GSE162441
MLL-fusion-mediated activation of FLT3-ITD lesions promotes leukemogenesis
MLL-rearranged (MLL-r) leukemia, a particularly intractable disease, depends on DOT1L-mediated H3K79 methylation. Depletion of this transcriptionally activating mark by DOT1L loss-of-function or high concentrations of highly-specific inhibitor pinometostat (=1 mM EPZ5676) leads to the downregulation of HOXA9 and MEIS1, and consequent reduction leukemia survival. .  Yet, some MLL-r cell lines are inexplicably susceptible to low-dose pinometostat, below the threshold for downregulating these canonical oncogenic drivers. Here we define alternative pathways disrupted by low-dose pinometostat (10 nM), a concentration that reduces proliferation of the MLL-r MV4;11 cell line without affecting HOXA9 and MEIS1 expression and downregulates a subset of MLL-fusion targets including FLT3, one of the most commonly mutated genes in leukemia. Using quantitative ICeChIP-seq, we observe profound H3K79me2 depletion at downregulated MLL-r targets, with resulting increases in transcriptionally activating H3K4me3 at promoters and reductions in repressive H3K27me3. The presence of co-occurring activating FLT3 mutations, portend greater cytotoxicity to inhibitor treatment. Although downregulation of polycomb components modestly contributes to reductions in proliferation, overexpression of constitutively active STAT5A, a target of FLT3-ITD-signalling, nearly completely rescues proliferation, accounting for the bulk of cytotoxicity from H3K79me2 depletion. We also observe a dependence of FLT3-STAT5A signaling on MLL function, suggesting that the FLT3 locus is exquisitely sensitivity to both H3K79me2 and H3K4me3 depletion and arguing that combinations of DOT1L, MLL1 and FLT3 inhibitors should be explored for treating the ~30% of all leukemias that carry FLT3 mutations. Overall design: Examination of histone modifications by calibrated ChIP-seq in MV4;11 cells with and without methyltransferase inhibitor