Immunotherapeutic Potential of m6A-Modifiers in Controlling Acute Myeloid Leukemia

Epigenetic alterations have contributed greatly to human carcinogenesis. Conventional epigenetic studies have been predominantly focused on DNA methylation, histone modifications and chromatin remodelling. However, recently, RNA modification (m6A-methylation) also termed ‘epitranscriptomics’ has emerged as a new layer of epigenetic regulation due to its diverse role in various biological processes. In this review, we have summarized the therapeutic potential of m6A-modifiers in controlling haematological disorders especially acute myeloid leukemia (AML). It is a type of blood cancer affecting specific subsets of blood-forming hematopoietic stem/progenitor cells (HSPCs) which proliferate rapidly and acquire self-renewable capacities with impaired terminal cell-differentiation and apoptosis leading to abnormal accumulation of white blood cells, and thus an alternative therapeutic approach is required urgently. Here, we have described how RNA m6A-modification machineries EEE (Editor/writer: Mettl3, Mettl14; Eraser/remover: FTO, ALKBH5 and Effector/reader: YTHDF-1/2) could be reformed into potential druggable candidate or as RNA modifying drug (RMD) to treat leukemia. Moreover, we have shed-light on the role of microRNA and suppressor of cytokine signalling (SOCS/CISH) in increasing anti-tumor immunity towards leukemia. We anticipate, our investigation will provide a fundamental knowledge in nurturing the potential of RNA modifiers in discovering novel therapeutics or immunotherapeutic procedures.


Introduction
There are several conventional drugs, including epigenetic-based drugs targeting DNA and histone modifications, have been already approved by the US food and drug administration (FDA) for the treatment of AML. However, targeting RNA-modification machineries as RNA modifying drugs (RMDs) refuelled the scientific interest to discover novel therapeutic drugs. Although RNA modification was discovered as early in 1970s [1,2] but it refuelled the passion of scientific research after the discovery of first m6A-demethylase in 2008 and 2011 [3,4].To date more than 150-170 chemical modifications have been identified [5,6] in methylating approximately 7,000 human transcripts; among them, 50% is conserved in mouse [7]. The abundance of total RNA in a single cell of 10-30 pg consisting of 85% r-RNA, 15% t-RNA and 1-5% m-RNA, where the majority of m6A-modification takes place in the coding region (CDS) of mRNA enriching near 3`UTR and 5`UTR stop codon requiring conserved DRACH (D = G/A/U; R = G/A; H = A/C/U) sequences [8][9][10] . The three major RNA epigenetic modifiers reforming the whole biological functions are writers, erasers and readers. The detailed mechanism of m6A-modification and gene regulation is well described in several excellent journals. However, this review mainly focuses on reforming RNA epigenetic machineries into druggable form to treat haematological disorders especially AML.

AML Therapeutics
AML is a group of neoplastic diseases involving bone marrow with or without the involvement of peripheral blood. It is the most common type of haematological disorder prevalent in children & adults and globally affecting nearly 3-million people every year [11]. For some time now, several conventional therapies have been employed to treat AML including chemotherapy and rarely surgery and radiation [11]. Cytotoxic chemotherapy or remission-induction with chemotherapeutic agents (anthracycline and cytarabine) followed by consolidation-therapy involving an allogeneic stem cell transfer (SCT), bone-marrow transplant (BMT) or hematopoietic stem cell transplantation (HSCT) have been used as a standard therapy regimen for prolonged remission of AML [12]. However, due to the delayed diagnosis of AML and the therapy-related morbidity and mortality, these conventional means of treating AML have fallen short when it comes to "do no harm" approach. Recently, with the advancement of NGS and gene-mutational analysis, several new strategies have been under development to minimize the global effects of these conventional therapies [13,14]. One of these new strategies includes the 'targeted-drug therapy" which is cell, gene or marker specific and allows the option of treatment in patients where invasive chemotherapy is not feasible. This targeted therapy includes biological (monoclonal antibodies), epigenetic or combination therapies [15] ( Figure 1, Table 1). Several antigen-specific monoclonal antibodies have now been approved by the FDA for AML therapy. Among these, CD33-directed Gemtuzumab ozogamicin (GO) mAb was the first to be used for AML therapy [16]. In addition, several studies have now emerged marking DNA methylation as one of the hallmarks of AML carcinogenesis [17] involving DNA methyltransferases (DNMTs) and Ten-eleven-translocation (TET) dioxygenases [18]. Various DNMT inhibitors have been investigated for AML therapy, however only two of these (azacitidine and decitabine) are FDA approved [19]. On the other hand, any mutation in the TET family members results in altered DNA methylation [20]. For example, isocitrate dehydrogenases (IDH1 and IDH2) can inhibit TET2 and can cause hypermethylation resulting in AML progression [21]. Therefore, IDH inhibition can resolve this TET2 reduction-induced hypermethylation [22]. The FDA has approved two oral IDH inhibitors: ivosidenib (AG-120) and enasidenib (AG-221) for AML treatment [23,24]. In addition to DNA modifiers, histone modifiers (HATs and HDACs) have also been implicated in AML treatment [25] by regulating tumor suppressor genes (TSGs) [26,27]. At present, four HDAC inhibitors have been approved by the US FDA including three as panHDACi: belinostat, vorinostat, panobinostat, and romidepsin as a selective HDACi [28]. In this review, we have summarized the therapeutic potential of m6A-modifiers in controlling AML. Furthermore, we have also summarized the current biopharmaceutical companies developing AML therapeutics as RNA modification drugs (RMDs) [6] Figure 1. Development of AML therapeutics. The major AML therapeutics are depicted in the diagram above which includes; Conventional therapy (chemotherapy, radiotherapy and surgery), Biological therapy (stem cell therapy or bone marrow transplant), Epigenetic drugs (DNA and histone modifier-based drugs), RNA-modifying drugs (writers/editors, erasers/removers and readers/effectors), MicroRNAs (miR-29b, miR-34a and miR-150) and Suppressor of cytokine signalling (SOCS1/CISH). Additionally, we have mentioned some biopharmaceutical companies developing RMDs and estimated phase-I clinical trials.

