
==== Front
Blood Sci
Blood Sci
BS9
Blood Science
2543-6368
Lippincott Williams & Wilkins Hagerstown, MD

BLS-24-042
00008
10.1097/BS9.0000000000000206
3
Review Article
Epigenetic modifications in hematopoietic ecosystem: a key tuner from homeostasis to acute myeloid leukemia
Yao Shuxin abc
Guo Rongxia d
Tian Wen abc
Zheng Yanbing c
Hu Jin c
Han Guoqiang bc
Yin Rong bc
Zhou Fuling b*
Zhang Haojian abce*
a State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, China
b Department of Hematology, Zhongnan Hospital, Medical Research Institute, Wuhan University, Wuhan, China
c Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China
d Department of Laboratory Medicine, Zhongnan Hospital of Wuhan University, Wuhan, China
e Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan, China
*Address correspondence: Haojian Zhang, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, No.185, East Lake Road, Wuchang District, Wuhan, Hubei 430071, China. E-mail address: haojian_zhang@whu.edu.cn (H. Zhang); Fuling Zhou, Department of Hematology, Zhongnan Hospital, Wuhan University, No.185, East Lake Road, Wuchang District, Wuhan, Hubei 430071, China. E-mail address: zhoufuling@whu.edu.cn (F. Zhou).
12 9 2024
10 2024
6 4 e00206e00206
12 5 2024
20 8 2024
Copyright © 2024 The Authors. Published by Wolters Kluwer Health Inc., on behalf of the Chinese Medical Association (CMA) and Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College (IHCAMS).
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Hematopoietic stem cells (HSCs) maintain homeostasis in the hematopoietic ecosystem, which is tightly regulated at multiple layers. Acute myeloid leukemia (AML) is a severe hematologic malignancy driven by genetic and epigenetic changes that lead to the transformation of leukemia stem cells (LSCs). Since somatic mutations in DNA methylation-related genes frequently occur in AML, DNA methylation is widely altered and functions as a starting engine for initiating AML. Additionally, RNA modifications, especially N6-methyladenosine (m6A), also play an important role in the generation and maintenance of the hematopoietic ecosystem, and AML development requires reprogramming of m6A modifications to facilitate cells with hallmarks of cancer. Given the complex pathogenesis and poor prognosis of AML, it is important to fully understand its pathogenesis. Here, we mainly focus on DNA methylation and RNA m6A modification in hematopoiesis and AML and summarize recent advances in this field.

Acute myeloid leukemia
DNA methylation
Hematopoiesis
Hematopoietic stem cells
Leukemia stem cells
RNA modification
OPEN-ACCESSTRUE
==== Body
pmc1. INTRODUCTION

During the entire lifespan, hematopoietic stem cells (HSCs) maintain homeostasis of the hematopoietic ecosystem through self-renewal and differentiation into all lineages of blood and immune cells.1 These processes are tightly controlled by a series of regulatory mechanisms in multiple layers, including metabolism, protein synthesis, and epigenetic regulation.2–4 These mechanisms function as HSC quality-control systems to maintain the hematopoietic ecosystem; dysregulation of these quality-control systems interrupts homeostasis of the hematopoietic ecosystem, which subsequently leads to abnormal hematopoiesis and blood diseases, such as leukemia and myelodysplastic syndrome.

Genetic and epigenetic changes drive the development of many hematological malignancies. Acute myeloid leukemia (AML) is a fatal disorder characterized by abnormal expansion and arrested differentiation ability of myeloid progenitor cells5; AML development origins from leukemia stem cells (LSCs).6 Currently, chemotherapy remains the standard treatment for most newly diagnosed patients with AML, and the 5-year overall survival rate remains lower (~30%), as many AML patients eventually relapse after reaching remission or directly fail to achieve remission after primary therapy and unfortunately develop into refractory AML.7 Thus, a comprehensive understanding of AML pathogenesis remains an important task in this field.

Chemical modification is a highly specific and efficient way to regulate the functions of biological macromolecules (eg, DNA, RNA, and proteins).8 DNA methylation is one of the major types of epigenetic modifications that refers to changes in regulating gene expression without affecting the genetic sequence.9 RNA can also be modified using more than 170 different chemical modifications.10 Many studies have clearly demonstrated the implication of these nucleic acid modifications in the hematopoietic ecosystem,2 and alterations in these modifications could disrupt gene expression and cause the development of hematologic malignancies, especially AML.11 In this review, we mainly focus on DNA methylation and RNA N6-methyladenosine (m6A) modification in hematopoiesis and AML and summarize recent advances in this field.

