Background: Targeting cell cycle checkpoints has the potential to enhance the efficacy of chemoradiation therapy. Tumor cells commonly have an abnormal G1 checkpoint due to mutations in the p53 pathway making them reliant on the G2 checkpoint to repair DNA damage. In our preclinical studies, WEE1 inhibition with AZD1775 abrogates the G2 checkpoint and sensitizes pancreatic cancer cell lines and xenografts to chemoradiation. Additionally, AZD1775 attenuates homologous recombination repair and promotes replication stress in cancer cells. Given our preclinical findings, we designed a phase I dose escalation study of the WEE1 inhibitor AZD1775 with gemcitabine and radiation in patients with unresectable pancreatic cancer (NCT02037230). Methods: The primary objective of our phase I study is to determine the MTD (maximum tolerated dose) of AZD1775 when combined with gemcitabine and radiation in patients with locally advanced pancreatic cancer. Our secondary objectives are to estimate the efficacy of this regimen at the target dose and to determine if WEE1 is inhibited by AZD1775 at or below its target dose in surrogate tissues (hair follicles). Patients with unresectable, non-metastatic pancreatic cancer are eligible for the study. Protocol therapy consists of the administration of AZD1775 and gemcitabine at the assigned dose levels in accordance with a Time-to-Event Continual Reassessment Method. All patients on the study are treated with four cycles of therapy consisting of AZD1775 given orally on days 1, 2 and 8, 9 of a 21 day cycle with gemcitabine given intravenously over 30 minutes on day 1 and day 8. Cycles 2 and 3 are administered with concurrent radiation therapy, 52.5 Gy in 25 fractions to the primary pancreatic tumor. The MTD will be determined by the development of dose limiting toxicities (DLT) within the first 4 cycles of therapy with a target DLT rate of 25%. Blood samples obtained at baseline and after cycles 1, 2, and 4 will be used for correlative studies on circulating tumor cells and tumor derived exosomes. Our estimated accrual is 36 patients. To date we have enrolled 21 patients. Supported by P50 CA130810, R01 CA163895, Cancer Center Core grant P30 CA46592, and the Taubman Institute. Clinical trial information: NCT02037230