Epitranscriptomics in AML therapy
The development of advanced molecular techniques potentiates the opportunities to discover immune cell-based therapeutics over conventional pharmacological-based treatments. This section describes the strategies to treat haematological disorders, especially AML by targeting intracellular RNA-modification machineries, like EEE (Editor/writer: Mettl3, Mettl14; Eraser/remover: FTO, ALKBH5 and Effector/reader: YTHDF-1, YTHDF-2), microRNAs and Suppressor of Cytokine Signalling family of proteins (SOCS/CISH) and as a personalized medicines ( Figure 1).

Mettl3 in AML:
The author has demonstrated the role of m6A writer enzyme 'Mettl3' in AML progression [29] supported by Barbieri et. al., [30] and enlightened the molecular mechanism to control leukaemia. He found that 'Mettl3' is normally expressed in CD34+ hematopoietic stem/progenitor cells (HSPCs) but aberrantly expressed in leukemia cells as compared to the other tumor-types. However, Mettl3-silencing (shRNA) in HSPCs promoted HSPCs differentiation and apoptosis but inhibited self-renewal capacity & proliferation. Conversely, Mettl3 overexpression inhibited cell-differentiation and apoptosis but allowed cell-proliferation and self-renewal capacities. These results clearly suggest that Mettl3 facilitates AML cell growth but resist cell-differentiation. Furthermore, it was also reported that the adoptive transfer of Mettl3-deficient (shMettl3) human AML cell line (MOLM-13) into immunodeficient (recipient) mice induces cell-differentiation and apoptosis but remarkably delayed leukemia progression. This result further supports the key role of Mettl3 in AML progression. Mechanistically, he justified (via m6A-mapping and ribosome profiling) that Mettl3 increases the expression of c-Myc, Bcl2 (apoptosis regulator) and pTEN (a proto-oncogene or AKT regulator) genes by m6A-modification in human leukemic MOLM-1 cell-line. Moreover, Mettl3 regulates AKT-RICTOR signalling (PI3K-AKT) pathway and contributes to AML pro-gression by increasing cell-proliferation and decreasing cell-differentiation capacities. However, the loss of Mettl3 activates pTEN-mediated AKT phosphorylation and controls AML by reducing cell-proliferation and promoting cell-differentiation, confirmed after treating AML cells with PI3K-inhibitor in blocking cell-differentiation under Mettl3-deficient state [29]. Taken together, these investigations confirm the key role of Mettl3 in facilitating AML growth and survival and potentiate the therapeutic value of Mettl3 in controlling AML by means of either specific inhibition or selective intracellular silencing in AML. These findings were further supported by Konstantinos et. al., 2019[31]; by synthesizing two small molecule compounds of Mettl3-inhibitor in inhibiting AML cell expansion (at 30mg/kg oral/intraperitoneal dose in mouse model with no clinical toxicity and weight loss) Table 2 Weng et. al., 2018 [34]; demonstrated the role of m6A-writer enzyme 'Mettl14' in AML progression, and enlightened the intrinsic mechanism to control AML by targeting Mettl14. The authors have found that 'Mettl14' is aberrantly expressed in AML patients confirmed by Cancer Genome Atlas (TCGA)-datasheet, human leukemia cell lines (MM6 and NB4), immortalized BM-progenitor cells (MA9, AE9a, PML-RARa) and AML cells carrying t(11q23), t(15;17), or t(8;21) translocation)-mutations, and gradually started decreasing during myeloid differentiation, suggesting the prominent role of Mettl14-mediated m6A-modification in leukemia progression. Experimentally he showed that, the in-vivo depletion of Mettl14 in recipient conditional knockout mice (tamoxifen-induced silencing) Mettl14 cKO or Mettl14 fl/fl -CRE ERT mice decreases cell proliferation and self-renewal capacity of CD45.2 + (of Mettl14 cKO ) in peripheral blood as compared to the CD45.1 + CD45.2 + (competitor) cells. However, in-vitro depletion of Mettl14 ('shM14' pLKO.1 lentiviral-mediated silencing) in human HSPCs/-CD34 + promoted cell-differentiation, which was confirmed by increase of differentiation marker (CD11b-monocyte and CSF1R-macrophage). This suggests that Mettl14 induces leukemia growth by increasing cell-proliferation and decreasing terminal cell-differentiation. Conversely, the addition of differentiation inducing agent; OP9 culture medium [35], PMA (0.5ng/ml/48h) [36] and all-trans retinoic acid (ATRA 500nM/72h) [37] significantly decreased the expression of Mettl14 and overall m6A-abundance in AML cells. Reciprocally, Mettl14 overexpression (Mettl14-R298P vector) inhibits myeloid differentiation as compared to the empty vector. These results clearly suggest that Mettl14 is playing a significant role in AML progression by decreasing cell-differentiation and increasing cell-proliferation, and thus designating 'Mettl14' as a potential therapeutic target for AML treatment. Mechanistically, they have justified that (via RNA-sequencing) Mettl14-methylated methylation of Myb and Myc increase mRNA stability [38] leading to highly upregulated level of these transcription factors (TcFs) in leukaemia's and lymphomas [39-41], confirmed by reduced half-life due to decreased polyribosomes 40S, 60S, 80S (ribosome profiling) and eukaryotic initiation factor-3A (EIF3A)-binding