Rac GTPase Survival Signaling Through Anti-Apoptotic Bcl-2 Proteins Reveals a Target for Combination Therapy in MLL-AF9 Acute Myeloid Leukemia

Abstract 1536 The Rac family of small Rho GTPases has attracted interest as a therapeutic target in hematologic malignancies due to their central role in coordinating diverse cellular processes such as adhesion, cytoskeletal organization, proliferation, and survival. Rac activity is increased in MLL-AF9 (MA9) acute myeloid leukemia, and Rac inhibition using the small-molecule NSC23766 induces apoptosis in MA9 cells. We recently found that loss of Rac2 delays development of MA9 leukemia in a murine genetic model. Furthermore, latency of disease can be rescued by ectopic expression of Bcl-xL in the Rac2 knockout cells implicating Rac2 regulation of pro-survival Bcl-2 proteins in MA9 leukemogenesis. Whether Rac survival signaling through Bcl-2 proteins can be exploited therapeutically in the developed MA9 leukemia is untested. We hypothesized that Rac2 signaling is critical for MA9 leukemia cell survival, and that inhibition of Rac or downstream Bcl-2 proteins could be an effective therapeutic strategy alone or in combination. We tested our hypothesis in MA9 cells derived from human CD34+ umbilical cord blood (UCB) cells expressing MA9 by retroviral transduction. Lentiviral knockdown was used to determine the specific contribution of Rac2 to MA9 cell survival and maintenance. Decreased levels of Bcl-xL and Bcl-2 were seen in MA9 cells expressing Rac2-targeting shRNA, compared to non-targeting control. Rac2 knockdown induced apoptosis and impaired growth of MA9 cells in culture. Furthermore, Rac2 deficiency reduced in vitro colony-forming ability indicating impairment of the clonogenic MA9 cell. MA9 cells expressing two different Rac2 shRNA vs. non-targeting control were injected into NOD/LtSz-scid-SGM3 (NSS) mice to determine whether Rac2 deficiency impairs engraftment and progression of the MA9 leukemia stem cell in a xenotransplantation assay. Flow cytometric analysis of bone marrow aspirates showed markedly reduced MA9 engraftment in the Rac2 knockdown groups. Whereas all mice in the control group eventually died of MA9 leukemia (N=5), only one death from MA9 cells expressing Rac2 shRNA was seen in the knockdown groups (N=10). We next evaluated the effects of direct inhibition of Bcl-2 proteins downstream from Rac using the BH3-mimetic ABT-737. Three different MA9 cell lines, as well as the THP-1 cell line bearing an MLL-AF9 fusion, were highly sensitive (IC50 ∼30 nM) to ABT-737, with no toxicity seen in control UCB cells in the dose range tested. To determine in vivo efficacy, ABT-737 was administered to NSS mice engrafted with human MA9 cells. End of treatment aspirates showed a marked decrease in leukemia engraftment in the ABT-737 treatment group compared to vehicle control ( in vitro and in vivo , in association with decreased expression of downstream pro-survival Bcl-2 proteins, and identify a pathway that can be exploited therapeutically. We conclude that survival signaling through Rac and Bcl-2 family proteins can be effectively targeted with small-molecule inhibitors alone and in combination in MLL-AF9 acute myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.