Abstract ZFTA-RELA ependymomas are highly aggressive brain tumors with significant mortality. These tumors are characterized by the oncogenic fusion of a putative chromatin remodeler ZFTA and the NFκB effector RELA. Using a comprehensive metabolic screen, we discovered that ZFTA-RELA cells generate itaconate, a metabolite linked to the TCA cycle. Although itaconate is a well-known immunomodulatory metabolite produced by macrophages, its production and function within tumor cells have been unclear. We found that itaconate is synthesized by Aconitate Decarboxylase-1 (ACOD1), and that ZFTA-RELA induces ACOD1 expression in an NFκB-dependent manner. Itaconate production in turn supports a coupled metabolic-epigenetic feed-forward loop that sustains pathogenic ZFTA-RELA fusion expression through H3K4me3-dependent, epigenetic activation. To provide the metabolic input required for itaconate synthesis, ZFTA-RELA tumors suppress PTEN expression to activate PI3K/AKT signaling pathway. The increased glutaminolysis in these tumors supplied the carbon needed for itaconate generation. As a result, inhibiting glutamine metabolism reduces pathogenic ZFTA-RELA levels and shows strong therapeutic efficacy in multiple in vivo models. Moreover, combining glutamine antagonists with PI3K/mTOR inhibitors prevents spinal metastasis. Overall, our findings show that ZFTA-RELA ependymomas hijack the macrophage-associated itaconate metabolic pathway to epigenetically reinforce expression of the ZFTA-RELA fusion driver, identifying itaconate as an oncometabolite. These results highlight itaconate upregulation as an unrecognized driver of ZFTA-RELA ependymoma and point to new therapeutic avenues for children affected by this devastating disease, while broadening our understanding of oncometabolites as a distinct class of cancer dependencies. Citation Format: Siva Kumar Natarajan, Joanna Lum, James Haggerty-Skeans, Minal Nenwani, Sanjana Eyunni, Mateus Mota, Jill Bayliss, Akash Deogharkar, Erin Hamanishi, Simon Hoffman, Eleanor Young, Qiuyang Zhang, Rijul Mehta, Abhijit Parolia, Peter Sajjakulnukit, Robert Doherty, Carl Koschmann, Arul M. Chinnaiyan, Costas Andreas Lyssiotis, Deepak Nagrath, Sriram Venneti. Itaconate acts as an oncometabolite to drive lethal pediatric ependymomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3285.

PDGFRA alterations define a high-risk subset of high-grade glioma (HGG), yet targeted therapies have yielded limited and transient benefit. Here, we show that the CNS-penetrant PDGFRA inhibitor avapritinib induces sustained MAPK pathway activation at supratherapeutic dosing, revealing a therapy-induced adaptive vulnerability. High-dimensional kinome profiling (>900 nodes) and in vivo studies demonstrate robust, dose-dependent ERK activation following avapritinib treatment. This response is enriched in cycling oligodendrocyte precursor cell-like (OPC-like) tumor populations and promotes survival through ERK-dependent stabilization of the anti-apoptotic protein MCL-1. Modeling of clinically relevant PDGFRA variants reveals that D842V-mutant tumor cells exhibit heightened MAPK activation and potential for MAPK co-targeting. Rational combination strategies suppress this adaptive signaling, with MEK inhibition producing durable pathway suppression and significant survival benefit in vivo. Translation to patients demonstrates feasibility and early clinical activity, including a sustained complete regression in an unresected PDGFRA-mutant HGG treated with avapritinib and the MEK1/2 inhibitor selumetinib. Together, these findings identify adaptive MAPK reactivation as a targetable liability and support combined PDGFRA-MAPK inhibition as a therapeutic strategy.

