Citations

Abstract Background Tumor heterogeneity underlies resistance and disease progression in glioblastoma (GBM), and tumors most commonly recur adjacent to the surgical resection margins in contrast non-enhancing (NE) regions. To date, no targeted therapies have meaningfully altered overall patient survival in the up-front setting. The aim of this study was to characterize intratumoral heterogeneity in recurrent GBM using bulk samples from primary resection and recurrent samples taken from contrast-enhancing (EN) and contrast NE regions. Methods Whole exome and RNA sequencing were performed on matched bulk primary and multiple recurrent EN and NE tumor samples from 16 GBM patients who received standard of care treatment alone or in combination with investigational clinical trial regimens. Results Private mutations emerge across multi-region sampling in recurrent tumors. Genomic clonal analysis revealed increased enrichment in gene alterations regulating the G2M checkpoint, Kras signaling, Wnt signaling, and DNA repair in recurrent disease. Subsequent functional studies identified augmented PI3K/AKT transcriptional and protein activity throughout progression, validated by phospho-protein levels. Moreover, a mesenchymal transcriptional signature was observed in recurrent EN regions, which differed from the proneural signature in recurrent NE regions. Conclusions Subclonal populations observed within bulk resected primary GBMs transcriptionally evolve across tumor recurrence (EN and NE regions) and exhibit aberrant gene expression of common signaling pathways that persist despite standard or targeted therapy. Our findings provide evidence that there are both adaptive and clonally mediated dependencies of GBM on key pathways, such as the PI3K/AKT axis, for survival across recurrences.

Abstract Purpose: Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization. Experimental Design: PDXs were established from glioblastoma, IDH-wildtype (n = 93), glioblastoma, IDH-mutant (n = 2), diffuse midline glioma, H3 K27M-mutant (n = 1), and both primary (n = 60) and recurrent (n = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, n = 83), RNA-sequencing (n = 68), and genome-wide methylation profiling (n = 76). WES data from 24 patient tumors was compared with derivative models. Results: PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of TERT, EGFR, PTEN, TP53, BRAF, and IDH1, most at frequencies comparable with human glioblastoma. However, PDGFRA amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. MGMT promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including EGFR amplification. However, in four patient–PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection. Conclusions: Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.

Abstract Glioblastoma multiforme (GBM) remains the most common and deadly primary brain tumor with minimal advances in therapeutic development for pharmacological treatment. Tumor resection followed by temozolomide (TMZ) and radiotherapy adds months to survival; however, tumor recurrence usually occurs. The fibroblast growth factor-inducible 14 (Fn14) receptor has been shown to be up-regulated in the GBM cells left behind after surgery and contributes to an invasive and pro-survival phenotype, providing a desirable molecule to target after removal of the primary tumor. Previously, L524-0366 was reported by our lab to be the first antagonist of Fn14 signaling; however, minimal efficacy/potency has limited this drug from progressing from a useful research tool to preclinical studies. Structure activity relationship (SAR) studies using the parent L524-0366 compound gave rise to K78, which was found to bind to Fn14 with enhanced affinity, while showing no binding to the structurally-related TNFα receptor. Furthermore, K78 was able to abate the downstream signaling effects initiated by the Fn14 ligand, tumor necrosis factor-like weak inducer of apoptosis (TWEAK), as well as sensitize GBM cells to the cytotoxic effects of TMZ. TWEAK-induced cellular migration was also mitigated with co-treatment of K78 in GBM cells, compared to a vehicle control. It is anticipated that further development of Fn14 antagonists will be of great importance for the advancement of GBM patient treatment and warrant further investigation as primary and combination therapy, especially for the prevention of recurrent disease. Citation Format: Bryan Harder, Chris Sereduk, Harshil Dhruv, Nghia Millard, Anthony Kim, Jeffrey Winkles, Graeme Woodworth, Holly Yin, Nhan L. Tran. Development of an antagonist to the TWEAK-Fn14 ligand-receptor interaction with enhanced efficacy/potency in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4910.

