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Purpose: Chk1 inhibitors, such as AZD7762, are in clinical development in combination with cytotoxic agents for the treatment of solid tumors, including pancreatic cancers. To maximize the likelihood of their clinical success, it is essential to optimize drug scheduling as well as pharmacodynamic biomarkers in preclinical models.Experimental Design: We tested multiple schedules of administration of gemcitabine and AZD7762 on the survival of pancreatic cancer cells. Potential pharmacodynamic biomarkers including pChk1, pChk2, pHistone H3, and caspase-3 were evaluated in vitro, followed by assessment of promising candidate biomarkers in vivo. We then went on to determine the contributions of PP2A and DNA damage to the mechanism(s) of induction of the identified biomarker, pS345 Chk1.Results: AZD7762 given during and after or after gemcitabine administration produced maximum chemosensitization. In vivo, AZD7762 significantly inhibited the growth of pancreatic tumor xenografts in response to gemcitabine. Of the biomarkers assessed, pS345 Chk1 was most consistently increased in response to gemcitabine and AZD7762 in tumors and normal tissues (hair follicles). pS345 Chk1 induction in response to gemcitabine and AZD7762 occurred in the presence of PP2A inhibition and in association with elevated γH2AX, suggesting that DNA damage is an underlying mechanism.Conclusions: AZD7762 sensitizes pancreatic cancer cells and tumors to gemcitabine in association with induction of pS345 Chk1. Together these data support the clinical investigation of AZD7762 with gemcitabine in pancreatic cancer under a dosing schedule in which gemcitabine is administered concurrent with or before AZD7762 and in conjunction with skin biopsies to measure pS345 Chk1. Clin Cancer Res; 17(11); 3706–15. ©2011 AACR.

Supplementary Figures S1-S5; Supplementary Tables S1-S2.

Supplementary Figures 1-3, Tables 1-3 from Mechanism of Radiosensitization by the Chk1/2 Inhibitor AZD7762 Involves Abrogation of the G2 Checkpoint and Inhibition of Homologous Recombinational DNA Repair

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 G2 checkpoint as well as with inhibition of Rad51 focus formation, inhibition of homologous recombination repair, and persistent γ-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 G2 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. Cancer Res; 70(12); 4972–81. ©2010 AACR.

Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation but the immune mechanisms leading to the diverse clinical manifestations of cGVHD remain unknown. In this study, we examined regulatory T cells (Tregs) in 57 transplant recipients (30 with cGVHD and 27 without active cGVHD) and 26 healthy donors. Phenotypic studies demonstrated decreased frequency of CD4+CD25+ T cells in patients with cGVHD compared with patients without cGVHD (P < .001) and healthy individuals (P < .001). Gene expression of Treg transcription factor FOXP3 was reduced in cGVHD patients compared with patients without cGVHD (P = .009) or healthy donors (P = .01). T-cell receptor excision circle (TREC) assays for the evaluation of thymus activity revealed fewer TRECs in both transplant groups compared with healthy donors (P < .001 and P = .02, respectively) although no difference was observed between patients with or without cGVHD (P = .13). When tested in functional assays, Tregs from both patient cohorts and healthy individuals mediated equivalent levels of suppression. Collectively, these studies indicate that patients with active cGVHD have reduced frequencies of Tregs but the function of these cells remains normal. These findings support the development of new strategies to increase the number of Tregs following allogeneic hematopoietic stem cell transplantation to prevent or correct cGVHD. (Blood. 2005; 106:2903-2911)