ZFTA-RELA+ ependymomas are malignant brain tumours defined by fusions formed between the putative chromatin remodeller ZFTA and the NF-κB mediator RELA1. Here we show that ZFTA-RELA+ cells produce itaconate, a key macrophage-associated immunomodulatory metabolite2. Itaconate is generated by cis-aconitate decarboxylase 1 (ACOD1; also known as IRG1). However, the production of itaconate by tumour cells and its tumour-intrinsic role are not well established. ACOD1 is upregulated in a ZFTA-RELA-dependent manner. Functionally, itaconate enables a feed-forward system that is crucial for the maintenance of pathogenic ZFTA-RELA levels. Itaconate epigenetically activates ZFTA-RELA transcription by enriching for activating H3K4me3 via inhibition of the H3K4 demethylase KDM5. ZFTA-RELA+ tumours enhance glutamine metabolism to supply carbons for itaconate synthesis. Antagonism of ACOD1 or glutamine metabolism reduces pathogenic ZFTA-RELA levels and is potently therapeutic in multiple in vivo models. Mechanistically, ZFTA-RELA epigenetically suppresses PTEN expression to upregulate PI3K-mTOR signalling, a known driver of glutaminolysis. Finally, suppression of ACOD1 or a combination of glutamine antagonism with PI3K-mTOR inhibition abrogates spinal metastasis. Our data demonstrate that ZFTA-RELA+ ependymomas subvert a macrophage-like itaconate metabolic pathway to maintain expression of the ZFTA-RELA driver, which implicates itaconate as a candidate oncometabolite. Taken together, our results position itaconate upregulation as a previously unappreciated driver of ZFTA-RELA+ ependymomas. Our work has implications for future drug development to reduce pathogenic ZFTA-RELA expression for this brain tumour, and will advance our understanding of oncometabolites as a new class of therapeutic dependencies in cancers.

Abstract Background Diffuse midline glioma (DMG) is a lethal pediatric brain tumor driven by the H3K27M oncohistone, which disrupts epigenetic regulation and promotes tumor proliferation. While prior studies show that H3K27M is essential for tumor initiation, its role in established tumors, tumor microenvironment (TME) regulation, and therapeutic response remain unclear. Methods Here, we developed inducible and reversible H3.3K27M and H3.1K27M cell and mouse models to study oncohistone-dependent effects on tumor growth, recurrence, and the immune/stromal microenvironment. We generated a tetracycline-inducible PiggyBac-based oncohistone expression cassette in patient- and murine-derived models and validated inducible and reversible H3K27M expression. Results Re-expression of H3K27M in knockout cells induced morphological changes and suppressed astrocytic markers. Chromatin accessibility profiling revealed distinct states between ON, OFF, and OFF–ON groups, including PD1-mediated immunosuppressive mechanisms associated with H3K27M expression. Single-cell RNA sequencing demonstrated that the oncohistone reshapes the TME. H3K27M expression promotes tumor–neuron interactions, enhances neuronal excitability, excitatory/inhibitory imbalance, and synaptic connectivity that supports tumor proliferation. These effects are associated with increased glutamatergic signaling and enhanced tumor–neuron coupling through glutamate transport and receptor pathways, including EAAT1 ( SLC1A3 ) and AMPARs ( GRIA3 ). Conversely, H3K27M inhibition reduces neuronal excitation, disrupts tumor-associated signaling, and partially restores neuron–neuron and neuron–immune communications. These findings identify H3K27M as a key driver of excitatory neuron-to-tumor coupling and immunosuppression in DMG. Conclusions Overall, our findings demonstrate that H3K27M extensively reshapes TME in DMG and support direct oncohistone targeting as a potential therapeutic strategy, including potential CRISPR-based or small-molecule approaches for patients with H3K27M-mutant DMG. Key Points We developed inducible and reversible H3K27M DMG models to investigate the role of H3K27M in the tumor microenvironment. H3K27M promotes tumor–neuron communication, while its inhibition disrupts these interactions, supporting H3K27M-targeted therapies for DMG. Importance of Study Diffuse midline glioma (DMG) remains one of the deadliest pediatric brain tumors, with limited effective treatment options and poor patient survival. Although the H3K27M oncohistone is recognized as a key driver of tumor initiation, its role in maintaining tumor progression and shaping the tumor microenvironment is unclear. In this study, we developed inducible and reversible H3.3K27M and H3.1K27M murine and patient-derived DMG cell- and mouse-models that enabled precise control of the oncohistone expression. Using these models, we demonstrate that H3K27M actively promotes tumor–neuron interactions, neuronal excitability, and glutamatergic signaling pathways that support tumor growth. Importantly, inhibition of H3K27M disrupted these tumor-associated signaling networks and partially restored neuron–immune communication within the tumor microenvironment. Together, these findings demonstrate that H3K27M extensively reshapes the tumor microenvironment in these Diffuse Midline Gliomas and provides strong rationale for directly targeting the oncohistone as a therapeutic strategy for patients with H3K27M-mutant DMG. Lay Summary Diffuse Midline Glioma (DMG) is a devastating childhood brain cancer. Despite decades of research, radiation remains the primary treatment and provides only temporary benefit. Most DMGs carry a mutation called H3K27M, which is an attractive target for new treatments such as directly inhibiting or removing this mutation using gene-editing. However, it remains unclear whether inhibiting H3K27M alone will be sufficient to stop the growth of established tumors. In this study, we developed human and mouse models that allow H3K27M to be turned on and off. We found that H3K27M helps tumors communicate with surrounding cells, particularly neurons. Inhibiting H3K27M disrupted tumor-promoting interactions and partially restored normal communication, supporting direct H3K27M-targeted therapies as a promising strategy for children with DMG.

