Patient-derived Glioma Cell Lines Research Articles

Primary murine high-grade glioma cells derived from RCAS/tv-a diffuse glioma model reprogram naive T cells into immunosuppressive regulatory T lymphocytes

High-grade gliomas (HGGs) and glioblastomas (GBMs) are the most aggressive and lethal brain tumors. The current standard of care (SOC) includes gross safe surgical resection followed by chemoradiotherapy. The main chemotherapeutic agents are the DNA-alkylating agent temozolomide (TMZ) and adjuvants. Due to the outdated therapeutic protocols and lack of specific treatments, there is an urgent and rising need to improve ourunderstanding of tumor biology and design more effective therapeutic strategies. Invitro models are essential for investigating glioma biology and testing novel therapeutic approaches. While using commercially available and patient-derived glioma cell lines for invitro studies is common practice, they exhibit several limitations, including failing to maintain the genetic and phenotypic diversity of primary tumors, undergo genetic drift over time, and often lacking the invasive and stem-like characteristics of patient tumors. These limitations can lead to inconsistent and non-reproducible results, hampering translational research progress. In this study, we established a novel primary murine HGG cell line, isolated from an immunocompetent HGG-bearing RCAS/T-va mouse. We characterized the transcriptome and phenotype to ensure that this cell line resembles the nature of HGGs and retains the ability to reprogram primary murine T lymphocytes.

Multi-omics and pharmacological characterization of patient-derived glioma cell lines

Glioblastoma (GBM) is the most common brain tumor and remains incurable. Primary GBM cultures are widely used tools for drug screening, but there is a lack of genomic and pharmacological characterization for these primary GBM cultures. Here, we collect 50 patient-derived glioma cell (PDGC) lines and characterize them by whole genome sequencing, RNA sequencing, and drug response screening. We identify three molecular subtypes among PDGCs: mesenchymal (MES), proneural (PN), and oxidative phosphorylation (OXPHOS). Drug response profiling reveals that PN subtype PDGCs are sensitive to tyrosine kinase inhibitors, whereas OXPHOS subtype PDGCs are sensitive to histone deacetylase inhibitors, oxidative phosphorylation inhibitors, and HMG-CoA reductase inhibitors. PN and OXPHOS subtype PDGCs stably form tumors in vivo upon intracranial transplantation into immunodeficient mice, whereas most MES subtype PDGCs fail to form tumors in vivo. In addition, PDGCs cultured by serum-free medium, especially long-passage PDGCs, carry MYC/MYCN amplification, which is rare in GBM patients. Our study provides a valuable resource for understanding primary glioma cell cultures and clinical translation and highlights the problems of serum-free PDGC culture systems that cannot be ignored.

Click-chemistry mediated synthesis of OTBN-1,2,3-Triazole derivatives exhibiting STK33 inhibition with diverse anti-cancer activities

There is a continuous and pressing need to establish new brain-penetrant bioactive compounds with anti-cancer properties. To this end, a new series of 4′-((4-substituted-4,5-dihydro-1H-1,2,3-triazol-1-yl)methyl)-[1,1′-biphenyl]-2-carbonitrile (OTBN-1,2,3-triazole) derivatives were synthesized by click chemistry. The series of bioactive compounds were designed and synthesized from diverse alkynes and N3-OTBN, using copper (II) acetate monohydrate in aqueous dimethylformamide at room temperature. Besides being highly cost-effective and significantly reducing synthesis, the reaction yielded 91–98 % of the target products without the need of any additional steps or chromatographic techniques. Two analogues exhibit promising anti-cancer biological activities. Analogue 4l shows highly specific cytostatic activity against lung cancer cells, while analogue 4k exhibits pan-cancer anti-growth activity. A kinase screen suggests compound 4k has single-digit micromolar activity against kinase STK33. High STK33 RNA expression correlates strongly with poorer patient outcomes in both adult and pediatric glioma. Compound 4k potently inhibits cell proliferation, invasion, and 3D neurosphere formation in primary patient-derived glioma cell lines. The observed anti-cancer activity is enhanced in combination with specific clinically relevant small molecule inhibitors. Herein we establish a novel biochemical kinase inhibitory function for click-chemistry-derived OTBN-1,2,3-triazole analogues and further report their anti-cancer activity in vitro for the first time.

