Search

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

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

Supplementary methods and figures

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

Glioblastoma (GBM) contains self-renewing GBM stem cells (GSC) potentially amenable to immunologic targeting, but chimeric antigen receptor (CAR) T-cell therapy has demonstrated limited clinical responses in GBM. Here, we interrogated molecular determinants of CAR-mediated GBM killing through whole-genome CRISPR screens in both CAR T cells and patient-derived GSCs. Screening of CAR T cells identified dependencies for effector functions, including TLE4 and IKZF2. Targeted knockout of these genes enhanced CAR antitumor efficacy. Bulk and single-cell RNA sequencing of edited CAR T cells revealed transcriptional profiles of superior effector function and inhibited exhaustion responses. Reciprocal screening of GSCs identified genes essential for susceptibility to CAR-mediated killing, including RELA and NPLOC4, the knockout of which altered tumor–immune signaling and increased responsiveness of CAR therapy. Overall, CRISPR screening of CAR T cells and GSCs discovered avenues for enhancing CAR therapeutic efficacy against GBM, with the potential to be extended to other solid tumors.Significance:Reciprocal CRISPR screening identified genes in both CAR T cells and tumor cells regulating the potency of CAR T-cell cytotoxicity, informing molecular targeting strategies to potentiate CAR T-cell antitumor efficacy and elucidate genetic modifications of tumor cells in combination with CAR T cells to advance immuno-oncotherapy.This article is highlighted in the In This Issue feature, p. 995

Supplementary figures 1-8 and figures legends.

Type I interferons (IFN), which activate many IFN-stimulated genes (ISG), are known to regulate tumorigenesis. However, little is known regarding how various ISGs coordinate with one another in developing antitumor effects. Here, we report that the ISG UBA7 is a tumor suppressor in breast cancer. UBA7 encodes an enzyme that catalyzes the covalent conjugation of the ubiquitin-like protein product of another ISG (ISG15) to cellular proteins in a process known as “ISGylation.” ISGylation of other ISGs, including STAT1 and STAT2, synergistically facilitates production of chemokine-receptor ligands to attract cytotoxic T cells. These gene-activation events are further linked to clustering and nuclear relocalization of STAT1/2 within IFN-induced promyelocytic leukemia (PML) bodies. Importantly, this coordinated ISG–ISGylation network plays a central role in suppressing murine breast cancer growth and metastasis, which parallels improved survival in patients with breast cancer. These findings reveal a cooperative IFN-inducible gene network in orchestrating a tumor-suppressive microenvironment.Significance:We report a highly cooperative ISG network, in which UBA7-mediated ISGylation facilitates clustering of transcription factors and activates an antitumor gene-expression program. These findings provide mechanistic insights into immune evasion in breast cancer associated with UBA7 loss, emphasizing the importance of a functional ISG–ISGylation network in tumor suppression.This article is highlighted in the In This Issue feature, p. 327

CRISPR screening reads on CAR T cells and GSCs

Supplementary tables

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

Supplementary Table

Supplementary figures 1-7, supplementary tables 1-4.

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.

Purpose: Normal stem cells tightly control self-renewal and differentiation during development, but their neoplastic counterparts, cancer stem cells (CSCs), sustain tumorigenicity both through aberrant activation of stemness and evasion of differentiation. Although regulation of CSC stemness has been extensively studied, the molecular mechanisms suppressing differentiation remain unclear.Experimental Design: We performed in silico screening and in vitro validation studies through Western blotting, qRT-PCR for treatment of WNT and SHH signaling inhibitors, and BMP signaling inducer with control and ID1-overexpressing cells. We also performed in vivo drug treatment assays with Balb/c nude mice.Results: Inhibitor of differentiation 1 (ID1) abrogated differentiation signals from bone morphogenetic protein receptor (BMPR) signaling in glioblastoma stem cells (GSCs) to promote self-renewal. ID1 inhibited BMPR2 expression through miRNAs, miR-17 and miR-20a, which are transcriptional targets of MYC. ID1 increases MYC expression by activating WNT and SHH signaling. Combined pharmacologic blockade of WNT and SHH signaling with BMP treatment significantly suppressed GSC self-renewal and extended survival of tumor-bearing mice.Conclusions: Collectively, our results suggested that ID1 simultaneously regulates stemness through WNT and SHH signaling and differentiation through BMPR-mediated differentiation signaling in GSCs, informing a novel therapeutic strategy of combinatorial targeting of stemness and differentiation. Clin Cancer Res; 24(2); 383–94. ©2017 AACR.