ZFTA-RELA ependymomas are malignant brain tumors that are frequently lethal. They are defined by fusions formed between the putative chromatin remodeler ZFTA and the NFκB-mediator-RELA. Through a comprehensive metabolic screen, we identified that ZFTA-RELA cells produced itaconate, a TCA-cycle related metabolite. Itaconate is a key macrophage-associated immunomodulator metabolite. However, itaconate production by tumor cells and its tumor-intrinsic role are not well-established. Itaconate is synthesized by the enzyme Aconitate Decarboxylase-1 (ACOD1) and ZFTA-RELA upregulated ACOD1 in an NFκB-dependent manner. Additionally, itaconate production enabled an integrated metabolic/epigenetic feed-forward system that maintained pathogenic ZFTA-RELA fusion expression through epigenetic activation. To supply the metabolic fuel needed to generate itaconate, ZFTA-RELA tumors epigenetically activated PI3K/mTOR signaling to enhance glutaminolysis, which provided the carbons necessary for itaconate synthesis. Consequently, antagonizing glutamine metabolism lowered pathogenic ZFTA-RELA levels and was potently therapeutic in multiple in vivo models. Finally, combining glutamine antagonism with PI3K/mTOR inhibition abrogated spinal metastasis. Our data demonstrate that ZFTA-RELA ependymomas subvert a macrophage-like itaconate metabolic pathway to epigenetically maintain expression of the ZFTA-RELA fusion driver, implicating itaconate as an oncometabolite. Taken together, our results position itaconate upregulation as a previously unappreciated driver of ZFTA-RELA ependymomas. This study, therefore, has implications for future drug development for children with this devastating brain tumor and will further our understanding of oncometabolites as a novel class of therapeutic dependencies in cancers.

Background Radiotherapy (RT) is the primary treatment for diffuse midline glioma (DMG), a lethal pediatric malignancy defined by histone H3 lysine 27-to-methionine (H3K27M) mutation. Based on the loss of H3K27 trimethylation producing broad epigenomic alterations, we hypothesized that H3K27M causes a functional double-strand break (DSB) repair defect that could be leveraged therapeutically with PARP inhibitor and RT for selective radiosensitization and antitumor immune response. Methods H3K27M isogenic DMG cells and orthotopic brainstem DMG tumors in immune deficient and syngeneic, immune competent mice were used to evaluate the efficacy and mechanisms of PARP1/2 inhibition by olaparib or PARP1-selective inhibition by AZD9574 with concurrent RT. Results H3K27M mutation caused a homologous recombination repair (HRR) defect characterized by impaired RT-induced K63-linked polyubiquitination of histone H1 and inhibition of HRR protein recruitment. H3K27M DMG cells were selectively radiosensitized by olaparib in comparison to isogenic controls, and this effect translated to efficacy in H3K27M orthotopic brainstem tumors. Olaparib and RT induced an innate immune response and induction of NK cell (NKG2D) activating ligands leading to increased NK cell-mediated lysis of DMG cells. In immunocompetent syngeneic orthotopic DMG tumors, either olaparib or AZD9574 in combination with RT enhanced intratumoral NK cell infiltration and activity in association with NK cell-mediated therapeutic responses and favorable activity of AZD9574. Conclusions The HRR deficiency in H3K27M DMG can be therapeutically leveraged with PARP inhibitors to radiosensitize and induce an NK cell-mediated antitumor immune response selectively in H3K27M DMG, supporting the clinical investigation of PARP1 inhibitors with RT in DMG patients.