Abstract 2130: Glioblastoma-derived extracellular vesicle cargo as a reporter of drug delivery and effect

Abstract The selectivity of the blood-brain-barrier (BBB) can prevent therapeutics from reaching the brain, and this may contribute to the high failure rate of new drugs for tumors like glioblastoma (GBM) and brain metastasis. The capacity to detect BBB penetrance in real-time in a noninvasive manner would arguably aid the rate of therapeutic development. Extracellular vesicles, which are membrane bound particles secreted by all cell types in the body, have potential as a neuro pharmacodynamic reporter given that their protein and RNA cargo reflect the originating cell population. We hypothesized that the exposure of GBM cells to therapy results in altered EV cargo, and that the detection of this shift can be leveraged as a liquid biopsy from patients during early clinical trials to determine BBB penetrance and tumor engagement. To identify cargo shifts indicative of treatment, we exposed four short-term patient derived glioma cell lines to arsenic trioxide (an idiopathic toxin), MLN4924 (a neddylation inhibitor), and the alkylating agent temozolomide, then harvested EVs for unbiased mass spectrometry and RNA sequencing. Interestingly, we identified conserved upregulated and downregulated protein and RNA species across cell lines and across drug treatments, despite the diverse mechanisms of action. Candidate proteins were further validated by mesoscale immunoassays and exoview evaluation of tetraspanin-captured EVs. RNA species were validated by qRT-PCR. Overall, we demonstrate that GBM-derived EV cargo changes in response to therapy, supporting the development of EVs as a reporter of drug delivery in GBM. Ongoing and future work is focused on identifying enrichment handles in order to selectively evaluate GBM-derived EV cargo from patient biofluids. Thus far, B7H3, PTPRZ1, EGFRvII and IL13Ra2 have been screened as putative handles. Success in this pursuit could substantially enhance findings and accelerate drug testing in early-stage clinical trials in brain tumor patients by reporting drug accessibility to the tumor and engagement with drug targets. Citation Format: Valerie DeLuca, Nanyun Tang, Nathaniel Hansen, Anjali Raju, Charles Shaffer, Yue Hao, Terry C. Burns, Jann N. Sarkaria, Ian Parney, Patrick Pirrotte, Kendall Van Keuren-Jensen, Michael E. Berens. Glioblastoma-derived extracellular vesicle cargo as a reporter of drug delivery and effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2130.

DDDR-24. SINGLE-CELL FUNCTIONAL TISSUE PROFILING IDENTIFIES PROMISING CANDIDATES FOR DRUG REPURPOSING AGAINST GLIOBLASTOMA

Abstract There remains an unmet need for novel treatment strategies against glioblastoma that are more effective than the current standard of care. However, the complex tumor microenvironment and genetic heterogeneity of these tumors require novel technologies enabling high-throughput assessment of cellular responses to treatments on a functional level in complex tumor samples with an intact cellular microenvironment. Here, we used a novel single-cell functional tissue profiling platform based on high—throughput microscopy followed by computer vision and machine learning algorithms to screen 70 clinically-approved drugs with good blood-brain-barrier penetration in 35 glioblastoma patient samples that were freshly obtained from surgery. This revealed prognostically relevant subtypes of glioma cell morphologies and promising candidates for drug repurposing. Among the drugs with potent anti-glioma activity, we identified the antidepressant Vortioxetine, that had strong anti-glioma activity across patients independent of the promoter methylation status of O6-methylguanine-DNA methyltransferase (MGMT). Complementrary in vitro studies using patient-derived glioma cell lines and orthotopic glioma xenograft mouse models confirmed the strong anti-glioma activity of Vortioxetine, leading to a notable fraction of long-term surviving mice. Mechanistically, multiplexed transcriptomics and proteomics unveiled that Vortioxetine induced an upregulation of the Activator protein 1 (AP-1) transcription factor family, along with the B-cell translocation gene (BTG) tumor suppressor genes. These findings serve as the cornerstone for future clinical applications of the single-cell functional tissue profiling platform and the potential clinical translation of Vortioxetine as a promising drug-repurposing candidate for glioblastoma patients.

CTNI-23. SINGLE CELL TRANSCRIPTOMICS, PHARMACOKINETICS, AND PHARMACODYNAMICS OF COMBINED CDK4/6 AND MTOR INHIBITION IN A PHASE 0 TRIAL OF RECURRENT HIGH-GRADE GLIOMA