to the Myb and Myc transcript in Mettl14-silenced (shMettl14) MM6 and NB4 (human leukemic) cell lines as compared to the control (shCON) transfections. Furthermore, m6A mapping and immunoprecipitation (miCLIP) revealed increased m6A-binding to the Myb and Myc transcripts relative to the IgG iso-control, suggesting the significant role of Mettl14-mediated m6A-modification in increasing Myb and Myc level and further downstream signalling cascades rather than decaying respective mRNAs. However, they did not find significant involvement of YTHDF2 reader protein in regulating TcFs by mRNA-decay mechanism [38] in Mettl14-deficient AML cells. Collectively, these investigations suggests that Mettl14 promotes AML progression by increasing the activity of Myb (in increasing cell proliferation [42]) and Myc (in inhibiting cell differentiation [43,44]) gene functions. Moreover, they found that PU.1 (Spl1 encoded transcription factor, also known as master regulator of myeloid cells, or an oncogene product) binds to Mettl14 at 5kb upstream of TSS and negative regulates its expression, confirmed by chromatin immunoprecipitation (ChIP-seq) followed by luciferase reporter assays. Furthermore, Spl1-knockdown increases both mRNA and protein level of Mettl14. Conversely, Spl1 overexpression decreases Mettl14 expression. These results further suggests that Mettl14 itself is targeted by 'Spl1' and an additional therapeutic target to control Mettl14 expression. In support of this Konstantinos et. al., 2019; recently developed a small molecule 'inhibitor of Mettl3' by structure-guided medicinal chemistry tool and confirmed m6A-inhibition and respective cellular effects comparable to the Spl1-inhibitor, This study further support the therapeutic potential of Mettl3-inhibitor in controlling AML progression [31]. Taken together, Mettl14 showed its immunotherapeutic potential by regulating Spl1-Mettl14-Myb/Myc signalling axis, and therefore intracellular silencing of Mettl14 in combination with standard ATRA (differentiation inducing agent) therapy would be a promising approach to control AML with the other forms of therapeutic approaches (radio/chemo/BM-transplantation) [34], ( Figure 2, Table  2).  Li, et. al., 2017 [47]; have demonstrated the oncogenic role of FTO, an m6A-demethylase, in facilitating leukemogenesis by promoting cell-proliferation (self-renewal) and reducing cell-differentiation and apoptotic processes, and enlightened the molecular mechanism to control APL by targeting FTO. They found that, FTO is aberrantly expressed in a specific subset of hematopoietic stem/progenitor cells (HSPCs/CD34 + CD38 -) carrying t(11q23)/MLL-rearrangement and t(15;17)/PML-RARA mutations, called acute promyelocytic leukemia (APL), confirmed in one hundred 'affected' and nine 'control' human samples of (CD34 + CD33 + and MNC) cells with Affymetrix GeneChip and qRT-PCR assays. In order to investigate the causative mechanism and the potential target of FTO in facilitating leukemogenesis, they overexpressed FTO in human leukemic 'MONOMAC-6' cell lines via retrovirus expression system (MSCV-PIG-FTO and MSCV-PIG-CON) and voted two stable clones under 0.5µg/ml puromycin selection marker. Before sequencing, they confirmed increase and decrease of FTO and m6A-abundancy by western blot and dot-blot/methylene blue assays. Interestingly, the sequencing result revealed two potential target of FTO, namely ankyrin repeat and SOCS box protein 2 (ASB2), involved in controlling leukemia [48] by suppressor of cytokine signalling (SOCS/CISH)-mediated mechanism, and retinoic acid receptor alfa (RARA), a hallmark feature of APL [49], selected on the basis of MACS and exomePeak methods [50,51]. Moreover, conserved m6A-methylation motif 'GGAC' was also confirmed enriching >87% CDS as well as near stop codon vicinity. The authenticity of this potential target gene was validated by dual-luciferase reporter coupled with mutagenesis assays, where, [FTO cloning: pMIRNA1-FTO (wild-type), pMIRNA1-FTO-Mut (mutant) and pMIRNA1-Ctrl (empty vector); and target gene cloning: pMIR-REPORT-ASB2-3`UTR containing wild-type 'GGAC' and mutant 'GGTC' with four m6A-conserved motifs 464bp; RARA-3`UTR with five conserved motif 474bp and RARA-5`UTR with six conserved motif 395bp and transfected into HEK-293T cells/24h] the wild-type FTO significantly decreased the luciferase activity of ASB2 and RARA fragments containing wild-type m6A-methylated 'GGAC' motifs as compared to the other combinations of mutant and control transfections, indicating the requirement of m6A-methylation site for the regulation of FTO-mediated demethylase activity [52,53]. Furthermore, the FTO target was again validated by silencing FTO (shFTO) in MA9/FLT3-ITD leukemic cell lines in hyper-methylating (increased m6A-modification) ASB2 and RARA genes via m6A-seq and RNA-seq respectively. These results clearly suggest that (i) FTO regulate its target gene expression by its m6A-demethylase activity and (ii) the target gene must be m6A-modified. However, under hypo-methylated condition the exact mechanism of FTO-mediated negative regulation of its target gene expression was further justified by mRNA stability testing, where FTO overexpressing cell line (5µg/mL actinomycin D for 0, 3 and 