HOXA9 Represses Bim Expression in MLL Rearranged Leukemia: Implications for Drug Therapy.

MLL translocations can be found in roughly 10% of acute leukemias, including over 70% of infant leukemias. These leukemias are often associated with a poor prognosis, making new therapeutics for this subtype of leukemias a priority. HoxA cluster genes have been shown to be upregulated by MLL fusion proteins, and make up a portion of a self-renewal associated gene expression signature found in murine models of MLL-AF9-expressing leukemia stem cells. The role of HoxA cluster genes in MLL-fusion mediated leukemia is not clearly understood, mainly due to lack of knowledge of HoxA downstream targets. One possible role for HoxA proteins in MLL-fusion induced leukemia is enhancement of growth and survival pathways. Evidence for this possibility can be found in C.elegans, where the pro-apoptotic BH3-only gene egl-1 is regulated by homeobox genes. Therefore we sought to determine whether a HoxA cluster gene could in fact regulate survival/apoptosis machinery. Here we demonstrate that HoxA9 serves as a repressor of the pro-apoptotic BH3-only family member Bim. Knockdown of HoxA9 using shRNA in the MLL-AF9 expressing AML cell line Molm14 leads to upregulation of Bim, and rapid induction of apoptosis. Interestingly, knockdown of Meis1 or Pbx2, known binding partners of HoxA9, showed only an intermediate effect, with slower growth, but without an increase in Bim levels. Other BH3-only family members Noxa and Puma showed no significant change in expression after HoxA9 suppression as determined by quantitative RT-PCR. Using a FLAG-HOXA9 expressing Molm14 cell line, we were able to determine by chromatin immunoprecipitation that HOXA9 is present at a DNA region containing a canonical HOX-PBX binding site (TGATTTAT) roughly 100bp downstream of the first exon-intron junction in the human Bim gene. As histone deacetylases (HDACs) are generally transcription repressors, it is plausible that HOXA9 recruits HDACs to the Bim gene, thus regulating its expression. Since HDAC inhibitors also activate BIM, we assessed BIM activation in Molm14 cells after treatment with an HDAC inhibitor, and noted similar kinetics of BIM activation, suggesting a possible link between HoxA9 mediated suppression of BIM and HDACs. We are now assessing HOXA9/HDAC interactions in MLL rearranged leukemia cells. These data suggest that HOXA9 ensures survival of leukemia cells by directly inhibiting transcription of the pro-apoptotic gene Bim, and that interfering with this mechanism may improve treatment for MLL-fusion bearing leukemias.