Glioblastoma Response Research Articles

Transcriptome analysis evinces anti-neoplastic mechanisms of hypericin: A study on U87 glioblastoma cell line

AimsHypericin (HYP) from Hypericum perforatum has cytotoxic effects on a variety of malignant cell types, but the pattern of gene expression mediating the effect is largely unknown. Here we sought to analyze the response of U87 glioblastoma (GBM) cell lines in response to HYP. Materials and methodsU87 cell line was treated by HYP. Cytotoxicity was assessed using MTT and Annexin V/PI assays. Gene expression profile was obtained using high-throughput sequencing. Enrichment analysis was performed on differentially expressed genes (DEGs). Upstream transcription factors and microRNAs regulating DEGs were predicted. The effects of DEGs on survival of GBM patients were calculated. Protein-protein interaction analysis was conducted to obtain key altered genes. The possible effect of HYP treatment on immunity response was evaluated. Key findingsThe IC50 of HYP on U87 cell line was determined to be 1.5 μg/ml. The main type of cell death was apoptosis. A total of 312 DEGs were found. Affected Gene Ontology terms and pathways were identified. Analysis of upstream modulators of DEGs pointed out to transcription factors that significantly overlap with GBM stem cell transcription factor. Survival analysis suggested that HYP works best for the mesenchymal subtype patients. Tumor infiltration analysis predicted that HYP may affect Treg and macrophage infiltration in vivo. Using expression pattern of GBM patients and HYP-induced DEGs we suggested Fedratinib as a complementary drug to HYP. SignificanceOur study represents the response of U87 cell line to HYP, with analyses on survival, transcription factors and personalization according to GBM subtype.

Quantitative CEST and MT at 1.5T for monitoring treatment response in glioblastoma: early and late tumor progression during chemoradiation.

Quantitative MRI (qMRI) was performed using a 1.5T protocol that includes a novel chemical exchange saturation transfer/magnetization transfer (CEST/MT) approach. The purpose of this prospective study was to determine if qMRI metrics at baseline, at the 10th and 20th fraction during a 30 fraction/6week standard chemoradiation (CRT) schedule, and at 1month following treatment could be an early indicator of response for glioblastoma (GBM). The study included 51 newly diagnosed GBM patients. Four regions-of-interest (ROI) were analyzed: (i) the radiation defined clinical target volume (CTV), (ii) radiation defined gross tumor volume (GTV), (iii) enhancing-tumor regions, and (iv) FLAIR-hyperintense regions. Quantitative CEST, MT, T1 and T2 parameters were compared between those patients progressing within 6.9months (early), and those progressing after CRT (late), using mixed modelling. Exploratory predictive modelling was performed to identify significant predictors of early progression using a multivariable LASSO model. Results were dependent on the specific tumor ROI analyzed and the imaging time point. The baseline CEST asymmetry within the CTV was significantly higher in the early progression cohort. Other significant predictors included the T2 of the MT pools (for semi-solid at fraction 20 and water at 1month after CRT), the exchange rate (at fraction 20) and the MGMT methylation status. We observe the potential for multiparametric qMRI, including a novel pulsed CEST/MT approach, to show potential in distinguishing early from late progression GBM cohorts. Ultimately, the goal is to personalize therapeutic decisions and treatment adaptation based on non-invasive imaging-based biomarkers.

IMMU-27. SINGLE CELL RNA-SEQUENCING IDENTIFIES NOVEL BONE MARROW DERIVED MYELOID CELLS IN GLIOBLASTOMA ASSOCIATED WITH TUMOR AGGRESSION