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

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

Type I interferons (IFN), which activate many IFN-stimulated genes (ISG), are known to regulate tumorigenesis. However, little is known regarding how various ISGs coordinate with one another in developing antitumor effects. Here, we report that the ISG UBA7 is a tumor suppressor in breast cancer. UBA7 encodes an enzyme that catalyzes the covalent conjugation of the ubiquitin-like protein product of another ISG (ISG15) to cellular proteins in a process known as “ISGylation.” ISGylation of other ISGs, including STAT1 and STAT2, synergistically facilitates production of chemokine-receptor ligands to attract cytotoxic T cells. These gene-activation events are further linked to clustering and nuclear relocalization of STAT1/2 within IFN-induced promyelocytic leukemia (PML) bodies. Importantly, this coordinated ISG–ISGylation network plays a central role in suppressing murine breast cancer growth and metastasis, which parallels improved survival in patients with breast cancer. These findings reveal a cooperative IFN-inducible gene network in orchestrating a tumor-suppressive microenvironment. Significance: We report a highly cooperative ISG network, in which UBA7-mediated ISGylation facilitates clustering of transcription factors and activates an antitumor gene-expression program. These findings provide mechanistic insights into immune evasion in breast cancer associated with UBA7 loss, emphasizing the importance of a functional ISG–ISGylation network in tumor suppression. This article is highlighted in the In This Issue feature, p. 327

Glioblastoma (GBM), a lethal primary brain tumor, contains glioma stem cells (GSCs) that promote malignant progression and therapeutic resistance. SOX2 is a core transcription factor that maintains the properties of stem cells, including GSCs, but mechanisms associated with posttranslational SOX2 regulation in GSCs remain elusive. Here, we report that DNA-dependent protein kinase (DNA-PK) governs SOX2 stability through phosphorylation, resulting in GSC maintenance. Mass spectrometric analyses of SOX2-binding proteins showed that DNA-PK interacted with SOX2 in GSCs. The DNA-PK catalytic subunit (DNA-PKcs) was preferentially expressed in GSCs compared to matched non-stem cell tumor cells (NSTCs) isolated from patient-derived GBM xenografts. DNA-PKcs phosphorylated human SOX2 at S251, which stabilized SOX2 by preventing WWP2-mediated ubiquitination, thus promoting GSC maintenance. We then demonstrated that when the nuclear DNA of GSCs either in vitro or in GBM xenografts in mice was damaged by irradiation or treatment with etoposide, the DNA-PK complex dissociated from SOX2, which then interacted with WWP2, leading to SOX2 degradation and GSC differentiation. These results suggest that DNA-PKcs-mediated phosphorylation of S251 was critical for SOX2 stabilization and GSC maintenance. Pharmacological inhibition of DNA-PKcs with the DNA-PKcs inhibitor NU7441 reduced GSC tumorsphere formation in vitro and impaired growth of intracranial human GBM xenografts in mice as well as sensitized the GBM xenografts to radiotherapy. Our findings suggest that DNA-PK maintains GSCs in a stem cell state and that DNA damage triggers GSC differentiation through precise regulation of SOX2 stability, highlighting that DNA-PKcs has potential as a therapeutic target in glioblastoma.

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

Glioblastoma ranks among the most lethal of primary brain malignancies, with glioblastoma stem cells (GSCs) at the apex of tumor cellular hierarchies. Here, to discover novel therapeutic GSC targets, we interrogated gene expression profiles from GSCs, differentiated glioblastoma cells (DGCs), and neural stem cells (NSCs), revealing EYA2 as preferentially expressed by GSCs. Targeting EYA2 impaired GSC maintenance and induced cell cycle arrest, apoptosis, and loss of self-renewal. EYA2 displayed novel localization to centrosomes in GSCs, and EYA2 tyrosine (Tyr) phosphatase activity was essential for proper mitotic spindle assembly and survival of GSCs. Inhibition of the EYA2 Tyr phosphatase activity, via genetic or pharmacological means, mimicked EYA2 loss in GSCs in vitro and extended the survival of tumor-bearing mice. Supporting the clinical relevance of these findings, EYA2 portends poor patient prognosis in glioblastoma. Collectively, our data indicate that EYA2 phosphatase function plays selective critical roles in the growth and survival of GSCs, potentially offering a high therapeutic index for EYA2 inhibitors.