2. DNA METHYLATION

DNA methylation was first discovered in bacteria in the 1920s and has been investigated in a vast range of organisms.12 DNA methylation occurs at the cytosine 5 carbon position of the genomic cytosines preceding guanines (CpG) dinucleotide by the incorporation of a methyl group via a covalent bond.13 The reaction is catalyzed by DNA methyltransferases (DNMTs).14 Among the DNMT family members, DNMT1 is mainly responsible for maintaining methylation patterns, whereas DNMT3A and DNMT3B are responsible for adding new methylation patterns (Fig. 1). However, the other two family members, DNMT2 and DNMT3L, do not exhibit catalytic activity. DNMT3L interacts with and stabilizes DNMT3A to facilitate DNA methylation.15,16 DNMT2 is an RNA methyltransferase that functions as a catalyst for the methylation of aspartate cytosine sites on transfer RNAs (tRNAs).17 DNA demethylation is catalyzed by DNA demethylases, in which methylated bases are removed in the presence of DNA glycosidases. This is equivalent to repairing damaged DNA catalyzed by glycosidases and base-free nuclease cleavage couplings. So far, active DNA demethylases include ten-eleven translocation (TET) methylcytosine dioxygenase, activation-induced cytidine deaminase/apolipoprotein B microRNA (mRNA) editing enzyme complex (AID/APOBEC), and thymine DNA glycosylase (TDG)18–20 (Fig. 1). In addition, methylated CpG-binding proteins, such as MBD2, have demethylase activity.21–24

Figure 1. DNA methylation pathways. The methyl group of 5mC can be modified by the addition of a hydroxyl group mediated by Tet enzymes to generate 5hmC. 5hmC can also be chemically modified by AID/APOBEC. AID/APOBEC can deaminate 5hmC to produce 5hmU. Eventually, the 5hmU can be recognized and cleaved off to replace with a naked cytosine mediated by TDG. DNMT1 maintains methylation patterns, and DNMT3A/3B add new methylation. Isocitrate dehydrogenases (IDH1/2) are responsible for converting isocitrate to αKG. Mutant IDH proteins acquire a neomorphic enzymatic activity converting αKG to 2HG and inducing global DNA hypermethylation. Accumulation of 2HG inhibits TET proteins. 2HG = 2-hydroxyglutarate, 5hmC = 5-hydroxymethyl-cytosine, 5hmU = 5-hydroxymethyl-uracil, 5mC = 5-methylcytosine, αKG = α-ketoglutarate, AID/APOBEC = activation-induced cytidine deaminase/apolipoprotein B mRNA editing enzyme complex, DNMT = DNA methyltransferase, IDH = isocitrate dehydrogenase, TCA = Tricarboxylic acid, TDG = thymine DNA glycosylase, TET = ten-eleven translocation.

2.1. DNA methylation maintains HSC function via the distinct roles of DNMTs in establishing and maintaining methylation patterns

The spatiotemporal dynamics of DNA methylation and its synergistic action with transcription factors are essential for maintaining the HSC state and the hematopoietic ecosystem. First, the functional programs of HSCs are governed by gradual differences in methylation levels, and constitutive methylation is essential for HSC self-renewal, but dispensable for cell cycle control, homing, and suppression of apoptosis. For instance, conditional deletion of DNMT1 in the hematopoietic system severely reduces the genomic methylation levels of HSCs and impairs HSC self-renewal, niche retention, and differentiation potential.25 Secondly, DNMT1 regulates distinct patterns of methylation and expression of discrete gene families in HSCs and progenitors. HSC with reduced DNMT1 activity can differentiate into myeloerythroid but not lymphoid progeny,26 while DNMT1 deficiency in myeloid progenitor cells enhances cell cycling and causes inappropriate expression of mature lineage genes.25

DNMT3A and DNMT3B are de novo DNMTs responsible for establishing DNA methylation patterns. Loss of Dnmt3a results in HSC expansion and impaired differentiation.27 Although the predominant Dnmt3b isoform in adult HSCs is catalytically inactive, its residual activity in Dnmt3a-null HSCs can drive their differentiation and generates the paradoxical hypermethylation of CpG islands. Thus, the combined loss of Dnmt3a and Dnmt3b is synergistic, resulting in enhanced HSC self-renewal and a more severe block in differentiation than in Dnmt3a-null cells,28 indicating that DNMT3A and DNMT3B have both overlapping and distinct functions in HSCs.

Interestingly, the TET family enzymes remove DNA methylation by oxidizing 5mC to 5hmC, which is also essential for hematopoietic homeostasis.29 TET2 is abundantly expressed in HSCs. Tet2 deficiency leads to decreased genomic levels of 5hmC and increases the size of the hematopoietic stem and progenitor cell (HSPC) pool in a cell-autonomous manner. Tet2-deficient HSCs are capable of multilineage reconstitution and possess a competitive advantage over wild-type HSCs, resulting in enhanced hematopoiesis into both the lymphoid and myeloid lineages.30 In vitro, Tet2 deficiency delays HSC differentiation and skews development toward the monocyte/macrophage lineage.30 Notably, the actions of DNMTs and TETs cause opposite results: the addition or removal of methylation; however, they are also epistatic, as hydroxymethylation depends on the presence of the 5mC marks introduced by DNMTs. DNMT3A and TET2 can compete and cooperate to repress lineage-specific transcription factors (eg, Klf1, Epor) in HSCs, as DNMT3A and TET2 both repress these factors, suggesting a model of cooperative inhibition by epigenetic modifiers.31 Together, these studies indicate that DNA methylation levels tuned up by DNMTs and TETs play a key role in controlling the functional programs of HSCs.