Abstract Glioblastoma Multiforme (GBM) is the most common malignant brain tumor in adults. Most GBM patients succumb to the disease less than one-year post diagnosis due to the highly invasive nature of the tumor, which prevents complete surgical resection and gives rise to tumor recurrence. The invasive phenotype also confers radio and chemoresistant properties to the tumor cells; therefore, there is a need to develop new therapeutics that target drivers of GBM invasion. Amplification of EGFR is observed in over 50% of GBM tumors, of which half concurrently overexpress the variant EGFRvIII, and expression of both receptors confers a worse prognosis. EGFR and EGFRvIII cooperate to promote tumor progression and invasion, in part, through activation of the Stat-signaling pathway. Here we report that EGFRvIII activates Stat5 and GBM invasion, in part, by inducing the expression of a previously established mediator of glioma cell invasion and survival, fibroblast growth factor-inducible 14 (Fn14). EGFRvIII-mediated induction of Fn14 expression is dependent upon Stat5 and requires activation of Src, whereas EGFR regulation of Fn14 is dependent upon MEK/ERK-Stat3 activation. Notably, treatment of EGFRvIII expressing GBM cells with the FDA approved Stat5 inhibitor pimozide blocked Stat5 phosphorylation, Fn14 expression, and cell migration. Since EGFR inhibitors display limited therapeutic efficacy in GBM patients, we hypothesize that the EGFRvIII-Stat5-Fn14 signaling pathway represents a node of vulnerability in the invasive GB cell population and that targeting critical effectors in this pathway will limit GBM tumor dispersion, mitigate therapeutic resistance, and increase survival. Citation Format: Alison Roos, Harshil D. Dhruv, Sen Peng, Landon J. Inge, Serdar Tuncali, Michael Pineda, Nghia Millard, Jeffrey A. Winkles, Joseph C. Loftus, Nhan L. Tran. EGFRvIII-Stat5 signaling enhances glioblastoma cell invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3462.

Abstract While genomic profiling and therapeutic selection support individualized GBM treatment, such therapeutic decision-making is usually made with reference to tumor obtained from the enhancing core region. GBM is known to be heterogeneous and exhibits a high resistance to standard therapies. To address whether non-enhancing tumor (representing the majority of tumor left behind after surgery) shows distinct genomic characteristics and therapeutic targets compared to the enhancing tumor core, we performed genome-wide exome-sequencing and RNA-sequencing for 12 patients with matched enhancing region and at least one non-enhancing region. Non-enhancing biopsies show a surprisingly high level of tumor content, with a median of 28% tumor cells and 6 of the 22 samples having greater than 50% tumor cells. Cognate non-enhancing and enhancing specimens demonstrated overall concordance in therapeutically actionable alterations (single nucleotide variants) and copy number alterations. However, non-enhancing regions were not genetically identical and did reveal additional and distinct variants compared to enhancing cores. For example, the non-enhancing region of patient 1 showed two nonsense NF1 mutations (R1534X; R2517X) while the enhancing region showed an NF1 frameshift mutation (F1247fs). Clonality analysis by LumosVar also indicated that 7 out of 12 patients harbored dissimilar cellular prevalence patterns between enhancing and non-enhancing regions. In addition, comparison of alternative polyadenylation between enhancing and non-enhancing regions uncovered distinct 3' UTR usage: e.g. SGMS2 and TOB1 tended to have longer 3' UTR in enhancing regions whereas longer 3' UTR of SYNPO and NOS1AP were prevalent in non-enhancing regions. We posit that the enhancing component of glioblastoma probably underrepresents the genomic alterations in patients' tumors. Given non-enhancing tumor is left behind after surgical debulking, genomic profiling of this region would potentially reveal more accurate tumor vulnerabilities and lead to more effective therapy. Supported by a grant from the Ben & Catherine Ivy Foundation. Citation Format: Sen Peng, Rebecca Halperin, Harshil Dhruv, Sara Byron, Christophe Legendre, Joanna Phillips, Michael Prados, Michael Berens, Nhan Tran. Probing the non-enhancing component of glioblastoma: Targeting what is left behind [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 289.