6h) showed increased mRNA-decay (i.e. decreased half-life t1/2= In2/Kdecay) compared to the FTO silencing, suggesting the mechanism of target gene control by destabilizing the mRNA stabilities. These investigations clearly suggests that, under diseased state, the aberrantly expressed FTO negatively regulate its target gene expression; neither by directly decreasing m6A-level (hypo-methylation) of the target mRNA nor by m6A-effector protein YTHDF2-mRNA decay-dependent mechanisms, but by directly affecting the mRNA stability via its demethylase activity [38] . However, the possibility of m6A-reader protein (YTHDF1 and YTHDF2)-mediated regulation of ASB2 and RARA also cannot be denied completely due to the finding of increased target gene translation after YTHDF1 knockdown. Nevertheless, the FTO-mediated APL progression is not only by targeting mRNA destabilization, but also by affecting the efficacy of 'ATRA-mediated therapy' [54,55] justified by analysing NB4 cell (human acute promyelocytic leukemia cell line) differentiation marker. Where, the overexpression of FTO significantly inhibits ATRA-induced cell-differentiation of NB4 cells [56] characterized by decreased (differentiated: CD11b + CD14 + ) and increased (un-differentiated: CD11b -CD14 -) markers compared to the FTO-silenced (shFTO) cells in reversing the both phenotypes. Conversely, the induction of 500mM ATRA significantly decreased FTO and thereby rescued/increased the expression of its target ASB2 and RARA genes [57,58]. Moreover, forced expression of ASB2 and RARA also increases cell-differentiation. Collectively, these results authenticate that FTO is playing a crucial role in promoting acute promyelocytic leukemia (i) by inhibiting the efficacy of AT-RA-mediated induction of cell-differentiation and apoptosis, and (ii) by suppressing its target gene expression. Taken together, these studies revealed the oncogenic property of FTO in promoting APL. Therefore, targeting FTO would be a recommendable approach to control APL with other forms of chemotherapeutic drug combinations [47,59] ( Figure  3, Table 2, 3).  Shen et. al., 2020 [61]; have demonstrated the role of other m6A-eraser protein 'α-ketoglutarate-dependent dioxygenase AlkB homolog 5' (ALKBH5) in facilitating leukemia stem/initiating cells (LSCs/LICs) progression, a subsets of AML, characterized by high self-renewal capacity, and enlightened intrinsic mechanism to control LSCs by targeting ALKBH5. They found that, likewise the other m6A-eraser protein FTO [47] , ALKBH5 also facilitates leukemogenesis, evidenced by significantly higher expression of ALKBH5 in various subtypes of AML cells carrying t(15;17), Inv(16), t(8;21) and t(11q23) mutations, independent of specific TP53-mutation (MONOMAC-6/MMC6, NOMO1 and NB4) and TP53-wild type (MA9.3-ITD and MOLM13) human cell lines, confirmed via GSE42519, GSE13159 Affymetrix, TCGA-sequencing and western blot analysis, as compared to the normal HSCs. However, in vitro silencing (lentiviral shRNA) of ALKBH5 in NOMO1 and MMC6 human cell lines significantly promotes apoptosis (AnnexinV hi PI hi ) and restrict cell growth/proliferation with overall decreased % survival of TCGA-AML datasheet [62]. Furthermore, ex-vivo conditional knockout of ALKBH5 in MOLM13 (MOLM13-iKD) cells (doxycycline-inducible conditional knockdown model: where addition of doxycycline depletes ALKBH5, as similar to stable knockout) also promoted apoptosis and reduced cell growth/proliferation as compared to the KO (MOLM13-iCas9) cells. Conversely, overexpression of wild-type ALKBH5 (A5-WT) reversed this effect and promoted AML-cell progression as compared to the mutant (H204A) ALKBH5, suggesting the crucial role of ALKBH5 in facilitating leukemogenesis. In addition to this, significantly increased abundance of 'global m6A-level' was observed in the bone marrow of ALKBH5-depleted (Exon-1 depletion by CRISPR-Cas9) mice compared to the wild-type mice. Moreover, marked increase in global m6A-level was also noticed in both in vitro/ex vivo ALKBH5-depleted cell lines confirmed by LC-MS/MS and dot-blot analysis as well. These results clearly suggest that (i) the m6A-demethylase ALKBH5 facilitate AML progression and required for its growth and survival. (ii) ALKBH5-depletion enhances global m6A-level. Next, to further validate the requirement of ALKBH5 in the promotion/transformation of AML cells, they used MLL-AF9 (MA9)-induced leukemogenesis model (where, MLL-rearranged fusion protein alone is sufficient to transform normal HSPCs into leukemic cells [63]) coupled with ALKBH5 knockout (ALKBH5 KO ) model. Interestingly, ALKBH5-depletion significantly inhibited MA9-mediated cell immortalization. On the contrary, forced expression of ALKBH5 (A5-WT) significantly promoted MA9-mediated cell immortalization compared to the mutant ALKBH5 (A5-Mut), confirmed by in vitro colony formation/immortalization assays. Furthermore, adoptive transfer (or bone marrow transplantation-BMT) of ALHBH5-deficient immune population (donor: CD45.2 + Lin -) co-cultured with MLL-AF9 cells into recipient (CD45.1) mice significantly delayed leukemia progression and prolonged survival with decreased splenomegaly, white blood cells count and immature blast cell (CD11b (Mac-1) + c-Kit + ) populations.