Abstract BACKGROUND Glioma associated myeloid cells (GAMs) can be induced to adopt an immunosuppressive phenotype that can lead to inhibition of anti-tumor responses in glioblastoma (GBM). Understanding the composition and phenotypes of GAMs is essential to modulating the myeloid compartment as a therapeutic adjunct to improve anti-tumor immune response. METHODS We performed single-cell RNA-sequencing (sc-RNAseq) of 435,400 myeloid and tumor cells to identify transcriptomic and phenotypic differences in GAMs across glioma grades. We further correlated the heterogeneity of the GAM landscape with tumor cell transcriptomics to investigate interactions between GAMs and tumor cells. RESULTS sc-RNAseq revealed a diverse landscape of myeloid-lineage cells in gliomas with an increase in preponderance of bone marrow derived myeloid cells (BMDMs) with increasing tumor grade. We identified two populations of BMDMs unique to GBMs; Mac-1and Mac-2. Mac-1 demonstrates upregulation of immature myeloid gene signature and altered metabolic pathways. Mac-2 is characterized by expression of scavenger receptor MARCO. Pseudotime and RNA velocity analysis revealed the ability of Mac-1 to transition and differentiate to Mac-2 and other GAM subtypes. We further found that the presence of these two populations of BMDMs are associated with the presence of tumor cells with stem cell and mesenchymal features. Bulk RNA-sequencing data demonstrates that gene signatures of these populations are associated with worse survival in GBM. CONCLUSION We used sc-RNAseq to identify a novel population of immature BMDMs that is associated with higher glioma grades. This population exhibited altered metabolic pathways and stem-like potentials to differentiate into other GAM populations including GAMs with upregulation of immunosuppressive pathways. Our results elucidate unique interactions between BMDMs and GBM tumor cells that potentially drives GBM progression and the more aggressive mesenchymal subtype. Our discovery of these novel BMDMs have implications in new therapeutic targets in improving the efficacy of immune-based therapies in GBM.

TMOD-17. ONCOLYTIC POLIOVIRUS AS A PROBE FOR MECHANISMS OF IMMUNE RESISTANCE IN GLIOBLASTOMA

Abstract Glioblastoma is an immunologically cold tumor that employs multiple resistance mechanisms to escape activation of both innate and adaptive immunity. Transient immune activation and responses induced by immunotherapies in glioblastoma are almost always suppressed by potent intrinsic and extrinsic resistance mechanisms, limiting therapeutic efficacy. PVSRIPO is a recombinant poliovirus/rhinovirus chimera that elicits robust innate immune activation through infecting and lysing glioblastoma cells expressing the human poliovirus receptor (CD155), and through sublethal infection of tumor associated macrophages, setting the stage for lingering viral replication. We have constructed a pathological model using murine CT2ACD155 intracranial tumors stereotactically infused with a single dose of PVSRIPO (5×107 pfu) or mock control to study the response of glioblastoma in an immune competent in vivo tumor microenvironment. Tumor bearing brains were harvested before therapy and on days 2, 4, 6, 9, 12 post therapy for histology. On day 4 post therapy, histopathology showed > 90% shrinkage of intracranial tumor diameter (n= 6/6). Residual tumor is surrounded by a necrotic rim on histology with profound infiltration of F4/80+ macrophages and substantially reduced expression of Ki-67 on IHC compared to controls. Other markers including CD3, CD8 and Cleaved-Caspase 3 showed no difference in tumors of both groups. Interestingly, most residual tumor progressed on day 6 post therapy and caught up with the size of control tumor by day 9. Long-term remission of tumor occurred in only 10% of mice. This model is valuable in studying the mechanisms of intrinsic resistance and the timeline of resistance to develop in order to obtain more durable immunotherapy responses in glioblastoma. Future steps are to track inflammation induced by viral infection by RNAseq, and to find potential therapeutic interventions (including blocking PD1:PD-L1 axis) to mitigate therapy resistance in this preclinical model.

CTNI-34. INITIAL ANALYSIS OF PHASE-III TRIAL - STANDARD CHEMOTHERAPY VS. CHEMOTHERAPY GUIDED BY A CANCER STEM CELL TEST IN RECURRENT GLIOBLASTOMA (NCT03632135)

Abstract BACKGROUND ChemoID is a cancer stem cell (CSC) cytotoxicity assay for guiding personalized treatment in the clinical trial NCT03632135 using standard-of-care drugs for improving clinical outcomes. The ChemoID assay is a functional test that determines the response to chemotherapy treatments from a panel of FDA approved drugs or their combinations. The trial aims to determine the clinical utility of the ChemoID assay as a predictor of clinical response in recurrent glioblastoma (GBM). METHODS The study has been designed as a parallel group controlled clinical trial where participants with recurrent GBM amenable to surgery or biopsy are randomized at a ratio of 1:1 to either standard-of-care chemotherapy chosen by the physician or ChemoID-guided therapy. Response to therapy is measured by MRI imaging (RANO 1.1). The primary endpoint is median overall survival (OS) and secondary endpoints are OS at 6, 9, and 12 months, median progression-free survival (PFS), PFS at 4, 6, 9, and 12 months, objective tumor response, time to recurrence, and quality of life. RESULTS A total of 41 participants (29 males, 12 females) have been accrued to the trial thus far with a median age of 61yo. Data from 38 participants (27 males, 11 females) has reached maturity for analysis. 21 participants have been randomized to the assay-guided arm and 17 to the physician-choice arm. From an initial analysis, we observed that 71% (15/21) of the participants in the ChemoID-guided arm are alive and 29% (6/21) are deceased. We also observed that 41% (7/17) of the participants in the physician-choice arm are alive and 59% (10/17) are deceased. This gives the odds of death for the ChemoID-guided arm to be 72% lower than on the physician-guided arm (OR=0.28; (0.07–1.08); p=0.065). CONCLUSIONS Our preliminary results indicate that the ChemoID assay has the potential to improve survival of recurrent GBM patients.

