The human genome produces thousands of long non-coding RNAs (lncRNAs) – transcripts >200 nucleotides long that do not encode proteins. While critical roles in normal biology and disease have been revealed for a subset of lncRNAs, the function of the vast majority remains untested. Here, we developed a CRISPR interference (CRISPRi) platform targeting 16,401 lncRNA loci in 7 diverse cell lines including 6 transformed cell lines and human induced pluripotent stem cells (iPSCs). Large-scale screening identified 499 lncRNA loci required for robust cellular growth, of which 89% showed growth modifying function exclusively in one cell type. We further found that lncRNA knockdown can perturb complex transcriptional networks in a cell type-specific manner. These data underscore the functional importance and cell type-specificity of many lncRNAs.

BackgroundTumor-associated macrophages (TAMs) are abundant in gliomas and immunosuppressive TAMs are a barrier to emerging immunotherapies. It is unknown to what extent macrophages derived from peripheral blood adopt the phenotype of brain-resident microglia in pre-treatment gliomas. The relative proportions of blood-derived macrophages and microglia have been poorly quantified in clinical samples due to a paucity of markers that distinguish these cell types in malignant tissue.ResultsWe perform single-cell RNA-sequencing of human gliomas and identify phenotypic differences in TAMs of distinct lineages. We isolate TAMs from patient biopsies and compare them with macrophages from non-malignant human tissue, glioma atlases, and murine glioma models. We present a novel signature that distinguishes TAMs by ontogeny in human gliomas. Blood-derived TAMs upregulate immunosuppressive cytokines and show an altered metabolism compared to microglial TAMs. They are also enriched in perivascular and necrotic regions. The gene signature of blood-derived TAMs, but not microglial TAMs, correlates with significantly inferior survival in low-grade glioma. Surprisingly, TAMs frequently co-express canonical pro-inflammatory (M1) and alternatively activated (M2) genes in individual cells.ConclusionsWe conclude that blood-derived TAMs significantly infiltrate pre-treatment gliomas, to a degree that varies by glioma subtype and tumor compartment. Blood-derived TAMs do not universally conform to the phenotype of microglia, but preferentially express immunosuppressive cytokines and show an altered metabolism. Our results argue against status quo therapeutic strategies that target TAMs indiscriminately and in favor of strategies that specifically target immunosuppressive blood-derived TAMs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1362-4) contains supplementary material, which is available to authorized users.

Noncoding mutations in cancer genomes are frequent but challenging to interpret. PVT1 encodes an oncogenic lncRNA, but recurrent translocations and deletions in human cancers suggest alternative mechanisms. Here, we show that the PVT1 promoter has a tumor-suppressor function that is independent of PVT1 lncRNA. CRISPR interference of PVT1 promoter enhances breast cancer cell competition and growth in vivo. The promoters of the PVT1 and the MYC oncogenes, located 55 kb apart on chromosome 8q24, compete for engagement with four intragenic enhancers in the PVT1 locus, thereby allowing the PVT1 promoter to regulate pause release of MYC transcription. PVT1 undergoes developmentally regulated monoallelic expression, and the PVT1 promoter inhibits MYC expression only from the same chromosome via promoter competition. Cancer genome sequencing identifies recurrent mutations encompassing the human PVT1 promoter, and genome editing verified that PVT1 promoter mutation promotes cancer cell growth. These results highlight regulatory sequences of lncRNA genes as potential disease-associated DNA elements.

