The N6-Adenine Methyltransferase METTL14 Plays an Oncogenic Role in Acute Myeloid Leukemia

Abstract

N6-methyladenosine (m6A) modification is the most abundant internal RNA modification in eukaryotes. Recent studies have shown that the dynamic and reversible regulation of m6A modifications in mRNAs or non-coding RNAs plays critical roles in tissue development, stem cell self-renewal and differentiation, control of heat shock response, and circadian clock controlling, as well as in RNA metabolism and processing. However, little is known about the functions of m6A and m6A regulators in malignant hematopoiesis.METTL14 is a major m6A writer which together with METTL3 forms the core of the methyltransferase complex that catalyzes the conversion of adenosine (A) to m6A. Through qPCR assays, we found that METTL14 was aberrantly up-regulated in mononuclear cells (MNC) from acute myeloid leukemia (AML) patients with t(11q23), t(15;17), or t(8;21) relative to those from healthy donors.To investigate the pathological role of METTL14 in AML, we transduced lineage negative (Lin-) bone marrow (BM) progenitor cells from Mettl14fl/flCreERT mice with MLL-AF9, AML1-ETO9a, or PML-RARa fusion genes and performed colony-forming/replating assays with or without addition of 4-hydroxytamoxifen (4-OHT). Induction of genetic knockout of Mettl14 by 4-OHT treatment remarkably impaired the colony-forming ability of all these AML-related fusion genes after replating. After the first round of plating, we harvested MLL-AF9-transduced cells that were not treated with 4-OHT and transplanted them into lethally irradiated recipient mice. As expected, tamoxifen (TAM) treatment of transplanted mice significantly delayed leukemogenesis compared to mice treated with vehicle (MLL-AF9+TAM, with median survival of 91 days; MLL-AF9+vehicle, with median survival of 71 days; P=0.0012) (Fig.1A). In addition, specific knockdown of Mettl14 with shRNAs showed similar patterns to Mettl14 knockout. Thus our data demonstrate that Mettl14 is crucial for cell transformation and leukemogenesis. Further, to determine the role of Mettl14 in the maintenance of leukemia, we transduced leukemic BM cells from primary MLL-AF9 leukemic mice with shRNAs against Mettl14 or scramble shRNA and transplanted these cells into lethally irradiated recipient mice. Again, a significantly prolonged survival was observed in Mettl14 knockdown groups compared to that in the control group (MLL-AF9+shRNA1, with median survival of 32 days; MLL-AF9+shRNA2, with median survival of 32 days; MLL-AF9+shScramble, with median survival of 23.5 days; P< 0.001 for both knockdown groups) (Fig.1B). Noticeable, mice in Mettl14 knockdown groups showed less c-kit+ cells in BM than mice in the control group (Fig.1C).In addition to the mouse model, we used human leukemia cell lines to investigate the function of METTL14 in human AML cells. Silencing of METTL14 with shRNAs significantly inhibited cell viability, induced apoptosis as well as terminal differentiation of MONOMAC6 and NB4 cell lines (Fig.1D, E, F). Moreover, xenograft model showed that repression of METTL14 significantly inhibited the engraftment of MONOMAC6 cells and thus delayed the onset of leukemia in NSG-SGM3 (NSGS) immunodeficient mice (Fig.1G). Furthermore, knockdown of METTL14 sensitized MONOMAC cells to ATRA or PMA-induced differentiation. Taken together, our results support the oncogenic role of METTL14 in AML and highlight METTL14 as a novel therapeutic target in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.