Inhibition of the Rho GTPase, Rac, Differentially Inhibits Proliferation of AML Cell Lines and May Serve as a Novel Molecular Target.

The Rho GTPases are a family of signal transduction proteins that have important functions in cell growth and survival, gene transcription, adhesion, motility, and formation of the actin cytoskeleton. These proteins cycle between active GTP-bound and inactive GDP-bound states. Members of the Rho GTPase family have been found to be overexpressed or mutated in a wide variety of malignancies. The Rac GTPases have been shown to play an essential role in hematopoietic cell proliferation and survival and in myeloid cell function (Gu et al, Science 2003). We hypothesized that activation of Rac may be dysregulated in myeloid leukemia and may contribute to the leukemic phenotype. We first analyzed the expression and activity of Rac in leukemic cell lines. We used the PAK-effector pull-down assay, which detects the active GTP-bound Rac proteins, and found that all four AML cell lines tested exhibited an elevated Rac activity when compared with normal human bone marrow derived CD34+ cells. The abnormal activity varied considerably between the cell lines tested. | Cells Tested | Ratio of GTP Rac/Total Rac | |:-------------------------:| -------------------------- | | THP1 (monocytic) | 0.83 | | ML2 (myelomonocytic) | 0.50 | | U937 (monocytic) | 0.43 | | HL60 (promyelocytic) | 0.55 | | Bone marrow derived CD34+ | 0.13 | Next we examined if a novel small molecule Rac inhibitor, NSC 23766 (Gao et al., PNAS 2004), could be used to pharmacologically inhibit Rac activity and reverse the proliferation phenotype of the cells. NSC 23766 interferes with the guanine nucleotide exchange factor binding of the Rac GTPases and prevents the GTP loading of Rac, but not Cdc42 or RhoA. Biochemical analysis demonstrated a dose-dependent inhibition of Rac in all cell lines except HL60. Cell lines were treated with increasing doses of NSC 23766 to determine the effect of inhibition of Rac activity on cell growth. The growth of U937 cells, which had the lowest level of Rac activity at baseline, was modestly inhibited, while HL60 cell growth did not appear to be inhibited by NSC 23766. Growth of both THP1 and ML2, which had high levels of Rac activity, was significantly inhibited by NSC 23766 in a dose dependent fashion. We next determined if we could lower the level of GTP-bound Rac proteins in HL60 and ML2 by retroviral transduction of the cells with two dominant negative Rac mutants (Rac D57N and Rac N17). Both of these dominant negative Rac constructs lowered the Rac activity in ML2 but not in HL60. Additional studies focused on ML2 cells which showed the greatest response to NSC 23766. Proliferation, which was assessed by [3H] thymidine incorporation, was decreased by over 500% when comparing untreated to cells treated with 40 μM of NSC 23766 (P<0.001). Cell cycle analysis by propidium iodine staining, showed a significant increase in G0/G1 in cells treated with NSC 23766 (38.9% vs. 79.8% G0/G1, non-treated vs. treated). Apoptosis, as analyzed by Annexin V/7AAD, increased from 0.87% to 11.27% after NSC 23766 treatment. Our studies demonstrate that there is a wide variability in activation of Rac GTPases in myeloid leukemia cell lines. NSC 23766 suppresses Rac activity of AML cells in a dose dependent fashion and inhibits growth in sensitive cells by cell cycle arrest and apoptosis. These results also suggest the possibility that Rac may prove to be a novel therapeutic target in AML.