Abstract Glioblastoma multiforme (GBM) is the most common brain malignancies in adults. Most GBM patients succumb to the disease less than 1 year after diagnosis due to the highly invasive nature of the tumor, which prevents complete surgical resection and gives rise to tumor recurrence. The invasive phenotype also confers radioresistant and chemoresistant properties to the tumor cells; therefore, there is a critical need to develop new therapeutics that target drivers of GBM invasion. Amplification of EGFR is observed in over 50% of GBM tumors, of which half concurrently overexpress the variant EGFRvIII, and expression of both receptors confers a worse prognosis. EGFR and EGFRvIII cooperate to promote tumor progression and invasion, in part, through activation of the Stat signaling pathway. Here, it is reported that EGFRvIII activates Stat5 and GBM invasion by inducing the expression of a previously established mediator of glioma cell invasion and survival: fibroblast growth factor-inducible 14 (Fn14). EGFRvIII-mediated induction of Fn14 expression is Stat5 dependent and requires activation of Src, whereas EGFR regulation of Fn14 is dependent upon Src–MEK/ERK–Stat3 activation. Notably, treatment of EGFRvIII-expressing GBM cells with the FDA-approved Stat5 inhibitor pimozide blocked Stat5 phosphorylation, Fn14 expression, and cell migration and survival. Because EGFR inhibitors display limited therapeutic efficacy in GBM patients, the EGFRvIII–Stat5–Fn14 signaling pathway represents a node of vulnerability in the invasive GBM cell populations. Implications: Targeting critical effectors in the EGFRvIII–Stat5–Fn14 pathway may limit GBM tumor dispersion, mitigate therapeutic resistance, and increase survival. Mol Cancer Res; 16(7); 1185–95. ©2018 AACR.

Abstract Glioblastoma is the most frequent primary brain tumor in adults and a highly lethal malignancy with a median survival of about 15 months. The aggressive invasion of the surrounding normal brain makes complete surgical resection impossible, increases the resistance to radiation and chemotherapy, and assures tumor recurrence. Thus, there is an urgent need to develop innovative therapeutics to target the invasive tumor cells for improved treatment outcomes of this disease. Expression of TROY (TNFRSF19), a member of the tumor necrosis factor (TNF) receptor family, increases with increasing glial tumor grade and inversely correlates with patient survival. Increased expression of TROY stimulates glioblastoma cell invasion in vitro and in vivo and increases resistance to temozolomide and radiation therapy. Conversely, silencing TROY expression inhibits glioblastoma cell invasion, increases temozolomide sensitivity, and prolongs survival in an intracranial xenograft model. Here, a novel complex is identified between TROY and EGFR, which is mediated predominantly by the cysteine-rich CRD3 domain of TROY. Glioblastoma tumors with elevated TROY expression have a statistically positive correlation with increased EGFR expression. TROY expression significantly increases the capacity of EGF to stimulate glioblastoma cell invasion, whereas depletion of TROY expression blocks EGF stimulation of glioblastoma cell invasion. Mechanistically, TROY expression modulates EGFR signaling by facilitating EGFR activation and delaying EGFR receptor internalization. Moreover, the association of EGFR with TROY increases TROY-induced NF-κB activation. These findings substantiate a critical role for the TROY–EGFR complex in regulation of glioblastoma cell invasion. Implications: The TROY–EGFR signaling complex emerges as a potential therapeutic target to inhibit glioblastoma cell invasion. Mol Cancer Res; 16(2); 322–32. ©2017 AACR.