Abstract Adult High-grade gliomas (HGGs) often exhibit deregulation of RB-CDK4/6 and mTOR signaling pathways. This study investigated tumor single cell transcriptomics, pharmacokinetics, and pharmacodynamics immediately following combined CDK4/6 (ribociclib) and mTOR (everolimus) inhibition in recurrent HGG. 24 patients with recurrent gliomas harboring RB+, CDKN2A/B deletion or CDK4/6 amplification, and PTEN loss or PIK3CA mutations were enrolled in a phase 0 trial. Patients received ribociclib treatment for 5 days before surgery, including six who received ribociclib (400mg QD) plus everolimus (2.5mg QD), and six dose-escalation cohorts (n = 3 each) reaching ribociclib 600mg QD plus everolimus 70mg QW. No dose-limiting toxicities were observed. Validated LC-MS/MS methods determined total and unbound drug concentrations. Preoperative MRI revealed a marked decrease in gadolinium-enhancement in all patients. In gadolinium-nonenhancing tumor regions, the median unbound concentration of ribociclib was 561 nM, while no unbound everolimus concentrations were detected. Ribociclib’s pharmacodynamic effects in surgical specimens were compared with matched archival tissue using IHC. Across IDH-wild-type tumors, there was significant decrease in MIB1 levels between the matched archival and surgical specimens (p < 0.05) suggesting drug-associated cell cycle inhibition. Single nuclei RNAseq (snRNA) analyses were performed on 17 IDH-wild-type recurrent samples treated with ribociclib plus everolimus and 29 IDH-wild-type recurrences treated with standard therapies. Cellular state analysis based on snRNAseq data demonstrated a decreased neural progenitor-like (NPC-like) malignant cell state compared with standard therapies (p < 0.01), indicating inhibition of the CDK4-driven neural progenitor-like cell state. This observation was validated using snRNA and patient-derived glioma cell lines exposed to ribociclib, showing a consistent decrease in NPC-like proportions and cycling cells (p < 0.05). This study highlights the value of integrating pharmacokinetics/pharmacodynamics and snRNA data to assess treatment effects in phase 0 surgical tissues. These findings support further development of ribociclib, but not everolimus, for the treatment of glioma patients.

DDDR-09. FUNCTIONAL GENOMICS AND CHEMOTHERAPY RESISTANCE SCREENING IDENTIFIES BIOMARKERS AND TARGETABLE MECHANISMS OF CCNU RESISTANCE IN GLIOMAS

Abstract The bifunctional alkylator lomustine (CCNU) remains widely but heterogeneously used as one of the few effective treatments in glioblastoma (GBM) and IDH mutant gliomas. Despite its widespread use, mechanisms of response and resistance to this agent are controversial and patients who benefit are poorly understood. To address this with next-generation patient models and functional genomics approaches, we generated and characterized CCNU response in a large cohort of 71 patient-derived glioma cell lines (PDCLs, spheroids/organoids). Models had fully defined clinical and molecular characteristics (WES, RNAseq, and patient and model MGMT). Patient models, including those derived from patients who acquired resistance to CCNU, exhibited a surprisingly wide range of differential response to CCNU (median IC50: 21.66uM; range: 1.52uM-159uM) using metabolic (CTG) as well as cell death readouts, with no lines showing complete resistance (EC100 < 291uM). MGMT promoter methylation status but not mismatch repair (MMR) trended, but did not reach, statistical significance as a predictor of CCNU sensitivity (p=0.053), in striking contrast to TMZ (p=0.01). Chronic CCNU exposure of two PDCLs led to acquired resistance to CCNU (IC50 increase from 5uM to 50uM on average). RNA sequencing showed 194 significantly differentially expressed genes while no mutations acquired in resistant models were identified in DNA sequencing. PRISM pooled screening of over 900 barcoded cancer cell lines and correlation with DepMap whole genome CRISPR-knockout data showed unique patterns of CCNU response and strong upregulation of DNA repair targets in the resistant state, which differed from TMZ. Shared targets were also identified in a CRISPR-activator screen performed on a CCNU sensitive cell line in the presence and absence of CCNU and converged on multiple mTOR pathway genes as top enriched targets and biomarkers of resistance in CCNU-resistant models. These druggable targets are currently being investigated for combination therapies with CCNU and potential to overcome resistance.

Aloe-Emodin Overcomes Anti-Cancer Drug Resistance to Temozolomide and Prevents Colony Formation and Migration in Primary Human Glioblastoma Cell Lines NULU and ZAR.

Glioblastoma, the most dangerous and aggressive type of CNS tumor, appears resistant to many chemotherapy drugs. In the patient-derived glioma cell lines NULU and ZAR, which exhibit drug-resistant phenotypes, we investigated the effect of combined AE (Aloe-emodin) and TMZ (temozolomide) and found a significant additive inhibitory effect on cell growth and a promising cytotoxic effect on both cell lines compared to treatment with single agents. We also examined the effect of combined AE and TMZ treatment on the drug-resistance protein MGMT. The results suggest that using AE combined with traditional drugs restores drug resistance in both primary resistant cell lines (NULU and ZAR). Furthermore, migration assays and scratch tests showed that the combined use of AE and TMZ can slow down the colony formation and migration of glioblastoma cells. These convincing results suggest that AE could be a natural adjuvant agent to potentiate the effects of traditional drugs (TMZ) and overcome drug resistance in glioblastoma cells.

Vitamin D3 suppresses the cholesterol homeostasis pathway in patient-derived glioma cell lines.

