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PDF file-31KB, Synthesis of octyl-�-ketoglutarate

PDF file-23KB, D-2HG in cell line lysate samples, as assessed by LC-MS/M

PDF file-22KB, D-2HG in primary human glioma tissue samples, as assessed by LC-MS/MS

PDF file-101KB, Mutant IDH1 in the absence of wild type IDH1 is not sufficient to efficiently produce D-2HG in HCT116 cells

Despite the strikingly grave prognosis for older patients with glioblastomas, significant variability in patient outcome is experienced. To explore the potential for developing improved prognostic capabilities based on the elucidation of potential biological relationships, we did analyses of genes commonly mutated, amplified, or deleted in glioblastomas and DNA microarray gene expression data from tumors of glioblastoma patients of age >50 for whom survival is known. No prognostic significance was associated with genetic changes in epidermal growth factor receptor (amplified in 17 of 41 patients), TP53 (mutated in 11 of 41 patients), p16INK4A (deleted in 15 of 33 patients), or phosphatase and tensin homologue (mutated in 15 of 41 patients). Statistical analysis of the gene expression data in connection with survival involved exploration of regression models on small subsets of genes, based on computational search over multiple regression models with cross-validation to assess predictive validity. The analysis generated a set of regression models that, when weighted and combined according to posterior probabilities implied by the statistical analysis, identify patterns in expression of a small subset of genes that are associated with survival and have value in assessing survival risks. The dominant genes across such multiple regression models involve three key genes—SPARC (Osteonectin), Doublecortex, and Semaphorin3B—which play key roles in cellular migration processes. Additional analysis, based on statistical graphical association models constructed using similar computational analysis methods, reveals other genes which support the view that multiple mediators of tumor invasion may be important prognostic factor in glioblastomas in older patients.

Supplementary Tables 1-2 from Gene Expression Profiling and Genetic Markers in Glioblastoma Survival

Glioblastoma multiforme is the most prevalent type of adult brain tumor and one of the deadliest tumors known to mankind. The genetic understanding of glioblastoma multiforme is, however, limited, and the molecular mechanisms that facilitate glioblastoma multiforme cell survival and growth within the tumor microenvironment are largely unknown. We applied digital karyotyping and single nucleotide polymorphism arrays to screen for copy-number changes in glioblastoma multiforme samples and found that the most frequently amplified region is at chromosome 7p11.2. The high resolution of digital karyotyping and single nucleotide polymorphism arrays permits the precise delineation of amplicon boundaries and has enabled identification of the minimal region of amplification at chromosome 7p11.2, which contains two genes, EGFR and SEC61γ. SEC61γ encodes a subunit of a heterotrimeric protein channel located in the endoplasmic reticulum (ER). In addition to its high frequency of gene amplification in glioblastoma multiforme, SEC61γ is also remarkably overexpressed in 77% of glioblastoma multiforme but not in lower-grade gliomas. The small interfering RNA–mediated knockdown of SEC61γ expression in tumor cells led to growth suppression and apoptosis. Furthermore, we showed that pharmacologic ER stress agents induce SEC61γ expression in glioblastoma multiforme cells. Together, these results indicate that aberrant expression of SEC61γ serves significant roles in glioblastoma multiforme cell survival likely via a mechanism that is involved in the cytoprotective ER stress–adaptive response to the tumor microenvironment. [Cancer Res 2009;69(23):9105–11]

Purpose: A major mechanism of resistance to methylating agents, including temozolomide, is the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT). Preclinical data indicates that defective DNA mismatch repair (MMR) results in tolerance to temozolomide regardless of AGT activity. The purpose of this study was to determine the role of MMR deficiency in mediating resistance in samples from patients with both newly diagnosed malignant gliomas and those who have failed temozolomide therapy. Experimental Design: The roles of AGT and MMR deficiency in mediating resistance in glioblastoma multiforme were assessed by immunohistochemistry and microsatellite instability (MSI), respectively. The mutation status of the MSH6 gene, a proposed correlate of temozolomide resistance, was determined by direct sequencing and compared with data from immunofluorescent detection of MSH6 protein and reverse transcription-PCR amplification of MSH6 RNA. Results: Seventy percent of newly diagnosed and 78% of failed-therapy glioblastoma multiforme samples expressed nuclear AGT protein in ≥20% of cells analyzed, suggesting alternate means of resistance in 20% to 30% of cases. Single loci MSI was observed in 3% of patient samples; no sample showed the presence of high MSI. MSI was not shown to correlate with MSH6 mutation or loss of MSH6 protein expression. Conclusions: Although high AGT levels may mediate resistance in a portion of these samples, MMR deficiency does not seem to be responsible for mediating temozolomide resistance in adult malignant glioma. Accordingly, the presence of a fraction of samples exhibiting both low AGT expression and MMR proficiency suggests that additional mechanisms of temozolomide resistance are operational in the clinic.

