IFI35 regulates non-canonical NF-κB signaling to maintain glioblastoma stem cells and recruit tumor-associated macrophages.

Glioblastoma (GBM) is the most aggressive malignant primary brain tumor characterized by a highly heterogeneous and immunosuppressive tumor microenvironment (TME). The symbiotic interactions between glioblastoma stem cells (GSCs) and tumor-associated macrophages (TAM) in the TME are critical for tumor progression. Here, we identified that IFI35, a transcriptional regulatory factor, plays both cell-intrinsic and cell-extrinsic roles in maintaining GSCs and the immunosuppressive TME. IFI35 induced non-canonical NF-kB signaling through proteasomal processing of p105 to the DNA-binding transcription factor p50, which heterodimerizes with RELB (RELB/p50), and activated cell chemotaxis in a cell-autonomous manner. Further, IFI35 induced recruitment and maintenance of M2-like TAMs in TME in a paracrine manner. Targeting IFI35 effectively suppressed in vivo tumor growth and prolonged survival of orthotopic xenograft-bearing mice. Collectively, these findings reveal the tumor-promoting functions of IFI35 and suggest that targeting IFI35 or its downstream effectors may provide effective approaches to improve GBM treatment.

Lymphatic endothelial-like cells promote glioblastoma stem cell growth through cytokine-driven cholesterol metabolism.

Glioblastoma is the most lethal primary brain tumor with glioblastoma stem cells (GSCs) atop a cellular hierarchy. GSCs often reside in a perivascular niche, where they receive maintenance cues from endothelial cells, but the role of heterogeneous endothelial cell populations remains unresolved. Here, we show that lymphatic endothelial-like cells (LECs), while previously unrecognized in brain parenchyma, are present in glioblastomas and promote growth of CCR7-positive GSCs through CCL21 secretion. Disruption of CCL21-CCR7 paracrine communication between LECs and GSCs inhibited GSC proliferation and growth. LEC-derived CCL21 induced KAT5-mediated acetylation of HMGCS1 on K273 in GSCs to enhance HMGCS1 protein stability. HMGCS1 promoted cholesterol synthesis in GSCs, favorable for tumor growth. Expression of the CCL21-CCR7 axis correlated with KAT5 expression and HMGCS1K273 acetylation in glioblastoma specimens, informing patient outcome. Collectively, glioblastomas contain previously unrecognized LECs that promote the molecular crosstalk between endothelial and tumor cells, offering potentially alternative therapeutic strategies.

Circular extrachromosomal DNA promotes tumor heterogeneity in high-risk medulloblastoma.

Circular extrachromosomal DNA (ecDNA) in patient tumors is an important driver of oncogenic gene expression, evolution of drug resistance and poor patient outcomes. Applying computational methods for the detection and reconstruction of ecDNA across a retrospective cohort of 481 medulloblastoma tumors from 465 patients, we identify circular ecDNA in 82 patients (18%). Patients with ecDNA-positive medulloblastoma were more than twice as likely to relapse and three times as likely to die within 5 years of diagnosis. A subset of tumors harbored multiple ecDNA lineages, each containing distinct amplified oncogenes. Multimodal sequencing, imaging and CRISPR inhibition experiments in medulloblastoma models reveal intratumoral heterogeneity of ecDNA copy number per cell and frequent putative 'enhancer rewiring' events on ecDNA. This study reveals the frequency and diversity of ecDNA in medulloblastoma, stratified into molecular subgroups, and suggests copy number heterogeneity and enhancer rewiring as oncogenic features of ecDNA.

EGFR promotes ALKBH5 nuclear retention to attenuate N6-methyladenosine and protect against ferroptosis in glioblastoma.

Growth factor receptors rank among the most important oncogenic pathways, but pharmacologic inhibitors often demonstrate limited benefit as monotherapy. Here, we show that epidermal growth factor receptor (EGFR) signaling repressed N6-methyladenosine (m6A) levels in glioblastoma stem cells (GSCs), whereas genetic or pharmacologic EGFR targeting elevated m6A levels. Activated EGFR induced non-receptor tyrosine kinase SRC to phosphorylate the m6A demethylase, AlkB homolog 5 (ALKBH5), thereby inhibiting chromosomal maintenance 1 (CRM1)-mediated nuclear export of ALKBH5 to permit sustained mRNA m6A demethylation in the nucleus. ALKBH5 critically regulated ferroptosis through m6A modulation and YTH N6-methyladenosine RNA binding protein (YTHDF2)-mediated decay of the glutamate-cysteine ligase modifier subunit (GCLM). Pharmacologic targeting of ALKBH5 augmented the anti-tumor efficacy of EGFR and GCLM inhibitors, supporting an EGFR-ALKBH5-GCLM oncogenic axis. Collectively, EGFR reprograms the epitranscriptomic landscape through nuclear retention of the ALKBH5 demethylase to protect against ferroptosis, offering therapeutic paradigms for the treatment of lethal cancers.