Glioblastoma is one of the most common malignant brain tumors. Vitamin D, primarily its hormonally active form calcitriol, has been reported to have anti-cancer activity. In the present study, we used patient-derived glioma cell lines to examine the effect of vitamin D3 and calcitriol on glioblastoma. Surprisingly, vitamin D3 showed a more significant inhibitory effect than calcitriol on cell viability and proliferation. Vitamin D receptor (VDR) mediates most of the cellular effects of vitamin D, and thus we examined the expression level and function of VDR via gene silencing and gene knockout experiments. We observed that VDR does not affect the sensitivity of patient-derived glioma cell lines to vitamin D3, and the gene encoding VDR is not essential for growth of patient-derived glioma cell lines. RNA sequencing data analysis and sterolomics analysis revealed that vitamin D3 inhibits cholesterol synthesis and cholesterol homeostasis by inhibiting the expression level of 7-dehydrocholesterol reductase, which leads to the accumulation of 7-dehydrocholesterol and other sterol intermediates. In conclusion, our results suggest that vitamin D3, rather than calcitriol, inhibits growth of patient-derived glioma cell lines via inhibition of the cholesterol homeostasis pathway.

Biomarker-Guided Drug Repurposing and Molecular Validation of Angiotensin-2 Receptor Type-1 in Brain Tumor

Glioma, the most frequent and malignant brain tumor, is one of the most lethal forms of cancer. Among a wide range of aberrantly expressed genes, Angiotensin-2 Receptor Type-1 (AGTR1) is reported to be up-regulated in glioma and associated with aggressive tumor traits and progression. The current study evaluates the anticancer potential of commonly used angiotensin receptor blocker (ARB) telmisartan. A total of eleven, Food and Drug Administration (FDA) approved ARBs were selected, and eight drugs were docked against the AGTR1 receptor. The antihypertensive drug with the highest docking score was selected for in vitro experimentation. Half maximal inhibitory concentration was determined and subsequently applied to patient-derived glioma cell lines. Quantitative gene expression post-drug treatment was determined through real-time polymerase chain reaction (PCR). In vitro growth inhibitory assays revealed that telmisartan at a dose of 45±0.06 μM was able to inhibit 50% of the cell population in malignant glioma U87 cell lines. The PCR results show that AGTR1 expression in the untreated sample was high, as evidenced by 2-ΔΔCt values (342.4, 138.5, 1467.3) for sample IK148-Glioblastma multiform (GBM), IK163-low grade glioma (LGG), and IK231-Medulloblastoma (MDB), respectively. The comparison was made with positive control U-87 cell lines. Following the drug treatment, an increase in the cycle threshold (Ct) mean value for AGTR1 was observed, coinciding with a decrease in mean fold changes of 0.06±0.8, 0.5±2.0, and 0.02±0.02 for GBM, LGG, and MDB samples, respectively. Based on the findings of this study, it can be concluded that telmisartan exhibits successful inhibitory effects against AGTR1 expression in glioma cell lines.

CSIG-40. STEAROYL-COA DESATURASE 1 (SCD1) IS REQUIRED FOR WNT SIGNALING TO INDUCE AN APOPTOSIS IN IDH MUTANT GLIOMA

Abstract BACKGROUND AND HYPOTHESES SCD1, a major enzyme of saturated fatty acids, has been implicated to be important for tumor metabolic reprograming. Our previous study show that a high level of SCD1 mRNA is associated with IDH1mut lower grade gliomas. IDH1mut glioma cells are more sensitive to SFA induced apoptosis. However, the underlying mechanism remains unclear. In this study, we investigate the functions of SCD1 in IDHmut glioma and the potential contribution of SCD1 for cancer therapy of glioma. STUDY DESIGN AND METHODS The genetically engineered IDH wild-type, IDHmut and patient derived glioma cell lines were used to evaluate SCD1 functions. The expression of proteins were checked by Western-blotting assay. SCD1 was silenced by CRISPR or siRNA. The transcriptome change after SCD1 knockdown was profiled by RNA-seq or single cell RNA-seq (scRNA-seq). RESULTS AND CONCLUSIONS SCD1 transient silencing slowed down the cell growth, suggesting that SCD1 may possess an oncogenic property. RNA-seq analysis revealed that SCD1 inhibition decreased the expression of wnt-signaling pathway genes in IDH-1mut cells. scRNA confirmed that CRISPR SCD1 significantly decreased wnt signaling in the patient cell line Ts603. Therefore, we activated wnt pathway using a small chemical compound, BML-2838. Consistent with recent studies, wnt pathway induction led to a dramatically suppression of glioma cells growth. However, SCD1 silencing reversed this inhibitory effect. Further investigation revealed that SCD1 inhibition reduced the nucleus translocation of phosphorylated beta-catenin. Overall, the results suggest that SCD1 is vital for the onset of wnt pathway in glioma cells. High level of SCD1 expression may render the IDHmut glioma cells more sensitive to wnt pathway induced apoptosis. RELEVANCE AND IMPORTANCE In clinical, the 5-years survival rate of glioma remains low. SCD1 have been considered as a target for glioma therapy, recently. Our data provides a new insight on the strategy to target SCD1 in clinical.