IMMU-17. SINGLE-CELL RNA-SEQ REVEALS HIGH-GRADE GLIOMA ASSOCIATED MICROGLIA WITH PROINFLAMMATORY AND STEM-LIKE FEATURES SUPPORT TUMOR PROGRESSION

Abstract Poor response of human glioblastoma to current therapies are influenced by tumor microenvironment. Although glioblastoma is recognized by large enrichment of microglia, characterization of diverse cell subsets and their functions remain challenging because of high heterogenicity. Here, we analyzed single-cell transcriptomics to comprehensively map the cell populations and determine the roles of microglia in IDH1/2 wild-type (IDH-wt) glioblastoma progression. Besides finding microglia were significantly enriched in IDH-wt glioblastoma compared to IDH1/2 mutant (IDH-mut) gliomas, we identified a unique high-grade glioma microglia (HGAM) subtype characterized by proinflammatory and stem-like features. In particular, HGAM’s pro-tumoral IL1β secretion is mediated via ApoE-induced activation of NLRP1 inflammasome. HGAM phagocytosed OPC-like malignant cells forming the neoplastic microglia, which presented the stem-like potential giving rise to activated microglia. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation. Additionally, an intricated evaluation of glioma patients revealed that SETD2 mutation/low-expression correlated with adverse prognosis. Further analysis showed that SETD2 -dificient tumor cells presented hypersensitivity to HGAM-derived IL1β via epigenetic dysregulation of PHF6. Also, SETD2 -deficient tumor cells produced TGF-β1 contributing to microglia activation. Finally, targeting the TGF-β1/TβRI signaling impaired HGAM activation and tumor growth. Our studies identify a unique neoplastic microglia subpopulation and establish cellular basis of interactions with tumor cells important for disease progression.

BIOM-22. EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) AS A MOLECULAR DETERMINANT OF GLIOBLASTOMA RESPONSE TO DOPAMINE RECEPTOR 2 (DRD2) INHIBITORS

Abstract BACKGROUND There are ongoing clinical trials exploring the efficacy of dopamine receptor 2 (DRD2) inhibition against glioblastomas, the most common primary brain tumor. Here we examine potential molecular determinants of this efficacy. METHODS The Cancer Genome Atlas (TCGA) glioblastoma database and other published mRNA profiles were used to analyze the DRD2 and EGFR expression pattern. In vitro and in vivo responses to DRD2 inhibitors were determined using patient derived xenograft (PDX) glioblastoma models. Immunohistochemical studies were performed on clinically annotated glioblastoma samples derived from patients treated with ONC201, a DRD2 inhibitor. RESULTS Analysis of clinical glioblastoma specimens derived from independent patient cohorts revealed an inverse correlation between EGFR and DRD mRNA expression, with implication that signaling mediated by these proteins shares overlapping functions. In independent panels of PDX glioblastoma lines, high EGFR expression was associated with poor in vitro and in vivo response to DRD2 inhibitors, including haloperidol and ONC201. Moreover, ectopic expression of a constitutively active EGFR, EGFRvIII, suppressed glioblastoma sensitivity to ONC201. DRD2 expression positively correlated with expression of rate-limiting enzymes for dopamine synthesis as well as dopamine secretion, suggesting contribution of autocrine DRD2 signaling. Analysis of specimens from patients treated with ONC201 (n = 15) showed an inverse correlation between the intensity of EGFR staining and clinical response. The median overall survival for patients with high and low EGFR staining was 162 and 373 days, respectively (p = 0.037). CONCLUSIONS High EGFR expression is a determinant of poor glioblastoma response to DRD2. This finding should inform future clinical trial designs.

Improving Radiation Response in Glioblastoma Using ECO/siRNA Nanoparticles Targeting DNA Damage Repair.

