Remote neuronal activity drives glioma progression through SEMA4F.

The tumour microenvironment plays an essential role in malignancy, and neurons have emerged as a key component of the tumour microenvironment that promotes tumourigenesis across a host of cancers1,2. Recent studies on glioblastoma (GBM) highlight bidirectional signalling between tumours and neurons that propagates a vicious cycle of proliferation, synaptic integration and brain hyperactivity3-8; however, the identity of neuronal subtypes and tumour subpopulations driving this phenomenon is incompletely understood. Here we show that callosal projection neurons located in the hemisphere contralateral to primary GBM tumours promote progression and widespread infiltration. Using this platform to examine GBM infiltration, we identified an activity-dependent infiltrating population present at the leading edge of mouse and human tumours that is enriched for axon guidance genes. High-throughput, in vivo screening of these genes identified SEMA4F as a key regulator of tumourigenesis and activity-dependent progression. Furthermore, SEMA4F promotes the activity-dependent infiltrating population and propagates bidirectional signalling with neurons by remodelling tumour-adjacent synapses towards brain network hyperactivity. Collectively our studies demonstrate that subsets of neurons in locations remote to primary GBM promote malignant progression, and also show new mechanisms of glioma progression that are regulated by neuronal activity.

Remote neuronal activity drives glioma infiltration via Sema4f.

The tumor microenvironment (TME) plays an essential role in malignancy and neurons have emerged as a key component of the TME that promotes tumorigenesis across a host of cancers. Recent studies on glioblastoma (GBM) highlight bi-directional signaling between tumors and neurons that propagates a vicious cycle of proliferation, synaptic integration, and brain hyperactivity; however, the identity of neuronal subtypes and tumor subpopulations driving this phenomenon are incompletely understood. Here we show that callosal projection neurons located in the hemisphere contralateral to primary GBM tumors promote progression and widespread infiltration. Using this platform to examine GBM infiltration, we identified an activity dependent infiltrating population present at the leading edge of mouse and human tumors that is enriched for axon guidance genes. High-throughput, in vivo screening of these genes identified Sema4F as a key regulator of tumorigenesis and activity-dependent infiltration. Furthermore, Sema4F promotes the activity-dependent infiltrating population and propagates bi-directional signaling with neurons by remodeling tumor adjacent synapses towards brain network hyperactivity. Collectively, our studies demonstrate that subsets of neurons in locations remote to primary GBM promote malignant progression, while revealing new mechanisms of tumor infiltration that are regulated by neuronal activity.

Glioma epileptiform activity and progression are driven by IGSF3-mediated potassium dysregulation.

Seizures are a frequent pathophysiological feature of malignant glioma. Recent studies implicate peritumoral synaptic dysregulation as a driver of brain hyperactivity and tumor progression; however, the molecular mechanisms that govern these phenomena remain elusive. Using scRNA-seq and intraoperative patient ECoG recordings, we show that tumors from seizure patients are enriched for gene signatures regulating synapse formation. Employing a human-to-mouse in vivo functionalization pipeline to screen these genes, we identify IGSF3 as a mediator of glioma progression and dysregulated neural circuitry that manifests as spreading depolarization (SD). Mechanistically, we discover that IGSF3 interacts with Kir4.1 to suppress potassium buffering and found that seizure patients exhibit reduced expression of potassium handlers in proliferating tumor cells. In vivo imaging reveals that dysregulated synaptic activity emanates from the tumor-neuron interface, which we confirm in patients. Our studies reveal that tumor progression and seizures are enabled by ion dyshomeostasis and identify SD as a driver of disease.

Medium-Chain Acyl-CoA Dehydrogenase Protects Mitochondria from Lipid Peroxidation in Glioblastoma.

