S-nitrosoglutathione reductase (GSNOR) enhances vasculogenesis by mesenchymal stem cells.

Although nitric oxide (NO) signaling promotes differentiation and maturation of endothelial progenitor cells, its role in the differentiation of mesenchymal stem cells (MSCs) into endothelial cells remains controversial. We tested the role of NO signaling in MSCs derived from WT mice and mice homozygous for a deletion of S-nitrosoglutathione reductase (GSNOR(-/-)), a denitrosylase that regulates S-nitrosylation. GSNOR(-/-) MSCs exhibited markedly diminished capacity for vasculogenesis in an in vitro Matrigel tube-forming assay and in vivo relative to WT MSCs. This decrease was associated with down-regulation of the PDGF receptorα (PDGFRα) in GSNOR(-/-) MSCs, a receptor essential for VEGF-A action in MSCs. Pharmacologic inhibition of NO synthase with L-N(G)-nitroarginine methyl ester (L-NAME) and stimulation of growth hormone-releasing hormone receptor (GHRHR) with GHRH agonists augmented VEGF-A production and normalized tube formation in GSNOR(-/-) MSCs, whereas NO donors or PDGFR antagonist reduced tube formation ∼50% by murine and human MSCs. The antagonist also blocked the rescue of tube formation in GSNOR(-/-) MSCs by L-NAME or the GHRH agonists JI-38, MR-409, and MR-356. Therefore, GSNOR(-/-) MSCs have a deficient capacity for endothelial differentiation due to downregulation of PDGFRα related to NO/GSNOR imbalance. These findings unravel important aspects of modulation of MSCs by VEGF-A activation of the PDGFR and illustrate a paradoxical inhibitory role of S-nitrosylation signaling in MSC vasculogenesis. Accordingly, disease states characterized by NO deficiency may trigger MSC-mediated vasculogenesis. These findings have important implications for therapeutic application of GHRH agonists to ischemic disorders.

NADPH oxidase-2 inhibition restores contractility and intracellular calcium handling and reduces arrhythmogenicity in dystrophic cardiomyopathy.

Duchenne muscular dystrophy may affect cardiac muscle, producing a dystrophic cardiomyopathy in humans and the mdx mouse. We tested the hypothesis that oxidative stress participates in disrupting calcium handling and contractility in the mdx mouse with established cardiomyopathy. We found increased expression (fivefold) of the NADPH oxidase (NOX) 2 in the mdx hearts compared with wild type, along with increased superoxide production. Next, we tested the impact of NOX2 inhibition on contractility and calcium handling in isolated cardiomyocytes. Contractility was decreased in mdx myocytes compared with wild type, and this was restored toward normal by pretreating with apocynin. In addition, the amplitude of evoked intracellular Ca(2+) concentration transients that was diminished in mdx myocytes was also restored with NOX2 inhibition. Total sarcoplasmic reticulum (SR) Ca(2+) content was reduced in mdx hearts and normalized by apocynin treatment. Additionally, NOX2 inhibition decreased the production of spontaneous diastolic calcium release events and decreased the SR calcium leak in mdx myocytes. In addition, nitric oxide (NO) synthase 1 (NOS-1) expression was increased eightfold in mdx hearts compared with wild type. Nevertheless, cardiac NO production was reduced. To test whether this paradox implied NOS-1 uncoupling, we treated cardiac myocytes with exogenous tetrahydrobioterin, along with the NOX inhibitor VAS2870. These agents restored NO production and phospholamban phosphorylation in mdx toward normal. Together, these results demonstrate that, in mdx hearts, NOX2 inhibition improves the SR calcium handling and contractility, partially by recoupling NOS-1. These findings reveal a new layer of nitroso-redox imbalance in dystrophic cardiomyopathy.

Harnessing multi-source data for individualized care in Hodgkin Lymphoma.

Hodgkin lymphoma is a rare, but highly curative form of cancer, primarily afflicting adolescents and young adults. Despite multiple seminal trials over the past twenty years, there is no single consensus-based treatment approach beyond use of multi-agency chemotherapy with curative intent. The use of radiation continues to be debated in early-stage disease, as part of combined modality treatment, as well as in salvage, as an important form of consolidation. While short-term disease outcomes have varied little across these different approaches across both early and advanced stage disease, the potential risk of severe, longer-term risk has varied considerably. Over the past decade novel therapeutics have been employed in the retrieval setting in preparation to and as consolidation after autologous stem cell transplant. More recently, these novel therapeutics have moved to the frontline setting, initially compared to standard-of-care treatment and later in a direct head-to-head comparison combined with multi-agent chemotherapy. In 2018, we established the HoLISTIC Consortium, bringing together disease and methods experts to develop clinical decision models based on individual patient data to guide providers, patients, and caregivers in decision-making. In this review, we detail the steps we followed to create the master database of individual patient data from patients treated over the past 20 years, using principles of data science. We then describe different methodological approaches we are taking to clinical decision making, beginning with clinical prediction tools at the time of diagnosis, to multi-state models, incorporating treatments and their response. Finally, we describe how simulation modeling can be used to estimate risks of late effects, based on cumulative exposure from frontline and salvage treatment. The resultant database and tools employed are dynamic with the expectation that they will be updated as better and more complete information becomes available.