Abstract We sought to delineate, and target integrated metabolic and epigenetic pathways in cMYC-driven Group-3 medulloblastoma (MB). In a comprehensive screen, we identified upregulation of wild type isocitrate dehydrogenase 1 (IDH1) in Group-3 MB. Surprisingly, genetic and pharmacologic targeting of IDH1 reduced cMYC expression levels and was therapeutic in three independent (patient-derived D283, p = 0.0004; D341, p = 0.0008 and syngeneic murine Group-3 MB p = 0.0005) Group-3 MB animal models in vivo, but not in non-group 3 ONS76 animals (p = 0.1771). Mechanistically, IDH1 inhibition epigenetically suppressed cMYC expression by increasing repressive methylation marks at the cMYC locus. Additionally, we noted that IDH1 inhibition triggered a novel copper-dependent form of cell death termed cuproptosis. This mechanism was via cMYC-dependent downregulation of dihydrolipoyl transacetylase (DLAT), the E2-subunit of pyruvate dehydrogenase complex (PDC). Forced in vivo increase in tumor cellular copper levels by treating with Elesclomol, a blood-brain-barrier penetrant copper ionophore reduced tumor burden and increased overall survival in three independent (patient-derived D283, p = 0.0009; D341, p = 0.0188 and syngeneic murine Group-3 MB p = 0.0002) Group-3 MB animal models in vivo, but not in non-group 3 ONS76 animals (p = 0.1307). Our work establishes an integrated metabolic/epigenetic pathway driven by wild type IDH1 which serves as a gatekeeper to regulate cMYC expression and downstream copper metabolism in Group-3 MB. This novel pathway can be therapeutically leveraged using the blood-brain-barrier penetrant copper ionophore elesclomol that is currently in clinical trials for adult cancers.

Diffuse midline gliomas (DMGs) are lethal pediatric high-grade brain tumors with a prognosis of less than two years from diagnosis, the majority of which harbor the H3K27M histone mutation. A subgroup of DMGs harbor alterations in epidermal growth factor receptor (EGFR), including wildtype amplification (WT) and mutations in the extracellular domain (exon 7, e.g. A289T) or kinase domain (exon 20, e.g. 767delins). EGFR-targeting TKIs have shown limited efficacy against brain tumors but have not been developed to address the unique biology and EGFR alterations specific to H3K27M-DMG. Further, EGFR alterations are being validated as a biomarker of resistance to ONC201 suggesting that targeting EGFR may have broad utility for H3K27M-DMG. To this end, we established multiple human and murine models of H3K27M-DMG tumor cells with isogenic EGFR alterations (WT/A289T/767delins) and systematically evaluated the efficacy of the novel TKI BLU5082 (Blueprint Medicines). BLU5082 was uniquely effective in vitro at nanomolar concentrations against human H3K27M-DMG cells that express these EGFR alterations. Notably, the IC50 value of <0.2 µM against 767delins distinguished BLU5082. In cell-free kinase inhibition assays BLU5082 reduced EGFR WT and T790M activity by 50% at ~1 nM. We applied high-throughput kinase-activity mapping (HT-KAM) to detect the catalytic activity of >900 kinase-substrate nodes in EGFR-driven H3K27M-DMG cells. BLU5082 induced significant modulation of key oncogenic pathways in EGFR-767delins cells, including decreased MAPK family activity but persistence of AKT-driven metabolic pathways, denoting possible combinatorial partners which are being studied. In vivo testing demonstrated that daily 50 mg/kg BLU5082 treatment resulted in tumor eradication of isogenic EGFR-mutant (A289T/767delins) human H3K27M-DMG flank xenograft models within four weeks without any signs of toxicity. Pharmacokinetic analysis of 50 mg/kg BLU5082 resulted in peak brain concentrations of ~2 µM. Further assessment of the efficacy of BLU5082 against multiple intracranial H3K27M-DMG models is ongoing. Collectively, these preclinical studies highlight the potential of BLU5082 as a novel, targeted TKI therapeutic for EGFR-driven H3K27M-DMGs.

