 and miR-155 lead to disruption of the expression of epigenetic genes such as DNMT3A , TET2 , or EZH2 , which are involved in the regulation of methylation status and chromatin status of the cell [ 43 – 45 ].

LncRNAs have a more complex function and can act as scaffolds or guides for epigenetic molecules. Some lncRNAs, such as HOX transcript antisense intergenic RNA (HOTAIR) or metastasis associated lung adenocarcinoma transcript 1 (MALAT1), interact with chromatin remodeling complexes such as PRC2 (Polycomb Repressive Complex 2) or LSD1/CoREST/HDAC and direct them to specific genes. This guidance leads to histone methylation and repression of tumor suppressor genes [ 46 , 47 ]. For example, in AML [ 48 ] and chronic lymphocytic leukemia (CLL) [ 49 ], increased expression of HOTAIR is associated with decreased expression of differentiation genes. LncRNAs, such as nuclear-enriched abundant transcript 1 (NEAT1) [ 50 ] or long intergenic non-protein coding RNA, p53 induced transcript (LINC-PINT) [ 51 ], can also interact with mRNA-binding proteins and affect the regulation of translation of mRNAs important in the cell cycle or apoptosis. As a result, epigenetic modifications on non-coding RNAs not only cause changes in gene expression but also in translation regulation and chromatin remodeling, and these pathways are considered novel targets for the treatment of blood cancers.

Blueprints of disorders: chromatin remodeling in hematological malignancies

Chromatin remodeling is a key epigenetic mechanism involved in regulating DNA accessibility and, consequently, gene expression. In hematological malignancies, alterations in chromatin remodeling proteins can lead to persistent changes in gene expression involved in blood cell differentiation, proliferation, and apoptosis. Chromatin is influenced by remodeling complexes such as ISWI, SWI/SNF, INO80, and CHD. These complexes use ATP energy to alter nucleosome structure and facilitate or restrict access of transcription factors to DNA [ 52 , 53 ]. Mutations or loss of expression of components of these complexes (e.g., SMARCA4, ARID1A, CHD2) have been observed in many hematological malignancies such as AML, ALL, and lymphomas [ 54 – 56 ].

For example, reduced SWI/SNF function can lead to activation of cell growth pathways and repression of tumor suppressor genes. On the other hand, the PRC2 complex (including EZH2), which causes histone H3K27 methylation, is hyperactivated in some lymphomas, such as DLBCL, leading to repression of differentiation genes. Changes in chromatin remodeling are often accompanied by other epigenetic changes, such as DNA methylation and histone modifications, and act as part of a complex network of gene expression regulation. These pathways are not only involved in the development of hematological malignancies, but are also potential targets for epigenetic therapies [ 53 ].

RNA rewritten: epitranscriptomics in hematological malignancies

Epitranscriptomic regulation refers to a set of reversible chemical modifications on RNA that control RNA function, stability, and translation without altering its sequence. This type of regulation, which has gained attention in recent years, also plays an important role in hematological malignancies. The most common of these changes is N6-adenosine methylation, or m6A, which affects mRNA, lncRNA, and miRNA [ 57 ].

Writer enzymes such as Methyltransferase-like 3 (METTL3) and METTL14 add this m6A mark to RNA [ 58 ], while eraser such as FTO and ALKBH5 remove it. Reader proteins such as YTHDF1/2/3 also recognize these marks and regulate RNA fate pathways such as stability, translation, and translation [ 59 ]. In malignancies such as AML, increased expression of METTL3 leads to increased m6A on mRNAs related to cell proliferation and decreased differentiation, whereas its deletion can inhibit leukemia growth [ 60 ].

Changes in m6A regulation can also affect the processing of miRNAs and the function of lncRNAs, thereby leading to indirect epigenetic regulation. These changes affect not only gene expression but also drug response and drug resistance. Therefore, epitranscriptomic regulation, as a new layer of epigenetic regulation, plays an important role in the development and progression of blood cancers and could be an emerging target for targeted therapies [ 61 ].

In this regard, epigenetic changes are recognized as one of the fundamental axes in the pathobiology of hematological malignancies, which profoundly affect gene expression without changing the DNA sequence. Understanding these changes have opened new horizons in diagnosis, prognosis, and therapeutic targeting. Therapeutic interventions based on epigenetic modification provide a dynamic and reversible approach to inhibit disease progression. These strategies have found a promising place, especially in treatment-resistant cancers. Overall, epigenetics has created a bridge between molecular biology and individualized therapies in hematological malignancies.

