Abstract 3240: Pre-clinical investigation of the Wee1 inhibitor, MK1775, in glioblastoma multiforme.
Authors: Pokorny Jenny , Pucci Vincenzo , Shumway Stuart , Ma Bennett , Bakken Katrina , Schroeder Mark , Carlson Brett , Sarkaria Jann N. 
Published: August 2014

Abstract
Wee1 is a key regulator of both G2/M transition and DNA replication fork stability. In this study, the efficacy of the Wee1 inhibitor MK1775 was evaluated in the Mayo primary glioblastoma multiforme (GBM) xenograft model. Based on western blot expression of Wee1, GBM22, GBM12 and GBM6 were selected for study based on low, intermediate and high-level expression, respectively. Wee1-mediated phosphorylation of Y15-Cdc2 was robustly suppressed in vitro in both GBM22 and GBM6 at 300 nM MK1775. At this concentration, MK1775 had marked anti-proliferative effects on GBM22 with 80±2.5% suppression of absorbance in a CyQUANT cell proliferation assay, while similar treatment of GBM6 had minimal effect on growth (26±21% suppression of absorbance). In a similar primary neurosphere formation assay, 300 nM completely inhibited neurosphere formation in GBM22 and GBM12, while having no effect on GBM6 neurosphere formation. Consistent with the cytotoxicity data, single agent treatment of GBM22 with MK1775 resulted in a marked S-phase arrest 24 hours after treatment (60% vs. 40% cells in S with MK1775 vs. control, respectively) and subsequent accumulation of cells in G2/M 48 hours after treatment (95% vs. 9.5%, respectively). In contrast, relatively minimal cell cycle perturbations were observed with MK1775 in GBM6 (24 hour S-phase accumulation 27% vs. 21%, respectively). Combination studies with MK1775 and temozolomide (TMZ) in a neurosphere formation assay demonstrated enhancement of treatment benefit only in GBM22 and GBM12 but not GBM6 (relative neurosphere numbers for TMZ, MK1775, or combined treatment compared to untreated were 34%, 16% and 7%, respectively for GBM22, 41%, 95% and 15%, respectively, for GBM12; while the same combination had no effect on GBM6 neurosphere formation). In anticipation of evaluating treatment efficacy in animal models, pharmacokinetic modeling of MK1775 brain delivery was performed in nude mice. Following a single MK1775 dose of 50 mg/kg, time to maximum blood concentration was 1.0 hours, AUC was 29.8 microM·h, and Cmax was 7.8 microM in whole blood and 0.24 microM in brain parenchyma. To evaluate whether limitations in brain delivery affected efficacy, parallel in vivo GBM12 heterotopic and orthotopic xenograft efficacy studies were performed with a 5 day dosing regimen of TMZ and MK1775 repeated every 28 days for 3 cycles. In the orthotopic model, MK1775 provided no survival benefit with median survival being 16, 15, 88 and 92 days for placebo, MK1775 alone, TMZ alone, or MK1775 + TMZ treatment, respectively. In contrast, there is some suggestion of MK1775 efficacy in the flank tumor model with median time to reach 1000 mm3 being 45, 56, 101 and 112 days, respectively. Collectively, these data suggest that despite promising in vitro activity for MK1775, limited drug delivery into the brain may reduce the efficacy of MK1775 therapy in an orthotopic GBM xenograft model.
Citation Format: Jenny Pokorny, Vincenzo Pucci, Stuart Shumway, Bennett Ma, Katrina Bakken, Mark Schroeder, Brett Carlson, Jann N. Sarkaria. Pre-clinical investigation of the Wee1 inhibitor, MK1775, in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3240. doi:10.1158/1538-7445.AM2013-3240