The Pim-1 Kinase Is a Direct Target of the Hoxa-9 Homeodomain Protein and a Mediator of Hoxa-9’s Effects on Hematopoiesis.

The Hoxa-9 homeodomain protein exerts dramatic effects in hematopoiesis. Loss of Hoxa-9 function in blood cells severely inhibits proliferation of primitive hematopoietic cells and impairs in vivo hematopoietic stem cell (HSC) function. The defects in Hoxa-9−/− animals are more striking than any other single or compound Hox mutation reported to date, and more dramatic than that seen in animals lacking the entire Hoxb cluster of homeobox genes, presumably because Hoxa-9 is one of the most highly expressed members of the 39-member Hox gene family in HSC. Conversely, enforced expression of Hoxa-9 markedly enhances the proliferation of primitive blood cells, expands HSC and leads inevitably to a fatal acute myeloid leukemia in mice. Despite these potent biological effects, the pathways by which Hoxa-9 acts in blood cells are unknown, and although Hoxa-9 is a DNA-binding transcription factor, no direct physiologic target genes have been identified in hematopoietic cells. Our previous gene expression profiling studies suggested that Hoxa-9 positively regulates the Pim-1 serine-threonine kinase, an oncogene with important antiapoptotic and pro-proliferative effects in blood cells. In addition, the hematological phenotypes of Hoxa-9 and Pim-1 deficient animals are strikingly similar, and include a blunted response to myeloid cytokines, T- and B-cell defects, and microcytosis of red blood cells. Using chromatin immunoprecipitation assays, we show that Hoxa-9 protein binds directly to the Pim-1 promoter/enhancer region in hematopoietic cells. Enforced expression of Hoxa-9 significantly induces Pim-1 mRNA and protein in blood cells in a DNA-binding dependent manner. Furthermore, Pim-1 protein levels are diminished in lin- blood cells from Hoxa-9−/ − fetal liver, showing that Hoxa-9 is required to maintain normal levels of Pim-1 in primitive hematopoietic cells. By real-time PCR, we find that Hoxa-9 and Pim-1 mRNA levels track together in highly purified subpopulations of long-term and short-term HSC and in common myeloid and granulocyte-monocytes progenitors (CMP/GMP). Induction of Pim-1 protein by Hoxa-9 significantly increases the phosphorylation and inactivation of the pro-apoptotic BAD protein, a known target of the Pim-1 kinase. Hoxa-9−/ − marrow cells show increased levels of apoptosis and decreased cell cycle progression and proliferation compared to wild-type cells, and these defects are significantly improved by the re-introduction of Pim-1. These data provide strong evidence that Pim-1 is a direct transcriptional target of Hoxa-9 and a key mediator of its antiapoptotic and pro-proliferative effects on primitive hematopoietic cells. Since Hoxa-9 is upregulated in many cases of human acute myeloid leukemia, Pim-1 may serve as a useful therapeutic target in Hoxa-9 driven leukemias.