DNA methylation also coordinates with other mechanisms (eg, histone modifications, higher-order chromatin structure, and RNA splicing) to regulate HSCs. For instance, a recent study showed that DNMT3A and the spliceosome govern HSCs exit from the stem state toward differentiation.32 DNMT3A coordinates splicing by recruiting the core spliceosome protein SF3B1 to RNA polymerase and mRNA, and the loss of DNMT3A leads to impaired splicing during stem cell turnover. Interestingly, in this process, the DNA methylation function of DNMT3A is not required.32 Additionally, TET2 modulates the spatial redistribution of H3K9me3-marked heterochromatin to mediate the upregulation of endogenous retroviruses and interferon-stimulated genes, thus contributing to the functional decline in aged HSCs.33 Large DNA methylation nadirs can form long anchor chromatin loops between repressive elements and form genomic subcompartments to maintain HSC identity.34 Overall, these studies suggest that DNA methylation is necessary for maintaining the hematopoietic ecosystem by regulating the functional programs of HSCs.

2.2. DNA methylation in AML pathogenesis

Alterations in DNA methylation are common in patients with AML. Mutations in DNA methylation-related genes, such as DNMT3A, TET2, and isocitrate dehydrogenase (IDH)1/2, lead to aberrant methylation patterns, contributing to leukemogenesis. These mutations often coexist with other genetic alterations, thereby compounding their effects.

Given the important role of DNA methylation in regulating the HSC program, aberrant DNA methylation contributes substantially to AML pathogenesis. The genomic and epigenomic landscapes of adult de novo AML were constructed using 200 clinically annotated cases (50 cases for whole-genome sequencing and 150 cases for whole-exome sequencing).35 Unsupervised analysis of changes in DNA methylation revealed significant differences across human AML samples, with 67% of the 160,519 CpG loci resulting in a gain of methylation, and 33% resulting in a loss. In addition, mutations in DNA methylation-related genes were detected in approximately 44% of cases35 (Fig. 2). Thus, these data indicate that alterations in DNA methylation may act as a key driver of AML pathogenesis.

Figure 2. Aberrant DNA methylation drives AML development. Aberrant DNA methylation caused by recurrent mutations in epigenetic modifiers function as a driving force to control the fate of HSPCs and initiate AML development by tuning up a series of cellular and molecular programs including the transcriptional programs, chromatin modifications, RNA m6A modification, and metabolism networks. 2HG = 2-hydroxyglutarate, AML = acute myeloid leukemia, BCAA = branched-chain amino acid, DNMT = DNA methyltransferase, HSPC = hematopoietic stem and progenitor cell, HSC = hematopoietic stem cell, IDH = isocitrate dehydrogenase, LSC = leukemia stem cell, TET = ten-eleven translocation.

Among DNA methylation-related genes, somatic mutations in DNMT3A account for approximately 22% of AML.36,37 Multiple missense mutations in the coding region of DNMT3A were identified, the most common of which was predicted to affect amino acid R882 (R882H or R882C) in the structural domain of the methyltransferase. Other types of mutations resulting in frameshift, nonsense, or alternative splicing in DNMT3A were also identified.36 DNMT3A mutations frequently co-occur with the other four most commonly mutated genes (FLT3, NPM1, IDH1, and IDH2) in AML.38 DNMT3A mutations are associated with poor event-free survival and shorter overall survival; this association is independent of the presence of FLT3 or NPM1 mutations, and regardless of the mutation type or genetic location.36 Analysis of global DNA methylation using LC-MS did not reveal a significant difference in 5-methylcytosine (5mC) levels in DNMT3A-mutant AMLs compared with those in AML genomes without DNMT3A mutations.36 Notably, DNMT3A haploinsufficiency is sufficient to contribute to myeloid transformation because DNMT3A mutations occur in one allele in the vast majority of AML patients.36

TET2 is another gene frequently mutated in myeloid malignancies (Fig. 2). Somatic deletions and inactivating mutations in TET2 have been identified in 10% to 20% of MPN and MDS cases and in 7% to 23% of AML cases.39,40 In another study, 131 somatic TET2 mutations were identified in 27.4% of AML patients (87/318).41 TET2 mutations concomitantly occurred with mutations in other genes (eg, NPM1, FLT3-ITD, FLT3-TKD, JAK2, RUNX1, CEBPA, and CBL). Similarly, TET2 mutations also act as important prognostic biomarkers in AML and are associated with a poorer prognosis in de novo AML patients with normal cytogenetics (CN-AML).41–43 TET2 mutations are heterozygous and the wild-type allele is retained in most AML patients, suggesting that TET2 is a haploinsufficient tumor suppressor in leukemia. Another family member TET1 was identified as a fusion partner of the mixed lineage leukemia (MLL) gene in AML with t(10;11)(q22;q23).44 Interestingly, TET1 is a direct target of MLL-fusion proteins and is significantly upregulated in MLL-rearranged leukemia, leading to a global increase in 5-hydroxymethylcytosine level.45

Recurring mutations in isocitrate dehydrogenases IDH1 and IDH2 have been frequently identified in AML.46,47 While wild-type IDH1/2 are responsible for converting isocitrate to α-ketoglutarate (αKG), mutant IDH proteins acquire a neomorphic enzymatic activity converting αKG to 2-hydroxyglutarate (2HG) and inducing global DNA hypermethylation. This is most likely due to the inhibitory effect of 2HG on TET protein activity. Interestingly, IDH1/2 mutations are mutually exclusive of TET2 mutations. Thus, IDH1/2 mutants cause epigenetic defects similar to TET2 loss-of-function mutations,48 implying that IDH1/2 and TET2 may, to certain extent, share some relevant mechanisms in driving AML pathogenesis.

