Abstract Background Oligodendrogliomas were previously defined by histology, but since 2016, the WHO has integrated molecular signatures to create the entity of IDH-mutant 1p/19q-codeleted oligodendroglioma. These tumors harbor recurrent genetic alterations—including IDH mutations, CIC and FUBP1 loss-of-function mutations, and PI3K-activating mutations—that collectively define a genetic profile distinct from IDH-mutant astrocytoma and IDH wild-type glioblastoma. The paucity of faithful model systems has impeded progress in advancing mechanistic insights and therapeutic development for oligodendroglioma. Results To test whether recurrent mutations found in oligodendroglioma patients would drive tumor formation in mice, we utilized in utero electroporation to introduce combinations of gain- and loss-of-function mutations into the developing cortex. Initial tests that included expression of IDH-and PIK3CA-mutants with Cic and Fubp1 loss (termed Oligo) were non-tumorigenic but biased cells towards glial lineages. Introducing Cdkn2a-loss, which is normally suppressed by IDH-associated epigenetic changes in human tumors, to the Oligo combination (termed Oligo-Cdkn2a) resulted in fully penetrant tumor formation. Histopathological review showed the Oligo Cdkn2a-loss mouse model resembled grade II/III oligodendroglioma with rounded nuclei and open chromatin. If Trp53-loss is used (termed Oligo-Trp53) in place of Cdkn2a-loss, tumor histology shifts to a more astrocytic phenotype with angulated nuclei with denser chromatin, demonstrating a direct impact of Trp53-loss on histological features. Bulk RNA-sequencing verifies metabolic alterations driven by the presence of IDH-mutations in Oligo-Cdkn2a mouse models compared to an IDH wild-type GBM model. Single-cell transcriptional profiling shows that Oligo-Cdkn2a tumors contain an increased population of tumor cells that resemble more differentiated oligodendrocytes. Examination of the tumor immune microenvironment within Oligo-Cdkn2a and IDHWT GBM mouse models is being performed to determine changes in immune cell infiltration and transcriptional programs. Conclusion We have created, to our knowledge, the first murine model of IDH-mutant 1p/19q-codeleted oligodendroglioma that incorporates Cic loss. This immune-competent mouse model of IDH-mutant 1p/19q-codeleted oligodendroglioma offers a powerful new tool for interrogating tumor biology and evaluating preclinical therapeutic strategies.

Oligodendroglioma is a primary central nervous system tumor classified by the presence of isocitrate dehydrogenase (IDH) mutations and codeletion of 1p/19q. Here we describe the generation of an IDH-mutant 1p/19q-codeleted oligodendroglioma mouse model using in utero electroporation. We identified IDH1R132H, PIK3CAE545K, Cic KO, Fubp1 KO and Cdkn2a KO as the optimal combination (termed OligoCdkn2a) to drive fully penetrant tumors that histologically resemble human grade II/III IDH-mutant, 1p/19q-codeleted oligodendroglioma. Replacing Cdkn2a with Trp53 loss in this mouse model shifted tumor histology towards high grade astrocytoma. OligoCdkn2a tumors displayed metabolic and transcriptional changes associated with IDH and CIC mutations, and single cell sequencing identified a bias towards oligodendrocyte differentiation compared to an IDH wild-type glioblastoma mouse model. OligoCdkn2a tumors represent the first mouse model system to recapitulate the genetic, histological and transcriptional features of human IDH-mutant 1p/19q-codeleted oligodendrogliomas, offering a platform to further dissect tumor biology and test new therapeutic strategies.

