Pharmacological Inhibition Of The SIRT1 Deacetylase With The Small Molecule Inhibitor Tenovin-6 Enhances Ablation Of FLT3-ITD+ LSC In Combination With TKI Treatment

Acute myeloid leukemia (AML) is propagated by small populations of leukemia stem cells (LSC). Internal tandem duplication (ITD) in the FMS-like tyrosine kinase-3 (FLT3) is the most frequent mutation seen in AML patients, and is associated with poor prognosis. However, FLT3 tyrosine kinase inhibitors (TKI) demonstrate only moderate clinical activity in FLT3-ITD AML patients. Persistent FLT3-ITD+ AML LSC may represent a source of relapse after treatment. Additional therapeutic strategies are required to target LSC and improve outcomes for FLT3-ITD+ AML patients. We have shown that the stress-related deacetylase SIRT1 is expressed at high levels in FLT3-ITD+ AML stem/progenitor cells and in cord blood CD34+ cells ectopically expressing FLT3-ITD. Inhibition of SIRT1 using RNAi or a potent small molecule inhibitor Tenovin-6 (TV-6) induces apoptosis in FLT3-ITD+ AML progenitors via p53-dependent mechanisms while sparing normal CD34+ cells suggesting a potential role for SIRT1 in FLT3-ITD+ AML LSC maintenance (Blood 2012,120: A1368). We now show that treatment of FLT3-ITD+ AML CD34+ cells with the TKI AC220 (20 nM) did not significantly reduce SIRT1 levels, increase p53 acetylation, or increase p53 target gene expression (p21, Bax, Puma, DR5, Noxa), suggesting that kinase independent mechanisms maintain SIRT1 expression and activity in TKI-treated cells. The combination of TV-6 (1μM) with AC220 (20nM) significantly reduced survival of primary human FLT3-ITD+ AML CD34+ cells compared with AC220 or TV-6 alone (combination 47±8% inhibition, AC220 alone 21±7%, TV-6 alone 25±5%, p=0.03, n=7), and significantly increased inhibition of AML CD34+ cell growth compared to AC220 and TV-6 alone (TV-6 44±7% inhibition; AC220 30±6%, TV+AC220 59±8%, p=0.04, n=7). TV-6 treatment increased p53 expression and activity in AC220-treated FLT3-ITD+ AML CD34+ cells, but did not affect FLT3 phosphorylation or STAT5 activation. Interestingly, TV-6 treatment also reduced survival of FLT3-ITD+ AML CD34+ cells expressing the D835Y mutation associated with TKI resistance (AC220 5±5% inhibition, TV 33±8%, p=0.04, n=3], suggesting SIRT1 inhibition can target TKI-resistant FLT3-ITD+ AML cells. We next tested the effect of TV-6, AC220 and the combination on primary cells from one untreated cytogenetically normal and FLT3-ITD+ patient engrafted in NSG mice (2×10⁶ cells/mouse).12 weeks after transplantation, mice were treated with vehicle, AC220 (10mg/kg, gavage), TV-6 (100mg/kg, intraperitoneal), or the combination (n=7 per group) for 4 weeks. Treatment with TV reduced human cell engrafted in NSG mice (TV-6 2.9*10⁷±1.2*10⁷ human CD45+ cells/2 femurs vs. Control 5.3*10⁷±2.6*10⁷, after 4 weeks treatment, p=0.04). Importantly, the combination of TV and AC220 significantly reduced AML cell engraftment compared with AC220 alone (AC220 2.4*10⁷±0.9*10⁷ human CD45+ cells. Combination 1.2*10⁷±0.6*10⁷, p=0.01). Residual functional LSC were assessed by transplantation into secondary recipients. BM cells from the combination treatment group demonstrated significantly reduced engraftment of leukemia cells after secondary transplantation compared to AC220 treatment alone (16 weeks after secondary transplantation, AC220 1.8*10⁶±0.6*10⁶ human CD45+ cells/2 femurs, Combination 0.4*10⁶±0.1*10⁶, p=0.02, n=6). To determine the potential of this combination for FLT3-ITD+ AML patients refractory to standard chemotherapy, AML xenografts established using cells from a chemotherapy-resistant patient (n = 36) were treated with TV-6, AC220, or the combination vs. Standard of care (SOC, Ara-C + DNR), and vehicle control. Molecular imaging of primary patient xenografts with fluorescently labeled monoclonal antibodies after 4 weeks of therapy revealed significant differences between combination and single arm groups (vs. TV-6, p=0.0005, n=8; vs. AC220, p=0.003, n=7). Furthermore, TV-6 alone increased survival over controls (TV-6 vs. Control, p=0.02, n=6), and the combination demonstrated significantly improved survival over AC220 (p=0.02) and SOC (p=0.006. n=6). In conclusion, our studies indicate that elevated SIRT1 expression in FLT3-ITD AML cells is maintained after FLT3 TKI treatment. Inhibition of SIRT1 with TV-6 can enhance ablation of FLT3-ITD+ AML LSC in combination with FLT3 TKI treatment. Our results support further evaluation of inhibition of SIRT1 as a therapeutic strategy in FLT3-ITD+ AML.