2.3. Aberrant DNA methylation functions as a starting engine for initiating AML by changing multiple cellular and molecular programs

Over the past 15 years, extensive attention has been paid to how alterations in DNA methylation drive the progression of normal hematopoietic ecosystems to hematological diseases. Mutations in DNA methylation-related genes are considered founder mutations and early events in AML development (Fig. 2). A recent study provided key insights into the cellular context and functional consequences of the earliest genetic lesions of human AML and established the sequential order of mutation acquisition; DNMT3Amut occurs before NPM1c and FLT3-ITD.49 In addition, ancestral pre-leukemic HSCs have been identified. These cells enable regeneration of the entire hematopoietic hierarchy while possessing a competitive repopulation advantage over non-leukemic HSCs; they survive induction chemotherapy and persist in the bone marrow at remission.49 Thus, the cell of origin for DNMT3Amut AML is the HSC, and the DNMT3A mutation confers a pre-leukemic state with clonal expansion ability. This leukemogenic process was confirmed by retroviral transduction and bone marrow transplantation to establish a murine model of DNMT3AR882H hotspot mutation.50 DNMT3AR882H enhances the sensitivity of HSPCs and transforms them into leukemia-initiating cells (LIC) in cooperation with NRAS mutations. DNMT3AR882H induces focal epigenetic alterations and transactivates stemness genes (MEIS1, MN1, and HOXA genes) (Fig. 2). Inhibition of DOT1L represses DNMT3AR882H-induced gene expression programs, providing an attractive therapeutic strategy for DNMT3A-mutated leukemias.50 Similarly, a mouse conditional knock-in model of Cre-mediated expression of the mutant Dnmt3aR878H was established.51 Dnmt3a mutation results in enlarged Lin−Sca1+cKit+ cells and causes hypomethylation that contributes to mammalian target of rapamycin (mTOR) upregulation, in turn leading to aberrantly increased CDK1 expression.51 Mechanistically, hotspot mutations in DNMT3A act in a dominant-negative manner, and DNMT3A mutations disrupt the formation of a DNMT3A-associated tetramer complex required for efficient DNA methylation. For instance, hotspot DNMT3AR882 mutations promote polymerization and a dominant-negative effect, leading to aberrant DNA methylation at specific sites.52 The polymerization capacity of DNMT3AR882mut relies on the heterodimerization motif but not on its various chromatin-binding domains. Mutation of the heterodimerization motif interferes with DNMT3AR882mut binding to endogenous wild-type DNMT proteins and partially reverses the CpG hypomethylation phenotype caused by DNMT3AR882mut.53 Thus, DNMT3A acts in a dominant-negative manner during leukemia transformation.

In addition, altered DNA methylation of Hi-C and whole-genome sequencing have identified recurrent and subtype-specific 3D genome alterations in human AML. Altered DNA methylation contributes to these pathogenic chromatin interactions by displacing CCCTC-binding factor (CTCF) binding. For instance, multiple chromatin loops were detected in AML samples across the lost CTCF-binding sites, including a loop linking WDR66 promoter to distal regions54 (Fig. 2). These data revealed a delicate relationship between DNA methylation, CTCF binding, and 3D genome structure. Interestingly, a recent study identified 58 tumor-specific antigens (TSAs) by analyzing the major histocompatibility complex class I (MHC-I)-associated immunopeptidome in human AML samples and found that mutations in epigenetic modifiers correlated with TSA expression, which causes expansion of cognate T cell receptor clonotypes and accumulation of activated cytotoxic T cells,55 indicating that epigenetic alterations in leukemia cells also change the hematopoietic ecosystem.

Mutations in both TET2 and IDH1/2 result in DNA hypermethylation and are considered founders for AML transformation. Genetic inactivation of Tet2 in the mouse hematopoietic system confers HSPCs with a competitive advantage and increased susceptibility to cellular transformation; however, cooperative mutations (eg, FLT3, KIT) are necessary to achieve full malignant transformation. In a mouse model of human AML1-ETO-induced AML, the loss of Tet2 leads to a genome-wide increase in the DNA methylation of active enhancers, especially for several tumor suppressor genes (eg, Mtss1, Las2, Lxn, Ctdspl, Grap2), and this effect is progressive.56 Similarly, mutations in Tet2 and Flt3 that result in fully penetrant and lethal AML, such as Flt3ITD mutations and Tet2 loss, cooperatively remodel site-specific DNA methylation and gene expression that are not observed with either mutant allele alone.57 TET2 activity also shapes the local chromatin environment at enhancers to facilitate TF binding. Genome-wide mapping of TET2 binding sites reveals that TET2 localizes to regions of open chromatin and cell type-specific enhancers, and the deletion of Tet2 attenuates the binding of the basic helix-loop-helix (bHLH) TF family within these regions.58 Additionally, Tet2 deficiency may regulate RNA methylation. A recent study found that TET2 deficiency results in the accumulation of methyl-5-cytosine modification in TSPAN13 mRNA, which stabilizes the TSPAN13 mRNA recognized by YBX1. Increased expression of TSPAN13 activates the CXCR4/CXCL12 signaling, promoting LSCs homing/migration into the BM niche.59 Because the three TET proteins exhibit functional redundancy, a mouse model of inducible deletion of all three Tet genes (iTKO) was generated recently.60 Interestingly, these iTKO mice developed rapid and fatal AML within 4 to 5 weeks, and a striking increase in the expression of all members of the stefin/cystatin gene cluster was observed. Independent of DNA methylation, the increased expression of stefin/cystatin cluster genes was associated with a heterochromatin-to-euchromatin compartment switch, indicating that TET deficiency can also change the 3D genomic structure (Fig. 2).