Diffuse midline glioma (DMG) is an aggressive pediatric brain tumor driven by the H3K27M histone mutation and represents the leading cause of cancer-related death in children. These tumors are highly infiltrative and can occasionally migrate to distant CNS regions. To uncover migration dependencies, we developed a novel two-step pooled whole-genome CRISPR-migration screen in metastatic H3K27M-DMG stem cells (n=3). Genes involved in focal adhesion (ITGB1 [integrin beta-1], CRKL, PARVA, PTK2, FERMT2) significantly restricted migration across all models; notably, only ITGB1 knockout (ITGB1-KO) completely abrogated migration. In H3K27M-DMG patient samples, unlike other brain tumor types, expression of ITGB1 correlates with higher glioma grade and worse survival. ITGB1-KO models demonstrated a reduction in expression of MYC target genes, including MYC-regulated metabolic genes involved in purine biosynthesis (e.g., IMPDH2). Further integrated RNA/metabolomic analyses revealed that loss of ITGB1 downregulates purine metabolism and the citric acid (TCA) cycle. Importantly, in in vivo models, ITGB1 deficiency significantly prolonged survival (UMPED83: 100 vs. 163.5 days, p=0.0003; pSCG-SVZ: 49 vs. 68 days, p=0.0095). Spatial transcriptomic and proteomic analyses of ITGB1-KO orthotopic H3K27M tumors showed widespread reduction in MYC target genes and depletion of precursor, undifferentiated (OPC-like), and an increase in differentiated (OC-like) K27M cells in the infiltrating edge. Direct pharmacological targeting of ITGB1 (anti-ITGB1 antibody, CNS delivered) significantly extended survival in DMG models (UMPED83: 100 vs. 125.5 days, p=0.0169; pSCG-SVZ: 49 vs. 72 days, p=0.0384). However, ITGB1-deficient pSCG-SVZ tumors exhibited compensatory alternative integrins upregulation (i.e., ITGB3, ITGB5). Promisingly, co-treatment with anti-ITGB1 antibody and cilengitide (ITGB3/5 inhibitor) further improved survival and resulted in 75% long-term survivors, free of disease. Strikingly, this combinatorial strategy failed to confer any survival benefit in adult glioblastoma (H3WT) models. Overall, these findings highlight integrin targeting as a promising therapeutic avenue in H3K27M-DMG, capable of disrupting tumor-specific migration, MYC-driven purine biosynthesis, and stemness programs.

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.

Abstract PDGFRA is a frequently altered gene in pHGG, driving aggressive behavior and worse prognoses. Avapritinib, a potent CNS-penetrant PDGFRA inhibitor, has shown promise in vitro, in vivo, and in pHGG patients. Given the failure of single-agent trials in targeting PDGFRA-altered HGG, combinatorial therapy is likely needed with other targetable pathways for treatment. We performed a high-throughput kinase-activity mapping (HT-KAM) screen to detect the catalytic activity of > 900 kinase-substrate nodes in our pHGG models. These results demonstrated that supraphysiological doses of > =1uM avapritinib treatment of PDGFRA-altered pHGG cells in vitro results in sustained activation of the MAPK pathway. Specifically, short-term avapritinib treatment with > =1uM doses resulted in MEK/ERK (MEK2) and MEK/JNK (MKK4/7) activation, and long-term treatment resulted in sustained MEK/ERK (MEK2) activation across all models. Dose-dependent pERK upregulation in response to avapritinib was confirmed in multiple pHGG in vitro and in vivo models. Single-cell RNA-seq analysis of avapritinib-treated pHGG tumors in vivo demonstrated that cycling OPC-like cells were primarily responsible for increased expression of MAPK pathway genes. Furthermore, upregulation of the ERK-driven anti-apoptotic protein MCL-1 was found in short-term avapritinib-treated pHGG cells in vitro. Combinatorial treatment of pHGG models with MEK (trametinib), ERK (ulixertinib) and integrated stress/ERK inhibitors (ONC201, ONC206) in vitro eradicated pERK activity. Trametinib demonstrated the strongest combinatorial survival benefit among preliminary results in PDGFRA-driven pHGG models in vivo. We subsequently showed in vitro synergy between avapritinib and trametinib in a tumor-derived organoid from a pediatric patient with PDGFRA D842V-mutant metastatic CNS sarcoma that grew on avapritinib. This patient was later treated with this combination and demonstrated stability for five months. Combinatorial therapy with avapritinib and a MEK inhibitor was performed in four additional PDGFRA-driven pHGG patients. In light of sustained MAPK activation identified in our study, dual avapritinib-MAPK targeted treatment may be an effective approach for PDGFRA-driven pHGG.

