Figure 4. Tet2 deletion is associated with accelerated fork progression and impaired DSB repair resulting in accumulation of DSBs in PARPi +/−OTKi treated Flt3^ITD-positive cells.

Flt3^ITD (F), Flt3^ITD;Tet2^−/− (FT), Flt3^ITD;Dnmt3a^−/− (FD) and Flt3^ITD;Tet2^−/−;Dnmt3a^−/− (FTD) murine Lin-cKit+ BMC were tested. (A) Cells were treated with 10 μM olaparib (O) +/− 100 nM quizartinib (Q) for 24 and 48 hrs. DSBs were assessed by γ-H2AX immunofluorescence (upper panels) and neutral comet assay (lower panels). Results represent mean % of control ± SD from triplicates. (B) DNA fibers from cells untreated or treated with 2.5 μM olaparib (O) for 24 hrs. Results represent forks speed; p value was calculated using Mann-Whitney rank sum test. (C) HR, D-NHEJ and Alt-NHEJ activities were measured using DR-GFP, EJ-GFP and EJ2-GFP reporter cassettes, respectively. Results represent mean % ± SD from triplicates. *p<0.05 in comparison to Flt3^ITD cells. (D) Western blot analysis of the nuclear lysates obtained from indicated cells; DDR = DNA damage response, HR = homologous recombination, D-NHEJ = DNA-PK –mediated non-homologous end-joining, and (Alt-NHEJ) = alternative NHEJ.