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Radiotherapy is a major component of standard-of-care treatment for gliomas, the most prevalent type of brain tumor. However, resistance to radiotherapy remains a major concern. Identification of mechanisms governing radioresistance in gliomas could reveal improved therapeutic strategies for treating patients. Here, we report that mitochondrial metabolic pathways are suppressed in radioresistant gliomas through integrated analyses of transcriptomic data from glioma specimens and cell lines. Decreased expression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α), the key regulator of mitochondrial biogenesis and metabolism, correlated with glioma recurrence and predicted poor prognosis and response to radiotherapy of patients with glioma. The subpopulation of glioma cells with low-mitochondrial-mass exhibited reduced expression of PGC1α and enhanced resistance to radiotherapy treatment. Mechanistically, PGC1α was phosphorylated at serine (S) 636 by DNA-dependent protein kinase in response to irradiation. Phosphorylation at S636 promoted the degradation of PGC1α by facilitating its binding to the E3 ligase RNF34. Restoring PGC1α activity with expression of PGC1α S636A, a phosphorylation-resistant mutant, or a small-molecule PGC1α activator ZLN005 increased radiosensitivity of resistant glioma cells by reactivating mitochondria-related reactive oxygen species production and inducing apoptotic effects both in vitro and in vivo. In summary, this study identified a self-protective mechanism in glioma cells in which radiotherapy-induced degradation of PGC1α and suppression of mitochondrial biogenesis play a central role. Targeted activation of PGC1α could help improve response to radiotherapy in patients with glioma. Significance: Glioma cells reduce mitochondrial biogenesis by promoting PGC1α degradation to promote resistance to radiotherapy, indicating potential therapeutic strategies to enhance radiosensitivity.

Glioblastoma (GBM) constitutes the most lethal primary brain tumor for which immunotherapy has provided limited benefit. The unique brain immune landscape is reflected in a complex tumor immune microenvironment (TIME) in GBM. Here, single-cell sequencing of the GBM TIME revealed that microglia were under severe oxidative stress, which induced nuclear receptor subfamily 4 group A member 2 (NR4A2)–dependent transcriptional activity in microglia. Heterozygous Nr4a2 (Nr4a2+/−) or CX3CR1+ myeloid cell–specific Nr4a2 (Nr4a2fl/flCx3cr1Cre) genetic targeting reshaped microglia plasticity in vivo by reducing alternatively activated microglia and enhancing antigen presentation capacity for CD8+ T cells in GBM. In microglia, NR4A2 activated squalene monooxygenase (SQLE) to dysregulate cholesterol homeostasis. Pharmacologic NR4A2 inhibition attenuated the protumorigenic TIME, and targeting the NR4A2 or SQLE enhanced the therapeutic efficacy of immune-checkpoint blockade in vivo. Collectively, oxidative stress promotes tumor growth through NR4A2–SQLE activity in microglia, informing novel immune therapy paradigms in brain cancer. Significance: Metabolic reprogramming of microglia in GBM informs synergistic vulnerabilities for immune-checkpoint blockade therapy in this immunologically cold brain tumor. This article is highlighted in the In This Issue feature, p. 799

Glioblastoma (GBM) is a complex ecosystem that includes a heterogeneous tumor population and the tumor-immune microenvironment (TIME), prominently containing tumor-associated macrophages (TAM) and microglia. Here, we demonstrated that β2-microglobulin (B2M), a subunit of the class I major histocompatibility complex (MHC-I), promotes the maintenance of stem-like neoplastic populations and reprograms the TIME to an anti-inflammatory, tumor-promoting state. B2M activated PI3K/AKT/mTOR signaling by interacting with PIP5K1A in GBM stem cells (GSC) and promoting MYC-induced secretion of transforming growth factor-β1 (TGFβ1). Inhibition of B2M attenuated GSC survival, self-renewal, and tumor growth. B2M-induced TGFβ1 secretion activated paracrine SMAD and PI3K/AKT signaling in TAMs and promoted an M2-like macrophage phenotype. These findings reveal tumor-promoting functions of B2M and suggest that targeting B2M or its downstream axis may provide an effective approach for treating GBM. Significance: β2-microglobulin signaling in glioblastoma cells activates a PI3K/AKT/MYC/TGFβ1 axis that maintains stem cells and induces M2-like macrophage polarization, highlighting potential therapeutic strategies for targeting tumor cells and the immunosuppressive microenvironment in glioblastoma.

Supplemental Figure S4 describes that NR4A2-knockdown affects lipid metabolism and promotes antigen presentation of microglia.

