Supplementary Information:Supplementary Figures 1-6 with Figure Legends, Supplementary Table 1 (reference only), Supplementary Table 2, Supplementary Methods, Supplementary References

Supplementary Table 1: NCI-H295R RNA-seq FPKM table, Related to Figures 2-4, 6.

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.

H3K27M diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), exhibit cellular heterogeneity comprising less-differentiated oligodendrocyte precursors (OPC)-like stem cells and more differentiated astrocyte (AC)-like cells. Here, we establish in vitro models that recapitulate DMG-OPC-like and AC-like phenotypes and perform transcriptomics, metabolomics, and bioenergetic profiling to identify metabolic programs in the different cellular states. We then define strategies to target metabolic vulnerabilities within specific tumor populations. We show that AC-like cells exhibit a mesenchymal phenotype and are sensitized to ferroptotic cell death. In contrast, OPC-like cells upregulate cholesterol biosynthesis, have diminished mitochondrial oxidative phosphorylation (OXPHOS), and are accordingly more sensitive to statins and OXPHOS inhibitors. Additionally, statins and OXPHOS inhibitors show efficacy and extend survival in preclinical orthotopic models established with stem-like H3K27M DMG cells. Together, this study demonstrates that cellular subtypes within DMGs harbor distinct metabolic vulnerabilities that can be uniquely and selectively targeted for therapeutic gain. Pediatric brain cancers are lethal malignancies driven by less-differentiated stem-like cells. Here the authors show that these cells exhibit distinct mitochondrial metabolism programs with targetable vulnerabilities.

Abstract Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent β-catenin–activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific β-catenin–containing complexes, and the epigenome. On chromatin, β-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, β-catenin bound histone methyltransferase EZH2. SF1/β-catenin and EZH2/β-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/β-catenin from chromatin and favored EZH2/β-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities. Significance: Oncogenic β-catenin can use tissue-specific partners to regulate cellular differentiation programs that can be reversed by epigenetic therapies, identifying epigenetic control of differentiation as a viable target for β-catenin–driven cancers.

Abstract Ependymomas are fatal brain malignancies with very few treatment options. More than 70% of supratentorial (ST) ependymomas harbor fusions of the zinc-finger containing, chromatin modifier ZFTA and the transcriptional activator of NF-κB signaling, RELA. Oncogene-driven metabolic reprogramming is a fundamental hallmark of cancer that enables sustained tumor proliferation. ZFTA-RELA fusion protein is essential for tumorigenesis and our goal is to determine how it drives metabolism in ST-ependymomas. To address this, we developed an in vitro isogenic system by expressing the ZFTA-RELA fusion protein in immortalized mouse neural stem cells (ZFTA-RELAFUS). Using this system, we show that these tumor cells selectively upregulate expression of the glutamine transporter (SLC1A5), glutaminase (GLS), and many downstream enzymes in the glutamine metabolic pathway. Therefore, we hypothesized that the ZFTA-RELA fusion drives glutamine metabolism in ST-ependymomas. We further demonstrate that ZFTA-RELAFUS tumor cells utilize glutamine to maintain redox homeostasis and show marked cell death upon its withdrawal. Moreover, JHU-083, a specific pharmacologic inhibitor of glutaminase, killed ZFTA-RELAFUS tumor cells in vitro and in vivo. To summarize, our results suggest that the ZFTA-RELA fusion expressing tumor cells exhibit strong glutamine dependence, and targeting it has significant therapeutic relevance. Citation Format: Siva Kumar Natarajan, James Haggerty-Skeans, Joanna Lum, Pranav Narayanan, Stefan Sweha, Sushanth Sunil, Pooja Panwalkar, Jill Bayliss, Peter Sajjakulnukit, Derek Dang, Abhinav Achreja, Deepak Nagrath, Costas Lyssiotis, Sriram Venneti. ZFTA-RELA fusion aberrantly drives glutamine metabolism in lethal pediatric ependymomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6047.