Hyperactivation of glucose metabolism to lactate is a metabolic hallmark of cancer. However, the functional role of lactate in pediatric diffuse midline glioma (DMG) cells is unclear. Here, using stable isotope tracing and loss-of-function studies in clinically relevant patient-derived DMG models, we show that the oncogenic histone H3K27M mutation epigenetically up-regulates the rate-limiting glycolytic enzyme phosphoglycerate kinase 1 (PGK1) and drives lactate production from [U-13C]-glucose in DMGs. Mechanistically, lactate posttranslationally activates the nucleoside diphosphate kinase NME1 through lactylation and facilitates the synthesis of nucleoside triphosphates that are essential for DNA replication and tumor proliferation. This mechanistic link between glycolysis and nucleotide biosynthesis provides the opportunity for deuterium metabolic imaging of tumor growth and response to therapy. Spatially mapping 2H-lactate production from [6,6-2H]-glucose allows visualization of the metabolically active tumor lesion and provides an early readout of response to standard of care and targeted therapy that precedes extended survival and reflects pharmacodynamic alterations in tumor tissues in preclinical DMG models in vivo at clinical field strength (3 T). Overall, we have identified an H3K27M-lactate-NME1 axis that drives DMG proliferation and facilitates noninvasive in vivo metabolic imaging of DMGs.

Background The 1p/19q codeletion is a hallmark of oligodendrogliomas. The goal of this study was to exploit the metabolic vulnerabilities induced by the 1p/19q codeletion for the treatment and imaging of oligodendrogliomas.Methods We used stable isotope tracing, mass spectrometry, and genetic and pharmacological approaches to interrogate [U-13C]-glucose metabolism in patient-derived oligodendroglioma models (SF10417, BT88, BT54, TS603, NCH612). We examined whether tracing [6,6 '-2H]-glucose metabolism using deuterium metabolic imaging (DMI) provided an early readout of treatment response.Results The glycolytic enzyme enolase 1 (ENO1; chromosome 1p36.23) was downregulated in patient-derived oligodendroglioma cells and patient tissue due to the 1p/19q codeletion and histone hypermethylation. Conversely, inactivation of the CIC transcriptional repressor, driven by activated mitogen-activated protein kinase (MAPK) signaling, upregulated the ENO2 isoform specifically in oligodendrogliomas. Genetic ablation of ENO2 or pharmacological inhibition using POMHEX inhibited proliferation with nanomolar potency but was not cytotoxic to oligodendroglioma cells. Mechanistically, ENO2 loss abrogated [U-13C]-glucose metabolism to lactate but shunted glucose toward biosynthesis of serine and purine nucleotides, an effect that was driven by the rate-limiting enzyme for serine synthesis, phosphoglycerate dehydrogenase (PHGDH). Importantly, combining the PHGDH inhibitor D8 with POMHEX resulted in synthetic lethality in vitro and induced tumor regression in vivo. Furthermore, DMI of lactate production from [6,6 '-2H]-glucose provided an early readout of response to combination therapy that preceded MRI-detectable alterations and reflected extended survival.Conclusions We have identified ENO2 and PHGDH as metabolic vulnerabilities induced by the 1p/19q codeletion in oligodendrogliomas and [6,6 '-2H]-glucose as a noninvasive tracer of early response to therapy.

Abstract ►Diffuse midline gliomas (DMGs) are uniformly lethal pediatric brain tumors that are driven by the H3K27M mutation. Since the brain is intrinsically deficient in amino acids, including serine, we investigated whether the H3K27M mutation rewires metabolism to facilitate tumor growth within this nutrient­restricted environment. Across murine and patient-derived DMG models, as well as patient tissue, we found that the H3K27M mutation upregulated the rate-limiting enzyme in serine synthesis, phosphoglycerate dehydrogenase (PHGDH). Both genetic ablation of PHGDH and treatment with a first-in-class molecular glue degrader (LXH-3-71) abolished serine synthesis from [U13C]-glucose in DMG cells. Loss of serine production, in turn, depleted nucleotides, sphingolipids, phospholipids, glutathione, and alpha-ketoglutarate. Mechanistically, the reduction in alpha-ketoglutarate, which is an obligate cofactor for histone demethylases, restored histone hypermethylation, thereby counteracting a key epigenetic consequence of the H3K27M mutation. In addition, the broader metabolic collapse led to oxidative stress and lipid peroxidation, resulting in ferroptotic cell death. Using in vivo stable-isotope tracing and spatial metabolomics, we confirmed that LXH-3-71 crossed the blood-brain barrier and abrogated serine synthesis in mice bearing intracranial DMG xenografts. Importantly, LXH-3-71 induced marked tumor regression and significantly prolonged survival in multiple intracranial DMG models in vivo. Finally, to develop a clinically translatable pharmacodynamic and response biomarker, we paired our therapy with non-invasive imaging. Deuterium metabolic imaging of glucose metabolism provided an early readout of response to LXH-3-71, preceding MRI-detectable changes and predicting survival benefit, in mice bearing intracranial DMG xenografts. Since deuterium metabolic imaging is clinically implementable and LXH-3-71 is brain penetrant, our therapy and imaging biomarker have the potential for clinical translation. Collectively, our studies mechanistically validate PHGDH as an H3K27M-induced metabolic vulnerability and present an integrated metabolic therapy and imaging strategy for DMGs.