Glioblastoma (GBM) is highly resistant to chemotherapies, immune-based therapies, and targeted inhibitors. To identify novel drug targets, we screened orthotopically implanted, patient-derived glioblastoma sphere-forming cells using an RNAi library to probe essential tumor cell metabolic programs. This identified high dependence on mitochondrial fatty acid metabolism. We focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids (MCFA), due to its consistently high score and high expression among models and upregulation in GBM compared with normal brain. Beyond the expected energetics impairment, MCAD depletion in primary GBM models induced an irreversible cascade of detrimental metabolic effects characterized by accumulation of unmetabolized MCFAs, which induced lipid peroxidation and oxidative stress, irreversible mitochondrial damage, and apoptosis. Our data uncover a novel protective role for MCAD to clear lipid molecules that may cause lethal cell damage, suggesting that therapeutic targeting of MCFA catabolism may exploit a key metabolic feature of GBM. SIGNIFICANCE: MCAD exerts a protective role to prevent accumulation of toxic metabolic by-products in glioma cells, actively catabolizing lipid species that would otherwise affect mitochondrial integrity and induce cell death. This work represents a first demonstration of a nonenergetic role for dependence on fatty acid metabolism in cancer.This article is highlighted in the In This Issue feature, p. 2659.

Identification of diverse tumor endothelial cell populations in malignant glioma.

BACKGROUND: Glioblastoma is the most common and aggressive type of primary brain tumor, as most patients succumb to the disease less than two years after diagnosis. Critically, studies demonstrate that glioma recruits surrounding blood vessels, while some work suggests that tumor stem cells themselves directly differentiate into endothelial cells, yet the molecular and cellular dynamics of the endothelium in glioma are poorly characterized. The goal of this study was to establish molecular and morphological benchmarks for tumor associated vessels (TAVs) and tumor derived endothelial cells (TDECs) during glioblastoma progression. METHODS: Using In-Utero Electroporation and CRISPR/Cas9 genome engineering to generate a native, immunocompetent mouse model of glioma, we characterized vascular-tumor dynamics in three dimensions during tumor progression. We employed bulk and single-cell RNA-Sequencing to elucidate the relationship between TAVs and TDECs. We confirmed our findings in a patient derived orthotopic xenograft (PDOX) model. RESULTS: Using a mouse model of glioma, we identified progressive alteration of vessel function and morphogenesis over time. We also showed in our mouse model that TDECs are a rare subpopulation that contributes to vessels within the tumor, albeit to a limited degree. Furthermore, transcriptional profiling demonstrates that both TAVs and TDECs are molecularly distinct, and both populations feature extensive molecular heterogeneity. Finally, the distinct molecular signatures of these heterogeneous populations are also present in human glioma. CONCLUSIONS: Our findings show extensive endothelial heterogeneity within the tumor and tumor microenvironment and provide insights into the diverse cellular and molecular mechanisms that drive glioma vascularization and angiogenesis during tumorigenesis.

PIK3CA variants selectively initiate brain hyperactivity during gliomagenesis.

Glioblastoma is a universally lethal form of brain cancer that exhibits an array of pathophysiological phenotypes, many of which are mediated by interactions with the neuronal microenvironment1,2. Recent studies have shown that increases in neuronal activity have an important role in the proliferation and progression of glioblastoma3,4. Whether there is reciprocal crosstalk between glioblastoma and neurons remains poorly defined, as the mechanisms that underlie how these tumours remodel the neuronal milieu towards increased activity are unknown. Here, using a native mouse model of glioblastoma, we develop a high-throughput in vivo screening platform and discover several driver variants of PIK3CA. We show that tumours driven by these variants have divergent molecular properties that manifest in selective initiation of brain hyperexcitability and remodelling of the synaptic constituency. Furthermore, secreted members of the glypican (GPC) family are selectively expressed in these tumours, and GPC3 drives gliomagenesis and hyperexcitability. Together, our studies illustrate the importance of functionally interrogating diverse tumour phenotypes driven by individual, yet related, variants and reveal how glioblastoma alters the neuronal microenvironment.

PRKCI promotes immune suppression in ovarian cancer.