Adrenocortical carcinoma (ACC) is a rare cancer of the adrenal cortex without curative medical therapies. CIMP-high is an aggressive ACC molecular subtype defined by global CpG island hypermethylation with paradoxical activation of adrenal differentiation (driven by master transcription factor SF1) and stemness (driven by β-catenin). We show DNA hypermethylation redistributes histone methyltransferase EZH2 and its mark, H3K27me3. EZH2 inhibition remains lethal to CIMP-high ACC cells, erasing transcriptional programs without altering DNA methylation. We reconcile this phenomenon by discovery of two nuclear complexes, SF1/β-catenin and EZH2/β-catenin, present in physiology and persistent through advanced ACC. We find SF1/β-catenin is a chromatin-bound complex that controls the ACC super-enhancer landscape, while EZH2/β-catenin is restricted to off-chromatin pools. EZH2 inhibition purges SF1/β-catenin from chromatin, sparing EZH2/β-catenin, inducing dedifferentiation and restraining ACC growth in vitro and in vivo. Our studies illustrate how cell-of-origin programs dictate cancer evolution, exposing differentiation as an therapeutic vulnerability. Citation Format: Dipika R. Mohan, Kleiton S. Borges, Isabella Finco, Christopher R. LaPensee, Juilee Rege, Donald W. Little, Tobias Else, Madson Q. Almeida, Derek Dang, James Haggerty-Skeans, Ana Claudia Latronico, Berenice B. Mendonca, Richard J. Auchus, William E. Rainey, Suely K. Marie, Thomas J. Giordano, Sriram Venneti, Maria Candida B. Fragoso, David T. Breault, Antonio M. Lerario, Gary D. Hammer. Epigenetic dedifferentiation as a therapeutic strategy in adrenal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1501.

Medulloblastomas (MB) are the most common pediatric brain malignancy. Transcriptomic, genomic, and epigenomic insights have stratified these tumors into four distinct subtypes: SHH, WNT, Group 3 and Group 4. Of the four subtypes, Group 3 tumors bear the worst prognosis. Each MB subtype is distinguished by their unique transcriptome profile and epigenetic landscape. Metabolic reprogramming is a hallmark of cancer and allows cells to actively promote the utilization of nutrients to support their uncontrolled proliferation. We identified distinct transcriptional metabolic profiles in medulloblastomas. We found that Group 3 MB show upregulation of key anabolic pathways. We then cross-referenced these data from comprehensive single-cell RNA sequencing profiles of cerebellar developmental niches. We found that Group 3 MB exhibits metabolic signature similar that of early progenitors, rhombic lip progenitors, and cerebellar ventricular zone derivatives. Finally, we identify DLAT, the E2 subunit of the pyruvate dehydrogenase complex, as a uniquely upregulated gene in Group 3 MB. Knockdown of DLAT inhibited tumor growth in-vitro and in-vivo suggesting a potential targets for future therapeutic regimens. Citation Format: Derek Dang, Kyle S. Smith, Pooja Panwalkar, John McKolay, Olamide Animasahun, Abhinav Achreja, Deepak Nagrath, Paul A. Northcott, Sriram Venneti. Investigating metabolic dependencies of group 3 medulloblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6041.

Recent clinical trials for H3K27-altered diffuse midline gliomas (DMGs) have shown much promise. We present a consensus roadmap and identify three major barriers: (1) refinement of experimental models to include immune and brain-specific components; (2) collaboration among researchers, clinicians, and industry to integrate patient-derived data through sharing, transparency, and regulatory considerations; and (3) streamlining clinical efforts including biopsy, CNS-drug delivery, endpoint determination, and response monitoring. We highlight the importance of comprehensive collaboration to advance the understanding, diagnostics, and therapeutics for DMGs.

Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent β-catenin activating mutations. Here, we demonstrate that ACC differentiation is maintained by a balance between nuclear, tissue-specific β-catenin-containing complexes and the epigenome. On chromatin, β-catenin binds master adrenal transcription factor SF1 and hijacks the adrenocortical super-enhancer landscape to maintain differentiation. Off chromatin, β-catenin binds histone methyltransferase EZH2, which is redistributed by the CIMP-high DNA methylation signature. SF1/β-catenin and EZH2/β-catenin complexes exist in normal adrenals and are selected for through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC favors EZH2/β-catenin assembly and purges SF1/β-catenin from chromatin, erasing differentiation and restraining cancer growth in vitro and in vivo . Our studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities. ### Competing Interest Statement The authors have declared no competing interest.

Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent β-catenin activating mutations. Here, we demonstrate that ACC differentiation is maintained by a balance between nuclear, tissue-specific β-catenin-containing complexes and the epigenome. On chromatin, β-catenin binds master adrenal transcription factor SF1 and hijacks the adrenocortical super-enhancer landscape to maintain differentiation. Off chromatin, β-catenin binds histone methyltransferase EZH2, which is redistributed by the CIMP-high DNA methylation signature. SF1/β-catenin and EZH2/β-catenin complexes exist in normal adrenals and are selected for through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC favors EZH2/β-catenin assembly and purges SF1/β-catenin from chromatin, erasing differentiation and restraining cancer growth in vitro and in vivo. Our studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities.