Simple SummaryGlioblastoma (GBM) is the most common form of brain cancer and among the most lethal of human cancers. Radiation therapy is a mainstay in the standard of care for GBM, killing tumor cells by creating DNA damage. Inhibiting DNA damage repair (DDR) proteins enhances radiation therapy by not allowing tumor cells to repair the DNA damage caused by radiation. The aim of our study was to investigate whether the novel nanoparticle material, ECO, could be used to deliver small interfering RNA (siRNA) to GBM tumor cells and temporarily reduce the production of DDR proteins to improve radiation therapy outcomes. SiRNAs can be designed to target an innumerable number of genes and with the right delivery vehicle can be used in a variety of disease settings. Our work provides support for the use of the novel ECO material for delivery of siRNA in GBM.Radiation therapy is a mainstay in the standard of care for glioblastoma (GBM), thus inhibiting the DNA damage response (DDR) is a major strategy to improve radiation response and therapeutic outcomes. Small interfering RNA (siRNA) therapy holds immeasurable potential for the treatment of GBM, however delivery of the siRNA payload remains the largest obstacle for clinical implementation. Here we demonstrate the effectiveness of the novel nanomaterial, ECO (1-aminoethylimino[bis(N-oleoylcysteinylaminoethyl) propionamide]), to deliver siRNA targeting DDR proteins ataxia telangiectasia mutated and DNA-dependent protein kinase (DNApk-cs) for the radiosensitzation of GBM in vitro and in vivo. ECO nanoparticles (NPs) were shown to efficiently deliver siRNA and silence target protein expression in glioma (U251) and glioma stem cell lines (NSC11, GBMJ1). Importantly, ECO NPs displayed no cytotoxicity and minimal silencing of genes in normal astrocytes. Treatment with ECO/siRNA NPs and radiation resulted in the prolonged presence of γH2AX foci, indicators of DNA damage, and increased radiosensitivity in all tumor cell lines. In vivo, intratumoral injection of ECO/siDNApk-cs NPs with radiation resulted in a significant increase in survival compared with injection of NPs alone. These data suggest the ECO nanomaterial can effectively deliver siRNA to more selectively target and radiosensitize tumor cells to improve therapeutic outcomes in GBM.

PTRF/cavin-1 remodels phospholipid metabolism to promote tumor proliferation and suppress immune responses in glioblastoma by stabilizing cPLA2.

Metabolism remodeling is a hallmark of glioblastoma (GBM) that regulates tumor proliferation and the immune microenvironment. Previous studies have reported that increased polymerase 1 and transcript release factor (PTRF) levels are associated with a worse prognosis in glioma patients. However, the biological role and the molecular mechanism of PTRF in GBM metabolism remain unclear. The relationship between PTRF and lipid metabolism in GBM was detected by nontargeted metabolomics profiling and subsequent lipidomics analysis. Western blotting, quantitative real-time PCR, and immunoprecipitation were conducted to explore the molecular mechanism of PTRF in lipid metabolism. A sequence of in vitro and in vivo experiments (both xenograft tumor and intracranial tumor mouse models) were used to detect the tumor-specific impacts of PTRF. Here, we show that PTRF triggers a cytoplasmic phospholipase A2 (cPLA2)-mediated phospholipid remodeling pathway that promotes GBM tumor proliferation and suppresses tumor immune responses. Research in primary cell lines from GBM patients revealed that cells overexpressing PTRF show increased cPLA2 activity-resulting from increased protein stability-and exhibit remodeled phospholipid composition. Subsequent experiments revealed that PTRF overexpression alters the endocytosis capacity and energy metabolism of GBM cells. Finally, in GBM xenograft and intracranial tumor mouse models, we showed that inhibiting cPLA2 activity blocks tumor proliferation and prevents PTRF-induced reduction in CD8+ tumor-infiltrating lymphocytes. The PTRF-cPLA2 lipid remodeling pathway promotes tumor proliferation and suppresses immune responses in GBM. In addition, our findings highlight multiple new therapeutic targets for GBM.

Machine Learning Based on a Multiparametric and Multiregional Radiomics Signature Predicts Radiotherapeutic Response in Patients with Glioblastoma.