A key feature of high-grade serous ovarian carcinoma (HGSOC) is frequent amplification of the 3q26 locus harboring PRKC-ι (PRKCI). Here, we show that PRKCI is also expressed in early fallopian tube lesions, called serous tubal intraepithelial carcinoma. Transgenic mouse studies establish PRKCI as an ovarian cancer-specific oncogene. Mechanistically, we show that the oncogenic activity of PRKCI relates in part to the up-regulation of TNFα to promote an immune-suppressive tumor microenvironment characterized by an abundance of myeloid-derived suppressor cells and inhibition of cytotoxic T-cell infiltration. Furthermore, system-level and functional analyses identify YAP1 as a downstream effector in tumor progression. In human ovarian cancers, high PRKCI expression also correlates with high expression of TNFα and YAP1 and low infiltration of cytotoxic T cells. The PRKCI-YAP1 regulation of the tumor immunity provides a therapeutic strategy for highly lethal ovarian cancer.

Genetic events that limit the efficacy of MEK and RTK inhibitor therapies in a mouse model of KRAS-driven pancreatic cancer.

Mutated KRAS (KRAS*) is a fundamental driver in the majority of pancreatic ductal adenocarcinomas (PDAC). Using an inducible mouse model of KRAS*-driven PDAC, we compared KRAS* genetic extinction with pharmacologic inhibition of MEK1 in tumor spheres and in vivo. KRAS* ablation blocked proliferation and induced apoptosis, whereas MEK1 inhibition exerted cytostatic effects. Proteomic analysis evidenced that MEK1 inhibition was accompanied by a sustained activation of the PI3K-AKT-MTOR pathway and by the activation of AXL, PDGFRa, and HER1-2 receptor tyrosine kinases (RTK) expressed in a large proportion of human PDAC samples analyzed. Although single inhibition of each RTK alone or plus MEK1 inhibitors was ineffective, a combination of inhibitors targeting all three coactivated RTKs and MEK1 was needed to inhibit proliferation and induce apoptosis in both mouse and human low-passage PDAC cultures. Importantly, constitutive AKT activation, which may mimic the fraction of AKT2-amplified PDAC, was able to bypass the induction of apoptosis caused by KRAS* ablation, highlighting a potential inherent resistance mechanism that may inform the clinical application of MEK inhibitor therapy. This study suggests that combinatorial-targeted therapies for pancreatic cancer must be informed by the activation state of each putative driver in a given treatment context. In addition, our work may offer explanative and predictive power in understanding why inhibitors of EGFR signaling fail in PDAC treatment and how drug resistance mechanisms may arise in strategies to directly target KRAS.

Somatic mutations of PIK3R1 promote gliomagenesis.

The phosphoinositide 3-kinase (PI3K) pathway is targeted for frequent alteration in glioblastoma (GBM) and is one of the core GBM pathways defined by The Cancer Genome Atlas. Somatic mutations of PIK3R1 are observed in multiple tumor types, but the tumorigenic activity of these mutations has not been demonstrated in GBM. We show here that somatic mutations in the iSH2 domain of PIK3R1 act as oncogenic driver events. Specifically, introduction of a subset of the mutations identified in human GBM, in the nSH2 and iSH2 domains, increases signaling through the PI3K pathway and promotes tumorigenesis of primary normal human astrocytes in an orthotopic xenograft model. Furthermore, we show that cells that are dependent on mutant P85α-mediated PI3K signaling exhibit increased sensitivity to a small molecule inhibitor of AKT. Together, these results suggest that GBM patients whose tumors carry mutant PIK3R1 alleles may benefit from treatment with inhibitors of AKT.

Integrative functional genomics identifies RINT1 as a novel GBM oncogene.

Large-scale cancer genomics efforts are identifying hundreds of somatic genomic alterations in glioblastoma (GBM). Distinguishing between active driver and neutral passenger alterations requires functional assessment of each gene; therefore, integrating biological weight of evidence with statistical significance for each genomic alteration will enable better prioritization for downstream studies. Here, we demonstrate the feasibility and potential of in vitro functional genomic screens to rapidly and systematically prioritize high-probability candidate genes for in vivo validation. Integration of low-complexity gain- and loss-of-function screens designed on the basis of genomic data identified 6 candidate GBM oncogenes, and RINT1 was validated as a novel GBM oncogene based on its ability to confer tumorigenicity to primary nontransformed murine astrocytes in vivo. Cancer genomics-guided low-complexity genomic screens can quickly provide a functional filter to prioritize high-value targets for further downstream mechanistic and translational studies.