Integrating POLE/POLD1 mutated for immunotherapy treatment planning of advanced stage non-small cell lung cancer.

BACKGROUND: In this study, we evaluated the potential of DNA polymerase epsilon (POLE) and DNA polymerase delta 1 (POLD1) as prognostic biomarkers for immune checkpoint inhibitor (ICI) treatment in patients with advanced stage non-small cell lung cancer (NSCLC). METHODS: Disease stage, PD-L1 positivity, histological subtypes, POLE/POLD1 mutation status, tumor mutation burden (TMB), and response to ICIs in NSCLC cases were derived from AACR GENIE dataset (n = 24 120), TCGA-Pan Lung Cancer dataset (n = 1144), AACR GENIE BPC NSCLC v2.0-public (n = 2004), and Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets dataset (n = 350). The smoking history from TCGA and AACR GENIE datasets was grouped into current, former or never-smokers. RESULTS: POLE and POLD1 genetic alterations were identified in 5% and 2.6% of NSCLC patients, respectively. Current smokers had 9% and 4% of POLE/POLD1 mutations, respectively, versus 1.7% for both POLE and POLD1 mutations prevalence in never-smokers. POLE/POLD1 mutations were associated with elevated mutation counts than those with wild-type (median mutation counts 16 vs. 7, p < 0.0001), more advanced disease stages (stage I disease 15.19% vs. 29.42%), more prevalent squamous histology subtype (21.69% vs. 9.05%, p = 0.0427), and a higher percentage of PD-L1 positivity (66.67% vs. 43.87%, p < 0.001). Treatment with ICIs improved survival in patients with both POLE/POLD1 mutated and those with TMB > 18 (p < 0.001). CONCLUSION: Current smokers have a five-fold increased risk of having POLE mutations than never-smokers. POLE/POLD1 mutation status and TMB > 18 can be a composite biomarker for selecting NSCLC patients with survival benefits to ICI treatment.

Clinical presentation of Warburg effect in aggressive lymphoma: a case report.

BACKGROUND: The Warburg effect is a rare condition in tumor biology, illustrated by significant lactate production in the presence of oxygen. The Warburg effect is associated with very poor prognosis in patients with malignancy. CASE PRESENTATION: We report a 76-year-old Caucasian woman with double-expressor diffuse large B cell lymphoma who presented with severe lactic acidosis and extreme hypoglycemia with normal mentation. Her lactic acidosis was initially controlled with a bicarbonate infusion, and the patient was started promptly on steroids, followed by chemotherapy, but her clinical course was complicated by tumor lysis syndrome, acute renal failure requiring hemodialysis, and progressive liver failure. She manifested a temporary clinical response to chemotherapy but eventually died of complications. CONCLUSIONS: This case demonstrates the importance of prompt recognition of the Warburg effect, aggressive supportive measures, and early initiation of chemotherapy. Future studies are needed to characterize the role of hemodialysis in this setting.

Ipilimumab and nivolumab induced immune-related adverse events in metastatic mucosal melanoma.

Mucosal melanoma is a rare subtype of melanoma and represents a unique diagnosis and treatment challenge. Immune-checkpoint inhibitors (ICIs) have revolutionised metastatic melanoma treatment, and one of the leading regimens is the combination of ipilimumab (anti-cytotoxic T lymphocyte-associated antigen 4: CTLA4) and nivolumab (anti-programmed cell death protein 1: PD1). We report a case of a patient with metastatic mucosal melanoma treated with ipilimumab and nivolumab who developed multiple immune-related adverse events (irAEs) including uveitis, type I diabetes complicated by diabetic ketoacidosis, destructive thyroiditis, hepatitis and vitiligo. Endocrinopathies including type 1 diabetes and hypothyroidism were treated with insulin and levothyroxine. Hepatitis was responsive to steroids. She had sustained complete response 12 months after discontinuation of the combination therapy. With the wide usage of ICIs in multiple types of malignancies, it is important for general practioners to recognise common and serious irAEs due to ICIs.