Supplementary Figures 1-7 and the figure legends

Glioblastoma (GBM) is the most lethal primary brain cancer characterized by therapeutic resistance, which is promoted by GBM stem cells (GSC). Here, we interrogated gene expression and whole-genome CRISPR/Cas9 screening in a large panel of patient-derived GSCs, differentiated GBM cells (DGC), and neural stem cells (NSC) to identify master regulators of GSC stemness, revealing an essential transcription state with increased RNA polymerase II–mediated transcription. The YY1 and transcriptional CDK9 complex was essential for GSC survival and maintenance in vitro and in vivo. YY1 interacted with CDK9 to regulate transcription elongation in GSCs. Genetic or pharmacologic targeting of the YY1–CDK9 complex elicited RNA m6A modification–dependent interferon responses, reduced regulatory T-cell infiltration, and augmented efficacy of immune checkpoint therapy in GBM. Collectively, these results suggest that YY1–CDK9 transcription elongation complex defines a targetable cell state with active transcription, suppressed interferon responses, and immunotherapy resistance in GBM.Significance:Effective strategies to rewire immunosuppressive microenvironment and enhance immunotherapy response are still lacking in GBM. YY1-driven transcriptional elongation machinery represents a druggable target to activate interferon response and enhance anti–PD-1 response through regulating the m6A modification program, linking epigenetic regulation to immunomodulatory function in GBM.This article is highlighted in the In This Issue feature, p. 275

Radiotherapy is a major component of standard-of-care treatment for gliomas, the most prevalent type of brain tumor. However, resistance to radiotherapy remains a major concern. Identification of mechanisms governing radioresistance in gliomas could reveal improved therapeutic strategies for treating patients. Here, we report that mitochondrial metabolic pathways are suppressed in radioresistant gliomas through integrated analyses of transcriptomic data from glioma specimens and cell lines. Decreased expression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α), the key regulator of mitochondrial biogenesis and metabolism, correlated with glioma recurrence and predicted poor prognosis and response to radiotherapy of patients with glioma. The subpopulation of glioma cells with low-mitochondrial-mass exhibited reduced expression of PGC1α and enhanced resistance to radiotherapy treatment. Mechanistically, PGC1α was phosphorylated at serine (S) 636 by DNA-dependent protein kinase in response to irradiation. Phosphorylation at S636 promoted the degradation of PGC1α by facilitating its binding to the E3 ligase RNF34. Restoring PGC1α activity with expression of PGC1α S636A, a phosphorylation-resistant mutant, or a small-molecule PGC1α activator ZLN005 increased radiosensitivity of resistant glioma cells by reactivating mitochondria-related reactive oxygen species production and inducing apoptotic effects both in vitro and in vivo. In summary, this study identified a self-protective mechanism in glioma cells in which radiotherapy-induced degradation of PGC1α and suppression of mitochondrial biogenesis play a central role. Targeted activation of PGC1α could help improve response to radiotherapy in patients with glioma.Significance:Glioma cells reduce mitochondrial biogenesis by promoting PGC1α degradation to promote resistance to radiotherapy, indicating potential therapeutic strategies to enhance radiosensitivity.

Glioblastoma (GBM) constitutes the most lethal primary brain tumor for which immunotherapy has provided limited benefit. The unique brain immune landscape is reflected in a complex tumor immune microenvironment (TIME) in GBM. Here, single-cell sequencing of the GBM TIME revealed that microglia were under severe oxidative stress, which induced nuclear receptor subfamily 4 group A member 2 (NR4A2)–dependent transcriptional activity in microglia. Heterozygous Nr4a2 (Nr4a2+/−) or CX3CR1+ myeloid cell–specific Nr4a2 (Nr4a2fl/flCx3cr1Cre) genetic targeting reshaped microglia plasticity in vivo by reducing alternatively activated microglia and enhancing antigen presentation capacity for CD8+ T cells in GBM. In microglia, NR4A2 activated squalene monooxygenase (SQLE) to dysregulate cholesterol homeostasis. Pharmacologic NR4A2 inhibition attenuated the protumorigenic TIME, and targeting the NR4A2 or SQLE enhanced the therapeutic efficacy of immune-checkpoint blockade in vivo. Collectively, oxidative stress promotes tumor growth through NR4A2–SQLE activity in microglia, informing novel immune therapy paradigms in brain cancer.Significance:Metabolic reprogramming of microglia in GBM informs synergistic vulnerabilities for immune-checkpoint blockade therapy in this immunologically cold brain tumor.This article is highlighted in the In This Issue feature, p. 799

Table S3. Overlapping between chromatin interactions and YY1 binding peaks in glioblastoma stem cells.

