Dhruv, Harshil

Abstract Glioblastoma (GBM) is the most common primary tumor of the CNS and carries a dismal prognosis. The aggressive invasion of GBM cells into the surrounding normal brain makes complete resection impossible, significantly increases resistance to the standard therapy regimen, and virtually assures tumor recurrence. Median survival for newly diagnosed GBM is 14.6 months and declines to 8 months for patients with recurrent GBM. New therapeutic strategies that target the molecular drivers of invasion are required for improved clinical outcome. We have demonstrated that TROY (TNFRSF19), a member of the TNFR super-family, plays an important role in GBM invasion and resistance. TROY expression increases with glial tumor grade and inversely correlates with patient survival. TROY stimulates GBM cell invasion and increases resistance to temozolomide (TMZ) and radiation treatment. Conversely, knockdown of TROY expression inhibits GBM cell invasion, increases sensitivity to temozolomide, and prolongs survival in an intracranial xenograft model. Propentofylline (PPF), an atypical synthetic methylxanthine compound, has been extensively studied in Phase II and Phase III clinical trials for Alzheimer's disease and vascular dementia where it has demonstrated blood-brain permeability and minimal adverse side effects. In this study, we demonstrated that PPF decreases TROY protein expression in glioma cells, and subsequently suppress activation downstream signaling effectors including AKT, NF-κB, and Rac1. PPF treatment of glioma cells also suppressed glioma cell migration and invasion, demonstrated by transwell migration assay and reduced membrane ruffling. Finally, PPF treatment increased vulnerability of glioma cells to Temozolomide (TMZ) and radiation. In summary, this study demonstrates that PPF provides a pharmacologic approach to target TROY to inhibit glioma cell invasion and reduce therapeutic resistance to TMZ and radiation. Citation Format: Harshil D. Dhruv, Serdar Tuncali, Alison Roos, Patrick Tomboc, Nathan Jameson, Ashley Chavez, Joseph Loftus, Michael E. Berens, Nhan L. Tran. Propentofylline inhibits TROY/TNFRSF19 signaling to enhance therapeutic efficacy in invasive glioblastoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5366. doi:10.1158/1538-7445.AM2015-5366

Abstract Glioblastoma (GBM) is a highly lethal CNS malignancy in adults with a median survival of about 15 months. Effective therapeutic control of GBM is thwarted by the invasive nature of the tumor, which prevents complete surgical removal and results in tumor recurrence. Thus, there is a dire need to develop innovative approaches to target the invasive tumor cells for improved treatment of this disease. Expression of TROY, a member of the TNFR family, increases with glial tumor grade and inversely correlates with patient survival. Increased expression of TROY stimulates glioblastoma cell invasion and increases resistance to temozolomide (TMZ) and radiation treatment. We have demonstrated that TROY forms a novel complex with EGFR and modulates EGFR survival signaling. IHC analysis of GBM specimens showed that tumors with elevated TROY expression had a statistically positive correlation with increased EGFR expression. TROY expression enhanced EGFR phosphorylation, stabilized EGFR surface expression in the presence of ligand, and significantly increased EGF-stimulated GBM cell invasion. Consistent with invasion, co-expression of TROY with EGFR increased cell survival to TMZ. Moreover, we have shown that in cells treated with EGF, the TROY and ErbB4 receptor are recruited to the EGFR complex. Although the role of ErbB4 in GBM is not well defined, inhibition of ErbB4 expression suppressed tumor growth and invasion in multiple tumor subtypes, decreased EGFR surface retention, and limited EGF-induced cell migration. These data suggest that the TROY-EGFR-ErbB4 complex may represent an unappreciated therapeutic target to inhibit glioma invasion and decrease therapeutic resistance. Citation Format: Alison Roos, Zachary Mayo, Jean Kloss, Serdar Tuncali, Harshil Dhruv, Michael E. Berens, Joseph C. Loftus, Nhan L. Tran. TROY-EGFR signaling complex mediates glioblastoma cells invasion and survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 506. doi:10.1158/1538-7445.AM2015-506

Abstract Deregulation of the TWEAK-Fn14 signaling pathway is observed in many diseases including inflammation, autoimmune diseases, and cancer, notably glioblastoma. Activation of Fn14 signaling by TWEAK binding triggers glioma cell invasion and survival and therefore represents an attractive pathway for therapeutic intervention. Based on structural studies of the TWEAK-binding cysteine rich domain of Fn14, several homology models of TWEAK were built to investigate plausible modes of TWEAK-Fn14 interaction. Two promising models, centered on different anchoring residues of TWEAK [Tyrosine 176 (Y176) and Tryptophan 231 (W231)], were prioritized using a data-driven strategy. Site-directed mutagenesis of TWEAK at Y176, but not W231, resulted in the loss of TWEAK binding to Fn14 substantiating Y176 as the anchoring residue. Importantly, mutation of TWEAK at Y176 did not disrupt TWEAK trimerization, but failed to induce Fn14 mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. The validated structural models were utilized in a virtual screen to design a targeted library of small molecules predicted to disrupt the TWEAK-Fn14 interaction. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. One specific compound, L524-0366 binds specifically to the Fn14 receptor and disrupt TWEAK-Fn14 interaction. Treatment of L524-0366 suppresses TWEAK-induced cell migration and survival in glioblastoma cells. In summary, we present a data-driven in silico study revealing key structural elements of the TWEAK-Fn14 interaction, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor interaction. Our results provide the foundation for further exploration utilizing L524-0366 as a small molecule inhibitor to the TWEAK-Fn14 signaling axis. Citation Format: Harshil D. Dhruv, Joseph C. Loftus, Pooja Narang, Joachim L. Petit, Donald Chow, Holly Yin, Michael Berens, Nathalie Meurice, Nhan L. Tran. Identification and characterization of L524-0366 as a small molecule inhibitor that disrupt TWEAK-Fn14 signaling in glioblastoma. [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 1780. doi:10.1158/1538-7445.AM2014-1780

