CRISPRi-based radiation modifier screen identifies long non-coding RNA therapeutic targets in glioma.

BackgroundLong 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.ResultsWe 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.ConclusionsThese 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

Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma

Summary High-grade gliomas (HGG) are a devastating group of cancers, representing the leading cause of brain tumor-related death in both children and adults. Therapies aimed at mechanisms intrinsic to the glioma cell have translated to only limited success; effective therapeutic strategies will need to also target elements of the tumor microenvironment that promote glioma progression. We recently demonstrated that neuronal activity robustly promotes the growth of a range of molecularly and clinically distinct HGG types, including adult glioblastoma (GBM), anaplastic oligodendroglioma, pediatric GBM, and diffuse intrinsic pontine glioma (DIPG)1. An important mechanism mediating this neural regulation of brain cancer is activity-dependent cleavage and secretion of the synaptic molecule neuroligin-3 (NLGN3), which promotes glioma proliferation through the PI3K-mTOR pathway1. However, neuroligin-3 necessity to glioma growth, proteolytic mechanism of secretion and further molecular consequences in glioma remain to be clarified. Here, we demonstrate a striking dependence of HGG growth on microenvironmental neuroligin-3, elucidate signaling cascades downstream of neuroligin-3 binding in glioma and determine a therapeutically targetable mechanism of secretion. Patient-derived orthotopic xenografts of pediatric GBM, DIPG and adult GBM fail to grow in Nlgn3 knockout mice. Neuroligin-3 stimulates numerous oncogenic pathways, including early focal adhesion kinase activation upstream of PI3K-mTOR, and induces transcriptional changes including upregulation of numerous synapse-related genes in glioma cells. Neuroligin-3 is cleaved from both neurons and oligodendrocyte precursor cells via the ADAM10 sheddase. ADAM10 inhibitors prevent release of neuroligin-3 into the tumor microenvironment and robustly block HGG xenograft growth. This work defines a promising strategy for targeting neuroligin-3 secretion, which could prove transformative for HGG therapy

The oncolytic virus Delta-24-RGD elicits an antitumor effect in pediatric glioma and DIPG mouse models

Pediatric high-grade glioma (pHGG) and diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors in desperate need of a curative treatment. Oncolytic virotherapy is emerging as a solid therapeutic approach. Delta-24-RGD is a replication competent adenovirus engineered to replicate in tumor cells with an aberrant RB pathway. This virus has proven to be safe and effective in adult gliomas. Here we report that the administration of Delta-24-RGD is safe in mice and results in a significant increase in survival in immunodeficient and immunocompetent models of pHGG and DIPGs. Our results show that the Delta-24-RGD antiglioma effect is mediated by the oncolytic effect and the immune response elicited against the tumor. Altogether, our data highlight the potential of this virus as treatment for patients with these tumors. Of clinical significance, these data have led to the start of a phase I/II clinical trial at our institution for newly diagnosed DIPG (NCT03178032)

Single-cell RNA-seq supports a developmental hierarchy in human oligodendroglioma

Although human tumours are shaped by the genetic evolution of cancer cells, evidence also suggests that they display hierarchies related to developmental pathways and epigenetic programs in which cancer stem cells (CSCs) can drive tumour growth and give rise to differentiated progeny. Yet, unbiased evidence for CSCs in solid human malignancies remains elusive. Here we profile 4,347 single cells from six IDH1 or IDH2 mutant human oligodendrogliomas by RNA sequencing (RNA-seq) and reconstruct their developmental programs from genome-wide expression signatures. We infer that most cancer cells are differentiated along two specialized glial programs, whereas a rare subpopulation of cells is undifferentiated and associated with a neural stem cell expression program. Cells with expression signatures for proliferation are highly enriched in this rare subpopulation, consistent with a model in which CSCs are primarily responsible for fuelling the growth of oligodendroglioma in humans. Analysis of copy number variation (CNV) shows that distinct CNV sub-clones within tumours display similar cellular hierarchies, suggesting that the architecture of oligodendroglioma is primarily dictated by developmental programs. Subclonal point mutation analysis supports a similar model, although a full phylogenetic tree would be required to definitively determine the effect of genetic evolution on the inferred hierarchies. Our single-cell analyses provide insight into the cellular architecture of oligodendrogliomas at single-cell resolution and support the cancer stem cell model, with substantial implications for disease management

Integrated Molecular Meta-Analysis of 1,000 Pediatric High-Grade and Diffuse Intrinsic Pontine Glioma.