Dual Role of CXCL8 in Maintaining the Mesenchymal State of Glioblastoma Stem Cells and M2-Like Tumor-Associated Macrophages.

PURPOSE: The dynamic interplay between glioblastoma stem cells (GSC) and tumor-associated macrophages (TAM) sculpts the tumor immune microenvironment (TIME) and promotes malignant progression of glioblastoma (GBM). However, the mechanisms underlying this interaction are still incompletely understood. Here, we investigate the role of CXCL8 in the maintenance of the mesenchymal state of GSC populations and reprogramming the TIME to an immunosuppressive state. EXPERIMENTAL DESIGN: We performed an integrative multi-omics analyses of RNA sequencing, GBM mRNA expression datasets, immune signatures, and epigenetic profiling to define the specific genes expressed in the mesenchymal GSC subsets. We then used patient-derived GSCs and a xenograft murine model to investigate the mechanisms of tumor-intrinsic and extrinsic factor to maintain the mesenchymal state of GSCs and induce TAM polarization. RESULTS: We identified that CXCL8 was preferentially expressed and secreted by mesenchymal GSCs and activated PI3K/AKT and NF-κB signaling to maintain GSC proliferation, survival, and self-renewal through a cell-intrinsic mechanism. CXCL8 induced signaling through a CXCR2-JAK2/STAT3 axis in TAMs, which supported an M2-like TAM phenotype through a paracrine, cell-extrinsic pathway. Genetic- and small molecule-based inhibition of these dual complementary signaling cascades in GSCs and TAMs suppressed GBM tumor growth and prolonged survival of orthotopic xenograft-bearing mice. CONCLUSIONS: CXCL8 plays critical roles in maintaining the mesenchymal state of GSCs and M2-like TAM polarization in GBM, highlighting an interplay between cell-autonomous and cell-extrinsic mechanisms. Targeting CXCL8 and its downstream effectors may effectively improve GBM treatment.

Sterol regulatory element-binding protein 2 maintains glioblastoma stem cells by keeping the balance between cholesterol biosynthesis and uptake.

BACKGROUND: Glioblastomas (GBMs) display striking dysregulation of metabolism to promote tumor growth. Glioblastoma stem cells (GSCs) adapt to regions of heterogeneous nutrient availability, yet display dependency on de novo cholesterol biosynthesis. The transcription factor Sterol Regulatory Element-Binding Protein 2 (SREBP2) regulates cholesterol biosynthesis enzymes and uptake receptors. Here, we investigate adaptive behavior of GSCs under different cholesterol supplies. METHODS: In silico analysis of patient tumors demonstrated enrichment of cholesterol synthesis associated with decreased angiogenesis. Comparative gene expression of cholesterol biosynthesis enzymes in paired GBM specimens and GSCs were performed. In vitro and in vivo loss-of-function genetic and pharmacologic assays were conducted to evaluate the effect of SREBP2 on GBM cholesterol biosynthesis, proliferation, and self-renewal. Chromatin immunoprecipitation quantitative real-time PCR was leveraged to map the regulation of SREBP2 to cholesterol biosynthesis enzymes and uptake receptors in GSCs. RESULTS: Cholesterol biosynthetic enzymes were expressed at higher levels in GBM tumor cores than in invasive margins. SREBP2 promoted cholesterol biosynthesis in GSCs, especially under starvation, as well as proliferation, self-renewal, and tumor growth. SREBP2 governed the balance between cholesterol biosynthesis and uptake in different nutrient conditions. CONCLUSIONS: SREBP2 displays context-specific regulation of cholesterol biology based on its availability in the microenvironment with induction of cholesterol biosynthesis in the tumor core and uptake in the margin, informing a novel treatment strategy for GBM.

Sorting nexin 10 sustains PDGF receptor signaling in glioblastoma stem cells via endosomal protein sorting.