Methods The MRI images, genetic data, and clinical data of 152 patients with GBM were analyzed. 122 patients from the TCIA dataset (training set: n = 82; validation set: n = 40) and 30 patients from local hospitals were used as an independent test dataset. Radiomics features were extracted from multiple regions of multiparameter MRI. Kaplan-Meier survival analysis was used to verify the ability of the imaging signature to predict the response of GBM patients to radiotherapy before an operation. Multivariate Cox regression including radiomics signature and preoperative clinical risk factors was used to further improve the ability to predict the overall survival (OS) of individual GBM patients, which was presented in the form of a nomogram. Results The radiomics signature was built by eight selected features. The C-index of the radiomics signature in the TCIA and independent test cohorts was 0.703 (P < 0.001) and 0.757 (P = 0.001), respectively. Multivariate Cox regression analysis confirmed that the radiomics signature (HR: 0.290, P < 0.001), age (HR: 1.023, P = 0.01), and KPS (HR: 0.968, P < 0.001) were independent risk factors for OS in GBM patients before surgery. When the radiomics signature and preoperative clinical risk factors were combined, the radiomics nomogram further improved the performance of OS prediction in individual patients (C‐index = 0.764 and 0.758 in the TCIA and test cohorts, respectively). Conclusion This study developed a radiomics signature that can predict the response of individual GBM patients to radiotherapy and may be a new supplement for precise GBM radiotherapy.

Sexually dimorphic radiogenomic models identify distinct imaging and biological pathways that are prognostic of overall survival in glioblastoma.

Recent epidemiological studies have suggested that sexual dimorphism influences treatment response and prognostic outcome in glioblastoma (GBM). To this end, we sought to (i) identify distinct sex-specific radiomic phenotypes-from tumor subcompartments (peritumoral edema, enhancing tumor, and necrotic core) using pretreatment MRI scans-that are prognostic of overall survival (OS) in GBMs, and (ii) investigate radiogenomic associations of the MRI-based phenotypes with corresponding transcriptomic data, to identify the signaling pathways that drive sex-specific tumor biology and treatment response in GBM. In a retrospective setting, 313 GBM patients (male = 196, female = 117) were curated from multiple institutions for radiomic analysis, where 130 were used for training and independently validated on a cohort of 183 patients. For the radiogenomic analysis, 147 GBM patients (male = 94, female = 53) were used, with 125 patients in training and 22 cases for independent validation. Cox regression models of radiomic features from gadolinium T1-weighted MRI allowed for developing more precise prognostic models, when trained separately on male and female cohorts. Our radiogenomic analysis revealed higher expression of Laws energy features that capture spots and ripple-like patterns (representative of increased heterogeneity) from the enhancing tumor region, as well as aggressive biological processes of cell adhesion and angiogenesis to be more enriched in the "high-risk" group of poor OS in the male population. In contrast, higher expressions of Laws energy features (which detect levels and edges) from the necrotic core with significant involvement of immune related signaling pathways was observed in the "low-risk" group of the female population. Sexually dimorphic radiogenomic models could help risk-stratify GBM patients for personalized treatment decisions.

A novel hypoxic tumor microenvironment signature for predicting the survival, progression, immune responsiveness and chemoresistance of glioblastoma: a multi-omic study.

The hypoxic tumor microenvironment (TME) was reported to promote the aggressive phenotype, progression, recurrence, and chemoresistance of glioblastoma (GBM). We developed and validated a hypoxia gene signature for individualized prognostic prediction in GBM patients. In total, 259 GBM-specific hypoxia-related genes (HRGs) were obtained in hypoxic cultured GBM cells compared with normoxic cells. By applying the k-means algorithm, TCGA GBM patients were divided into two subgroups, and the patients in Cluster 1 exhibited high HRG expression patterns, older age, and poor prognosis, which was validated in the CGGA cohort. Cox regression analyses were performed to generate an HRG-based risk score model consisting of five HRGs, which could reliably discriminate the overall survival (OS) and progression-free survival (PFS) of high- and low-risk patients in both the TCGA training and CGGA validation cohorts. Then, nomograms with the hypoxia signature for OS and PFS prediction were constructed for individualized survival prediction, better treatment decision-making, and follow-up scheduling. Finally, functional enrichment, immune infiltration, immunotherapy response prediction and chemotherapy resistance analyses demonstrated the vital roles of the hypoxic TME in the development, progression, multitherpy resistance of GBM. The hypoxia gene signature could serve as a promising prognostic predictor and potential therapeutic target to combat chemoresistant GBM.

Investigating Glioblastoma Response to Hypoxia.

