Intact Rac Signaling Is Important for Leukemia Cell Survival
2010-11-19

Abstract Abstract 2885 Rho GTPases, Ras-related small G proteins, regulate multiple cell processes in hematopoietic cells. There is growing evidence that acute myeloid leukemia (AML) blasts and particularly MLL-rearranged AML blasts, rely on Rac activity (Mulloy JC et al, Blood, 2010). However, little is known about the role of these GTPases in acute lymphoblastic leukemia (ALL) and particularly precursor B cell ALL. To investigate the role of Rac and potential compensation by other GTPases in ALL, we first assessed the protein expression and activation of Rac in a number of B-ALL cell lines (SEM; RS4,11; REH; Nalm 6; Raji), compared with a T-ALL cell line (Jurkat) and several AML cell lines (ML2; MV4,11). Of these cell lines SEM; RS4,11; ML2 and MV4,11 are characterized by MLL-fusion genes. Jurkat and MLL-rearranged AML cell lines show higher expression of Rac proteins compared to B cell leukemia lines (Table 1). Overall, B-ALL cell lines exhibit highly variable levels of Rac expression and activity with no obvious correlation to the presence of MLL-fusion proteins. We then investigated proliferation and apoptosis in cell lines treated with the small molecule inhibitor NSC23766 (NSC), which blocks interaction of a subset of guanine exchange factors (GEFs) with Rac and thus inhibits its activation. Treatment with NSC led to ∼2-fold increase in cells arrested at G0/G1 and induced apoptosis in a dose-dependent fashion at NSC concentrations previously demonstrated to be non-toxic in normal hematopoietic cells (Muller LUW et al., Leukemia, 2008) (Table 2). The lymphoid cell lines Jurkat, Raji and SEM appeared less responsive to NSC with no increased apoptosis at 40μM NSC. There was no correlation between NSC response and baseline expression or activation status of Rac. However, cell lines resistant to NSC exhibited a paradoxical and transient early increase in Rac activation, suggesting the existence of compensatory activation mechanisms. To determine if the relative resistance observed in some cell lines was related to dependence on GEFs not targeted by NSC and to validate that the inhibitory effect of NSC was specifically due to Rac inhibition in sensitive cells, shRNAs were utilized to knock-down different members of the Rac subfamily. Effective shRNA-mediated knockdown was validated by western blot. Knockdown of Rac1 or Rac2 consistently induced apoptosis compared to non-targeting vector controls in NSC sensitive cell lines ML2 and Nalm6, with ML2 cells appearing slightly more sensitive to knock-down of Rac2 (Table 3). Knock-down of either Rac1 or Rac2 had little effect upon Jurkat cells which are resistant to NSC treatment. These data suggest that Jurkat cells are not dependent upon Rac signaling for survival; however we cannot discount the possibility that some compensation may occur between Rac1 and Rac2. These experiments demonstrate the importance of intact Rac signaling pathways for the survival of the majority of leukemia cell lines tested and demonstrate that dependence on Rac signaling is not restricted to leukemias characterized by MLL-rearrangements. Our observations also suggest that activation of different Rac isoforms may influence sensitivity towards pharmacological Rac inhibition. Table 1: Baseline Expression of Rac assessed by Western blot Cell line Jurkat ML-2 MV-4,11 RS-4,11 SEM Nalm 6 REH Raji Rac/b-actin expression* 1.6 2.5 1.7 0.5 0.7 0.8 1.0 1.0 (*arbitrary units, italics indicate cell lines carrying MLL-rearrangements) Table 2: % AnnexinV+ cells after treatment of the different cell lines with increasing doses the Rac-specific inhibitor NSC Cell line Jurkat ML-2 MV-4,11 RS-4,11 SEM Nalm 6 REH Raji control 6%+1.4 6%+1.3 9%+0.3 12%+3.6 9%+1.9 7%+1.5 9%+2 13%+2.3 20uM NSC 6%+1.4 9%+1.3 15%+0.3** 21%+8.5 8%+1.5 6%+1.9 25%+6.4 16%+3 40uM NSC 7%+1.8 24%+9.1 60%+4** 52%+11* 10%+1.3 10%+3.4 39%+11 16%+1.9 80uM NSC 15%+3.5* 73%+14.7** 97%+0.4** 80%+4** 17%+1.2* 46%+10.5** 62%+12.3* 22%+4 (Mean±SEM; n=5; * p&lt;0.05; ** p&lt;=0.01 versus control, bolded columns indicate increased NSC sensitivity) Table 3: % AnnexinV+ cells 7 days after lentiviral transduction of the different cell lines with Rac1 and Rac2-specific shRNA Cell line Jurkat ML-2 Nalm 6 non targeting control 4.3%+0.3 14.2%+8 11.4%+2.2 Rac1 shRNA* 8.0%+3.5 26.3%+7.9 36.8%+8.5 non targeting control 9.6%+4.2 8.1%+4.0 16.2%+3.1 Rac2 shRNA* 18.7%+4.5 35.5%+12.9 43.7%+7.1 (Mean±SEM; n=6; * second set of Rac1 and Rac2 shRNAs gave comparable results) Disclosures: No relevant conflicts of interest to declare.