Rac GTPases Are Potential Therapeutic Targets in p210-BCR-ABL-Induced Myeloproliferative Disease (MPD).
Authors: Thomas Emily K. , Cancelas Jose A. , Chae Heedon , Cox Adrienne D. , Keller Patricia J. , Perrotti Danilo , Neviani Paolo , Druker Brian J. , Zheng Yi , Harris Chad E. , Williams David A. 
Published: October 2019

The p210-BCR-ABL fusion protein is a constitutively active tyrosine kinase that is necessary and sufficient for the development of chronic myelogenous leukemia (CML). ABL-kinase inhibitors such as imatinib mesylate (Gleevec, STI571) potently block BCR-ABL activation, but the continued presence of leukemic stem cells and the emergence of imatinib-resistant BCR-ABL mutants suggest that ABL kinase inhibitors alone cannot completely eradicate disease. Rac GTPases have been implicated in BCR-ABL-mediated proliferation in cell lines and regulate many of the same signaling pathways as BCR-ABL, suggesting that these proteins could be additional therapeutic targets in CML. We have found that Rac1, Rac2, and, to a lesser extent, Rac3 were hyperactivated in CD34+ cells purified from the peripheral blood of two CML patients. To better study the role of Rac in BCR-ABL disease development, murine hematopoietic stem cells (HSC) genetically deficient in Rac1 and/or Rac2 were transduced with a retroviral vector expressing p210-BCR-ABL. Wild type (WT) and Rac1−/− mice experienced similar disease progression [median survival 23 ± 6 days (n=30) and 22 ± 4 days (n=8), respectively], Rac2−/− mice exhibited significantly attenuated development of BCR-ABL-mediated MPD [median survival 43 ± 27 days (n=18); p<0.001], and Rac1−/−;Rac2−/− animals showed markedly prolonged survival [median survival 92 ± 34 days (n=19); p<0.001]. p210-BCR-ABL WT, Rac1−/−, and Rac2−/− mice had elevated circulating myeloblasts 30 days post-transplant, while Rac1−/−;Rac2−/− mice had normal peripheral blood morphology. Attenuation of disease in Rac2- and Rac1/Rac2-deficient animals correlated with severely diminished activation of BCR-ABL-induced signaling pathways, including p44/42 and p38 ERK, JNK, CrkL, and Akt. The leukemogenesis impairment induced by Rac deficiency did not appear to be due to loss of p210-BCR-ABL vector integration, as clonal analysis of leukemic bone marrow from mice in each genotype by LAM-PCR showed similar, oligoclonal reconstitution of p210-BCR-ABL expressing cells. Interestingly, bone marrow cells obtained from Rac1/Rac2-deficient animals that developed late leukemia showed marked hyperactivation of Rac3 and initiated disease in recipients with a latency of three weeks, suggesting that leukemia-initiating cells were able to engraft, in spite of Rac1/Rac2 deficiency. Treatment of BCR-ABL-expressing murine HSC with NSC23766, a rationally-designed Rac-specific small molecule antagonist, potently inhibited cell proliferation in vitro and increased the survival of leukemic animals treated in vivo, compared to PBS control-treated animals (p<0.05). NSC23766 also inhibited the growth of an imatinib-resistant p210-BCR-ABL-T315I-expressing Ba/F3 leukemic cell line by 90%, compared to <5% by imatinib alone, blocked the growth of primary human chronic phase Rac-hyperactivated CML blast colonies by 80% in vitro, and inhibited survival of these cells in NOD-SCID mice. These results suggest that individual Rac proteins play both unique and combinatorial roles in stem cell transformation and may represent unique targets for therapy of BCR-ABL-persistent and imatinib-resistant CML.