Glioblastoma (GB) is the most common and deadly type of primary malignant brain tumor with an average patient survival of only 15–17 months. GBs typically have hypoxic regions associated with aggressiveness and chemoresistance. Using patient derived GB cells, we characterized how GB responds to hypoxia. We noted a hypoxia-dependent glycolytic switch characterized by the up-regulation of HK2, PFKFB3, PFKFB4, LDHA, PDK1, SLC2A1/GLUT-1, CA9/CAIX, and SLC16A3/MCT-4. Moreover, many proangiogenic genes and proteins, including VEGFA, VEGFC, VEGFD, PGF/PlGF, ADM, ANGPTL4, and SERPINE1/PAI-1 were up-regulated during hypoxia. We detected the hypoxic induction of invasion proteins, including the plasminogen receptor, S100A10, and the urokinase plasminogen activator receptor, uPAR. Furthermore, we observed a hypoxia-dependent up-regulation of the autophagy genes, BNIP-3 and DDIT4 and of the multi-functional protein, NDRG1 associated with GB chemoresistance; and down-regulation of EGR1 and TFRC (Graphical abstract). Analysis of GB patient cohorts’ revealed differential expression of these genes in patient samples (except SLC16A3) compared to non-neoplastic brain tissue. High expression of SLC2A1, LDHA, PDK1, PFKFB4, HK2, VEGFA, SERPINE1, TFRC, and ADM was associated with significantly lower overall survival. Together these data provide important information regarding GB response to hypoxia which could support the development of more effective treatments for GB patients.

Epidermal growth factor receptor as a molecular determinant of glioblastoma response to dopamine receptor D2 inhibitors.

There are ongoing clinical trials exploring the efficacy of dopamine receptor D2 (DRD2) inhibition against glioblastomas, the most common primary brain tumor. Here we examine potential molecular determinants of this efficacy. The Cancer Genome Atlas glioblastoma database and other published mRNA profiles were used to analyze the DRD2 and epidermal growth factor receptor (EGFR) expression pattern. In vitro and in vivo responses to DRD2 inhibitors were determined using patient-derived xenograft (PDX) glioblastoma models. Immunohistochemical studies were performed on clinically annotated glioblastoma samples derived from patients treated with ONC201. Analysis of clinical glioblastoma specimens derived from independent patient cohorts revealed an inverse correlation between EGFR and DRD2 mRNA expression, with implication that signaling mediated by these proteins shares overlapping functions. In independent panels of PDX glioblastoma lines, high EGFR expression was associated with poor in vitro and in vivo response to DRD2 inhibitors, including haloperidol and ONC201. Moreover, ectopic expression of a constitutively active EGFR, variant (v)III, suppressed glioblastoma sensitivity to ONC201. DRD2 expression positively correlated with expression of rate-limiting enzymes for dopamine synthesis as well as dopamine secretion, suggesting contribution of autocrine DRD2 signaling. Analysis of specimens from patients treated with ONC201 (n = 15) showed an inverse correlation between the intensity of EGFR staining and clinical response. The median overall survival for patients with high and low EGFR staining was 162 and 373 days, respectively (0.037). High EGFR expression is a determinant of poor glioblastoma response to DRD2. This finding should inform future clinical trial designs.

β-arrestin 1 transfection induced cell death in high grade glioma in vitro

ABSTRACT The best known functions of β-arrestins (β-arr) are to regulate G protein-coupled receptors (GPCR) signaling through receptor desensitization and internalization. Many reports also suggest that β-arrs play important role in immune regulation and inflammatory responses, under physiological and pathological conditions. Recent studies have shown that β-arr 1 silencing halts proliferation and increases temozolomide (TMZ) response in glioblastoma (GBM) cells. The focus of this paper is to analyze the role of β-arr 1 overexpression in the 18 high grade glioma (HGG) cell line in terms of viability and their response to TMZ treatment. For this reason, the cell line was transfected with β-arr 1 and the effect was analyzed after 24 h, 48 h and 72 h in terms of proliferation and treatment response. We observed that β-arr 1 overexpression induced a time and dose dependant inhibition in the HGG cells. Unexpectedly, β-arr transfection resulted in a very mild increase in TMZ toxicity after 24 h, becoming non-statistically significant at 72 h. In conclusion, we showed that β-arr 1 overexpression inhibits cell proliferation in the 18 cell line but only has a very modest effect on treatment response with the alkylating agent TMZ.

OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to ionizing radiation

Glioblastoma contains a rare population of self-renewing brain tumor stem cells (BTSCs) which are endowed with properties to proliferate, spur the growth of new tumors, and at the same time, evade ionizing radiation (IR) and chemotherapy. However, the drivers of BTSC resistance to therapy remain unknown. The cytokine receptor for oncostatin M (OSMR) regulates BTSC proliferation and glioblastoma tumorigenesis. Here, we report our discovery of a mitochondrial OSMR that confers resistance to IR via regulation of oxidative phosphorylation, independent of its role in cell proliferation. Mechanistically, OSMR is targeted to the mitochondrial matrix via the presequence translocase-associated motor complex components, mtHSP70 and TIM44. OSMR interacts with NADH ubiquinone oxidoreductase 1/2 (NDUFS1/2) of complex I and promotes mitochondrial respiration. Deletion of OSMR impairs spare respiratory capacity, increases reactive oxygen species, and sensitizes BTSCs to IR-induced cell death. Importantly, suppression of OSMR improves glioblastoma response to IR and prolongs lifespan.

Abstract 1249: SetD2 histone methyltransferase mutation status predicts treatment response in glioblastoma: Strategies to overcome chemoresistance

Abstract Purpose: Glioblastoma (GBM) is a highly aggressive primary brain tumor. A major challenge in GBM treatment is tumor resistance to radiation and chemotherapy. An important hallmark of GBM is the frequent mutation of epigenetic modifiers resulting in alteration of epigenetic signaling pathways. However, the effect of epigenetic signaling on chemotherapy response in GBM remains to be elucidated. SETD2 is a unique histone methyl transferase that facilitates H3K36 tri-methylation. Here, we unveil the role of SETD2 mutations that frequently occur in GBM in tumor resistance to temozolomide chemotherapy. Method: We have generated CRISPR/Cas9-mediated SETD2-KO GBM cell lines to study the role of SETD2 in temozolomide response. DNA methylome and whole exome sequencing data from the TCGA databank were analyzed to determine the MGMT (methyl-guanine methyl transferase) promotor methylation status and SETD2 mutations, respectively. H3K36me3 and MSH6 levels were determined in GBM patient samples by immunohistochemical staining. SETD2 mutation status was determined in GBM patient samples by targeted sequencing of the SETD2 gene using the Agilent SureSelect Target technique. Results: Our TCGA database analysis reveals that SETD2 mutations are associated with reduced overall survival in patients with methylated MGMT promotor who received temozolomide suggesting that mutation of SETD2 is a predictive marker for chemotherapy resistance in GBM. Consequently, we demonstrate that loss of SETD2 results in reduced H3K36me3 levels and a profound resistance to temozolomide in GBM cells. MGMT-deficient tumors can acquire chemoresistance due to disrupted mismatch repair (MMR), a DNA repair pathway that plays a critical role in converting primary temozolomide-induced DNA lesions into toxic DNA double-strand breaks. Strikingly, we found that SETD2 loss abrogates the expression of the MMR factor MSH6 indicating that chemoresistance in SETD2-deficient cells is due to disrupted MMR. Accordingly, low H3K36me3 levels in GBM tumor samples correlate with loss of MSH6 expression. Mechanistically, we show that SETD2 regulates MMR by promoting transcription of the MSH6 gene in GBM. Finally, we establish a strategy to re-sensitize SETD2-deficient GBM cells to temozolomide. Epigenetic modifiers have specific antagonists capable of reversing chromatin alterations induced by these modifiers. This provides a unique opportunity to restore chemotherapy response in SETD2-mutant GBM by targeting the antagonists of SETD2. We demonstrate that combined targeting of H3K36me3-specific histone de-methylases KDM4A and NO66 restores H3K36me3 levels along with MSH6 expression and sensitivity to temozolomide in SETD2-deficient GBM cells. Conclusions: Our findings establish SETD2 mutation as a novel molecular marker predictive of chemotherapy response in GBM and provide a framework for a novel approach to overcome chemotherapy resistance in this malignant brain tumor by targeting a specific epigenetic signaling pathway. Fig.1: Immunohistochemical staining of H3K36me3 and MSH6 in GBM tumor sections demonstrating that tumor areas with low H3K36me3 expression lack MSH6 expression. Citation Format: Nishanth Gabriel, Sonika Dahiya, Xiaowei Wang, Michael Goldstein. SetD2 histone methyltransferase mutation status predicts treatment response in glioblastoma: Strategies to overcome chemoresistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1249.

Dual MGMT inactivation by promoter hypermethylation and loss of the long arm of chromosome 10 in glioblastoma.