Epigenetic therapies in hematology

In recent decades, it has been established that tumors are formed as a result of the accumulation of a series of molecular changes at the genome level. However, tumor formation is not limited to genetic changes alone, and epigenetic changes are also involved in this phenomenon. Factors that modify the chromatin structure regulate epigenetic indicators, which, despite their stability and heritability, are completely dynamic and reversible. By changing the parts of chromatin, epigenetic mechanisms change the structure of chromatin, which in turn changes the way genes are expressed [ 62 , 63 ]. A normal hematopoietic progenitor cell follows a series of tightly controlled sequential changes in its maturation path. If molecules stray from this path, it can change the way genes are expressed in these cells, which can turn the normal process of development into leukogenesis. In fact, hematological malignancies often involve a spectrum of abnormalities in factors that control transcription, acetylation, methylation, and histone structure [ 64 ]. Mutations in genes encoding epigenetic modifiers are a frequent occurrence in hematological malignancies and are important in cancer initiation and progression. Epigenetic changes are also often reversible, providing excellent opportunities for therapeutic intervention. Epigenetic therapies aim to reverse epigenetic abnormalities, restore epigenetic balance, and return malignant cells to their normal state [ 65 ]. In this section, we discuss agents and drug therapies that target epigenetic regulators, as well as the challenges and limitations posed by each therapeutic target (Table 1 ).

Current epigenetic drugs

Researchers have studied and evaluated DNA methylation and histone modifications more than other epigenetic pathways. DNA methylation plays a prominent role in the occurrence of hematological malignancies [ 66 ]. Epigenetic modifications allow cancer cells to adapt to changes in their surrounding microenvironment. However, tumor suppressor genes that have become dormant due to hypermethylation can be reactivated with specific drugs. An increasing number of chemical compounds have been identified that exhibit hypomethylating activity. These agents were initially employed for epigenetic research and are currently being investigated for use in cancer therapy [ 63 ]. The basis of these therapeutic approaches is since following promoter methylation, the nucleotide sequence of the inactivated gene remains intact and, if re-expressed, can express the target protein with full efficiency. Therefore, in leukemic blasts, the return of a tumor suppressor gene that has become epigenetically inactive would be likely [ 35 , 67 ]. The following is an introduction to the approach of some of the drugs introduced with the aforementioned mechanisms (Table 1 ).

DNA methyltransferase inhibitors (e.g., azacitidine, decitabine)

As mentioned, DNA methylation in the promoter region of tumor suppressor genes is considered one of the most important mechanisms for the development of hematological malignancies. DNA methylation is carried out by DNMTs enzymes, the most important of which is DNMT1 [ 68 ]. Aberrant hypermethylation of the promoter of tumor suppressor genes (TSG) by the DNMT1 enzyme causes the silencing of these genes and the formation of leukemias [ 69 ]. Given the early occurrence of epigenetic changes and the reversibility of these changes, several studies have been directed toward identifying DNMT1 inhibitors. 5-Azacytidine (5AC) and its derivative 5-aza-2'-deoxycytidine (decitabine; DAC) are two DNA hypomethylating agents approved by the Food and Drug Administration (FDA) for the treatment of acute leukemia [ 70 , 71 ] and myelodysplastic syndrome [ 72 ]. The drug 5-azacytidine is a major inhibitor of DNMT enzymes, which is a simple derivative of the nucleoside cytidine. This compound can be incorporated into DNA like cytidine and, as a base analog, is placed alongside other major DNA bases. DNMTs methylate both DNA cytosines and the incorporated 5-azacytidine analogs. However, when these enzymes bind to 5-azacytidine, they are no longer able to dissociate from DNA and are thus blocked. Decitabine is a ribose sugar analog of 5-azacytidine that is much more specific and may be less toxic than 5-azacytidine. This compound has specific activity in myeloid malignancies, MDS, and acute and chronic myeloid leukemia [ 73 , 74 ]. The above azanucleoside drugs have limited utility in the treatment of hyperproliferative leukemias due to their non-specific DNA binding activity, limited efficacy in the S cell phase, and inappropriate use of these drugs. For this reason, the field has progressed toward synthetic non-nucleoside inhibitors such as procainamide, although these inhibitors also have negligible hypomethylation potency. Therefore, the use of natural substances that have a hypomethylation effect on DNA has received great attention, including curcumin and green tea polyphenols [ 75 , 76 ].

In 1984, azacitidine was evaluated in MDS patients with encouraging results and was associated with overall survival. Based on these results, the FDA approved azacitidine in 2004 for the treatment of all subtypes of MDS. Evaluation of azacitidine in multiple phase I clinical trials to assess overall survival (OS), complete or partial remission rates, and side effects showed that the drug has therapeutic value in patients with lower-risk MDS, such as those without poor cytogenetics [ 77 ]. However, with decitabine in hematological malignancies, the results of using this drug at low doses were promising. In contrast, at high doses, it had strong and long-lasting suppressive effects on the myeloid lineage. However, it is worth noting that survival was significantly higher in MDS patients who responded to this drug than in those who did not respond adequately [ 73 , 78 , 79 ]. This led the FDA to introduce decitabine as a treatment option for Chronic myelomonocytic leukemia (CMML) patients in the MDS category in 2006 [ 77 ].

ASTX727 is a novel oral DNMTi drug that combines cedazuridine (a cytidine deaminase (CDA) inhibitor) and decitabine, where the degradation of decitabine by CDA in the gastrointestinal tract is limited, and its bioavailability is increased. ASTX727 was approved by the FDA in 2020 for use in MDS patients [ 80 , 81 ]. An oral form of azacitidine, CC-486, also showed promising results in a randomized trial, showing no relapse of AML in elderly patients undergoing chemotherapy. This oral drug is FDA-approved for AML, not MDS. However, clinical trials of CC-486 in MDS patients are ongoing and have shown promising results [ 82 , 83 ]. ASTX030, a combination of cedazuridine and oral azacitidine, is currently being studied and evaluated in clinical trial NCT04256317 [ 77 ].

