Human cancers are complex ecosystems composed of cells with distinct phenotypes, genotypes and epigenetic states, but current models do not adequately reflect tumor composition in patients. We used single cell RNA-seq to profile 430 cells from five primary glioblastomas, which we found to be inherently variable in their expression of diverse transcriptional programs related to oncogenic signaling, proliferation, complement/immune response and hypoxia. We also observed a continuum of stemness-related expression states that enabled us to identify putative regulators of stemness in vivo. Finally, we show that established glioblastoma subtype classifiers are variably expressed across individual cells within a tumor and demonstrate the potential prognostic implications of such intratumoral heterogeneity. Thus, we reveal previously unappreciated heterogeneity in diverse regulatory programs central to glioblastoma biology, prognosis, and therapy.Tumor heterogeneity poses a major challenge to cancer diagnosis and treatment. It can manifest as variability between tumors, wherein different stages, genetic lesions or * Corresponding authors: Bernstein.bradley@mgh.harvard.edu (BEB), aregev@broadinstitute.org (AR), Suva.Mario@mgh.harvard.edu (MLS). † These authors contributed equally to this work. ‡ These authors contributed equally to this work. Glioblastoma is an archetypal example of a heterogeneous cancer and one of the most lethal human malignancies (9, 10). Intratumoral heterogeneity and redundant signaling routes likely underlie the inability of conventional and targeted therapies to achieve long-term remissions (11-13). These tumors contain cellular niches enriched for distinct phenotypic properties, including transient quiescence and self-renewal (14-16), adaptation to hypoxia (17), and resistance to radiation induced DNA damage (18,19). DNA and RNA profiles of bulk tumors have enabled genetic and transcriptional classification of glioblastomas (20,21). However, the relationships between different sources of intratumoral heterogeneitygenetic, transcriptional and functional -remain obscure.Single cell transcriptome analysis by 23) should in principle enable functional characterization from landmark genes and annotated gene sets, relate in vivo states to in vitro models, inform transcriptional classifications based on bulk tumors, and even capture genetic information for expressed transcripts. To interrogate intratumoral heterogeneity systematically, we isolated individual cells from five freshly resected and dissociated human glioblastomas and generated single cell full-length transcriptomes using SMART-seq (96-192 cells/tumor, total 672 cells; Fig. 1A). Prior to sorting, the suspension was depleted for CD45 + cells to remove inflammatory infiltrate. As a control, we also generated population (bulk) RNA-seq profiles from the CD45-depleted tumor samples. All tumors were IDH1/2 wild type primary glioblastomas ( Fig. S1) and three were EGFR amplified as determined by routine clinical tests (Table S1). We exclude...

Diverse genetic, epigenetic, and developmental programs drive glioblastoma, an incurable and poorly understood tumor, but their precise characterization remains challenging. Here, we use an integrative approach spanning single-cell RNA-sequencing of 28 tumors, bulk genetic and expression analysis of 401 specimens from the The Cancer Genome Atlas (TCGA), functional approaches, and single-cell lineage tracing to derive a unified model of cellular states and genetic diversity in glioblastoma. We find that malignant cells in glioblastoma exist in four main cellular states that recapitulate distinct neural cell types, are influenced by the tumor microenvironment, and exhibit plasticity. The relative frequency of cells in each state varies between glioblastoma samples and is influenced by copy number amplifications of the CDK4, EGFR, and PDGFRA loci and by mutations in the NF1 locus, which each favor a defined state. Our work provides a blueprint for glioblastoma, integrating the malignant cell programs, their plasticity, and their modulation by genetic drivers.

Summary Developmental fate decisions are dictated by master transcription factors (TFs) that interact with cis-regulatory elements to direct transcriptional programs. Certain malignant tumors may also depend on cellular hierarchies reminiscent of normal development but superimposed on underlying genetic aberrations. In glioblastoma (GBM), a subset of stem-like tumor-propagating cells (TPCs) appears to drive tumor progression and underlie therapeutic resistance, yet remain poorly understood. Here, we identify a core set of neurodevelopmental TFs (POU3F2, SOX2, SALL2, OLIG2) essential for GBM propagation. These TFs coordinately bind and activate TPC-specific regulatory elements, and are sufficient to fully reprogram differentiated GBM cells to ‘induced’ TPCs, recapitulating the epigenetic landscape and phenotype of native TPCs. We reconstruct a network model that highlights critical interactions and identifies novel therapeutic targets for eliminating TPCs. Our study establishes the epigenetic basis of a developmental hierarchy in GBM, provides detailed insight into underlying gene regulatory programs, and suggests attendant therapeutic strategies.

