Mutant Gliomas Research Articles

TRIM67 drives tumorigenesis in oligodendrogliomas through Rho GTPase-dependent membrane blebbing.

IDH mutant gliomas are grouped into astrocytomas or oligodendrogliomas depending on the codeletion of chromosome arms 1p and 19q. Although the genomic alterations of IDH mutant gliomas have been well described, transcriptional changes unique to either tumor type have not been fully understood. Here, we identify Tripartite Motif Containing 67 (TRIM67), an E3 ubiquitin ligase with essential roles during neuronal development, as an oncogene distinctly upregulated in oligodendrogliomas. We used several cell lines, including patient-derived oligodendroglioma tumorspheres, to knock down or overexpress TRIM67. We coupled high-throughput assays, including RNA sequencing, total lysate-mass spectrometry (MS), and coimmunoprecipitation (co-IP)-MS with functional assays including immunofluorescence (IF) staining, co-IP, and western blotting (WB) to assess the in vitro phenotype associated with TRIM67. Patient-derived oligodendroglioma tumorspheres were orthotopically implanted in mice to determine the effect of TRIM67 on tumor growth and survival. TRIM67 overexpression alters the abundance of cytoskeletal proteins and induces membrane bleb formation. TRIM67-associated blebbing was reverted with the nonmuscle class II myosin inhibitor blebbistatin and selective ROCK inhibitor fasudil. NOGO-A/Rho GTPase/ROCK2 signaling is altered upon TRIM67 ectopic expression, pointing to the underlying mechanism for TRIM67-induced blebbing. Phenotypically, TRIM67 expression resulted in higher cell motility and reduced cell adherence. In orthotopic implantation models of patient-derived oligodendrogliomas, TRIM67 accelerated tumor growth, reduced overall survival, and led to increased vimentin expression at the tumor margin. Taken together, our results demonstrate that upregulated TRIM67 induces blebbing-based rounded cell morphology through Rho GTPase/ROCK-mediated signaling thereby contributing to glioma pathogenesis.

CTNI-23. PRELIMINARY SAFETY AND PHARMACOKINETICS DATA FOR A PHASE 1B TRIAL OF TELAGLENASTAT IN COMBINATION WITH RADIATION THERAPY AND TEMOZOLOMIDE IN PATIENTS WITH IDH-MUTANT GRADE 2/3 ASTROCYTOMA (NCI-10218)

Abstract IDH mutant gliomas depend on glutaminase for glutamate/glutathione generation from glutamine because R-2-hydroxyglutarate inhibits branched chain amino acid transaminase mediated glutamate biosynthesis. Telaglenastat (CB-839 HCl) is a potent glutaminase-1 specific inhibitor which depletes tumor glutamate in orthotopic IDH mutant glioma PDX models and extends survival in these orthotopic models when added to radiation/temozolomide. NCI-10218 (NCT03528642) is a phase I clinical trial investigating the safety and tolerability of telaglenastat administered orally concurrently with standard doses of radiation (50.4 Gy, grade 2; 59.4 Gy, grade 3) and temozolomide (75 mg/m2 orally daily) in patients (age 16+) with previously untreated IDH mutant grade 2/3 astrocytoma. Telaglenastat dose was escalated in cohorts (400-800 mg twice daily) based on a standard 3 + 3 design to determine the recommended phase 2 dose (RP2D). Toxicities were graded per CTCAE v5.0. An expansion cohort additionally incorporated a seven-day run-in of telaglenastat monotherapy at RP2D prior to radiation to evaluate the pharmacodynamic impact of telaglenastat on plasma and tumor metabolites. 23 patients with IDH mutant astrocytoma (WHO grade 2, n = 5; WHO grade 3, n = 18) were accrued between December 2018 and January 2022 (Dose Escalation: 16; Dose Expansion: 7). Median age was 32 years (range 23-69 years). 61% were male and 70% were ECOG 0. No dose-limiting toxicities were observed. Grade 3/4 adverse events (independent of attribution) included: lymphopenia (3), neutropenia (2), leukocytosis (2), alanine transaminase elevation (2), thrombocytopenia (1), leukopenia (1), maculopapular rash (1), hyperglycemia (1), hyponatremia (1). The RP2D of concurrent telaglenastat was defined as 800 mg twice daily. Following peak absorption on Day 15 at RP2D, the mean (%CV) terminal elimination half-life in the plasma was 4.2 (53.5%) hours (range 2.1-7.1 hours). The Cmax, Tmax, oral clearance, and AUC were 1496 ng/mL, 4.0 hr, 93.6 L/hr/m2, and 7430 ng/mL*hr, respectively.