Other DNMTi-based compounds include venetoclax (a BCL-2 inhibitor) in combination with decitabine or azacitidine, which has shown good response in older patients with AML and was approved by the FDA in 2018 [ 84 ]. The combination of sorafenib (a FLT3 inhibitor) [ 85 ] and ivosidenib (an IDH1 inhibitor) [ 86 ] with azacitidine has also been shown to be effective in older AML patients who have not responded adequately to chemotherapy. Gilteritinib, a FLT3 inhibitor, is being studied in combination with azacitidine in an ongoing clinical trial ( NCT02752035 ) [ 77 ].

Histone deacetylase inhibitors (e.g., Vorinostat, romidepsin)

Another type of inhibitor used in epigenetic changes associated with hematological malignancies is histone deacetylase inhibitors, which have emerged as a promising group of antineoplastic agents due to their ability to modulate gene expression epigenetically [ 87 , 88 ]. By inhibiting HDACs, chromatin is relaxed, resulting in the activation of transcription of genes involved in proliferation, cell cycle regulation, and apoptosis [ 89 ]. HDAC inhibitors also inhibit angiogenesis through epigenetic mechanisms by eliminating the signaling pathway associated with angiogenesis and modulating genes involved in angiogenesis, such as VEGF, angiopoietin, and eNOS in endothelial cells, thereby preventing tumor progression. Both injection and oral routes administer HDAC inhibitor drugs. The oral route is absorbed in the gastrointestinal tract and may be metabolized by the liver before reaching the bloodstream. However, the injectable type enters the bloodstream directly, resulting in rapid and complete absorption of the drug. HDACi come in different categories, including hydroxamic acids, cyclic peptides, benzamides, and short-chain fatty acids [ 90 , 91 ]. The most prominent HDAC inhibitors include the following:

Romidepsin (FK228) is an inhibitor of class I HDACs, specifically HDAC1 and HDAC2 [ 92 ]. It, therefore, inhibits the cell cycle by increasing the apoptotic pathway through the increase in proapoptotic proteins [ 93 ]. Romidepsin also reduces the activity of DNMTs, followed by demethylation and activation of methylated and inactive tumor suppressor genes. Therefore, romidepsin can be considered a dual epigenetic therapeutic approach [ 94 ]. This drug was initially used for peripheral T-cell lymphomas (PTCL) and cutaneous T-cell lymphomas (CTCL) but was later considered for clinical use in various forms of leukemia [ 95 , 96 ]. Another HDACi is belinostat, which has been introduced as a pan-HDAC inhibitor and inhibits a wide range of HDACs, including three classes of HDACs, HDACs 1, 2, and 4 [ 97 ]. It inhibits cancer by arresting the cell cycle, inducing apoptosis, and inhibiting angiogenesis [ 98 , 99 ]. Belinostat, like romidepsin, has been proposed as a treatment option for PTCL but is currently being studied for its therapeutic effects in other leukemias [ 99 ].

Vorinostat, or suberoylanilide hydroxamic acid (SAHA), like Belinostat, is a pan-HDACi [ 100 ]. This would provide similar effects to those reported for Belinostat in patients with CTCL [ 101 , 102 ]. A hydroxamic acid derivative, givinostat or ITF2357, has been introduced as an HDACi that inhibits class I and IIb isoforms and induces apoptosis in leukemic cells [ 103 ]. Studies are underway in patients with MDS and AML treated with givinostat [ 91 ]. Another HDACi is Panobinostat or LBH589, which, like belinostat and Vorinostat, inhibits class I, II, and IV HDACs but is more potent than other HDACi [ 104 ]. The extensive hyperacetylation that Panobinostat induces in cancer cells inhibits proliferation and cell cycle progression and induces apoptosis. It was first proposed in 2015 for the treatment of multiple myeloma [ 105 ] but has now been proposed for ALL [ 106 ]. In fact, this histone deacetylase inhibitor has a synergistic effect in suppressing cancerous T and B-lymphocyte cells with the monoclonal antibody bortezomib [ 106 , 107 ]. Chidamide (CS055/HBI-8000) is from the benzamide class that inhibits HDACs class 1, 2, 3, and 10 [ 108 ]. However, entinostat (MS-275, SNDX-275) is a synthetic derivative of the benzamide class and inhibits only HDAC1 [ 109 ]. Entinostat has shown promising results in the treatment of CLL [ 110 ] and AML [ 111 ]. Due to the deacetylation of genes involved in the cell cycle and apoptosis, proteins involved in cell cycle arrest and apoptosis induction are produced, preventing the progression of cells to cancer. In studies, the combination of entinostat and azacitidine has been proposed as a potential treatment for leukemias [ 111 ].