STEM-15. THERAPY-INDUCED CHANGES BY BRAF AND MEK INHIBITORS IN BRAF V600E-MUTATED GLIOMA MODELS PROVIDE POTENTIAL NOVEL THERAPEUTIC OPPORTUNITIES

Abstract Combinations of the MEK inhibitor trametinib, and BRAF inhibitor dabrafenib (BRAFi+MEKi) show rapid and sustained responses in patients with BRAF V600E-mutated low-grade glioma, but tumor rebound after treatment discontinuation is frequent. Moreover, a lack of response is common in patient with high-grade glioma raising the need for further research into BRAFi+MEKi effects on tumors. We showed previously that BRAF V600E-mutated glioma cells positive for CD133 (Prominin-1), a marker of brain tumor stem cells, show decreased sensitivity to BRAFi, indicative of their role in promoting therapy resistance. BRAF V600E-mutated murine and patient-derived glioma cell lines (STN-10049, SU-aGBM5) were generated and together with established BRAF V600E-mutated cell lines (DBTRG, AM38) were analyzed for changes in gene expression in response to 48 hrs treatment with BRAFi dabrafenib and MEKi trametinib. Cells were analyzed by RNA-seq and gene enrichment analyses while cell culture supernatant was analyzed for cytokine production using an ELISA. Syngeneic, orthotopic BRAF V600E-mutated tumor allograft-bearing mice were treated with BRAFi+MEKi, with therapeutic antibodies against immune checkpoint molecules (anti-PD-L1 and anti-CTLA-4) and with combination of all four agents, and tumors were analyzed by mass cytometry and immunofluorescence for stem and T cell markers. BRAFi+MEKi treatment induced an interferon gamma (IFNg) response gene signature in BRAF V600E-mutated glioma cells and increased HLA gene expression. The frequency of tumor-infiltrating CD4+ CD8+ T cells in syngeneic BRAF V600E-mutated tumor allografts increased with BRAFi+MEKi treatment. Combining BRAFi+MEKi with anti-PD-L1 and anti-CTLA-4 treatment decreased CD133+ cells more effectively than either therapy alone, and resulted in a T cell-dependent survival benefit of mice with orthotopic BRAF V600E-mutated high-grade glioma. Combination of BRAFi+MEKi with immune checkpoint inhibition should be further explored as a viable option to prevent tumor rebound and therapy resistance in patients with BRAF V600E-mutated glioma.

TMET-36. ACID CERAMIDASE INHIBITION EXPLOITS SPHINGOLIPID VULNERABILITIES IN IDH MUTANT GLIOMAS

Abstract The presence of the IDH mutation in gliomas is a major classifier of brain tumor subtypes and has several important implications for cancer growth. Our recent work uncovered that IDH-mutant tumors are susceptible to increased apoptosis via alterations of the sphingolipid pathway due to their excess production of pro-apoptotic ceramides over pro-proliferative sphingosine 1-phosphate (S1P). To that end, we proposed that this rheostat can be modulated to induce cell death in IDHmut tumors by targeting acid ceramidase, a critical sphingolipid enzyme in gliomas. We hypothesize that pharmacological inhibition of acid ceramidase will increase ceramide levels and therefore induce apoptosis in IDHmutgliomas. Using a preliminary drug screen, we have identified a group of haloacetate C2-ceramide derivatives known as SOBRACs that potently inhibit acid ceramidase. We selected five candidate compounds from this family and assessed the effectiveness of each drug in 3 I IDHmut (BT142, TS603, & U251mut) and 3 IDHmut (GSC923, GSC827, U251wt) patient-derived glioma cell lines, as well as non-immortalized normal human astrocytes, using the CCK8 cell viability assay. Our results indicate that the SOBRAC drugs are nearly 10 times more potent in IDH-mutant tumors compared to IDHmut cell lines. Additionally, the SOBRAC drugs are more effective than other known acid ceramidase inhibitors, making them attractive as potential novel therapeutics. To date, azide-SOBRAC is the most potent drug in the family, with EC50 value of 300 nM in BT142 cells (IDHmutmut

