Determining venous thromboembolism risk in patients with adult-type diffuse glioma.

Venous thromboembolism (VTE) is a life-threating condition that is common in patients with adult-type diffuse gliomas, yet thromboprophylaxis is controversial because of possible intracerebral hemorrhage. Effective VTE prediction models exist for other cancers, but not glioma. Our objective was to develop a VTE prediction tool to improve glioma patient care, incorporating clinical, blood-based, histologic, and molecular markers. We analyzed preoperative arterial blood, tumor tissue, and clinical-pathologic data (including next-generation sequencing data) from 258 patients with newly diagnosed World Health Organization (WHO) grade 2 to 4 adult-type diffuse gliomas. Forty-six (17.8%) experienced VTE. Tumor expression of tissue factor (TF) and podoplanin (PDPN) each positively correlated with VTE, although only circulating TF and D-dimers, not circulating PDPN, correlated with VTE risk. Gliomas with mutations in isocitrate dehydrogenase 1 (IDH1) or IDH2 (IDHmut) caused fewer VTEs; multivariable analysis suggested that this is due to IDHmut suppression of TF, not PDPN. In a predictive time-to-event model, the following predicted increased VTE risk in newly diagnosed patients with glioma: (1) history of VTE; (2) hypertension; (3) asthma; (4) white blood cell count; (5) WHO tumor grade; (6) patient age; and (7) body mass index. Conversely, IDHmut, hypothyroidism, and MGMT promoter methylation predicted reduced VTE risk. These 10 variables were used to create a web-based VTE prediction tool that was validated in 2 separate cohorts of patients with adult-type diffuse glioma from other institutions. This study extends our understanding of the VTE landscape in these tumors and provides evidence-based guidance for clinicians to mitigate VTE risk in patients with glioma.

Tumor-induced peripheral immunosuppression promotes brain metastasis in patients with non-small cell lung cancer.

INTRODUCTION: Brain metastases are a significant source of morbidity and mortality for patients with lung cancer. Lung cancer can induce local and systemic immunosuppression, promoting tumor growth and dissemination. One mechanism of immunosuppression is tumor-induced expansion of programmed death-ligand 1 (PD-L1) expressing myeloid cells. Here, we investigate peripheral blood immune phenotype in NSCLC patients with or without brain metastasis. METHODS: Peripheral blood was collected from patients with lung metastatic brain tumors and pre-metastatic lung cancer. Immunosuppressive monocytes, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs) were quantified through flow cytometry. T cell reactivity was analyzed via ELISpot. Brain metastasis conditioned media was collected from tumor-derived cell cultures and analyzed for cytokines by ELISA. Naïve monocytes were stimulated with brain metastasis conditioned media to evaluate PD-L1 stimulation. RESULTS: Patients with brain metastatic lung carcinoma demonstrated increased peripheral monocyte PD-L1, MDSC abundance, and Treg percentage compared to early stage pre-metastatic patients and healthy controls. Patients with elevated peripheral monocyte PD-L1 had less reactive T cells and worse survival. Brain metastasis conditioned media stimulation increased monocyte PD-L1, and conditioned media IL-6 levels correlated with PD-L1 induction. Treatment with anti-IL-6 or anti-IL-6 receptor antibodies reduced PD-L1 expression. In summary, patients with lung cancer and brain metastases exhibit multiple markers of peripheral immunosuppression. CONCLUSIONS: The frequency of PD-L1+ myeloid cells correlated with the presence of brain metastases. Tumor-derived IL-6 was capable of inducing PD-L1+ myeloid cells in vitro, suggesting that monitoring of immunosuppressive factors in peripheral blood may identify new targets for therapeutic intervention in selected patients.

Red Blood Cell Dysfunction Induced by High-Fat Diet: Potential Implications for Obesity-Related Atherosclerosis.

BACKGROUND: High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). METHODS AND RESULTS: A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC(-/-) mice. In RBCs from HFD-fed wild-type and DARC(-/-) mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. CONCLUSIONS: RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic.