INTRODUCTION Tumor fitness, evolution, and resistance to therapy are governed by selection of malignant cells with specific genotypes, by expression programs related to cellular phenotypes, and by influences of the tumor microenvironment (TME). Although bulk tumor analysis can interrogate the genetic state of tumor cells with high precision, bulk expression profiles average the diverse cells within each tumor, thereby masking critical differences and providing limited insight into cancer cell programs and TME influences. Single-cell RNA sequencing (scRNA-seq) can help to address those challenges but incurs financial and logistic considerations, including the time required to accrue large cohorts of fresh tumor specimen for single-cell analysis. RATIONALE We reasoned that scRNA-seq of a limited number of representative tumors could be combined with bulk data from large cohorts to decipher differences between tumor subclasses. In this approach, bulk samples collected for large cohorts, such as from The Cancer Genome Atlas (TCGA), are first used to define the combined effects of differences in cancer cell genotypes, phenotypes, and the composition of the TME. Single-cell analysis of a limited set of representative tumors is then used to distinguish those effects. We applied this approach to understand the differences between two types of isocitrate dehydrogenase (IDH)-mutant gliomas: astrocytoma (IDH-A) and oligodendroglioma (IDH-O). IDH-A and IDH-O are distinguished by co-occurring signature genetic events and by histopathology and are thought to recapitulate distinct glial lineages. By combining 9879 scRNA-seq profiles from 10 IDH-A tumors, 4347 scRNA-seq profiles from six IDH-O tumors, and 165 TCGA bulk RNA profiles, we could decipher differences between these two tumor types at single-cell resolution. RESULTS We find that differences in bulk expression profiles between IDH-A and IDH-O are primarily explained by the impact of signature genetic events and TME composition, but not by distinct expression programs of glial lineages in the malignant cells. We infer that both IDH-A and IDH-O share the same developmental hierarchy, consisting in each case of three subpopulations of malignant cells: nonproliferating cells differentiated along the astrocytic and oligodendrocytic lineages, and proliferative undifferentiated cells that resemble neural stem/progenitor cells. By analyzing tumors of different clinical grades, we observe that higher-grade tumors present enhanced proliferation, larger pools of undifferentiated glioma cells, and an increase in macrophage over microglia programs in the TME. CONCLUSION Our approach provides a general framework to decipher differences between classes of human tumors by decoupling cancer cell genotypes, phenotypes, and the composition of the TME. The shared glial lineages and developmental hierarchies observed in IDH-A and IDH-O suggest a common progenitor for all IDH-mutant gliomas, shedding light on a longstanding debate in gliomagenesis. In contrast to the similarity in gl...

Summary Heterozygous mutation of IDH1 in cancers modifies IDH1 enzymatic activity, reprogramming metabolite flux and markedly elevating 2-hydroxyglutarate (2-HG). Here, we found that 2-HG depletion did not inhibit growth of several IDH1 mutant solid cancer types. To identify other metabolic therapeutic targets, we systematically profiled metabolites in endogenous IDH1 mutant cancer cells after mutant IDH1 inhibition and discovered a profound vulnerability to depletion of the coenzyme NAD+. Mutant IDH1 lowered NAD+ levels by downregulating the NAD+ salvage pathway enzyme nicotinate phosphoribosyltransferase (Naprt1), sensitizing to NAD+ depletion via concomitant nicotinamide phosphoribosyltransferase (NAMPT) inhibition. NAD+ depletion activated the intracellular energy sensor AMPK, triggered autophagy and resulted in cytotoxicity. Thus, we identify NAD+ depletion as a metabolic susceptibility of IDH1 mutant cancers.