EZH2 inhibitors (e.g., tazmetostat)

EZH2 is a catalytic subunit of the core Polycomb repressive complex 2 (PRC2) that methylates lysine 9 and 27 of histone H3, silencing downstream genes and repressing transcription. EZH2 increases tumor cell proliferation and maintains stem cell pluripotency. EZH2 is also abnormally expressed in a variety of malignant cancers [ 112 ]. Follicular lymphoma is one of the hematological malignancies whose pathogenesis is highly dependent on the disruption of epigenetic regulators. Follicular lymphoma is associated with germinal centers that produce lymphocytes. In these centers, the EZH2 factor plays a very prominent role. Methylation of lysine 27 of histone H3 regulates the inactivation of genes involved in the maturation of B lymphocytes. If a mutation occurs in EZH2, lymphomagenicity occurs [ 113 , 114 ]. Tazmetostat acts as an EZH2i or EZH2 inhibitor. In several studies, it was found that tazmetostat has significant efficacy on the mutant form of the EZH2 gene more than its wild-type form, and the only reported non-hematological adverse effect was fatigue. So far, the phase I clinical trial of this combination has been successfully completed, and phase II is underway on 500 patients in combination with rituximab [ 113 , 115 , 116 ]. Of course, in a phase I study, the combination of lenalidomide, tazmetostat, and Obinutuzumab was used for efficacy on FL patients, which, while having a favorable efficacy, i.e., complete response or CR was about 72%, the toxicity caused was unacceptable [ 117 ].

On the other hand, deletion of EZH2 increases the number of natural killer cells (NK cells), which increases the capacity to fight tumor cells. In an in vivo study on mice engineered to knock out the EZH2 gene, it has been seen that characteristic such as IL-15Rβ and NKG2D, which have a high potential for NK cell killing, increase [ 118 ]. It is worth mentioning that Extranodal NK/T-cell lymphoma, nasal type (ENKTL), a type of malignancy involving NK cells associated with the Epstein–Barr virus, is actually a malignancy associated with hypermethylation [ 119 ]. On the other hand, EZH2 methylation causes the silencing of regulatory genes associated with tumor suppression. It has been shown that treatment of ENKTL cells with tazmetostat failed to differentiate malignant NK cells along with other hypomethylating agents effectively. These studies indicated that EZH2 is considered a clinical target for the treatment of hematological disorders, but the inhibitory effect of tazmetostat still needs to be further investigated [ 120 ].

Other studies have been conducted on the association between the EZH2 gene and oncogenicity, chemotherapy resistance, immune evasion, poor prognosis, and relapse of AML. This has made EZH2 a promising target for the treatment of AML [ 121 ]. However, monotherapy treatments have not yielded effective results, so most studies have investigated the simultaneous combination of several drugs for the treatment of AML. For example, it has been shown that the combination of vitamin C with DZNeP and venetoclax with DZNeP increased the apoptosis and killing of CD117 + blasts, respectively, which in each of these two therapeutic approaches inhibited the progression of leukemia. Other multidrug approaches can be used to use EZH2 inhibitors along with other drugs such as BCL-2 inhibitor Venetoclax, histone deacetylase inhibitor Panobinostat, 5 Azacytidine, ATRA, doxorubicin, Ara-C, and FLT3 inhibitors to advance the treatment of AML [ 112 , 122 ]. More recent studies have shown that EZH2 function is also dependent on the cancer tissue and the stage of AML. In fact, it acts as a suppressor protein in the induction phase of AML, and if mutated, it leads to AML [ 123 ]. However, according to AML-related treatment studies based on the use of EZH2 inhibitors, what is largely agreed upon is the use of combination therapies to increase the effectiveness of AML treatment. The effectiveness of EZH2 inhibitors is still in the preclinical stages and requires larger trial studies regarding their use in AML patients.

Emerging therapies

Despite the relative lack of effective treatments and new drug approvals for hematological disorders such as AML and MDS, as well as the identification of new biological complexities and mutations involved in the development of hematological malignancies, promising new targeted therapies have recently been adopted, including CRISPR technology and the use of BET inhibitors.