Meningiomas arising from the meningothelial central nervous system lining are the most common primary intracranial tumors, and a significant cause of neurologic morbidity and mortality 1 . There are no effective medical therapies for meningioma patients 2,3 , and new treatments have been encumbered by limited understanding of meningioma biology. DNA methylation profiling provides robust classification of central nervous system tumors 4 , and can elucidate targets for molecular therapy 5 . Here we use DNA methylation profiling on 565 meningiomas integrated with genetic, transcriptomic, biochemical, and single-cell approaches to show meningiomas are comprised of 3 epigenetic groups with distinct clinical outcomes and biological features informing new treatments for meningioma patients. Merlin-intact meningiomas (group A, 34%) have the best outcomes and are distinguished by a novel apoptotic tumor suppressor function of NF2/Merlin. Immune-enriched meningiomas (group B, 38%) have intermediate outcomes and are distinguished by immune cell infiltration, HLA expression, and lymphatic vessels. Hypermitotic meningiomas (group C, 28%) have the worst outcomes and are distinguished by convergent genetic mechanisms misactivating the cell cycle. Consistently, we find cell cycle inhibitors block meningioma growth in cell culture, organoids, xenografts, and patients. Our results establish a framework for understanding meningioma biology, and provide preclinical rationale for new therapies to treat meningioma patients.

Background: Long non-coding RNAs (lncRNAs) exhibit highly cell type-specific expression and function, making this class of transcript attractive for targeted cancer therapy. However, the vast majority of lncRNAs have not been tested as potential therapeutic targets, particularly in the context of currently used cancer treatments. Malignant glioma is rapidly fatal, and ionizing radiation is part of the current standard-of-care used to slow tumor growth in both adult and pediatric patients. Results: We use CRISPR interference (CRISPRi) to screen 5689 lncRNA loci in human glioblastoma (GBM) cells, identifying 467 hits that modify cell growth in the presence of clinically relevant doses of fractionated radiation. Thirty-three of these lncRNA hits sensitize cells to radiation, and based on their expression in adult and pediatric gliomas, nine of these hits are prioritized as lncRNA Glioma Radiation Sensitizers (lncGRS). Knockdown of lncGRS-1, a primate-conserved, nuclear-enriched lncRNA, inhibits the growth and proliferation of primary adult and pediatric glioma cells, but not the viability of normal brain cells. Using human brain organoids comprised of mature neural cell types as a three-dimensional tissue substrate to model the invasive growth of glioma, we find that antisense oligonucleotides targeting lncGRS-1 selectively decrease tumor growth and sensitize glioma cells to radiation therapy. Conclusions: These studies identify lncGRS-1 as a glioma-specific therapeutic target and establish a generalizable approach to rapidly identify novel therapeutic targets in the vast non-coding genome to enhance radiation therapy.

Meningiomas are the most common primary intracranial tumors, but the molecular drivers of meningioma tumorigenesis are poorly understood. We hypothesized that investigating intratumor heterogeneity in meningiomas would elucidate biologic drivers and reveal new targets for molecular therapy. To test this hypothesis, here we perform multiplatform molecular profiling of 86 spatially-distinct samples from 13 human meningiomas. Our data reveal that regional alterations in chromosome structure underlie clonal transcriptomic, epigenomic, and histopathologic signatures in meningioma. Stereotactic co-registration of sample coordinates to preoperative magnetic resonance images further suggest that high apparent diffusion coefficient (ADC) distinguishes meningioma regions with proliferating cells enriched for developmental gene expression programs. To understand the function of these genes in meningioma, we develop a human cerebral organoid model of meningioma and validate the high ADC marker genes CDH2 and PTPRZ1 as potential targets for meningioma therapy using live imaging, single cell RNA sequencing, CRISPR interference, and pharmacology.

Long non‐coding RNAs (lncRNAs) have emerged as important regulators of cell biology. The mechanisms by which lncRNAs function are likely numerous, and most are poorly understood. Currently, the mechanisms of functional lncRNAs include those that directly involve the lncRNA transcript, the process of their own transcription and splicing, and even underlying transcriptional regulatory elements within the genomic DNA that encodes the lncRNA. As our understanding of lncRNA biology evolves, so have the methods that are utilized to elucidate their functions. In this review, we survey a collection of different methods used to modulate lncRNA expression levels for the assessment of biological function. From RNA‐targeted strategies, genetic deletions, to engineered gene regulatory systems, the advantages and caveats of each method will be discussed. Ultimately, the selection of tools will be guided by which potential lncRNA mechanisms are being investigated, and no single method alone will likely be sufficient to reveal the function of any particular lncRNA.

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