ALKBH5 in AML:
Moreover, ALKBH5-deletion inhibited the engraftment of MA9-transformed donor cells in the peripheral blood as compared to the ALHBH5-wild type donor under lethal irradiation conditions. These investigations clearly suggest that 'ALKBH5' plays a key role in promoting leukemogenesis of a specific subset of AML cells. The relevant experiments on leukemia xenograft mouse model further potentiate the involvement of ALKBH5 in self-renewal and maintenance of AML-cells. Next, In order to understand the molecular mechanism, ALKBH5-depleted MOLM13 and NO-MO1 cells were sent for RNA sequencing (RNA-seq), which revealed TACC3 (transforming acidic coiled-coil-containing protein 3) as a positive target of ALKBH5. . Surprisingly, from a total of 18 (12 positive and 6 negative targets of ALKBH5) only 'TACC3' was significant, which was not in-case of FTO, suggesting different target pattern of these two m6A-demethylaes/eraser proteins in AML. Furthermore, m6A-sequencing (m6A-seq) revealed enrichment of m6A-abundance at TACC3 mRNA (from a total of 510 transcripts with maximum enrichment around protein coding region and 3`-UTR in ALKBH5-depleted AML cells) confirmed by RNA immunoprecipitation & sequencing (RIP-seq) followed by gene-specific m6A-qRT-PCR. Mechanistically they proved that, the ALKBH5 silencing (shALKBH5) decreases the expression of TACC gene, due to hyper-methylation of m6A, confirmed by decreased half-life of TACC3 (in MOLM13: 2.35h -1.56h and NOMO1: 3.4h -1.55h) compared to control (shNS) transfections. Conversely, forced expression of ALKBH5 (A5-WT) increases the half-life (3.63h -7.47h) of TACC3 in NOMO1 cells compared to mutant (A5-Mut) transfections. Moreover, TACC3 has been seen targeting 'Myc' and 'P-21' genes as well [64,65], confirmed by decreased Myc and increased P-21 protein levels in ALKBH5-silenced NOMO1 and MMC6 cells (Shen et . al., 2020 figure-6).These result clearly suggests that (i) TACC3 is the bona fide target of ALKBH5. (ii) ALKBH5/m6A/TACC3 axis regulates p53 apoptotic pathways in AML. Next, the functional analysis of TACC3 showed similar phenotypic effect like ALKBH5 justified by increased leukemia cell apoptosis (AnnexinV hi PI hi ) and decreased cell-proliferation after silencing TACC3 (shTACC3) in NOMO1 and MMC6 cells confirmed by FACS and colony-formation and replanting capabilities. Collectively, these studies clearly suggests that the m6A-eraser protein ALKBH5 as well as also FTO promotes leukemogenesis, and therefore targeting ALKBH5 by means of either selective inhibitor of specific intracellular silencing could be a potential approach to control AML in humans ( Figure 4, Table 2  The m6A-demethylase 'ALKBH5' is aberrantly expressed in AML, especially in leukemia stem/initiating cells (LSC/LIC) and facilitates its progression, indicating its requirement for development, self-renewal, maintenance and propagation. Biologically, the enhanced ALKBH5 in disease-state increases the expression of transforming acidic coiled-coil-containing protein 3 (TACC3) gene expression by m6A-mediated mechanism, resulting in increased self-renewal capacity and thereby increased progression of LSC/LICs. (B) Therapeutic model: Anti-ALKBH5 therapy: The selective inhibition of ALKBH5 decreases m6A methylation-mediated expression of TACC3 gene resulting in decreased self-renewal and increased apoptosis is well-efficient to control AML subsets.