Glioblastoma is the most malignant primary brain tumor, the prognosis of which remains dismal even with aggressive surgical, medical, and radiation therapies. Glioblastoma stem cells (GSCs) promote therapeutic resistance and cellular heterogeneity due to their self-renewal properties and capacity for plasticity. To understand the molecular processes essential for maintaining GSCs, we performed an integrative analysis comparing active enhancer landscapes, transcriptional profiles, and functional genomics profiles of GSCs and non-neoplastic neural stem cells (NSCs). We identified sorting nexin 10 (SNX10), an endosomal protein sorting factor, as selectively expressed in GSCs compared with NSCs and essential for GSC survival. Targeting SNX10 impaired GSC viability and proliferation, induced apoptosis, and reduced self-renewal capacity. Mechanistically, GSCs utilized endosomal protein sorting to promote platelet-derived growth factor receptor ß (PDGFRß) proliferative and stem cell signaling pathways through posttranscriptional regulation of the PDGFR tyrosine kinase. Targeting SNX10 expression extended survival of orthotopic xenograft-bearing mice, and high SNX10 expression correlated with poor glioblastoma patient prognosis, suggesting its potential clinical importance. Thus, our study reveals an essential connection between endosomal protein sorting and oncogenic receptor tyrosine kinase signaling and suggests that targeting endosomal sorting may represent a promising therapeutic approach for glioblastoma treatment.

ß2-Microglobulin Maintains Glioblastoma Stem Cells and Induces M2-like Polarization of Tumor-Associated Macrophages.

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 ß2-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-ß1 (TGFß1). Inhibition of B2M attenuated GSC survival, self-renewal, and tumor growth. B2M-induced TGFß1 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: ß2-microglobulin signaling in glioblastoma cells activates a PI3K/AKT/MYC/TGFß1 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.

PDGF signaling inhibits mitophagy in glioblastoma stem cells through N6-methyladenosine.

Dysregulated growth factor receptor pathways, RNA modifications, and metabolism each promote tumor heterogeneity. Here, we demonstrate that platelet-derived growth factor (PDGF) signaling induces N6-methyladenosine (m6A) accumulation in glioblastoma (GBM) stem cells (GSCs) to regulate mitophagy. PDGF ligands stimulate early growth response 1 (EGR1) transcription to induce methyltransferase-like 3 (METTL3) to promote GSC proliferation and self-renewal. Targeting the PDGF-METTL3 axis inhibits mitophagy by regulating m6A modification of optineurin (OPTN). Forced OPTN expression phenocopies PDGF inhibition, and OPTN levels portend longer survival of GBM patients; these results suggest a tumor-suppressive role for OPTN. Pharmacologic targeting of METTL3 augments anti-tumor efficacy of PDGF receptor (PDGFR) and mitophagy inhibitors in vitro and in vivo. Collectively, we define PDGF signaling as an upstream regulator of oncogenic m6A regulation, driving tumor metabolism to promote cancer stem cell maintenance, highlighting PDGF-METTL3-OPTN signaling as a GBM therapeutic target.

Glioblastoma stem cells reprogram chromatin in vivo to generate selective therapeutic dependencies on DPY30 and phosphodiesterases.

Glioblastomas are universally fatal cancers and contain self-renewing glioblastoma stem cells (GSCs) that initiate tumors. Traditional anticancer drug discovery based on in vitro cultures tends to identify targets with poor therapeutic indices and fails to accurately model the effects of the tumor microenvironment. Here, leveraging in vivo genetic screening, we identified the histone H3 lysine 4 trimethylation (H3K4me3) regulator DPY30 (Dpy-30 histone methyltransferase complex regulatory subunit) as an in vivo­specific glioblastoma dependency. On the basis of the hypothesis that in vivo epigenetic regulation may define critical GSC dependencies, we interrogated active chromatin landscapes of GSCs derived from intracranial patient-derived xenografts (PDXs) and cell culture through H3K4me3 chromatin immunoprecipitation and transcriptome analyses. Intracranial-specific genes marked by H3K4me3 included FOS, NFκB, and phosphodiesterase (PDE) family members. In intracranial PDX tumors, DPY30 regulated angiogenesis and hypoxia pathways in an H3K4me3-dependent manner but was dispensable in vitro in cultured GSCs. PDE4B was a key downstream effector of DPY30, and the PDE4 inhibitor rolipram preferentially targeted DPY30-expressing cells and impaired PDX tumor growth in mice without affecting tumor cells cultured in vitro. Collectively, the MLL/SET1 (mixed lineage leukemia/SET domain-containing 1, histone lysine methyltransferase) complex member DPY30 selectively regulates H3K4me3 modification on genes critical to support angiogenesis and tumor growth in vivo, suggesting the DPY30-PDE4B axis as a specific therapeutic target in glioblastoma.

Transcription Elongation Machinery Is a Druggable Dependency and Potentiates Immunotherapy in Glioblastoma Stem Cells.

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.