Supplementary Figures and Figure legends

Table S2 describes information of key research agents, data deposition and software versions.

Table S7. Clinical information of 95 GBM patients

Supplementary Data from Natural Coevolution of Tumor and Immunoenvironment in Glioblastoma

Supplementary Figure from Natural Coevolution of Tumor and Immunoenvironment in Glioblastoma

Isocitrate dehydrogenase (IDH) wild-type glioblastoma (GBM) has a dismal prognosis. A better understanding of tumor evolution holds the key to developing more effective treatment. Here we study GBM's natural evolutionary trajectory by using rare multifocal samples. We sequenced 61,062 single cells from eight multifocal IDH wild-type primary GBMs and defined a natural evolution signature (NES) of the tumor. We show that the NES significantly associates with the activation of transcription factors that regulate brain development, including MYBL2 and FOSL2. Hypoxia is involved in inducing NES transition potentially via activation of the HIF1A–FOSL2 axis. High-NES tumor cells could recruit and polarize bone marrow–derived macrophages through activation of the FOSL2–ANXA1–FPR1/3 axis. These polarized macrophages can efficiently suppress T-cell activity and accelerate NES transition in tumor cells. Moreover, the polarized macrophages could upregulate CCL2 to induce tumor cell migration.Significance:GBM progression could be induced by hypoxia via the HIF1A–FOSL2 axis. Tumor-derived ANXA1 is associated with recruitment and polarization of bone marrow–derived macrophages to suppress the immunoenvironment. The polarized macrophages promote tumor cell NES transition and migration.This article is highlighted in the In This Issue feature, p. 2711

Glioblastoma ranks among the most aggressive and lethal of all human cancers. Functionally defined glioma stem cells (GSC) contribute to this poor prognosis by driving therapeutic resistance and maintaining cellular heterogeneity. To understand the molecular processes essential for GSC maintenance and tumorigenicity, we interrogated the superenhancer landscapes of primary glioblastoma specimens and in vitro GSCs. GSCs epigenetically upregulated ELOVL2, a key polyunsaturated fatty-acid synthesis enzyme. Targeting ELOVL2 inhibited glioblastoma cell growth and tumor initiation. ELOVL2 depletion altered cellular membrane phospholipid composition, disrupted membrane structural properties, and diminished EGFR signaling through control of fatty-acid elongation. In support of the translational potential of these findings, dual targeting of polyunsaturated fatty-acid synthesis and EGFR signaling had a combinatorial cytotoxic effect on GSCs.Significance:Glioblastoma remains a devastating disease despite extensive characterization. We profiled epigenomic landscapes of glioblastoma to pinpoint cell state–specific dependencies and therapeutic vulnerabilities. GSCs utilize polyunsaturated fatty-acid synthesis to support membrane architecture, inhibition of which impairs EGFR signaling and GSC proliferation. Combinatorial targeting of these networks represents a promising therapeutic strategy.See related commentary by Affronti and Wellen, p. 1161.This article is highlighted in the In This Issue feature, p. 1143

Supplementary figures 1-8 and figures legends.

Supplementary Information

Supplementary Data

Supplementary methods

Supplementary Table

Glioblastomas are highly lethal cancers, containing self-renewing glioblastoma stem cells (GSC). Here, we show that GSCs, differentiated glioblastoma cells (DGC), and nonmalignant brain cultures all displayed robust circadian rhythms, yet GSCs alone displayed exquisite dependence on core clock transcription factors, BMAL1 and CLOCK, for optimal cell growth. Downregulation of BMAL1 or CLOCK in GSCs induced cell-cycle arrest and apoptosis. Chromatin immunoprecipitation revealed that BMAL1 preferentially bound metabolic genes and was associated with active chromatin regions in GSCs compared with neural stem cells. Targeting BMAL1 or CLOCK attenuated mitochondrial metabolic function and reduced expression of tricarboxylic acid cycle enzymes. Small-molecule agonists of two independent BMAL1–CLOCK negative regulators, the cryptochromes and REV-ERBs, downregulated stem cell factors and reduced GSC growth. Combination of cryptochrome and REV-ERB agonists induced synergistic antitumor efficacy. Collectively, these findings show that GSCs co-opt circadian regulators beyond canonical circadian circuitry to promote stemness maintenance and metabolism, offering novel therapeutic paradigms.Significance:Cancer stem cells are highly malignant tumor-cell populations. We demonstrate that GSCs selectively depend on circadian regulators, with increased binding of the regulators in active chromatin regions promoting tumor metabolism. Supporting clinical relevance, pharmacologic targeting of circadian networks specifically disrupted cancer stem cell growth and self-renewal.This article is highlighted in the In This Issue feature, p. 1469