Activation of carbonic anhydrase IX by alternatively spliced tissue factor under late-stage tumor conditions.

Molecules of the coagulation pathway predispose patients to cancer-associated thrombosis and also trigger intracellular signaling pathways that promote cancer progression. The primary transcript of tissue factor, the main physiologic trigger of blood clotting, can undergo alternative splicing yielding a secreted variant, termed asTF (alternatively spliced tissue factor). asTF is not required for normal hemostasis, but its expression levels positively correlate with advanced tumor stages in several cancers, including pancreatic adenocarcinoma. The asTF-overexpressing pancreatic ductal adenocarcinoma cell line Pt45.P1/asTF+ and its parent cell line Pt45.P1 were tested for growth and mobility under normoxic conditions that model early-stage tumors, and in the hypoxic environment of late-stage cancers. asTF overexpression in Pt45.P1 cells conveys increased proliferative ability. According to cell cycle analysis, the major fraction of Pt45.P1/asTF+ cells reside in the dividing G2/M phase of the cell cycle, whereas the parental Pt45.P1 cells are mostly confined to the quiescent G0/G1 phase. asTF overexpression is also associated with significantly higher mobility in cells plated under either normoxia or hypoxia. A hypoxic environment leads to upregulation of carbonic anhydrase IX (CAIX), which is more pronounced in Pt45.P1/asTF+ cells. Inhibition of CAIX by the compound U-104 significantly decreases cell growth and mobility of Pt45.P1/asTF+ cells in hypoxia, but not in normoxia. U-104 also reduces the growth of Pt45.P1/asTF+ orthotopic tumors in nude mice. CAIX is a novel downstream mediator of asTF in pancreatic cancer, particularly under hypoxic conditions that model late-stage tumor microenvironment.

Mutant IDH1 and thrombosis in gliomas.

Mutant isocitrate dehydrogenase 1 (IDH1) is common in gliomas, and produces D-2-hydroxyglutarate (D-2-HG). The full effects of IDH1 mutations on glioma biology and tumor microenvironment are unknown. We analyzed a discovery cohort of 169 World Health Organization (WHO) grade II-IV gliomas, followed by a validation cohort of 148 cases, for IDH1 mutations, intratumoral microthrombi, and venous thromboemboli (VTE). 430 gliomas from The Cancer Genome Atlas were analyzed for mRNAs associated with coagulation, and 95 gliomas in a tissue microarray were assessed for tissue factor (TF) protein. In vitro and in vivo assays evaluated platelet aggregation and clotting time in the presence of mutant IDH1 or D-2-HG. VTE occurred in 26-30 % of patients with wild-type IDH1 gliomas, but not in patients with mutant IDH1 gliomas (0 %). IDH1 mutation status was the most powerful predictive marker for VTE, independent of variables such as GBM diagnosis and prolonged hospital stay. Microthrombi were far less common within mutant IDH1 gliomas regardless of WHO grade (85-90 % in wild-type versus 2-6 % in mutant), and were an independent predictor of IDH1 wild-type status. Among all 35 coagulation-associated genes, F3 mRNA, encoding TF, showed the strongest inverse relationship with IDH1 mutations. Mutant IDH1 gliomas had F3 gene promoter hypermethylation, with lower TF protein expression. D-2-HG rapidly inhibited platelet aggregation and blood clotting via a novel calcium-dependent, methylation-independent mechanism. Mutant IDH1 glioma engraftment in mice significantly prolonged bleeding time. Our data suggest that mutant IDH1 has potent antithrombotic activity within gliomas and throughout the peripheral circulation. These findings have implications for the pathologic evaluation of gliomas, the effect of altered isocitrate metabolism on tumor microenvironment, and risk assessment of glioma patients for VTE.

Apolipoprotein A-I and Paraoxonase-1 Are Potential Blood Biomarkers for Ischemic Stroke Diagnosis.