Additionally, mutations of IDH1/2 provide an important metabolic input for driving AML pathogenesis, as IDH1/2 are the key regulator of intercellular αKG homeostasis. αKG serves as a critical cofactor for many dioxygenases, such as prolyl hydroxylases (PHDs), Egl-9 family hypoxia-inducible factor 1 (EGLN1), and TET enzymes.61 Thus, IDH1/2 mutations reduce the level of αKG accompanied with increased production of 2HG, which inhibit the activity of TET enzymes. AML with IDH1/2 mutations displays a specific DNA hypermethylation signature, impairs hematopoietic differentiation, and increases the expression of stem/progenitor cell markers.48 A recent study revealed another key metabolic pathway, the branched-chain amino acid (BCAA) pathway, which regulates αKG homeostasis in AML stem cells.62 Cytosolic BCAA transaminase 1 (BCAT1) transfers the α-amino group from BCAAs to αKG, yielding glutamate and the respective branched-chain α-keto acid (BCKA). BCAT1 is overexpressed in AML stem cells and restricts αKG levels, leading to IDHmut-like DNA hypermethylation. By contrast, knockdown of BCAT1 causes αKG accumulation that leads to EGLN1-mediated hypoxia-inducible factor-1α (HIF1α) protein degradation, subsequently abrogating leukemia-initiating potential.62 IDH mutations can also affect fatty acid metabolism. The comprehensive metabolomic analysis identifies an IDH1mut-specific reprogramming of fatty acid metabolism, which provides a targetable synthetic lethal vulnerability for IDH1mut AML63 (Fig. 2).

Overall, aberrant DNA methylation caused by recurrent mutations function as a starting engine to control the fate of HSPCs and initiates AML development by tuning up a series of cellular and molecular programs, including the transcriptional programs, chromatin modifications, genomic structures, and metabolic networks.

3. RNA MODIFICATION

According to the central law of genetics, RNA is a key mediator in the transfer of genetic information from DNA to proteins. Over 170 types of RNA modifications have been identified over several decades; however, the revival of this field should be attributed to the discovery of RNA modification effects in regulating gene expression and to the technological developments for characterizing these modifications at the transcriptome-wide level.64 Currently, it is known that RNA modifications substantially affect RNA fate by regulating its structure, splicing, stability, or translation. In the following paragraphs, we mainly focus on m6A briefly discussing the modifications of pseudouridine (Ψ), 5-methylcytosine (m5C), and N1-methyladenosine (m1A).

3.1. The features of pseudouridine, m5C, and m1A

Different RNA modifications confer distinct properties and play important roles in almost all biological processes. Pseudouridine (Ψ) is the first RNA modification identified in the 1950s,65 and widely presents in most RNA species. A class of enzymes called pseudouridine synthases (PUSes) catalyze uridine into Ψ,66,67 and the specific cellular localization of these enzymes confers them the potential to target their unique RNA species. For example, PUS1 predominantly localizes to the nucleus and modifies tRNA, mRNA, small nucleolar RNAs (snRNAs), and non-coding RNAs (ncRNAs).68 Notably, the eraser and readers for Ψ remain unclear. It plays important roles in RNA biogenesis, structure, stability, and gene expression. Pseudouridine in ribosomal RNA (rRNA) is required for translational fidelity,69 and Ψ in tRNA maintains its structure and stability.70 However, the effect of pseudouridine on mRNA expression remains unclear.

m5C is a rare RNA modification that was first observed in Escherichia coli RNA.71 This methylation occurs at position 5 of the cytidine residue on tRNA, mRNA, rRNA, mRNA, or enhancer RNA (eRNA). The NOL1/NOP2/SUN domain (NSUN) family including NSUN1-7 has been identified as m5C writers, and NSUN2 mainly catalyzes m5C in mRNA.72 Interestingly, DNMT2 can also catalyze the formation of m5C on RNA,73 and TET proteins not only oxidize 5-mC in DNA but also act as erasers of RNA m5C by oxidizing m5C to 5-hydroxymethylcytidine (hm5C).74,75 ALKBH1, a 2-oxoglutarate/Fe2+-dependent dioxygenase, is another m5C demethylase.76 About the readers of m5C, the mammalian mRNA export adaptor ALYREF was the first identified as a mRNA m5C reader.72 Interestingly, the RNA-binding protein (RBP) YBX1 has also been identified as another m5C reader. YBX1 can read m5C-modified mRNAs via its indole ring in the cold-shock domain.77 Currently, the protein machinery of RNA m5C remains to be investigated. Similar to other RNA modifications, m5C plays a distinct role in these RNA subtypes. For instance, m5C affects tRNA or rRNA structure,78 and m5C marks regulates mRNA export mediated by the ALYREF reader.72

