RNAi-Mediated Inhibition of Mcl-1 Expression Enhances Apoptosis in Imatinib-Treated CML Progenitors

Abstract 1669

Tyrosine kinase inhibitor (TKI) treatment inhibits proliferation in CML stem/progenitor cells, but only modestly increases apoptosis. Residual leukemia stem cells remain a potential source of disease relapse in IM-treated patients. The Bcl-2 family of anti-apoptotic proteins plays a central role in the regulation of apoptosis. Several Bcl-2 inhibitors are being evaluated in preclinical and clinical studies and there is considerable interest in evaluating their ability to induce apoptosis in CML stem and progenitor cells. However these agents have considerable toxicity possibly related to lack of selectivity for individual family members. We performed a functional siRNA screen to determine the role of individual Bcl-2 family members in maintaining survival of in CML and normal CD34+ cells. CML and normal CD34+ cells were transfected with siRNAs targeting Bcl-2, Bcl-2L1, Bcl-2L2, Bcl-2L10, Mcl-1 and Bcl2A1. In this screen Mcl-1 knockdown resulted in significant reduction in viability of CML CD34+ cells, with or without co-treatment with IM (1uM). Significant reduction in normal CD34+ viability was not seen. These results were validated using different siRNA sequences to knockdown Mcl1 expression. Increased apoptosis of CML but not normal CD34+ cells was seen (23±8% for CML vs. 4.2±1.5% for normal CD34+ cells, n=3, p<0.5). CML CD34+ cell apoptosis was further enhanced by combination of Mcl-1 inhibition with IM treatment (48±15% for CML vs. 7.2±3% for CB progenitors, p<0.1). To further evaluate the role of Mcl-1 in regulating CML CD34+ cell growth, an anti-Mcl-1 shRNA construct was cloned into the pHIV7-SF-RFP lentivirus vector. Cord blood and CML CD34+ cells were transduced with Mcl-1 specific or control, non-specific shRNA expressing vectors. Western blotting demonstrated effective knockdown of Mcl-1 protein levels in Mcl-1 shRNA transduced CD34+cells (82% reduction in CML and 78% in normal CD34+ cells). CD34+ RFP+ cells were selected by flow cytometry and cultured in presence and absence of IM. A significant increase in apoptosis was seen in Mcl-1 knockdown CML CD34+ cells compared with control shRNA-transduced cells, and further increase in apoptosis was seen following IM treatment (4.7±0.5 for control shRNA-transduced cells VS 25.7±2.1 for Mcl-1 knockdown cells). Mcl-1 knockdown CML CD34+ cells generated fewer colonies in methylcellulose progenitor culture (93 colonies for control siRNA transduced cells vs. 31 colonies for Mcl-1 knockdown cells) and demonstrated reduced cell expansion following culture with growth factor (SCF; IL3; GM-CSF and G-CSF) compared with control shRNA transduced cells (383,750± 172,476 for control shRNA-transduced cells 224,250± 87,044 for Mcl-1 knockdown cells). Cell expansion was further reduced with IM treatment. Mcl-1 knockdown resulted in complete loss of erythroid colony formation. Analysis of cell differentiation by flow cytometry after culture for 4 or 7 days revealed that Mcl-1 knockdown resulted in reduced generation of both erythroid (GPA+) and myeloid (CD33+ and CD14+) cells. In contrast to the results of the initial siRNA studies, shRNA-mediated Mcl-1 knockdown also resulted in significantly increased apoptosis of normal CD34+ cells (12.6± 1.6% for control shRNA-transduced cells and 24.5± 0.9% for Mcl-1 knockdown cells) associated with reduced colony formation and reduced growth in culture (1.265e+006± 273,892 for control shRNA-transduced cells 589,000 ± 188,082 for Mcl-1 knockdown cells). We conclude that RNAi-mediated Mcl-1 knockdown inhibits CML CD34+ cell survival and proliferation and enhances apoptosis after IM treatment, but also reduces viability of normal CD34+ cells. Since Mcl-1 protein expression is subject to multiple levels of regulation, our results suggest that strategies to selectively target Mcl-1 regulatory mechanisms active in CML but not normal progenitors may be less toxic and have greater clinical utility than direct targeting of the protein.