We collated data from 157 unpublished cases of pediatric high-grade glioma and diffuse intrinsic pontine glioma and 20 publicly available datasets in an integrated analysis of >1,000 cases. We identified co-segregating mutations in histone-mutant subgroups including loss of FBXW7 in H3.3G34R/V, TOP3A rearrangements in H3.3K27M, and BCOR mutations in H3.1K27M. Histone wild-type subgroups are refined by the presence of key oncogenic events or methylation profiles more closely resembling lower-grade tumors. Genomic aberrations increase with age, highlighting the infant population as biologically and clinically distinct. Uncommon pathway dysregulation is seen in small subsets of tumors, further defining the molecular diversity of the disease, opening up avenues for biological study and providing a basis for functionally defined future treatment stratification

Epigenetic targeting of Hedgehog pathway transcriptional output through BET bromodomain inhibition

Hedgehog signaling drives oncogenesis in several cancers and strategies targeting this pathway have been developed, most notably through inhibition of Smoothened. However, resistance to Smoothened inhibitors occurs via genetic changes of Smoothened or other downstream Hedgehog components. Here, we overcome these resistance mechanisms by modulating GLI transcription via inhibition of BET bromodomain proteins. We show the BET bromodomain protein, BRD4, regulates GLI transcription downstream of SMO and SUFU and chromatin immunoprecipitation studies reveal BRD4 directly occupies GLI1 and GLI2 promoters, with a substantial decrease in engagement of these sites upon treatment with JQ1, a small molecule inhibitor targeting BRD4. Globally, genes associated with medulloblastoma-specific GLI1 binding sites are downregulated in response to JQ1 treatment, supporting direct regulation of GLI activity by BRD4. Notably, patient- and GEMM-derived Hedgehog-driven tumors (basal cell carcinoma, medulloblastoma and atypical teratoid/rhabdoid tumor) respond to JQ1 even when harboring genetic lesions rendering them resistant to Smoothened antagonists

Functional diversity and co-operativity between subclonal populations of paediatric glioblastoma and diffuse intrinsic pontine glioma cells

The failure to develop effective therapies for pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) is in part due to their intrinsic heterogeneity. We aimed to quantitatively assess the extent to which this was present in these tumors through subclonal genomic analyses and to determine whether distinct tumor subpopulations may interact to promote tumorigenesis by generating subclonal patient-derived models in vitro and in vivo. Analysis of 142 sequenced tumors revealed multiple tumor subclones, spatially and temporally coexisting in a stable manner as observed by multiple sampling strategies. We isolated genotypically and phenotypically distinct subpopulations that we propose cooperate to enhance tumorigenicity and resistance to therapy. Inactivating mutations in the H4K20 histone methyltransferase KMT5B (SUV420H1), present in <1% of cells, abrogate DNA repair and confer increased invasion and migration on neighboring cells, in vitro and in vivo, through chemokine signaling and modulation of integrins. These data indicate that even rare tumor subpopulations may exert profound effects on tumorigenesis as a whole and may represent a new avenue for therapeutic development. Unraveling the mechanisms of subclonal diversity and communication in pGBM and DIPG will be an important step toward overcoming barriers to effective treatments