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

Abstract Glioblastoma multiforme (GBM) is the most common primary central nervous system tumor accounting for approximately 40% of all primary malignant brain tumors. The mechanism driving the development and recurrence of GBM is still largely unknown which greatly limits the successful treatment of this disease. The tumor necrosis factor receptor superfamily member TNFRSF19 (TROY) is a type I cell surface receptor protein containing the highly conserved TNFR cysteine-rich motifs in the extracellular domain and a tumor necrosis factor-receptor-associated factor (TRAF) - binding sequence in the cytoplasmic domain. We recently demonstrated that increased expression of TROY stimulated glioma cell migration in vitro and increased cell invasion in an organotypic brain slice model. Conversely, siRNA mediated knockdown of TROY expression inhibited glioma cell migration. In addition, profiling of TROY in brain tumor samples indicated that TROY mRNA expression was significantly increased in GBM samples, directly correlated with increasing glial tumor grade, and inversely correlated with patient outcome suggesting that TROY expression may play a role in GBM invasion and is a good indicator of survival outcome. In the current study, we investigated the role of TROY in therapeutic resistance and survival signaling. We report that TROY protein expression was significantly increased in patient GBM tumor samples with TROY mRNA exhibiting increased expression in the invasive cell population. Aberrant expression of TROY in mouse astrocytes in situ using glial-specific gene transfer in transgenic mice induced astrocyte migration within the brain supporting an important role for TROY in glioma cell migration. Notably, increased TROY expression did not increase cell proliferation but increased resistance of glioma cells to both radiation and temozolomide induced apoptosis while knockdown of TROY increased temozolomide sensitivity. TROY induced resistance to TMZ was dependent upon Akt and NF-κB activation. We also report that TROY induced NF-κB phosphorylation and stimulation of migration required the membrane proximal region of the TROY cytoplasmic domain and that knockdown of TROY expression increased survival in a xenograft model. The current results further support a role for TROY in GBM and suggest that targeting TROY and its signaling pathway represents a potential approach to increase tumor vulnerability and improve the therapeutic response of glioblastoma. Citation Format: Harshil D. Dhruv, Serdar Tuncali, Jean Kloss, Zhongbo Yang, Cassie Schumacher, Bart Williams, Julianna Ross, Nhan Tran, Joseph Loftus. TNFRSF19 (TROY) promotes glioma cell survival signaling and therapeutic resistance. [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 2621. doi:10.1158/1538-7445.AM2013-2621

Glioma cell migration correlates with Pyk2 activity, but the intrinsic mechanism that regulates the activity of Pyk2 is not fully understood. Previous studies have supported a role for the N-terminal FERM domain in the regulation of Pyk2 activity as mutations in the FERM domain inhibit Pyk2 phosphorylation. To search for novel protein-protein interactions mediated by the Pyk2 FERM domain, we utilized a yeast two-hybrid genetic selection to identify the mammalian Ste20 homolog MAP4K4 as a binding partner for the Pyk2 FERM domain. MAP4K4 coimmunoprecipitated with Pyk2 and was a substrate for Pyk2 but did not coimmunoprecipitate with the closely related focal adhesion kinase FAK. Knockdown of MAP4K4 expression inhibited glioma cell migration and effectively blocked Pyk2 stimulation of glioma cell. Increased expression of MAP4K4 stimulated glioma cell migration; however, this stimulation was blocked by knockdown of Pyk2 expression. These data support that the interaction of MAP4K4 and Pyk2 is integrated with glioma cell migration and suggest that inhibition of this interaction may represent a potential therapeutic strategy to limit glioblastoma tumor dispersion.

Abstract Of the features that characterize glioblastoma, arguably none is more clinically relevant than the propensity of malignant glioma cells to aggressively invade into the surrounding normal brain tissue. These invasive cells render complete resection impossible, confer significant resistance to chemo- and radiation-therapy, and virtually assure tumor recurrence. Expression of TROY (TNFRSF19), a member of the TNF receptor superfamily, inversely correlates with patient survival and stimulates glioblastoma cell migration and invasion in vitro. In this study, we report that TROY is overexpressed in glioblastoma tumor specimens and TROY mRNA expression is increased in the invasive cell population in vivo. In addition, inappropriate expression of TROY in mouse astrocytes in vivo using glial-specific gene transfer in transgenic mice induces astrocyte migration within the brain, validating the importance of the TROY signaling cascade in glioblastoma cell migration and invasion. Knockdown of TROY expression in primary glioblastoma xenografts significantly prolonged survival in vivo. Moreover, TROY expression significantly increased resistance of glioblastoma cells to both IR- and TMZ-induced apoptosis via activation of Akt and NF-κB. Inhibition of either Akt or NF-κB activity suppressed the survival benefits of TROY signaling in response to TMZ treatment. These findings position aberrant expression and/or signaling by TROY as a contributor to the dispersion of glioblastoma cells and therapeutic resistance. Implications: Targeting of TROY may increase tumor vulnerability and improve therapeutic response in glioblastoma. Mol Cancer Res; 11(8); 865–74. ©2013 AACR.