Purpose: Primary central nervous system (CNS) tumors represent a diverse group of tumor types with heterogeneous molecular mechanisms that underlie their formation and maintenance. CNS tumors depend on angiogenesis and often display increased activity of ErbB-associated pathways. Current nonspecific therapies frequently have poor efficacy in many of these tumor types, so there is a pressing need for the development of novel targeted therapies. Experimental Design: ZD6474 is a novel, orally available low molecular weight inhibitor of the kinase activities associated with vascular endothelial growth factor receptor-2 and epidermal growth factor receptor. We hypothesized that ZD6474 may provide benefit in the treatment of several CNS tumor types. Results: In mice bearing established s.c. tumor xenografts of CNS tumors (malignant glioma and ependymoma) or rhabdomyosarcoma, a limited course of ZD6474 treatment produced significant tumor growth delays and a high rate of partial tumor regression in most models examined. Mice with i.c. malignant glioma xenografts treated with ZD6474 experienced a significant prolongation of survival. Tumors from mice treated with ZD6474 displayed a lower proliferative index and disrupted tumor vascularity. Notably, some of these models are insensitive to low molecular weight kinase inhibitors targeting only vascular endothelial growth factor receptor-2 or epidermal growth factor receptor functions, suggesting that the combined disruption of both epidermal growth factor receptor and vascular endothelial growth factor receptor-2 activities may significantly increase tumor control. Conclusions: In conclusion, ZD6474 shows significant activity against xenograft models of several primary human CNS tumor types. Consideration for clinical development in this disease setting seems warranted.

Despite the strikingly grave prognosis for older patients with glioblastomas, significant variability in patient outcome is experienced. To explore the potential for developing improved prognostic capabilities based on the elucidation of potential biological relationships, we did analyses of genes commonly mutated, amplified, or deleted in glioblastomas and DNA microarray gene expression data from tumors of glioblastoma patients of age >50 for whom survival is known. No prognostic significance was associated with genetic changes in epidermal growth factor receptor (amplified in 17 of 41 patients), TP53 (mutated in 11 of 41 patients), p16INK4A (deleted in 15 of 33 patients), or phosphatase and tensin homologue (mutated in 15 of 41 patients). Statistical analysis of the gene expression data in connection with survival involved exploration of regression models on small subsets of genes, based on computational search over multiple regression models with cross-validation to assess predictive validity. The analysis generated a set of regression models that, when weighted and combined according to posterior probabilities implied by the statistical analysis, identify patterns in expression of a small subset of genes that are associated with survival and have value in assessing survival risks. The dominant genes across such multiple regression models involve three key genes—SPARC (Osteonectin), Doublecortex, and Semaphorin3B—which play key roles in cellular migration processes. Additional analysis, based on statistical graphical association models constructed using similar computational analysis methods, reveals other genes which support the view that multiple mediators of tumor invasion may be important prognostic factor in glioblastomas in older patients.