Glioma

Gliomas are primary brain tumours that are thought to develop from neural stem or progenitor cells that carry tumour-initiating genetic alterations. Based on microscopic appearance and molecular characteristics, they are classified according to the WHO classification of central nervous system (CNS) tumours and graded into CNS WHO grades 1-4 from a low to high grade of malignancy. Diffusely infiltrating gliomas in adults comprise three tumour types with distinct natural course of disease, response to treatment and outcome: isocitrate dehydrogenase (IDH)-mutant and 1p/19q-codeleted oligodendrogliomas with the best prognosis; IDH-mutant astrocytomas with intermediate outcome; and IDH-wild-type glioblastomas with poor prognosis. Pilocytic astrocytoma is the most common glioma in children and is characterized by circumscribed growth, frequent BRAF alterations and favourable prognosis. Diffuse gliomas in children are divided into clinically indolent low-grade tumours and high-grade tumours with aggressive behaviour, with histone 3 K27-altered diffuse midline glioma being the leading cause of glioma-related death in children. Ependymal tumours are subdivided into biologically and prognostically distinct types on the basis of histology, molecular biomarkers and location. Although surgery, radiotherapy and alkylating agent chemotherapy are the mainstay of glioma treatment, individually tailored strategies based on tumour-intrinsic dominant signalling pathways have improved outcome in subsets of patients

Diffusion-Weighted MRI Derived Apparent Diffusion Coefficient Identifies Prognostically Distinct Subgroups of Pediatric Diffuse Intrinsic Pontine Glioma

While pediatric diffuse intrinsic pontine gliomas (DIPG) remain fatal, recent data have shown subgroups with distinct molecular biology and clinical behavior. We hypothesized that diffusion-weighted MRI can be used as a prognostic marker to stratify DIPG subsets with distinct clinical behavior. Apparent diffusion coefficient (ADC) values derived from diffusion-weighted MRI were computed in 20 consecutive children with treatment-naïve DIPG tumors. The median ADC for the cohort was used to stratify the tumors into low and high ADC groups. Survival, gender, therapy, and potential steroid effects were compared between the ADC groups. Median age at diagnosis was 6.6 (range 2.3-13.2) years, with median follow-up seven (range 1-36) months. There were 14 boys and six girls. Seventeen patients received radiotherapy, five received chemotherapy, and six underwent cerebrospinal fluid diversion. The median ADC of 1,295 × 10(-6) mm(2)/s for the cohort partitioned tumors into low or high diffusion groups, which had distinct median survivals of 3 and 13 months, respectively (log-rank p \u3c 0.001). Low ADC tumors were found only in boys, whereas high ADC tumors were found in both boys and girls. Available tissue specimens in three low ADC tumors demonstrated high-grade histology, whereas one high ADC tumor demonstrated low-grade histology with a histone H3.1 K27M mutation and high-grade metastatic lesion at autopsy. ADC derived from diffusion-weighted MRI may identify prognostically distinct subgroups of pediatric DIPG

Diffusion-Weighted MRI Derived Apparent Diffusion Coefficient Identifies Prognostically Distinct Subgroups of Pediatric Diffuse Intrinsic Pontine Glioma

While pediatric diffuse intrinsic pontine gliomas (DIPG) remain fatal, recent data have shown subgroups with distinct molecular biology and clinical behavior. We hypothesized that diffusion-weighted MRI can be used as a prognostic marker to stratify DIPG subsets with distinct clinical behavior. Apparent diffusion coefficient (ADC) values derived from diffusion-weighted MRI were computed in 20 consecutive children with treatment-naïve DIPG tumors. The median ADC for the cohort was used to stratify the tumors into low and high ADC groups. Survival, gender, therapy, and potential steroid effects were compared between the ADC groups. Median age at diagnosis was 6.6 (range 2.3-13.2) years, with median follow-up seven (range 1-36) months. There were 14 boys and six girls. Seventeen patients received radiotherapy, five received chemotherapy, and six underwent cerebrospinal fluid diversion. The median ADC of 1,295 × 10(-6) mm(2)/s for the cohort partitioned tumors into low or high diffusion groups, which had distinct median survivals of 3 and 13 months, respectively (log-rank p \u3c 0.001). Low ADC tumors were found only in boys, whereas high ADC tumors were found in both boys and girls. Available tissue specimens in three low ADC tumors demonstrated high-grade histology, whereas one high ADC tumor demonstrated low-grade histology with a histone H3.1 K27M mutation and high-grade metastatic lesion at autopsy. ADC derived from diffusion-weighted MRI may identify prognostically distinct subgroups of pediatric DIPG