Glioblastoma (GBM) contains self-renewing GBM stem cells (GSC) potentially amenable to immunologic targeting, but chimeric antigen receptor (CAR) T-cell therapy has demonstrated limited clinical responses in GBM. Here, we interrogated molecular determinants of CAR-mediated GBM killing through whole-genome CRISPR screens in both CAR T cells and patient-derived GSCs. Screening of CAR T cells identified dependencies for effector functions, including TLE4 and IKZF2. Targeted knockout of these genes enhanced CAR antitumor efficacy. Bulk and single-cell RNA sequencing of edited CAR T cells revealed transcriptional profiles of superior effector function and inhibited exhaustion responses. Reciprocal screening of GSCs identified genes essential for susceptibility to CAR-mediated killing, including RELA and NPLOC4, the knockout of which altered tumor–immune signaling and increased responsiveness of CAR therapy. Overall, CRISPR screening of CAR T cells and GSCs discovered avenues for enhancing CAR therapeutic efficacy against GBM, with the potential to be extended to other solid tumors. Significance: Reciprocal CRISPR screening identified genes in both CAR T cells and tumor cells regulating the potency of CAR T-cell cytotoxicity, informing molecular targeting strategies to potentiate CAR T-cell antitumor efficacy and elucidate genetic modifications of tumor cells in combination with CAR T cells to advance immuno-oncotherapy. This article is highlighted in the In This Issue feature, p. 995

Glioblastoma (GBM) is the most lethal primary brain cancer characterized by resistance to chemotherapy and radiotherapy, which is driven by GBM stem cells (GSCs). Here, we interrogated gene expression profiles and whole genome CRISPR/Cas9 screening in a large panel of patient-derived GSCs, differentiated glioblastoma cells (DGCs), and neural stem cells (NSCs) to identify master regulators of GSC stemness, revealing a dependency on Yin Yang 1 (YY1) in GSCs. YY1 controlled a cell state with increased RNA polymerase II-mediated transcription and RNA processing through interaction with transcriptional cyclin-dependent kinases (CDKs) and regulating chromatin loop formation in GSCs. YY1 and transcriptional CDKs were essential in GSC survival and stemness maintenance in vitro and in vivo. Moreover, YY1 knockdown or simultaneously targeting transcriptional CDKs elicited interferon responses and augmented efficacy of immune checkpoint therapy. Collectively, these results suggest that YY1-mediated chromatin regulation defines a targetable cell state with active transcription and immunotherapy resistance in glioblastoma.

Posterior fossa A (PFA) ependymomas are lethal malignancies of the hindbrain in infants and toddlers. Lacking highly recurrent somatic mutations, PFA ependymomas are proposed to be epigenetically driven tumors for which model systems are lacking. Here we demonstrate that PFA ependymomas are maintained under hypoxia, associated with restricted availability of specific metabolites to diminish histone methylation, and increase histone demethylation and acetylation at histone 3 lysine 27 (H3K27). PFA ependymomas initiate from a cell lineage in the first trimester of human development that resides in restricted oxygen. Unlike other ependymomas, transient exposure of PFA cells to ambient oxygen induces irreversible cellular toxicity. PFA tumors exhibit a low basal level of H3K27me3, and, paradoxically, inhibition of H3K27 methylation specifically disrupts PFA tumor growth. Targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma. Hypoxia reprograms the cellular metabolome and epigenome to promote growth of the most lethal ependymomas.