BET inhibitors and their potential in hematologic malignancies

Bromodomains are highly regulated transcription factors that play an important role in the formation of blood cells, thus playing a role in the differentiation of red blood cells or platelets, and play a very important role in the regulation of cytokines. BET binds to the ends of acetylated histones and plays an important role in the epigenetic regulation of transcription [ 124 , 125 ]. In fact, it recruits transcriptional regulators to its binding site. Several proteins cooperate with BET, including BRD4. The first known BET inhibitor to be identified was JQ1, which inhibits angiogenesis by reducing angiogenic factors such as VEGF and c-MYC. JQ1 binds to the BRD4 protein and prevents its function in the angiogenic process by disrupting the recruitment of P-TEFb [ 126 , 127 ]. In vitro and in vivo studies have shown that BET inhibitors have significant antitumor activity in AML and MDS patients [ 128 ]. The transcription factor RUNX1, which is highly expressed in AML, controls the expression of several genes. BET inhibitors have a suppressive effect on the mutated RUNX1 gene, which subsequently exerts its therapeutic effect on AML with RUNX1 mutations [ 129 , 130 ]. Myelofibrosis is a hematological disorder defined by an overactive JAK-STAT pathway, which ultimately leads to increased cytokine production. However, other pathways are involved in the progression to myelofibrosis, the most important of which is NF-κB. Here, BET inhibitors target it and help reduce the burden of cytokine production, thereby reversing the inhibition of red blood cell and platelet differentiation that occurs in this disease [ 124 ]. BET are also implicated in the development of non-Hodgkin lymphomas and may activate BCL2 and MYC-related pathways [ 131 ]. BET inhibitors, including birabresib, have not been effective as monotherapy in the treatment of DLBCL but have shown promising results in combination with rituximab and venetoclax in DLBCL patients [ 132 ]. In a phase I clinical trial, OTX015, a BET inhibitor, was administered to patients with AML and MDS, achieving near-blast clearance in two patients and complete remission in three patients. Side effects included increased serum bilirubin and fatigue. Other BET inhibitors currently in clinical trials include GSK525762, TEN-010, and CPI-0610 [ 124 , 129 ].

It has also been reported that BRD4 expression is increased in multiple myeloma (MM) relative to stem cells. Therefore, using JQ1, c-MYC was reduced, and subsequently, the proliferation of malignant plasma cells was inhibited [ 131 , 133 ]. OTX015 (MK-8628), as a BET inhibitor with its anti-angiogenic effects, has potential effects on hematological malignancies [ 134 ]. Of course, BET inhibitors, in combination with other therapeutic approaches, are also currently being used in studies.

CRISPR-based epigenetic editing tools

As mentioned, epigenetic mechanisms regulate cellular processes without changing the genetic content of the cell. In this regard, any of them that undergo undesirable changes can lead cells to malignancy. Technological advances have made it possible to monitor these changes in malignant blood cells with greater accuracy and depth. These new technologies include the CRISPR technique and gene editing [ 135 ]. So far, this technology has been used to study epigenetic changes in AML leukemia [ 135 ] and beta-thalassemia [ 136 ]. As mentioned previously, histone changes, including methylation and acetylation of histone H3 at lysines 4 and 27, respectively, play an effective role in post-translational changes and transcriptional regulation [ 137 ]. One of the most important histone modifications that is widely known for its special role in hematopoiesis is the methylation of lysine 79 of histone H3, which is carried out by the methyltransferase DOT1L. In a study conducted in mice, it was found that deficiency and deficiency of the enzyme DOT1L sometimes cause severe and fatal anemia [ 138 , 139 ]. Most of the proteins involved in DNA methylation are multi-subunit complexes, so if the protein is completely deleted, it cannot be determined which subunit is involved in epigenetic changes and turning genes on or off. To overcome this limitation, CRISPR-Cas9 technology has been used, which creates point mutations in the protein in the suspected subunits, and, as a result, its functional changes are analyzed. This has led CRISPR to genome editing. In addition, this technology has been used to remove or add effective changes to correct mutations in enzymes involved in epigenetic pathways using a modified form of Cas9 called “dead Cas9” (dCas9) [ 140 ]. For example, in an in vitro study on HEK293T cells that have hemoglobin enhancers knocked out, dCas9-p300 increased the levels of acetylation of lysine 27 of histone H3, which subsequently enhanced the transcription of the hemoglobin gene [ 141 ]. This CRISPR system, also known as CRISPRa (CRISPR activation) and CRISPRi (CRISPR inhibition), can also be used to screen for resistance to chemotherapy in AML patients [ 142 ]. In an in-vivo study on mouse embryos, dCas9 combined with the DNMT3A enzyme in T lymphocytes helped to successfully methylate target regions, including the RUNX1 gene, which is involved in the development of AML. This has caused the inhibition of leukogenesis [ 135 , 143 ].

It was stated that CRISPR technology has also been used in beta-thalassemia. Beta-thalassemia can be caused by 300 different mutations, which reduce the expression of the beta-globin chain gene, and subsequently, the alpha chain accumulates in the red blood cell [ 144 ]. These unstable accumulations of alpha chains cause the production of reactive oxygen species, which results in hemolysis. Therefore, if an approach is adopted that reduces alpha chain production, the adverse clinical effects of these patients can be reduced [ 145 ]. Therapeutic targets have been introduced by targeting epigenetic regulatory pathways, including induction of methylation and inhibition of lysine acetylation of histones related to alpha-globulin gene regulation. IOX1 is a pan-histone demethylase inhibitor that, in an in vitro study, showed that histone lysine demethylation was reduced in primary erythroid cells [ 146 ]. Vorinostat also acts as a histone deacetylase inhibitor, reduces alpha-chain gene expression, and increases gamma-globin chain expression in erythroid cells. These two compounds have shown promising results in the laboratory and could be considered a therapeutic option for early clinical development [ 147 ].

