Abstract

RNA N6-Methyladenosine (m6A) modification is an abundant modification of internal mRNAs in eukaryotes and some viruses, which is dynamically and reversibly fine-tuned during normal and pathological bioprocesses. Recent studies have shown that m6A methyltransferases, METTL3 and METTL14, play important roles in maintaining self-renewal capacity of hematopoietic stem/progenitor cells (HSPCs) and promoting acute myeloid leukemia (AML) development (Barbiori et al., Nature, 2017; Vu et al., Nature Method, 2017; Weng et al. Cell Stem Cell, 2018). The m6A demethylase, FTO, was also shown to promote leukemic cell transformation and leukemogenesis in various type of AML (Li et al., Cancer Cell, 2017). However, little is known about the functions of m6A readers in malignant hematopoiesis.

We recently reported that Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) is an specific m6A binding protein, which recognize m6A transcripts through the K Homology (KH) domains to stabilize and promote translation of its target mRNAs (Huang et al., Nature Cell Biology, 2018). In analysis of TCGA AML dataset (n=157), we found that a higher expression level of IGF2BP3 is significantly associated with a poor prognosis in AML patients (p<0.001; Median overall survival: IGF2BP3-High vs. IGF2BP3-Low = 11 months vs. 31 months). In addition, we analyzed our in-house microarray profiling of 113 AML patient samples and found that IGF2BP3 is highly expressed in mononuclear cells (MNC) from MLL-rearranged leukemia patients as compared to those from healthy donors (p<0.05) or non-MLL-rearranged leukemic patients (p<0.001). Consistent with the overexpression of IGF2BP3 in human MLL-rearranged AML, MLL-AF9 or MLL-AF10 transformed mouse hematopoietic stem/progenitor cells (HSPCs; herein mouse lineage negative (Lin-) bone marrow cells) showed a >10 fold increase in expression level of Igf2bp3, compare to the non-transformed counterpart HSPCs. Furthermore, in analysis of 562 samples from adult patients with AML (GSE37642), we found that within cytogenetically normal human AML, patients carrying FLT3-ITD mutation showed a significantly higher level of IGF2BP3 expression than those without FLT3-ITD mutation (p<0.01).

To investigate the potential oncogenic role of IGF2BP3 in AML, we cotransduced mouse Lin- BM progenitor cells with MLL-AF9 and three individual shRNAs targeting Igf2bp3 or a scrambled control shRNA and performed colony-forming/replating assays. Knockdown of Igf2bp3 significantly (p<0.05) reduced the colony-forming capacity of MLL-AF9-transduced HSPCs to 20-50% of that of the control group. Conversely, forced expression of wild-type IGF2BP3 significantly (p<0.05) promoted colony formation of MLL-AF9-transduced Lin- BM progenitor cells. Such promotion was almost completely impaired when KH3-4 domain of IGF2BP3 was mutated or when Mettl14 was depleted, suggesting that IGF2BP3 exerts its oncogenic function as an m6A reader through an m6A-dependent mechanism.

We further used human leukemia cell lines to investigate the function of IGF2BP3 in human AML cells. Silencing of IGF2BP3 by two shRNAs significantly inhibited cell viability and proliferation and induced cell apoptosis (p<0.01) in MonoMac6 AML cell line which harbors the t(9;11) translocation. In Molm13 and MV4-11 AML cells which are heterozygous and homozygous for the FLT3-ITD mutation, respectively, a further decrease of cell viability and increase of apoptotic cells upon IGF2BP3 knockdown was observed compared to MonoMac6 with wild-type FLT3. Mechanically, through cross-linking immunoprecipitation sequencing (CLIP-seq), we showed that IGF2BP3 targets mRNAs in cell cycle, DNA replication and protein synthesis pathways. Taken together, these results demonstrated the oncogenic role of the new m6A reader protein IGF2BP3 in AML. Given the fact that expression of IGF2BP3 correlates with an overall poor prognosis in AML, IGF2BP3 is likely a promising therapeutic target for AML treatment.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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