High-volume facilities are not always low risk: comparing risk-standardized mortality rates versus facility volume as quality measures in surgical neuro-oncology.

OBJECTIVE: Risk-standardized mortality rates (RSMRs) have recently been shown to outperform facility case volume as a proxy for surgical quality in lung and gastrointestinal cancer. The aim of this study was to investigate RSMR as a surgical quality metric in primary CNS cancer. METHODS: This retrospective observational cohort study used data from the National Cancer Database, a population-based oncology outcomes database sourced from more than 1500 institutions in the United States, and included adult patients 18 years of age and older who were diagnosed with glioblastoma, pituitary adenoma, or meningioma and were treated with surgery. For each group, RSMR quintiles and annual volume were calculated in a training set (2009-2013) and these thresholds were applied to the validation set (2014-2018). In this paper, the authors compared the effectiveness and efficiency of facility volume-based versus RSMR-based hospital centralization models and evaluated the overlap between the two systems. A patterns-of-care analysis was also performed to explore socioeconomic predictors of being treated at better-performing treating facilities. RESULTS: A total of 37,838 meningioma, 21,189 pituitary adenoma, and 30,788 glioblastoma patients were surgically treated from 2014 to 2018. There were substantial differences between RSMR and facility volume classification schemes among all tumor types. In an RSMR-based centralization model, an average of 36 patients undergoing glioblastoma surgery would need to relocate to a low-mortality hospital to prevent one 30-day mortality following surgery, whereas 46 would need to relocate to a high-volume hospital. For pituitary adenoma and meningioma, both metrics were inefficient in centralizing care to reduce surgical mortality. Additionally, overall survival for glioblastoma patients was better modeled in an RSMR classification scheme. Analyses to investigate the impact of care disparities found that Black and Hispanic patients, patients earning less than $38,000, and uninsured patients were more likely to be treated at high-mortality hospitals. CONCLUSIONS: RSMR is more effective and efficient than a traditional volume-based approach for preventing early postoperative death in glioblastoma surgery. These data have important implications for future quality-related studies in neurosurgical oncology and may be relevant for healthcare/insurance payments, hospital evaluation assessments, healthcare disparities, and the standardization of care across hospitals.

Clinical Importance of the lncRNA NEAT1 in Cancer Patients Treated with Immune Checkpoint Inhibitors.

PURPOSE: mAbs targeting the PD-1/PD-L1 immune checkpoint are powerful tools to improve the survival of patients with cancer. Understanding the molecular basis of clinical response to these treatments is critical to identify patients who can benefit from this immunotherapy. In this study, we investigated long noncoding RNA (lncRNA) expression in patients with cancer treated with anti-PD-1/PD-L1 immunotherapy. EXPERIMENTAL DESIGN: lncRNA expression profile was analyzed in one cohort of patients with melanoma and two independent cohorts of patients with glioblastoma (GBM) undergoing anti-PD-1/PD-L1 immunotherapy. Single-cell RNA-sequencing analyses were performed to evaluate lncRNA expression in tumor cells and tumor-infiltrating immune cells. RESULTS: We identified the lncRNA NEAT1 as commonly upregulated between patients with melanoma with complete therapeutic response and patients with GBM with longer survival following anti-PD-1/PD-L1 treatment. Gene set enrichment analyses revealed that NEAT1 expression was strongly associated with the IFNγ pathways, along with downregulation of cell-cycle-related genes. Single-cell RNA-sequencing analyses revealed NEAT1 expression across multiple cell types within the GBM microenvironment, including tumor cells, macrophages, and T cells. High NEAT1 expression levels in tumor cells correlated with increased infiltrating macrophages and microglia. In these tumor-infiltrating myeloid cells, we found that NEAT1 expression was linked to enrichment in TNFα/NFκB signaling pathway genes. Silencing NEAT1 suppressed M1 macrophage polarization and reduced the expression of TNFα and other inflammatory cytokines. CONCLUSIONS: These findings suggest an association between NEAT1 expression and patient response to anti-PD-1/PD-L1 therapy in melanoma and GBM and have important implications for the role of lncRNAs in the tumor microenvironment.

Oncogene Amplification in Growth Factor Signaling Pathways Renders Cancers Dependent on Membrane Lipid Remodeling.

