Role of the SIRT1 Deacetylase in Survival and Imatinib Resistance of CML CD34+ Progenitors.

Abstract 189 [][1] Imatinib mesylate (IM) treatment has primarily anti-proliferative effects on CML progenitors, and only modest induction of apoptosis is observed. Quiescent, primitive progenitors are especially insensitive to IM induced apoptosis. Identification and targeting of mechanisms of resistance to IM is required to allow enhanced elimination of residual CML progenitors in IM treated patients. SIRT1 is a NAD+ dependent deacetylase that regulates activity of several proteins involved in stress responses. We have observed significantly increased expression of SIRT1 mRNA and protein in CML compared to normal CD34+ progenitors. Here we investigated the effect of inhibition of SIRT1 expression on growth, survival and IM sensitivity of CML progenitors. CML and normal CD34+ cells were transduced with lentivirus vectors expressing SIRT1 shRNAs (Si-1 or Si-2) or control shRNA (Ctrl). Inhibition of SIRT1 expression in Si-1 (95% inhibition) and Si-2 (80% inhibition) transduced CD34+ cells was confirmed on Western blotting. SIRT1 inhibition resulted in modest increase in apoptosis of CML progenitors (Si-1 16±9%, Si-2 10±5% and Ctrl 8±4% apoptosis), and significantly enhanced sensitivity of CML progenitors to IM (2.5μM) induced apoptosis (Si-1, 27±12%, Si-2, 15±4% and Ctrl, 14±5%, Si-1 versus Ctrl, p=0.04, n=4). SIRT1 inhibition did not induce apoptosis in normal progenitors or increase their sensitivity to IM (Si-1 14±3%, Si-2, 13±4% and Ctrl, 11±2% without IM; and Si-1, 14±2%, Si-2, 12±3% and Ctrl, 12±2% with IM). Importantly SIRT1 inhibition significantly enhanced apoptosis of non-dividing (CFSE bright) CML progenitors treated with IM (Si-1, 49±16% and Ctrl, 33±4%, p=0.02, n=4). SIRT1 knock down inhibited proliferation of CML progenitors measured by CFSE labeling (Si-1, 46±5% and Si-2, 14±3% inhibition versus Ctrl, p<0.01, n=4) and enhanced inhibition of CML progenitors in combination with IM (Si-1, 69±11% and Si-2, 53±12% inhibition versus Ctrl, 44±9%, p<0.05, n=4). Inhibition of normal progenitor proliferation was also seen, but was significantly less than that of CML progenitors (Si-1, 30±6% and Si-2, 8±3% inhibition compared with Ctrl, p<0.05, n=4; in combination with IM, Si-1, 33±5% and Si-2, 9±4% inhibition compared with Ctrl, 8±2% inhibition). SIRT1 inhibition also reduced the number of cells generated in culture from CML progenitors (20-fold reduction in cells generated at day 14 with Si-1 compared to controls). Relatively greater inhibition of myeloid compared with erythroid cell growth was seen (at day 14, CD33+ cells with Si-1 0.42±0.21×106 and Ctrl 1.72 ± 0.61 ×106; CD11b+ cells with Si-1 0.32±0.19 ×106 and Ctrl 9.46 ±1.33 ×106). SIRT-1 inhibition also reduced the number of colonies generated from CML CD34+ cells in methylcellulose progenitor culture by itself (17±6% CFC with Si-1 compared to controls, p=0.005, n=3), and in combination with IM (3±1% CFC with Si-1 compared to 23±8% with Ctrl, p=0.03, n=4). The greater effects of Si-1 compared to Sir-2 shRNA in the above studies suggest that near complete knock-down of SIRT-1 is required to alter CML progenitor proliferation and survival. The above results indicate that the stress response gene SIRT1 contributes to enhanced survival and proliferation of CML progenitors and protects CML progenitors from IM-induced apoptosis. SIRT1 inhibition may provide a novel and effective strategy to eradicate residual CML progenitors in combination with IM. Disclosures: No relevant conflicts of interest to declare. [1]: #fn-2