EXTH-95. UNCOVERING THERAPEUTIC VULNERABILITIES IN MISMATCH REPAIR-DEFICIENT GLIOMAS

Abstract Approximately 25% of recurrent gliomas exhibit hypermutation, which is most often acquired in chemotherapy sensitive gliomas post-treatment with standard of care alkylating agent temozolomide, Intriguingly, nearly all of these recurrent hypermutant tumors harbor deficiencies in mismatch repair (MMR) genes. Unlike other MMR-deficient cancers, hypermutant gliomas do not exhibit detectable microsatellite instability (MSI) at the population level, and do not share similar dependencies. Thus, strategies to therapeutically target MMR-deficient and hypermutant gliomas are urgently needed, and likely to impact many patients. To evaluate vulnerabilities associated with MMR-deficient gliomas, we have generated isogenic MMR-deficient models by systematic ablation of core MMR genes MSH2, MSH6, MLH1, and PMS2, in multiple patient-derived glioma cell lines using a robust all-in-one CRISPR-Cas9 system. We characterized these isogenic MMR-deficient glioma lines in comparison to respective MMR-proficient control line by performing differential gene expression analysis and subsequent gene set enrichment analyses. Our analyses reveal in an unbiased fashion an enrichment of several hallmark gene sets including cell cycle control and DNA repair in the MMR-deficient cell lines. We performed a CRISPR-Cas9 knockout screen in these MMR deficient and hypermutant models and identified candidate genes involved in DNA repair, cell cycle, RNA metabolism and other pathways as preferential dependencies in the MMR-deficient glioma cells. We also screened multiple MMR-deficient isogenic models against the high-throughput drug repurposing (REPO) chemical library and our preliminary results reveal a number of compounds from a variety of drug classes that selectively kill the MMR-deficient glioma cells, indicating that the loss of MMR confers differential dependencies to these small molecule inhibitors. The candidate drugs and genes identified from these high-throughput assays are currently being subjected to further validation, and they possibly will help us identify novel therapeutic strategies or improve existing therapeutic strategies to target recurrent MMR-deficient gliomas.

Abstract LB188: Identification of intrinsic molecular vulnerabilities in inherited and treatment-related hypermutant patient-derived glioma cell line models

Abstract Background: Hypermutant gliomas in children are caused by inherited or therapy related replication repair deficiency (RRD), the latter typically following treatment with the alkylating agent temozolomide (TMZ). Recently, immune checkpoint inhibitors (ICIs) revealed clinical responses and prolonged survival in 30% of pediatric-inherited hypermutant gliomas but none of the treatment-related adult hypermutant gliomas. We hypothesized that high-throughput screening of hypermutant and RRD patient derived glioma cell lines from both glioma types (PDGCL) would reveal specific molecular vulnerabilities immediately translatable as targeted therapies to be used in combination with ICIs. Methods: We screened two pediatric hypermutant and RRD PDGCLs (1806, 1260); one pediatric non-hypermutant non-RRD PDGCL (477); an adult non-hypermutant non-RRD PDGCL (189EW); matched recurrent post-TMZ treatment hypermutant RRD PDGCL (248z, 248xy) from patient 189 against a library of 3336 bioactive compounds, including 1500 FDA approved drugs supplemented with 16 additional compounds (BET, PLK1, EZH2, PARP1 inhibitors) at 100nM.Each plate screened contained negative (0.2% DMSO) and positive controls (50nM bortezomib). Cell viability was calculated at 96hr by ATP measurement. Raw values from negative and positive control wells were aggregated and used to calculate Z’-factors for each assay plate, as a measure of assay performance and data quality, with a Z’-factor >0.5 representing high quality data. Only drugs scoring Normalized Percentage of Inhibition (NPI) >50% and z-score>3 were considered potential hits for follow-up evaluation, focusing on those enriched in the pediatric and adult hypermutant PDGCLs. Results: We identified 45 drugs active in all three of the pediatric PDGCLs and 23 common hits in the adult cell lines. Broad target classes were identified such as microtubule associated inhibitors, heat shock protein inhibitors for PDGCLs and proteasome inhibitors for adult cell lines. Unique targets were also identified. Importantly, the inherited and treatment related mismatch repair deficient glioma cells (MMR1260,248z, 248xy) and the hypermutant pediatric cell line (1806) exhibited significant response to PI3K/mTOR inhibitors. Ultrahypermutant RRD cell line and the treatment-related hypermutant cell lines (1806 248z 248xy ) displayed sensitivity to topoisomerase I inhibitors. Preliminary in vitro data confirm sensitivity to topotecan of hypermutant pediatric and adult cell lines. Conclusion: We have identified both mechanism specific and common intrinsic vulnerabilities in hypermutant PDGCLs that can be tested as adjuvant therapies for ICI. Functional validation of the lead candidates is ongoing and will be reported. Citation Format: Laura Scolaro, Nuno Miguel Nunes, Michelle Kushida, David C. Schultz, Sara Cherry, Kanupriya Whig, Peter Dirks, Uri Tabori, John M. Maris. Identification of intrinsic molecular vulnerabilities in inherited and treatment-related hypermutant patient-derived glioma cell line models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB188.

