Sensitization of Pancreatic Cancers to Gemcitabine Chemoradiation by WEE1 Kinase Inhibition Depends on Homologous Recombination Repair.

To improve the efficacy of chemoradiation therapy for locally advanced pancreatic cancer and begin to establish patient selection criteria, we investigated the combination of the WEE1 inhibitor AZD1775 with gemcitabine-radiation in homologous recombination (HR) repair proficient and deficient pancreatic cancers. Sensitization to gemcitabine-radiation by AZD1775 was assessed in pancreatic cancer cells by clonogenic survival and in patient-derived xenografts by tumor growth. The contributions of HR repair inhibition and G2 checkpoint abrogation to sensitization were assessed by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistone H3 flow cytometry, respectively. We found that AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type but not BRCA2 mutant pancreatic cancer cells. In all cells, AZD1775 caused inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. However, sensitization by AZD1775 was associated with persistent γH2AX and inhibition of RAD51 focus formation. In HR-proficient (BRCA2 wild-type) or -deficient (BRAC2 null) isogenic cells, AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type, but not in BRCA2 null cells, despite significant G2 checkpoint abrogation. In patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibited tumor growth and impaired RAD51 focus formation in response to gemcitabine-radiation. In conclusion, WEE1 inhibition by AZD1775 is an effective strategy for sensitizing pancreatic cancers to gemcitabine chemoradiation. Although this sensitization is accompanied by inhibition of CDK1 phosphorylation and G2 checkpoint abrogation, this mechanism is not sufficient for sensitization. Our findings demonstrate that sensitization to chemoradiation by WEE1 inhibition results from inhibition of HR repair and suggest that patient tumors without underlying HR defects would benefit most from this therapy.

Kausar T ，Schreiber JS ，Karnak D ，Parsels LA ，Parsels JD ，Davis MA ，Zhao L ，Maybaum J ，Lawrence TS ，Morgan MA ... -  《NEOPLASIA》

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

Morgan MA ，Parsels LA ，Zhao L ，Parsels JD ，Davis MA ，Hassan MC ，Arumugarajah S ，Hylander-Gans L ，Morosini D ，Simeone DM ，Canman CE ，Normolle DP ，Zabludoff SD ，Maybaum J ，Lawrence TS ... -  《-》

Combined inhibition of Wee1 and PARP1/2 for radiosensitization in pancreatic cancer.

While the addition of radiation to chemotherapy improves survival in patients with locally advanced pancreatic cancer, more effective therapies are urgently needed. Thus, we investigated the radiosensitizing efficacy of the novel drug combination of Wee1 and PARP1/2 inhibitors (AZD1775 and olaparib, respectively) in pancreatic cancer. Radiosensitization of AsPC-1 or MiaPaCa-2 human pancreatic cancer cells was assessed by clonogenic survival and tumor growth assays. Mechanistically, the effects of AZD1775, olaparib, and radiation on cell cycle, DNA damage (γH2AX), and homologous recombination repair (HRR) were determined. Treatment of AsPC-1 and MiaPaCa-2 cells with either AZD1775 or olaparib caused modest radiosensitization, whereas treatment with the combination significantly increased radiosensitization. Radiosensitization by the combination of AZD1775 and olaparib was associated with G2 checkpoint abrogation and persistent DNA damage. In addition, AZD1775 inhibited HRR activity and prevented radiation-induced Rad51 focus formation. Finally, in vivo, in MiaPaCa-2-derived xenografts, olaparib did not radiosensitize, whereas AZD1775 produced moderate, yet significant, radiosensitization (P < 0.05). Importantly, the combination of AZD1775 and olaparib produced highly significant radiosensitization (P < 0.0001) evidenced by a 13-day delay in tumor volume doubling (vs. radiation alone) and complete eradication of 20% of tumors. Taken together, these results demonstrate the efficacy of combined inhibition of Wee1 and PARP inhibitors for radiosensitizing pancreatic cancers and support the model that Wee1 inhibition sensitizes cells to PARP inhibitor-mediated radiosensitization through inhibition of HRR and abrogation of the G2 checkpoint, ultimately resulting in unrepaired, lethal DNA damage and radiosensitization. Clin Cancer Res; 20(19); 5085-96. ©2014 AACR.

Karnak D ，Engelke CG ，Parsels LA ，Kausar T ，Wei D ，Robertson JR ，Marsh KB ，Davis MA ，Zhao L ，Maybaum J ，Lawrence TS ，Morgan MA ... -  《-》

Sensitization of pancreatic cancer to chemoradiation by the Chk1 inhibitor MK8776.

The combination of radiation with chemotherapy is the most effective therapy for unresectable pancreatic cancer. To improve upon this regimen, we combined the selective Checkpoint kinase 1 (Chk1) inhibitor MK8776 with gemcitabine-based chemoradiation in preclinical pancreatic cancer models. We tested the ability of MK8776 to sensitize to gemcitabine-radiation in homologous recombination repair (HRR)-proficient and -deficient pancreatic cancer cells and assessed Rad51 focus formation. In vivo, we investigated the efficacy, tumor cell selectivity, and pharmacodynamic biomarkers of sensitization by MK8776. We found that MK8776 significantly sensitized HRR-proficient (AsPC-1, MiaPaCa-2, BxPC-3) but not -deficient (Capan-1) pancreatic cancer cells to gemcitabine-radiation and inhibited Rad51 focus formation in HRR-proficient cells. In vivo, MiaPaCa-2 xenografts were significantly sensitized to gemcitabine-radiation by MK8776 without significant weight loss or observable toxicity in the small intestine, the dose-limiting organ for chemoradiation therapy in pancreatic cancer. We also assessed pChk1 (S345), a pharmacodynamic biomarker of DNA damage in response to Chk1 inhibition in both tumor and small intestine and found that MK8776 combined with gemcitabine or gemcitabine-radiation produced a significantly greater increase in pChk1 (S345) in tumor relative to small intestine, suggesting greater DNA damage in tumor than in normal tissue. Furthermore, we demonstrated the utility of an ex vivo platform for assessment of pharmacodynamic biomarkers of Chk1 inhibition in pancreatic cancer. Together, our results suggest that MK8776 selectively sensitizes HRR-proficient pancreatic cancer cells and xenografts to gemcitabine-radiation and support the clinical investigation of MK8776 in combination with gemcitabine-radiation in locally advanced pancreatic cancer.

Engelke CG ，Parsels LA ，Qian Y ，Zhang Q ，Karnak D ，Robertson JR ，Tanska DM ，Wei D ，Davis MA ，Parsels JD ，Zhao L ，Greenson JK ，Lawrence TS ，Maybaum J ，Morgan MA ... -  《-》

Augmented antitumor activity by olaparib plus AZD1775 in gastric cancer through disrupting DNA damage repair pathways and DNA damage checkpoint.

Lin X ，Chen D ，Zhang C ，Zhang X ，Li Z ，Dong B ，Gao J ，Shen L ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》