FLT3-ITD+ AML Stem/Progenitor Cells Demonstrate Increased SIRT1 Expression and Enhanced Sensitivity to SIRT1 Inhibition

Abstract 1368

In acute myeloid leukemia (AML) small populations of leukemia stem and progenitor cells are resistant to available therapies and represent an important barrier to response and cure. There is pressing need to develop new treatment approaches to target AML stem/progenitor cells. The NAD-dependent deacetylase SIRT1 plays an important role in protection of stem cells from stress. We have recently shown that SIRT1 inhibition enhances elimination of CML LSC via acetylation and activation of p53 (Cancer Cell 2012, 21:266). Here we evaluated the role of SIRT1 in growth and maintenance of AML stem/progenitor cells. We observed higher levels of SIRT1 expression in AML compared to normal CD34⁺CD38⁻ and CD34⁺CD38⁺ cell, with highest expression levels in samples from patients with poor-risk cytogenetic abnormalities. We tested the effects of the SIRT1 inhibitor Tenovin-6 (TV-6) on CD34⁺ cells from high-risk AML patients (n=12) and healthy controls (n=4). AML CD34⁺ cells demonstrated varying degrees of sensitivity to TV-6, but TV-6 treatment did not significantly reduce viability of AML CD34⁺ cells taken as a group, compared to normal cells (p=0.8). However, we observed significantly reduced viability of a subset of samples bearing the FLT3-ITD mutation following TV-6 treatment (p=0.01). We therefore tested the effects of TV-6 on a larger set of poor risk AML samples with the FLT3-ITD mutation (n=11), or wild-type FLT3 (FLT3-WT, n=11). The FLT3-ITD mutation was associated with significantly enhanced sensitivity of AML CD34⁺ cells to TV-6 induced apoptosis (LD50 for FLT3-ITD=1.9μM; FLT3-WT=3.9μM, p=0.009), and inhibition of cell growth measured using the CellTitre Glo luminescent cell viability assay (FLT3-ITD IC50=0.73μM; versus FLT3-WT IC50=1.4μM, p=0.01). Therefore the presence of the FLT3-ITD mutation identifies a subset of AML samples that are very sensitive to SIRT1 inhibition. Tenovin-6 treatment increased acetylated as well as total p53 levels in FLT3-ITD+ AML CD34⁺ cells. Tenovin-6 also induced apoptosis and inhibited growth of the FLT3-ITD+ and p53 wild type cell line MV4-11. SIRT1 knock-down reduced TV-6-induced apoptosis and growth inhibition in MV4-11 cells confirming that TV-6 effects are related to SIRT1 inhibition. The growth inhibitory and pro-apoptotic effects of TV-6 in MV4-11 cells were also significantly reduced by p53 knock-down (TV-6 1μM, p53 shRNA 39±8% inhibition, control ShRNA 66±2% inhibition, p=0.04, n=3). Taken together these results support a potential role for p53 activation in mediating the effects of TV-6 treatment in FLT3-ITD+ AML cells. Sequencing studies indicated that only one sample in the FLT3-WT group harbored a p53 mutation, suggesting that their resistance to SIRT1 inhibitors is related to mechanisms other than p53 mutation. Flow cytometry analysis demonstrated increased SIRT1 levels in FLT3-ITD (n=7) compared to FLT3-WT (n=9) AML CD34⁺ cells (p=0.01). We used lentivirus vectors to ectopically express a FLT3-ITD construct in human cord blood CD34⁺ cells. Expression of FLT3-ITD resulted in significantly increased SIRT1 expression levels compared to FLT3-WT or vector controls. Consistent with results obtained with samples from AML patients, FLT3-ITD transformed CB CD34⁺ cells demonstrated enhanced sensitivity to TV-6 mediated apoptosis (TV6 1μM, FLT3-ITD 44±3% survival, n=5; FLT3-WT 59±2% survival, n=5, p=0.04; empty vector 64±4% survival, p=0.02, n=5) and cell growth inhibition. These results indicate that FLT3-ITD-mediated SIRT1 overexpression may contribute to the increased sensitivity of FLT3-ITD AML CD34⁺ cells to SIRT1 inhibition. In conclusion, our studies indicate that AML stem/progenitor cells demonstrate varying degrees of sensitivity to SIRT1 inhibition reflecting underlying genetic lesions. Importantly the poor prognosis FLT3-ITD mutation is associated with increased SIRT1 expression and enhanced sensitivity to SIRT1 inhibition. In view of the limited success of small molecule FLT3 inhibitors so far, the use of SIRT1 inhibitors may offer an attractive approach to target FLT3-ITD+ AML stem/progenitor cells.