BACKGROUND: Blood biomarkers for ischemic and hemorrhagic stroke diagnosis remain elusive. Recent investigations suggested that apolipoprotein (Apo), matrix metalloproteinase (MMP), and paraoxonase-1 may be associated with stroke. We hypothesized that Apo A-I, Apo C-I, Apo C-III, MMP-3, MMP-9, and paraoxonase-1 are differentially expressed in ischemic stroke, hemorrhagic stroke, and controls. METHODS: In a single-center prospective observational study, consecutive stroke cases were enrolled if blood samples were obtainable within 12 hours of symptom onset. Age- (±5 years), race-, and sex-matched controls were recruited. Multiplex assays were used to measure protein levels. The Wilcoxon signed-rank test and the Mann-Whitney U-test were used to compare biomarker values between ischemic stroke patients and controls, hemorrhagic stroke patients and controls, and ischemic and hemorrhagic stroke patients. The 95% confidence intervals (CIs) for the difference of 2 medians were calculated. RESULTS: Fourteen ischemic stroke case-control pairs and 23 intracerebral hemorrhage (ICH) case-control pairs were enrolled. Median Apo A-I levels were lower in ischemic stroke cases versus controls (140 mg/dL versus 175 mg/dL, difference of 35 mg/dL, 95% CI -54 to -16) and in ischemic stroke versus ICH cases (140 mg/dL versus 180 mg/dL, difference of 40 mg/dL, 95% CI -57 to -23). Median paraoxonase-1 was lower in ischemic stroke cases than in both ICH cases and matched controls. Median Apo C-I was slightly lower in ischemic stroke cases than in ICH cases. There were no differences between groups for MMP-3, MMP-9, and Apo C-III. CONCLUSION: Apo A-I and paraoxonase-1 levels may be clinically useful for ischemic stroke diagnosis and for differentiating between ischemic and hemorrhagic strokes.

Levels of Alternatively Spliced Tissue Factor in the Plasma of Patients with Pancreatic Cancer May Help Predict Aggressive Tumor Phenotype.

BACKGROUND: Circulating ('blood-borne') tissue factor (TF) is implicated in the pathogenesis of several chronic conditions, most notably cardiovascular disease, diabetes, and cancer. Full-length TF is an integral membrane protein, while alternatively spliced TF (asTF) can be secreted and, owing to its unique C-terminus, selectively detected in bio-specimens. The predictive and/or prognostic value of asTF in the circulation is unknown. In a retrospective study, we measured levels of circulating asTF in healthy subjects and individuals with acute coronary syndrome (ACS), diabetes mellitus (DM), ongoing ACS + DM, and pancreatic ductal adenocarcinoma (PDAC). METHODS: The prototype-tailored procedure (Diagnostica Stago) was used to measure asTF in plasma from 205 subjects. RESULTS: There was no significant difference between the proportion of healthy subjects with asTF ≥200 pg/mL and those with ACS, DM, or ACS + DM. The proportion of pancreatic cancer patients (n = 43; PDAC: 42; pancreatic neuroendocrine tumor: 1) with asTF levels ≥200 pg/mL was significantly higher than in healthy subjects; asTF levels ≥200 pg/mL were detected more often in patients with unresectable disease irrespective of initial evaluation and/or preoperative carbohydrate antigen 19-9 (CA19-9) levels. CONCLUSIONS: While asTF levels ≥200 pg/mL are not observed with increased frequency in patients with ACS and/or DM, they do occur more frequently in the plasma of patients with pancreatic cancer and are associated with lower likelihood of tumor resectability, irrespective of the preoperative diagnosis. asTF may thus have utility as a novel marker of aggressive pancreatic tumor phenotype.

Alternatively spliced tissue factor contributes to tumor spread and activation of coagulation in pancreatic ductal adenocarcinoma.