DDDR-39. IDENTIFICATION OF A NOVEL THERAPEUTIC TARGET THAT IS SYNTHETICALLY LETHAL WITH MUTANT IDH INHIBITOR IN GLIOMA USING THE CRISPR/CAS9 GENOME EDITING TECHNOLOGY

Abstract Diffuse glioma is the most frequent and malignant neuroepithelial tumor in the central nervous system. IDH mutations are the most frequent genetic mutations in adult gliomas. The WHO Classification of Tumors, 5th edition, volume 6: Central Nervous System Tumours revised in 2021, defines the presence of IDH mutations in gliomas as astrocytoma IDH-mutant and oligodendroglioma IDH-mutant and 1p/19q-codeleted. In addition, more than 90% of IDH mutations found in gliomas are IDH1 R132H mutation. Several clinical trials of mutant IDH1 inhibitors targeting the IDH1 R132H mutation have shown their efficacy, but it has also been reported that IDH1 inhibitors alone may not provide sufficient tumor suppression. In this study, we performed genome-wide knockout screening using CRISPR/Cas9 genome editing technology to search for new drug targets that are synergistic with the mutant IDH1 inhibitor DS-1001b. The results of CRISPR screening and RNA sequence analysis revealed that several metabolism-related genes are factors that define resistance to mutant IDH inhibitor. In a tumor suppression study, treatment of two IDH1 R132H mutant glioma cell lines (MGG152 and BT142) with mutant IDH1 inhibitor suppressed production of the oncometabolite D2-HG, but did not significantly alter cell growth. However, when mutant IDH1 inhibitor were combined with inhibitor for metabolism-related genes, cell survival was significantly reduced, and the effects were synergistic. In this study, we found that administration of mutant IDH inhibitors to IDH mutant gliomas significantly altered intratumor metabolism using various molecular approaches. We then identified targets that, when combined with mutant IDH inhibitor, provide synergistic tumor suppression. These results are expected to provide a new therapeutic approach for a subset of patients who have difficulty responding to mutant IDH1 inhibitors.

EXTH-69. ASTROCYTOMA CELLS HARBORING MUTANT IDH1 ARE UNIQUELY SENSITIVE TO INHIBITION OF OXIDATIVE PHOSPHORYLATION

Abstract BACKGROUND Diffuse gliomas are the most common primary brain tumor in adults, affecting ~20,000 people in the United States each year. Current treatment modalities, which include surgery, radiation, and chemotherapy, are relatively ineffective against these tumors and the prognosis is poor. Although most cancer cells rely on glycolysis to generate ATP, IDH1 mutant gliomas show enhanced dependence on oxidative phosphorylation. Thus, we evaluated the effect of IACS-010759, a potent and selective inhibitor of complex I that interferes with cellular respiration, in multiple patient-derived glioma cells. METHODS In the present study, we used six different patient-derived glioma cell cultures including four that are IDH1 wild type (wt) (GSC 20, GSC 23, LN 18 and SF 268) and two that are IDH1R132H mutant (BT142 & 21238-IDH1R132H). All cells were cultured as neurospheres to maintain glioma stem cell (GSC) culture conditions. The toxicity of IACS-010759 was also evaluated in immortal non-transformed mouse astrocytes. Neurospheres and astrocytes were treated with IACS-010759 (0.1 µM-100 µM) and cell viability was evaluated after 72 hours using CellTiter-Glo. RESULTS Inhibition of oxidative phosphorylation significantly reduced cell proliferation in all glioma cells evaluated. The IC50 of IACS-010759 ranged from 0.7 nM-100 µM but was lowest in IDH1R132H astrocytoma cells, supporting the hypothesis that IDH1 mutant astrocytoma cells have enhanced dependence on oxidative phosphorylation. Importantly, immortal, non-transformed astrocytes were relatively resistant to IACS-010759 suggesting that this strategy may have an effective therapeutic window in this disease. CONCLUSION In summary, IACS-010759-induced inhibition of oxidative phosphorylation elicited pronounced anti-neoplastic responses in vitro, especially in astrocytomas expressing mutant IDH1. IACS-010759 may be a promising therapy for both IDH1 wt and mutant gliomas, and we are currently evaluating the efficacy of this strategy in glioma mouse models.