Advances in DNA sequencing technologies have reshaped our understanding of the molecular basis of cancer, providing a precise genomic view of tumors. Complementary biochemical and biophysical perspectives of cancer point toward profound shifts in nutrient uptake and utilization that propel tumor growth and major changes in the structure of the plasma membrane of tumor cells. The molecular mechanisms that bridge these fundamental aspects of tumor biology remain poorly understood. Here, we show that the lysophosphatidylcholine acyltransferase LPCAT1 functionally links specific genetic alterations in cancer with aberrant metabolism and plasma membrane remodeling to drive tumor growth. Growth factor receptor-driven cancers are found to depend on LPCAT1 to shape plasma membrane composition through enhanced saturated phosphatidylcholine content that is, in turn, required for the transduction of oncogenic signals. These results point to a genotype-informed strategy that prioritizes lipid remodeling pathways as therapeutic targets for diverse cancers.

A cell type-selective apoptosis-inducing small molecule for the treatment of brain cancer.

Glioblastoma multiforme (GBM; grade IV astrocytoma) is the most prevalent and aggressive form of primary brain cancer. A subpopulation of multipotent cells termed GBM cancer stem cells (CSCs) play a critical role in tumor initiation, tumor maintenance, metastasis, drug resistance, and recurrence following surgery. Here we report the identification of a small molecule, termed RIPGBM, from a cell-based chemical screen that selectively induces apoptosis in multiple primary patient-derived GBM CSC cultures. The cell type-dependent selectivity of this compound appears to arise at least in part from redox-dependent formation of a proapoptotic derivative, termed cRIPGBM, in GBM CSCs. cRIPGBM induces caspase 1-dependent apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) and acting as a molecular switch, which reduces the formation of a prosurvival RIPK2/TAK1 complex and increases the formation of a proapoptotic RIPK2/caspase 1 complex. In an orthotopic intracranial GBM CSC tumor xenograft mouse model, RIPGBM was found to significantly suppress tumor formation in vivo. Our chemical genetics-based approach has identified a drug candidate and a potential drug target that provide an approach to the development of treatments for this devastating disease.

Glioblastoma cellular cross-talk converges on NF-κB to attenuate EGFR inhibitor sensitivity.

In glioblastoma (GBM), heterogeneous expression of amplified and mutated epidermal growth factor receptor (EGFR) presents a substantial challenge for the effective use of EGFR-directed therapeutics. Here we demonstrate that heterogeneous expression of the wild-type receptor and its constitutively active mutant form, EGFRvIII, limits sensitivity to these therapies through an interclonal communication mechanism mediated by interleukin-6 (IL-6) cytokine secreted from EGFRvIII-positive tumor cells. IL-6 activates a NF-κB signaling axis in a paracrine and autocrine manner, leading to bromodomain protein 4 (BRD4)-dependent expression of the prosurvival protein survivin (BIRC5) and attenuation of sensitivity to EGFR tyrosine kinase inhibitors (TKIs). NF-κB and survivin are coordinately up-regulated in GBM patient tumors, and functional inhibition of either protein or BRD4 in in vitro and in vivo models restores sensitivity to EGFR TKIs. These results provide a rationale for improving anti-EGFR therapeutic efficacy through pharmacological uncoupling of a convergence point of NF-κB-mediated survival that is leveraged by an interclonal circuitry mechanism established by intratumoral mutational heterogeneity.

mTORC2 Regulates Amino Acid Metabolism in Cancer by Phosphorylation of the Cystine-Glutamate Antiporter xCT.

Mutations in cancer reprogram amino acid metabolism to drive tumor growth, but the molecular mechanisms are not well understood. Using an unbiased proteomic screen, we identified mTORC2 as a critical regulator of amino acid metabolism in cancer via phosphorylation of the cystine-glutamate antiporter xCT. mTORC2 phosphorylates serine 26 at the cytosolic N terminus of xCT, inhibiting its activity. Genetic inhibition of mTORC2, or pharmacologic inhibition of the mammalian target of rapamycin (mTOR) kinase, promotes glutamate secretion, cystine uptake, and incorporation into glutathione, linking growth factor receptor signaling with amino acid uptake and utilization. These results identify an unanticipated mechanism regulating amino acid metabolism in cancer, enabling tumor cells to adapt to changing environmental conditions.

An LXR-Cholesterol Axis Creates a Metabolic Co-Dependency for Brain Cancers.

Small-molecule inhibitors targeting growth factor receptors have failed to show efficacy for brain cancers, potentially due to their inability to achieve sufficient drug levels in the CNS. Targeting non-oncogene tumor co-dependencies provides an alternative approach, particularly if drugs with high brain penetration can be identified. Here we demonstrate that the highly lethal brain cancer glioblastoma (GBM) is remarkably dependent on cholesterol for survival, rendering these tumors sensitive to Liver X receptor (LXR) agonist-dependent cell death. We show that LXR-623, a clinically viable, highly brain-penetrant LXRα-partial/LXRß-full agonist selectively kills GBM cells in an LXRß- and cholesterol-dependent fashion, causing tumor regression and prolonged survival in mouse models. Thus, a metabolic co-dependency provides a pharmacological means to kill growth factor-activated cancers in the CNS.

