SIRT1 Inhibition Induces Apoptosis In Human CML Progenitors by Enhancing p53 Acetylation and Activation

Abstract 200

Imatinib mesylate (IM) treatment is effective in inhibiting CML primitive progenitor growth but induces only modest levels of apoptosis. Improved approaches to enhance elimination of residual CML progenitors in IM-treated patients are required. The NAD+ dependent deacetylase SIRT1 is a stress-response gene that is expressed at higher levels in CML compared to normal CD34+ progenitors. We have shown that inhibition of SIRT1 expression using lentivirus-mediated SIRT1 shRNA expression results in modest induction of apoptosis in CML progenitors and significantly enhanced apoptosis in combination with IM (Blood 2009, 114: 189). SIRT1 inhibition does not induce apoptosis in normal progenitors or increase their sensitivity to IM. SIRT1 can potentially regulate the acetylation of several transcription factors, including the p53 tumor suppressor protein. In contrast to several other cancers, p53 mutations are rare in CP CML, suggesting that p53 may still be subject to activation in CML progenitors. However we have observed that p53 levels are reduced in IM-treated CML CD34+ progenitors. We were therefore interested in investigating whether increased apoptosis of CML progenitors following SIRT1 inhibition was related to enhancement of p53 activity via protein acetylation. We observed that inhibition of SIRT1 using shRNA resulted in increased acetylation of p53 in CML CD34+ cells without increase in total p53 expression on both western blotting and flow cytometry. SIRT1 inhibition also increased p53 acetylation in IM-treated cells. Acetylated p53 was observed to localize to the nuclei of CML CD34+ cells on immunofluorescence microscopy. Q-PCR analysis revealed increased expression of the p53 transcriptional targets, GFI-1 and Necdin, in SIRT1 knockdown CML CD34+ cells (Necdin, Si versus Ctrl, 2.7±0.4 fold, p<0.05, n=3; GFI-1, Si versus Ctrl, 2.4±0.4 fold, p<0.05, n=3). These results suggest that SIRT1 inhibition results in increased p53 acetylation, nuclear localization and transcriptional activity in CML CD34+ cells. To further investigate the role of p53 in mediating the effects of SIRT1 inhibition we concomitantly knocked down both p53 and SIRT1 in CML CD34+ cells. Inhibition of p53 expression by lentivirus mediated delivery of p53 shRNA significantly enhanced growth and reduced apoptosis of SIRT1 knockdown CML CD34+ cells (14±2% apoptosis with SIRT1 knockdown, 7±2% apoptosis with combined SIRT1 and p53 knockdown, p<0.05, n=3). These results confirm an important role for p53 in SIRT1 mediated effects in CML progenitors. SIRT1 inhibition did not inhibit growth or induce apoptosis in CML blast crisis K562 cells, which are p53 null. To further determine the specific role of p53 acetylation in mediating SIRT1 effects, we expressed both wild type and acetylation-deficient p53 constructs in K562 cells. K562 cells ectopically expressing the wild type p53 gene demonstrated significant growth inhibition and apoptosis following SIRT1 knockdown (SIRT1 shRNA, 18±5% versus Ctrl shRNA, 8±3%, p<0.05), increased levels of acetylated p53, and enhanced transactivation of a p53 reporter containing the mdm2 promoter cloned upstream of the luciferase gene (p<0.05). In contrast, K562 cells transfected with an acetylation-defective p53 gene (with all eight acetylation sites mutated) did not demonstrate significant growth inhibition or apoptosis following SIRT1 inhibition. These results indicate that the inhibitory effect of SIRT1 on CML cells is dependent on p53 acetylation. We conclude that inhibition of SIRT1 enhances p53 acetylation and transcriptional activity resulting in enhanced apoptosis of CML progenitors. SIRT1 is a potentially druggable target, and several groups are actively developing SIRT1 inhibitory compounds. Activation of p53 via SIRT1 inhibition represents an attractive approach to eradicate CML stem cells in combination with IM or other treatments.