Berens, Michael E.

Abstract Glioblastoma multiforme (GBM) is the most common and most lethal primary malignant brain tumor, affecting 25,000 patients per year. Approximately 87% of GBMs have abnormalities in the p53 pathway that affects G2 checkpoint. In the absence of p53, Wee1 kinase has been shown to regulate G2 checkpoint upon chemotherapeutic insult. Wee1 inhibits CDC2 function, thereby promoting G2 phase cell cycle arrest, thus preventing damaged cells from entering into premature mitosis without repairing the DNA. We hypothesize that in p53-deficient tumor cells, inhibitors of Wee1 would be effective to heighten DNA damage due to genetic and pharmacologic loss of both the G1 and G2 checkpoints, respectively. MK-1775 (Merck) is a potent and selective small molecule inhibitor of the Wee1 kinase that has demonstrated enhanced cytotoxic effect in combination with several chemotherapeutic agents in preclinical studies in vitro and in vivo. Here, we selected an array of short term cultures of glioma patient derived xenograft (PDX) models (GBM12, GBM43, GBM79, GBM84) and long term established glioma cell lines (U87, SF767, T98G) with aberrant and wild type G1 checkpoint pathways and investigated cytotoxic effects of MK-1775 as a single agent and in combination with Temozolomide (TMZ). Our results demonstrated differential sensitivity to MK-1775 and TMZ as single agents for different glioma cell lines in vitro. However, no correlation between MK-1775, TMZ sensitivity and p53 status of the cell lines was observed. Combination therapy with MK-1775 and TMZ showed a synergistic effect for one PDX model, GBM43, with combination index (CI) of 0.4. Combination therapy of all other glioma cell lines either showed an additive effect or antagonistic effect with CI of >0.75 irrespective of their p53 status. Our results demonstrated that synergistic effects of MK-1775 in combination with chemotherapy (TMZ) are not solely dependent on p53 status of glioma cells and require a more comprehensivce understanding of the molecular mechanism that dictates GBM's dependence on Wee1 for escaping DNA damage therapy. Supported by the Ben & Catherine Ivy Foundation Citation Format: Harshil D. Dhruv, Andrew D. Nelson, Brock Armstrong, Julie L. Boerner, Jann N. Sarkaria, Nhan L. Tran, Michael Berens. Vulnerability and resistance in glioblastoma to G2-specific drugs. [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 1319. doi:10.1158/1538-7445.AM2014-1319

Targeting a kinase common to both the EGFR and TNFα signaling pathways may prevent drug resistance in glioblastoma.

Abstract Malignant dispersion of glioma cells is a nemesis to clinical management, provoking the need for a better understanding of the underlying molecular drivers of invasion. The changes in signal transduction and consequent alterations in activation/de-activation of Transcription Factors (TFs) in glioma cells were investigated coincident to migration-engagement or impaired FAK kinase function by small molecule inhibition (PF-562,271). Earlier work showed that FAK and Pyk2 affect proliferation and migration in reciprocal ways in glioma cells (Lipinski CA, et al 2008; Loftus JC, et al 2009). The interaction of FAK and Pyk2 with specific downstream signaling effectors and the resultant activation of disparate signaling pathways is likely to script a signaling “signature” that will reveal to what extent glioma cell malignant behaviors are dependent on these kinases. Inhibition of FAK leads to diminution of signaling by pSrc, pJnk, and pNFkB, and variable activation of pAkt, and pp38. Of the 20 TFs assayed (Marligen, Inc.), the activation state of 7 TFs showed significant and consistent patterns of change across 6 primary GBM cell strains following FAK inhibition. Across a panel of 6 human GBM xenograft lines SP1, p53, E2F1-5, and NFκB evidenced strong activation (heightened nuclear localization), while AR (androgen receptor), Myc-Max, and CREB activation levels dropped by more than 2-fold following PF-562,271 treatment. Separate studies of established glioma cell lines under conditions of sparse plating (migration) and of dense crowded plating (migration-constrained), showed that p53, E2F1-5, and NFκB were standouts for activation accompanying migration, also associated by strong reduced activation of AR, Myc-Max, and CREB. (Because glioma cells following FAK knockdown or FAK inhibition show reduced entry into cell cycle, the signaling linkage between FAK and Myc-Max activation and also between FAK signaling and CREB activation suggest mechanistic ties to phenotype. E2F1 is a transcription factor that regulates genes involved in cell proliferation and cell survival and has even been suggested to fit a paradigm of oncogene addiction in glioma cells. Our findings demonstrate phenotypic responses in glioma cells downstream of FAK signaling linked to the activation or inactivation of specific transcription factors. If these patterns of TF activation state, and the signaling pathways upstream of that activation, associate (or account for) patterns of glioma cell invasion or proliferation in situ, they will suggest points of vulnerability in the underlying pathological processes of this disease. Supported by NIH NS042262 (Berens) and NIH CA103956 (Loftus). 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 1250.

Abstract The invasion of malignant glioma cells into the surrounding normal brain precludes effective clinical treatment. In this report, we investigated the role of the NH2-terminal FERM domain in the regulation of the promigratory function of Pyk2. We report that the substitution of residues that constitute a small cleft on the surface of the F3 module of the FERM domain do not significantly alter Pyk2 expression but result in the loss of Pyk2 phosphorylation. A monoclonal antibody, designated 12A10, specifically targeting the Pyk2 FERM domain was generated and recognizes an epitope located on the β5C-α1C surface of the F3 module of the FERM domain. Amino acid substitutions in the F3 module that resulted in the loss of Pyk2 phosphorylation also inhibited the binding of 12A10, suggesting that the 12A10 epitope overlaps a site that plays a role in Pyk2 activity. Conjugation of 12A10 to a membrane transport peptide led to intracellular accumulation and inhibition of glioma cell migration in a concentration-dependent manner. A single chain Fv fragment of 12A10 was stable when expressed in the intracellular environment, interacted directly with Pyk2, reduced Pyk2 phosphorylation, and inhibited glioma cell migration in vitro. Stable intracellular expression of the 12A10 scFv significantly extended survival in a glioma xenograft model. Together, these data substantiate a central role for the FERM domain in regulation of Pyk2 activity and identify the F3 module as a novel target to inhibit Pyk2 activity and inhibit glioma progression. [Mol Cancer Ther 2009;8(6):1505–14]

Abstract Glial tumors progress to malignant grades by heightened proliferation and relentless dispersion throughout the central nervous system. Understanding genetic and biochemical processes that foster these behaviors is likely to reveal specific and effective targets for therapeutic intervention. Our current report shows that the fibroblast growth factor-inducible 14 (Fn14), a member of the tumor necrosis factor (TNF) receptor superfamily, is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Forced Fn14 overexpression stimulates glioma cell migration and invasion, and depletion of Rac1 by small interfering RNA inhibits this cellular response. Activation of Fn14 signaling by the ligand TNF-like weak inducer of apoptosis (TWEAK) stimulates migration and up-regulates expression of Fn14; this TWEAK effect requires Rac1 and nuclear factor-κB (NF-κB) activity. The Fn14 promoter region contains NF-κB binding sites, which mediate positive feedback causing sustained overexpression of Fn14 and enduring glioma cell invasion. Furthermore, Fn14 gene expression levels increase with glioma grade and inversely correlate with patient survival. These results show that the Fn14 cascade operates as a positive feedback mechanism for elevated and sustained Fn14 expression. Such a feedback loop argues for aggressive targeting of the Fn14 axis as a unique and specific driver of glioma malignant behavior. (Cancer Res 2006; 66(19): 9535-42)