Figure 3.
Figure 3. JAK2i ruxolitinib reduced HRR and D-NHEJ activity and enhanced the anti-MPN effect of PARPi olaparib. (Panel A) Parental cell lines and those expressing JAK2(V617F)+EpoR, CALR(del52)+MPL(wt), or MPL(W515L) were untreated (C) or treated with 5 μM olaparib (O) or 400 nM ruxolitinib (R) or ruxolitinib plus olaparib (R+O) in the presence of IL-3 plus Epo for 24 hours (γ-H2AX) and 96 hours (cell survival). DSBs were detected by γ-H2AX immunofluorescence overlapping with 4′,6-diamidino-2-phenylindole (DAPI) (top panel), and living cells were counted in Trypan blue (bottom panel; percentage of living cells in comparison with untreated control). Results represent means plus or minus SD from 3 independent experiments. *P < .05 in comparison with C using the Student t test; **P ≤ .001 in comparison with R and O groups using the response additivity approach. (Panel B) Western analysis of the indicated proteins in cells expressing JAK2(V617F)+EpoR, CALR(del52)+MPL and MPL(W515L), and in BaF3-EpoR cells (Parental) after 24-hour incubation with 400 nM ruxolitinib in the presence of IL-3 plus Epo. Proteins downregulated by ruxolitinib are in red boxes. (Panel C) HRR and D-NHEJ activities in JAK2(V617F)+ cells untreated (−) or treated for 24 hours with 400 nM ruxolitinib (+). Top panel, Western blots, Middle panel, D-NHEJ activity. S indicates linearized plasmid substrate; P indicates ligated plasmid products; results show the percentage of P. Bottom panel, HRR activity measured by restoration of EGFP expression. Results show the percentage of GFP+ cells; *P ≤ .01. (Panel D) Number of proliferating Lin−CD34+CD38−CTVlow and quiescent Lin−CD34+CD38−CTVmax cells from individual JAK2(V617F)+ MPN samples left untreated (C) or treated with ruxolitinib (R; 25 nM), olaparib (O; 1.25 μM), and ruxolitinib plus olaparib (R+O). (Panel E) Cumulative percentages from samples examined in panel D. *P < .001 in comparison with R or O groups using the Student t test; **P < .01 in comparison with R and O groups using the response additivity approach.

JAK2i ruxolitinib reduced HRR and D-NHEJ activity and enhanced the anti-MPN effect of PARPi olaparib. (Panel A) Parental cell lines and those expressing JAK2(V617F)+EpoR, CALR(del52)+MPL(wt), or MPL(W515L) were untreated (C) or treated with 5 μM olaparib (O) or 400 nM ruxolitinib (R) or ruxolitinib plus olaparib (R+O) in the presence of IL-3 plus Epo for 24 hours (γ-H2AX) and 96 hours (cell survival). DSBs were detected by γ-H2AX immunofluorescence overlapping with 4′,6-diamidino-2-phenylindole (DAPI) (top panel), and living cells were counted in Trypan blue (bottom panel; percentage of living cells in comparison with untreated control). Results represent means plus or minus SD from 3 independent experiments. *P < .05 in comparison with C using the Student t test; **P ≤ .001 in comparison with R and O groups using the response additivity approach. (Panel B) Western analysis of the indicated proteins in cells expressing JAK2(V617F)+EpoR, CALR(del52)+MPL and MPL(W515L), and in BaF3-EpoR cells (Parental) after 24-hour incubation with 400 nM ruxolitinib in the presence of IL-3 plus Epo. Proteins downregulated by ruxolitinib are in red boxes. (Panel C) HRR and D-NHEJ activities in JAK2(V617F)+ cells untreated (−) or treated for 24 hours with 400 nM ruxolitinib (+). Top panel, Western blots, Middle panel, D-NHEJ activity. S indicates linearized plasmid substrate; P indicates ligated plasmid products; results show the percentage of P. Bottom panel, HRR activity measured by restoration of EGFP expression. Results show the percentage of GFP+ cells; *P ≤ .01. (Panel D) Number of proliferating Lin−CD34+CD38−CTVlow and quiescent Lin−CD34+CD38−CTVmax cells from individual JAK2(V617F)+ MPN samples left untreated (C) or treated with ruxolitinib (R; 25 nM), olaparib (O; 1.25 μM), and ruxolitinib plus olaparib (R+O). (Panel E) Cumulative percentages from samples examined in panel D. *P < .001 in comparison with R or O groups using the Student t test; **P < .01 in comparison with R and O groups using the response additivity approach.

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