Figure 6. HDAC8 inhibition or SIRT6 knockdown sensitizes AML to KPT-9274 by suppression of HR and D-NHEJ pathways or mono-ADP-ribosylation of PARP1.

A) Combined treatment of AR-42 and KPT-9274 synergistically increases unrepaired DNA damage sites as indicated by the accumulation of phosphorylated H2A.X. MOLM13, IDH2^WT TF-1 and IDH2^mut TF-1 cells were incubated with vehicle control, 0.8 μM AR-42 alone, 0.1 μM KPT-2974 alone or drug combination for 48 hours. Thereafter, cells were fixed, permeabilized and stained with BV421 anti-H2A.X (pS139) (Clone N1-431). Stained cells were analyzed by flow cytometry. Results are shown as mean ± SEM of duplicates. *p<0.05. N.S.: not significant. B) Left panel: schematic illustration of I-SceI-based EJDR reporter system; Right panel: Quantification of OCI-AML3-DR and -EJ reporter assays. AR-42 blocks HR and NHEJ repairs, while KPT-9274 impairs NHEJ repair. Results are shown as mean ± SEM of duplicates. *p<0.05;**p<0.01. C) Exposure of MOLM13 cells to AR-42 and KPT-9274 results in a decrease in activities of HR, NHEJ and ATM signaling, and a concomitant increase in phospho-H2A.X (pS139) (Clone 20E3), consistent with the loss of multiple pathway-mediated DSB repairs. MOLM13 cells were treated with vehicle control, 0.4 μM or 0.8 μM AR-42 alone, 0.1 μM or 0.25 μM KPT-2974 alone or drug combination for 24 hours before being subject to Western blotting analysis of HR, NHEJ and ATM markers. GAPDH serves as loading control. Results are representative of two independent experiments. D) Knockdown of HDAC8 reduces the levels of HR pathway mediators and abolishes the activities of D-NHEJ and ATM signaling in cooperation with KPT-9274. Scrambled or shHDAC8-transduced MOLM13 cells were treated with vehicle, 0.1 μM or 0.25 μM KPT-2974 before being subject to immunoblotting analysis of DNA repair targets. GAPDH serves as loading control. Results are representative of two independent experiments. E) shSIRT6 and KPT-9274 synergistically increase unrepaired DNA damage sites as indicated by the accumulation of phosphorylated H2A.X. Results are shown as mean ± SEM of duplicates. **p<0.01. F) Co-IP showing that shSIRT6 decreases mono-ADP-ribosylation of PAPR1 in response to KPT-9274 treatment. PAPR1 was immunoprecipitated and mono-ADP-ribosylation was detected by western blotting. Results are representative of two independent experiments. G) Schematic illustration of possible mechanisms of sensitizing AML cells to NAMPT inhibition by suppression of DNA repairs.