Unraveling a Novel Mechanism of Stat5 Regulation by FAK and Rac1 in Flt3ITD Induced Leukemogenesis

Abstract 858

Multiple genetic checks and balances regulate the complex process of hematopoiesis. Despite these measures, mutations in crucial regulatory genes are still known to occur, which in some cases results in abnormal hematopoiesis, including leukemogenesis and/or myeloproliferative neoplasms (MPN). An example of a mutated gene that contributes to leukemogenesis is the FMS- like tyrosine kinase 3 (Flt3) that encodes a receptor tyrosine kinase, which plays an essential role in normal hematopoiesis. Interestingly, Flt3 is one of the most frequently mutated genes (∼30%) in acute myeloid leukemia (AML). Although various pathways downstream of Flt3 activation that lead to leukemic transformation have been extensively studied, effective treatment options for Flt3ITD mediated leukemogenesis is still warranted. In this study we used genetic, pharmacological and biochemical approaches to identify a novel role of Focal adhesion kinase (FAK) in Flt3ITD induced leukemogenesis. We observed hyperactivation of FAK in Flt3ITD expressing human and mouse cell. Treatment with FAK specific small molecule inhibitors F-14 and Y-11, inhibited proliferation and induced cell death of Flt3ITD expressing cells. Similarly, treatment of primary AML patient samples (n=9) expressing Flt3ITD mutations with F-14 inhibited their proliferation. Consistently expression of a dominant negative domain of FAK (FRNK) inhibited hyperproliferation and induced death of Flt3ITD bearing cells. Further, low-density bone marrow (LDBM) cells derived from FAK−/− mice transduced with Flt3ITD showed significantly reduced growth compared to wild-type (WT) LDBM cells transduced with Flt3ITD. We also observed hyperactivation of Rac1 in Flt3ITD expressing cells downstream of FAK, which was downregulated upon treatment with FAK inhibitor F-14 and Y11. Moreover, expression of dominant negative Rac1N17, or treatment with Rac1 inhibitor NSC23766 inhibited hyperproliferation of ITD bearing cells. We next wanted to ascertain the underlying mechanism of FAK mediated activation of Rac1 in Flt3ITD expressing cells. Toward this end, we found RacGEF Tiam1 to be hyperactive in Flt3ITD expressing cells, which was downregulated upon pharmacological inhibition of FAK. A Tiam1-Rac1 complex was also co-immunoprecipitated from Flt3ITD bearing cells, and this association was perturbed upon pharmacological inhibition of FAK. While, Stat5 a key molecule in Flt3ITD mediated leukemic progression, is activated and recruited to the nucleus to express Stat5 responsive genes; however the mechanism of Stat5 translocation to the nucleus is unknown. We observed a novel mechanism involving FAK and Rac1GTPase, in regulating the nuclear translocation of active Stat5. Pharmacological inhibition of FAK and Rac1 resulted in reduced Rac1 and STAT5 translocation into the nucleus, indicating a role of FAK-Rac-STAT5 signaling in Flt3ITD induced leukemogenesis. More importantly, expression of Flt3ITD in Rac1−/− or FAK−/− LDBM cells, showed inhibition of Stat5 activation and its failure to translocate into the nucleus when compared to Flt3ITD expression in WT-LDBM cells. We also observed association between active Rac1 and active Stat5 in the nucleus and in whole cell lysates of Flt3ITD bearing cells, and also in human AML patient samples (n=3), which was attenuated upon pharmacological inhibition of FAK. To determine the effect of FAK inhibition in vivo on Flt3ITD induced MPN, syngeneic transplantation was performed, and mice were treated with vehicle or with FAK inhibitor F-14. While vehicle treated mice developed MPN within 30 days, mice treated with F-14 showed significant overall survival (*p<0.02) and over 50% F-14 treated mice survived till 60 days post transplantation.

Inhibition of kinases, and other signaling molecules, that are deregulated in cancer is an exciting new therapeutic strategy. This study indicate an essential role of FAK and Rac1 molecules in Flt3ITD mediated proliferation, survival and leukemogenesis, and demonstrates a novel mechanistic role of FAK/Rac1 in translocating active Stat5 into the nucleus and regulates transformation. To our knowledge, this is also the first time a role of RacGEF Tiam1 is observed in Flt3ITD induced leukemogenesis. Overall, this study demonstrates inhibition of FAK and Rac1 as potentially novel targets, and provides an alternative approach in treating humans suffering from Flt3-ITD induced AML.