Alternatively spliced tissue factor (asTF) promotes neovascularization and monocyte recruitment via integrin ligation. While asTF mRNA has been detected in some pancreatic ductal adenocarcinoma (PDAC) cell lines and increased asTF expression can promote PDAC growth in a subcutaneous model, the expression of asTF protein in bona fide PDAC lesions and/or its role in metastatic spread are yet to be ascertained. We here report that asTF protein is abundant in lesional and stromal compartments of the five studied types of carcinoma including PDAC. Analysis of 29 specimens of PDAC revealed detectable asTF in >90% of the lesions with a range of staining intensities. asTF levels in PDAC lesions positively correlated with the degree of monocyte infiltration. In an orthotopic model, asTF-overexpressing high-grade PDAC cell line Pt45P1/asTF+ produced metastases to distal lymph nodes, which stained positive for asTF. PDAC cells stimulated with and/or overexpressing asTF exhibited upregulation of genes implicated in PDAC progression and metastatic spread. Pt45P1/asTF+ cells displayed higher coagulant activity compared to Pt45P1 cells; the same effect was observed for cell-derived microparticles (MPs). Our findings demonstrate that asTF is expressed in PDAC and lymph node metastases and potentiates PDAC spread in vivo. asTF elicits global changes in gene expression likely involved in tumor progression and metastatic dissemination, and it also enhances the procoagulant potential of PDAC cells and cell-derived MPs. Thus, asTF may comprise a novel therapeutic target to treat PDAC and, possibly, its thrombotic complications.

Human coronary artery perivascular adipocytes overexpress genes responsible for regulating vascular morphology, inflammation, and hemostasis.

Inflammatory cross talk between perivascular adipose tissue and the blood vessel wall has been proposed to contribute to the pathogenesis of atherosclerosis. We previously reported that human perivascular (PV) adipocytes exhibit a proinflammatory phenotype and less adipogenic differentiation than do subcutaneous (SQ) adipocytes. To gain a global view of the genomic basis of biologic differences between PV and SQ adipocytes, we performed genome-wide expression analyses to identify differentially expressed genes between adipocytes derived from human SQ vs. PV adipose tissues. Although >90% of well-expressed genes were similarly regulated, we identified a signature of 307 differentially expressed genes that were highly enriched for functions associated with the regulation of angiogenesis, vascular morphology, inflammation, and blood clotting. Of the 156 PV upregulated genes, 59 associate with angiogenesis, vascular biology, or inflammation, noteworthy of which include TNFRSF11B (osteoprotegerin), PLAT, TGFB1, THBS2, HIF1A, GATA6, and SERPINE1. Of 166 PV downregulated genes, 21 associated with vascular biology and inflammation, including ANGPT1, ANGPTL1, and VEGFC. Consistent with the emergent hypothesis that PV adipocytes differentially regulate angiogenesis and inflammation, cell culture-derived adipocyte-conditioned media from PV adipocytes strongly enhanced endothelial cell tubulogenesis and monocyte migration compared with media from SQ adipocytes. These findings demonstrate that PV adipocytes have the potential to significantly modulate vascular inflammatory crosstalk in the setting of atherosclerosis by their ability to signal to both endothelial and inflammatory cells.

Docetaxel targets aggressive methylation profiles and serves as a radiosensitizer in high-risk meningiomas.

BACKGROUND: Meningioma is the most common primary intracranial tumor in adults. A subset of these tumors recur and invade the brain, even after surgery and radiation, resulting in significant disability. There is currently no standard-of-care chemotherapy for meningiomas. As genomic DNA methylation profiling can prognostically stratify these lesions, we sought to determine whether any existing chemotherapies might be effective against meningiomas with high-risk methylation profiles. METHODS: A previously published dataset of meningioma methylation profiles was used to screen for clinically significant CpG methylation events and associated cellular pathways. Based on these results, patient-derived meningioma cell lines were used to test candidate drugs in vitro and in vivo, including efficacy in conjunction with radiotherapy. RESULTS: We identified 981 genes for which methylation of mapped CpG sites was related to progression-free survival in meningiomas. Associated molecular pathways were cross-referenced with FDA-approved cancer drugs, which nominated Docetaxel as a promising candidate for further preclinical analyses. Docetaxel arrested growth in 17 meningioma cell sources, representing all tumor grades, with a clinically favorable IC50 values ranging from 0.3 nM to 10.7 mM. The inhibitory effects of this medication scaled with tumor doubling time, with maximal benefit in fast-growing lesions. The combination of Docetaxel and radiation therapy increased markers of apoptosis and double-stranded DNA breaks, and extended the survival of mice engrafted with meningioma cells relative to either modality alone. CONCLUSIONS: Global patterns of DNA methylation may be informative for the selection of chemotherapies against meningiomas, and existing drugs may enhance radiation sensitivity in high-risk cases.

