Figure 6.
Pharmacodynamic and therapeutic efficacy of PARP and WEE1 inhibition in Jak2V617F/+Trp53–/– PEL. (A) Western blot analysis of the indicated proteins was performed in duplicate in c-Kit+ spleen cells from WT, JVF, JVFP+/−, and PEL JVFP−/− mice. Side-by side duplicates for each murine genotype are presented. (B) PB counts and spleen weights of PEL JVFP−/− from mice treated with vehicle (Veh), olaparib (Ola), adavosertib (Ada), and combination of olaparib and adavosertib (Ola + Ada) after 2 weeks of treatment. N = 4 to 5 mice for each arm. White blood cell (WBC): Ola + Ada vs Veh, P < .0001; Ola + Ada vs Ola, P = .0016; Ola + Ada vs Ada, P = .0075. Platelet (PLT): Ola + Ada vs Veh, P = .0032; Ola + Ada vs Ada, P = .0047. Spleen weight: Ola + Ada vs Veh, P < .0001; Ola + Ada vs Ola, P = .0171; Ola + Ada vs Ada, P < .0001. (C) Percentage of leukemia cells (CD45–CD3–B220–Gr1–CD11b–c-Kit+) in PB (left) and BM (right) from mice treated with Veh, Ola, Ada, and the Ola + Ada combination after 2 weeks of treatment. N = 4 to 5 mice for each arm. PB: Ola + Ada vs Veh, P = .0017; Ola + Ada vs Ola, P = .0059; Ola + Ada vs Ada, P = .0122. BM: Ola + Ada vs Veh, P = .002; Ola + Ada vs Ola, P = .0133; Ola + Ada vs Ada, P = .0723. (D) Whole spleen specimens and histopathologic hematoxylin and eosin (H&E) sections of BM, spleen, and liver from representative PEL JVFP−/− mice after 2 weeks of treatment with Veh, Ola, Ada, and the Ola + Ada combination. Red arrows indicate blasts, yellow arrow indicates megakaryocytes, blue arrow indicates lymphoid follicle, and black arrow indicates hepatocytes. Magnification, 400×. (E) Kaplan-Meier comparative survival analysis of PEL JVFP−/− mice after 2 weeks of treatment with Veh, Ola, Ada, or the Ola + Ada combination. P value was determined by using the log-rank test. N = 5 mice for each arm. Ola + Ada vs Veh, P = .0039; Ola + Ada vs Ola, P = .0027; Ola + Ada vs Ada, P = .0019. (F) Western blot analysis of CDC2 and PAR in c-Kit+ spleen cells from JVFP−/− PEL mice treated with Veh, Ola, Ada, or the Ola + Ada combination after 3 and 6 days. (G) PEL JVFP−/− mice were randomized and treated with Veh, Ola, Ada, or the Ola + Ada combination and were euthanized after 3 or 6 days. Whole BM cells were stained with CD45, CD3, B220, Gr1, CD11b, c-Kit, and Annexin V (left), P-γH2AX (middle), or P-HH3 (right) to analyze the apoptosis and DDR in leukemia cells. N = 3 mice for each arm. Annexin V, Day 3: Ola + Ada vs Veh, P < .0001; Ola + Ada vs Ola, P = .0015; Ola + Ada vs Ada, P = .0042; Day 6: Ola + Ada vs Veh, P = .0232; Ola + Ada vs Ola, P = .0241; Ola + Ada vs Ada, P = .0167. P-γH2AX, Day 3: Ola + Ada vs Veh, P < .0001; Ola + Ada vs Ola, P = .0005; Ola + Ada vs Ada, P = .0008; Day 6: Ola + Ada vs Veh, P < .0001; Ola + Ada vs Ola, P < .0001; Ola + Ada vs Ada, P < .0001. P-HH3, Day 3: Ola + Ada vs Veh, P < .0001; Ola + Ada vs Ola, P = .0002; Ola + Ada vs Ada, P = .0025; Day 6: Ola + Ada vs Veh, P < .0001; Ola + Ada vs Ola, P = .009; Ola + Ada vs Ada, P = .0041. Data are represented as mean ± standard error of the mean (SEM). The unpaired t test was used to compare the mean of 2 groups in panels B, C, and G. *P < .05, **P ≤ .01, ***P ≤ .001. HRR, homologous recombination repair.