Cancer cells rely on telomerase or the alternative lengthening of telomeres (ALT) pathway to overcome replicative mortality. ALT is mediated by recombination and is prevalent in a subset of human cancers, yet whether it can be exploited therapeutically remains unknown. Loss of the chromatin remodeling protein ATRX associates with ALT in cancers. Here, we show that ATRX loss compromises cell-cycle regulation of the telomeric non-coding RNA TERRA and leads to persistent association of replication protein A (RPA) with telomeres after DNA replication, creating a recombinogenic nucleoprotein structure. Inhibition of the protein kinase ATR, a critical regulator of recombination recruited by RPA, disrupts ALT and triggers chromosome fragmentation and apoptosis in ALT cells. Importantly, the cell death induced by ATR inhibitors is highly selective for cancer cells that rely on ALT, , suggesting that such inhibitors may be useful for treatment of ALT-positive cancers.Cancer cells overcome replicative senescence by activating telomerase or the alternative lengthening of telomeres (ALT) pathway (1-3). ALT is used in ~5-15% of all human Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript cancers and is prevalent in specific cancer types, including osteosarcoma and glioblastoma (4). Currently, there are no therapies specifically targeting ALT. ALT relies on recombination to elongate telomeres (3), but how the recombinogenic state of ALT telomeres is established remains elusive. In contrast to cancer cells defective for homologous recombination (HR) and susceptible to Poly(ADP-ribose) polymerase (PARP) inhibition (5, 6), ALT-positive cells are HR-proficient (7). Thus, the reliance of ALT on recombination raises an important question as to whether recombination can be exploited in ALT-positive cancers as a means for targeted therapy.Single-stranded DNA (ssDNA) coated by replication protein A (RPA) is a key intermediate in both DNA replication and HR (8). RPA transiently associates with telomeres during DNA replication, but is released from telomeres after S phase (9, 10). The release of RPA may be an important mechanism to suppress HR at telomeres. The association of RPA with telomeres in S phase is facilitated by TERRA, the telomere repeat-containing RNA, which is also present at telomeres during this period (9,(11)(12)(13). To investigate how ALT is established, we determined whether the association of TERRA with telomeres is altered in ALT cells. TERRA colocalized with the telomere-binding protein TRF2 in telomerasepositive HeLa cervical cancer cells ( fig. S1) (9). However, in both HeLa and telomerasepositive SJSA1 osteosarcoma cells ( fig. S24B), the number of TERRA foci declined from S phase to G2 ( Considering that RPA is released from telomeres in G2/M when TERRA is repressed by ATRX (9), we examined whether ATRX is required for the release of RPA. In HeLa cells, numerous small replication-associated RPA foci (type-A RPA foci) were detected in S phase (Fig. S7). As cells progressed from S to ...

Glioblastoma, the most malignant type of primary brain tumor, is one of the solid cancers where cancer stem cells have been isolated, and studies have suggested resistance of those cells to chemotherapy and radiotherapy. Here, we report the establishment of CSC-enriched cultures derived from human glioblastoma specimens. They grew as neurospheres in serumfree medium with epidermal growth factor and fibroblast growth factor 2, varied in the level of CD133 expression and very efficiently formed highly invasive and/or vascular tumors upon intracerebral implantation into immunodeficient mice. As a novel therapeutic strategy for glioblastoma-derived cancer stem-like cells (GBM-SC), we have tested oncolytic herpes simplex virus (oHSV) vectors. We show that although ICP6 (UL39)-deleted mutants kill GBM-SCs as efficiently as wild-type HSV, the deletion of g34.5 significantly attenuated the vectors due to poor replication. However, this was significantly reversed by the additional deletion of a47. Infection with oHSV G47# (ICP6À ) not only killed GBMSCs but also inhibited their self-renewal as evidenced by the inability of viable cells to form secondary tumor spheres. Importantly, despite the highly invasive nature of the intracerebral tumors generated by GBM-SCs, intratumoral injection of G47# significantly prolonged survival. These results for the first time show the efficacy of oHSV against human GBM-SCs, and correlate this cytotoxic property with specific oHSV mutations. This is important for designing new oHSV vectors and clinical trials. Moreover, the new glioma models described in this study provide powerful tools for testing experimental therapeutics and studying invasion and angiogenesis. [Cancer Res 2009;69(8):3472-81]

The poor prognosis of patients with aggressive and invasive cancers combined with toxic effects and short half-life of currently available treatments necessitate development of more effective tumor selective therapies. Mesenchymal stem cells (MSCs) are emerging as novel cell-based delivery agents; however, a thorough investigation addressing their therapeutic potential and fate in different cancer models is lacking. In this study, we explored the engineering potential, fate, and therapeutic efficacy of human MSCs in a highly malignant and invasive model of glioblastoma. We show that engineered MSC retain their ''stem-like'' properties, survive longer in mice with gliomas than in the normal brain, and migrate extensively toward gliomas. We also show that MSCs are resistant to the cytokine tumor necrosis factor apoptosis ligand (TRAIL) and, when engineered to express secreted recombinant TRAIL, induce caspase-mediated apoptosis in established glioma cell lines as well as CD133-positive primary glioma cells in vitro. Using highly malignant and invasive human glioma models and employing real-time imaging with correlative neuropathology, we demonstrate that MSC-delivered recombinant TRAIL has profound anti-tumor effects in vivo. This study demonstrates the efficacy of diagnostic and therapeutic MSC in preclinical glioma models and forms the basis for developing stem cell-based therapies for different cancers.gliomas ͉ in vivo imaging ͉ TRAIL