Gliomas are devastating brain tumors in adults and children. The metabolic milieu of the brain shapes the immune microenvironment, which is dominated by tumor-associated myeloid cells (TAMs; both blood-derived macrophages and brain-resident microglia). The brain is unique, with >25-fold higher concentration of fructose in the cerebrospinal fluid relative to the plasma. However, whether fructose influences glioma growth is unclear. Therefore, our goal was to delineate the role of fructose in gliomas. Quantitative flow cytometry showed that TAMs from murine gliomas expressed high levels of the fructose transporter SLC2A5 (>150,000 molecules/cell), unlike tumor cells, astrocytes, T cells, or endothelial cells (<2,000). Human TAMs generated by exposing human microglia to conditioned media from patient-derived glioma cells also upregulated SLC2A5, confirming clinical relevance. Stable isotope tracing demonstrated lactate production from 13C-fructose in murine and human TAMs, but not tumor or other non-neoplastic cells. Notably, these findings were oncogene-independent and conserved across adult (glioblastoma, IDH mutant) and pediatric (H3K27M, BRAFV600E) gliomas. Strikingly, genetic or pharmacological ablation of SLC2A5 in murine and human TAMs abrogated lactate production and reduced T cell suppression in vivo. Mechanistically, lactate activated immunosuppressive gene expression (HMOX1, LGALS3, TGFB1, CD274, HAVCR2) in TAMs via promoter histone H3K18 lactylation. Next, we questioned whether deuterium metabolic imaging, which is a clinical-stage method of tracing metabolism, could be used for imaging immunosuppressive TAMs. Human and murine TAMs polarized to an immunosuppressive phenotype produced lactate from [6,6-2H]-fructose, unlike those polarized to a proinflammatory state. Importantly, depleting TAMs using the CSF1R inhibitor sotuletinib or rewiring them to a proinflammatory state using an anti-TIM3 antibody resulted in the loss of lactate production in vivo, confirming that [6,6-2H]-fructose is a unique tracer of immunosuppressive TAMs in gliomas. Collectively, we leverage a mechanistic understanding of fructose metabolism for non-invasive metabolic imaging of TAM-driven immunosuppression in vivo.