Abstract 4170: Integrative transcriptomic and proteomic analysis reveals immunogenic and spatially-conserved HLA-binding neoantigen targets derived from tumor-specific alternative splicing events

Abstract BACKGROUND: While immunotherapy is profoundly efficacious in certain cancers, its success is limited in cancers with lower mutational burden, such as gliomas. Therefore, investigating neoantigens beyond those from somatic mutations can expand the repertoire of immunotherapy targets. Recent studies detected alternative-splicing (AS) events in various cancer types that could potentially translate into tumor-specific proteins. Our study investigates AS within glioma to identify novel MHC-I-presented neoantigen targets through an integrative transcriptomic and proteomic computational pipeline, complemented by an extensive spatiotemporal analysis of the AS candidates. METHODS: Bulk RNA-seq of high tumor purity TCGA-GBM/LGG (n=429) were analyzed through a novel systematic pipeline, and tumor-specific splicing junctions (neojunctions) were identified in silico by cross-referencing with bulk RNA-seq of GTEx normal tissue (n=9,166). Two HLA-binding prediction algorithms were subsequently incorporated to predict peptide sequences with high likelihood for HLA-presentation. Investigation of the tumor-wide clonality and temporal stability of the candidates was performed on extensive RNA-seq data from our spatially mapped intratumoral samples and longitudinally collected tumor tissue RNA-seq. Proteomic validation was conducted through mass-spec analysis of the Clinical Proteomic Tumor Analysis Consortium (CPTAC)-GBM repository (n=99). RESULTS: Our analysis of TCGA-GBM/LGG bulk RNA-seq identified 249 putative neojunctions that translate into 222 cancer-specific peptide sequences which confer 21,489 tumor-specific n-mers (8-11 amino acids in length). Both prediction algorithms concurrently identified 271 n-mers likely to bind and be presented by HLA*A0101, HLA*A0201, HLA*A0301, HLA*A1101, or HLA*A2402. We confirmed the expression of 15 out of 58 HLA*A0201-binding candidates in RNA-seq of two HLA*A0201+ patient-derived glioma cell lines with a subset of candidates found tumor-wide. Analysis of CPTAC-GBM mass-spec data detected 23 tumor-specific peptides with 5 containing detected n-mers highly predicted to be HLA-presented. CONCLUSION: Tumor-specific neojunctions identified in our unique integrative pipeline present novel candidate immunotherapy targets for gliomas and offer a new avenue in neoantigen discovery across cancer types. Citation Format: Darwin Kwok, Takahide Nejo, Aidan Du, Maggie Colton, James Woo, Joseph Costello, Hideho Okada. Integrative transcriptomic and proteomic analysis reveals immunogenic and spatially-conserved HLA-binding neoantigen targets derived from tumor-specific alternative splicing events [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4170.

HGG-51. Uncovering therapeutic vulnerabilities in mismatch repair-deficient gliomas

INTRODUCTION: We have observed that approximately 26% of recurrent gliomas acquire hypermutation following treatment with temozolomide (TMZ). Intriguingly, 91% of these tumors harbor mutations in mismatch repair (MMR) genes. Since MMR deficiency confers resistance to TMZ, strategies to target MMR-deficient gliomas stand to impact many patients. METHODS: We ablated the MMR genes MSH2, MSH6, MLH1, and PMS2 using an all-in-one sgRNA-CRISPR/Cas9 expression vector to generate isogenic MMR knockouts in patient-derived glioma cell lines. We characterized the gene expression profiles of these MMR-deficient glioma models and leveraged high-throughput drug screens and genome-scale CRISPR/Cas9 dropout screens to identify therapeutic vulnerabilities induced by loss of MMR. RESULTS: We show that loss of each major MMR gene confers resistance to TMZ. Gene set enrichment analysis of our MMR-deficient knockouts shows enrichment of several hallmark gene sets including DNA repair and G2M checkpoint signatures, and our genome-wide CRISPR dropout screen reveals that MMR-deficient cells are preferentially dependent on a number of genes involved in DNA repair and cell cycle, along with several other pathways. Lastly, the high-throughput drug repurposing (REPO) screen shows that loss of MMR confers differential dependencies to small molecule inhibitors. CONCLUSIONS: Using CRISPR/Cas9 to knock out individual MMR pathway members allows us to systematically study the response of MMR-deficient cells to alkylating agents in an isogenic context. Importantly, these isogenic models reveal that MMR-deficient glioma cells possess novel genetic dependencies and sensitivities to small molecules, which may inform future therapies for MMR-deficient tumors.

Poly-guanidine shows high cytotoxicity in glioma cell cultures and glioma stem cells

Glioblastoma multiforme (GBM) is a malignant CNS tumor with a poor prognosis. GBM shows aberrant glycosylation with hypersialylation. This property is a potential target for therapy. This study investigates the growth inhibitory efficacy of poly-guanidine (GuaDex), with an affinity for sialic acid (Sia). Glioma cell cultures and patient-derived glioma cell lines (PDGCLs) expressing Prominin-1 (CD133) were used. Human fibroblasts and astrocyte-derived cells were used as controls. Temozolomide (standard GBM drug, TMZ) and DMSO were used as a comparison. GuaDex at 1–10 µM concentrations, were incubated for 3.5–72 h and with PDGCLs cells for 6–24 h. The cytotoxicity was estimated with a fluorometric cytotoxicity assay (FMCA). Fluorescence-labelled GuaDex was used to study the cell interactions. Sia expression was confirmed with a fluorescence labelled Sia binding lectin. Expression of glial fibrillary acidic protein was determined. GuaDex induction of growth inhibition was fast, showing after less than 5 min incubation while the control cells were not affected even after 50 min incubation. The growth inhibitory effect on PDGCLs spheroids was persistent still showing after 4 weeks post-treatment. The growth inhibition of GuaDex was induced at low µM concentrations while TMZ induced only a slight inhibition at mM concentrations. GuaDex efficacy appears significant and warrants further studies.

