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Background Glioblastoma (GBM) is the most common primary brain tumor in adults with a median survival of approximately 15 months; therefore, more effective treatment options for GBM are required. To identify new drugs targeting GBMs, we performed a high-throughput drug screen using patient-derived neurospheres cultured to preferentially retain their glioblastoma stem cell (GSC) phenotype. Methods High-throughput drug screening was performed on GSCs followed by a dose-response assay of the 5 identified original “hits.” A PI3K/mTOR dependency to a proteasome inhibitor (carfilzomib), was confirmed by genetic and pharmacologic experiments. Proteasome Inhibition Response Signatures were derived from proteomic and bioinformatic analysis. Molecular mechanism of action was determined using three-dimensional (3D) GBM-organoids and preclinical orthotopic models. Results We found that GSCs were highly sensitive to proteasome inhibition due to an underlying dependency on an increased protein synthesis rate, and loss of autophagy, associated with PTEN loss and activation of the PI3K/mTOR pathway. In contrast, combinatory inhibition of autophagy and the proteasome resulted in enhanced cytotoxicity specifically in GSCs that did express PTEN. Finally, proteasome inhibition specifically increased cell death markers in 3D GBM-organoids, suppressed tumor growth, and increased survival of mice orthotopically engrafted with GSCs. As perturbations of the PI3K/mTOR pathway occur in nearly 50% of GBMs, these findings suggest that a significant fraction of these tumors could be vulnerable to proteasome inhibition. Conclusions Proteasome inhibition is a potential synthetic lethal therapeutic strategy for GBM with proteasome addiction due to a high protein synthesis rate and autophagy deficiency.

Neddylation is a specific pathway within the ubiquitin/proteasome system that is overactive in GBM, and whose upregulation has been associated with glioma progression and worse survival. Pevonedistat (MLN4924) is a first-in-class small-molecule neddylation inhibitor shown to inhibit growth of GBM cells by impacting protein degradation in culture and orthotopic xenografts. However, the determinants of vulnerability are not fully understood. Because the molecular heterogeneity within and across GBM patients obscures therapeutic targets and obfuscates signals of efficacy in clinical trials, we pursue the use of molecular “signatures of vulnerability” to targeted agents in subsets of preclinical models. Selective vulnerability to pevonedistat was shown in a subset of GBM; notably, models with mutations or copy number deletions of PTEN are associated with de novo resistance to pevonedistat. Time-course studies of sensitive and non-sensitive GBM cells using transcriptomics and proteomics/phosphoproteomics uncovered additional determinants of response to pevonedistat. Our results demonstrate that in GBM, resistance to pevonedistat is driven by reduced PTEN-chromatin binding (loss-of-function or lower expression) that is also independent of PTEN’s lipid phosphatase activity (i.e., PI3K/AKT signaling). Across 25 glioma cell lines, we found that PTEN signaling, DNA replication, and chromatin instability pathways are the most significant differentiators between pevonedistat sensitive vs. non-sensitive models. In GBM models with modest to low sensitivity to pevonedistat, TOP2A expression was elevated. Combination treatment with the TOP2A inhibitor, etoposide, proved synergistic with pevonedistat. We report that PTEN status both serves as a novel biomarker for GBM sensitivity to pevonedistat and reveals a synergistic vulnerability to TOP2A inhibitors in combination with pevonedistat. Paired use of GBM PDX models of varying sensitivity with drug development testing allows the advancement of a promising agent as well as a patient-enrollment “signature of vulnerability” likely to increase the likelihood of demonstrating therapeutic efficacy in early stage clinical trials.

