An Assessment of Glioblastoma Metabolism Reveals Pathway-Specific Targets for Therapy

Glioblastoma (GBM) remains the most common and lethal primary brain tumor in adults despite concerted efforts to establish more effective treatments. The oncogenic-associated alterations that make GBM cells metabolically distinct from surrounding tissue also represent prime targets for the development of novel therapies. Due to the interconnectivity of signaling networks and the overall heterogeneity of the disease, identifying key metabolic pathways that drive neoplastic pathogenesis is essential to establishing more effective therapeutic strategies. In this dissertation, we performed expression analysis and unbiased metabolomics to better characterize metabolic differences between a cohort of patient-derived isocitrate dehydrogenase 1 (IDH1) mutant and wildtype gliomasphere cultures. This analysis revealed clear, cell type-specific differences in glucose metabolism, nucleotide synthesis utilization, and DNA repair capacity following radiation that could be exploited for therapy. Furthermore, we investigated the ability of GBM cells to oxidize fatty acids (FAO) and ketone bodies to support tumor growth, while also interrogating the effectiveness of the ketogenic diet (KD) as an adjuvant therapy for GBM. We discovered extensive FAO utilization throughout the GBM metabolome, identified CPT1A as a potential therapeutic target, and determined that the KD can have adverse effects on tumor growth

Maternal Inflammation Contributes to Brain Overgrowth and Autism-Associated Behaviors through Altered Redox Signaling in Stem and Progenitor Cells

A period of mild brain overgrowth with an unknown etiology has been identified as one of the most common phenotypes in autism. Here, we test the hypothesis that maternal inflammation during critical periods of embryonic development can cause brain overgrowth and autism-associated behaviors as a result of altered neural stem cell function. Pregnant mice treated with low-dose lipopolysaccharide at embryonic day 9 had offspring with brain overgrowth, with a more pronounced effect in PTEN heterozygotes. Exposure to maternal inflammation also enhanced NADPH oxidase (NOX)-PI3K pathway signaling, stimulated the hyperproliferation of neural stem and progenitor cells, increased forebrain microglia, and produced abnormal autism-associated behaviors in affected pups. Our evidence supports the idea that a prenatal neuroinflammatory dysregulation in neural stem cell redox signaling can act in concert with underlying genetic susceptibilities to affect cellular responses to environmentally altered cellular levels of reactive oxygen species

Maternal inflammation contributes to brain overgrowth and autism-associated behaviors through altered redox signaling in stem and progenitor cells.

A period of mild brain overgrowth with an unknown etiology has been identified as one of the most common phenotypes in autism. Here, we test the hypothesis that maternal inflammation during critical periods of embryonic development can cause brain overgrowth and autism-associated behaviors as a result of altered neural stem cell function. Pregnant mice treated with low-dose lipopolysaccharide at embryonic day 9 had offspring with brain overgrowth, with a more pronounced effect in PTEN heterozygotes. Exposure to maternal inflammation also enhanced NADPH oxidase (NOX)-PI3K pathway signaling, stimulated the hyperproliferation of neural stem and progenitor cells, increased forebrain microglia, and produced abnormal autism-associated behaviors in affected pups. Our evidence supports the idea that a prenatal neuroinflammatory dysregulation in neural stem cell redox signaling can act in concert with underlying genetic susceptibilities to affect cellular responses to environmentally altered cellular levels of reactive oxygen species

BET Bromodomain Degradation Disrupts Function but Not 3D Formation of RNA Pol2 Clusters