Diffuse midline gliomas (DMGs) are lethal pediatric cancers. Histone H3.3 (H3-3A) is the most commonly mutated gene in DMG; up to 80% exhibit a gain-of-function mutation at lysine 27 (K27M). K27M mutations often co-occur with loss of ATRX, a chromatin remodeler. We generated isogenic mouse and human cell lines to characterize the interaction between H3.3K27M and ATRX co-mutations. Interestingly, CUT&RUN-seq demonstrates that ATRX loss drives global loss of H3K4me3 at promoters in murine K27M cells when compared to H3.3WT or H3.3G34R models. Integration of RNA- and ATAC-seq shows that on a K27M background, ATRX loss downregulates differentiation-related gene sets, consistent with the role of ATRX in healthy brain differentiation. Gene loci for oligodendrocyte precursor genes OLIG2 and CSPG4 exhibit both H3K4me3 loss and decreased chromatin accessibility with ATRX loss. Separately, RNA- and ATAC-seq integration show ATRX loss leads to dysregulation of cell cycle genes in H3K27M cells. p16, a critical G1/S checkpoint regulator which has been shown to be decreased in H3K27M cells, is further decreased with ATRX loss. ATRX deficiency leads to loss of H3K4me3 binding and further decrease in chromatin accessibility at the p16 promoter uniquely in H3K27M cells. This leads to: (1) absent p16 expression, (2) inappropriate release of cell cycle checkpoints after radiation, and (3) heightened sensitivity to CDK4/6 inhibitors in ATRX-H3K27M co-mutant cells. Ongoing work includes determining the impact of ATRX on H3K4 demethylase function and association of H3K4me3 distribution with established K27M “poised” promoters. We are additionally pursuing in vivo CDK4/6 inhibitor studies by H3 and ATRX status, as well as assembling anecdotal use of abemaciclib in human DMG patients with H3.3/ATRX mutation. In summary, ATRX loss leads to a unique epigenetic landscape in H3K27M tumor cells, with global H3K4me3 loss at promoters, resulting in pathways that may be therapeutically targetable.

MYC-driven group-3 medulloblastomas (MBs) are malignant pediatric brain cancers without cures. To define actionable metabolic dependencies, we identify upregulation of dihydrolipoyl transacetylase (DLAT), the E2-subunit of pyruvate dehydrogenase complex (PDC) in a subset of group-3 MB with poor prognosis. DLAT is induced by c-MYC and targeting DLAT lowers TCA cycle metabolism and glutathione synthesis. We also note upregulation of isocitrate dehydrogenase 1 (IDH1) gene expression in group-3 MB patient tumors and suppression of IDH1 epigenetically reduces c-MYC and downstream DLAT levels in multiple c-MYC amplified cancers. DLAT is a central regulator of cuproptosis (copper-dependent cell death) induced by the copper ionophore elesclomol. DLAT expression in group-3 MB cells correlates with increased sensitivity to cuproptosis. Elesclomol is brain-penetrant and suppresses tumor growth in vivo in multiple group-3 MB animal models. Our data uncover an IDH1/c-MYC dependent vulnerability that regulates DLAT levels and can be targeted to kill group-3 MB by cuproptosis.

MYC-driven Group-3 medulloblastomas (MB) are deadly and malignant pediatric brain cancers and we sought to define actionable metabolic dependencies in these tumors. To identify uniquely upregulated genes in Group-3 MB, we performed transcriptomic analysis on two previously published medulloblastoma RNA-seq datasets. To elucidate the relationship between c-MYC/IDH1/DLAT and assess impact on tumor metabolism, we performed metabolic and transcriptional profiling of Group-3 MB cell lines that were either untreated or were subjected to shRNA-mediated knockdown of DLAT or treatment with IDH1 inhibitor. We also treated Group-3 MB cell lines containing varying levels of DLAT expression with copper ionophore elesclomol and assessed its ability to induce toxicity. Finally, we established in vivo models of Group-3 MB via orthotopic implantation to assess the effect of DLAT knockdown, IDH1 inhibition, and cuproptosis induction on tumor growth and survival outcomes. We identified upregulation of dihydrolipoyl transacetylase (DLAT), the E2-subunit of pyruvate dehydrogenase complex (PDC) in a subset of Group-3 MB. DLAT was induced by c-MYC and targeting DLAT lowered TCA-cycle metabolism and glutathione synthesis in Group-3 MB cells. We also noted upregulation of isocitrate dehydrogenase 1 (IDH1) in Group-3 MB. Remarkably, genetic and pharmacologic suppression of IDH1 epigenetically reduced c-MYC and downstream DLAT levels. DLAT is a central regulator of cuproptosis, a copper-dependent cell death mechanism induced by the copper ionophore elesclomol. DLAT expression in Group-3 MB cells correlated with increased sensitivity to cuproptosis. Elesclomol was CNS-penetrant and suppressed tumor growth in vivo in Group-3 MB animal models. Our data uncover an IDH1/c-MYC dependent vulnerability that regulates DLAT levels and can be targeted to kill Group-3 MB by cuproptosis.