m1A, which is methylated at position 1 of adenosine, was first identified in mammalian and plant RNA in the 1960s.79,80 The known writer of m1A is a complex containing tRNA methyltransferase 6 non-catalytic subunit (TRMT6) and RNA methyltransferase catalytic 61A (TRMT61A).81,82 RNA m1A also shares some regulators (eg, YTHDF2) with m6A but with a relatively low affinity.83 ALKBH1 and ALKBH3 are m1A erasers. ALKBH3 is the only known eraser of m1A.84,85 In addition to m5C demethylation, ALKBH1 also mediates the demethylation of m1A in tRNAs.86 m1A primarily affects the structures and functions of tRNA and rRNA. Since m1A is preferentially enriched around the start codon upstream of the first splice site, it is associated with translation initiation and affects translation efficiency.87

The roles of these RNA modifications in the hematopoietic system and related malignancies remain elusive. A recent study revealed that RNA m5C methyltransferases (eg, NSUN1/3) may be associated with leukemia cell drug sensitivity.88 ALKBH3 is dispensable for HSC maintenance and differentiation; however, ALKBH3 overexpression may rectify HSC aging.89 Therefore, it is necessary to further explore the role of these RNA modifications under various physiological and pathological conditions.

3.2. RNA m6A modification

RNA m6A is the most abundant and best-characterized modification in mammalian mRNA. It is known that m6A modification is reversible, highly dynamic, and controlled by m6A modifiers, including writers, erasers, and readers. The writer is a multicomponent complex composed of two core methyltransferase-like proteins (METTL3 and METTL14) and multiple regulatory proteins (WTAP, VIRMA, CBLL1, RBM15/RBM15B, and ZC3H13).90–94 In this complex, METTL3 acts as the sole catalytical protein for the transfer of a methyl group from S-adenosylmethionine (SAM) to the sixth N atom of RNA adenosine, and METTL14 maintains the complex structural stability.95 Other subunits regulate the activity and specificity of this complex.91 Both FTO and ALKBH5 are two main α-KG/Fe-dependent m6A demethylases.96,97 The m6A readers include YTH domain-containing protein 1-2 (YTHDC1-2), YTH domain-containing family member 1-3 (YTHDF1-3), and insulin-like growth factor-2 mRNA-binding protein (IGF2BP) family IGF2BP1-3.98–101 These readers recognize m6A sites and perform key functions in the regulation of distinct mRNA fates. YTHDF1 and YTHDF3 mainly regulate the translation of their targets102; in contrast, YTHDF2 facilitates the degradation of its m6A targets.103 YTHDC1 is involved in the export of m6A-tagged mRNAs from the nucleus to the cytoplasm,98,104–106 whereas YTHDC2 regulates the translation and stability of m6A targets.107–109 Another group, IGF2BPs, mainly stabilizes their m6A targets.101 Our group revealed that the function of IGF2BPs requires YBX1 participation, as YBX1 facilitates IGF2BPs to recognize and stabilize m6A-tagged transcripts.110,111 In the future, identifying the cofactors of m6A modifiers may be much more important.

3.3. RNA m6A modification is essential for the generation and maintenance of hematopoietic ecosystem

HSCs are generated from embryonic precursors such as hemogenic endothelial cells and pre-HSCs during mid-gestation and progressively migrate into the fetal liver for expansion.112,113 Before birth, HSCs migrate to the bone marrow, where they maintain a hematopoietic ecosystem via self-renewal and differentiation. Increasing evidence has shown that RNA m6A plays a key role in the generation and maintenance of HSCs.

RNA m6A governs HSCs generation and expansion during hematopoietic development. m6A functions as a rheostat to control endothelial-to-hematopoietic transition (EHT). Activation of Notch1 signaling maintains endothelial cell identity and represses HSPC programming during EHT. m6A inhibits Notch1a expression by promoting its degradation mediated by YTHDF2.114 The m6A modification is also involved in innate immune responses. Double-stranded RNA (dsRNA) from foreign pathogens such as viruses triggers the activation of the cellular innate immune response. Interestingly, the loss of m6A modification results in the aberrant formation of endogenously derived dsRNA in HSCs, which induces an aberrant innate immune response and subsequently causes hematopoietic failure and perinatal lethality.115 These studies have revealed the key role of m6A in embryonic hematopoiesis.