Genomic sequencing has driven precision-based oncology therapy; however, the genetic drivers of many malignancies remain unknown or non-targetable, so alternative approaches to the identification of therapeutic leads are necessary. Ependymomas are chemotherapy-resistant brain tumours, which, despite genomic sequencing, lack effective molecular targets. Intracranial ependymomas are segregated on the basis of anatomical location (supratentorial region or posterior fossa) and further divided into distinct molecular subgroups that reflect differences in the age of onset, gender predominance and response to therapy1,2,3. The most common and aggressive subgroup, posterior fossa ependymoma group A (PF-EPN-A), occurs in young children and appears to lack recurrent somatic mutations2. Conversely, posterior fossa ependymoma group B (PF-EPN-B) tumours display frequent large-scale copy number gains and losses but have favourable clinical outcomes1,3. More than 70% of supratentorial ependymomas are defined by highly recurrent gene fusions in the NF-κB subunit gene RELA (ST-EPN-RELA), and a smaller number involve fusion of the gene encoding the transcriptional activator YAP1 (ST-EPN-YAP1)1,3,4. Subependymomas, a distinct histologic variant, can also be found within the supratetorial and posterior fossa compartments, and account for the majority of tumours in the molecular subgroups ST-EPN-SE and PF-EPN-SE. Here we describe mapping of active chromatin landscapes in 42 primary ependymomas in two non-overlapping primary ependymoma cohorts, with the goal of identifying essential super-enhancer-associated genes on which tumour cells depend. Enhancer regions revealed putative oncogenes, molecular targets and pathways; inhibition of these targets with small molecule inhibitors or short hairpin RNA diminished the proliferation of patient-derived neurospheres and increased survival in mouse models of ependymomas. Through profiling of transcriptional enhancers, our study provides a framework for target and drug discovery in other cancers that lack known genetic drivers and are therefore difficult to treat., This work was supported by an Alex's Lemonade Stand Young Investigator Award (S.C.M.), The CIHR Banting Fellowship (S.C.M.), The Cancer Prevention Research Institute of Texas (S.C.M., RR170023), Sibylle Assmus Award for Neurooncology (K.W.P.), the DKFZ-MOST (Ministry of Science, Technology & Space, Israel) program in cancer research (H.W.), James S. McDonnell Foundation (J.N.R.) and NIH grants: CA154130 (J.N.R.), R01 CA169117 (J.N.R.), R01 CA171652 (J.N.R.), R01 NS087913 (J.N.R.) and R01 NS089272 (J.N.R.). R.C.G. is supported by NIH grants T32GM00725 and F30CA217065. M.D.T. is supported by The Garron Family Chair in Childhood Cancer Research, and grants from the Pediatric Brain Tumour Foundation, Grand Challenge Award from CureSearch for Children’s Cancer, the National Institutes of Health (R01CA148699, R01CA159859), The Terry Fox Research Institute and Brainchild. M.D.T. is also supported by a Stand Up To Cancer St. Baldrick’s Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113).

Glioblastomas are lethal cancers defined by angiogenesis and pseudopalisading necrosis. Here, we demonstrate that these histological features are associated with distinct transcriptional programs, with vascular regions showing a proneural profile and hypoxic regions a mesenchymal pattern. As these regions harbor glioma stem cells (GSCs), we investigated the epigenetic regulation of these two niches. Proneural, perivascular GSCs activated EZH2, whereas mesenchymal GSCs in hypoxic regions expressed BMI1 protein, which promoted cellular survival under stress, due to downregulation of the E3 ligase, RNF144A. Using both genetic and pharmacologic inhibition, we found that proneural GSCs are preferentially sensitive to EZH2 disruption, whereas mesenchymal GSCs are preferentially sensitive to BMI1 inhibition. Given that glioblastomas contain both proneural and mesenchymal GSCs, combined EZH2 and BMI1 targeting proved more effective than either agent alone both in culture and in vivo, suggesting that strategies that simultaneously target multiple epigenetic regulators within glioblastomas may be necessary to overcome resistance to therapies caused by intratumoral heterogeneity.

