Tran, Nhan L.

Abstract Pancreatic ductal adenocarcinoma (PDAC) cell growth is dependent on altered biochemical and cellular activities that facilitate increased metabolic dependencies. We determined the role of mutationally activated KRAS, found in ~95% of PDAC, in supporting autophagy. Surprisingly, acute KRAS suppression, which blocks proliferation, was associated with increased rather than decreased autophagic flux. Similarly, KRAS transformation of immortalized human pancreatic epithelial cells showed decreased autophagic flux. Pharmacologic inhibition of ERK MAPK phenocopied the genetic silencing of KRAS and also increased autophagic flux. Addressing a mechanism for ERK suppression increased autophagy, we found increased AMPK activity, decreased mTOR activity, and increased expression of autophagic proteins. We speculated that increased autophagy may be a compensatory mechanism caused by the loss of KRAS- and ERK-dependent metabolic processes. To address this possibility, we performed RNA-seq analyses to monitor gene expression changes in response to ERK inhibition. We observed increased transcription of genes responsible for the metabolic processes of autophagy and β-oxidation, with concurrent decreased transcription of glycolysis and mitochondrial-associated genes. These changes were validated in metabolomic analyses following either genetic silencing of KRAS or ERK inhibition. Thus, the loss of ERK-driven metabolic processes may induce compensatory mechanisms to increase autophagy. We then addressed whether ERK inhibition increased PDAC dependence on autophagy. Supporting this possibility, we found that cotreatment with the autophagy inhibitor chloroquine synergistically enhanced ERK inhibitor mediated antiproliferative activity. Similarly, genetic or pharmacologic inhibition of specific regulators of autophagy also enhanced ERK inhibitor activity. We conclude that concurrent suppression of multiple metabolic processes, to block compensatory rebound activities, will be needed for effective PDAC treatment. Citation Format: Kirsten Bryant, Sen Peng, Andrey Tikunov, Mariaelena Pierobon, Venugopal Gunda, Garima Tomar, Pankaj Singh, Emanuel Petricoin, Jeffrey Macdonald, Nhan Tran, Alec Kimmelman, Channing Der. Inhibition of ERK MAPK signaling increases pancreatic cancer dependency on autophagy [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 5492.

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 Purpose: Glioblastoma is an aggressive and molecularly heterogeneous cancer with few effective treatment options. We hypothesized that next-generation sequencing can be used to guide treatment recommendations within a clinically acceptable time frame following surgery for patients with recurrent glioblastoma. Experimental Design: We conducted a prospective genomics-informed feasibility trial in adults with recurrent and progressive glioblastoma. Following surgical resection, genome-wide tumor/normal exome sequencing and tumor RNA sequencing were performed to identify molecular targets for potential matched therapy. A multidisciplinary molecular tumor board issued treatment recommendations based on the genomic results, blood–brain barrier penetration of the indicated therapies, drug–drug interactions, and drug safety profiles. Feasibility of generating genomics-informed treatment recommendations within 35 days of surgery was assessed. Results: Of the 20 patients enrolled in the study, 16 patients had sufficient tumor tissue for analysis. Exome sequencing was completed for all patients, and RNA sequencing was completed for 14 patients. Treatment recommendations were provided within the study's feasibility time frame for 15 of 16 (94%) patients. Seven patients received treatment based on the tumor board recommendations. Two patients reached 12-month progression-free survival, both adhering to treatments based on the molecular profiling results. One patient remained on treatment and progression free 21 months after surgery, 3 times longer than the patient's previous time to progression. Analysis of matched nonenhancing tissue from 12 patients revealed overlapping as well as novel putatively actionable genomic alterations. Conclusions: Use of genome-wide molecular profiling is feasible and can be informative for guiding real-time, central nervous system–penetrant, genomics-informed treatment recommendations for patients with recurrent glioblastoma. Clin Cancer Res; 24(2); 295–305. ©2017 AACR. See related commentary by Wick and Kessler, p. 256

Abstract Archival tumor samples present a rich resource of annotated specimens for genomics research. However, standard variant calling approaches require a matched normal sample from the same individual, which is often not available with archival tissue. Without a matched normal sample, it is very difficult to distinguish between true somatic variants and germline variants that are private to the individual. Archival FFPE sections often contain adjacent normal tissue, but this normal tissue is often contaminated with infiltrating tumor cells. Comparative somatic variant callers are designed to exclude variants present in the normal sample, so a novel approach is required to leverage sequencing of adjacent normal tissue. Here we present LumosVar 2.0, a software package designed to jointly analyze multiple samples from the same patient. The approach is based on the concept that the allelic fraction of somatic variants, but not germline variants, would be diluted by the presence of normal tissue. Given the allele specific copy number state and expected tumor fraction of each sample, one can model the expected allelic fractions of somatic and germline variants. LumosVar estimates allele specific copy number and tumor sample fractions from the data, and uses the model to call somatic and germline variants. LumosVar is also able to detect sub clonal events and spatial heterogeneity between samples. To evaluate this approach, we used a glioblastoma dataset, where enhancing (high tumor content) and non-enhancing (low tumor content) samples were available for each patient, as well as the matched normal sample to define the true somatic variants. We show that both sensitivity and positive predictive value are improved by analyzing the high tumor and low tumor jointly compared to analyzing the samples individually or in silico pooling of the two samples. Finally, we apply this approach to a set of breast and prostate archival FFPE tumor samples where normal samples were not available for germline sequencing. Citation Format: Rebecca F. Halperin, Winnie S. Liang, Erica Tassone, Jonathan Adkins, Megan Russell, Nhan Tran, Nicole Hank, James Newell, Sandeep Gupta, Michael Berens, Ronald Korn, David W. Craig, Sara A. Byron. Leveraging spatial heterogeneity in tumor purity for improved somatic variant calling of archival tumor-only samples [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 LB-007. doi:10.1158/1538-7445.AM2017-LB-007

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