A critical role for the RNA m6A methylation complex in myeloid leukemia

N6-methyladenosine (m6A) is a well-known RNA modification that can affect mRNA splicing, stability and translation (D. Dominissini et al., 2012; X. Wang et al. 2015; Y. Wang et al. 2014; J. Zhou et al. 2015; J. Choi et al. 2016), and the processing of miRNA precursors (C.R. Alarcon et al. 2015). In mammals, the m6A writer is a multicomponent complex composed of the two methyltransferases METTL3 and METTL14 (J. Liu et al. 2014), and the regulatory protein WTAP (XL. Ping et al. 2014). Post-transcriptional m6A RNA modification is indispensable for cell viability and development, yet its role in cell differentiation and disease are still poorly understood. Notably, WTAP protein is an oncogene overexpressed in Acute Myeloid Leukemia (AML) compared to healthy control cells (H. Bansal et al., 2014). Moreover, METTL3, METTL14, and RBM15 are highly expressed in AML compared with other cancers (S.R. Jaffrey and M G. Kharas, 2017). We analysed the expression of the m6A RNA methylation complex components in AML observing that both METTL3 and METTL14 mRNAs are upregulated in AML samples (TCGA) respect to fully differentiated myeloid cells (GeoDatasets). Conversely, WTAP mRNA has lower levels in AML, even if the protein is upregulated. We showed that in AML and Chronic Myeloid Leukemia (CML) cell lines METTL3 is localized in both nucleus and cytoplasm. In cytoplasm METTL3 binds WTAP mRNA enhancing its translation independently from its catalytic activity, possibly explaining, at least partially, WTAP protein increased levels observed in AML cells. Moreover, we investigated the role of m6A RNA methylation complex components in myeloid leukemia cells, confirming that WTAP affects proliferation and differentiation of AML cells acting as an oncogene and demonstrating that METTL3 and METTL14 are essential proteins in these cells. Indeed, depletion of the two methyltransferases leads to a marked cellular mortality of AML cells. Notably, METTL3 affects viability and proper myeloid differentiation also in hematopoietic stem cells CD34+ derived from healthy umbilical cord. In CML K562 cells METTL3 and METTL14 depletion does not lead to apoptosis but could affect proper ribosome biogenesis, leading to an evident slowdown of cellular proliferation. Notably, decreased levels of METTL3 and METTL14 in K562 cells cause a downregulation of MYC protein, as recently reported in AML cells (L.P. Vu et al., 2017). Analysing ENCODE MYC ChIP- seq data we observed strong MYC peaks on the promoter of all the genes critical for ribosome biogenesis that are affected by METTL3 and METTL14 depletion, suggesting a MYC-mediated effect. In conclusion, we analysed some functional aspects of m6A RNA chemical modification in both acute and chronic myeloid leukemia cells, identifying differential m6A-regulated pathways between these two kinds of pathologies. Moreover, we also highlighted a more complex and double role for METTL3 protein in these cells, being one of the two essential m6A methyltransferase in the nucleus, and an RNA binding protein promoting translation in cytoplasm. Altogether, these data indicate a link between m6A factors and leukemogenesis and pave the way to possible future therapies targeting the RNA m6a methylation complex components.