The Cancer Genome Atlas (TCGA) Network recently catalogued recurrent genomic abnormalities in glioblastoma (GBM). This genomic profiling led to the molecular classification of GBMs into four subtypes: Proneural, Neural, Classical and Mesenchymal. The importance of identifying these subtypes stands to help researchers and clinicians to better understand GBMs with the potential to personalize treatment options and explore different therapeutic approaches that each subtype may require. As such, the development of patient derived xenograft models that represent each TCGA subtype serves to expedite the testing of targeted molecular therapeutic strategies for the treatment of GBMs. For this project, we established xenografts using patient derived tissue and passaged these xenografts until reliable growth characteristics were obtained. Established GBM xenografts were classified into TCGA-defined subtypes first by obtaining global gene expression data using the GeneChip Human Genome U133A 2.0 array from Affymetrix. Microarray data was then quantile normalized and probes were summarized as gene expression levels using RMA. Then data was log2 transformed and genes median centered. Xenografts were subsequently classified into one of four previously defined subtypes as described using Classification to the Nearest Centroid (ClaNC) with the TCGA GBM data as the training dataset. Using established xenografts from each subtype, a panel of standard of care treatment agents was assessed by delay in tumor growth and by tumor regression in athymic mice bearing subcutaneous tumors. Statistical analysis was performed using a personalized SAS statistical analysis program, the Wilcoxon rank order test for growth delay, and Fisher's exact test for tumor regression. To date we have identified at least 2 patient derived xenografts for each TCGA-defined subtype. These xenografts lines have realiable growth patterns with a tumor take of 80+%. Drug testing has been completed and includes tumor growth responses to the following single agents: temozolomide, bevacizumab, BCNU, cytoxan, irinotecan, carboplatin, erlotinib, CCNU, procabazine and vincristine. The identification of these valid xenograft models represents an important contribution toward the ability of studying GBM subtypes, in particular for modeling and predicting therapeutic response. We have currently completed the first round of drug testing within each subtype and look to expand testing to include additional agents as well as select combinations. Citation Format: Stephen T. Keir, B. Ahmed Rasheed, Katherine A. Hoadley, Martin A. Roskoski, Danuta Gasinski, Madan M. Kwatra, Henry S. Friedman, Darell D. Bigner. Identification and treatment data of xenografts representing TCGA-defined glioblastoma subtypes. [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 2435A. doi:10.1158/1538-7445.AM2015-2435A

The Cancer Genome Atlas (TCGA) Network recently catalogued recurrent genomic abnormalities in glioblastoma (GBM). This genomic profiling lead to the molecular classification of GBMs into four subtypes: Proneural, Neural, Classical and Mesenchymal. The importance of identifying these subtypes helps researchers and clinicians to better understand GBMs with the potential to personalize treatment options and explore different therapeutic approaches that each subtype may require. In addition, the TCGA identified possible mechanisms that can cause some GBM tumors to become resistant to therapy, including the standard of care alkylating agent temozolomide (TMZ). For this project, we established xenografts using patient derived tissue and passaged these xenografts until reliable growth characteristics were obtained. Established GBM xenografts were classified into TCGA-defined subtypes first by obtaining global gene expression data using the GeneChip Human Genome U133A 2.0 array from Affymetrix. Microarray data was then quantile normalized and probes were summarized as gene expression levels using RMA. Then data was log2 transformed and genes median centered. Xenografts were subsequently classified into one of four previously defined subtypes as described using Classification to the Nearest Centroid (ClaNC) with the TCGA GBM data as the training dataset. Using established xenografts from each subtype, a panel of standard of care treatment agents (including TMZ) was assessed by delay in tumor growth and by tumor regression. Statistical analysis was performed using a SAS statistical analysis program, the Wilcoxon rank order test for growth delay, and Fisher's exact test for tumor regression. Data regarding each xenografts BRAF, EGFR, EGFRvIII, IDH1, PIK3CA, PIK3R1, PTEN, RB1, TERT and TP53 status is reported within. The identification of these valid xenograft models represents an important contribution toward the ability of studying GBM subtypes, in particular for modeling and predicting therapeutic response. Citation Format: Stephen T. Keir, B Ahmed Rasheed, Katherine A. Hoadley, Martin A. Roskoski, Danuta Gasinski, Patrick J. Killela, Hai Yan, Madan M. Kwatra, Henry S. Friedman, Darell D. Bigner. Establishment, identification and treatment data of TCGA glioblatoma xenograft subtypes. [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 838. doi:10.1158/1538-7445.AM2014-838