Purpose: Chk1 inhibitors, such as AZD7762, are in clinical development in combination with cytotoxic agents for the treatment of solid tumors, including pancreatic cancers. To maximize the likelihood of their clinical success, it is essential to optimize drug scheduling as well as pharmacodynamic biomarkers in preclinical models. Experimental Design: We tested multiple schedules of administration of gemcitabine and AZD7762 on the survival of pancreatic cancer cells. Potential pharmacodynamic biomarkers including pChk1, pChk2, pHistone H3, and caspase-3 were evaluated in vitro, followed by assessment of promising candidate biomarkers in vivo. We then went on to determine the contributions of PP2A and DNA damage to the mechanism(s) of induction of the identified biomarker, pS345 Chk1. Results: AZD7762 given during and after or after gemcitabine administration produced maximum chemosensitization. In vivo, AZD7762 significantly inhibited the growth of pancreatic tumor xenografts in response to gemcitabine. Of the biomarkers assessed, pS345 Chk1 was most consistently increased in response to gemcitabine and AZD7762 in tumors and normal tissues (hair follicles). pS345 Chk1 induction in response to gemcitabine and AZD7762 occurred in the presence of PP2A inhibition and in association with elevated γH2AX, suggesting that DNA damage is an underlying mechanism. Conclusions: AZD7762 sensitizes pancreatic cancer cells and tumors to gemcitabine in association with induction of pS345 Chk1. Together these data support the clinical investigation of AZD7762 with gemcitabine in pancreatic cancer under a dosing schedule in which gemcitabine is administered concurrent with or before AZD7762 and in conjunction with skin biopsies to measure pS345 Chk1. Clin Cancer Res; 17(11); 3706–15. ©2011 AACR.

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 G2 checkpoint as well as with inhibition of Rad51 focus formation, inhibition of homologous recombination repair, and persistent γ-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 G2 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. Cancer Res; 70(12); 4972–81. ©2010 AACR.

Apoptotic and antiproliferative activities of small heterodimer partner (SHP) nuclear receptor ligand (E)-4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC), which was derived from 6-[3'-(1-adamantyl)-4'-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN), and several carboxyl isosteric or hydrogen bond-accepting analogues were examined. 3-Cl-AHPC continued to be the most effective apoptotic agent, whereas tetrazole, thiazolidine-2,4-dione, methyldinitrile, hydroxamic acid, boronic acid, 2-oxoaldehyde, and ethyl phosphonic acid hydrogen bond-acceptor analogues were inactive or less efficient inducers of KG-1 acute myeloid leukemia and MDA-MB-231 breast, H292 lung, and DU-145 prostate cancer cell apoptosis. Similarly, 3-Cl-AHPC was the most potent inhibitor of cell proliferation. 4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorophenyltetrazole, (2E)-5-{2-[3'-(1-adamantyl)-2-chloro-4'-hydroxy-4-biphenyl]ethenyl}-1H-tetrazole, 5-{4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorobenzylidene}thiazolidine-2,4-dione, and (3E)-4-[3'-(1-adamantyl)-2-chloro-4'-hydroxy-4-biphenyl]-2-oxobut-3-enal were very modest inhibitors of KG-1 proliferation. The other analogues were minimal inhibitors. Fragment-based QSAR analyses relating the polar termini with cancer cell growth inhibition revealed that length and van der Waals electrostatic surface potential were the most influential features on activity. 3-Cl-AHPC and the 3-chlorophenyltetrazole and 3-chlorobenzylidenethiazolidine-2,4-dione analogues were also able to inhibit SHP-2 protein-tyrosine phosphatase, which is elevated in some leukemias. 3-Cl-AHPC at 1.0 microM induced human microvascular endothelial cell apoptosis but did not inhibit cell migration or tube formation.