On the other hand, CRISPR-based genome editing of the alpha globin chain gene has also reduced alpha chain expression in beta-thalassemia patients in the laboratory. A clinical trial ( NCT03655678 ) was conducted on a woman with beta-thalassemia with CRISPR/Cas9-based genome editing of the enhancer region of BCL11A in CD34 + stem cells. The results showed an increase and stability in gamma-globin gene expression, thereby reducing the need for transfusions and blood transfusions [ 148 ]. CRISPR/Cas9 genome editing has also been shown in hematopoietic stem cells by targeting the alpha chain on the HUDEP-2 β0 cell line, resulting in reduced alpha chain deposition in red blood cells and subsequent reduction in the complications of beta-thalassemia [ 149 , 150 ]. Therefore, the use of CRISPR technology in the treatment of beta-thalassemia provides a promising approach for these patients.

Combination therapies targeting epigenetic and genetic alteration

Several studies have investigated the use of combination therapy regimens to increase treatment efficacy and minimize challenges such as off-target effects and drug resistance. The most important of these is the combination of HDACi with TKi (tyrosine kinase inhibitors), which has shown synergistic effects in inhibiting proliferation and increasing apoptosis of leukemia cells. The combination of these two groups of inhibitors has been very effective, mainly on the BCR-ABL, JAK-STAT and FLT3 signaling pathways [ 151 – 153 ]. The combination of HDAC inhibitors with gilteritinib, quizartinib, sorafenib and midostaurin has been proposed to suppress the FLT3 pathway in leukemia treatment [ 154 , 155 ]. Another combination therapy, including chemotherapeutic drugs such as cytarabine, vincristine and doxorubicin with HDAC inhibitors, has been shown to significantly improve leukemia patients' response to treatment and survival [ 156 ].

Also, the use of HDACi together with radiotherapy reduces metastasis and overcomes resistance to treatment. Radiation therapy alone does not have a strong therapeutic potential in leukemias [ 157 ]. However, when combined with HDACi, HDACi induce an active state of chromatin expression, making it highly sensitive to ionizing radiation. On the other hand, HDACi reduce angiogenesis and subsequently blood supply to the cancer tissue, thereby improving the efficacy of radiotherapy. However, this therapeutic strategy is still used to a limited extent [ 158 , 159 ]. With the introduction of immunotherapy, its application in emerging therapies has increased. The combined approach of immunotherapy and HDACi has been effective for solid tumors, but few studies support it for leukemias [ 160 ]. Indeed, with the influence of HDACi, the tumor environment provides a suitable place for the presentation of tumor antigens to immune cells. Important components of this tumoral microenvironment include tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) [ 161 – 163 ]. Finally, other combination therapies between epigenetic HDAC inhibitors with BET or DNMTs also enhance antitumor effects. Several preclinical and clinical studies have been conducted on the efficacy of combination therapy with epigenetic inhibitors, confirming the reduction in oncogenes by inhibiting proliferation and inducing apoptosis (Table 2 ) [ 164 – 166 ].

In addition to its major effects on epigenetic regulation, CRISPR-related technology also exerts its effects on the genome, which will bring more beneficial results. Other technologies related to epigenetics and genetics include single-cell epigenomics, which has been used in hematopoietic processes and the study of leukemic cells. Epigenetic approaches at the genome level, such as assessment of chromatin accessible to transposase factors (ATAC-seq), chromatin immunoprecipitation (ChIPseq), and bisulfite sequencing (BS-seq), each of which provides easy access to DNA, histone modifications, and the study of histone methylation patterns, respectively. These technologies contribute significantly to understanding the approach of hematological malignancies such as AML [ 135 ].

Challenges and limitations

However, challenges remain in developing efficient and safe delivery strategies to specifically target hematological disorders, enhance therapeutic outcomes, and minimize off-target effects and toxicity of any selected therapy. Further extensive preclinical and clinical research in the future will help overcome these challenges. The above azanucleoside drugs have limited utility in the treatment of hypo-proliferative leukemias due to their non-specific DNA binding activity, limited efficacy in the S cell phase, and inappropriate use of these drugs. For this reason, the field has progressed toward synthetic non-nucleoside inhibitors such as procainamide, although these inhibitors also have negligible hypomethylation potency. Therefore, the use of natural substances that have a hypomethylation effect on DNA has received significant attention, including curcumin and green tea polyphenols [ 179 ]. Although not wholly curable, DNMTis is a significant treatment approach for patients with AML and chronic myelomonocytic leukemia-related hematologic neoplasms of the MDS category who are not candidates for BMT or chemotherapy [ 77 ].

A significant limitation of the clinical management of patients with DNMTi-based therapies is that several months of treatment with these drugs are required before the therapeutic efficacy can be assessed, thus exposing these patients to high treatment costs and side effects such as cytopenia and losing significant opportunities to evaluate other new therapies promptly. There are few alternative treatments in some patients who do not respond to DNMTi, and these patients generally have a poor prognosis [ 77 ]. In a study of patients with MDS treated with decitabine and azacitidine, the median overall survival (OS) was 44 and 31 months, respectively [ 180 ], compared with 15 months after discontinuation of hypomethylating agents (HMA) [ 181 ] (Table 3 ).

