In the past few years, the molecular characterization of AML has aroused the researchers’ interest in better understanding of pathogenesis, prognosis prediction, treatment stratification, development of new targeted therapies and assistance in MRD detection.
An increasing number of researches have focused on the crucial role of mRNA modifiers in progression of leukemia. METTL3 is one of these mRNA modifications that have been associated with pathogenesis of AML . The methylation process of the mRNA nucleotide by METTL3 occurs regularly in approximately 20% of human cellular mRNAs. However, alteration of this methylation process in AML cells has been associated with high abnormal level of METTL3 mRNA and protein and leads to marked alteration in cell differentiation and developing of myeloid hematological malignancies .
In this work, we aimed to measure METTL3 mRNA expression level by qRT-PCR in newly diagnosed AML cases and study this level in relation to clinical, laboratory and prognostic markers.
Forty newly diagnosed AML patients were enrolled in our study along with 20 age and sex-matched control subjects. We found that METTL3 is significantly overexpressed in AML patients with median value 6.95 compared to median 1.18 in control group (p value < 0.001). Some recent studies have demonstrated similar results where overexpression of METTL3 was shown in AML cells compared to the normal Hematopoietic Stem/Progenitor Cells (HSPCs) [6, 12].
Similarly, another study was done by Vu et al.  to assess the alteration of METTL3 mRNA expression in leukemia in which they compared the METTL3 mRNA expression in AML samples to other cancers based on the cancer genome atlas database. They found that METTL3 mRNA expression is significantly higher in AML than in other cancer types with p value < 0.00001. They further assessed the relative abundance of METTL3 in myeloid leukemia and examined both METTL3 mRNA and protein levels in multiple leukemia cell lines in comparison with primary HSPCs cord blood-derived CD34+ cells. They found that both METTL3 mRNA and METTL3 protein were more abundant in AML cell lines. However, there was no significant difference in METTL3 expression across multiple subtypes of AML in the blood pool database .
In our study, we divided the 40 AML patients into 2 prognostic subgroups according to the American Cancer Society (2021 guidelines). We found that most of our AML patients exhibited good prognostic criteria. However, we didn't find significant association between any prognostic criterion and the METTL3 gene expression level.
In the same context, a cohort study was carried on 191 AML patients and detected mutations of m6A regulatory genes in 2.6% (5/191) and variation in gene copy number in 10.5% (20/191) of patients. They studied whether mutations and copy number variations (CNVs) of m6A regulatory genes were associated with clinical and molecular features (older age > 60 years, white blood cell count > median (15,200/mm3), unfavorable cytogenetic risk and mutations of DNMT3A and TP53). They observed that mutations and/or CNVs of METTL3, METTL14, YTHDF1, YTHDF2, FTO and ALKBH5 as a group were significantly associated with poorer cytogenetic risk in AML (p < 0.0001). Additionally, they detected a marked increase in TP53 mutations (p < 0.0001). However, these mutations and/or CNVs were not associated with older age (> 60 years) or high white blood cell count > median .
METTL3 gene expression pretreatment level was statistically significantly higher in AML cases with HSM (p value 0.014). There was no statistically significant relation between METTL3 gene expression level and any other parameter (age, gender, initial TLC count, hemoglobin, platelet count or initial BM blast count).
Similarly, a study of METTL3 and METTL14 expressions in 37 ALL patients investigated the relation between METTL3 and METTL14 expressions with clinical features. They didn't find association between the expression level of METTL3 and METTL14 with gender, age, initial TLC and blast percentage, indicating that these two genes may not be associated with tumor burden . The association between METTL3 expression level and clinical data in AML patients' needs further studies to be evaluated.
Another recent study of METTL3 expression level in solid tumors was carried on 340 patients with oral squamous cell carcinoma reported that a higher METTL3 expression level was significantly positively associated with advanced tumor stage, advanced clinical stage and lymph node metastasis, but no differences in other features, such as sex and age, were observed .
Our study revealed that no significant association was found between the higher METTL3 expression levels and specific AML subtypes. The same finding was reported by Vu et al.  during their study of METTL3 mRNA and protein levels in multiple leukemia cell lines. They found that METTL3 mRNA was more abundant in AML cell lines with no significant difference in METTL3 expression across multiple subtypes of AML.
As regards the cytogenetic abnormalities in our study, they were not statistically related to METTL3 gene expression level. Contrarily, a recent study of the molecular function of m6A RNA methylation in cancer has reported that METTL3 and METTL14 are highly expressed in AML cells carrying t(11q23), t(15;17) or t(8;21) and are down-regulated during myeloid differentiation . This controversy may be attributed to the few numbers of cases exhibiting these abnormalities in our study.
Another study by Weng et al.  found that METTL14 is highly expressed in normal HSCs and AML and is down-regulated during myeloid differentiation. In particular, METTL14 was found to be overexpressed in AML cells carrying 11q23 alterations, t(15;17) or t(8;21). Analysis of the Cancer Genome Atlas (TCGA) data revealed that AML blast cells expressed higher mRNA levels of both METTL3 and METTL14 than most cancer types, and genetic alteration of those genes has significantly correlated with poorer prognosis .