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

Mack et al. defined active chromatin landscapes of glioblastoma stem cells (GSCs) and primary tumor specimens, revealing novel transcriptional regulatory circuits and therapeutic targets. Super-enhancers identified essential transcription factors that underlie GSC identity and intertumoral diversity, potentially informing precision medicine., Glioblastoma is an incurable brain cancer characterized by high genetic and pathological heterogeneity. Here, we mapped active chromatin landscapes with gene expression, whole exomes, copy number profiles, and DNA methylomes across 44 patient-derived glioblastoma stem cells (GSCs), 50 primary tumors, and 10 neural stem cells (NSCs) to identify essential super-enhancer (SE)–associated genes and the core transcription factors that establish SEs and maintain GSC identity. GSCs segregate into two groups dominated by distinct enhancer profiles and unique developmental core transcription factor regulatory programs. Group-specific transcription factors enforce GSC identity; they exhibit higher activity in glioblastomas versus NSCs, are associated with poor clinical outcomes, and are required for glioblastoma growth in vivo. Although transcription factors are commonly considered undruggable, group-specific enhancer regulation of the MAPK/ERK pathway predicts sensitivity to MEK inhibition. These data demonstrate that transcriptional identity can be leveraged to identify novel dependencies and therapeutic approaches., Graphical Abstract

Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Here, we trace metabolic aberrations in GSCs to link core genetic mutations in glioblastoma to dependency on de novo pyrimidine synthesis. Targeting the pyrimidine synthetic rate-limiting step enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase (CAD) or the critical downstream enzyme dihydroorotate dehydrogenase (DHODH) inhibited GSC survival, self-renewal, and in vivo tumor initiation through the depletion of the pyrimidine nucleotide supply in rodent models. Mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of metabolic activity of pyrimidine synthesis and GSC tumorigenic capacity in vitro. Higher expression of pyrimidine synthesis genes portends poor prognosis of patients with glioblastoma. Collectively, our results demonstrate a therapeutic approach of precision medicine through targeting the nexus between driver mutations and metabolic reprogramming in cancer stem cells.

Glioblastomas co-opt stem cell regulatory pathways to maintain brain tumor-initiating cells (BTICs), also known as cancer stem cells. NOTCH signaling has been a molecular target in BTICs, but NOTCH antagonists have demonstrated limited efficacy in clinical trials. Recombining binding protein suppressor of hairless (RBPJ) is considered a central transcriptional mediator of NOTCH activity. Here, we report that pharmacologic NOTCH inhibitors were less effective than targeting RBPJ in suppressing tumor growth. While NOTCH inhibitors decreased canonical NOTCH gene expression, RBPJ regulated a distinct profile of genes critical to BTIC stemness and cell cycle progression. RBPJ was preferentially expressed by BTICs and required for BTIC self-renewal and tumor growth. MYC, a key BTIC regulator, bound the RBPJ promoter and treatment with a bromodomain and extraterminal domain (BET) family bromodomain inhibitor decreased MYC and RBPJ expression. Proteomic studies demonstrated that RBPJ binds CDK9, a component of positive transcription elongation factor b (P-TEFb), to target gene promoters, enhancing transcriptional elongation. Collectively, RBPJ links MYC and transcriptional control through CDK9, providing potential nodes of fragility for therapeutic intervention, potentially distinct from NOTCH.

Ependymomas are malignant glial tumors that occur throughout the central nervous system. Of the nine distinct molecular types of ependymoma, Posterior Fossa A(PFA) ependymomas, found in the hindbrain of infants and young children, are the most prevalent type. They rarely harbor recurrent somatic single nucleotide or copy number aberrations, but rather display epigenetic dysregulations. Transcriptional analyses show that PFAs, but not other molecular ependymoma variants, have elevated hypoxic signaling. We hypothesized that the metabolic environment of the developing human fetal hindbrain contributes to PFA ependymomas through the intermediary metabolic mechanism. Hypoxia microenvironment is essential for PFA survival and upholding their epigenetic dysregulation. Hypoxia blocks methyltransferase activity by upregulating EZHIPs expression and restricting SAM abundance. Fine-tuning the abundance of a-KG and acetyl-CoA, hypoxia fuels demethylase, and acetyltransferase activity which collectively resulting in H3K27 hypomethylation and hyperacetylation. Genome-wide essentially screen further underscore that minimal basal level of H3K27me3 is essential for PFA survival and further attenuation of H3K27me3 decreases the fitness of PFAs. PFA Ependymomas have a unique epigenome, suggesting a model in which they thrive in a narrow Goldilocks zone, with deviation to either increased or decreased H3K27me3 levels leading to diminished cellular fitness. PFAs are maintained under hypoxia, associated with restricted availability of specific metabolites to diminish histone methylation and increase histone demethylation and acetylation at H3K27. PFAs initiate from a cell lineage in the first trimester of human development that resides in restricted oxygen. Unlike other ependymomas, transient exposure of PFA cells to ambient oxygen induces irreversible cellular toxicity. PFA tumors exhibit a low basal level of H3K27me3 and, paradoxically, inhibition of H3K27 methylation specifically disrupts PFA growth. Microenvironmental regulation of PFA ependymoma epigenome appears to play a major role in tumorigenesis. Targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma.