Myeloid-rich microenvironment is a hallmark of glioblastoma (GBM), the most aggressive and lethal brain malignancies in adults, leading to profound immunosuppression and therapy resistance. Metabolic dysregulation in tumor-associated myeloid cells (TAMCs) has been recognized as a key player driving immune evasion and supporting their pro-tumorigenic roles. Our single-cell RNA sequencing analysis and multiplex immunofluorescence have demonstrated high overexpression of monocarboxylate transporter 4 (MCT4), a key lactate transporter, in TAMCs in both murine and human GBMs, which may greatly contribute to TAMC-mediated GBM immunosuppression. To enable a myeloid-specific targeting of MCT4 in GBM, we have developed MCT4 nano-therapeutics using our antibody-directed immune targeting (ADIT) nano-platform. Lipid nanoparticles were surface functionalized with anti-PD-L1 antibody and encapsulated with small interfering RNA (siRNA) that targets MCT4. Our data indicated a robust and durable MCT4 inhibition, achieving over 90% gene knockdown efficiency. In vitro, MCT4 nano-therapeutics effectively targeted and reprogrammed immunosuppressive TAMCs, and restored CD8+ T cell proliferation and activation. In vivo, MCT4 nano-therapeutics through intracranial administration significantly improved the anti-glioma effectiveness of radiotherapy in CT-2A glioma-bearing C57 mice, leading to enhanced brain tumor infiltration of effector CD8+ T cells and CD103+ dendritic cells as well as much prolonged animal survival. When further combined with anti-CTLA-4 immune checkpoint therapy, over 60% of mice were cured from CT-2A glioma, and the long-term survivor animals developed anti-glioma immunological memory. Besides intracranial injections, we have also demonstrated the effectiveness of MCT4 nano-therapeutics through a systemic intranasal delivery route. These findings altogether may highlight a great potential of targeting lactate transport for modulating myeloid-mediated GBM immunosuppression and create a new immunotherapeutic approach to improve the existing standard treatments and immunotherapies for GBM.

Neuronal activity and synchrony in both normal and pathological states are regulated by excitatory and inhibitory synaptic inputs, determined by ion channel permeability across the cell membrane. This study investigates neuron-glioma cross-talk in chloride flux and the role of chloride, the most abundant neuronal anion, in influencing neuronal excitability and glioblastoma (GBM) pathophysiology. We hypothesized that GBM’s aggressive proliferative and invasive phenotype is linked to chloride dysregulation and that inhibiting glioma chloride flux could reduce tumor proliferation and glioma-induced neuronal hyperexcitability. The direct effects of chloride flux on glioma growth and invasion were tested using optogenetic stimulation of chloride opsin-expressing glioma cells both in vitro and implanted in vivo in patient-derived xenograft (PDX) mice. To explore chloride cross-talk mechanisms between neurons and glioma cells, we performed single-nucleus RNA sequencing (sNuc-seq) of GBM-neuron co-cultures, analyzing 54,000 cells, and revealed the upregulation of genes involved in chloride homeostasis, including the voltage-gated ClC-3 chloride channel and the sodium-potassium-chloride cotransporter 1 (NKCC1). Mass spectrometry-based proteomic analysis of culture supernatant was performed to identify mechanistic targets and paracrine factors mediating chloride transfer. Light-activated chloride pumping into glioma cells significantly reduced tumor proliferation in vitro and in vivo, underscoring the link between intracellular chloride concentration and tumor growth. sNuc-seq also showed upregulation of NKCC1 and the ligand-gated chloride channel GABAA receptor in tumor-associated neurons co-cultured with NKCC1-overexpressing glioma cells, suggesting shared chloride signaling mechanisms in the tumor microenvironment. Proteomic profiling of conditioned media identified glioma-derived MK2 (MAPKAPK2) as a driver of neuronal NKCC1 upregulation and chloride dysregulation. Pretreatment of GBM cells with the MK2 inhibitor ralimetinib restored chloride balance and reduced neuronal hyperexcitability. Collectively, these findings reveal a paracrine mechanism of chloride transfer between glioma cells and neurons, highlighting chloride signaling as a novel therapeutic target to inhibit neuronal hyperexcitability and tumor proliferation in glioblastoma multiforme (GBM).