Does denosumab offer survival benefits? -Our experience with denosumab in metastatic non-small cell lung cancer patients treated with immune-checkpoint inhibitors.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of non-small-cell lung cancer (NSCLC). Denosumab is a humanized monoclonal antibody to RANK ligand used to prevent skeletal-related events of bone metastases in solid tumors. We are reporting the clinical outcomes in our NSCLC patients who received RANKL inhibitor in combination with ICIs. METHODS: This observational study used retrospective data from a tertiary cancer center from 2015-2020. Stage IV non-small cell lung cancer patients who received denosumab within 30 days of ICIs (pembrolizumab, nivolumab, atezolizumab, ipilimumab) were included. Kaplan-Meier curves were obtained for survival analysis. RESULTS: We identified 69 patients and all had skeletal metastases, and 37.7% had brain metastases. Median OS was 6.3 months and median PFS was 2.8 months, with overall response rate (ORR) of 18.8% and disease control rate (DCR) of 40.6%. Median OS in patients with concomitant denosumab and ICIs more than 3 months was 11.5 months, comparing to 3.6 months in patients with <3 months of concomitant therapy (P=0.0005). OS and PFS did not differ with respect to brain metastases or number of skeletal metastases. However, the duration of ICIs and denosumab overlap was associated with improved OS and PFS. Among the 18.8% of patients who achieved complete response (CR) and partial response (PR), six-month survival rate was 100% and one-year survival rate was 69.2%. Most of the patients tolerated denosumab well, and hypocalcemia was the most commonly reported side effect. CONCLUSIONS: Patients receiving combination therapy did not perform poorly comparing to published studies despite of poor prognostic features such as brain metastases and numerous skeletal metastases. Although we did notice potential benefit of the longer duration of concomitant use of ICI and denosumab, future prospective clinical trials are needed to evaluate the synergistic effect of RANKL inhibitors/ICI and if duration of RANKL inhibitors matters.

GSNOR Deficiency Enhances In Situ Skeletal Muscle Strength, Fatigue Resistance, and RyR1 S-Nitrosylation Without Impacting Mitochondrial Content and Activity.

AIM: Nitric oxide (NO) plays important, but incompletely defined roles in skeletal muscle. NO exerts its regulatory effects partly though S-nitrosylation, which is balanced by denitrosylation by enzymes such as S-nitrosoglutathione reductase (GSNOR), whose functions in skeletal muscle remain to be fully deciphered. RESULTS: GSNOR null (GSNOR-/-) tibialis anterior (TA) muscles showed normal growth and were stronger and more fatigue resistant than controls in situ. However, GSNOR-/- lumbrical muscles showed normal contractility and Ca2+ handling in vitro, suggesting important differences in GSNOR function between muscles or between in vitro and in situ environments. GSNOR-/- TA muscles exhibited normal mitochondrial content, and capillary densities, but reduced type IIA fiber content. GSNOR inhibition did not impact mitochondrial respiratory complex I, III, or IV activities. These findings argue that enhanced GSNOR-/- TA contractility is not driven by changes in mitochondrial content or activity, fiber type, or blood vessel density. However, loss of GSNOR led to RyR1 hypernitrosylation, which is believed to increase muscle force output under physiological conditions. cGMP synthesis by soluble guanylate cyclase (sGC) was decreased in resting GSNOR-/- muscle and was more responsive to agonist (DETANO, BAY 41, and BAY 58) stimulation, suggesting that GSNOR modulates cGMP production in skeletal muscle. INNOVATION: GSNOR may act as a "brake" on skeletal muscle contractile performance under physiological conditions by modulating nitrosylation/denitrosylation balance. CONCLUSIONS: GSNOR may play important roles in skeletal muscle contractility, RyR1 S-nitrosylation, fiber type specification, and sGC activity. Antioxid. Redox Signal. 26, 165-181.

S-Nitrosoglutathione Reductase Deficiency Enhances the Proliferative Expansion of Adult Heart Progenitors and Myocytes Post Myocardial Infarction.