NIMG-38. 2-HYDROXYGLUTARATE MAGNETIC RESONANCE SPECTROSCOPY IN ADULT BRAINSTEM GLIOMA PATIENTS

Abstract INTRODUCTION The onco-metabolite, 2-hydroxyglutarate (2HG), is non-invasive biomarker detected by magnetic resonance spectroscopy (MRS), indicating isocitrate dehydrogenase (IDH) mutant glioma. Especially in patients with brainstem lesions where biopsies are at high risk of causing neurological deficit, 2HG-MRS may be useful. Here, we examined the utility of 2HG-MRS for diagnosing IDH mutant adult diffuse brainstem glioma and the ability of prognosis prediction. METHODS Three tesla -MRS (3T-MRS) was performed on patients with radiographically confirmed brainstem tumor. A single-voxel was set in the lesion with reference to the T2 / FLAIR image, and analyzed according to the 2HG-tailored MRS protocol (Philips, Achieva, PRESS, TE 35 ms). All patients underwent biopsy using an intraoperative navigation system (Brain LABTM) or stereotactic biopsy system (Komai’s CT-stereotactic frame) before initial treatment. IDH and H3K27M mutation status were confirmed by immunohistochemistry and DNA sequence. We examined the relationship between 2HG values and patient survival period. RESULTS Ten patients (7 male and 3 female, median age 33.5) underwent 2HG-MRS and biopsy. Four had H3K27M mutation and 4 had IDH mutation (1 R132H, 2 R132S, and 1 R132G). Two had neither H3K27M nor IDH mutation. H3-K27 and IDH mutations were mutually exclusive. All tumor located at pons mainly. There was no significant radiological difference between H3K27M and IDH mutant in conventional MRI sequence. Pearson's chi-square test demonstrated that 2HG concentrations > 1.5 mM were 100% in sensitivity and 83.3% in specificity for IDH mutant glioma (p = 0.0098). The median overall survival was 130.5 months in IDH mutant glioma (n=4) and 25 months in IDH wild-type glioma (n=6), respectively. CONCLUSIONS 3T-MRS was able to detect 2HG in adult brainstem glioma. A noninvasive 2HG-MRS study may be helpful in the diagnosis and predicting prognosis of adult diffuse brainstem glioma patient.

DNAR-03. TARGETING POLY (ADP-RIBOSE) SIGNALING TO INDUCE LETHAL DNA DAMAGE IN IDH MUTANT GLIOMA

Abstract Isocitrate dehydrogenase (IDH) 1 or 2 mutant gliomas have unique metabolic characteristics and DNA damage response. Radiotherapy and chemotherapy has been proven to be an effective treatment for IDH mutant gliomas. A key mediator of DNA damage response, Poly(ADP-ribose) (PAR) is regulated by opposing enzymes, poly(ADP-ribose) polymerases (PARPs) that synthesizes PAR through enzymatic polymerization of nicotinamide adenine dinucleotide (NAD), and poly(ADP-ribose) glycohydrolase (PARG) that breaks down and recycles PAR. We recently demonstrated that combining PARG inhibitor (PARGi) and Temozolomide (TMZ) in IDH mutant cells drove an imbalanced sequestration of NAD into PAR, resulting in metabolic lethality through NAD depletion. Here, we sought to characterize the effect of combining PARGi and radiation. Isogenic pairs of IDH1-mutant glioma cells, alongside multiple IDH mutant patient-derived glioma cells, were treated with TMZ, radiation, PARPi, PARGi, and combination thereof. In contrast to PARPi which can have a “trapping” effect, PARGi monotherapy displayed no significant difference between IDH mutant and wild-type isogenic cells. However, we found a substantial augmentation of radiation cytoxicity when combined with PARGi, that was genotype-specific in IDH mutant glioma models, mirroring that seen with PARPi and radiation (Wang et al, Science Advances 2020). Unlike TMZ+PARGi, NAD was not depleted with PARGi and radiation. Conversely, the cytotoxic effect was partially reversed with either PARPi or IDH inhibitor, indicative of a key contribution of PAR mediated DNA damage signaling in the IDH mutant context. Therefore, these results support a model of converging vulnerabilities in IDH mutant cells, with PAR-mediated DNA repair mechanisms independent from NAD metabolism.In summary, this study demonstrates that the combination of PARGi and radiation regulates DNA repair pathways with cytoxicity specific to IDH mutant gliomas. Targeting PAR can potentially be a promising treatment for clinical translation in IDH mutant gliomas, minimizing the radiation effect to surrounding normal cells.

CNSC-24. THE EPILEPTIC LANDSCAPE OF IDH MUTANT GLIOMAS

Abstract Tumor-associated epilepsy (TAE) is a frequent complication of diffusely infiltrative gliomas, and impairs quality-of-life. We previously showed that isocitrate dehydrogenase 1 mutant (IDHmut) gliomas were associated with preoperative seizures, and that D2HG increased synchronized bursts in cultured neurons (PMID: 28404805). But the mechanism whereby this occurs, whether IDHmut inhibitors can block this, and whether seizure risk varies by IDHmut status post-operatively, are unknown. We discovered that exogenous 3 mM D2HG had a 150% greater effect on the firing rate of cultured mouse cortical neurons when nonneoplastic glia cells were present versus when they were absent (P=0.002). Coculture with patient-derived IDHmut glioma cells increased the firing rate of human cortical neuron/astrocyte spheroids by up to 272%, and the IDHmut inhibitor AG881 reduced the excitatory effect of IDHmut glioma cells on spheroids by 79% (P=0.0008). Using a novel in vivo model of TAE, wherein EEG recordings were taken of immunocompetent mice engrafted with isogenic mouse glioma lines (NRAS/ATRX/TP53mut ± IDHmut), we found that IDHmut gliomas produced 7.6-fold more epileptiform spikes than IDHwt gliomas (P=0.004). RNA-Seq analysis of the peritumoral mouse brain tissue showed that this increase in spikes was associated with significantly increased expression of key genes known to be upregulated in epilepsy, including SLC12A5, THSB1, VEGFA, FOSL2, and SYNPO. Treatment with 5 mg/kg AG881 by daily oral gavage reduced those spikes in IDHmut-engrafted mice by 51% within three days (P=0.027), whereas vehicle control had no effect (P=0.33). Among 247 patients with grade 2–4 adult-type diffuse gliomas, multivariable time-to-event analysis showed that post-operative seizure risk was positively associated with pre-operative seizures, subtotal resection, and IDHmut astrocytoma. Together, these data show that (i) the D2HG product of IDHmut gliomas increases neuronal excitation in a glial-dependent manner; (ii) IDHmut also affects post-operative seizure risk; (iii) IDHmut inhibitors may improve TAE control.