Supplementary reagents showing information of reagents used in this manuscript

Glioblastoma is a universally lethal cancer driven by glioblastoma stem cells (GSC). Here, we interrogated N6-methyladenosine (m6A) mRNA modifications in GSCs by methyl RNA immunoprecipitation followed by sequencing and transcriptome analysis, finding transcripts marked by m6A often upregulated compared with normal neural stem cells (NSC). Interrogating m6A regulators, GSCs displayed preferential expression, as well as in vitro and in vivo dependency, of the m6A reader YTHDF2, in contrast to NSCs. Although YTHDF2 has been reported to destabilize mRNAs, YTHDF2 stabilized MYC and VEGFA transcripts in GSCs in an m6A-dependent manner. We identified IGFBP3 as a downstream effector of the YTHDF2–MYC axis in GSCs. The IGF1/IGF1R inhibitor linsitinib preferentially targeted YTHDF2-expressing cells, inhibiting GSC viability without affecting NSCs and impairing in vivo glioblastoma growth. Thus, YTHDF2 links RNA epitranscriptomic modifications and GSC growth, laying the foundation for the YTHDF2–MYC–IGFBP3 axis as a specific and novel therapeutic target in glioblastoma.Significance:Epitranscriptomics promotes cellular heterogeneity in cancer. RNA m6A landscapes of cancer and NSCs identified cell type–specific dependencies and therapeutic vulnerabilities. The m6A reader YTHDF2 stabilized MYC mRNA specifically in cancer stem cells. Given the challenge of targeting MYC, YTHDF2 presents a therapeutic target to perturb MYC signaling in glioblastoma.This article is highlighted in the In This Issue feature, p. 211

Supplementary Methods 2

Supplementary figures and legends

Meningiomas are the most common primary intracranial tumor with current classification offering limited therapeutic guidance. Here, we interrogated meningioma enhancer landscapes from 33 tumors to stratify patients based upon prognosis and identify novel meningioma-specific dependencies. Enhancers robustly stratified meningiomas into three biologically distinct groups (adipogenesis/cholesterol, mesodermal, and neural crest) distinguished by distinct hormonal lineage transcriptional regulators. Meningioma landscapes clustered with intrinsic brain tumors and hormonally responsive systemic cancers with meningioma subgroups, reflecting progesterone or androgen hormonal signaling. Enhancer classification identified a subset of tumors with poor prognosis, irrespective of histologic grading. Superenhancer signatures predicted drug dependencies with superior in vitro efficacy to treatment based upon the NF2 genomic profile. Inhibition of DUSP1, a novel and druggable meningioma target, impaired tumor growth in vivo. Collectively, epigenetic landscapes empower meningioma classification and identification of novel therapies.Significance:Enhancer landscapes inform prognostic classification of aggressive meningiomas, identifying tumors at high risk of recurrence, and reveal previously unknown therapeutic targets. Druggable dependencies discovered through epigenetic profiling potentially guide treatment of intractable meningiomas.This article is highlighted in the In This Issue feature, p. 1611

Supplementary Table

Information of TAMs harboring tumor-derived mutations

Supplementary Figures S1-7

Tumor-associated macrophages (TAM) play a crucial role in immunosuppression. However, how TAMs are transformed into immunosuppressive phenotypes and influence the tumor microenvironment (TME) is not fully understood. Here, we utilized single-cell RNA sequencing and whole-exome sequencing data of glioblastoma (GBM) tissues and identified a subset of TAMs dually expressing macrophage and tumor signatures, which were termed double-positive TAMs. Double-positive TAMs tended to be bone marrow–derived macrophages (BMDM) and were characterized by immunosuppressive phenotypes. Phagocytosis of glioma cells by BMDMs in vitro generated double-positive TAMs with similar immunosuppressive phenotypes to double-positive TAMs in the GBM TME of patients. The double-positive TAMs were transformed into M2-like macrophages and drove immunosuppression by expressing immune-checkpoint proteins CD276, PD-L1, and PD-L2 and suppressing the proliferation of activated T cells. Together, glioma cell phagocytosis by BMDMs in the TME leads to the formation of double-positive TAMs with enhanced immunosuppressive phenotypes, shedding light on the processes driving TAM-mediated immunosuppression in GBM.Significance:Bone marrow–derived macrophages phagocytose glioblastoma cells to form double-positive cells, dually expressing macrophage and tumor signatures that are transformed into M2-like macrophages and drive immunosuppression.

