Brock Armstrong

TROY (TNFRSF19) is Overexpressed in Advanced Glial Tumors and Promotes Glioblastoma Cell Invasion via Pyk2-Rac1 Signaling

A critical problem in the treatment of malignant gliomas is the extensive infiltration of individual tumor cells into adjacent brain tissues. This invasive phenotype severely limits all current therapies, and to date, no treatment is available to control the spread of this disease. Members of the tumor necrosis factor (TNF) ligand superfamily and their cognate receptors regulate various cellular responses including proliferation, migration, differentiation, and apoptosis. Specifically, the TNFRSF19/TROY gene encodes a type I cell surface receptor that is expressed on migrating or proliferating progenitor cells of the hippocampus, thalamus, and cerebral cortex. Here, we show that levels of TROY mRNA expression directly correlate with increasing glial tumor grade. Among malignant gliomas, TROY expression correlates inversely with overall patient survival. In addition, we show that TROY overexpression in glioma cells activates Rac1 signaling in a Pyk2-dependent manner to drive glioma cell invasion and migration. Pyk2 coimmunoprecipitates with the TROY receptor, and depletion of Pyk2 expression by short hairpin RNA interference oligonucleotides inhibits TROY-induced Rac1 activation and subsequent cellular migration. These findings position aberrant expression and/or signaling by TROY as a contributor, and possibly as a driver, of the malignant dispersion of glioma cells. Mol Cancer Res; 8(11); 1558–67. ©2010 AACR.

Reciprocal Activation of Transcription Factors Underlies the Dichotomy between Proliferation and Invasion of Glioma Cells

Histology of malignant glioma depicts dense proliferative areas rich in angiogenesis as well as dissemination of neoplastic cells into adjacent brain tissue. Although the mechanisms that trigger transition from proliferative to invasive phenotypes are complex, the dichotomy of cell proliferation and migration, the “Go or Grow” hypothesis, argues for specific and coordinated regulation of these phenotypes. We investigated transcriptional elements that accompany the phenotypes of migration and proliferation, and consider the therapeutic significance of the “Go or Grow” hypothesis. Interrogation of matched core and rim regions from human glioblastoma biopsy specimens in situ (n = 44) revealed higher proliferation (Ki67 labeling index) in cells residing at the core compared to the rim. Profiling activated transcription factors in a panel of migration-activated versus migration-restricted GBM cells portrayed strong NF-κB activity in the migratory cell population. In contrast, increased c-Myc activity was found in migration-restricted proliferative cells. Validation of transcriptional activity by NF-κB- or c-Myc-driven GFP or RFP, respectively, showed an increased NF-κB activity in the active migrating cells, whereas the proliferative, migration restricted cells displayed increased c-Myc activity. Immunohistochemistry on clinical specimens validated a robust phosphorylated c-Myc staining in tumor cells at the core, whereas increased phosphorylated NF-κB staining was detected in the invasive tumor cells at the rim. Functional genomics revealed that depletion of c-Myc expression by siRNA oligonucleotides reduced cell proliferation in vitro, but surprisingly, cell migration was enhanced significantly. Conversely, inhibition of NF-κB by pharmacological inhibitors, SN50 or BAY-11, decreased both cell migration in vitro and invasion ex vivo. Notably, inhibition of NF-κB was found to have no effect on the proliferation rate of glioma cells. These findings suggest that the reciprocal and coordinated suppression/activation of transcription factors, such as c-Myc and NF-κB may underlie the shift of glioma cells from a “growing-to-going” phenotype.