Postoperative risk of IDH-mutant glioma-associated seizures and their potential management with IDH-mutant inhibitors.

Seizures are a frequent complication of adult-type diffuse gliomas, and are often difficult to control with medications. Gliomas with mutations in isocitrate dehydrogenase 1 or 2 (IDHmut) are more likely than IDH-wild type (IDHwt) gliomas to cause seizures as part of their initial clinical presentation. However, whether IDHmut is also associated with seizures during the remaining disease course, and whether IDHmut inhibitors can reduce seizure risk, are unclear. Clinical multivariable analyses showed that preoperative seizures, glioma location, extent of resection, and glioma molecular subtype (including IDHmut status) all contributed to postoperative seizure risk in adult-type diffuse glioma patients, and that postoperative seizures were often associated with tumor recurrence. Experimentally, the metabolic product of IDHmut, d-2-hydroxyglutarate, rapidly synchronized neuronal spike firing in a seizure-like manner, but only when non-neoplastic glial cells were present. In vitro and in vivo models recapitulated IDHmut glioma-associated seizures, and IDHmut inhibitors currently being evaluated in glioma clinical trials inhibited seizures in those models, independent of their effects on glioma growth. These data show that postoperative seizure risk in adult-type diffuse gliomas varies in large part by molecular subtype, and that IDHmut inhibitors could play a key role in mitigating such risk in IDHmut glioma patients.

A first-in-human phase 0 clinical study of RNA interference-based spherical nucleic acids in patients with recurrent glioblastoma.

Glioblastoma (GBM) is one of the most difficult cancers to effectively treat, in part because of the lack of precision therapies and limited therapeutic access to intracranial tumor sites due to the presence of the blood-brain and blood-tumor barriers. We have developed a precision medicine approach for GBM treatment that involves the use of brain-penetrant RNA interference-based spherical nucleic acids (SNAs), which consist of gold nanoparticle cores covalently conjugated with radially oriented and densely packed small interfering RNA (siRNA) oligonucleotides. On the basis of previous preclinical evaluation, we conducted toxicology and toxicokinetic studies in nonhuman primates and a single-arm, open-label phase 0 first-in-human trial (NCT03020017) to determine safety, pharmacokinetics, intratumoral accumulation and gene-suppressive activity of systemically administered SNAs carrying siRNA specific for the GBM oncogene Bcl2Like12 (Bcl2L12). Patients with recurrent GBM were treated with intravenous administration of siBcl2L12-SNAs (drug moniker: NU-0129), at a dose corresponding to 1/50th of the no-observed-adverse-event level, followed by tumor resection. Safety assessment revealed no grade 4 or 5 treatment-related toxicities. Inductively coupled plasma mass spectrometry, x-ray fluorescence microscopy, and silver staining of resected GBM tissue demonstrated that intravenously administered SNAs reached patient tumors, with gold enrichment observed in the tumor-associated endothelium, macrophages, and tumor cells. NU-0129 uptake into glioma cells correlated with a reduction in tumor-associated Bcl2L12 protein expression, as indicated by comparison of matched primary tumor and NU-0129-treated recurrent tumor. Our results establish SNA nanoconjugates as a potential brain-penetrant precision medicine approach for the systemic treatment of GBM.

Beyond thrombosis: the impact of tissue factor signaling in cancer.

