Targeting FAcilitates Chromatin Transcription complex inhibits pleural mesothelioma and enhances immunotherapy.

BACKGROUND: Diffuse pleural mesothelioma (DPM) is an aggressive therapy-resistant cancer with unique molecular features. Numerous agents have been tested, but clinically effective ones remain elusive. Herein, we propose to use a small molecule CBL0137 (curaxin) that simultaneously suppresses nuclear factor-κB (NF-κB) and activates tumor suppressor p53 via targeting FAcilitates Chromatin Transcription (FACT) complex, a histone chaperone critical for DNA repair. METHODS: We used DPM cell lines, murine models (xeno- and allo-grafts), plus DPM patient samples to characterize anti-tumor effects of CBL0137 and to delineate specific molecular mechanisms. RESULTS: We verified that CBL0137 induced cell cycle arrest and apoptosis. We also discovered that DPM is a FACT-dependent cancer with overexpression of both subunits structure-specific recognition protein 1 (SSRP1), a poor prognosis indicator, and suppressor of Ty 16 (SUPT16H). We defined several novel uses of CBL0137 in DPM therapy. In combination with cisplatin, CBL0137 exhibited additive anti-tumor activity compared to monotherapy. Similarly, CBL0137 (systemic) could be combined with other novel agents like microRNA-215 (intrapleural) as a more effective regimen. Importantly, we established that CBL0137 induces immunogenic cell death that contributes to activating immune response pathways in DPM. Therefore, when CBL0137 is combined with dual immune checkpoint inhibitors DPM tumor growth is significantly suppressed. CONCLUSIONS: We identified an unrecognized molecular vulnerability of DPM based on FACT dependency. CBL0137 alone and in several combinations with different therapeutics showed promising efficacy, including that of improved anti-tumor immunity. Overall, these preclinical findings suggest that CBL0137 could be ideally suited for use in DPM clinical trials.

Inhibition of HSP 90 is associated with potent anti-tumor activity in Papillary Renal Cell Carcinoma.

BACKGROUND: There is no universally accepted treatment for patients with advanced papillary renal cell carcinoma (PRCC). The presence of activating mutations in MET, as well as gain of chromosome 7, where the MET gene is located, are the most common genetic alterations associated with PRCC, leading to the clinical evaluation of MET tyrosine kinase inhibitors (TKIs) in this cancer. However, TKIs targeting MET selectively, as well as multitargeted TKIs with activity against MET demonstrate modest efficacy in PRCC and primary and secondary treatment failure is common; other approaches are urgently needed to improve outcomes in these patients. METHODS: High throughput screening with small molecule libraries identified HSP90 inhibitors as agents of interest based on antitumor activity against patient derived PRCC cell lines. We investigated the activity of the orally available HSP90 inhibitor, SNX2112 in vitro, using 2D/3D PRCC cell culture models and in vivo, in mice tumor xenograft models. The molecular pathways mediating antitumor activity of SNX2112 were assessed by Western blot analysis, Flow cytometry, RNA-seq analysis, Real Time qPCR and imaging approaches. RESULTS: SNX2112 significantly inhibited cellular proliferation, induced G2/M cell cycle arrest and apoptosis in PRCC lines overexpressing MET. In contrast to TKIs targeting MET, SNX2112 inhibited both MET and known downstream mediators of MET activity (AKT, pAKT1/2 and pERK1/2) in PRCC cell lines. RNAi silencing of AKT1/2 or ERK1/2 expression significantly inhibited growth in PRCC cells. Furthermore, SNX2112 inhibited a unique set of E2F and MYC targets and G2M-associated genes. Interestingly, interrogation of the TCGA papillary RCC cohort revealed that these genes were overexpressed in PRCC and portend a poor prognosis. Finally, SNX-2112 demonstrated strong antitumor activity in vivo and prolonged survival of mice bearing human PRCC xenograft. CONCLUSIONS: These results demonstrate that HSP90 inhibition is associated with potent activity in PRCC, and implicate the PI3K/AKT and MEK/ERK1/2 pathways as important mediators of tumorigenesis. These data also provide the impetus for further clinical evaluation of HSP90, AKT, MEK or E2F pathway inhibitors in PRCC.

Surface-fill hydrogel attenuates the oncogenic signature of complex anatomical surface cancer in a single application.

