Smad4 as a Therapeutic Target in Nup98-HoxA9-Induced Leukemia.

Hematopoietic stem cells (HSC) are subject to complex regulation by many signal transduction pathways and transcription factors. In leukemia, these regulatory circuits are abnormal and generate dysregulated growth of HSC and their progeny. Among the prominent transcription factors that are dysregulated in leukemia are the homeobox (Hox) transcription factors or fusion-proteins derived from Hox proteins. The Nup98- HoxA9 oncogenic fusion protein is found to enforce expression of HoxA9 target genes that immortalize and block the differentiation of HSC. TGFβ inhibits Nup98-HoxA9 transformation of bone marrow cells, through direct interaction between Smad4 and the fusion protein, in a fashion which is surprisingly independent of active Smad signaling (EMBO J, 2006). Our hypothesis is that an interaction between the TGFβ and the Hox pathways is involved in leukemogenesis. To directly address this issue, we used our knockout mouse model (J.Exp Med, 2007) to determine HSC behavior in Smad4-/- cells and analyze interconnection between the TGFβ signaling pathway and the HoxA9 oncogenic activity. Using retroviral vectors to generate enforced expression of Nup98- HoxA9 in Smad4 null HSC, the transforming function was found increased approximately 4-fold in vitro. Transplantation of these transduced cells into recipient mice confirms that a block of the TGFβ pathway leads to a fast development of leukemia. At the molecular level, a 5-fold accumulation of Smad4 protein is found in wt HSC transduced by the Nup98-HoxA9, suggesting that the oncogenic potential involves a sequestration of Smad4 and a block of the TGFβ pathway. To reactivate the TGFβ pathway, one therapeutic strategy would be to induce a re-allocation of the Smad4 protein accumulated in these cells. To directly address this issue, we generated retroviral constructs that enable expression of different portions of the MH1 part of the Smad4 protein, which has been described to interact physically with the Nup98-HoxA9 oncoprotein. The retroviral transduction of one of this portions encoding a 20aa-peptide, allows release of the endogenous sequestrated- Smad4 by competition, which leads to reactivation of the TGFβ pathway and the expansion of leukemic cells is prevented in secondary recipient mice. While, administration of TGFβ remains inconceivable, because of secondary effects, the findings from these studies might consequently be used to design novel therapies for leukemia induced by abnormal Hox proteins.