Pancreatic cancer remains among the most lethal cancers with an overall 5-year survival of less than 5%. Chk1 inhibition has emerged as a promising approach to sensitize pancreatic cancer to gemcitabine (Gem), the current standard therapy for both local control and metastatic disease. Here, we characterized the ability of AZD7762, a small molecule Chk1/2 inhibitor currently in Phase I trials, to sensitize pancreatic cancer cells to Gem both in vitro and in vivo. We first evaluated the in vitro schedule-dependence of AZD7762-mediated Gem sensitization by clonogenic assay. We found that AZD7762 effectively sensitized pancreatic cancer cell lines to Gem-induced loss of clonogenicity, with shifts in IC50 from 3- to 38-fold, compared to cells treated with Gem alone. At low concentrations of Gem (≤IC50), AZD7762 was most effective when present during Gem treatment. At higher concentrations of Gem, however, giving AZD7762 24 h after Gem resulted in the greatest sensitization. We next assessed the molecular mechanisms of AZD7762-mediated chemosensitization in one of these cell lines, MiaPaCa2, by immunoblot, flow cytometry and immunofluorescent staining. Gemcitabine sensitization was associated with inhibition of Chk1, rather than Chk2 and abrogation of the S-M and G2-M checkpoints. Despite checkpoint abrogation, we found that Gem/AZD7762-treated cells were unable to exit S-phase and, over time, AZD7762 inhibited DNA synthesis, as measured by BrdUrd incorporation. This inhibition was associated with pan γ-H2AX staining in Gem-treated cells, in addition to disruption of Rad51 foci and inhibition of homologous recombination repair activity. We next evaluated the effects of AZD7762 in two different human pancreatic cancer xenograft models; MiaPaCa2, and a patient-derived early passage pancreatic cancer xenograft, EPX-J. AZD7762 had anti-tumor activity both as a single agent, and in combination with Gem in both MiaPaCa2 and EPX-J, with average times to tumor doubling of 37 and 49 days, respectively, in tumors treated with both Gem/AZD7762, compared to 18 and 29 days for tumors treated with Gem alone (*p≤0.05). Chk1 inhibition, as determined by S296- and S345-Chk1 phosphorylation status, correlated with in vivo response. Finally, we found increased S345-Chk1 phosphorylation in normal tissues from mice treated with Gem/AZD7762, suggesting this may be a useful surrogate marker for AZD7762 activity in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1765.

The combination of gemcitabine and radiation is superior to gemcitabine alone for locally advanced pancreatic cancer however the median survival is only approximately one year. We have previously demonstrated that inhibition of Chk1 (checkpoint kinase 1) sensitizes pancreatic cancer cell lines, as well as patient-derived tumor xenografts, to both radiation and gemcitabine-radiation. The exact mechanism(s) for this sensitization, however, remains unclear. Based on the abilities of Chk1 to induce cell cycle arrest and promote HRR (homologous recombination repair) in response to DNA damage, we set out to determine the relevance of these mechanisms to radiosensitization by AZD7762, an AstraZenca Chk1/2 inhibitor currently in Phase I clinical trials. We tested the effects of AZD7762 on survival, cell cycle checkpoints, Rad51 focus formation and HRR activity in response to radiation and gemcitabine-radiation in two pancreatic cancer cell lines, Panc-1 and MiaPaCa-2. We began this study with the observation that AZD7762 sensitized MiaPaCa-2 cells to radiation and gemcitabine-radiation with enhancement ratios of 1.5 ± 0.08 (AZD7762), 1.2 ± 0.07 (50 nM gemcitabine), and 1.9 ± 0.16 (AZD7762 + 50 nM gemcitabine) but did not sensitize Panc-1 cells where enhancement ratios were 1.0 ± 0.03 (AZD7762), 1.5 ± 0.11 (100 nM gemcitabine), and 1.4 ± 0.05 (AZD7762 + 100 nM gemcitabine). AZD7762 inhibited Chk1 in both cell lines as evidenced by accumulation of Cdc25A and abrogation of the radiation-induced G2 arrest. To begin to determine the contribution of HRR to radiosensitization by AZD7762, we utilized a DR-GFP reporter assay which measures homology directed repair of an I-SceI endonuclease-induced DNA double strand break in an integrated GFP reporter gene in MiaPaCa-2 and Panc-1 cells. Treatment of cells expressing the DR-GFP reporter with 100 nM AZD7762 produced a 2- to 3-fold (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 491.