Pharmacodynamic and therapeutic efficacy of PARP and WEE1 inhibition in Jak2V617F/+Trp53–/– PEL. (A) Western blot analysis of the indicated proteins was performed in duplicate in c-Kit+ spleen cells from WT, JVF, JVFP+/−, and PEL JVFP−/− mice. Side-by side duplicates for each murine genotype are presented. (B) PB counts and spleen weights of PEL JVFP−/− from mice treated with vehicle (Veh), olaparib (Ola), adavosertib (Ada), and combination of olaparib and adavosertib (Ola + Ada) after 2 weeks of treatment. N = 4 to 5 mice for each arm. White blood cell (WBC): Ola + Ada vs Veh, P < .0001; Ola + Ada vs Ola, P = .0016; Ola + Ada vs Ada, P = .0075. Platelet (PLT): Ola + Ada vs Veh, P = .0032; Ola + Ada vs Ada, P = .0047. Spleen weight: Ola + Ada vs Veh, P < .0001; Ola + Ada vs Ola, P = .0171; Ola + Ada vs Ada, P < .0001. (C) Percentage of leukemia cells (CD45–CD3–B220–Gr1–CD11b–c-Kit+) in PB (left) and BM (right) from mice treated with Veh, Ola, Ada, and the Ola + Ada combination after 2 weeks of treatment. N = 4 to 5 mice for each arm. PB: Ola + Ada vs Veh, P = .0017; Ola + Ada vs Ola, P = .0059; Ola + Ada vs Ada, P = .0122. BM: Ola + Ada vs Veh, P = .002; Ola + Ada vs Ola, P = .0133; Ola + Ada vs Ada, P = .0723. (D) Whole spleen specimens and histopathologic hematoxylin and eosin (H&E) sections of BM, spleen, and liver from representative PEL JVFP−/− mice after 2 weeks of treatment with Veh, Ola, Ada, and the Ola + Ada combination. Red arrows indicate blasts, yellow arrow indicates megakaryocytes, blue arrow indicates lymphoid follicle, and black arrow indicates hepatocytes. Magnification, 400×. (E) Kaplan-Meier comparative survival analysis of PEL JVFP−/− mice after 2 weeks of treatment with Veh, Ola, Ada, or the Ola + Ada combination. P value was determined by using the log-rank test. N = 5 mice for each arm. Ola + Ada vs Veh, P = .0039; Ola + Ada vs Ola, P = .0027; Ola + Ada vs Ada, P = .0019. (F) Western blot analysis of CDC2 and PAR in c-Kit+ spleen cells from JVFP−/− PEL mice treated with Veh, Ola, Ada, or the Ola + Ada combination after 3 and 6 days. (G) PEL JVFP−/− mice were randomized and treated with Veh, Ola, Ada, or the Ola + Ada combination and were euthanized after 3 or 6 days. Whole BM cells were stained with CD45, CD3, B220, Gr1, CD11b, c-Kit, and Annexin V (left), P-γH2AX (middle), or P-HH3 (right) to analyze the apoptosis and DDR in leukemia cells. N = 3 mice for each arm. Annexin V, Day 3: Ola + Ada vs Veh, P < .0001; Ola + Ada vs Ola, P = .0015; Ola + Ada vs Ada, P = .0042; Day 6: Ola + Ada vs Veh, P = .0232; Ola + Ada vs Ola, P = .0241; Ola + Ada vs Ada, P = .0167. P-γH2AX, Day 3: Ola + Ada vs Veh, P < .0001; Ola + Ada vs Ola, P = .0005; Ola + Ada vs Ada, P = .0008; Day 6: Ola + Ada vs Veh, P < .0001; Ola + Ada vs Ola, P < .0001; Ola + Ada vs Ada, P < .0001. P-HH3, Day 3: Ola + Ada vs Veh, P < .0001; Ola + Ada vs Ola, P = .0002; Ola + Ada vs Ada, P = .0025; Day 6: Ola + Ada vs Veh, P < .0001; Ola + Ada vs Ola, P = .009; Ola + Ada vs Ada, P = .0041. Data are represented as mean ± standard error of the mean (SEM). The unpaired t test was used to compare the mean of 2 groups in panels B, C, and G. *P < .05, **P ≤ .01, ***P ≤ .001. HRR, homologous recombination repair.

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