EXTH-39. BIOMARKER DRIVEN TARGETING OF RECURRENT IDH MUTANT GLIOMA WITH ABEMACICLIB

Abstract Despite initial responsiveness to standard treatments like radiation and chemotherapy, IDH mutant gliomas inevitably recur, become more clinically aggressive, and lead to untimely death. The aggressive change in clinical outcome is driven by insensitivity to conventional methods of treatment, necessitating an improved therapeutic strategy at this stage of the disease. Greater than 20% of recurrent IDH mutant gliomas exhibit homozygous loss of CDKN2A, leading to the loss of the endogenous CDK4/6 inhibitor p16 and aberrant signaling through the CDK-RB pathway. We hypothesized that CDKN2A loss leads to enhanced sensitivity to CDK4/6 inhibitors which have been approved for use in a variety of other cancer types. We examined the sensitivity of a panel of patient-derived IDH mutant gliomas with either endogenous CDKN2A loss or CRISPR-mediated gene deletion to treatment with CDK4/6 inhibitors. We observed a correlation between CDKN2A loss and enhanced cytotoxicity following abemaciclib treatment in these glioma models. We have characterized the cell cycle profile following abemaciclib treatment by flow cytometry and western blotting in both sensitive and resistant lines. Further, abemaciclib treatment resulted in significant slowing of tumor growth rate in a subcutaneous patient-derived IDH mutant glioma model. Survival studies in an intracranial model are ongoing. These preclinical results provide foundational data to support a biomarker-driven clinical trial of CDK4/6 inhibitors in patients with recurrent IDH mutant glioma.

TMET-03. STEAROYL-COA DESATURASE INHIBITOR SUPPRESSES IDH MUTANT GLIOMA GROWTH VIA ENHANCING LIPOLYSIS

Abstract BACKGROUND Mutant isocitrate dehydrogenase (IDH) produces 2-hydroxyglutarate (D2HG) and causes metabolic reprograming, but so far, little is known about the role of mutant IDH and D2HG in de novo lipogenesis and fatty acids synthesis. In this study, to develop a feasible drug for IDH mutant glioma, we targeted Stearoyl-CoA desaturase 1 (SCD1) catalyzing the biosynthesis of monosaturated fatty acids (MUFA) to suppress IDH mutant glioma progression. Materials and METHODS We prepared genetically engineered glioma cell lines (U251 wild type: U251WT and U251 IDHR132H mutant: U251RH), normal human astrocytes (empty vector induced-NHA: NHAEV and IDHR132H mutant: NHARH) and patient derived cell lines. Lipid metabolic analysis was conducted by using LC-MS, and functional analysis for the role of SCD1 expression was investigated by RNA sequence and Western-blotting. RESULTS LC-MS analysis of extracted Endoplasmic Reticulum revealed that there was significantly higher amount of MUFA in IDH mutant than wild type. SCD1 expression was increased in IDH mutant compared to wild type due to D2HG-mediated upregulation of SCD1 in IDH mutant. Therefore, IDH mutant in which SCD1 expression level was higher than wild type indicated high sensitivity to SCD inhibitor, and apoptosis was highly induced in IDH mutant compared to wild type. RNA sequencing was performed in U251RH treated with SCD inhibitor compared to U251RH treated with DMSO, and lipid droplet metabolism-associated RNA expression was significantly changed in SCD inhibitor-treated U251RH. Based on the RNA sequence data, we checked lipid droplet in U251RH with presence or absence of SCD inhibitor, and lipolysis was induced by SCD inhibitor treatment, suggesting that SCD inhibition is associated with the apoptosis in IDH mutant via enhanced lipolysis mechanism. CONCLUSIONS D2HG produced in IDH mutant glioma directly induces SCD1 expression and enhances sensitivity to SCD inhibitor, which suggests that SCD inhibitor would be IDH mutant glioma-specific treatment strategy.