Fusion-positive rhabdomyosarcoma (FP-RMS) is driven by a translocation that creates the chimeric transcription factor PAX3-FOXO1 (P3F), which assembles de novo super enhancers to drive high levels of transcription of other core regulatory transcription factors (CRTFs). P3F recruits co-regulatory factors to super enhancers such as BRD4, which recognizes acetylated lysines via BET bromodomains. In this study, we demonstrate that inhibition or degradation of BRD4 leads to global decreases in transcription, and selective downregulation of CRTFs. We also show that the BRD4 degrader ARV-771 halts transcription while preserving RNA Polymerase II (Pol2) loops between super enhancers and their target genes, and causes the removal of Pol2 only past the transcriptional end site of CRTF genes, suggesting a novel effect of BRD4 on Pol2 looping. We finally test the most potent molecule, inhibitor BMS-986158, in an orthotopic PDX mouse model of FP-RMS with additional high-risk mutations, and find that it is well tolerated in vivo and leads to an average decrease in tumor size. This effort represents a partnership with an FP-RMS patient and family advocates to make preclinical data rapidly accessible to the family, and to generate data to inform future patients who develop this disease

Large-scale assessment of the gliomasphere model system.

BackgroundGliomasphere cultures are widely utilized for the study of glioblastoma (GBM). However, this model system is not well characterized, and the utility of current classification methods is not clear.MethodsWe used 71 gliomasphere cultures from 68 individuals. Using gene expression-based classification, we performed unsupervised clustering and associated gene expression with gliomasphere phenotypes and patient survival.ResultsSome aspects of the gene expression-based classification method were robust because the gliomasphere cultures retained their classification over many passages, and IDH1 mutant gliomaspheres were all proneural. While gene expression of a subset of gliomasphere cultures was more like the parent tumor than any other tumor, gliomaspheres did not always harbor the same classification as their parent tumor. Classification was not associated with whether a sphere culture was derived from primary or recurrent GBM or associated with the presence of EGFR amplification or rearrangement. Unsupervised clustering of gliomasphere gene expression distinguished 2 general categories (mesenchymal and nonmesenchymal), while multidimensional scaling distinguished 3 main groups and a fourth minor group. Unbiased approaches revealed that PI3Kinase, protein kinase A, mTOR, ERK, Integrin, and beta-catenin pathways were associated with in vitro measures of proliferation and sphere formation. Associating gene expression with gliomasphere phenotypes and patient outcome, we identified genes not previously associated with GBM: PTGR1, which suppresses proliferation, and EFEMP2 and LGALS8, which promote cell proliferation.ConclusionsThis comprehensive assessment reveals advantages and limitations of using gliomaspheres to model GBM biology, and provides a novel strategy for selecting genes for future study

Large-scale assessment of the gliomasphere model system

BACKGROUND: Gliomasphere cultures are widely utilized for the study of glioblastoma (GBM). However, this model system is not well characterized, and the utility of current classification methods is not clear. METHODS: We used 71 gliomasphere cultures from 68 individuals. Using gene expression-based classification, we performed unsupervised clustering and associated gene expression with gliomasphere phenotypes and patient survival. RESULTS: Some aspects of the gene expression-based classification method were robust because the gliomasphere cultures retained their classification over many passages, and IDH1 mutant gliomaspheres were all proneural. While gene expression of a subset of gliomasphere cultures was more like the parent tumor than any other tumor, gliomaspheres did not always harbor the same classification as their parent tumor. Classification was not associated with whether a sphere culture was derived from primary or recurrent GBM or associated with the presence of EGFR amplification or rearrangement. Unsupervised clustering of gliomasphere gene expression distinguished 2 general categories (mesenchymal and nonmesenchymal), while multidimensional scaling distinguished 3 main groups and a fourth minor group. Unbiased approaches revealed that PI3Kinase, protein kinase A, mTOR, ERK, Integrin, and beta-catenin pathways were associated with in vitro measures of proliferation and sphere formation. Associating gene expression with gliomasphere phenotypes and patient outcome, we identified genes not previously associated with GBM: PTGR1, which suppresses proliferation, and EFEMP2 and LGALS8, which promote cell proliferation. CONCLUSIONS: This comprehensive assessment reveals advantages and limitations of using gliomaspheres to model GBM biology, and provides a novel strategy for selecting genes for future study