Bcl-XL Is a Critical Mediator of Rac Signaling in MLL-AF9-Induced Acute Myeloid Leukemia.

Abstract 1971 Poster Board I-994 Rac GTPases have been found to play an important role in MLL-AF9 (MA9) acute myeloid leukemia. Rac activity is increased in MA9 leukemia cells, and inhibition of Rac by shRNA or the Rac specific inhibitor [NSC23766][1] induces rapid apoptosis in MA9 cells but not in normal cord blood cells or cells expressing the t(8;21) fusion protein AML1-ETO (Wei et al, Cancer Cell 2008). More recently, we demonstrated that MA9-mediated leukemogenesis is significantly delayed in Rac2 knockout (Rac2 KO) low-density bone marrow (LDBM) cells in contrast to the rapid development of leukemia seen after transplantation of wild-type (WT) murine LDBM expressing MA9. Loss of Rac2 has previously been shown to affect Bcl-2 family expression, specifically resulting in decreased levels of the pro-survival protein Bcl-xL and increased levels of the pro-apoptotic protein Bad (Yang et al, Immunity 2000). Here we investigate the role of Bcl-xL in mediating the effects of Rac signaling in MA9 leukemia. Interestingly, treatment with [NSC23766][1] inhibits Bcl-xL expression in MA9 cells. This effect was also seen in Bcl-xL-transduced MA9 cells, implicating a role for Rac in post-transcriptional regulation of Bcl-xL expression in addition to the transcriptional effect that has been previously reported. Significantly fewer Annexin V+ cells were found in [NSC23766][1]-treated MA9 cells expressing ectopic Bcl-xL protein when compared to control MA9-pBabe cells, demonstrating that excess Bcl-xL expression can partially rescue the phenotype associated with Rac inhibition. To target the Bcl-2 survival signals in MA9 cells, we used the small-molecule pan-Bcl-2 inhibitor GX15-070 and found a significant inhibition of survival and growth of treated MA9 cells at much lower doses than required for control cells not expressing MA9. Similarly, shRNA knockdown of Bcl-xL in MA9 cells inhibited growth. To determine whether ectopic expression of Bcl-xL could compensate for loss of Rac2 in vivo and restore short latency MA9-mediated leukemogenesis, LDBM was harvested from Rac2 KO mice and transduced with a retroviral vector expressing MA9 and a second retrovirus to express either Bcl-xL or empty vector control. Unsorted cells were transplanted into congenic CD45.1 mice to also establish a competitive assay between singly transduced cells expressing MA9 alone and doubly transduced cells co-expressing both MA9 and Bcl-xL. All recipients of Rac2 KO cells from the MA9+empty vector transduction group remain alive at 300 days from transplant. All recipients of Rac2 KO cells from the MA9+Bcl-xL transduction group died of leukemia with a mean latency of 184 days (range 102 — 280 days), and analyzable tumors showed co-expression of MA9 and Bcl-xL. Two mice that died later in this cohort (day 224 and day 280) also showed a second population of cells expressing MA9 without co-expression of Bcl-xL. Experiments are underway to determine which population has leukemogenic ability upon secondary transplant. Mice that received WT LDBM transduced with MA9+Bcl-xL died of leukemia with the expected mean latency of 190 days for this model (range 130 — 250 days). In this group, the number of WT MA9 tumors co-expressing Bcl-xL was in equal proportion to tumors co-expressing empty vector, suggesting the advantage bestowed by Bcl-xL overexpression is more pronounced in the context of Rac2 deficiency. Together, these findings suggest a critical role for Rac signaling through the Bcl-xL pathway in MA9 leukemia and support therapeutic approaches targeting this pathway in MLL leukemia. Disclosures: No relevant conflicts of interest to declare. [1]: /lookup/external-ref?link_type=GENPEPT&access_num=NSC23766&atom=%2Fbloodjournal%2F114%2F22%2F1971.atom