Abstract Wee1 kinase is a critical regulator of the G2/M checkpoint by initiating inhibitory phosphorylation of the conserved tyrosine-15 residue of cdk1/cdc2. Wee1 also phosphorylates tyrosine 15 of cdk2 and plays a crucial role in maintaining genome integrity during S-phase. MK1775 (NSC 754352) is a small molecule selective inhibitor of Wee1 kinase that is currently under clinical evaluation. We have developed a quantitative immunofluorescence assay to measure inhibition of Wee1 by detection of pY15 of cyclin-dependent kinases. A full analysis of the pharmacodynamic (PD) effects of Wee1 inhibition by MK1775, as well as the downstream mechanistic consequences of Wee1 inhibition on DNA repair, apoptosis, and premature mitotic entry markers was performed using two xenograft models: A673 Ewing sarcoma and U87-MG glioblastoma. Four DNA repair markers (pNbs1, γH2AX, Rad51, and pATR) were examined as well as the mitotic marker pHistone H3. Advanced quantitative image analysis was performed using Definiens software to measure changes in PD markers by two approaches: total nuclear area measurements and foci per nucleus. Definiens software enables enhanced analysis of the markers by enumeration of the nuclei in the imaged xenograft tissues over an entire data set with high-content capacity. Our data demonstrate greater than 80% inhibition of pY15-cdk in vivo at the clinically relevant dose of 60 mg/kg (180 mg/m2) MK1775 in two xenograft models. In addition, γH2AX induction was observed after multiple doses of MK1775 as a single agent. A PD biomarker time course was determined for MK1775 and gemcitabine (NSC 750927) as single agents to develop a drug administration schedule for combination studies in a Ewing sarcoma model. The time point at which the DNA damage response peaked following administration of the maximum tolerated dose of gemcitabine in the mouse was determined. This study elucidates a broad profile of PD marker response, as well as the corresponding levels of MK1775 in the xenografts. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Deborah F. Wilsker, Allison M. Marrero, Melinda Hollingshead, Scott M. Lawrence, Alice Chen, Shivaani Kummar, Joseph M. Covey, Ralph E. Parchment, Robert J. Kinders, Joseph E. Tomaszewski, James H. Doroshow. Pre-clinical investigation of the wee1 inhibitor MK-1775 using pharmacodynamic and mechanistic markers in diverse cancer models in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4689. doi:10.1158/1538-7445.AM2014-4689