Childhood posterior fossa group A ependymomas (PFAs) have limited treatment options and bear dismal prognoses compared to group B ependymomas (PFBs). PFAs overexpress the oncohistone-like protein EZHIP (enhancer of Zeste homologs inhibitory protein), causing global reduction of repressive histone H3 lysine 27 trimethylation (H3K27me3), similar to the oncohistone H3K27M. Integrated metabolic analyses in patient-derived cells and tumors, single-cell RNA sequencing of tumors, and noninvasive metabolic imaging in patients demonstrated enhanced glycolysis and tricarboxylic acid (TCA) cycle metabolism in PFAs. Furthermore, high glycolytic gene expression in PFAs was associated with a poor outcome. PFAs demonstrated high EZHIP expression associated with poor prognosis and elevated activating mark histone H3 lysine 27 acetylation (H3K27ac). Genomic H3K27ac was enriched in PFAs at key glycolytic and TCA cycle–related genes including hexokinase-2 and pyruvate dehydrogenase. Similarly, mouse neuronal stem cells (NSCs) expressing wild-type EZHIP (EZHIP-WT) versus catalytically attenuated EZHIP-M406K demonstrated H3K27ac enrichment at hexokinase-2 and pyruvate dehydrogenase, accompanied by enhanced glycolysis and TCA cycle metabolism. AMPKα-2, a key component of the metabolic regulator AMP-activated protein kinase (AMPK), also showed H3K27ac enrichment in PFAs and EZHIP-WT NSCs. The AMPK activator metformin lowered EZHIP protein concentrations, increased H3K27me3, suppressed TCA cycle metabolism, and showed therapeutic efficacy in vitro and in vivo in patient-derived PFA xenografts in mice. Our data indicate that PFAs and EZHIP-WT–expressing NSCs are characterized by enhanced glycolysis and TCA cycle metabolism. Repurposing the antidiabetic drug metformin lowered pathogenic EZHIP, increased H3K27me3, and suppressed tumor growth, suggesting that targeting integrated metabolic/epigenetic pathways is a potential therapeutic strategy for treating childhood ependymomas.

Dandy–Walker malformation (DWM) and Cerebellar vermis hypoplasia (CVH) are commonly recognized human cerebellar malformations diagnosed following ultrasound and antenatal or postnatal MRI. Specific radiological criteria are used to distinguish them, yet little is known about their differential developmental disease mechanisms. We acquired prenatal cases diagnosed as DWM and CVH and studied cerebellar morphobiometry followed by histological and immunohistochemical analyses. This was supplemented by laser capture microdissection and RNA-sequencing of the cerebellar rhombic lip, a transient progenitor zone, to assess the altered transcriptome of DWM vs control samples. Our radiological findings confirm that the cases studied fall within the accepted biometric range of DWM. Our histopathological analysis points to reduced foliation and inferior vermian hypoplasia as common features in all examined DWM cases. We also find that the rhombic lip, a dorsal stem cell zone that drives the growth and maintenance of the posterior vermis is specifically disrupted in DWM, with reduced proliferation and self-renewal of the progenitor pool, and altered vasculature, all confirmed by transcriptomics analysis. We propose a unified model for the developmental pathogenesis of DWM. We hypothesize that rhombic lip development is disrupted through either aberrant vascularization and/or direct insult which causes reduced proliferation and failed expansion of the rhombic lip progenitor pool leading to disproportionate hypoplasia and dysplasia of the inferior vermis. Timing of insult to the developing rhombic lip (before or after 14 PCW) dictates the extent of hypoplasia and distinguishes DWM from CVH.

Malignant Rhabdoid Tumors (MRT’s) are aggressive tumors characterized by loss of the SWI/SNF complex protein SMARCB1. They occur in the CNS (atypical teratoid/rhabdoid tumors-AT/RT) or outside (extra-CNS, eCNS). MRTs are defined by tumor cells that exhibit varying cell lineage phenotypes including neuroglial, mesenchymal and epithelial differentiation. The mechanisms that regulate polyphenotypic differentiation are not known. Because the SWI/SNF complex regulates differentiation, we hypothesized that heterogeneity in expression of various SWI/SNF components regulate multiple lineage differentiation. We examined expression of various BAF and PBAF SWI/SNF complex proteins in MRTs (24 AT/RT and 19 eCNS MRT) in relation to neuroglial, mesenchymal and epithelial differentiation. Surprisingly, we found that a subset of tumors co-expressed the mutually exclusive BAF complex components: ACTL6A and ACTL6B in eCNS MRTs and ARID1A and ARID1B in AT/RTs. ACTL6A and ACTL6B are critical regulators of neuronal differentiation. Accordingly, MRTs that co-expressed ACTL6A/ACTL6B exhibited greater neuronal differentiation. In contrast, tumors that did not express both ACTL6A/ACTL6B showed more epithelial and mesenchymal differentiation. Conversely, ARID1A and ARID1B are known to regulate epithelial differentiation and AT/RTs that showed expression of both ARID1A and ARID1B demonstrated greater epithelial and mesenchymal differentiation in comparison to tumors that did not. These data suggest that aberrant co-expression of specific mutually exclusive subunits may govern lineage differentiation in MRT. Finally, the SWI/SNF-PBAF subunits - ARID2, PBRM1 and BRD7 also showed heterogeneity in MRTs. PBRM1 regulates the immune tumor microenvironment and accordingly PBRM1 expression related inversely with cytotoxic CD8+-T cell levels and overall prognosis. Finally, tumors with high PBAF expression exhibited cell-cycle deregulation and worse prognosis. Together, our data suggest in addition to SMARCB1 deletion, the composition of residual BAF and PBAF subunits govern the biology of MRTs in relation to polyphenotypic differentiation and the immune tumor microenvironment.