Off-target effects and toxicity

Despite the promising results of HDACi, challenges such as off-target effects, drug resistance, and choosing the best dose and timing of drug administration remain [ 182 ]. Also, in terms of safety, various side effects, such as hematological and cardiac toxicity, gastrointestinal disorders, anemia, thrombocytopenia, and neutropenia, have been reported [ 183 ].

There are also limitations to the CRISPR technique. Genotoxicity is a major challenge when using this technique, which can lead to unwanted changes in other parts of the DNA content. In fact, off-target or incorrect binding of the sgRNA to a point other than the original target in the DNA can show the opposite result to what was expected. Of course, newer systems that use more precise Cas9 enzymes have primarily overcome this limitation. Also, CRISPR/Cas9 has caused it to be used much more cautiously in the clinic by activating proto-oncogenes or creating significant cuts or deletions in DNA. For example, in the case of reducing alpha-globin gene expression in beta-thalassemia patients, it is difficult to titrate the desired deletion or knockdown of the alpha-globin gene. Deleting 25 to 50% of the alpha-globin gene is sufficient to balance the alpha-to-beta chain ratio and improve symptoms. If the alpha chain is removed too much, it will cause even more severe anemia complications [ 184 ].

Also, some JAK2 inhibitors, including fedratinib, have reported side effects such as encephalopathy. Some other introduced drugs, including CEP-701, AZD1480, LY2784544, BMS-911543, and INCB039110, have been discontinued due to multiple toxicities in MPN patients. Hence, new JAK2 inhibitors have been developed to minimize off-target effects [ 185 ]. Therefore, all the mentioned therapeutic approaches in hematological disorders should be used widely in the clinic with great care and caution.

Resistance mechanisms to epigenetic therapies

Neoplasms have a high resistance rate to proposed anticancer therapies due to diverse biological changes in leukemic patients and intratumoral heterogeneity due to the acquisition of new changes, and therefore, pose a high risk of recurrence and mortality for these patients [ 186 ].

Several factors contribute to resistance to proposed drug therapies. Older patients may have a more significant number of epigenetic and genetic abnormalities. For example, mutations in the tumor suppressor protein P53 are important factors in resistance to chemotherapy agents and poor prognosis in leukemia patients [ 187 ]. Excessive and inappropriate activation of the Hedgehog, glioma-associated oncogene homolog 1 (GLI), and smoothened (SMO) signaling pathways are associated with malignant cell growth and migration and treatment resistance in AML patients [ 186 ]. A range of heterogeneous mutations in hematological disorders, along with other epigenetic regulatory factors such as DNMT3A, RAS/MAPK signaling, RNA splicing protein (SRSF2), and TET2, contribute to chemotherapy resistance and DNA damage in mutated malignant cells [ 188 ].

Thus, the accumulation of somatic mutations in a tumor setting leads to clonal diversity of malignant cells, resulting in treatment resistance, relapse, and poor prognosis in hematological cancers. A precise understanding of the nature of clonal diversity may move toward personalized therapy. Immunotherapy and single-cell DNA sequencing (scDNA-seq) have precisely discovered clonal diversity [ 186 , 189 , 190 ].

The era of personalized medicine has provided a major step toward identifying unknown pieces of the complex pathology puzzle of blood cancers and escaping drug resistance. Engineered CAR-T cells were introduced to implement personalized therapy [ 191 ].

Need for personalized approaches

Recent advances in sequencing technologies and understanding the genetic and epigenetic pathogenesis of leukemias, including CML and AML, have led to the emergence of precision medicine, a personalized treatment model for everyone. Precision medicine often proposes diagnostic tests to find optimal treatments based on changes in an individual's genetic and epigenetic content [ 192 ]. The successful development of personalized medicine has been demonstrated in studies by introducing IDH1/IDH2, BCL2, and FLT3 inhibitors for AML patients [ 186 ]. When used as monotherapy, these drugs have low to moderate antileukemic activity. However, combined with other targeted therapies targeting epigenetic and genetic signaling pathways, they increase the cytotoxicity of malignant cells. The emergence of personalized immunotherapies offers a promising prospect; however, it is necessary to identify the dose-dependent resistance and to identify and circumvent the factors contributing to the emergence of primary and secondary resistance to new-generation drugs for use in clinical trials [ 129 ].

The emergence of chimeric antigen receptor (CAR)-T cells is another type of immunotherapy approach that is FDA-approved for B-cell-related leukemias and is currently being evaluated for myeloid leukemias. In CML, a marker called IL1 receptor accessory protein (IL1RAP) is expressed, a common receptor for IL33 and IL1. This receptor is not expressed on the surface of normal HSCs, so it has been introduced as a suitable target for the CAR-T cell therapy approach, which explicitly destroys CML stem cells. This therapeutic approach has no off-target effects or tumor lysis syndrome that occurs following the breakdown of malignant cells [ 193 ]. Some patients who have become resistant to tyrosine kinase inhibitors have been able to limit this resistance with the help of CAR-T cell therapy against the CD19 marker. Thus, anti-CD19 CAR-T therapy combined with dasatinib, a TKI, has caused malignant cells to be first cleared by CD19 CAR-T and then re-sensitized to dasatinib. This highlights the importance of the immune system in more appropriate therapeutic responses against leukemias resistant to previously conventional treatments, which has led to more personalized new treatments [ 194 ].