BackgroundEpigenetic inactivation of O6‐methylguanine‐methyltransferase (MGMT) gene by methylation of its promoter is predictive of Temozolomid (TMZ) response in glioblastoma (GBM). MGMT is located on chromosome 10q26 and the loss of chromosome 10q is observed in 70% of GBMs. In this study, we assessed the hypothesis that the dual inactivation of MGMT, by hypermethylation of MGMT promoter and by loss the long arm of chromosome 10 (10q), may confer greater sensitivity to TMZ.MethodsA total of 149 tumor samples from patients diagnosed with GBM based on the WHO 2016 classification were included in this retrospective study between November 2016 and December 2018. Methylation status of MGMT promoter was evaluated by pyrosequencing and status of chromosome 10q was assessed by array comparative genomic hybridization.ResultsGlioblastoma patients with chromosome 10q loss associated with hypermethylation of MGMT promoter had significantly longer overall survival (OS) (P = .0024) and progression‐free survival (PFS) (P = .031). Indeed, median OS of patients with dual inactivation of MGMT was 21.5 months compared to 12 months and 8.1 months for groups with single MGMT inactivation by hypermethylation and by 10q loss, respectively. The group with no MGMT inactivation had 9.5 months OS. Moreover, all long‐term survivors with persistent response to TMZ treatment (OS ≥ 30 months) displayed dual inactivation of MGMT.ConclusionsOur data suggest that the molecular subgroup characterized by the dual inactivation of MGMT receives greater benefit from TMZ treatment. The results of our study may be of immediate clinical interest since chromosome 10q status and methylation of MGMT promoter are commonly determined in routine practice.

Rapid Processing and Drug Evaluation in Glioblastoma Patient-Derived Organoid Models with 4D Bioprinted Arrays

SummaryGlioblastoma is the most common and deadly primary brain malignancy. Despite advances in precision medicine oncology (PMO) allowing the identification of molecular vulnerabilities in glioblastoma, treatment options remain limited, and molecular assays guided by genomic and expression profiling to inform patient enrollment in life-saving trials are lacking. Here, we generate four-dimensional (4D) cell-culture arrays for rapid assessment of drug responses in glioblastoma patient-derived models. The arrays are 3D printed with thermo-responsive shape memory polymer (SMP). Upon heating, the SMP arrays self-transform in time from 3D cell-culture inserts into histological cassettes. We assess the utility of these arrays with glioblastoma cells, gliospheres, and patient derived organoid-like (PDO) models and demonstrate their use with glioblastoma PDOs for assessing drug sensitivity, on-target activity, and synergy in drug combinations. When including genomic and drug testing assays, this platform is poised to offer rapid functional drug assessments for future selection of therapies in PMO.

A co-formulation of interferons type I and II enhances temozolomide response in glioblastoma with unmethylated MGMT promoter status.

Temozolomide (TMZ) is a chemotherapeutic used for the treatment of glioblastoma. The MGMT repair enzyme (O'-(6)-methyl guanine-DNA-methyltransferase) promoter methylation is a predictive biomarker to TMZ response; interferons (IFNs) type I can downregulate MGMT expression improving survival in patients with unmethylated MGMT promoter. HeberFERON is a co-formulation of IFNs type I and II with higher antiproliferative effect over glioblastoma cell lines than individual IFNs. We investigated the proliferative response of patient-derived glioblastoma cultures to HeberFERON and its combination with TMZ in relation to MGMT promoter methylation and the regulation of MGMT transcript after HeberFERON treatment. Eleven glioblastoma-derived cultures, molecularly classified according to TCGA and MGMT promoter methylation, were assayed for proliferation inhibition with HeberFERON at low doses (1-25 IU/mL) [alone or combined with TMZ] or at higher doses (50-200 IU/mL) using CellTiter-Glo Luminescent Cell Viability Assay (Promega). Eight cultures were further treated with 100 IU/mL of HeberFERON for 72 h, total RNA purified (Qiagen) and converted to cDNA (Superscript III kit, Invitrogen) as quantitative PCR templates. Changes of MGMT&P53 transcripts level were monitored. Response of cultures to HeberFERON is variable, dose-dependent and apparently independent from TCGA classification and MGMT methylation status, based on the eight Classical cultures data. When combining HeberFERON with TMZ there was an increase in cell death for cultures, 2/4 with methylated and 5/5 with unmethylated MGMT promoter. In two out five cultures with unmethylated MGMT status, we observed a decrease of MGMT gene levels and an increase in P53 encoding gene levels. HeberFERON and TMZ combination should be further assayed in glioblastoma, mainly for those with unmethylated MGMT promoter.