EGFR-altered H3K27M-mutant diffuse midline glioma (H3K27M-DMG) represent a recently recognized yet poorly understood subpopulation of H3K27M-DMG characterized by increased malignancy. To better understand the role of EGFR signaling in this patient population, we generated isogenic H3K27M-DMG cell lines expressing wild-type and commonly observed activating mutant isoforms of EGFR (p.A289T, p.A767delinsASVG). EGFR alterations increased H3K27M-DMG cell proliferation in vitro and in in vivo mouse flank tumor xenograft models. Surprisingly, overexpression of EGFR alterations further decreased H3K27M-associated H3K27me3 and significantly downregulated “late” stemness genes (e.g., PDGFRA, SOX10, OLIG2). Furthermore, gene expression programs of EGFR-altered H3K27M-DMG mapped to “early” OPC-like-3 cells that are enriched in brainstem H3K27M-DMG tumors in single-cell RNA-sequencing datasets. Mechanistically, EGFR-altered H3K27M-DMG cells decrease chromatin accessibility and expression of lactate dehydrogenase A (LDHA), leading to decreased L-2-hydroxyglutarate (L-2HG) metabolite levels, a known inhibitor of the JmjC domain family of histone demethylases. Consistent with this, EGFR alterations in H3K27M-DMG cells conferred resistance to the ClpP agonist ONC201 in vitro, whose efficacy in H3K27M-DMG depends on LDHA-mediated production of L-2HG. Indeed, through secondary analysis of clinical outcomes and tumor sequencing from the Phase I ONC014 trial (NCT03416530), we identified high expression of wild-type EGFR and EGFR mutations as key biomarkers of ONC201 sensitivity. EGFRHIGH (“OPC-like-3”) expression was associated with negative radiographic responses while PDGFRAHIGH (“OPC-like-1”) expression correlated with positive radiographic responses. Pharmacologic inhibition of EGFR (EGFRi) variably restored H3K27me3, supporting the idea that EGFR signaling reinforces the hypomethylated state. Treatment of an in vivo orthotopic model of H3K27M-DMG resulted in combinatorial survival benefit with ONC201 and EGFRi, with multiple additional in vivo studies ongoing. Our findings demonstrate a previously undiscovered role of EGFR in the metabolic suppression of epigenetic differentiation, providing a novel pathway to therapeutically target EGFR-altered H3K27M-DMG and OPC-like-3 cell sub-populations of all H3K27M-DMG.

Oncogenes hyperactive lactate production, but the mechanisms by which lactate facilitates tumor growth are unclear. Here, we demonstrate that lactate is essential for nucleotide biosynthesis in pediatric diffuse midline gliomas (DMGs). The oncogenic histone H3K27M mutation upregulates phosphoglycerate kinase 1 (PGK1) and drives lactate production from [U- 13 C]-glucose in DMGs. Lactate activates the nucleoside diphosphate kinase NME1 via lactylation and promotes the synthesis of nucleoside triphosphates essential for tumor proliferation. Importantly, we show that this mechanistic link between glycolysis and nucleotide biosynthesis provides a unique opportunity for deuterium metabolic imaging of DMGs. Spatially mapping 2 H-lactate production from [6,6- 2 H]-glucose allows visualization of the metabolically active tumor lesion and provides an early readout of response to standard-of-care radiation and targeted therapy that precedes extended survival and reflects pharmacodynamic alterations at the tissue level in preclinical DMG models in vivo at clinical field strength (3T). In essence, we have identified an H3K27M-lactate-NME1 axis that promotes DMG proliferation and facilitates non-invasive metabolic imaging of DMGs. STATEMENT OF SIGNIFICANCE:This study establishes a role for lactate in driving nucleotide biosynthesis in DMGs. Importantly, imaging lactate production from glucose using DMI provides a readout of tumor proliferation and early response to therapy in clinically relevant DMG models. Our studies lay the foundation for precision metabolic imaging of DMG patients.