Abstract 1468: Characterization of patient-derived xenograft (PDX) models to evaluate clinical and therapeutic responses of glioblastoma multiforme

BACKGROUND/PURPOSE The Mayo Clinic has developed a large panel of patient-derived xenografts (PDX) from patients with glioblastoma multiforme (GBM). Here we report initial molecular profiling of the Mayo PDX panel and a comparison of patient and PDX molecular profile and response to therapy. METHODS & MATERIALS Clinical data was retrieved by retrospective chart review. Animal data from all PDX investigations conducted between 2004-2014 were retrieved from experimental logs and subsequently consolidated into a database for analysis. RESULTS From 1999 to 2014, 182 patient tumor samples of varying histological grades were attempted for xenograft with 73 resulting viable PDX lines. Viable xenografts were only produced from WHO grade IV tumor specimens, yielding an overall success rate of 49% for these tumors. GBM patients that produced viable xenografts compared to those that did not exhibited a trend for decreased overall survival (P = 0.18). There was no significant association between successful xenografting and whether tissue was from newly diagnosed (45/93%) or recurrent (20/40; 50%) tumors. Patient age > 45 at diagnosis was correlated with increased PDX viability in GBMs (p = 0.05). Of the viable PDX models analyzed, EGFR mutation was identified in 17 lines, TERT mutation was found in 13 lines, IDH mutation in 1 line, and MGMT hypermethylation in 25 lines. RNAseq was performed on orthotopic tumor samples from 53 PDX models. After excluding contaminating murine sequence reads, expression analysis demonstrated 32 models with a mesenchymal phenotype. Array comparative genomic hybridization was performed on 9 patient samples and derivative early, mid and late passage PDX tumors. Using unbiased hierarchical clustering, there was a high concordance between patient and xenograft models. Within the PDX panel, fractionated radiation (RT) alone and RT combined with temozolomide (TMZ) was tested in orthotopic tumors in 38 lines. The overall median survival benefit (ratio of median survival for treated vs. placebo) in PDX lines treated with RT only was 1.6 (range: 0.9-2.5) and with RT/TMZ was 2.5 (range: 1.1 - 8.9). There was a positive association between observed patient survival and the corresponding survival benefit in the PDX for subjects treated with RT/TMZ (r = 0.2; n = 17). Response to adjuvant TMZ was evaluated in 42 tumor lines, and response to bevacizumab was tested in 33 tumor lines, and correlations with clinical treatment response are being evaluated. CONCLUSIONS: The Mayo GBM PDX panel is widely used in the neuro-oncology community. The initial molecular analysis suggests a good correlation between patient and PDX at the level of genotypic characteristics and therapeutic sensitivity. Citation Format: Dioval A. Remonde, Brett L. Carlson, Mark A. Schroeder, Brock Armstrong, Sen Peng, Lisa Evers, Paul A. Decker, Jeanette Eckel Passow, Michael E. Berens, Nhan L. Tran, Robert B. Jenkins, Jann N. Sarkaria. Characterization of patient-derived xenograft (PDX) models to evaluate clinical and therapeutic responses of glioblastoma multiforme. [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 1468. doi:10.1158/1538-7445.AM2015-1468

Abstract 1319: Vulnerability and resistance in glioblastoma to G2-specific drugs

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 GBM9s 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 306: TWEAK functions as chemotactic factor for glioma cells via Lyn activation

The long-term survival of patients with glioblastoma multiforme is compromised by the tumor9s proclivity for local invasion into the surrounding normal brain. These invasive cells escape surgery and display resistance to chemotherapeutic- and radiation-induced apoptosis. We have previously shown that tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell invasion and survival via binding to the fibroblast growth factor-inducible 14 (Fn14) receptor and subsequent activation of the Rac1/NF-kappaB pathway. In addition, we have reported previously that Fn14 is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Here we demonstrate that TWEAK can act as a chemotactic factor for glioma cells, a potential process to drive cell invasion into the surrounding brain tissue. Specifically, we detected a chemotactic migration of glioma cells to the concentration gradient of TWEAK using the Modified Boyden chamber assay. Because TWEAK has been shown to be expressed in normal astrocytes, microglia, and endothelial cells, we neutralized TWEAK using a recombinant soluble murine Fn14-Fc decoy receptor and examined the effect on glioma cell invasion in the ex vivo rat brain slice model. Incubation of the brain slice with the Fn14-Fc decoy receptor reduced glioma cell invasion as compared to control IgG treated brain slice. Since Src family kinases (SFK) have been implicated in chemotaxis, we next determined whether TWEAK:Fn14 engagement activated these cytoplasmic tyrosine kinases. Our data shows that TWEAK stimulation of glioma cells results in a rapid phosphorylation of Src as detected by Western blot analysis using a pan-Src phosphorylation antibody. To determine which SFK member is phosphorylated by TWEAK, we applied the multiplex Luminex assay and identified Lyn as a candidate SFK. Immunoprecipitation of Lyn from TWEAK-treated cells verified that Lyn is phosphorylated as determined by phosphotyrosine immunoblot analysis. Immunodepletion of Lyn by siRNA oligonucleotides suppressed the chemoattractive effect of TWEAK on glioma cells. We hypothesize that TWEAK secretion by cells present in the glioma microenvironment induce invasion of glioma cells into the brain parenchyma. Understanding the function and signaling of the TWEAK-Fn14 ligand-receptor system may lead to development of novel therapies to therapeutically target invasive glioma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 306. doi:1538-7445.AM2012-306

