Mechanism of radiosensitization by the Chk1/2 inhibitor AZD7762 involves abrogation of the G2 checkpoint and inhibition of homologous recombinational DNA repair

Abstract

The median survival for patients with locally advanced pancreatic cancer treated with gemcitabine and radiation is approximately 1 year. To develop improved treatment, we have combined a Chk1/2-targeted agent, AZD7762, currently in phase I clinical trials, with gemcitabine and ionizing radiation in preclinical pancreatic tumor models. We found that in vitro AZD7762 alone or in combination with gemcitabine significantly sensitized MiaPaCa-2 cells to radiation. AZD7762 inhibited Chk1 autophosphorylation (S296 Chk1), stabilized Cdc25A, and increased ATR/ATM-mediated Chk1 phosphorylation (S345 Chk1). Radiosensitization by AZD7762 was associated with abrogation of the G(2) checkpoint as well as with inhibition of Rad51 focus formation, inhibition of homologous recombination repair, and persistent gamma-H2AX expression. AZD7762 was also a radiation sensitizer in multiple tumor xenograft models. In both MiaPaCa-2- and patient-derived xenografts, AZD7762 significantly prolonged the median time required for tumor volume doubling in response to gemcitabine and radiation. Together, our findings suggest that G(2) checkpoint abrogation and homologous recombination repair inhibition both contribute to sensitization by Chk1 inhibition. Furthermore, they support the clinical use of AZD7762 in combination with gemcitabine and radiation for patients with locally advanced pancreatic cancer.

Figure 1. Radiosensitization in response to Chk1 inhibition and gemcitabine

A, Schedule of treatments. B, MiaPaCa-2 cells were treated with gemcitabine (Gem; 50 nM), AZD7762 (AZD; 100 nM), and radiation (IR; 0–8 Gy) as illustrated (A). The plating efficiency of the untreated control cells was 41 %. C, MiaPaCa-2 cells were treated as illustrated (A) except with 7.5 Gy. Cells were collected for immunoblotting at t = 26 hours. D, Alternatively, cells were treated with Chk1 and/or Chk2 siRNA, irradiated 48 hours post-transfection, and then processed for clonogenic survival. The radiation enhancement ratios were normalized to the mean inactivation dose of the non-specific siRNA (NS) treated samples. Data are from a single experiment representative of 3 independent experiments (B, C) or the mean radiation enhancement ratio of 4 independent experiments ± standard error (B inset, D). For B, error bars are contained within the points. Statistically significant differences are indicated (*, P < 0.05).

Figure 2. The effects of AZD7762 on progression from S-phase in response to gemcitabine and radiation

MiaPaCa-2 cells were treated as illustrated in Fig. 1A with the exception that cells were pulsed with BrdU immediately prior to radiation (t = 24 h; 7.5 Gy). At times 26, 30, and 40 hours, cells were collected for flow cytometry. Untreated MiaPaCa-2 cells had a 20 hour cell doubling time. Data in the histograms represent only the BrdU positive cells from a single experiment with the percentages of cells in G1, S, and G2/M illustrated. Data are representative of 3 independent experiments.

Figure 3. The effects of AZD7762 on Rad51, HRR, and γ-H2AX

A, MiaPaCa-2 cells were treated as illustrated (Fig. 1A) and fixed for immunofluorescence at 30 hours. Cells were stained with DAPI (blue) and for Rad51 (green). B, Rad51 foci were analyzed at t = 26 and 30 hours. Data are the mean ± standard error of 3 independent experiments. C, MiaPaCa-2-DR-GFP cells were treated as illustrated and at t = 48 hours, the percentage of GFP positive cells was measured by flow cytometry. Data are expressed as the mean percentage of GFP positive cells ± standard error of n = 6 independent experiments. D, γ-H2AX was assessed by flow cytometry in MiaPaCa-2 cells treated with radiation (7.5 Gy) at t = 24 hours, according to the schedule illustrated in Fig. 1A, and collected at various time points following radiation (t = 24 – 48 hours). Data are expressed as the percentage of cells staining positive for γ-H2AX and are the mean ± standard error of n = 4 – 6 independent experiments. B–D, Statistical significance (P < 0.05) is indicated versus Control*, Gem^‡, IR^π, Gem IR^†.

Figure 4. The effects of AZD7762 on MiaPaCa-2 xenografts in response to gemcitabine and radiation

A, Schedule of treatments. B, Athymic nude mice bearing bilateral, subcutaneous MiaPaCa-2-derived xenografts were treated with gemcitabine (90 mg/kg), AZD7762 (20 mg/kg), and/or radiation (2 Gy/fraction) for 3 cycles as illustrated (A). Data are expressed as the proportion of tumors doubled in volume. Each treatment group contained 6 – 7 animals (12 – 14 tumors). For immunoblotting (C) or immunohistochemistry (D), tumors were harvested on day 1. Immunoblots are from a single experiment representative of 3 independent experiments. For S345 Chk1 immunohistochemistry, the average score obtained from 3 – 4 tumor specimens is illustrated where ‘ − ’ indicates no staining and ‘+++’, maximal staining.

Figure 5. The effects of AZD7762 on patient-derived xenografts in response to gemcitabine and radiation

Nod-scid mice bearing bilateral, subcutaneous xenografts derived from two different patient pancreatic tumors (patients ‘F’ and ‘J’) (B and C, respectively) were treated with gemcitabine (90 mg/kg), AZD7762 (20 mg/kg), and/or radiation (1.8 Gy/fraction) for 2 cycles as illustrated (A). Data are expressed as the proportion of tumors doubled in volume. Each treatment group contained 6 – 10 mice (12 – 20 tumors).