Tumours growing in a sheet-like manner on the surface of organs and tissues with complex topologies represent a difficult-to-treat clinical scenario. Their complete surgical resection is difficult due to the complicated anatomy of the diseased tissue. Residual cancer often responds poorly to systemic therapy and locoregional treatment is hindered by the limited accessibility to microscopic tumour foci. Here we engineered a peptide-based surface-fill hydrogel (SFH) that can be syringe- or spray-delivered to surface cancers during surgery or used as a primary therapy. Once applied, SFH can shape change in response to alterations in tissue morphology that may occur during surgery. Implanted SFH releases nanoparticles composed of microRNA and intrinsically disordered peptides that enter cancer cells attenuating their oncogenic signature. With a single application, SFH shows efficacy in four preclinical models of mesothelioma, demonstrating the therapeutic impact of the local application of tumour-specific microRNA, which might change the treatment paradigm for mesothelioma and possibly other surface cancers.

Kidney cancer: from genes to therapy.

Renal cell carcinoma incidence is rising worldwide with increasing subtype stratification by the World Health Organization. Each subtype has unique genetic alterations, cell biology changes and clinical findings. Such genetic alterations offer the potential for individualized therapeutic approaches that are rapidly progressing. This review highlights the most common subtypes of renal cell carcinoma, including both hereditary and sporadic forms, with a focus on genetic changes, clinical findings and ongoing clinical trials.

MicroRNA-206 suppresses mesothelioma progression via the Ras signaling axis.

Malignant pleural mesothelioma (MPM) is an incurable surface neoplasm with peculiar pathobiology. MPM proliferates by using the tyrosine-kinase-Ras pathway. Despite representing an attractive therapeutic target, there are no standard agent(s) specifically inhibiting Ras signaling adopted in clinical settings. We posited that biologic effects of microRNA (miRNA) can disrupt this molecular network. Using patient samples, cell lines, and murine tumor xenograft models, we confirmed specific genes in the Ras pathway are targeted by an MPM-associated miRNA and then examined its therapeutic effects. We verified significant and consistent downregulation of miR-206 in MPM tissues. When miR-206 is ectopically re-expressed in MPM cells and delivered to tumor xenografts in mice, it exerted significant cell killing by suppressing multiple components of the receptor-tyrosine-kinase-Ras-cell-cycle-signaling network; some of which were prognostic when overexpressed and/or have not been druggable. Of note, we validated CDK6 as a novel target of miR-206. Overall, this miR-206-targeting mechanism manifested as induced G1/S cell cycle arrest. In addition, we identified a novel MPM therapeutic combination by adding systemic-route abemaciclib with local-route miR-206, which showed additive efficacy translating to improved survival. Our pre-clinical study suggests a potential pathophysiologic role for, and therapeutic relevance of, miR-206 in MPM.

Increased inflammasome activity in subcutaneous adipose tissue of patients with metabolic syndrome.

AIMS: The metabolic syndrome (MetS) is an inflammatory disorder associated with an increased risk for diabetes and atherosclerotic cardiovascular disease (ASCVD). Studies in patients and animal models of obesity and diabetes have shown increased NOD-like receptor family pyrin domain containing 3 (NLPR3) inflammasome activity. However, there is scanty data on the activity of the NLRP3 inflammasome in patients with nascent MetS. The aim of this study was to determine the status of the inflammasome in subcutaneous adipose tissue (SAT) of patients with nascent MetS without concomitant diabetes, ASCVD and smoking. MATERIALS AND METHODS: Patients with nascent MetS and controls were recruited from Sacramento County. Fasting blood samples were collected for biomediators of inflammation and SAT was obtained by biopsy for immunohistochemical (IHC) staining for caspase 1, IL-1ß and IL-18. RESULTS: Caspase1, a marker of inflammasome activity and its downstream mediators IL-1ß and IL-18 were significantly increased in SAT of patients with MetS compared to controls. Significant positive correlations of caspase 1 were obtained with certain cardio-metabolic features, biomediators of inflammation and markers of angiogenesis and fibrosis in SAT. Both mast cell and eosinophil abundance but not macrophage density correlated with caspase1. CONCLUSIONS: We make the novel observation that the SAT of patients with nascent MetS displays increased NLRP3 inflammasome activity manifest by increased caspase 1 in SAT and this may contribute to increased insulin resistance, inflammation and SAT fibrosis in these patients.

Fetuin-A is also an adipokine.

Fetuin-A (FetA), which impairs insulin action is considered classically as a hepatokine. In patients with Metabolic Syndrome without the confounding of diabetes or cardiovascular diseases, we showed significant increases in both circulating and subcutaneous adipose tissue secreted Fet-A. Furthermore we showed in mice models increase mRNA and protein following a high fat diet and in a model of metabolic syndrome. This work was recently confirmed by another group of investigators. Hence we propose that Fet-A be considered also as an adipokine.