The associations between m6A and genetic alterations in AML were studied by Paris et al.  who reported that m6A promotes the translation of c-MYC, BCL2 and PTEN mRNAs in the human AML cell line. Moreover, loss of METTL3 leads to increased levels of phosphorylated AKT, which contributes to the differentiation-promoting effects of METTL3 depletion. Overall, these results provide a rationale for the therapeutic targeting of METTL3 in myeloid leukemia. In the same context, the molecular function of WTAP, which is a novel oncogenic protein in myeloid leukemia that acts as regulatory subunit of the m6A methylation complex, was evaluated by a group of researchers. Their results revealed a lack of association between WTAP levels and particular cytogenetic abnormalities, but a significant correlation was detected between some specific molecular mutations such as NPM1 and FLT3-ITD, and WTAP expression . WTAP is commonly upregulated in myeloid leukemia, but this upregulation alone is not enough to induce cell proliferation in the absence of a functioning METTL3 .
The patients enrolled in our preliminary study were de novo AML, with a mean age of 41.9 years and median TLC 36.05 × 109/L with the absence of unfavorable cytogenetic abnormalities (11q23 & t(9;22)). These data can collectively predict a good response to chemotherapy based on the updates of independent prognostic factors in AML . However, during the initial stages of the follow-up, 23/40 (57.5%) failed to achieve CR with induction therapy and by the end of the 6th month chemotherapy, 9/29 (31.1%) failed to maintain their CR. This may suggest the adverse prognostic role of METTL3 expression in AML.
In the current study, the initial response to chemotherapy was assessed morphologically at day 28 post-therapy and accordingly, based on the response to chemotherapy at day 28 post-therapy, AML patients were classified into responders (15/40; 37.5%) and non-responders (25/40; 62.5%). To evaluate the impact of METTL3 gene expression on achievement of hematological remission, METTL3 gene expression was studied in these 2 subgroups and surprisingly, responders revealed low normalized METTL3 gene expression level (median 2.28; IQR 1.87–2.58) while non-responders exhibited higher median gene expression (median 9.58; IQR 7.7–14.6).
ROC curve analysis was used to evaluate the ability of METTL3 gene expression level at diagnosis to anticipate response of AML patients to chemotherapy. A cutoff value of 4 was selected as discriminating point with sensitivity of 95.8%, specificity of 87.5%, PPV of 92%, NPV of 93.3% and a diagnostic accuracy of 98%. This cutoff value proves that AML patients with high gene expression levels have bad response to chemotherapy at day 28. Likewise, patients with low gene expression levels show good response to induction therapy.
Our study detected significant association between higher pretreatment level of METTL3 gene expression in AML cases at time of diagnosis and failure to maintain CR at 2nd month, 4th month and 6th month follow-up (p = 0.01, 0.02 and 0.003, respectively). On monitoring patients with high gene expression level, we found that one case died at day 28 and another two cases died at the 2nd month. Out of the 14 patients who expressed higher METTL3 level at diagnosis and could be followed till the end of the 6th month chemotherapy, eight patients (57.1%) did not achieve hematological remission and showed persistently high blast counts, compared to only 1/15 (6.67%) patients who expressed a low level of METTL3.
In the present study, we reassessed the METTL3 gene expression level (between the 2nd and 4th month post-treatment) in 15 cases who expressed higher pretreatment gene levels. We intended to monitor the short-term effect of chemotherapy on the gene expression levels and determine possible correlations with patients’ outcome. In comparison with the pretreatment levels, the gene level was found to increase after chemotherapy in 9/15 patients (60%). This elevation was associated with failure to maintain CR at 2nd m, 4th m and 6th m chemotherapy (p = 0.048, 0.015, 0.015, respectively). Moreover, 7/9 (77.8%) of AML cases with elevated gene level post-treatment failed to maintain hematological remission till the end of the 6th month; in contrast, none of the six AML cases with reduced gene expression level failed to maintain hematological remission. No related data in analogous studies are available in the literature. Nevertheless, these findings of gene levels monitoring comply with the poor prognostic effect obtained when analyzing the pretreatment gene levels. In addition, it is important to emphasize that despite the administration of chemotherapeutics, it can be speculated that this gene still has the ability to increase and exert its dismal prognostic effect.
Similarly, the poor prognosis of AML cases with m6A mutations was reported in a cohort study that was carried on 191 AML patients, where the authors found that mutation of any of the genes encoding m6A regulatory enzymes had a worse OS (p = 0.007) and EFS (p < 0.0001). Inferior OS and EFS were also evident in patients who had mutations and/or CNVs of these genes .
Recently, a therapeutic trial was done on small molecules that act as selective inhibitors of METTL3 in AML. Their anti-tumor effects were evaluated in patient-derived xenotransplantation experiments as well as transplantation experiments using an MLL-AF9-driven primary murine AML model. They reported that daily dosing of 30 mg/kg significantly inhibited AML expansion and reduced spleen weight compared to control, indicating that inhibition of METTL3 in vivo leads to strong anti-tumor effects in physiologically and clinically relevant models of AML .
Collectively, these studies highlight the prognostic role of both METTL3 in malignant hematopoietic cells and will encourage further epigenetic studies of target therapies in AML. These upcoming studies will reveal new insights regarding the molecular mechanisms regulating normal and malignant hematopoiesis and offer better opportunities for AML patients to improve their clinical outcomes.
In conclusion, our results have proved an association between high pretreatment gene expression level and bad response to chemotherapy. In addition, patients with a further increase in gene expression during the course of the disease were more likely to show failure to maintain hematological remission. Accordingly, an adverse prognostic impact of METTL3 expression on the outcome of AML adult patients can be concluded. However, since the small number of patients and the short follow-up time are two main limitations of this study, we strongly recommend large studies with longer follow-up periods to verify the proposed role of METTL3 gene expression in the pathogenesis and prognosis of AML.