BACKGROUND: Mammalian heart regenerative activity is lost before adulthood but increases after cardiac injury. Cardiac repair mechanisms, which involve both endogenous cardiac stem cells (CSCs) and cardiomyocyte cell-cycle reentry, are inadequate to achieve full recovery after myocardial infarction (MI). Mice deficient in S-nitrosoglutathione reductase (GSNOR(-/-)), an enzyme regulating S-nitrosothiol turnover, have preserved cardiac function after MI. Here, we tested the hypothesis that GSNOR activity modulates cardiac cell proliferation in the post-MI adult heart. METHODS AND RESULTS: GSNOR(-/-) and C57Bl6/J (wild-type [WT]) mice were subjected to sham operation (n=3 GSNOR(-/-); n=3 WT) or MI (n=41 GSNOR(-/-); n=65 WT). Compared with WT, GSNOR(-/-) mice exhibited improved survival, cardiac performance, and architecture after MI, as demonstrated by higher ejection fraction (P<0.05), lower endocardial volumes (P<0.001), and diminished scar size (P<0.05). In addition, cardiomyocytes from post-MI GSNOR(-/-) hearts exhibited faster calcium decay and sarcomeric relaxation times (P<0.001). Immunophenotypic analysis illustrated that post-MI GSNOR(-/-) hearts demonstrated enhanced neovascularization (P<0.001), c-kit(+) CSC abundance (P=0.013), and a ≈3-fold increase in proliferation of adult cardiomyocytes and c-kit(+)/CD45(-) CSCs (P<0.0001 and P=0.023, respectively) as measured by using 5-bromodeoxyuridine. CONCLUSIONS: Loss of GSNOR confers enhanced post-MI cardiac regenerative activity, characterized by enhanced turnover of cardiomyocytes and CSCs. Endogenous denitrosylases exert an inhibitory effect over cardiac repair mechanisms and therefore represents a potential novel therapeutic target.

S-nitrosoglutathione reductase-dependent PPARγ denitrosylation participates in MSC-derived adipogenesis and osteogenesis.

Bone marrow-derived mesenchymal stem cells (MSCs) are a common precursor of both adipocytes and osteoblasts. While it is appreciated that PPARγ regulates the balance between adipogenesis and osteogenesis, the roles of additional regulators of this process remain controversial. Here, we show that MSCs isolated from mice lacking S-nitrosoglutathione reductase, a denitrosylase that regulates protein S-nitrosylation, exhibited decreased adipogenesis and increased osteoblastogenesis compared with WT MSCs. Consistent with this cellular phenotype, S-nitrosoglutathione reductase-deficient mice were smaller, with reduced fat mass and increased bone formation that was accompanied by elevated bone resorption. WT and S-nitrosoglutathione reductase-deficient MSCs exhibited equivalent PPARγ expression; however, S-nitrosylation of PPARγ was elevated in S-nitrosoglutathione reductase-deficient MSCs, diminishing binding to its downstream target fatty acid-binding protein 4 (FABP4). We further identified Cys 139 of PPARγ as an S-nitrosylation site and demonstrated that S-nitrosylation of PPARγ inhibits its transcriptional activity, suggesting a feedback regulation of PPARγ transcriptional activity by NO-mediated S-nitrosylation. Together, these results reveal that S-nitrosoglutathione reductase-dependent modification of PPARγ alters the balance between adipocyte and osteoblast differentiation and provides checkpoint regulation of the lineage bifurcation of these 2 lineages. Moreover, these findings provide pathophysiological and therapeutic insights regarding MSC participation in adipogenesis and osteogenesis.

Conversion of 2-deoxyglucose-induced growth inhibition to cell death in normoxic tumor cells.

BACKGROUND: Inhibition of glucose metabolism has recently become an attractive target for cancer treatment. Accordingly, since 2-deoxyglucose (2-DG) competes effectively with glucose, it has come under increasing scrutiny as a therapeutic agent. The initial response of tumor cells to 2-DG is growth inhibition, which is thought to conserve energy and consequently protect cells from its ATP-lowering effects as a glycolytic inhibitor. However, since 2-DG also mimics mannose and thereby interferes with N-linked glycosylation, the question is raised of how this sugar analog inhibits tumor cell growth and whether the mechanism by which it protects cells can be manipulated to convert 2-DG-induced growth inhibition to cell death. METHODS: Cell growth and death were measured via counting viable and dead cells based on trypan blue exclusion. Markers of ATP reduction and the unfolded protein response (UPR) were detected by Western blot. Protein functions were manipulated through chemical compounds, siRNA and the use of gene-specific wild-type and knock-out mouse embryonic fibroblasts (MEFs). RESULTS: At 2-DG concentrations that can be achieved in human plasma without causing significant side effects, we find (a) It induces growth inhibition predominantly by interference with glycosylation, which leads to accumulation of unfolded proteins in the endoplasmic reticulum activating the UPR; (b) Inhibition of PERK (but not ATF6 or IRE1), a major component of the UPR, leads to conversion of 2-DG-induced growth inhibition to cell death and (c) secondarily to PERK, inhibition of GCN2, a kinase that is activated in response to low intracellular glutamine, increases 2-DG's cytotoxic effects in PERK -/- MEFs. CONCLUSIONS: Overall, these findings present a novel anticancer strategy that can be translated into therapeutic gain as they uncover the metabolic target PERK, and to a lesser degree GCN2, that when inhibited convert 2-DG's static effect to a toxic one in tumor cells growing under normoxia.