Mouse models of MLL-Af9 leukemia have been exploited to roughly determine leukemia initiating cell (LIC) characteristics and biology. Each colony formed by MLL-Af9 leukemic splenocytes is capable of initiating leukemia upon transplantation. MLL-fusion oncoproteins signal through elevated expression of HoxA9, Meis and Pbx proteins. The HoxA9 transcription factor is of critical interest in human AML because it is; 1) directly affected by chromosomal translocations, 2) upregulated by leukemia oncoproteins, and 3) the level of HoxA9 in cytogenetically normal human AML predicts outcome. However, the direct transcriptional targets of endogenous HoxA9 that mediate transformation remain largely unknown. The Growth factor independent-1 (Gfi1) transcriptional repressor is known to induce granulopoiesis and inhibit myeloid progenitor proliferation. GFI1 is mutated in patients with severe congenital neutropenia (SCN). SCN patients are at increased risk for AML. We have shown that Hox and Gfi1 orthologs antagonize each other during Drosophila anterior posterior patterning, and that in mammalian myeloid progenitors Gfi1 directly represses the expression of Hoxa9, Pbx1 and Meis1. Moreover, Gfi1 regulates the expression of miR-21 and miR-196b, and forced expression of these miR blocks G-CSF instructed granulopoiesis. Here we demonstrate that microRNA genes are the targets of endogenous HoxA9 versus Gfi1 antagonism and that this antagonism is relevant in the context of human leukemia. Moreover, we also show that miR-196b and miR-21 are activated by Hox-signaling leukemia oncoproteins. Next, in both murine leukemia models and primary human AML samples, antagomir-mediated inhibition of microRNA function specifically disrupts colony replating potential of Lin- bone marrow cells transformed by HoxA9, Nup98-HoxA9 and MLL-Af9, as well as bona fide MLL-Af9 leukemias. In contrast, cells transformed by AML-ETO (which does not signal through HoxA9) are not affected. In vivo, antagomir treatment blocked MLL-Af9-initiated leukemia (but not AML-ETO leukemia) lethality. Finally, limiting dilution analyses demonstrate that antagomirs inhibit Hox-based transformation by targeting the LIC. Antagomir treatment induces the re-expression of a histone lysine demethylase, downregulation of gene expression associated with maintenance of MLL-Af9 leukemia and associated H3K4me3 marks on these genes. Our data establish microRNA genes as functional downstream targets of endogenous HoxA9, and implicate epigenetic signaling as critical client/mediators of Hox-based leukemia oncoproteins in LIC maintenance.
Carroll:Sanofi Aventis Corporation: Research Funding; Kyowa Hakko Kirin Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding.
Asterisk with author names denotes non-ASH members.