YTHDF2 in AML: Paris et. al., 2019 [66]
; demonstrated the role of m6A-reader protein 'YTHDF2' in promoting AML, and enlightened the molecular mechanism to control AML by targeting YTHDF2 in HSPCs. They found that, although, YTHDF2 is required for the development of leukemia stem cells (LSC) but also initiates AML development. However, selective depletion of YTHDF2 inhibits the self-renewal capacity of leukemic cells and promotes cell-differentiation and apoptosis, suggesting the therapeutic value of YTHDF2 in controlling AML progression. Experimentally, they found that, YTHDF2 targets tumor necrosis factor-α (TNF-α), required for cell-necrosis and apoptosis [67], and inhibits its expression by m6A-mediated mRNA decay mechanism [38]. However, selective removal of YTHDF2 increased the expression of TNF-α by lowering mRNA decay, confirmed by increased half-life of the target mRNA, and thereby increased terminal cell differentiation and apoptosis. This result clearly suggests that YHTDF2 plays a crucial role in promoting leukemogenesis [66] was also witnessed by Li et. al., 2019 [68]. Therefore targeting YTHDF2 would be a potential approach to control AML by means of either intracellular silencing or via specific inhibitor ( Figure 5). : Li et. al., 2019 [68]; have demonstrated the therapeutic value of m6A-reader protein 'YTHDF2' in treating haematological disorders, especially AML including other types of cancer. The hematopoietic stem progenitor cell (HSPC/CD34 + CD38 -) population is believed to be major limiting issue during stem cell transplantation therapy, due to a lesser number of HSPCs populations from a single human umbilical cord blood (hUCB) donor. Therefore, ex-vivo expansion of HSPC population is a major and encouraging challenge for its widespread use. They found that under steady state, the YTHDF2 sequesters its target gene T-cell acute lymphocytic leukemia 1 (Tal1), which is required for the normal proliferation and self-renewal of HSPCs

Mettl3 Inhibitor:
Recently, Eliza et. al., 2021[72] have discovered the selective inhibitor of Mettl3 and Mettl14 (STM2457, IC50 = 16.9 nM) in controlling AML via high-throughput screening of 250,000 drug-like compounds. The binding affinity and specificity of STM2457 was confirmed by surface plasmon resonance (SPR) and X-ray crystallography as well as by intraperitoneal injection of STM2457 (50mg/kg) in selectively reducing m6A on poly-A+-enriched mRNA, with no effect on other mRNA modifications. Functionally, they showed significant effect of STM2457 in reducing clonogenic potential and inducing apoptosis in human and mouse AML model without affecting normal human cord blood (CD34 + /HSPCs) and non-leukemia (HPC7) hematopoietic cells. Moreover, in vivo studies on 3-human AML patient derived xenograft (PDX) model and primary murine MLL-AF9/Flt3 ltd/+ model showed convincing anti-leukemia effect compared to control STM2120 inhibitors [72]. Furthermore, the other researchers also suggested the significant effect of Mettl3/14-inhibitors as anti-leukemia effect (Table 3) [6,31,33]. : Huang et. al., 2019 [60]; have examined the therapeutic efficacy of two synthetic small molecule inhibitor of FTO (FB23 and FB23-2) in controlling AML.

FTO Inhibitor
They found that the m6A-demethylase 'FTO' is highly upregulated in certain AML-subtypes and facilitates leukemogenesis by promoting cell-proliferation and inhibiting terminal cell differentiation. The FTO inhibitor especially 'FB23-2' dramatically suppresses cell-proliferation and promotes differentiation and apoptosis of both the cell lines as well as the primary cells in xeno-transplanted mice. Mechanistically, the synthesized inhibitors directly bind to the FTO and selectively inhibit its m6A-demethylase activity, mimicking FTO depletion similar to in vivo knockout. This result suggests that these FTO inhibitors (FB23 and FB23-2) could be a potential druggable candidate to treat leukemia [60], supported by Yang et. al., 2019 [7] (Table 3). Su et. al., 2020 [73] have demonstrated the clinical value of two synthetic small-molecule inhibitor of FTO, designed on the basis structural guided tool, (CS1 and CS2) in controlling AML, especially leukemia stem cell (LSC) progression in the aspect of anti-tumor immunity. They found that, the abundantly expressed FTO facilitates LSCs progression by impairing T-cell activity via increasing the expression of an immune inhibitory checkpoint molecule/gene 'LILRB4'. The application CS1 and CS2 significantly controls LSC progression in mouse model with low drug toxicity. Mechanistically, CS1 and CS2 were found to be selectively binds to the FTO and inhibit its demethylase activity, resulting in decreased expression of 'LILRB4' via inhibiting YTHDF2-mediated mRNA-decay mechanism. Moreover, increased T-cell cytotoxicity and reduced self-renewal abilities of AML cells were noted in the treated mice, suggesting that CS1 and CS2 is a potent inhibitors of FTO and can be developed as a potential therapeutics against LSC by increasing anti-leukemia T-cell activity [73]. This hypothesis was further supported by Olsen et. al., 2020 [74], ( Figure 6). Additionally, some other FTO-inhibitors are well described in these articles [75,76] and [77,78] (Table 2, 3). Fig. 6. Therapeutic model targeting m6A-eraser 'FTO' in controlling LSC progression by enhancing anti-tumor immunity. (A) Biological mechanism: FTO has been found to be aberrantly expressed in AML, especially LSC populations, and facilitates its progression. Mechanistically, the FTO enhance the expression of its target LILRB4 (immune checkpoint) and other Myc and CEBPA genes via its m6A-demethylase activity by inhibiting m6A-reader protein YTHDF2-mediated mRNA-decay mechanism, causing decreased T-cell activity and increased self-renewal capacity, resulting in enhanced LSC progression. (B) Therapeutic model: Anti-FTO therapy: The two synthetic small-molecule inhibitor of FTO (CS1 and CS2) selectively binds to the FTO domain and inhibits its demethylase activity, leading to decreased expression of its target (LILRB4, Myc and CEBPA) mRNA via reducing YTHDF2-mediated mRNA-stability. The decreased expression of the targeted gene ultimately enhances T-cell cytotoxicity and immune evasion and thereby reduced self-renewal capacities, resulting in better control over LSC propagation.