Fibroblast Growth Factor 23 Predicts Mortality and End-Stage Renal Disease in a Canadian Asian Population with Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) is a leading cause of morbidity and mortality. Biomarkers that predict end-stage renal disease (ESRD) and/or mortality could usher in new therapeutics to halt this onslaught. While fibroblast growth factor (FGF)23 can predict both ESRD and mortality, it has not been studied in North American CKD patients of Asian ethnicity. METHOD: This is a prospective investigation about the role of FGF23 in 998 Canadian patients of Asian descent with CKD defined by an estimated glomerular filtration rate (eGFR) <60 mL/min/m2 and followed up for 3 years. RESULTS: The mean age of patients was 68.9 years and 68.3% were males. The mean (range) eGFR, and median FGF23 were 40.2 (11.0-59.0) mL/min and 154.1 (7.0-7,823.0) RU/mL, respectively. Over the 3 years, higher values of FGF23 levels at baseline were associated with higher risk of ESRD (hazard ratio [HR] for log[Fgf23] = 2.16 [95% CI 1.20-3.89]). Despite the short follow-up, 42 patients died due to cardiovascular diseases (38.8%), cancer (14.9%), and infections (12.7%). Log-FGF23 levels were independently associated with death, HR 1.94, 95% CI 1.24-3.03. Mortality risk increased in FGF23 subgroups from <100 to >400 RU/mL. In a time-changing covariate analysis, serial log-FGF23 levels over the 3 years predicted mortality with a HR of 2.66 (95% CI 1. 79-3.95). CONCLUSION: In a Canadian Asian population with CKD, FGF23 levels obtained at 6-monthly intervals for 3 years predicted ESRD and mortality suggesting that it is also a risk marker in Asians.

The effect of increasing body mass index on cardio-metabolic risk and biomarkers of oxidative stress and inflammation in nascent metabolic syndrome.

The effect of BMI defined obesity on cardio-metabolic features and biomarkers of oxidative stress and inflammation in patients with nascent metabolic Syndrome (MetS) is poorly defined. Hence the aim of this study was to examine the effect of increasing obesity on the cardio metabolic risk profile, pro-oxidant state and pro-inflammatory features in nascent MetS patients without Diabetes or CVD. MetS was diagnosed by ATPIII criteria using waist circumference (WC) as the measure of adiposity. Patients (n=58) were stratified into overweight, obese and extreme obesity groups using BMI cut offs of 25-29.9, 30-39.9kg/m2 and ≥40kg/m2 and cardio-metabolic features, circulating and cellular biomarkers of oxidative stress and inflammation were determined and correlated with BMI. None of the main cardio-metabolic features including blood pressure, blood glucose, HDL-cholesterol, triglycerides, HOMA-IR, free fatty acids were increased with increasing BMI. Also none of the biomarkers of oxidative stress (ox-LDL, nitrotyrosine and monocyte superoxide anion release) were increased with increasing BMI. However, significant increase in hsCRP, the soluble TNFR1 and sTNFR2 and leptin, were observed with increasing adiposity. Other inflammatory bio-mediators (IL-1ß, IL-6, IL-8, MCP-1, Toll-like receptors 2-4), endotoxin, LBP, sCD14 and HMGB1, adiponectin, and chemerin did not show significant increases with increasing BMI. Leptin, hsCRP, sTNFR1, and sTNFR2 correlated significantly with BMI. In conclusion, capturing the cardio-metabolic cluster of MetS that predisposed to both increased risk of diabetes and CVD, using waist circumference, as one of the 5 diagnostic criteria is sufficient and BMI does not appear to afford any major incremental benefit on the cardio-metabolic risk factors, increased oxidative stress and the majority of both cellular and circulating biomarkers of inflammation.

Gut Microbiome and Inflammation: A Study of Diabetic Inflammasome-Knockout Mice.

