ALBERT

Description: Abstract 3153 Leukemia stem cells (LSCs) are capable of limitless self-renewal and are responsible for the maintenance of leukemia. Since elimination of LSCs will be of therapeutic benefit, it is important to identify regulatory pathways that control their development. We studied LSCs in a Smad4-/- mouse model of acute myelogenous leukemia (AML) induced either by the HOXA9 gene or by the fusion oncogene NUP98-HOXA9. We first serially replated hematopoietic colonies from BM cells transduced with these oncogenes to analyze if loss of SMAD4 can increase immortalization of HSPCs. We observed that the immortalizing function of HOXA9 and NUP98-HOXA9 was increased in Smad4-/- HSPCs. The enhanced immortalization of SMAD4 deficient cells should be related to their incapacity to activate TGFβ signaling. However when we administrated a TGFβ receptor kinase inhibitor, the immortalization of Smad4-/- cells was still increased, compared to TGFb receptor-blocked Wt cells. Thereby, we conclude that the activation of the canonical TGFb signaling pathway is not required for the growth-reducing effect of HOXA9/NUP98-HOXA9 transduced progenitors mediated by SMAD4. We discovered that HOXA9/SMAD4 complexes accumulate in the cytoplasm of normal HSPCs transduced with HOXA9 or NUP98-HOXA9. In contrast there is no cytoplasmic accumulation of HOXA9 in SMAD4-/- HSPCs and as a consequence increased levels of HOXA9 accumulate in the nucleus leading to increased immortalization of HSPCs. Next, we investigated whether Smad4-/- HSPCs might exhibit increased frequency of BM transformation to induce leukemia in mice. Smad4 deficiency induces expansion of primitive LSK hematopoietic cells expressing HOXA9 or NUP98-HOXA9 in vivo, increases the frequency of leukemia stem cells and bone marrow transformation in mice. Accordingly, a larger fraction of mice transplanted with Smad4-/- transduced-HSPCs succumbed more rapidly to AML. Next, we developed an approach to activate the TGFβ pathway by restoring the subcellular distribution of the endogenous SMAD4 protein accumulated in the cytoplasm of HSPCs transduced with HOXA9 or NUP98-HOXA9. To this end, we first identified the best competitor by generating different constructs that enable expression of diverse portions of the MH1 domain of SMAD4 that bind HOXA9 (Wang et al., EMBO, 2007). All the retroviral vectors (RFP+) were tested by co-expression together with the NUP98-HOXA9 oncogene (GFP+). Co-transduced GFP+ RFP+ cells were sorted by FACS to assess the effect of MH1 overexpression by CFC assay. Interestingly, one specific portion of SMAD4 (MH1-c encoding a peptide of 20aa) dramatically reduced immortalization capacity of NUP98-HOXA9. The insensitivity of Smad4-/- cells shows notably that the specificity of this effect is SMAD4 dependent. Compared to the other truncated MH1 portions tested, the increased effect of MH1-c was correlated to its capacity to bind HOXA9 and to induce apoptosis. To understand the mechanism behind the effect of MH1-c, we looked at the localization of SMAD4 by immunostaining and confocal microscopy. Expression of MH1-c was observed to induce a robust nuclear translocation of the SMAD4 protein, while SMAD4 remained accumulated in the cytoplasm of cells transduced with the empty control vector (MH1-e). This clearly shows that MH1-c disrupts the physical interaction between HOXA9 and SMAD4. The SMAD4 subcellular distribution pattern was altered and the nuclear translocation clearly activated the TGFβ pathway and apoptosis of cells. Next, we asked whether MH1-c might affect the development of leukemia in vivo. When HSPCs were co-transduced with HOXA9 and the empty control vector and transplanted into the tail vein of lethally irradiated recipient mice, the transduced HSPCs developed AML. In contrast, HSPCs co-transduced with HOXA9 and MH1-c remained healthy over one year, without any detectable GFP+ RFP+ cells in BM when they were sacrificed. Taken together, these findings show that active SMAD4 that can freely translocate to the nucleus of HSPCs in vivo can prevent or profoundly delay the initiation of malignant transformation. In conclusion, targeting the association between SMAD4 and HOXA9 prevents/reduces immortalization in vitro and transformation to leukemia in vivo. These findings demonstrate how activation of a negative regulatory pathway that prevents leukemogenesis may be a feasible approach to improve leukemia treatment. Disclosures: Slovak: PerkinElmer: Employment.

Description: We studied leukemic stem cells (LSCs) in a Smad4−/− mouse model of acute myelogenous leukemia (AML) induced either by the HOXA9 gene or by the fusion oncogene NUP98-HOXA9. Although Hoxa9-Smad4 complexes accumulate in the cytoplasm of normal hematopoietic stem cells and progenitor cells (HSPCs) transduced with these oncogenes, there is no cytoplasmic stabilization of HOXA9 in Smad4−/− HSPCs, and as a consequence increased levels of Hoxa9 is observed in the nucleus leading to increased immortalization in vitro. Loss of Smad4 accelerates the development of leukemia in vivo because of an increase in transformation of HSPCs. Therefore, the cytoplasmic binding of Hoxa9 by Smad4 is a mechanism to protect Hoxa9-induced transformation of normal HSPCs. Because Smad4 is a potent tumor suppressor involved in growth control, we developed a strategy to modify the subcellular distribution of Smad4. We successfully disrupted the interaction between Hoxa9 and Smad4 to activate the TGF-β pathway and apoptosis, leading to a loss of LSCs. Together, these findings reveal a major role for Smad4 in the negative regulation of leukemia initiation and maintenance induced by HOXA9/NUP98-HOXA9 and provide strong evidence that antagonizing Smad4 stabilization by these oncoproteins might be a promising novel therapeutic approach in leukemia.