DDEL-07. DETERMINATION OF TOTAL AND UNBOUND LEVELS OF A POTENT INHIBITOR OF POLY ADP-RIBOSE POLYMERASE, NIRAPARIB, IN HUMAN PLASMA, CEREBROSPINAL FLUID AND BRAIN TUMOR BY A VALIDATED LC-MS/MS ASSAY

Abstract BACKGROUND Niraparib is an orally available inhibitor of poly ADP-ribose polymerase with high selectivity for PARP1 and PARP2. It is currently approved in the United States for the treatment of ovarian, fallopian tube, and primary peritoneal cancer. Herein we present the development and validation of an LC-MS/MS method for determination of total and unbound levels of niraparib in human plasma, cerebrospinal fluid (CSF) and glioblastoma tissue. METHODS Niraparib was extracted from human plasma, CSF and glioblastoma homogenate samples by protein precipitation with methanol. Unbound fractions of niraparib in plasma and brain tissues were determined by equilibrium dialysis. Validation was performed using Sciex ExionLC UHPLC system coupled with Sciex QTRAP 6500+ MS/MS detector using positive electrospray ionization mode. The method was validated according to FDA guidelines and CAP/CLIA regulations. RESULTS The method was validated for 1-10,000 nmol/L concentration range using plasma as matrix for all biospecimens. The chromatographic separation was performed on Phenomenex Kinetex™ PS C18 column with total run time of 3.5 min with gradient elution. Maximum coefficient of variation for intra- and inter-day precision was 10.6% and the accuracy was within 92.8-118.5% in all matrices. The analyte is stable in plasma and brain homogenate for at least 6 hours at room temperature (RT) and at least 32 days at -20°C. Stability of stock and working solutions was demonstrated for at least 21 hours (RT) and 41 days (4°C). The fraction unbound of niraparib in human plasma and brain were determined to be 0.15 and 0.05, respectively. CONCLUSIONS A sensitive and rapid bioanalytical method is developed and validated to quantify total and unbound niraparib levels in human plasma, CSF and glioblastoma tissue. This method is currently employed to assess plasma pharmacokinetics and CNS penetration of niraparib in newly-diagnosed glioblastoma and recurrent IDH1/2(+) ATRX mutant glioma patients (NCT05076513).

QOL-06. PREDICTING VENOUS THROMBOEMBOLISM IN PATIENTS WITH ADULT-TYPE DIFFUSE GLIOMAS

Abstract Venous thromboembolism (VTE) is a common paraneoplastic complication in patients with adult-type diffuse gliomas. VTE greatly diminishes quality-of-life, and can even be life-threatening. While effective VTE prediction models exist for other cancers, those models do not work for gliomas. Thus, there are no standard guidelines for thromboprophylaxis in glioma patients. To address this deficiency, arterial blood, tumor tissue, and comprehensive clinical-pathologic data (including next generation sequencing) were obtained from 258 newly diagnosed WHO grade 2-4 adult-type diffuse glioma patients at Northwestern Memorial Hospital. Forty-six out of 258 grade 2-4 glioma patients (17.8%) experienced a subsequent VTE. Intratumoral expression of two proteins that have been associated with VTE risk in other cancers, thromboplastin and podoplanin, was higher in IDH wild-type than IDH mutant gliomas. Tissue expression of both markers was associated with increased VTE risk, but in the circulation, only thromboplastin positively correlated with VTE. LASSO time-to-event analysis showed that older patient age, elevated body mass index, elevated WBC count, hypertension, a preoperative history of VTE, asthma, and higher WHO grade predicted increased postoperative VTE risk, whereas hypothyroidism, IDH mutation, and MGMT promoter methylation were negative risk factors. Together, these 10 variables generated a receiver operating characteristic curve for 3, 6, 9, and 12 months from original surgery resulting in estimated AUC value of 0.82 in the Northwestern discovery cohort, 0.72 in the UCLA validation cohort (N=68), and 0.68 in the Duke validation cohort (N=157). We created an online prediction tool based on this model, which estimates VTE risk up to 1 year from original surgery. To the best of our knowledge, this is the first externally validated VTE risk prediction tool designed specifically for adult-type diffuse glioma patients. This tool, based on readily obtainable information, provides treating physicians with more objective risk estimates on which to base thromboprophylaxis decision-making.

