Pharmacological Inhibition of the Stress-Related Deacetylase SIRT1 Enhances Eradication of CML stem Cells

Abstract 448

BCR-ABL tyrosine kinase inhibitors (TKI) are effective in inducing remissions and prolonging survival of CML patients, but fail to eradicate primitive leukemia stem cells (LSC) which remain a potential source of relapse. New strategies to enhance elimination of residual CML LSC in TKI-treated patients are required. We have previously reported that the stress-related deacetylase SIRT1 is expressed at high levels in CML stem/progenitor cells and that inhibition of SIRT1 expression using lentivirus-expressed shRNA induces apoptosis in CML progenitors and increases their sensitivity to imatinib (IM) by activating p53 signaling (Blood 2010, 116: 200A). These results support an important role for SIRT1 in CML LSC maintenance and TKI resistance, and as a potential molecular target for therapy directed against CML LSC. Tenovin-6 (TV) has been identified as a potent small molecule inhibitor of SIRT1 activity (Cancer Cell 2008, 13:454). Here we evaluated whether pharmacological inhibition of SIRT1 activity using TV could selectively inhibit CML stem/progenitor cells. As with shRNA-mediated knock-down of SIRT1, treatment with TV (0.5 μM) significantly increased apoptosis of CML CD34+ cells (TV16±7% vs. Control 3±2%, p=0.04, n=3), but not normal CD34+ cells (TV 6±2% vs. Control 4±2%, p=0.1, n=3). The combination of IM (2.5 μM) and TV induced significantly increased apoptosis in CML progenitors compared to IM alone, and to a significantly greater extent than in normal cells (CML, TV + IM 40±2% vs. IM 19±3%, p=0.009, n=3; CB, TV + IM 15±4% vs. IM 10±2%, p=0.04, n=3). TV (1 μM) increased apoptosis in both CML CD34+CD38− (TV 42±10% vs. Control 4±3%, p=0.04, n=3) and CD34+CD38+ cells (TV 35±7% vs. Control 8±2%, p=0.03, n=3). CFSE labeling indicated that treatment with TV resulted in increased apoptosis of undivided CML CD34+CD38− cells identified on the basis of high CFSE fluorescence (TV 20±7% vs. Control 2±1%, p=0.04, n=3). The combination of TV with IM resulted in a significant increase in apoptosis in CML CD34+CD38− CFSE^high cells compared to IM alone (TV plus IM 35±5% vs. IM 10±4%, p=0.03, n=3). Treatment with TV (0.5 μM) reduced CML CFC frequency (70±9% inhibition with TV compared to untreated controls, p=0.009, n=3) without affecting normal CFC frequency. Combination of TV (0.5 μM) with IM resulted in enhanced inhibition of CML CFC compared to IM alone, but did not enhance inhibition of normal CFC (CML: TV plus IM 82±6% inhibition vs. IM 57±10%, p=0.02, n=3; CB: TV plus IM 38±7% inhibition vs. IM 36±9%, p=0.1, n=3). TV treatment effectively inhibited the growth of Baf3 cells expressing T315I-mutated BCR-ABL, and significantly enhanced apoptosis of IM-resistant CML blast crisis CD34+ cells [TV (1 μm) 30±1% vs. Control 19±5%, p=0.04, n=3], suggesting SIRT1 inhibition can also target TKI-resistant CML cells. Ex vivo treatment with TV (1 μM) significantly reduced longer-term (12 weeks) engraftment of CML CD34+ cells in NSG mice following TV treatment (TV treated 0.2*10⁵±0.1*10⁵ human CD45+ cells in murine BM vs. Control 1.8*10⁵±0.6*10⁵, p=0.009, n=5). Significant reduction in engraftment of CD33+ (p=0.008) and CD14+ myeloid cells (p=0.009) was seen. Q-PCR and FISH analysis confirmed that engrafted human cells were leukemic in origin. Interestingly, engraftment of CB CD34+ cells was not reduced after treatment with TV (TV 2.7*10⁶±0.7*10⁶ human CD45+ cells in murine BM, vs. Control 2.4*10⁶±0.8*10⁶, p=0.2, n=6). These results show that SIRT1 inhibition by TV effectively targets primitive human CML cells with in vivo multi-lineage engraftment capacity. Treatment with TV significantly enhanced acetylated p53 levels in CML CD34+ cells, indicating effective inhibition of SIRT1 activity. TV treatment also increased total p53 levels, possibly related to reduced p53 degradation. TV treatment did not increase acetylated p53 or total p53 levels in normal CD34+ cells. Importantly shRNA-mediated knock-down of p53 resulted in significant reduction of TV-induced apoptosis in CML CD34+ cells (13±6% apoptosis with p53 shRNA; 33±7% apoptosis with control shRNA, p=0.04, n=3), indicating that the effects of TV on CML CD34+ cells are related to p53 acetylation and activation. In conclusion, our studies indicate that pharmacological inhibition of SIRT1 can activate p53 and enhance eradication of CML LSC in combination with TKI treatment, and support further evaluation of targeted inhibition of SIRT1 as a therapeutic strategy in CML.