Abstract Malignant cell transformation, invasion and metastasis are dependent on the coordinated rewiring of gene expression. A major component in the scaffold of these reprogramming events is one in which epithelial cells lose intercellular connections and polarity and adopt a motile mesenchymal phenotype, one that is largely supported by a robust transcriptional machinery consisting mostly of developmental transcription factors. We investigated the winged helix transcription factor, FOXQ1, identified from an oligonucleotide microarray expression analysis, as being concomitantly deregulated in both epithelial and stromal compartments. We found that FOXQ1 contributes to epithelial to mesenchymal transition (EMT), in part by directly affecting the transcription of Twist1, itself a key mediator of metastasis that transcriptionally regulates the expression of important molecules involved in EMT. Reporter gene assays and chromatin immunoprecipitation experiments showed that FOXQ1 regulated and directly interacted with the Twist1 promoter, leading to a suppression of E-cadherin and increased expression of mesenchymal markers. Forced expression and RNAi mediated silencing of FOXQ1 led to increased and decreased levels of Twist1 mRNA and protein levels, respectively, with concomitant effects on migration, invasion and in vivo metastasis of colorectal cancer cells. Moreover, FOXQ1 mRNA and protein expression correlated in a series of resected patient samples. Conclusively our results show that FOXQ1 is a novel mediator of colorectal cancer metastasis and joins the network of transcription factors that orchestrate EMT. Citation Format: Mohammed Abba, Nitin Patil, Kabeer Rasheed, Laura D. Nelson, Giridhar Mudduluru, Jörg H. Leupold, Heike Allgayer. A direct regulation of Twist1 by FOXQ1 promotes colorectal cancer metastasis. [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 2703. doi:10.1158/1538-7445.AM2013-2703

Myxoid liposarcoma exhibits a stable reciprocal translocation, leading to expression of FUS-CHOP chimeric oncogenic protein. Furthermore, studies have shown that this event promotes tumor formation in nude mice. However, the molecular mechanisms associated with FUS-CHOP as a putative transcription factor, and its ability to promote invasion and metastasis have never been investigated in detail. The present project was conducted to determine the role of FUS-CHOP in mediating invasion and metastasis, and to define potential target genes and molecular mediators. We found that stable overexpression of FUS-CHOP-I protein in SW872 liposarcoma and HT1080 fibrosarcoma cells leads to increased migration and invasion in matrigel assays. These results were supported by cell proliferation assays, showing no significant difference between FUS-CHOP stably over-expressing cells compared with mock-transfected cells. Based on our finding from microarray data gained from 3 resected liposarcoma patients, we investigated the expression of MMPs and observed that MMP-2, 9, and 7 mRNA were significantly up regulated (>3 fold), in both FUS-CHOP stably overexpressing cell lines. Comparing FUS-CHOP positive (n=14) and negative (n=17) liposarcoma patients, we confirmed MMP-2 expression to be significantly higher (p=0.002) in FUS-CHOP positive samples. Luciferase experiments using the basal promoter regions of MMP-2, −7, and −9 demonstrated that an overexpression of FUS-CHOP enhances the promoter activity of all 3 genes, with MMP-2 showing the highest inducibility. Similarly a 4X-AP1 Luc-reporter construct in both cell lines showed a more than 2-fold induction of reporter activity, indicating that FUS-CHOP is able to induce AP1-related transactivation. In contrast, a 6X-NFkB construct showed decreased reporter activity. In addition, chromatin-immunoprecipitation (ChIP) assays revealed an in vivo binding of FUS-CHOP to AP1 and C-EBP-β sites within the MMP-2 promoter after FUS-CHOP transfection. Finally, in vivo chicken embryo metastasis assays (CAM) showed a significantly increased metastasis to lungs and liver of the embryos in FUS-CHOP overexpressing liposarcoma cell lines. Furthermore, primary tumours excised from the upper CAM of this model showed an increase in MMP-2,−7 and −9 expression for both cell lines used. No significant increase in tumor weight was measured, suggesting that the ability of FUS-CHOP to induce metastasis is proliferation-independent. Taken together, these results for the first time suggest that FUS-CHOP overexpression leads to increased migration, invasion and metastasis of liposarcoma and fibrosarcoma cells, in part due to the transcriptional regulation of MMPs. Ongoing studies in our laboratory will elucidate more precise molecular mechanisms related to FUS-CHOP as a transcription factor. 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 LB-358. doi:10.1158/1538-7445.AM2011-LB-358

