Analysis of transcriptomic and epitranscriptomic effects of the acute megakaryoblastic leukemia fusion protein RBM15-MKL1
The RBM15-MKL1 (RM) fusion protein causes acute megakaryoblastic leukemia (AMKL) primarily in infants. RBM15 recruits the m6A writer complex and is essential for determining the m6A epitranscriptome. MKL1 is a transcriptional cofactor important to megakaryopoiesis. Although the functions of the component proteins are well described, the mechanism whereby the RM fusion protein induces AMKL is unknown. We performed a high-resolution multi-omics analysis to identify the direct transcriptional, RNA-binding, and epitranscriptional targets of RM compared to RBM15. While RM maintains the RNA-binding and m6A modification capabilities of wildtype RBM15, it also has distinct RNA targets, many of which are upregulated and stabilized in RM-expressing cells. Among the top targets that are differentially bound, m6A modified, and upregulated by RM are the Frizzled genes, which encode receptors for the Wnt signalling pathway. To assess the role of RM-mediated m6A modifications in a physiologically relevant model, we treated two murine AMKL cell lines with STM3675, a novel METTL3 inhibitor. In vitro, METTL3 inhibition causes murine AMKL cells to undergo differentiation and apoptosis. In vivo treatment with METTL3 inhibitor prolongs survival of mice transplanted with murine AMKL cells. We additionally show that treatment with STM3675 decreases expression of key RM target genes, including Frizzled genes. Knockdown of Frizzled genes in RM-driven murine AMKL cells decreases their growth and survival, suggesting that RM-specific binding, m6A modification, and aberrant upregulation of the Wnt signaling pathway contribute to leukemogenesis and may represent targetable therapeutic pathways for treatment of children with RM-AMKL. Overall design: We performed a multi-omics analysis of RBM15-MKL1 (RM)-AMKL using two cell models. (1) Human erythroleukemia (HEL) cells were stably transfected with empty vector or dox-inducible FLAG-RBM15, FLAG-RM. RNA-seq was performed on dox-treated and untreated cells in duplicate.  eCLIP-seq was performed on dox-treated cells in 2-3 replicates; pre-IP input was sequence for 1 replicate.  m6A eCLIP-seq was performed on dox-treated cells in 2-3 replicates.  TimeLapse-seq was performed on dox-treated, s4U-treated cells in triplicate with non-s4U-treated controls.  FLAG-MKL1 was included in RNA-seq experiments as an additional control.  (2) Two murine AMKL cell lines, 6133 (Mercher et al, 2009) and CAOM (this paper) were used in triplicate for RNAseq after vehicle or METTL3 inhibitor treatment.  We additionally performed RNAseq on sorted ERP (Lin-cKit+Sca-CD150+FcgRI/II-CD105+) and MKP (Lin-cKit+Sca-CD150+CD41+) from wildtype mice.