The enormous power of technological innovation in studies of genetic and epigenetic factors associated with blood disorders and any other type of cancer has helped to draw a molecular identity card for each human and tailor the treatment approach accordingly. This can treat blood cancers with the most favorable therapeutic response and the least side effects.

Future direction

The rapid evolution of epigenetic research in hematology has unveiled transformative opportunities, yet several frontiers remain unexplored. Below, we outline key directions poised to redefine therapeutic paradigms:

Next-generation epigenetic drugs with enhanced specificity

Current DNMT and HDAC inhibitors face limitations such as off-target effects and cytotoxicity. Future efforts should focus on designing isoform-specific inhibitors (e.g., targeting HDAC6 or DNMT3A) and leveraging nanotechnology for targeted drug delivery to malignant cells, minimizing systemic toxicity [ 195 , 196 ]. For instance, lipid-based nanoparticles encapsulating azacitidine have shown improved bioavailability in preclinical models [ 197 – 199 ].

Precision epigenome editing via CRISPR-derived tools

CRISPR-dCas9 systems, such as dCas9-DNMT3A or dCas9-TET1, enable locus-specific methylation or demethylation, offering a path to correct aberrant epigenetic marks without genetic disruption [ 200 ]. Recent advancements in base-editing CRISPR systems further allow single-nucleotide resolution, which could address mutations in epigenetic regulators like TET2 or IDH1/2 [ 135 , 201 ].

Synergistic combination therapies

Combining epigenetic agents with immune checkpoint inhibitors (e.g., PD-1/PD-L1 blockers) or CAR-T cell therapies may enhance antitumor immunity by reversing tumor microenvironment immunosuppression [ 160 , 202 ]. Preclinical studies demonstrate that HDAC inhibitors upregulate tumor-associated antigens, improving CAR-T efficacy in AML [ 203 ].

Overcoming resistance through epigenetic priming

Resistance mechanisms, such as compensatory histone modifications or TP53 mutations, necessitate dual targeting of epigenetic and genetic pathways. BET inhibitors paired with FLT3 or BCL-2 inhibitors are under investigation to circumvent resistance in refractory AML [ 204 ].

Integration of multi-omics for personalized therapy

Single-cell epigenomic profiling and artificial intelligence (AI)-driven predictive models will enable dynamic monitoring of clonal evolution and therapy response. For example, machine learning algorithms analyzing DNA methylation patterns in MDS patients have identified predictive biomarkers for hypomethylating agent responsiveness [ 205 – 207 ].

Exploiting non-coding RNAs as epigenetic modulators

Long non-coding RNAs (lncRNAs) and microRNAs regulate chromatin states and gene silencing. Targeting oncogenic lncRNAs like HOTAIR or restoring tumor-suppressive miRNAs (e.g., miR-29b) could complement existing therapies [ 208 , 209 ].

By bridging technological innovation with clinical insight, these strategies will propel epigenetic therapies toward curative potential in hematological malignancies.

Conclusion

Research on epigenetic mechanisms in hematology has revealed a complex and dynamic landscape that strongly influences the development and progression of hematological malignancies. As our understanding of the epigenetic changes that underlie these diseases grows, the potential for targeted therapies to reverse aberrant epigenetic changes is becoming clearer. However, the path to effective clinical use is full of challenges and current therapeutic strategies, including DNA methyltransferase inhibitors, histone deacetylase inhibitors, and emerging agents such as EZH2 inhibitors and BET, show promise in restoring normal gene expression and improving patient outcomes. Off-target effects, drug resistance and the inherent heterogeneity of hematological tumors complicate treatment regimens and reduce the effectiveness of existing therapies. Furthermore, the need for a personalized approach to treatment is essential to address the unique molecular nature of each patient's tumor. Advances in genomic and epigenetic profiling, combined with innovative therapeutic strategies such as CAR-T cell therapies, offer exciting avenues to overcome these barriers and improve treatment accuracy. In future, priority should be given to further research to clarify the complex interaction between genetic and epigenetic factors in hematological malignancies. By combining the insights of basic research with clinical applications, we can develop more effective and tailored treatment strategies that not only increase survival, but also improve the quality of life of patients struggling with these complex conditions. The ongoing development of epigenetic therapies is an important frontier in hematology, and holds the promise of transformative advances in our approach to the treatment and management of hematological malignancies.

Acknowledgements

Although the authors received no financial support, they would like to express their gratitude to the researchers whose articles were used in this study.

Authors’ contributions

Mahdis Abdar Esfahani, Nazli Servatian, Hamideh Rahmani Seraji, Ali Jihad Hemid Al-Athari and Elaf Salah Mehdi Khafaja wrote the main manuscript text and, Hamed Soleimani Samarkhazan Revise the manuscript. All authors reviewed the manuscript.

Funding

This research did not receive any financial support from public, commercial, or nonprofit organizations.

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.

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Associated Data

Data Availability Statement

No datasets were generated or analysed during the current study.