The RNA m6A Reader YTHDF2 Maintains Oncogene Expression and Is a Targetable Dependency in Glioblastoma Stem Cells.

Glioblastoma is a universally lethal cancer driven by glioblastoma stem cells (GSC). Here, we interrogated N 6-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.

The Meningioma Enhancer Landscape Delineates Novel Subgroups and Drives Druggable Dependencies.

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.

Three-dimensional bioprinted glioblastoma microenvironments model cellular dependencies and immune interactions.

Brain tumors are dynamic complex ecosystems with multiple cell types. To model the brain tumor microenvironment in a reproducible and scalable system, we developed a rapid three-dimensional (3D) bioprinting method to construct clinically relevant biomimetic tissue models. In recurrent glioblastoma, macrophages/microglia prominently contribute to the tumor mass. To parse the function of macrophages in 3D, we compared the growth of glioblastoma stem cells (GSCs) alone or with astrocytes and neural precursor cells in a hyaluronic acid-rich hydrogel, with or without macrophage. Bioprinted constructs integrating macrophage recapitulate patient-derived transcriptional profiles predictive of patient survival, maintenance of stemness, invasion, and drug resistance. Whole-genome CRISPR screening with bioprinted complex systems identified unique molecular dependencies in GSCs, relative to sphere culture. Multicellular bioprinted models serve as a scalable and physiologic platform to interrogate drug sensitivity, cellular crosstalk, invasion, context-specific functional dependencies, as well as immunologic interactions in a species-matched neural environment.

Targeting Glioblastoma Stem Cells through Disruption of the Circadian Clock.

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.

Glioblastoma stem cells: lessons from the tumor hierarchy in a lethal cancer.

Glioblastoma ranks among the most lethal of all human cancers. Glioblastomas display striking cellular heterogeneity, with stem-like glioblastoma stem cells (GSCs) at the apex. Although the original identification of GSCs dates back more than a decade, the purification and characterization of GSCs remains challenging. Despite these challenges, the evidence that GSCs play important roles in tumor growth and response to therapy has grown. Like normal stem cells, GSCs are functionally defined and distinguished from their differentiated tumor progeny at core transcriptional, epigenetic, and metabolic regulatory levels, suggesting that no single therapeutic modality will be universally effective against a heterogenous GSC population. Glioblastomas induce a systemic immunosuppression with mixed responses to oncoimmunologic modalities, suggesting the potential for augmentation of response with a deeper consideration of GSCs. Unfortunately, the GSC literature has been complicated by frequent use of inferior cell lines and a lack of proper functional analyses. Collectively, glioblastoma offers a reliable cancer to study cancer stem cells to better model the human disease and inform improved biologic understanding and design of novel therapeutics.

Reciprocal Signaling between Glioblastoma Stem Cells and Differentiated Tumor Cells Promotes Malignant Progression.

Glioblastoma is the most lethal primary brain tumor; however, the crosstalk between glioblastoma stem cells (GSCs) and their supportive niche is not well understood. Here, we interrogated reciprocal signaling between GSCs and their differentiated glioblastoma cell (DGC) progeny. We found that DGCs accelerated GSC tumor growth. DGCs preferentially expressed brain-derived neurotrophic factor (BDNF), whereas GSCs expressed the BDNF receptor NTRK2. Forced BDNF expression in DGCs augmented GSC tumor growth. To determine molecular mediators of BDNF-NTRK2 paracrine signaling, we leveraged transcriptional and epigenetic profiles of matched GSCs and DGCs, revealing preferential VGF expression by GSCs, which patient-derived tumor models confirmed. VGF serves a dual role in the glioblastoma hierarchy by promoting GSC survival and stemness in vitro and in vivo while also supporting DGC survival and inducing DGC secretion of BDNF. Collectively, these data demonstrate that differentiated glioblastoma cells cooperate with stem-like tumor cells through BDNF-NTRK2-VGF paracrine signaling to promote tumor growth.

Inhibition of ID1-BMPR2 Intrinsic Signaling Sensitizes Glioma Stem Cells to Differentiation Therapy.

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.

Transcription elongation factors represent in vivo cancer dependencies in glioblastoma.

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.

Messenger RNA Methylation Regulates Glioblastoma Tumorigenesis.

Messenger RNA (mRNA) modification provides an additional layer of gene regulation in cells. In this issue of Cancer Cell, Zhang et al. report that ALKBH5, a demethylase of the mRNA modification N6-methyladenosine, regulates proliferation and self-renewal of glioblastoma stem-like cells by modulating pre-mRNA stability and expression of the FOXM1 gene.