N6-methyladenosine (m6A) is the most abundant internal modification in messenger RNA (mRNA) mainly occurring at consensus motif of G[G>A]m6AC[U>A>C]. Despite the functional importance of m6A modification in various fundamental bioprocesses, the studies of m6A modification in cancer, especially in leukemia have largely been limited.Fat mass and obesity-associated protein (FTO), the first RNA demethylase,was known to be robustly associated with increased body mass and obesity in humans. However, the impact of FTO, especially as a RNA demethylase, in cancer development and progression has yet to be investigated. Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies with distinct geneticabnormalities and variable response to treatment.Here, we aim to definethe roleof FTO as an m6A demethylase in AML.
To access the potential effect of FTO, we analyzed its expression in AML patients with distinct genetic mutations. To determine the influence of FTO on transformation capacity/ cell viability and leukemogenesis, colony-forming/replating assay (CFA), MTT assays, cell apoptosis and bone marrow transplantation (BMT) were carried out. To identify potential targets of FTO, transcriptome-wide m6A-seq and RNA-seq were performed. To evaluate the function of FTO on m6A modification and mRNA metabolism,m6A dot blot, gene-specific m6A qPCR assays and RNA stability assays were conducted. To elucidate whether FTO-mediated regulation of its targets depends on its demethylase activity, gene-specific m6A qPCR assays and luciferase reporter and mutagenesis assays were carried out. To investigate the potential roles of FTO and its targets in hematopoiesis, ATRA-induced APL cell differentiation was used.
In analysis of AML datasets, we found FTO is highly expressed in AMLs with t(11q23)/MLL-rearrangements, t(15;17)/PML-RARA, FLT3-ITD and/or NPM1 mutations. Lentivirus-induced expression of wild-type FTO, but not mutant FTO (carrying two point mutations, H231A and D233A , which disrupt its enzymatic activity), significantly enhanced colony forming activities, promoted cell proliferation/transformation, restricted cell apoptosis and decreased global mRNA m6A levelin vitro. Forced expression of Fto significantly (p<0.05; log-rank test) accelerated MLL-AF9-induced leukemogenesis and decreased global m6A level in leukemic BM cells. The opposite is true when FTO/Fto was knocked down by shRNAs or genetically knocked out.
Via transcriptome-wide m6A-sequencingand RNA-sequencing (RNA-Seq) assays in MONOMAC-6 AML cells with or without overexpression or knockdown of FTO, we identified two functionally critical targets of FTO, ASB2 and RARA.Forced expression of wild-type FTO, but not mutant FTO, reduced expression of RARA and ASB2. Forced expression of either ASB2 or RARA largely recapitulated the phenotypes caused by FTO knockdown. Moreover, the effects of overexpression or knockdownof FTO can be largely rescued by that of RARA or ASB2, indicating that they are functional important targets of FTO.
Forced expression and knockdown of FTO reduced and increased, respectively, the m6A levels on ASB2 and RARA mRNA transcripts,and shortened and prolonged, respectively, the half-life of ASB2 and RARAmRNA transcripts in AML cells.Importantly, FTO reduced luciferase activity ofASB23'UTR, RARA3'UTR or RARA5'UTR constructs with intact m6A sites, while mutations in the m6A sites abrogated the inhibition, demonstrating that FTO-mediated gene regulation relies on its demethylase activity.
Upon ATRA treatment, FTO was significantly down-regulated, while RARA and ASB2were up-regulated in NB4 APL cells. Forced expression of FTO noticeably suppressed, while depletion of FTO enhanced, ATRA-induced cell differentiation.Forced expression of either RARA or ASB2 could also substantially enhance NB4 cell differentiation.
In summary, we provide compelling in vitroand in vivoevidence demonstrating that FTO, an m6A demethylase, plays a critical oncogenic role in cell transformation and leukemogenesis as well as in ATRA-mediated differentiation of leukemic cells, through reducing m6A levels in mRNA transcripts of its critical target genes such as ASB2 and RARA and thereby triggering corresponding signaling cascades. Our study highlights the functional importance of the m6A modification machinery in leukemia.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.