Tissue factor (TF) is the primary initiator of the coagulation cascade, though its effects extend well beyond hemostasis. When TF binds to Factor VII, the resulting TF:FVIIa complex can proteolytically cleave transmembrane G protein-coupled protease-activated receptors (PARs). In addition to activating PARs, TF:FVIIa complex can also activate receptor tyrosine kinases (RTKs) and integrins. These signaling pathways are utilized by tumors to increase cell proliferation, angiogenesis, metastasis, and cancer stem-like cell maintenance. Herein, we review in detail the regulation of TF expression, mechanisms of TF signaling, their pathological consequences, and how it is being targeted in experimental cancer therapeutics.

Methylation and transcription patterns are distinct in IDH mutant gliomas compared to other IDH mutant cancers.

Mutations in isocitrate dehydrogenases 1 and 2 (IDHmut) are present in a variety of cancers, including glioma, acute myeloid leukemia (AML), melanoma, and cholangiocarcinoma. These mutations promote hypermethylation, yet it is only a favorable prognostic marker in glioma, for reasons that are unclear. We hypothesized that the patterns of DNA methylation, and transcriptome profiles, would vary among IDHmut cancers, especially gliomas. Using Illumina 450K and RNA-Seq data from The Cancer Genome Atlas, we show that of 365,092 analyzed CpG sites, 70,591 (19%) were hypermethylated in IDHmut gliomas compared to wild-type (IDHwt) gliomas, and only 3%, 2%, and 4% of CpG sites were hypermethylated in IDHmut AML, melanoma, and cholangiocarcinoma, relative to each of their IDHwt counterparts. Transcriptome differences showed pro-malignant genes that appear to be unique to IDHmut gliomas. However, genes involved in differentiation and immune response were suppressed in all IDHmut cancers. Additionally, IDHmut caused a greater degree of hypermethylation in undifferentiated neural progenitor cells than in mature astrocytes. These data suggest that the extent and targets of IDHmut-induced genomic hypermethylation vary greatly according to the cellular context and may help explain why IDHmut is only a favorable prognostic marker in gliomas.

Methylation-dependent Tissue Factor Suppression Contributes to the Reduced Malignancy of IDH1-mutant Gliomas.

PURPOSE: Gliomas with isocitrate dehydrogenase 1 mutations (IDH1mut) are less aggressive than IDH1 wild-type (IDH1wt) gliomas and have global genomic hypermethylation. Yet it is unclear how specific hypermethylation events contribute to the IDH1mut phenotype. Previously, we showed that the gene encoding the procoagulant tissue factor (TF), F3, is among the most hypermethylated and downregulated genes in IDH1mut gliomas, correlating with greatly reduced thrombosis in patients with IDH1mut glioma. Because TF also increases the aggressiveness of many cancers, the current study explored the contribution of TF suppression to the reduced malignancy of IDH1mut gliomas.Experimental Design: TF expression was manipulated in patient-derived IDH1mut and IDH1wt glioma cells, followed by evaluation of in vitro and in vivo behavior and analyses of cell signaling pathways. RESULTS: A demethylating agent, decitabine, increased F3 transcription and TF-dependent coagulative activity in IDH1mut cells, but not in IDH1wt cells. TF induction enhanced the proliferation, invasion, and colony formation of IDH1mut cells, and increased the intracranial engraftment of IDH1mut GBM164 from 0% to 100% (P = 0.0001). Conversely, TF knockdown doubled the median survival of mice engrafted with IDH1wt/EGFRvIIIamp GBM6, and caused complete regression of IDH1wt/EGFRamp GBM12 (P = 0.001). In vitro and in vivo effects were linked to activation of receptor tyrosine kinases (RTK) by TF through a Src-dependent intracellular pathway, even when extracellular RTK stimulation was blocked. TF stimulated invasion predominately through upregulation of ß-catenin. CONCLUSIONS: These data show that TF suppression is a component of IDH1mut glioma behavior, and that it may therefore be an attractive target against IDH1wt gliomas.