TMET-09. LOSS OF MAT2A COMPROMISES METHIONINE METABOLISM AND REPRESENTS A VULNERABILITY IN H3K27M MUTANT GLIOMAS

Abstract H3K27-mutant diffuse midline gliomas (DMGs) are defined as grade IV tumors by the World Health Organization. DMGs are inoperable and resistant to chemo/radio therapies. Median survival ranges from 8-11 months, with 2% of patients surviving beyond 5 years. H3K27M mutations lead to global epigenetic and transcriptional reprogramming driven by global loss of negative transcriptional regulator H3K27 trimethylation (H3K27me3). Loss of H3K27me3 is an initiating event in gliomagenesis. This disease lacks appropriate models to predict disease biology and response to treatment. Therefore, we developed a novel syngeneic H3K27M mouse model. An unbiased integrated systems biology approach identified that H3K27M but not isogenic controls relied on the amino acid methionine and the enzyme Methionine Adenosyltransferase 2A (MAT2A). MAT2A is a central regulator of one-carbon metabolism by converting methionine to S-adenosylmethionine (SAM), the universal methyl-donor for protein and nucleotide methylation reactions. In complementary genetic approaches, we applied these findings to patient-derived cell lines with the H3K27M mutation. We hypothesize that MAT2A abrogation, genetic/pharmacological, would alter DMG viability by disrupting the methylome. The current MAT2A sensitivity paradigm is based on Methylthioadenosine Phosphorylase (MTAP) deletion through a synthetic lethal mechanism. We provide a novel mechanism whereby H3K27M cells are sensitive to MAT2A loss, independent of MTAP and through Adenosylmethionine Decarboxylase 1 (AMD1) overexpression disrupting MAT2A regulation. This results in H3K27M cells having lower MAT2A protein levels, conferring a sensitivity by inhibiting residual MAT2A. Genetic/pharmacological aberrations to MAT2A resulted in reduced proliferation. Parallel H3K36me3 ChIP and RNA-sequencing identified loss of oncogenic and developmental transcriptional programs associated with MAT2A loss. In vivo syngeneic and patient-derived xenograft models with both inducible MAT2A knockdown or methionine restricted diets showed extended survival. These results suggest novel interactions between methionine metabolism and the epigenome of H3K27M gliomas and provide evidence that MAT2A, presents exploitable therapeutic vulnerabilities in histone mutant gliomas.

CSIG-05. A GABP DOMINANT NEGATIVE APPROACH TO THE REVERSAL OF TUMOR IMMORTALITY

Abstract Immortality is a fundamental hallmark of human cancer cells and therapeutic reversal is of great interest. Telomerase Reverse Transcriptase promoter (TERTp) mutations reactivate TERT expression, the rate limiting step in telomerase activity and cellular immortality. TERTp mutations are the most common non-coding mutation across all cancer types, including glioblastoma (GBM), oligodendroglioma, medulloblastoma, and high-grade meningioma. While prior telomerase therapies lack tumor selectivity and are poorly tolerated, TERTp mutations and their regulation offer a unique opportunity for tumor specific reversal of cellular immortality. The mutations generate a de novo E26 Transformation Specific (ETS) binding motif that recruits the GA-Binding Protein (GABP) multimeric complex to reactivate TERT expression. Initially we found a GABP tetramer reactivates the mutant TERTp, suggesting targeting only the tetramer may induce telomere shortening and tumor cell death. However, through knocking out this tetramer in cell cultures, we found that the GABP dimer is upregulated and maintains TERT expression. In the absence of both the dimer and one tetramer isoform, many tumor cells senesce or undergo cell death. However, some tumor cells escape this fate via upregulation of a second GABP tetrameric paralogue that maintains TERT expression. Therefore, to block all GABP complexes with a single approach, we devised a transactivation domain null, putative dominant negative GABP subunit that ultimately could be delivered to patients via a replicating retroviral system. Introduction of the construct into TERTp mutant glioma cells significantly reduced TERT expression while not altering expression in TERTp wildtype GBM cells, suggesting tumor selectivity. TERT expression was also reduced in a second TERTp mutant tumor type. We are currently investigating the long-term effects of the dominant negative GABP on telomere length and tumor immortality. Dominant negative GABP is a promising candidate for tumor selective reversal of cellular immortality by inhibiting mutant TERTp activation by all GABP multimeric forms.

Single-nucleus transcriptomics of IDH1- and TP53-mutant glioma stem cells displays diversified commitment on invasive cancer progenitors

Glioma is a devastating brain tumor with a high mortality rate attributed to the glioma stem cells (GSCs) possessing high plasticity. Marker mutations in isocitrate dehydrogenase type 1 (IDH1) and tumor protein 53 (TP53) are frequent in gliomas and impact the cell fate decisions. Understanding the GSC heterogeneity within IDH1- and TP53- mutant tumors may elucidate possible treatment targets. Here, we performed single-nucleus transcriptomics of mutant and wild-type glioma samples sorted for Sox2 stem cell marker. For the first time the rare subpopulations of Sox2 + IDH1- and TP53-mutant GSCs were characterized. In general, GSCs contained the heterogeneity root subpopulation resembling active neural stem cells capable of asymmetric division to quiescent and transit amplifying cell branches. Specifically, double-mutant GSCs revealed the commitment on highly invasive oligodendrocyte- and astroglia-like progenitors. Additionally, double-mutant GSCs displayed upregulated markers of collagen synthesis, altered lipogenesis and high migration, while wild-type GSCs expressed genes related to ATP production. Wild-type GSC root population was highly heterogeneous and lacked the signature marker expression, thus glioblastoma treatment should emphasize on establishing differentiation protocol directed against residual GSCs. For the more differentiated IDH1- and TP53-mutant gliomas we suggest therapeutic targeting of migration molecules, such as CD44.