Background. Rhabdoid tumors (RTs) arise within (atypical teratoid/rhabdoid tumor [AT/RT]) or outside the brain (extra [e]CNS-RT) and are driven mainly by inactivation of the SWItch/sucrose nonfermentable (SWI/SNF) complex subunit SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1). A pathognomonic hallmark of RTs is heterogeneous multilineage differentiation, including anoma- lous neuronal differentiation in some eCNS-RTs. Because remodeling of the SWI/SNF complex regulates differentiation, we hypothesized that SWI/SNF Brahma-associated factors (BAF) and polybromo-associated BAF (PBAF) complex heterogeneity are related to both multilineage differentiation and clinical outcome. Methods. We performed an integrated analysis of SWI/SNF complex alterations in the developing kidney and cerebellum (most common regions of RT origin) in comparison to eCNS-RT (n = 14) and AT/RT (n =25) tumors. RT samples were interrogated using immunohistochemistry, DNA methylation, and gene expression analyses. Results. The SWI/SNF BAF paralogs actin-like protein (ACTL)6A and ACTL6B were expressed in a mutually exclusive manner in the developing cerebellum and kidney. In contrast, a subset of eCNS-RTs lost mutual exclusivity and coexpressed both subunits. These tumors showed aberrant DNA methylation of genes that regulate neuronal and renal development and demonstrated immunohistochemical evidence of neuronal differentiation. In addition, low expression of the PBAF subunit polybromo-1 (PBRM1) identified a group of AT/RTs in younger children with better overall prognosis. PBRM1-low AT/RT and eCNS-RTs showed altered DNA methylation and gene expression in immune-related genes. PBRM1 knockdown resulted in lowering immunosuppressive cytokines, and PBRM1 levels in tumor samples showed an inverse relationship with cluster of differentiation (CD)8 cytotoxicT-cell infiltration. Conclusions. Heterogeneity in SWI/SNF BAF (ACTL6A/ACTL6B) and PBAF (PBRM1) subunits is related to histogenesis, contributes to the immune microenvironment and prognosis in RTs, and may inform opportunities to develop immunotherapies.

Niemann–Pick type C disease is a fatal, progressive neurodegenerative disorder caused by loss-of-function mutations in NPC1, a multipass transmembrane glycoprotein essential for intracellular lipid trafficking. We sought to define the cellular machinery controlling degradation of the most common disease-causing mutant, I1061T NPC1. We show that this mutant is degraded, in part, by the proteasome following MARCH6-dependent ERAD. Unexpectedly, we demonstrate that I1061T NPC1 is also degraded by a recently described autophagic pathway called selective ER autophagy (ER-phagy). We establish the importance of ER-phagy both in vitro and in vivo, and identify I1061T as a misfolded endogenous substrate for this FAM134B-dependent process. Subcellular fractionation of I1061T Npc1 mouse tissues and analysis of human samples show alterations of key components of ER-phagy, including FAM134B. Our data establish that I1061T NPC1 is recognized in the ER and degraded by two different pathways that function in a complementary fashion to regulate protein turnover.

FOXC1 loss contributes to Dandy-Walker malformation (DWM), a common human cerebellar malformation. Previously, we found that complete Foxc1 loss leads to aberrations in proliferation, neuronal differentiation and migration in the embryonic mouse cerebellum (Haldipur et al., 2014). We now demonstrate that hypomorphic Foxc1 mutant mice have granule and Purkinje cell abnormalities causing subsequent disruptions in postnatal cerebellar foliation and lamination. Particularly striking is the presence of a partially formed posterior lobule which echoes the posterior vermis DW 'tail sign' observed in human imaging studies. Lineage tracing experiments in Foxc1 mutant mouse cerebella indicate that aberrant migration of granule cell progenitors destined to form the posterior-most lobule causes this unique phenotype. Analyses of rare human del chr 6p25 fetal cerebella demonstrate extensive phenotypic overlap with our Foxc1 mutant mouse models, validating our DWM models and demonstrating that many key mechanisms controlling cerebellar development are likely conserved between mouse and human.