EGFR Mutation Promotes Glioblastoma through Epigenome and Transcription Factor Network Remodeling.

Epidermal growth factor receptor (EGFR) gene amplification and mutations are the most common oncogenic events in glioblastoma (GBM), but the mechanisms by which they promote aggressive tumor growth are not well understood. Here, through integrated epigenome and transcriptome analyses of cell lines, genotyped clinical samples, and TCGA data, we show that EGFR mutations remodel the activated enhancer landscape of GBM, promoting tumorigenesis through a SOX9 and FOXG1-dependent transcriptional regulatory network in vitro and in vivo. The most common EGFR mutation, EGFRvIII, sensitizes GBM cells to the BET-bromodomain inhibitor JQ1 in a SOX9, FOXG1-dependent manner. These results identify the role of transcriptional/epigenetic remodeling in EGFR-dependent pathogenesis and suggest a mechanistic basis for epigenetic therapy.

Glucose-dependent acetylation of Rictor promotes targeted cancer therapy resistance.

Cancer cells adapt their signaling in response to nutrient availability. To uncover the mechanisms regulating this process and its functional consequences, we interrogated cell lines, mouse tumor models, and clinical samples of glioblastoma (GBM), the highly lethal brain cancer. We discovered that glucose or acetate is required for epidermal growth factor receptor vIII (EGFRvIII), the most common growth factor receptor mutation in GBM, to activate mechanistic target of rapamycin complex 2 (mTORC2) and promote tumor growth. Glucose or acetate promoted growth factor receptor signaling through acetyl-CoA-dependent acetylation of Rictor, a core component of the mTORC2 signaling complex. Remarkably, in the presence of elevated glucose levels, Rictor acetylation is maintained to form an autoactivation loop of mTORC2 even when the upstream components of the growth factor receptor signaling pathway are no longer active, thus rendering GBMs resistant to EGFR-, PI3K (phosphoinositide 3-kinase)-, or AKT (v-akt murine thymoma viral oncogene homolog)-targeted therapies. These results demonstrate that elevated nutrient levels can drive resistance to targeted cancer treatments and nominate mTORC2 as a central node for integrating growth factor signaling with nutrient availability in GBM.

A urokinase receptor-Bim signaling axis emerges during EGFR inhibitor resistance in mutant EGFR glioblastoma.

EGFR is the most common genetically altered oncogene in glioblastoma (GBM), but small-molecule EGFR tyrosine kinase inhibitors (TKI) have failed to yield durable clinical benefit. Here, we show that in two novel model systems of acquired resistance to EGFR TKIs, elevated expression of urokinase plasminogen activator (uPA) drives signaling through the MAPK pathway, which results in suppression of the proapoptotic BCL2-family member protein BIM (BCL2L11). In patient-derived GBM cells and genetic GBM models, uPA is shown to suppress BIM levels through ERK1/2 phosphorylation, which can be reversed by siRNA-mediated knockdown of uPA. TKI-resistant GBMs are resensitized to EGFR TKIs by pharmacologic inhibition of MEK or a BH3 mimetic drug to replace BIM function. A link between the uPA-uPAR-ERK1/2 pathway and BIM has not been previously demonstrated in GBM, and involvement of this signaling axis in resistance provides rationale for a new strategy to target EGFR TKI-resistant GBM.

mTOR complex 2 controls glycolytic metabolism in glioblastoma through FoxO acetylation and upregulation of c-Myc.

Aerobic glycolysis (the Warburg effect) is a core hallmark of cancer, but the molecular mechanisms underlying it remain unclear. Here, we identify an unexpected central role for mTORC2 in cancer metabolic reprogramming where it controls glycolytic metabolism by ultimately regulating the cellular level of c-Myc. We show that mTORC2 promotes inactivating phosphorylation of class IIa histone deacetylases, which leads to the acetylation of FoxO1 and FoxO3, and this in turn releases c-Myc from a suppressive miR-34c-dependent network. These central features of activated mTORC2 signaling, acetylated FoxO, and c-Myc levels are highly intercorrelated in clinical samples and with shorter survival of GBM patients. These results identify a specific, Akt-independent role for mTORC2 in regulating glycolytic metabolism in cancer.

The mTOR kinase inhibitors, CC214-1 and CC214-2, preferentially block the growth of EGFRvIII-activated glioblastomas.