Glioblastoma-Derived IL6 Induces Immunosuppressive Peripheral Myeloid Cell PD-L1 and Promotes Tumor Growth.

PURPOSE: Upregulation of programmed death-ligand 1 (PD-L1) on circulating and tumor-infiltrating myeloid cells is a critical component of GBM-mediated immunosuppression that has been associated with diminished response to vaccine immunotherapy and poor survival. Although GBM-derived soluble factors have been implicated in myeloid PD-L1 expression, the identity of such factors has remained unknown. This study aimed to identify factors responsible for myeloid PD-L1 upregulation as potential targets for immune modulation. EXPERIMENTAL DESIGN: Conditioned media from patient-derived GBM explant cell cultures was assessed for cytokine expression and utilized to stimulate naïve myeloid cells. Myeloid PD-L1 induction was quantified by flow cytometry. Candidate cytokines correlated with PD-L1 induction were evaluated in tumor sections and plasma for relationships with survival and myeloid PD-L1 expression. The role of identified cytokines on immunosuppression and survival was investigated in vivo utilizing immunocompetent C57BL/6 mice bearing syngeneic GL261 and CT-2A tumors. RESULTS: GBM-derived IL6 was identified as a cytokine that is necessary and sufficient for myeloid PD-L1 induction in GBM through a STAT3-dependent mechanism. Inhibition of IL6 signaling in orthotopic murine glioma models was associated with reduced myeloid PD-L1 expression, diminished tumor growth, and increased survival. The therapeutic benefit of anti-IL6 therapy proved to be CD8+ T-cell dependent, and the antitumor activity was additive with that provided by programmed death-1 (PD-1)-targeted immunotherapy. CONCLUSIONS: Our findings suggest that disruption of IL6 signaling in GBM reduces local and systemic myeloid-driven immunosuppression and enhances immune-mediated antitumor responses against GBM.

Radiosensitization by Histone H3 Demethylase Inhibition in Diffuse Intrinsic Pontine Glioma.

PURPOSE: Radiotherapy (RT) has long been and remains the only treatment option for diffuse intrinsic pontine glioma (DIPG). However, all patients show evidence of disease progression within months of completing RT. No further clinical benefit has been achieved using alternative radiation strategies. Here, we tested the hypothesis that histone demethylase inhibition by GSK-J4 enhances radiation-induced DNA damage, making it a potential radiosensitizer in the treatment of DIPG.Experimental Design: We evaluated the effects of GSK-J4 on genes associated with DNA double-strand break (DSB) repair in DIPG cells by RNA sequence, ATAC sequence, and quantitative real-time PCR. Radiation-induced DNA DSB repair was analyzed by immunocytochemistry of DSB markers γH2AX and 53BP1, DNA-repair assay, and cell-cycle distribution. Clonogenic survival assay was used to determine the effect of GSK-J4 on radiation response of DIPG cells. In vivo response to radiation monotherapy and combination therapy of RT and GSK-J4 was evaluated in patient-derived DIPG xenografts. RESULTS: GSK-J4 significantly reduced the expression of DNA DSB repair genes and DNA accessibility in DIPG cells. GSK-J4 sustained high levels of γH2AX and 53BP1 in irradiated DIPG cells, thereby inhibiting DNA DSB repair through homologous recombination pathway. GSK-J4 reduced clonogenic survival and enhanced radiation effect in DIPG cells. In vivo studies revealed increased survival of animals treated with combination therapy of RT and GSK-J4 compared with either monotherapy. CONCLUSIONS: Together, these results highlight GSK-J4 as a potential radiosensitizer and provide a rationale for developing combination therapy with radiation in the treatment of DIPG.

Modeling the diffusion of D-2-hydroxyglutarate from IDH1 mutant gliomas in the central nervous system.

