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.

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

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

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.

An in vivo RNA interference screening strategy in glioblastoma enabled the identification of a host of epigenetic targets required for glioblastoma cell survival that were not identified by parallel standard screening in cell culture, including the transcription pause–release factor JMJD6, and could be a powerful tool to uncover new therapeutic targets in cancer. Most high-throughput target discovery screens for glioblastoma have been limited to in vitro models with uncertain physiological relevance. Here, Jeremy Rich and colleagues perform two parallel RNA interference screens for transcriptional regulators, comparing an in vitro screen in cell lines to an in vivo screen that recapitulates the tumour microenvironment. They find several transcriptional elongation factors that are specifically required for glioblastoma cell survival in vivo, particularly the transcriptional pause release factor JMJD6 which is highly expressed in gliomas. This type of in vivo functional screen has the potential to uncover novel therapeutic targets for cancer that have not been identified in previous in vitro approaches. Glioblastoma is a universally lethal cancer with a median survival time of approximately 15 months1. 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 technology2,3,4 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.