Abstract 5068: Identification of key tumorigenic pathways in never-smoker lung adenocarcinoma using massively parallel DNA and RNA sequencing and methylation profiling

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Considered independently, lung cancer in never-smokers would rank among the ten most common causes of cancer mortality. Driver pathways and potential therapeutics must be identified for this clinically relevant subpopulation. We hypothesize that novel mutations and pathways identified by whole genome sequencing (WGS), whole transcriptome sequencing (WTS), and methylation profiling drive tumorigenesis in adenocarcinomas (AC) of never-smoker (NS) patients, and represent potential therapeutic targets. We have completed WGS, WTS, and methylation profiling on two lung ACs from female, never-smokers, one early-stage and one stage IV, and one female smoker (S) patient with early-stage lung AC. Approximately 100 short nucleotide variants (SNV) were discerned from the early-stage and stage IV NS lung ACs. Of interest, these NS patients lacked alterations in common genes associated with lung cancer such as EGFR and KRAS. In comparison, the lung AC from a smoker contained 78 SNVs, including a well-characterized KRAS mutation. Mutations in MAGEC1, a tumor marker in melanoma, were observed in common between the early-stage NS tumor and the smoker lung AC. The early-stage NS tumor contained a mutation in PIK3C3 and CSNK1E, a casein kinase involved Wnt signaling. The stage IV NS tumor demonstrated mutations in tumor suppressor genes such as p53, LATS2, and ATM. With relatively few mutations discerned from NS lung ACs, WTS and methylation profiling were performed. WTS showed 1,083 genes differentially regulated in the early-stage NS and Ingenuity Pathway Analysis implicated G-protein coupled receptor signaling. 2,000 genes were differentially expressed in the stage IV NS lung AC with sonic hedgehog implicated as a significantly regulated pathway. Besides gene expression, methylation profiling revealed ∼3,100 differentially methylated genes in the early-stage NS lung AC compared to normal lung DNA, with 65% hypo-methylated, including genes involved in lung carcinogenesis such as RET and BCL2. In the stage IV NS lung AC, ∼35,000 genes were differentially methylated compared to normal lung DNA (10X the early-stage NS) with ∼62% hypo-methylated. Thus, while an early-stage smoker lung AC displayed a classic driver mutation in KRAS, lung ACs from never-smokers had relatively quiet genomes. Despite few mutations, thousands of genes were differentially regulated at the mRNA level or by methylation. We continue to validate the genes implicated in NS lung AC by mutation, differential expression, and differential methylation, as well as integrating the data across platforms to discern significantly regulated pathways. We believe that the genes and pathways implicated by WGS, WTS, and methylation profiling will lead to a better understanding of NS lung AC and identify possible therapeutic interventions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5068. doi:1538-7445.AM2012-5068

Abstract 4917: Aligning xenograft models to glioblastoma (GB) patient tumors to assess chemovulnerability of patients