Simultaneous Integrated Boost Technique for Pediatric High-Grade Gliomas: A Single-Institution Experience

<h3>Purpose/Objective(s)</h3> The prognosis for pediatric patients with high grade glioma (HGG) remains dismal despite aggressive multidisciplinary treatment, and there is marked variability regarding the optimal radiation approach. We report outcomes from a retrospective review of pediatric patients with HGG treated using a simultaneous integrated boost (SIB) technique at a single institution. <h3>Materials/Methods</h3> The medical records of patients ages 0–22y with biopsy-proven non-DIPG localized HGG treated with radiotherapy (RT) from January 2006 to May 2021 were retrospectively reviewed on an IRB approved protocol. Endpoints were Kaplan-Meier estimates of overall survival (OS) and progression free survival (PFS), and patterns of failure. <h3>Results</h3> There were 32 patients in the cohort; 24 patients had glioblastoma without further molecular characterization, 2 had H3 K27M mutant diffuse midline gliomas, 2 had G34R mutant glioma, 2 had glioblastoma with IDH1 mutation, 1 had high-grade astrocytoma, and 1 had high-grade glial neoplasm. Median age at diagnosis was 14.9 (1.9–21.5) years. Prior to RT, 6 patients obtained both spinal MRIs and lumbar punctures, 5 obtained spinal MRIs, and 1 had a lumbar puncture; none had evidence of distant metastases. All patients were treated with intensity modulated radiotherapy (IMRT) using SIB with two dose levels. The most common (n=25) dose to the high dose volume was 60 Gy (range, 40–60 Gy); the most common (n=24) dose to the low dose volume was 50 Gy (range, 30–55 Gy). Treatment was most commonly (n=26) delivered in 30 fractions (range, 15-33). One patient did not complete planned RT due to tumor hemorrhage. Median follow up for all patients was 17 (0–164.4) months. For patients who completed planned RT, median PFS was 20.1 (1–140) months from RT start. Median OS was 25.9 months with one- and two-year survival rates of 80% and 56%, respectively. Of 19 patients with known recurrence, 8 recurred locally, 7 recurred in the distant brain, and 4 developed leptomeningeal dissemination. Three patients were alive at 10 years after initial diagnosis; none of these patients were known to have tumors with IDH mutations. <h3>Conclusion</h3> This study explores outcomes in a cohort of pediatric HGG patients treated with SIB technique, with encouraging initial results. Future efforts will be necessary to identify additional molecular and treatment factors that impact outcome.

Isocitrate dehydrogenase (IDH) mutant gliomas: A Society for Neuro-Oncology (SNO) consensus review on diagnosis, management, and future directions.

Isocitrate dehydrogenase (IDH) mutant gliomas are the most common adult, malignant primary brain tumors diagnosed in patients younger than 50, constituting an important cause of morbidity and mortality. In recent years, there has been significant progress in understanding the molecular pathogenesis and biology of these tumors, sparking multiple efforts to improve their diagnosis and treatment. In this consensus review from the Society for Neuro-Oncology (SNO), the current diagnosis and management of IDH-mutant gliomas will be discussed. In addition, novel therapies, such as targeted molecular therapies and immunotherapies, will be reviewed. Current challenges and future directions for research will be discussed.

Oncometabolite d-2HG alters T cell metabolism to impair CD8+ T cell function.

Gain-of-function mutations in isocitrate dehydrogenase (IDH) in human cancers result in the production of d-2-hydroxyglutarate (d-2HG), an oncometabolite that promotes tumorigenesis through epigenetic alterations. The cancer cell-intrinsic effects of d-2HG are well understood, but its tumor cell-nonautonomous roles remain poorly explored. We compared the oncometabolite d-2HG with its enantiomer, l-2HG, and found that tumor-derived d-2HG was taken up by CD8+ T cells and altered their metabolism and antitumor functions in an acute and reversible fashion. We identified the glycolytic enzyme lactate dehydrogenase (LDH) as a molecular target of d-2HG. d-2HG and inhibition of LDH drive a metabolic program and immune CD8+ T cell signature marked by decreased cytotoxicity and impaired interferon-γ signaling that was recapitulated in clinical samples from human patients with IDH1 mutant gliomas.