AIMS: Diabetes is a proinflammatory state, evidenced by increased pattern recognition receptors and the inflammasome (NOD-like receptor family pyrin domain (NLRP)) complex. Recent reports have elucidated the role of the gut microbiome in diabetes, but there is limited data on the gut microbiome in NLRP-KO mice and its effect on diabetes-induced inflammation. METHODS: Gut microbiome composition and biomarkers of inflammation (IL-18, serum amyloid A) were assessed in streptozotocin- (STZ-) induced diabetic mice on a NLRP3-knockout (KO) background versus wild-type diabetic mice. RESULTS: SAA and IL-18 levels were significantly elevated in diabetic mice (STZ) compared to control (WT) mice, and there was a significant attenuation of inflammation in diabetic NLRP3-KO mice (NLRP3-KO STZ) compared to control mice (p < 0.005). Principal coordinate analysis clearly separated controls, STZ, and NLRP3-KO STZ mice. Among the different phyla, there was a significant increase in the Firmicutes : Bacteroidetes ratio in the diabetic group compared to controls. When compared to the WT STZ group, the NLRP3-KO STZ group showed a significant decrease in the Firmicutes : Bacteroidetes ratio. Together, these findings indicate that interaction of the intestinal microbes with the innate immune system is a crucial factor that could modify diabetes and complications.

Investigation into the interference of the monoclonal antibody daratumumab on the free light chain assay.

OBJECTIVES: Recently monoclonal antibody therapy has been introduced in the treatment of multiple Myeloma (MM). One such efficacious therapy is the anti-CD38 monoclonal antibody, daratumumab (Dara). Since it is an Ig-G-kappa it can interfere with both the serum protein electrophoresis and immunofixation electrophoresis (IFE). The free light chain (FLC) assay is also useful in the diagnosis and therapeutic monitoring of MM. Hence we tested the effect of Dara on the FLC assay. METHODS: 30 serum samples from patients with known IgG-kappa (n=20) and non-IgG-kappa M -proteins (n=10) were spiked with Dara at a final concentration of 1.0mg/mL and the FLC performed on samples. On a further 20 samples we performed IFE to determine the migration of Dara. RESULTS: On IFE, Dara migrated in the same area of the gamma zone. In the 30 samples in which we assayed FLC there was no significant differences in levels of kappa, lambda and the ratio of kappa to lambda between untreated and Dara-spiked samples. CONCLUSION: Whilst Dara can interfere with the IFE to determine clinical responses the FLC assay can be useful in patients who have abnormal FLC ratios prior to Dara therapy to determine responses especially in IgG-kappa Myeloma.

Hyperglycemia Induces Toll-Like Receptor Activity Through Increased Oxidative Stress.

Hyperglycemia-induced oxidative stress and inflammation are central in the genesis of diabetic vascular complications. Toll-like receptors (TLRs) play a crucial role in promoting inflammatory responses and are known to be activated in diabetic patients. Also in animal models, they have been shown to have a role in the pathogenesis of diabetic vasculopathies. However, the mechanisms underlying this increase in TLR activity in diabetes are not well documented. Since increased reactive oxygen species (ROS) are also produced in various tissues under diabetic conditions, we postulated that ROS act as a potential activator of TLR. Several studies support our hypothesis that hyperglycemia-induced oxidative stress appears to be an important factor in promoting TLR activity in monocytes, both microvascular and macrovascular endothelial cells and cardiomyocytes and in animal models. Most importantly, the increase in ROS and TLR activity is ameliorated with antioxidant strategies. Thus, targeting ROS/NADPH oxidase with small molecular inhibitors could be a promising strategy to reduce both oxidative stress and TLR-mediated inflammation in diabetic vascular diseases.

The role of the high-mobility group box1 protein-Toll like receptor pathway in diabetic vascular disease.

OBJECTIVES: Increased Toll like receptors (TLRs) especially 2 and 4 have been demonstrated in obesity, metabolic syndrome (MetS) and diabetes resulting in increased cellular inflammation. Since we have shown increased TLR2 and 4 activities in both T1DM and T2DM and MetS, we wanted to elucidate the mechanisms of this sterile inflammation. In T1DM, T2DM and MetS we have shown that high mobility group box 1 protein (HMGB-1), a non-histone DNA binding protein is increased and could be a potential activator of TLRs since it has previously shown to activate TLR2, 4 and 9. We examined the role of HMGB-1 in patients and animal models of diabetes and MetS to determine how important it is as an activator of TLR mediated inflammation and its role in diabetic vascular complications. METHODS: A Medline search was conducted using the terms HMGB-1, TLRs and diabetes. RESULTS: HMGB-1 levels are increased in patients with diabetes and MetS, and associated with increased biomediators of inflammation. Furthermore data supported a role of HMGB-1 in both diabetic microvascular and macrovascular complications. CONCLUSIONS: HMGB-1 interaction with TLRs is implicated in diabetic complications and could be important therapeutic target.

Toll-like receptors 2 and 4 mediate hyperglycemia induced macrovascular aortic endothelial cell inflammation and perturbation of the endothelial glycocalyx.