Glioblastoma (GBM) is a complex ecosystem that includes a heterogeneous tumor population and the tumor-immune microenvironment (TIME), prominently containing tumor-associated macrophages (TAM) and microglia. Here, we demonstrated that β2-microglobulin (B2M), a subunit of the class I major histocompatibility complex (MHC-I), promotes the maintenance of stem-like neoplastic populations and reprograms the TIME to an anti-inflammatory, tumor-promoting state. B2M activated PI3K/AKT/mTOR signaling by interacting with PIP5K1A in GBM stem cells (GSC) and promoting MYC-induced secretion of transforming growth factor-β1 (TGFβ1). Inhibition of B2M attenuated GSC survival, self-renewal, and tumor growth. B2M-induced TGFβ1 secretion activated paracrine SMAD and PI3K/AKT signaling in TAMs and promoted an M2-like macrophage phenotype. These findings reveal tumor-promoting functions of B2M and suggest that targeting B2M or its downstream axis may provide an effective approach for treating GBM.Significance:β2-microglobulin signaling in glioblastoma cells activates a PI3K/AKT/MYC/TGFβ1 axis that maintains stem cells and induces M2-like macrophage polarization, highlighting potential therapeutic strategies for targeting tumor cells and the immunosuppressive microenvironment in glioblastoma.

Supplementary Figure 1. Induction of differentiation by BMP4 decreased expression of mevalonate synthesis enzymes (HMGCR, PMVK, MVK, MVD, IDI1 and FDPS). Supplementary Figure 2. Gene expression data from TCGA GBM patients revealed a high degree of co-expression of HMGCR, PMVK, MVK, MVD, IDI1 and FDPS. Supplementary Figure 3. Ivy glioblastomama atlas project (Ivy GAP) database analysis showed mevalonate pathway genes are enriched in invasive gliomas Supplementary Figure 4. Simvastatin induces cell cycle arrest in BTICs. Supplementary Figure 5. Simvastatin does not affect DGC proliferation. Supplementary Figure 6. Autophagy analysis showed that Simvastatin does not induce autophagy in BTICs. Supplementary Figure 7. Senescence analysis showed that Simvastatin does not cause senescence. Supplementary Figure 8. Apoptosis/ Necrosis analysis showed Simvastatin induces apoptosis in BTICs. Supplementary Figure 9. Farnesyltransferase inhibitor (FTI-277, 20 μM) and Geranylgeranyltransferase I inhibitor (GGTI-298, 10 μM) treatment decreased BTIC growth and self-renewal. Supplementary Figure 10. Farnesyl pyrophosphate (FPP, 20 μm/ml) can partially rescue the growth defect of BTICs induced by targeting MYC expression. Supplementary Figure 11. MYC and miR-33b expression were decreased when xenografted T3691 BTICs were treated with simvastatin (50mg/kg). Supplementary Figure 12. MiR-33b inhibitor treatment partially rescues the MYC protein levels and cellular growth defect caused by simvastatin.

Macrophages phagocytose tumor cells.

Supplementary Figure from β2-Microglobulin Maintains Glioblastoma Stem Cells and Induces M2-like Polarization of Tumor-Associated Macrophages

Glioblastoma (GBM) is the most lethal primary brain cancer characterized by therapeutic resistance, which is promoted by GBM stem cells (GSC). Here, we interrogated gene expression and whole-genome CRISPR/Cas9 screening in a large panel of patient-derived GSCs, differentiated GBM cells (DGC), and neural stem cells (NSC) to identify master regulators of GSC stemness, revealing an essential transcription state with increased RNA polymerase II–mediated transcription. The YY1 and transcriptional CDK9 complex was essential for GSC survival and maintenance in vitro and in vivo. YY1 interacted with CDK9 to regulate transcription elongation in GSCs. Genetic or pharmacologic targeting of the YY1–CDK9 complex elicited RNA m6A modification–dependent interferon responses, reduced regulatory T-cell infiltration, and augmented efficacy of immune checkpoint therapy in GBM. Collectively, these results suggest that YY1–CDK9 transcription elongation complex defines a targetable cell state with active transcription, suppressed interferon responses, and immunotherapy resistance in GBM. Significance: Effective strategies to rewire immunosuppressive microenvironment and enhance immunotherapy response are still lacking in GBM. YY1-driven transcriptional elongation machinery represents a druggable target to activate interferon response and enhance anti–PD-1 response through regulating the m6A modification program, linking epigenetic regulation to immunomodulatory function in GBM. This article is highlighted in the In This Issue feature, p. 275