Zika virus (ZIKV) causes microcephaly by killing neural precursor cells (NPCs) and other brain cells. ZIKV also displays therapeutic oncolytic activity against glioblastoma (GBM) stem cells (GSCs). Here we demonstrate that ZIKV preferentially infected and killed GSCs and stem-like cells in medulloblastoma and ependymoma in a SOX2-dependent manner. Targeting SOX2 severely attenuated ZIKV infection, in contrast to AXL. As mechanisms of SOX2-mediated ZIKV infection, we identified inverse expression of antiviral interferon response genes (ISGs) and positive correlation with integrin α(v) (ITGAV). ZIKV infection was disrupted by genetic targeting of ITGAV or its binding partner ITGB5 and by an antibody specific for integrin α(v)β(5). ZIKV selectively eliminated GSCs from species-matched human mature cerebral organoids and GBM surgical specimens, which was reversed by integrin α(v)β(5) inhibition. Collectively, our studies identify integrin α(v)β(5) as a functional cancer stem cell marker essential for GBM maintenance and ZIKV infection, providing potential brain tumor therapy.

Glioblastoma is a universally lethal cancer with a median survival time of approximately 15 months. Despite substantial efforts to define druggable targets, there are no therapeutic options that notably extend the lifespan of patients with glioblastoma. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology for use in orthotopic patient-derived xenograft models, creating a high-throughput negative-selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators needed for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause-release and elongation factors as one set of in vivo-specific cancer dependencies, and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause-release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, suggesting that targeting transcription elongation machinery may be an effective therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of 'cancer dependencies' not identified by previous in vitro approaches, and could supply new opportunities for therapeutic intervention.

Glioblastoma (GBM) is the most lethal primary brain tumor and is highly resistant to current treatments. GBM harbors glioma stem cells (GSCs) that not only initiate and maintain malignant growth but also promote therapeutic resistance including radioresistance. Thus, targeting GSCs is critical for overcoming the resistance to improve GBM treatment. Because the bone marrow and X-linked (BMX) nonreceptor tyrosine kinase is preferentially up-regulated in GSCs relative to nonstem tumor cells and the BMX-mediated activation of the signal transducer and activator of transcription 3 (STAT3) is required for maintaining GSC self-renewal and tumorigenic potential, pharmacological inhibition of BMX may suppress GBM growth and reduce therapeutic resistance. We demonstrate that BMX inhibition by ibrutinib potently disrupts GSCs, suppresses GBM malignant growth, and effectively combines with radiotherapy. Ibrutinib markedly disrupts the BMX-mediated STAT3 activation in GSCs but shows minimal effect on neural progenitor cells (NPCs) lacking BMX expression. Mechanistically, BMX bypasses the suppressor of cytokine signaling 3 (SOCS3)-mediated inhibition of Janus kinase 2 (JAK2), whereas NPCs dampen the JAK2-mediated STAT3 activation via the negative regulation by SOCS3, providing a molecular basis for targeting BMX by ibrutinib to specifically eliminate GSCs while preserving NPCs. Our preclinical data suggest that repurposing ibrutinib for targeting GSCs could effectively control GBM tumor growth both as monotherapy and as adjuvant with conventional therapies.

Pediatric tumors have enriched the understanding of germline genotype contribution to tumorigenesis. In this issue of Developmental Cell, Yin et al. (2018) describe genetic models of Sonic Hedgehog (SHH) subgroup of medulloblastoma with SUFU alterations, painting more nuanced roles for SUFU in tumorigenesis and maintenance of Gli2 transcription factor circuitries.

