Schroeder, Mark

Abstract Glioblastoma (GBM) is a rapidly fatal malignancy typically treated with radiation and temozolomide (TMZ), an alkylating chemotherapeutic. These cytotoxic therapies cause oxidative stress and DNA damage, yielding a senescent-like state of replicative arrest in surviving tumor cells. Unfortunately, recurrence is inevitable and may be driven by surviving tumor cells eventually escaping senescence. A growing number of so-called “senolytic” drugs have been recently identified that are defined by their ability to selectively eliminate senescent cells. A growing inventory of senolytic drugs is under consideration for several diseases associated with aging, inflammation, DNA damage, as well as cancer. Ablation of senescent tumor cells after radiation and chemotherapy could help mitigate recurrence by decreasing the burden of residual tumor cells at risk of recurrence. This strategy has not been previously explored for GBM. We evaluated a panel of 10 previously described senolytic drugs to determine whether any could exhibit selective activity against human GBM persisting after exposure to radiation or TMZ. Three of the 10 drugs have known activity against BCL-XL and preferentially induced apoptosis in radiated or TMZ-treated glioma. This senolytic activity was observed in 12 of 12 human GBM cell lines. Efficacy could not be replicated with BCL-2 inhibition or senolytic agents acting against other putative senolytic targets. Knockdown of BCL-XL decreased survival of radiated GBM cells, whereas knockdown of BCL-2 or BCL-W yielded no senolytic effect. Implications: These findings imply that molecularly heterogeneous GBM lines share selective senescence-induced BCL-XL dependency increase the significance and translational relevance of the senolytic therapy for latent glioma.

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

Abstract Heat shock protein (HSP) 90 is a molecular chaperone that modulates stability of several client proteins, many of which are key regulators of signaling pathways in glioblastoma multiforme (GBM). The potential for simultaneously targeting multiple effectors makes HSP90 an appealing therapeutic target. Here, we evaluated the biological activity of HSP990, a novel HSP90 inhibitor (Novartis Pharma AG) in a panel of adult glioma primary xenograft lines (n=15). HSP990 treatment resulted in a dose-dependent growth inhibition with IC50 values in the low nM range in each of the primary lines. HSP990 caused G2/M cell cycle arrest and apoptosis-induction within 72 hours of treatment. In addition, treatment with HSP990 resulted in up-regulation of HSP70, and decrease in activity of client proteins AKT, CDC2, CDC25C and activation of caspase 8 and 3, in tested cell lines. A range of sensitivity based on EGFR status was observed. EGFR-amplified cells showed depletion of client proteins and growth inhibition at lower dose of HSP990 (10nM) than wildtype EGFR cells (30nM). Furthermore, neurosphere formation assays revealed that HSP990 was effective against the growth of glioblastoma stem cells (GSCs) in 6 tested glioblastoma xenograft lines (IC50 less than 10nM).These results suggest that that both cell cycle effects and induction of apoptosis are mechanisms governing HSP990 activity against glioblastoma. Further studies are warranted to characterize the mechanisms by which HSP990 exerts its anti-glioma effects which in turn may provide valuable insights in developing HSP990 as a novel personalized GBM treatment based on specific molecular signatures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2898. doi:10.1158/1538-7445.AM2011-2898