In adult hematopoiesis, m6A balances self-renewal and differentiation of HSCs. Several studies have demonstrated that m6A loss blocks the normal differentiation and causes the accumulation of phenotypical HSCs with long-term hematopoietic disorders and impaired hematopoietic reconstitution potential.116–120 This effect is mediated by fine-tuning the expression of many key factors (eg, MYC, SON). MYC is a major determinant of HSC differentiation, and HSCs normally maintain very low levels of MYC protein. Interestingly, MYC is a direct target of m6A in HSCs, and Mettl3-deficient HSCs failed to upregulate MYC expression upon stimulation to differentiate.117,118 The SON RBP is a central component of the nuclear speckles. A recent study found that SON is another essential m6A target required for murine HSC self-renewal, symmetric commitment, and inflammation control.121 Similarly, the important role of m6A in adult HSCs was revealed by studying other key components of the m6A pathway. For instance, YTHDF2 depletion causes HSC expansion without skewing lineage differentiation preference.122,123 In contrast, the m6A eraser, AlKBH5, is not dispensable for adult hematopoiesis and HSC function.124,125 This might be due to the functional redundancy between ALKBH5 and FTO. Thus, these studies revealed a developmental stage-specific requirement for m6A in hematopoiesis, which requires the decoding of the dynamics of m6A modifications in the hematopoietic system.

To address this fundamental question, we recently delineated a comprehensive m6A landscape across the hematopoietic hierarchy by developing a super low-input m6A sequencing (SLIM-seq) strategy to profile the m6A landscape of HSCs and their progeny at the transcriptome-wide level. Interestingly, we observed a cell type-specific m6A landscape during hematopoiesis. m6A modifications arise mostly in the early stages of hematopoiesis and play distinct roles in determining mRNA fates in HSCs and committed progenitors. Furthermore, we confirmed that m6A is required to balance the quiescent active states of HSCs, which is mainly mediated by the m6A reader IGF2BP2. IGF2BP2 deficiency results in quiescence loss and impairs HSC function by increasing mitochondrial activity of HSCs.126

The interactions between hematopoietic cells and their niche are critical for hematopoietic ecosystem. m6A can also affect the HSC niche, which comprises several key cell types (eg, mesenchymal stromal cells [MSCs] and osteoblasts). Previously, epitranscriptomic programs have been shown to be involved in skeletal health and diseases, such as osteoporosis mese.127 A recent study indicated that the m6A-mediated epitranscriptomic program regulates the generation, but not maintenance, of the bone marrow HSC niche.128 Comparison of perinatal and adult bone marrow MSCs revealed that m6A-related genes are enriched in MSCs at the perinatal stage, whereas Mettl3 is rapidly downregulated after birth. Deletion of Mettl3 from developing MSCs resulted in a severe defect in HSC niche formation owing to excessive osteogenic differentiation, whereas deletion of Mettl3 from MSCs postnatally did not affect the HSC niche. Overall, m6A-mediated epitranscriptomic program controls the generation and maintenance of the hematopoietic ecosystem.

3.4. RNA m6A modification reprogramming is required for AML development

Most myeloid leukemias are initiated by LSCs that are transformed from HSPCs along with dysregulated programs (eg, aberrant DNA methylation). The role of RNA m6A in leukemia especially AML has gradually been recognized. For instance, the expression levels of m6A-related genes (eg, METTL3, METTL14, FTO, ALKBH5, YTHDF2, IGF2BP2) are obviously increased in AML and are associated with poor prognosis.120,125,129–131 Recently, we decoded the m6A landscape during AML development and observed obvious changes in the m6A methylome during leukemogenesis. Interestingly, we found that approximately 60% of the m6A targets identified in LICs were newly established and LIC-specific when compared to normal HSPCs, suggesting that reprogramming of RNA m6A modification occurs during cellular transformation.131,132 Importantly, these m6A-tagged targets are involved in the regulation of many hallmarks of cancer, such as sustaining proliferation, resisting cell death, metabolic adaptation, epigenetic reprogramming, and immune evasion (Fig. 3). In this section, we highlight three aspects.

Figure 3. RNA m6A modification regulates cancer hallmarks. m6A modification determines mRNA fates of many key regulators, which involves regulating AML properties including sustaining proliferation, resisting cell death, metabolic adaptation, epigenetic reprogramming, and immune evasion. AML = acute myeloid leukemia.

RNA m6A regulates various metabolic adaptations that are employed by AML cells to meet their metabolic requirements. For instance, we found that the m6A reader IGF2BP2 restricts the uptake of docosahexaenoic acid levels via the PRMT6-MFSD2A axis to balance the fatty acid metabolism in LSCs.131 In addition, IGF2BP2 controls glutamine metabolism by regulating the expression of critical targets (eg, MYC, GPT2, and SLC1A5) in an m6A-dependent manner.133 As discussed previously, IDHmut catalyzes the production of the oncometabolite R-2HG. Interestingly, R-2HG abrogated the FTO-m6A-YTHDF2-mediated post-transcriptional upregulation of phosphofructokinase platelet (PFKP) and lactate dehydrogenase B (LDHB), two critical glycolytic genes, thereby suppressing aerobic glycolysis in R-2HG-sensitive leukemia cells.134 Collectively, these studies revealed a key role of m6A in fine-tuning the metabolic adaptations of three key nutrients (glycolysis, amino acids, and lipids) in AML (Fig. 3).