// Qi Xie 1 , Qiulian Wu 1 , Stephen C. Mack 1 , Kailin Yang 1, 2 , Leo Kim 1 , Christopher G. Hubert 1 , William A. Flavahan 1, 3, 4, 5 , Chengwei Chu 1, 6 , Shideng Bao 1, 2 , Jeremy N. Rich 1, 2 1 Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA 2 Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA 3 Department of Pathology and Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA 4 Broad Institute of Harvard and Massachusetts Institute of Technology (MIT), Cambridge, MA 02142, USA 5 Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA 6 Division of Neurosurgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung 80756, Taiwan Correspondence to: Jeremy N. Rich, e-mail: drjeremyrich@gmail.com Keywords: cancer stem cell, glioblastoma, glioma, tumor initiating cell, CDC20 Received: March 05, 2015 Accepted: April 16, 2015 Published: April 28, 2015 ABSTRACT Glioblastoma is the most prevalent and lethal primary intrinsic brain tumor. Glioblastoma displays hierarchical arrangement with a population of self-renewing and tumorigenic glioma tumor initiating cells (TICs), or cancer stem cells. While non-neoplastic neural stem cells are generally quiescent, glioblastoma TICs are often proliferative with mitotic control offering a potential point of fragility. Here, we interrogate the role of cell-division cycle protein 20 (CDC20), an essential activator of anaphase-promoting complex (APC) E3 ubiquitination ligase, in the maintenance of TICs. By chromatin analysis and immunoblotting, CDC20 was preferentially expressed in TICs relative to matched non-TICs. Targeting CDC20 expression by RNA interference attenuated TIC proliferation, self-renewal and in vivo tumor growth. CDC20 disruption mediated its effects through induction of apoptosis and inhibition of cell cycle progression. CDC20 maintains TICs through degradation of p21 CIP1/WAF1 , a critical negative regulator of TICs. Inhibiting CDC20 stabilized p21 CIP1/WAF1 , resulting in repression of several genes critical to tumor growth and survival, including CDC25C, c-Myc and Survivin. Transcriptional control of CDC20 is mediated by FOXM1, a central transcription factor in TICs. These results suggest CDC20 is a critical regulator of TIC proliferation and survival, linking two key TIC nodes – FOXM1 and p21 CIP1/WAF1 — elucidating a potential point for therapeutic intervention.

Glioblastoma is a universally lethal cancer with a median survival of approximately 15 months. Despite substantial efforts to define druggable targets, there are no therapeutic options that meaningfully extend glioblastoma patient lifespan. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology for use in orthotopic patient-derived xenograft (PDX) models, creating a high-throughput negative selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators necessary for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause-release and elongation factors as one set of in vivo-specific cancer dependencies and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause-release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, supporting targeting the transcription elongation machinery as a therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of ‘cancer dependencies’ not identified by previous in vitro approaches, which could supply untapped opportunities for therapeutic intervention.

Purpose: Normal stem cells tightly control self-renewal and differentiation during development, but their neoplastic counterparts, cancer stem cells (CSCs), sustain tumorigenicity both through aberrant activation of stemness and evasion of differentiation. Although regulation of CSC stemness has been extensively studied, the molecular mechanisms suppressing differentiation remain unclear. Experimental Design: We performed in silico screening and in vitro validation studies through Western blotting, qRT-PCR for treatment of WNT and SHH signaling inhibitors, and BMP signaling inducer with control and ID1-overexpressing cells. We also performed in vivo drug treatment assays with Balb/c nude mice. Results: Inhibitor of differentiation 1 (ID1) abrogated differentiation signals from bone morphogenetic protein receptor (BMPR) signaling in glioblastoma stem cells (GSCs) to promote self-renewal. ID1 inhibited BMPR2 expression through miRNAs, miR-17 and miR-20a, which are transcriptional targets of MYC. ID1 increases MYC expression by activating WNT and SHH signaling. Combined pharmacologic blockade of WNT and SHH signaling with BMP treatment significantly suppressed GSC self-renewal and extended survival of tumor-bearing mice. Conclusions: Collectively, our results suggested that ID1 simultaneously regulates stemness through WNT and SHH signaling and differentiation through BMPR-mediated differentiation signaling in GSCs, informing a novel therapeutic strategy of combinatorial targeting of stemness and differentiation. Clin Cancer Res; 24(2); 383–94. ©2017 AACR.