Abstract Glioblastoma Multiforme (GBM) is the most common tumor of the CNS with a median survival of approximately 15 months. The highly aggressive invasion of malignant cells into the surrounding normal brain tissue renders complete surgical resection impossible, increases therapeutic resistance, and virtually assures tumor recurrence. We have previously demonstrated that TROY (TNFRSF19) has an important role in GBM invasion and resistance. TROY expression is correlated with glial tumor grade and inversely correlated with patient survival. Increased TROY expression stimulated cell migration/invasion in vitro and in vivo and confers resistance to both ionizing radiation- and temozolomide-induced apoptosis via activation of Akt and NF-κB. These data indicate that TROY stimulated migration/invasion is associated with signaling pathways that also increase survival and therapeutic resistance, however, the mechanistic basis of this signaling remains unclear. To investigate the mechanism by which TROY induces GBM cell invasion, we performed immunoprecipitation of the TROY receptor coupled with MALDI-TOF and MS/MS analysis and identified PDZ-RhoGEF as a binding partner for TROY. We validated the interaction of TROY with PDZ-RhoGEF by immunoprecipitation and western blotting, and demonstrated that PDZ-RhoGEF can exchange for both RhoA and RhoC in glioma cells and is implicated in GBM cell invasion, proliferation, and survival. In order to assess the specific role of PDZ-RhoGEF in TROY signaling, we examined the effects of PDZ-RhoGEF on TROY-mediated activation of NF-κB. While overexpression of TROY alone strongly stimulated NF-κB transcriptional activity in serum-free conditions, increased expression of PDZ-RhoGEF alone had no effect on NF-κB activity. Co-transfection of PDZ-RhoGEF with TROY synergistically promoted NF-κB activation. Mutation of the TRAF binding domain (TBD) in the cytoplasmic domain of TROY abolished its capacity to activate NF-κB, even in the presence of PDZ-RhoGEF, suggesting that the TBD is important for TROY induced NF-κB activation. Furthermore, knockdown of PDZ-RhoGEF by shRNA attenuated NF-κB activation in TROY expressing cells, blocked TROY mediated invasion of primary GBM xenograft cells and increased sensitivity to temozolomide. Collectively, these data indicate that PDZ-RhoGEF plays an important role in TROY signaling and provides insights into a potential node of vulnerability to limit GBM cell invasion and decrease therapeutic resistance. Citation Format: Zonghui Ding, Alison Roos, Jean Kloss, Harshil Dhruv, Marc Symons, Nhan L. Tran, Joseph C. Loftus. PDZ-RhoGEF functions as a critical signaling effector for TROY [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1878. doi:10.1158/1538-7445.AM2017-1878

Abstract Glioblastoma (GB) is the highest grade and most common form of primary adult brain tumors. Despite surgical removal followed by concomitant radiation and chemotherapy with the alkylating agent temozolomide, GB tumors develop treatment resistance and ultimately recur. Impaired response to treatment occurs rapidly, conferring a median survival of just fifteen months. Thus, it is necessary to identify the genetic and signaling mechanisms that promote tumor resistance to develop targeted therapies to combat this refractory disease. Previous observations indicated that SGEF (ARHGEF26), a RhoG-specific guanine nucleotide exchange factor (GEF), is overexpressed in GB tumors and plays a role in promoting TWEAK-Fn14–mediated glioma invasion. Here, further investigation revealed an important role for SGEF in glioma cell survival. SGEF expression is upregulated by TWEAK-Fn14 signaling via NF-κB activity while shRNA-mediated reduction of SGEF expression sensitizes glioma cells to temozolomide-induced apoptosis and suppresses colony formation following temozolomide treatment. Nuclear SGEF is activated following temozolomide exposure and complexes with the DNA damage repair (DDR) protein BRCA1. Moreover, BRCA1 phosphorylation in response to temozolomide treatment is hindered by SGEF knockdown. The role of SGEF in promoting chemotherapeutic resistance highlights a heretofore unappreciated driver, and suggests its candidacy for development of novel targeted therapeutics for temozolomide-refractory, invasive GB cells. Implication: SGEF, as a dual process modulator of cell survival and invasion, represents a novel target for treatment refractory glioblastoma. Mol Cancer Res; 14(3); 302–12. ©2016 AACR.