OBJECTIVES: Hyperglycemia-induced inflammation is central to the vascular complications in diabetes. Toll-like receptors (TLRs) are key players in regulating inflammatory responses. There are sparse data on the role of TLR2 and TLR4 in regulating human macrovascular aortic endothelial cells (HMAECs) inflammation and glycocalyx dysfunction under hyperglycemia. We examined the role of TLR2/4 in the above dysfunctions in HMAEC under high glucose (HG) conditions. METHODS: HMAECs were treated with high or normal glucose and TLR-2, TLR-4, MyD88, IRF3, TRIF, nuclear NF-κB p65, IL-8, IL-1ß, TNF-α, MCP-1, ICAM-1, sVCAM-1, monocyte adhesion to HMAECs, heparan sulfate and hyaluronic acid were measured. RESULTS: HG upregulated TLR2 and TLR4 mRNA and protein and increased both MyD88 and non-MyD88 pathways, NF-κB p65, inflammatory biomediators, and monocyte adhesion to HMAECs. Heparan sulfate protein expression was reduced and hyaluronic acid secretion was increased on HG exposure. Inhibition of TLR2 and TLR4 signaling by inhibitory peptides and knockdown of TLR-2 and TLR-4 gene expression by siRNA attenuated HG induced inflammation, leukocyte adhesion and glycocalyx dysfunction. An increase in ROS paralleled the increase in TLR-2/4 and antioxidants treatment reduced TLR-2/4 expression and downstream inflammatory biomediators. CONCLUSION: Thus hyperglycemia induces HMAEC inflammation and glycocalyx dysfunction through TLR-2/4 pathway activation via increased ROS.

Modulation of PBMC-decay accelerating factor (PBMC-DAF) and cytokines in rheumatoid arthritis.

Studies have suggested that abnormal expression of complement regulatory proteins and cytokines contribute significantly to the path-physiology of rheumatoid arthritis. In this context, Decay accelerating factor (DAF) a complement regulatory protein is gaining increased attention. With the notion that immune effecter mechanisms are all interlinked and circulating peripheral blood mononuclear cells (PBMCs) should have a role in a systemic disease like rheumatoid arthritis, we studied the modulation and significance of PBMC-DAF and cytokines in RA. Seventy-five RA patients and 75 healthy controls were recruited. Expression of DAF and cytokines (IFN-γ, IL-17A and IL-10) in the PBMCs of patients and controls was determined. Correlations among DAF, cytokines, and disease activity were evaluated by standard statistical methods. The effect of IFN-γ, IL-17A, and IL-10 on the expression of DAF in patients and controls was studied in vitro. Expression of PBMC-DAF declined in patients both at mRNA and surface level and correlated negatively with the disease activity. Expression of IFN-γ also declined in patients but correlated positively with DAF and negatively with disease activity. Expression of IL-17A and IL-10 was higher in patients. The levels correlated positively with disease activity and negatively with DAF both in patients and controls. In vitro studies indicated that IFN-γ up-regulated DAF expression in PBMCs, whereas IL-17A and IL-10 had negative effect on the same. The decline in the PBMC-DAF is a contributing factor in manifestations of RA. Cytokine environment contributes to this decline. These findings brought novel insights into the complement-cytokine axis in the path-physiology of RA.

Selective increase in monocyte p38 mitogen-activated protein kinase activity in metabolic syndrome.

OBJECTIVE: Metabolic syndrome is a common disorder that predisposes to both cardiovascular disease and diabetes. There is paucity of data on cellular signal transduction pathways in metabolic syndrome. This study determined monocyte mitogen-activated protein kinase activity in patients with metabolic syndrome. RESEARCH DESIGN AND METHODS: The p38, extracellular signal-regulated kinase-1/2 and Jun N-terminal kinase-mitogen-activated protein kinase activities were assayed in isolated monocytes from patients with metabolic syndrome and controls (n = 36 per group) and correlated with features of metabolic syndrome, inflammation and oxidative stress biomarkers. RESULTS: A significant increase in p38 mitogen-activated protein kinase activity was observed in metabolic syndrome even following adjustment for adiposity. There were no significant differences in extracellular signal-regulated kinase-1/2 and Jun N-terminal kinase activities. P38 mitogen-activated protein kinase activity correlated significantly with homeostasis model assessment-estimated insulin resistance and biomarkers of inflammation and oxidative stress. CONCLUSIONS: We are first to observe a selective increase in monocyte p38 mitogen-activated protein kinase activity in metabolic syndrome and suggest it as a pivotal molecular target for ameliorating insulin resistance and inflammation.