1. Condition MiaPaCa-2 Control Gem (100nm) AZD1775 (200nM) Gem + AZD1775 Panc-1 Control Gem (50nm) AZD1775 (200nM) Gem + AZD1775 Capan-1 Control Gem (50nm) AZD1775 (50nM) Gem + AZD1775 Supplementary Table 1. Summary of sensitization to gemcitabine-radiation by AZD1775 Supplementary Table 1. Summary of sensitization to gemcitabine-radiation by AZD1775. Cells were treated as described in Fig. 1. Data are the mean of mean ± standard error of 4-5 independent experiments with statistical significance indicated vs GemRT* (P<0.05). The mean inactivation dose (MID) used to calculate RER is shown. MID 2.7 ± 0.1 2.2 ± 0.4 2.4 ± 0.1 1.3 ± 0.1 3.7 ± 0.1 3.2 ± 0.1 3.4 ± 0.1 2.4 ± 0.2 2.3 ± 0.2 1.4 ± 0.2 2.2 ± 0.2 1.2 ± 0.1 Cytotoxicity 1.0 0.9 ± 0.1 1.1 ± 0.1 0.7 ± 0.3 1.0 0.9± 0.04 0.5 ± 0.05 1.0 0.9 ± 0.1 1.0 ± 0.1 0.8 ± 0.2 RER 1.0 1.3 ± 0.2 1.1 ± 0.1 2.1 ± 0.2* 1.0 1.1 ± 0.04 1.1 ± 0.03 1.5 ± 0.2* 1.0 1.8 ±0.2 1.0 ± 0.1 1.9 ± 0.2

2. Suppl. Fig. 1 Supplementary Figure 1. The effects of AZD1775 and gemcitabine-radiation on DNA damage response signaling. A, Panc-1 cells were treated with gemcitabine, AZD1775 and radiation (6Gy) as described in Fig. 1, except that cells were harvested for immunoblotting at 6 hours post-RT. B, pCDK1 (Y15) and pCHK1 (S345) protein levels from gemcitabine-radiation and AZD1775 treatment conditions (as described in Fig. 2) were quantitated from 3 independent experiments. Data are the mean fold change ± standard error in pCDK1 or pCHK1 protein levels in response to gemcitabine-radiation and increasing concentrations of AZD1775 relative to gemcitabine-radiation with no AZD1775 treatment. A. Panc1 pCHK1 (S345) pCDK1 (Y15) 01005020050200 GemRT 1000 CDK1 CHK1 GAPDH AZD1775 (nM): B.

3. Control pHistone H3 MiaPaCa-2 DNA content GemAZD1775GemAZD1775 RT DNA content GemRT DNA content AZD1775RT DNA content GemAZD1775RT t0t0 t2t2 t 48 t 24 AZD1775RTGem FACS M:1.4%M:1.2%M:2.7%M:2.3% M:0.5%M:0.3%M:1.4%M:2.8% pHistone H3 Suppl. Fig. 2 Supplementary Figure 2. Abrogation of the G2 checkpoint by AZD1775. MiaPaCa-2 cells were treated as described in Fig. 2C. Data are from a single representative experiment.

4. Suppl. Fig. 3 M:1.6%M:1.5% M:2.4% M:2.0% M:0.5% M:0.7% M:1.0% M:2.6% pHistone H3 AZD1775RTGem FACS t0t0 t2t2 t 48 t 24 Control DNA content GemAZD1775GemAZD1775 RT DNA content GemRT DNA content AZD1775RT DNA content GemAZD1775RT Capan-1 Supplementary Figure 3. Abrogation of the G2 checkpoint by AZD1775. Capan-1 cells were treated as described in Fig. 2D. Data are from a single representative experiment.

5. ConAZD1775GemGemAZD1775 RTGemRTAZD1775RTGemAZD1775RT Suppl. Fig. 4 Supplementary Figure 4. Induction of DNA damage by AZD1775. MiaPaCa-2 cells were treated as described in Fig. 3C with gemcitabine (50nM), AZD1775 (200nM) and radiation (6Gy). Cells were fixed for immunofluorescence at 16 hours post-RT. Cells were stained for γH2AX (red) and with DAPI (blue). Data are from a single representative experiment.

6. BRCA2 GAPDH DLD1 parentalDLD1 BRCA2-null Suppl. Fig. 5 Supplementary Figure 5. Characterization of BRCA2 isogenic DLD1 cells. DLD1 parental of BRCA2 null cells were subjected to immunoblotting for the indicated proteins. The absence of BRCA2 protein in BRCA2 null cells confirms genomic deletion of BRCA2.

7. Supplementary Table 2. Summary of sensitization to gemcitabine-radiation by AZD1775 Supplementary Table 2. Summary of sensitization to gemcitabine-radiation by AZD1775. Cells were treated as described in Fig. 4. Data are the mean ± standard error of 3 independent experiments with statistical significance indicated vs Control* (P<0.05). Condition DLD1 BRCA2 wild-type Gem (100nm) AZD1775 (200nM) Gem + AZD1775 DLD1 BRCA2 null Gem (50nm) AZD1775 (200nM) Gem + AZD1775 Cytotoxicity 0.9 ± 0.02 1.1 ± 0.03 0.7 ± 0.07 0.9 ± 0.14 0.8 ± 0.12 RER 1.3 ± 0.07 1.3 ± 0.03 1.5 ± 0.09* 0.9 ± 0.07 1.1 ± 0.08 1.0 ± 0.04

8. Suppl. Fig. 6 Supplementary Figure 6. RAD51 depletion sensitizes pancreatic cancer cells to gemcitabine- radiation. MiaPaCa-2 cells were plated at clonal densities and treated with non-specific (siNS) or RAD51 siRNA, followed 24 hours later by treatment with gemcitabine (100nM), AZD1775 (200nM), and radiation, according to the schedule illustrated in Fig. 1A. Data are from a single experiment that is representative of three independent experiments.

9. ConAZD1775 DLD1 BRCA2 wild-type GemAZD1775 RTGemRTAZD1775RTGemAZD1775RT Gem DLD1 BRCA2 null ConGemRT Suppl. Fig. 7 Supplementary Figure 7. Inhibition of RAD51 focus formation in DLD1 cells treated with AZD1775. DLD1 cells were treated as described in Fig. 4E. Cells were stained for RAD51 (red) and with DAPI (blue).

10. Suppl. Fig. 8 Supplementary Figure 8. The effects of AZD1775 and gemcitabine-radiation on DNA damage response signaling. Patient-derived xenografts were treated as described in Fig. 5A. Data are from 2-3 independent sets of tumors. Lines indicate where images were cropped for consistent arrangement of sample order. ConAZD1775Gem Gem+ AZD1775 ConAZD1775Gem Gem+ AZD1775 RT Gem+ AZD1775 ConAZD1775Gem Gem+ AZD1775 ConAZD1775Gem Gem+ AZD1775 RT GAPDH RAD51 pCHK1(S345) CHK1 CDK1 pCDK1(Y15)

11. Suppl. Fig. 9 ConAZD1775GemGemAZD1775 RTGemRTAZD1775RTGemAZD1775RT Supplementary Figure 9. Sensitization of patient-derived tumor xenografts to gemcitabine- radiation by AZD1775 involves inhibition of HR. Patient-derived xenografts (#08-444T) were treated as described in Fig. 5A. Xenografts were stained for RAD51 (green) and with DAPI (blue).