In 20% – 30% of all glioblastomas (GBMs), the most common and deadly form of glioma, gene rearrangements in amplified epidermal growth factor receptor (EGFR) cause amplification of a highly oncogenic variant of wild-type EGFR called EGFRvIII. A number of studies demonstrate that EGFRvIII amplification is detected predominantly in GBMs harboring wild-type EGFR amplification or those expressing the wild-type isoform of the tumor suppressor p53. However, it remains unclear why such genetic interactions occur or whether they are advantageous for tumor growth. Our limited understanding of these genetic interactions likely stems from an inability to study the function of amplified EGFRvIII in vitro using currently available model systems. Indeed, there are no immortalized GBM cell lines that express EGFRvIII because prolonged in vitro culture of EGFRvIII-positive GBM tumor cells invariably selects against EGFRvIII amplification. Moreover, the commonly used ectopic EGFRvIII expression models are unlikely to fully recapitulate the function, regulation, and downstream signaling activities of amplified EGFRvIII. Herein, we validate the use of short-term cell cultures derived from GBM xenografts expressing amplified EGFRvIII to study the endogenous function of amplified EGFRvIII. These cells exhibit competency in EGFR signaling, and maintain EGFRvIII expression for up to five months. Further characterization of these cells using small molecule inhibitor and short hairpin RNA approaches reveals three novel observations. First, loss of EGFRvIII expression and activity results in growth arrest and cellular senescence, indicating that EGFRvIII activity is required for cell growth. Second, specific depletion of wild-type EGFR significantly decreases EGFRvIII phosphorylation, demonstrating that high-level wild-type EGFR expression is required for maximal EGFRvIII activation. Third, p53 depletion inhibits growth arrest induced by loss of EGFRvIII activity, strongly suggesting that the growth-promoting activity of EGFRvIII is strictly dependent on p53 function. By showing that EGFRvIII functionally interacts with wild-type EGFR and wild-type p53 in a manner that promotes cell growth, these data provide a functional basis for the commonly observed genetic interactions between amplified EGFRvIII and amplified wild-type EGFR or wild-type p53. Furthermore, our data imply that with help from amplified wild-type EGFR, amplified EGFRvIII functions specifically to overcome the growth inhibitory activity of wild-type p53 in order to promote cell growth. Consistent with this insight, we provide preliminary evidence for the efficacy of a combination therapy involving EGFR and MDM2 inhibitors, which permits concomitant inhibition of EGFR signaling and potentiation of p53 function. 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 4417. doi:10.1158/1538-7445.AM2011-4417

Glioblastoma multiforme (GBM) is the most frequent and malignant human brain tumor. It is widely accepted that human cancer is a genetic disease caused by sequential accumulation of cancer gene mutations. Specifically, the gene or genes which initiate GBM formation has yet to be identified. Through collaboration with the Pakistani National Genetic Institute we have identified a consanguineous family presenting with a history of Pediatric GBMs to conduct comprehensive genomic analysis in hopes of identifying this “gatekeeper” gene. In this pedigree four children are deceased from GBM while an additional five infants have died within 4 days of birth without diagnosis. GBM has been the only diagnosed cancer in this family, indicating the existence of a GBM-specific germline mutation as a causative agent for the GBM clinical phenotype. Comprehensive genomic analysis has been conducted to elucidate a genetic aberration which is inherited and can be attributed to the GBM presenting clinical phenotype. We have collected blood and tumor DNAs for this family and performed sequencing on numerous genes including TP53, APC, IDH1, IDH2, PMS2, MLH1, MSH2, and, MSH6. Sequencing has successfully identified a germline frame shift mutation (p.Y181X) in PMS2. All affected patients have a homozygous mutation while their parents are both heterozygous carriers, suggesting the homozygous inheritance of this mutation is a causative agent for the GBM clinical phenotype. Our study suggests that the PMS2 inherited homozygous mutation could be a critical event in GBM formation. 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 1846.