Epidermal Growth Factor Receptor (EGFR) missense-mutations in glioblastoma (GBM) typically occur within the extracellular domain (ECD) and, to-date, have not shown a clinical impact. These ECD mutations, while not as common as wild-type amplification or EGFRvIII, do represent a distinct patient population in need of investigation. This study investigates the oncogenic effect of the most common EGFR ECD missense-mutations (R108K, A289D/T/V, and G598V) in a retrospective cohort of 260 de novo GBM patients from the University of Pennsylvania. Extrapolation of each individual missense-mutation revealed a significant reduction in patient survival for the EGFR(A289D/T/V) mutants (p=0.028). This effect was confirmed with a follow-up cohort of 111 patients. The Cancer Imaging Phenomics Toolkit (CaPTk – www.cbica.upenn.edu/captk) was used to extract 2104 quantitative imaging phenomic (QIP) features, from multiparametric magnetic resonance imaging, across the various tumor sub-regions comprising the enhancing and non-enhancing tumor core, as well as the peritumoral edematous/invaded tissue. Multivariate machine learning techniques integrated these features, revealing radiographic signatures suggestive of increased invasion and proliferation for patients bearing the A289 mutation. The underlying mechanism of EGFR(A289V) in tumor growth was examined using engineered U87 glioma cells and patient-derived HK281 glioma spheres simulating expression of wild-type-EGFR or EGFR(A289V). Corroborating our findings in patients, mice bearing intracranial tumors with EGFR(A289V) mutations revealed significantly worse survival accompanied by highly invasive tumors. Cells expressing EGFR(A289V) yielded a highly active EGFR/ERK signaling pathway, resulting in increased expression and functionality of the matrix metalloproteinase MMP1, which can be attenuated by the use of ERK pathway inhibitors. Collectively, the findings of this study highlight a highly invasive and proliferative phenotype associated with the EGFR(A289V) missense-mutation. Moreover, given the tumor-specific and extracellular nature of the mutation, we postulate that it may be amenable to targeted therapy. Key data presented is from a manuscript with preliminary acceptance to Cancer Cell. *equal contribution

Extensive neuropil invasion is a hallmark of glioma growth and a subset of tumors demonstrate recurrence in the contralateral hemisphere without a path of tumor spread detected by MR imaging. We used an integrated genomic and radiographic approach to study a patient with a right nonenhancing insular mass histologically diagnosed as IDH1/2 wild-type anaplastic astrocytoma (AA) and a punctate nonenhancing T2 hyperintense focus within the left middle frontal gyrus subcortical white matter. Follow-up MR imaging, six months after initiation of Temozolomide based chemoradiotherapy, demonstrated focal enhancement within the left middle frontal gyrus lesion. Six months later a rim enhancing centrally necrotic mass involving left frontal lobe white matter was observed and diagnosed as glioblastoma. Whole exome sequencing of both tumors and targeted Sanger sequencing of 7-9 samplings from each tumor identified a novel five amino acid in-frame insertion in the PTEN C2 domain in both tumors that potentially disrupts cell membrane interaction and activates phosphotidylinositol 3-kinase (PI3K) signaling. As wild-type PTEN inhibits cellular migration, this early PTEN mutation might also promote invasion and the seeding of metastatic foci remote from the initial tumor mass. The right insular AA harbored amplifications of EGFR, MDM4 and PDGFRA (amplified and mutated) that were not detected within the contralateral glioblastoma. Conversely, the glioblastoma acquired CDKN2A homozygous deletion, probable NF1 biallelic inactivation (mutation and heterozygous loss), and a second hit to PTEN (heterozygous loss). Thirty-six shared mutations (including PTEN) and two shared copy number alterations unequivocally indicate a common origin for these spatially and temporally separated tumors. Taken together, these integrated radiographic and genomic data strongly suggest that the cells seeding the left frontal lobe tumor migrated from the evolving right insular tumor at an early time point and then followed an independent evolutionary trajectory.

Glioblastoma (GBM) is the most lethal type of brain cancer, where deletions and/or mutations in the tumor suppressor gene PTEN (phosphatase and tensin homolog) are associated to responsiveness of cancer to radiotherapy and chemotherapy. Herein, we show that a PTEN-DAXX-H3.3 chromatin complex negatively regulates proliferation and tumor growth. PTEN directly regulates oncogene expression by coordinating DAXX and histone H3.3 deposition on chromatin and in turn, DAXX protects PTEN from acetylation and ubiquitination. Furthermore, DAXX disruption in PTEN-deficient glioblastoma patient derived xenograft models suppresses tumor growth and promotes therapy sensitivity. Since PTEN loss is a common alteration in cancer, DAXX inhibition presents an approach to ablate the growth of such cancers.