P12.14.A The role of onco-metabolite (R2hydroxyglutarate) in the IDH mutant glioblastoma microenvironment

Abstract Background Recently, we found that the reactive astrocytes in the IDH wild type glioblastoma contribute to anti-tumor immunity and support pro-oncogenic signaling. Here, we characterized the transcriptomic signature of IDH1/2-mutant glioma associated astrocytes and determined a unique inflammatory transformation, profoundly different to astrocytes in IDH wildtype glioma patients due to an onco-metabolite R-2-hydroxyglutarate using Next generation sequencing and human ex-vivo slice model. Material and Methods We purified and transcriptionally profiled astrocytes from 9 patients with confirmed IDH1-R132H mutation, by means of RNA-sequencing and the data were analyzed using the established pipelines. We also used spatial transcriptomics to evidently show the spatial distribution of astrocytes in IDH-mutated/wildtype glioma samples. We validated our findings using human organotypic slice model inoculated with IDH-mutant cell line or treated with oncometabolite 2-hydroxy glutarate. Additionally, LC-MS was further used to give us a chart of neurotransmitters due to altered microenvironment. Results Our results from RNA sequencing showed a transcriptional transformation of reactive astrocytes within the microenvironment of IDH-mutated tumors compared to wildtype glioma by means of RNA-sequencing of purified astrocytes. And, using our established human neocortical GBM model inoculated with IDH mutant tumor and R-2HG treatment, we showed that we were able to activate inflammatory transcriptional programs in astrocytes, mediated by the presence of microglia. Further, by spatially mapping the transcriptomic profiles of purified microglia, we were able to confirm that microglia also demonstrate inflammatory activation in IDH mutated glioma. This inflammatory astrocyte transformation is associated with a loss of neurotransmitter homeostasis (disrupted levels of glutamate) in the treated sections, as has been previously reported in IDH mutated tumors. Additionally, R-H2G increased neuronal spiking rate in, pointing to potential excitotoxicity. Conclusion Our work provides a crucial contribution towards understanding the role of R-2HG in the IDH mutant glioma microenvironment and sheds light on the significant microenvironmental differences to IDH wild-type glioma.

Using quantitative MRI to study the association of isocitrate dehydrogenase (IDH) status with oxygen metabolism and cellular structure changes in glioma

ObjectiveTo investigate the characteristics of oxygen metabolism and the cellular structure of glioma using quantitative MRI to predict the isocitrate dehydrogenase 1 (IDH1) status and to further understand the biological characteristics of gliomas. MethodsIn this retrospective study, 94 patients with gliomas eventually received quantitative MRI measures to study oxygen metabolism. The oxygen metabolism biomarker maps (oxygen extraction fraction [OEF] and cerebral metabolic rate of oxygen [CMRO2]) and the tissue-cellular-specific (R2t*) MRI relaxation parameter were evaluated in different regions of glioma. ResultsMRI results showed differences in oxygen metabolism measures in all patients with gliomas of different IDH1 statuses. Compared to patients with IDH1 mutant gliomas, patients with IDH1 wild type gliomas showed increased (P < 0.01) CMRO2, OEF, cerebral blood volume [CBF], and R2t* measures in tumor regions, while only OEF, CBF and R2t* were found to be increased (P < 0.05) in the peritumoral area. OEF achieved the best performance for distinguishing IDH1 wild type and mutant gliomas in the tumor area (AUC = 0.732, P < 0.001). R2t* values correlated with Ki-67(R = 0.35, P < 0.001) in the tumor area, while no significant correlations between Ki-67 and R2t* were found in the peritumoral area (R = 0.19, P = 0.072). ConclusionQuantitative MRI has potential applications in studying the tumor and peritumoral areas of glioma, and it has the ability to predict and reveal the characteristics of oxygen metabolism and cellular structure in different regions of gliomas.

Deciphering diffuse glioma immune microenvironment as a key to improving immunotherapy results.

Immunotherapeutic approaches have yet to demonstrate their clinical efficacy in diffuse gliomas. Evidence is mounting that the central nervous system is subject to immune surveillance, but brain tumours manage to escape due to factors intrinsic to their tumoral immune microenvironment (TME). This review aims to discuss the recently characterized molecular bases of the glioma TME and the potentially actionable targets to improve immunotherapeutic results in these hard-to-treat cancers. Single-cell studies defined the composition of the glioma immune TME and its peculiarities compared with other solid cancers. In isocitrate dehydrogenase (IDH) wildtype gliomas, the TME is enriched in myeloid cells (monocyte-derived macrophages and resident microglia) with mainly immunosuppressive functions. Lymphocytes can infiltrate the glioma TME, but are exposed to multiple immunomodulating signals that render them in a state of deep exhaustion. IDH mutant gliomas produce the oncometabolite D-2-hydroxyglutarate with negative effects on leukocyte recruitment and function, resulting in the induction of an 'immune-desert' TME. Several molecular pathways have been recently identified in the induction of an 'immune-hostile' microenvironment in diffuse gliomas, unravelling potential vulnerabilities to targeted immunotherapies.