Telomerase Reverse Transcriptase promoter (TERTp) mutations enable tumor cell immortality in millions of cancer patients annually. TERTp mutations are the most common non-coding mutations across all cancers, including glioblastoma, oligodendroglioma, medulloblastoma, and high-grade meningioma. A multitude of TERTp mutations, including the two hotspots, create de novo E26 transformation specific transcription factor binding sites. Among the 28 family members, we have shown that only the GA-binding protein (GABP, composed of DNA binding GABPA and transactivating GABPB subunits) activates the mutant TERTp. Prior therapies targeting the telomerase RNA component, TERC, lacked tumor selectivity and were poorly tolerated. The GABP-mediated reactivation of the mutated TERTp presents a unique therapeutic opportunity for tumor specific reversal of cellular immortality. Breakthroughs in the design of biological proteolysis-targeting chimera (bioPROTACs) offer a new approach to selectively degrade previously intractable targets such as transcription factors. We combined in silico protein-protein interaction modeling via AlphaFold and experimental validation to identify a minimal GABPB to bind GABPA and fused it with an E3 ubiquitin ligase. Introduction of this GABPA bioPROTAC into TERTp mutant glioblastoma cells depleted GABPA protein and eliminated GABPA binding to the mutant TERT promoter, reducing transcriptional activating (H3K4me3) and increasing suppressive (H3K27me3) histone marks. Reversion of mutant TERTp to an epigenetically silenced state reduced TERT expression by 73% to 95% and shortened tumor cell telomeres in a promoter mutation-specific manner. The GABP bioPROTAC reduced tumor growth and improved survival of mice bearing an orthotopic xenograft of TERTp mutated glioblastoma cells. The effects of the bioPROTAC were validated in vivo via magnetic resonance imaging of metabolic correlates of TERT expression. These data demonstrate that this GABP bioPROTAC potently degrades GABPA, leading to tumor-specific silencing of telomerase expression, and a reversal of glioblastoma tumor cell immortality. This artificial intelligence-guided approach may be applicable to other intractable cancer specific targets.

Understanding the mechanisms by which oncogenic events alter metabolism will help identify metabolic weaknesses that can be targeted for therapy. Telomerase reverse transcriptase (TERT) is essential for telomere maintenance in most cancers. Here, we show that TERT acts via the transcription factor forkhead box O1 (FOXO1) to upregulate glutamate-cysteine ligase (GCLC), the rate-limiting enzyme for de novo biosynthesis of glutathione (GSH, reduced) in multiple cancer models, including glioblastoma (GBM). Genetic ablation of GCLC or pharmacological inhibition using buthionine sulfoximine (BSO) reduces GSH synthesis from [U-13C]-glutamine in GBMs. However, GCLC inhibition drives de novo pyrimidine nucleotide biosynthesis by upregulating the glutamine-utilizing enzymes glutaminase (GLS) and carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotatase (CAD) in an MYC-driven manner. Combining BSO with the glutamine antagonist JHU-083 is synthetically lethal in vitro and in vivo and significantly extends the survival of mice bearing intracranial GBM xenografts. Collectively, our studies advance our understanding of oncogene-induced metabolic vulnerabilities in GBMs.

Mutations in isocitrate dehydrogenase (IDHm) define a distinct molecular class of gliomas. IDHm converts α-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (D-2HG), which drives tumorigenesis. The IDHm inhibitor vorasidenib suppresses D-2HG production and extends progression-free survival in some, but not all, IDHm glioma patients. Here, using clinically relevant patient-derived IDHm models and patient tissue, we show that phosphoglycerate dehydrogenase (PHGDH) drives intrinsic resistance to vorasidenib by promiscuously converting α-KG to D-2HG and maintaining D-2HG concentration despite IDHm inhibition. Silencing PHGDH sensitizes resistant models to vorasidenib, while conversely, overexpressing PHGDH induces vorasidenib resistance in sensitive models. Importantly, deuterium metabolic imaging of D-2HG production from diethyl-[3,3'- 2 H]-α-ketoglutarate provides an early readout of response and resistance to vorasidenib that is not available by anatomical imaging in vivo. Collectively, we have identified PHGDH-driven D-2HG production as an intrinsic mechanism of resistance to vorasidenib and diethyl-[3,3'- 2 H]α-ketoglutarate as a non-invasive tracer for interrogating intrinsic resistance in IDHm gliomas. STATEMENT OF SIGNIFICANCE:Vorasidenib, which suppresses D-2HG production, is the first precision therapy to be approved for IDHm glioma patients. We show that PHGDH-driven restoration of D-2HG production mediates intrinsic resistance to vorasidenib in IDHm gliomas. Importantly, deuterium metabolic imaging of D-2HG production from diethyl-[3,3'- 2 H]-α-ketoglutarate enables non-invasive assessment of resistance in IDHm gliomas.