Primary human tumor xenografts are able to capture the heterogeneity of Glioblastoma (GB) and can be exploited to identify patterns within genomic profiles that are indicative of vulnerability to specific treatments. The Ivy Genomics-Based Medicine (IGBM) project accessed 40 GB human xenografts in mice, and evaluated responses to 4 different treatments. Additionally, gene expression data for 301 GB patient samples from TCGA were investigated to find context-specific gene interactions within patient data. We discovered 12 sets of genes with corresponding subsets of samples with coherent expression profiles, termed contexts. Each context was tested for enrichment of known GB subtype and patient survival characteristics. Our goal is to align 39 xenografts to the 12 contexts, by establishing the similarity between the context samples and the mapped xenografts, and characterize the contexts for their potential therapeutic responses. We first eliminated confounding probe hybridization from mouse tissue. Removal of such “confounders” was inadequate to resolve gene expression disparity between xenografts and patients. We then used Kolmogorov-Smirnov test to determine a set of genes that retain similar distributions between the expression data sets, resulting in 1,650 genes. Initial clustering analysis indicated that xenografts could be aligned across GB patient samples, without any apparent segregation. Xenografts were mapped to individual contexts using various similarity metrics such as correlation or Mahalanobis distance based on expression data, and the quality of alignment was measured using Silhouette scores. We have also conducted preliminary survival analysis for the xenograft subgroups (untreated) and compared it to the patient data. Finally, we compared survival data of 12 treated xenografts to determine if certain contexts are associated with improved or decreased sensitivity to four different drug regimens (temozolomide (TMZ), TMZ PARP inhibitor, BEZ235 and Sorafenib). The results indicated that xenografts aligned to context enriched with classical subtype showed sensitivity to the TMZ drug regimens, whereas contexts enriched with proneural subtype were sensitive to TMZ PARP inhibitor and resistance to TMZ; xenografts not associated with a subtype showed resistance to Sorafenib. While this preliminary analysis needs to be strengthened with more xenografts, aligning xenografts to GB patients with specific gene expression patterns appears to be promising in finding drug targets for these patient groups. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government. It is also supported by the BenC 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4917. doi:1538-7445.AM2012-4917

Abstract 265: c-Myc and NFκB are reciprocal and contextual transcriptional regulators of glioma cell proliferation or invasion

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Classic histological features of glioblastoma include dense proliferative areas rich in angiogenesis as well as centripetal dissemination of neoplastic cells into adjacent brain tissue (most frequently white matter). The infiltrative dispersion patterns of malignant glioma preclude complete tumor resection; growth of satellite lesions causes significant neurological morbidity and mortality, and accounts for much of the post-treatment, recurrent (fatal) disease. Distinct transcriptomes are discernable between GBM cells at the tumor core and invasive rim, and many of the differentially-expressed genes are co-associated with migration and the collateral phenotype of cell survival; reciprocal downregulation of genes in invasive glioma cells are ontologically associated with proliferation. Our studies of glioma cells from paired core and rim human biopsy specimens reveal a higher proliferative index (Ki67 Mib-1 IHC score) at the core as compared to the rim (19 out of 35 specimens) p < 0.002. Analysis of activation states of transcription factors (Luminex multiplex assay) revealed that nuclear c-myc activity is up in the tumor core while nuclear NFκB activity is up at the invasive rim of the tumor. Depletion of c-myc (siRNA oligonucleotides) resulted in an increase in the migration rate of glioma cells in vitro, whereas inhibition of NFκB by SN50 resulted in a decrease in migration rate both in vitro and ex vivo (rat brain slice). Immunohistochemical validation using a glioma tissue microarray containing paired core and rim biopsy specimens showed that phosphorylated c-myc staining was higher in the core than in the rim for 23 out of 39 biopsy specimens scored, and that phosphorylated NFκB was higher in the rim than the core for 30 out of 43 biopsy specimens scored. The Go vs Grow hypothesis suggests cell proliferation and cell migration are temporally exclusive behaviors and tumor cells postpone cell division for migration. Our findings argue that differential suppression/activation of c-myc and NFκB underlie the shift of glioma cells from growing to going. 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 265. doi:10.1158/1538-7445.AM2011-265