Metabolic dysregulation promotes cancer growth through not only energy production, but also epigenetic reprogramming. Here, we report that a critical node in methyl donor metabolism, nicotinamide N-methyltransferase (NNMT), ranked among the most consistently overexpressed metabolism genes in glioblastoma relative to normal brain. NNMT was preferentially expressed by mesenchymal glioblastoma stem cells (GSCs). NNMT depletes S-adenosyl methionine (SAM), a methyl donor generated from methionine. GSCs contained lower levels of methionine, SAM, and nicotinamide, but they contained higher levels of oxidized nicotinamide adenine dinucleotide (NAD+) than differentiated tumor cells. In concordance with the poor prognosis associated with DNA hypomethylation in glioblastoma, depletion of methionine, a key upstream methyl group donor, shifted tumors toward a mesenchymal phenotype and accelerated tumor growth. Targeting NNMT expression reduced cellular proliferation, self-renewal, and in vivo tumor growth of mesenchymal GSCs. Supporting a mechanistic link between NNMT and DNA methylation, targeting NNMT reduced methyl donor availability, methionine levels, and unmethylated cytosine, with increased levels of DNA methyltransferases, DNMT1 and DNMT3A. Supporting the clinical significance of these findings, NNMT portended poor prognosis for glioblastoma patients. Collectively, our findings support NNMT as a GSC-specific therapeutic target in glioblastoma by disrupting oncogenic DNA hypomethylation.

Metabolic dysregulation drives tumor initiation in a subset of glioblastomas harboring isocitrate dehydrogenase (IDH) mutations, but metabolic alterations in glioblastomas with wild-type IDH are poorly understood. MYC promotes metabolic reprogramming in cancer, but targeting MYC has proven notoriously challenging. Here, we link metabolic dysregulation in patient-derived brain tumor–initiating cells (BTIC) to a nexus between MYC and mevalonate signaling, which can be inhibited by statin or 6-fluoromevalonate treatment. BTICs preferentially express mevalonate pathway enzymes, which we find regulated by novel MYC-binding sites, validating an additional transcriptional activation role of MYC in cancer metabolism. Targeting mevalonate activity attenuated RAS-ERK–dependent BTIC growth and self-renewal. In turn, mevalonate created a positive feed-forward loop to activate MYC signaling via induction of miR-33b. Collectively, our results argue that MYC mediates its oncogenic effects in part by altering mevalonate metabolism in glioma cells, suggesting a therapeutic strategy in this setting. Cancer Res; 77(18); 4947–60. ©2017 AACR.

Genetic defects in MOGS, the gene encoding mannosyl-oligosaccharide glucosidase (the first enzyme in the processing pathway of N-linked oligosaccharide), cause the rare congenital disorder of glycosylation type IIb (CDG-IIb), also known as MOGS-CDG. MOGS is expressed in the endoplasmic reticulum and is involved in the trimming of N-glycans. We evaluated two siblings with CDG-IIb who presented with multiple neurologic complications and a paradoxical immunologic phenotype characterized by severe hypogammaglobulinemia but limited clinical evidence of an infectious diathesis. A shortened immunoglobulin half-life was determined to be the mechanism underlying the hypogammaglobulinemia. Impaired viral replication and cellular entry may explain a decreased susceptibility to infections.

Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Here, we trace metabolic aberrations in GSCs to link core genetic mutations in glioblastoma to dependency on de novo pyrimidine synthesis. Targeting the pyrimidine synthetic rate-limiting step enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamyolase, dihydroorotase (CAD) or the critical downstream enzyme, dihydroorotate dehydrogenase (DHODH) inhibited GSC survival, self-renewal, and in vivo tumor initiation through the depletion of the pyrimidine nucleotide supply in rodent models. Mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of metabolic activity of pyrimidine synthesis and GSC tumorigenic capacity. Higher expression of pyrimidine synthesis genes portend poor prognosis of glioblastoma patients. Collectively, our results demonstrate a therapeutic approach of precision medicine through targeting the nexus between driver mutations and metabolic reprogramming in cancer stem cells.