MiR-150 in AML:
In addition to m6A-modifiers, microRNAs also showed promising outcome in the treatment of acute myeloid leukemia (AML). Fang and colleague, 2016 [79] have demonstrated the importance of miR-150 in controlling leukemia progression. They showed that miR-150 is down-regulated in AML and CML patients, but normalized after complete remission/treatment. However, miR-150 restoration therapy (miR-150 mimic) significantly inhibited the AML by reducing cell-proliferation and promoting apoptosis of the leukemia stem cells, leading to reduced tumorigenicity in xenograft leukemia model. The underlying mechanism evidenced that miR-150 targeted genes are mainly associated with RNA metabolism (synthesis, export, splicing and stability), transcriptional regulation, wnt-signalling and mTOR-signalling pathways. Interestingly, knockdown of any of these miR-150 downregulated targets (TET3, EIF4B, FOXO4B and PRKCA) showed anti-leukemia activity similar to miR-150 restoration therapy [80]. Conclusively, these results authenticate the druggable value of miR-150 in treating AML and as a novel candidate for therapeutic drug development [79].

MiR-34a in AML:
miR-34a was shown to be another microRNA playing a crucial role in controlling elderly AML, who were ineligible for conventional chemotherapy. The author [81] have found that, the combination of decitabine (dacogen/DAC; known as 5-aza-2'-deoxycytidine) and all-trans retinoic acid (ATRA; a well-known differentiation inducing agent) effectively control AML and prolong overall survival rate by 49.6% in a clinical trial of 36-elderly leukemia patients. The underlying mechanism demonstrates that the combination of DAC and ATRA inhibited the expression of DNA methyltransferase 1 (DNMT1) resulting in activation of miR-34a by hypo methylation. The activated miR-34a further inhibits the expression of Myc [43,44] resulting in cell cycle arrest and increased apoptosis in vitro. This result suggests that miR-34a could be a druggable candidate to control AML progression by modulating miR-34a/Myc axis [81,82]. ; demonstrated that miR-29b is also playing an important role in controlling (LSCs) leukemia stem cell progression, a subtype of acute myeloid leukemia, characterized by increased self-renewal capacity and decreased apoptosis, by targeting LSC-fucosylation. The author found that fucosyltransferase 4 (FUT4) is overexpressed in LSC population (CD34 + CD38 -) compared to the non-LSC (CD34 + CD38 + , CD34 -CD38 + , CD34 -CD38 -) populations isolated from MOLM13 and KG-1a cell lines determined by LTL lectin assays. However, selective depletion of FUT4 (shFUT4) significantly decreased cell-proliferation and induced apoptosis of the LSCs confirmed by reduced sphere formation ability (colony formation assay), reduced Ki67 cell-proliferation (immunofluorescence) and increased LSC apoptosis (TUNEL assays) indicated by increased cleavage of apoptotic (PARP and caspase3) markers. Moreover, in-vitro application of chemotherapeutic drug (ADR, Ara-C and Paclitaxel) in combination with shFUT4-LSC drastically reduced LSC cell-proliferation as compared to shFUT4 and drug effect alone. Furthermore, mouse xenograft studies showed decreased tumour growth when given in combination with shFUT4 and ADR relative to the individual treatments. This result clearly suggests that (i) FUT4-mediated fucosylation facilitate LSCs progression. (ii) FUT4 silencing overcome chemotherapeutic drug resistance in AML. Mechanistically, he proved that FUT4 is regulated by specificity protein 1 (Sp1) transcription factor confirmed by Sp1 binding to FUT4 promoter via chromatin immunoprecipitation (ChIP) and duel luciferase assays. Interestingly, miR-29b directly binds with Sp1 (bioinformatics analysis) and inhibits its expression along with Wnt/β-catenin activation, confirmed by miR29b-mimic in 'inhibiting' and anti-miR29b in 'promoting' Sp1 expression and thus influences FUT4 expression accordingly, was further validated by partial restoration of FUT4 expression by co-transfecting anti-miR29b with siSP1. Moreover, Wnt/β-catenin pathway inhibition by DKK inhibitor induces apoptosis and reduces cell-proliferation. Taken together, these investigations clearly suggest that miR-29b could be a potential candidate to control LSC progression by regulating miR-29b/Sp1/FUT4 axis [83].