GSK-3 Inhibition Is Cytotoxic in Glioma Stem Cells through Centrosome Destabilization and Enhances the Effect of Radiotherapy in Orthotopic Models.

Simple SummaryHigh-grade gliomas remain difficult-to-treat cancers. Novel treatment options include targeting glycogen synthase kinase 3 (GSK-3) to induce cell death but the mode of drug activity remains unknown and combination with conventional treatment including radiotherapy has not been explored. Here, we describe the effect of targeting GSK-3 with the inhibitor AZD2858 in in vitro and in vivo models of glioma. We established that AZD2858 exposure induces mitotic defects leading to cell death in patient-derived glioma cell lines and tumor growth delay in glioma xenografts. Co-administration also enhanced the effect of radiotherapy. We therefore propose AZD2858 as an adjuvant to radiotherapy in high-grade glioma.Background: Previous data on glycogen synthase kinase 3 (GSK-3) inhibition in cancer models support a cytotoxic effect with selectivity for tumor cells compared to normal tissue but the effect of these inhibitors in glioma has not been widely studied. Here, we investigate their potential as cytotoxics in glioma. Methods: We assessed the effect of pharmacologic GSK-3 inhibition on established (U87, U251) and patient-derived (GBM1, GBM4) glioblastoma (GBM) cell lines using cytotoxicity assays as well as undertaking a detailed investigation of the effect on cell cycle, mitosis, and centrosome biology. We also assessed drug uptake and efficacy of GSK-3 inhibition alone and in combination with radiation in xenograft models. Results: Using the selective GSK-3 inhibitor AZD2858, we demonstrated single agent cytotoxicity in two patient-derived glioma cell lines (GBM1, GBM4) and two established cell lines (U251 and U87) with IC50 in the low micromolar range promoting centrosome disruption, failed mitosis, and S-phase arrest. Glioma xenografts exposed to AZD2858 also showed growth delay compared to untreated controls. Combined treatment with radiation increased the cytotoxic effect of clinical radiation doses in vitro and in orthotopic glioma xenografts. Conclusions: These data suggest that GSK-3 inhibition promotes cell death in glioma through disrupting centrosome function and promoting mitotic failure and that AZD2858 is an effective adjuvant to radiation at clinical doses.

CSIG-02. NRF2-GUIDED GLUTATHIONE SYNTHESIS IS AN ESSENTIAL METABOLIC PATHWAY IN IN IDH1-MUTATED GLIOMA

Abstract BACKGROUND Isocitrate dehydrogenase (IDH) mutations are common genetic abnormalities in WHO Grade II/III glioma, which result in the reprogramming of cellular metabolism and redox homeostasis. Many lines of evidence showed that IDH mutations are critical for glioma formation, whereas the therapeutic options for IDH-mutated cancers remain limited. METHODS In the present study, we used the patient-derived glioma cell lines to investigate the role of nuclear factor erythroid 2-related factor 2 (NRF2) governed glutathione de novo synthesis. Further, we evaluated the therapeutic value of NRF2 inhibitors in IDH1-mutated cells and preclinical orthotopic models. RESULTS The neomorphic activity of mutant IDH reprogrammed the metabolic pathways involving enzyme cofactors such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). The depletion of NAD(P) in IDH1-mutated cells resulted in elevated oxidative stress and constitutive activation of NRF2-governed cytoprotective pathways through the decoupling of NRF2 from its E3 ligase Kelch-like ECH-associated protein 1 (Keap1). Activation of NRF2 enhanced glutathione synthesis by enhancing the gene transcription of GCLC, GCLM, and SLC7A11, which are the critical for glutathione de novo synthesis. Further, evidence from both in vitro assays and patient cohort indicated that NRF2 governed glutathione synthesis is important for maintaining the redox homeostasis and cell survival, especially in IDH1-mutated glioma. Finally, Blockade of the NRF2/glutathione metabolic pathway exhibited synergistic cytotoxicity with the metabolic stress in IDH1-mutated cells, which results in overwhelming oxidative damage, as well as a substantial reduction in tumor cell proliferation and xenograft expansion. CONCLUSION In this study, we highlighted that NRF2 plays critical roles in the disease progression of IDH1-mutated glioma by prompting glutathione synthesis. Targeting NRF2 governed glutathione metabolism could serve as a valuable synthetic lethality approach for IDH1-mutated malignancies.