RNA m6A interwines and cooperates with epigenetic alterations to promote AML development. By analyzing chromatin accessibility during leukemogenesis, we found that ALKBH5 was regulated by chromatin state alteration, which were mediated by the histone demethylase KDM4C. H3K9me3 around the ALKBH5 promoter region is removed by KDM4C to increase chromatin accessibility and recruitment of MYB and Pol II.125 ALKBH5 is required for LSCs and AML development, but not for adult hematopoiesis and HSC function. Alkbh5 loss significantly inhibits AML development and progression by affecting AXL mRNA stability in an m6A-dependent manner and the downstream PI3K/AKT pathway in AML. In addition, IGF2BP2 regulates the protein arginine methyltransferase PRMT6, which subsequently catalyzes the asymmetric dimethylation of histone H3R2 (H3R2me2a).131 Therefore, RNA m6A involves in the epigenetic reprogramming of AML cells (Fig. 3).

Immune evasion is another important hallmark of cancer, and m6A is involved in cancer immunology via multiple mechanisms. m6A modifiers upregulated in cancer cells may regulate the expression of the inhibitory immune checkpoint proteins (eg, PD-L1), or influence the function of immune cells in the tumor microenvironment (TME). For instance, FTO facilitates the immune surveillance escape of cancer cells through regulating glycolytic metabolism.135 In addition, FTO upregulates the expression of immune checkpoint genes including PD-L1 and LILRB4 in AML cells, thereby contributing to tumor immune evasion. Pharmacological inhibition of FTO sensitizes leukemia cells to CD8+ T cell cytotoxicity and overcomes hypomethylating agent-induced immune evasion.136 RNA m6A can also modulate the function of different immune cell populations (eg, T cells and dendritic cells) and shape the TME, which favors cancer growth. m6A controls CD8+ T cell differentiation and sustains Treg suppressive functions.137 m6A also regulates durable neoantigen-specific immunity. Loss of YTHDF1 in classical dendritic cells enhances cross-presentation of tumor antigens and cross-priming of CD8+ T cells.138 Thus, these data implicate RNA m6A in cancer immunology. Overall, the m6A modification plays an important role in modulating the programs that confer cancer hallmarks (Fig. 3).

4. CONCLUSION AND PERSPECTIVE

Epigenetic modifications act as key regulators of the hematopoietic system, and epigenetic alterations are widely considered as common genetic events in hematological malignancies. It should be noted that here we mainly discussed DNA methylation and RNA m6A modification, as DNA methylation alteration is the key driver of many myeloid malignancies, and the field of RNA m6A is exponentially growing. This striking evidence clearly shows the potential of targeting these epigenetic alterations in AML treatment.

Although their physiological and pathological roles have been well established, emerging interesting questions need to be elucidated. First, therapeutic strategies targeting epigenetic modifications are insufficient, and the development of more effective drugs is urgently needed for AML therapy. Second, it is necessary to further clarify the distinct roles of different m6A modifiers in different contexts, particularly how these modifiers sense various environments. Our recent work implies that RBPs or related cofactors in the regulatory machinery are key to determine their target specificity. Third, large gaps still exist between the current research and future clinical applications. The efficient translation of the findings on RNA m6A modifications and DNA methylation into real treatment strategies remains challenging. For instance, several inhibitors of RNA m6A modifiers (eg, STM2457, CS1, CS2, and CWI1-2) have been developed, but there is still a long way to go before the clinical application of these inhibitors.133,136,139 Collectively, epigenetics has attracted considerable attention, and it is promising to improve therapeutic efficiency by targeting epigenetic modifications.

ACKNOWLEDGMENTS

This work is supported by the grants to H.Z. from the National Key R&D Program of China (2022YFA0103200), and the National Natural Science Foundation of China (82325003, 82230007). This work is supported by the grants to R.G. from the Fundamental Research Funds for the Central Universities (2042023kf0057) and the National Natural Science Foundation (NSFC) (82301997). This work is also supported by the grants to R.Y. from the National Natural Science Foundation of China (82200188) and the Special Fund of China Postdoctoral Science Foundation (2022TQ0238). This work is also supported by the grants to H.Z. from the Fundamental Research Funds for the Central Universities (2042022dx0003 and 2042024kf1022).

We appreciate the members of our laboratory for discussion.

AUTHOR CONTRIBUTIONS

H.Z. wrote the manuscript with the help of S.Y. and R.G. All the other authors contributed to providing information, reviewing and revising this manuscript.

Conflict of interest: The authors declare that they have no conflict of interest.

S.Y., R.G., W.T. contributed equally to this work.

This work is supported by the grants to H.Z. from the National Key R&D Program of China (2022YFA0103200), and the National Natural Science Foundation of China (82325003, 82230007). This work is supported by the grants to R.G. from the Fundamental Research Funds for the Central Universities (2042023kf0057) and the National Natural Science Foundation (NSFC) (82301997). This work is also supported by the grants to R.Y. from the National Natural Science Foundation of China (82200188) and the Special Fund of China Postdoctoral Science Foundation (2022TQ0238). This work is also supported by the grants to H.Z. from the Fundamental Research Funds for the Central Universities (2042022dx0003 and 2042024kf1022).
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