PURPOSE: mTOR pathway hyperactivation occurs in approximately 90% of glioblastomas, but the allosteric mTOR inhibitor rapamycin has failed in the clinic. Here, we examine the efficacy of the newly discovered ATP-competitive mTOR kinase inhibitors CC214-1 and CC214-2 in glioblastoma, identifying molecular determinants of response and mechanisms of resistance, and develop a pharmacologic strategy to overcome it. EXPERIMENTAL DESIGN: We conducted in vitro and in vivo studies in glioblastoma cell lines and an intracranial model to: determine the potential efficacy of the recently reported mTOR kinase inhibitors CC214-1 (in vitro use) and CC214-2 (in vivo use) at inhibiting rapamycin-resistant signaling and blocking glioblastoma growth and a novel single-cell technology-DNA Encoded Antibody Libraries-was used to identify mechanisms of resistance. RESULTS: Here, we show that CC214-1 and CC214-2 suppress rapamycin-resistant mTORC1 signaling, block mTORC2 signaling, and significantly inhibit the growth of glioblastomas in vitro and in vivo. EGFRvIII expression and PTEN loss enhance sensitivity to CC214 compounds, consistent with enhanced efficacy in strongly mTOR-activated tumors. Importantly, CC214 compounds potently induce autophagy, preventing tumor cell death. Genetic or pharmacologic inhibition of autophagy greatly sensitizes glioblastoma cells and orthotopic xenografts to CC214-1- and CC214-2-induced cell death. CONCLUSIONS: These results identify CC214-1 and CC214-2 as potentially efficacious mTOR kinase inhibitors in glioblastoma, and suggest a strategy for identifying patients most likely to benefit from mTOR inhibition. In addition, this study also shows a central role for autophagy in preventing mTOR-kinase inhibitor-mediated tumor cell death, and suggests a pharmacologic strategy for overcoming it.

EGFR mutation-induced alternative splicing of Max contributes to growth of glycolytic tumors in brain cancer.

Alternative splicing contributes to diverse aspects of cancer pathogenesis including altered cellular metabolism, but the specificity of the process or its consequences are not well understood. We characterized genome-wide alternative splicing induced by the activating EGFRvIII mutation in glioblastoma (GBM). EGFRvIII upregulates the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 splicing factor, promoting glycolytic gene expression and conferring significantly shorter survival in patients. HnRNPA1 promotes splicing of a transcript encoding the Myc-interacting partner Max, generating Delta Max, an enhancer of Myc-dependent transformation. Delta Max, but not full-length Max, rescues Myc-dependent glycolytic gene expression upon induced EGFRvIII loss, and correlates with hnRNPA1 expression and downstream Myc-dependent gene transcription in patients. Finally, Delta Max is shown to promote glioma cell proliferation in vitro and augment EGFRvIII expressing GBM growth in vivo. These results demonstrate an important role for alternative splicing in GBM and identify Delta Max as a mediator of Myc-dependent tumor cell metabolism.

De-repression of PDGFRß transcription promotes acquired resistance to EGFR tyrosine kinase inhibitors in glioblastoma patients.

UNLABELLED: Acquired resistance to tyrosine kinase inhibitors (TKI) represents a major challenge for personalized cancer therapy. Multiple genetic mechanisms of acquired TKI resistance have been identified in several types of human cancer. However, the possibility that cancer cells may also evade treatment by co-opting physiologically regulated receptors has not been addressed. Here, we show the first example of this alternate mechanism in brain tumors by showing that EGF receptor (EGFR)-mutant glioblastomas (GBMs) evade EGFR TKIs by transcriptionally de-repressing platelet-derived growth factor receptor ß (PDGFRß). Mechanistic studies show that EGFRvIII signaling actively suppresses PDGFRß transcription in an mTORC1- and extracellular signal-regulated kinase-dependent manner. Genetic or pharmacologic inhibition of oncogenic EGFR renders GBMs dependent on the consequently de-repressed PDGFRß signaling for growth and survival. Importantly, combined inhibition of EGFR and PDGFRß signaling potently suppresses tumor growth in vivo. These data identify a novel, nongenetic TKI resistance mechanism in brain tumors and provide compelling rationale for combination therapy. SIGNIFICANCE: These results provide the fi rst clinical and biologic evidence for receptor tyrosinekinase (RTK) "switching" as a mechanism of resistance to EGFR inhibitors in GBM and provide a molecular explanation of how tumors can become "addicted" to a non amplified, nonmutated, physiologically regulated RTK to evade targeted treatment.