Background: Among diffusely infiltrative gliomas in adults, 20%-30% contain a point mutation in isocitrate dehydrogenase 1 (IDH1mut), which increases production of D-2-hydroxyglutarate (D2HG). This is so efficient that D2HG often reaches 30 mM within IDH1mut gliomas. Yet, while up to 100 µM D2HG can be detected in the circulating cerebrospinal fluid of IDH1mut glioma patients, the exposure of nonneoplastic cells within and surrounding an IDH1mut glioma to D2HG is unknown and difficult to measure directly. Methods: Conditioned medium from patient-derived wild type IDH1 (IDH1wt) and IDH1mut glioma cells was analyzed for D2HG by liquid chromatography-mass spectrometry (LC-MS). Mathematical models of D2HG release and diffusion around an IDH1mut glioma were independently generated based on fluid dynamics within the brain and on previously reported intratumoral and cerebrospinal D2HG concentrations. Results: LC-MS analysis indicates that patient-derived IDH1mut glioma cells release 3.7-97.0 pg D2HG per cell per week. Extrapolating this to an average-sized tumor (30 mL glioma volume and 1 × 108 cells/mL tumor), the rate of D2HG release by an IDH1mut glioma (SA) is estimated at 3.2-83.0 × 10-12 mol/mL/sec. Mathematical models estimate an SA of 2.9-12.9 × 10-12 mol/mL/sec, within the range of the in vitro LC-MS data. In even the most conservative of these models, the extracellular concentration of D2HG exceeds 3 mM within a 2 cm radius from the center of an IDH1mut glioma. Conclusions: The microenvironment of an IDH1mut glioma is likely being exposed to high concentrations of D2HG, in the low millimolar range. This has implications for understanding how D2HG affects nonneoplastic cells in an IDH1mut glioma.

Antibody-based targeting of alternatively spliced tissue factor: a new approach to impede the primary growth and spread of pancreatic ductal adenocarcinoma.

Alternatively spliced Tissue Factor (asTF) is a secreted form of Tissue Factor (TF), the trigger of blood coagulation whose expression levels are heightened in several forms of solid cancer, including pancreatic ductal adenocarcinoma (PDAC). asTF binds to ß1 integrins on PDAC cells, whereby it promotes tumor growth, metastatic spread, and monocyte recruitment to the stroma. In this study, we determined if targeting asTF in PDAC would significantly impact tumor progression. We here report that a novel inhibitory anti-asTF monoclonal antibody curtails experimental PDAC progression. Moreover, we show that tumor-derived asTF is able to promote PDAC primary growth and spread during early as well as later stages of the disease. This raises the likelihood that asTF may comprise a viable target in early- and late-stage PDAC. In addition, we show that TF expressed by host cells plays a significant role in PDAC spread. Together, our data demonstrate that targeting asTF in PDAC is a novel strategy to stem PDAC progression and spread.

Alternatively spliced tissue factor is not sufficient for embryonic development.

Tissue factor (TF) triggers blood coagulation and is translated from two mRNA splice isoforms, encoding membrane-anchored full-length TF (flTF) and soluble alternatively-spliced TF (asTF). The complete knockout of TF in mice causes embryonic lethality associated with failure of the yolk sac vasculature. Although asTF plays roles in postnatal angiogenesis, it is unknown whether it activates coagulation sufficiently or makes previously unrecognized contributions to sustaining integrity of embryonic yolk sac vessels. Using gene knock-in into the mouse TF locus, homozygous asTF knock-in (asTFKI) mice, which express murine asTF in the absence of flTF, exhibited embryonic lethality between day 9.5 and 10.5. Day 9.5 homozygous asTFKI embryos expressed asTF protein, but no procoagulant activity was detectable in a plasma clotting assay. Although the α-smooth-muscle-actin positive mesodermal layer as well as blood islands developed similarly in day 8.5 wild-type or homozygous asTFKI embryos, erythrocytes were progressively lost from disintegrating yolk sac vessels of asTFKI embryos by day 10.5. These data show that in the absence of flTF, asTF expressed during embryonic development has no measurable procoagulant activity, does not support embryonic vessel stability by non-coagulant mechanisms, and fails to maintain a functional vasculature and embryonic survival.