MIR142 Loss-of-Function Mutations Promote Leukemogenesis through Derepression of ASH1L Resulting in Increased HOX Gene Expression

Mutations of MIR142 have been identified in approximately 2% of de novo AML and in 20% of diffuse large B cell lymphoma (DLBCL). In AML, all of the mutations in MIR142 localize to the seed sequence of miRNA-142-3p, which is crucial in determining its target specificity. Previously, we showed that these mutations disrupt both miRNA-142-3p and miRNA-142-5p function, suggesting that loss of MIR142 plays a role in leukemic transformation (Yao et al, ASH abstract 1254, 2015). To test this hypothesis, we first characterized hematopoiesis in Mir142-/-mice. We previously reported that loss of Mir142 results in an expansion of myeloid progenitors with impaired erythropoiesis and lymphopoiesis (Yao, ASH abstract 1254, 2015). We now extend these analyses to investigate how loss of Mir142 promotes leukemic transformation. In the TCGA AML cohort, all 4 cases with somatic mutations in MIR142 also harbored mutations in either IDH1 or IDH2. To assess the functional importance of this association, we transduced wild-type or Mir142-/-hematopoietic stem/progenitor cells (HSPCs) with a retrovirus expressing the canonical IDH2 mutation, R172K. These cells were transplanted into lethally irradiated recipients and a tumor watch established. Loss of Mir142 alone was associated with mild splenomegaly, anemia, and leukopenia, but it was not sufficient to induce AML. Consistent with a prior report (Sasaki et al, Nature 2012), expression of IDH2 R172K alone induced a myeloproliferative disorder (MPD) characterized by increased myeloid cells, anemia, and splenomegaly. Concomitant loss of Mir142 did not affect the latency or penetrance of this MPD. However, the MPD in the double mutant mice was characterized by an increased percentage of CD34+ Gr1+ myeloblasts in the bone marrow and spleen plus a more severe anemia. To assess leukemia-initiating activity, we transplanted one million splenic cells into secondary recipients. Whereas IDH2 R172K alone cells rarely engrafted, Mir142-/- x IDH2 R172K cells efficiently engrafted and produced an MPD-like phenotype. These data suggest that loss of function mutations in MIR142 cooperate with IDH1/2 mutations to induce AML, possibly by increasing leukemic cell self-renewal. We examined several putative miR-142 target genes, eventually focusing on ASH1L. ASH1L is a member of the trithorax family of histone methyltransferases that has been recently implicated in MLL-associated leukemogenesis. The 39 UTR of ASH1L contains 4 putative binding sites for miRNA-142-3p, suggesting that this miRNA is critical in its post-transcriptional regulation. Indeed, in a luciferase assay with the ASH1L 39 UTR, MIR142 overexpression decreased translation by 80 percent. Consequently, Ash1l protein levels were 3 fold higher in Mir142-/- mice bone marrow compared to control mice. Since ASH1L is a key regulator of HOX gene expression, we examined HoxA9 and HoxA10 expression in Mir142-/- hematopoietic progenitor subsets. While HoxA9 and HoxA10 expression were not different in hematopoietic stem cells, they were markedly upregulated in myeloid progenitors. For example, in granulocyte-macrophage progenitors (GMPs), HoxA9 and HoxA10 expression were increased 2.86-fold and 34.4-fold, respectively in Mir142-/- versus control cells. Likewise, in megakaryocyte-erythroid progenitors (MEPs), HoxA9 and HoxA10 expression were increased 5.3-fold and 21.4-fold. Dysregulated HoxA9 and HoxA10 expression have been implicated in enhanced self-renewal capacity, and HoxA9 overexpression has been shown to cooperate with mutant IDH1 to induce AML in mice (Chaturvedi et al, Blood 2013). Collectively, these data suggest a model in which MIR142 mutations contribute to leukemogenesis by derepressing ASH1L expression, which, in turn, increases expression of HoxA9/10 and enhances self-renewal. Inhibitors targeting ASH1L may have therapeutic benefit in AML characterized by increased HOX gene expression. Disclosures No relevant conflicts of interest to declare.