Suppressor of cytokine signalling (SOCS/CISH) in AML
In addition to epigenetic modifiers (RMDs) and microRNAs; the role of cytokine-inducible SH2-domain containing protein (CISH or CIS) as anti-tumor and anti-leukemia activity, also, cannot be denied because of its versatile role in regulating cytokine signalling via sensitizing immune cells. For example, (i) CISH knockdown has been shown in increasing anti-tumor immunity by enhancing CD8 + T-cell effector function [84]. (ii) CISH-knockdown also increases NK-cell fitness and cytotoxicity [85] and thus playing a crucial role in protecting tumor metastasis [86]. (iii) More relevantly, CISH has been shown in maintaining T-cell homeostasis via Mettl3-mediated methylation mechanism [87,88] and (iv) The m6A-reader protein 'YTHDF2' is involved in controlling NK cell-mediated anti-tumor immunity [89]. Furthermore, the specific function of CISH in association with acute myeloid leukemia was demonstrated by Zhu et. al., [90], where the importance of CISH in NK cell-mediated anti-leukemia activity has been described. They have showed that selective depletion of CISH in NK cells (CISH -/-) derived from human iPSC (induced pluripotent stem cells) significantly enhance the expansion as well as survival in the tumor microenvironment (TME), confirmed by improved metabolic fitness of NK-cells, characterized by increased glycolytic capacity and mitochondrial respiration (OxPhos activity) via mammalian target of rapamycin (mTOR) signalling response. Moreover, increased NK-cell cytotoxicity was reported against multiple tumor cell lines as well as in in-vivo leukemia xenograft model [90]. This result additionally supports the role of CISH as an alternative approach to control AML by encompassing immune cell-based therapeutics. Nevertheless, the role of professional antigen presenting cells like, dendritic cells (DCs) also cannot be circumvent for its proven role in improving anti-tumor immunity by targeting CISH [91,92]. One more relevant research outcome showed the involvement of suppressor of cytokine signalling 1 (SOCS1), one of the members of CISH/SOCS family, in mimicking AML like phenotype. Where, the overexpression of SOCS1 in zebrafish model showed increased myelopoiesis with distorted kidney and splenic morphology, suggesting the therapeutic inference of AML by targeting SOCS1 (Figure 1) [93].

Conclusions
In this review, we have described the potential of m6A-modifiers in developing cancer precision medicines and explained how 'epitranscriptomics' plays a central role in regulating the crucial genes associated with AML. Among these, the m6A-writer protein Mettl3 and Mettl14 has been found to be aberrantly expressed in specific sub-types of AML and promotes leukemogenesis by regulating Myb, Myc, Bcl2, pTEN and PI3K-AKT pathways [29,30,34]. Likewise, m6A-methylases (writer); m6A-demethylases FTO and ALKBH5 (eraser) have also been found to be aberrantly expressed in APL & LSCs/LICs and promote leukemogenesis by targeting ASB2, RARA, LILRB4, CEBPA, TACC3, Myc and P-21 gene [47]. Nevertheless, FTO single nucleotide polymorphisms (SNPs) has been also found to be associated with other cancers [94]. Likewise, writer and eraser; the reader protein 'YTHDF2' has been also found in promoting leukemogenesis by inhibiting its target Tla1 [68] and TNF-α [66] through universal YTHDF2-mediated mRNA decay mechanism [38] . These inventions suggest the different mechanism of action 'of these epigenetics modifiers' in controlling AML by regulating respective gene targets. However, still some questions remained unanswered, how all these epigenetic modifiers are up-regulated simultaneously and facilitates AML development. For example, considering the balance mechanism of 'writer' and 'eraser' enzymes in regulating 'Myc' in the same cells by Mettl3-methylation as well as by FTO-mediated mechanism remains to be further justified. A justifiable answer seems to be possible by recruiting different gene set by inviting different RNA-machineries. Nevertheless, The microRNA; miR-150, miR-34a and miR-29b has also shown significant anti-leukemia effect by targeting wnt-signalling, mammalian target of rapamycin (mTOR)-signalling pathways [79] Myc [81] and Sp1/FUT4-fucosylations [83]. In addition to this, the immune-based therapeutics should also consider in AML treatment by utilizing dendritic cells, T-cells and NK cell-targeting CISH-mediated signalling [87,89,95]. Therefore, it is very critical to understand the stages of AML progression and so in implementing these exciting RMDs in eradicating AML with other forms of standard chemotherapeutic drugs.

Future Prospective
Targeting m6A-modification machineries has revolutionized the class of epigenetic research in discovering novel therapeutic drug targets. Co-targeting of intracellular genes along with m6A-modification machineries will improve the level of immunotherapy against those cancers which are resistant to the immune checkpoint-based therapeutics in patients with blood cancer. The future is not so far where targeted therapy in combination with other drugs which would become a universal panacea to control many diseases and other forms of cancer. Moreover, targeting suppressor of cytokine signalling family of proteins (SOCS/CISH) would further potentiate the immunotherapeutic potential of RNA modification inhibitors by targeting epigenetic regulator of dendritic cells. Pharmaceutical approaches pertaining to RNA epigenetic modification machinery are expected to shed light on the field of cancer immunotherapy for AML and other forms of blood cancer. Nevertheless, a human clinical trial is required under a medical setup to access the therapeutic value of these inhibitors by optimizing the initial doses or/if intracellular silencing (gene therapy). However, a proposed clinical trial is underway to conduct till 2021-2022 by several biopharmaceutical companies mentioned above.