National Cancer Institute Collaborative Workshop on Shaping the Landscape of Brain Metastases Research: challenges and recommended priorities.

Brain metastases are an increasing global public health concern, even as survival rates improve for patients with metastatic disease. Both metastases and the sequelae of their treatment are key determinants of the inter-related priorities of patient survival, function, and quality of life, mandating a multidimensional approach to clinical care and research. At a virtual National Cancer Institute Workshop in September, 2022, key stakeholders convened to define research priorities to address the crucial areas of unmet need for patients with brain metastases to achieve meaningful advances in patient outcomes. This Policy Review outlines existing knowledge gaps, collaborative opportunities, and specific recommendations regarding consensus priorities and future directions in brain metastases research. Achieving major advances in research will require enhanced coordination between the ongoing efforts of individual organisations and consortia. Importantly, the continual and active engagement of patients and patient advocates will be necessary to ensure that the directionality of all efforts reflects what is most meaningful in the context of patient care.

Dietary Antioxidants Significantly Attenuate Hyperoxia-Induced Acute Inflammatory Lung Injury by Enhancing Macrophage Function via Reducing the Accumulation of Airway HMGB1.

Mechanical ventilation with hyperoxia is the major supportive measure to treat patients with acute lung injury and acute respiratory distress syndrome (ARDS). However, prolonged exposure to hyperoxia can induce oxidative inflammatory lung injury. Previously, we have shown that high levels of airway high-mobility group box 1 protein (HMGB1) mediate hyperoxia-induced acute lung injury (HALI). Using both ascorbic acid (AA, also known as vitamin C) and sulforaphane (SFN), an inducer of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), we tested the hypothesis that dietary antioxidants can mitigate HALI by ameliorating HMGB1-compromised macrophage function in phagocytosis by attenuating hyperoxia-induced extracellular HMGB1 accumulation. Our results indicated that SFN, which has been shown to attenute HALI in mice exposed to hyperoxia, dose-dependently restored hyperoxia-compromised macrophage function in phagocytosis (75.9 ± 3.5% in 0.33 µM SFN versus 50.7 ± 1.8% in dimethyl sulfoxide (DMSO) control, p < 0.05) by reducing oxidative stress and HMGB1 release from cultured macrophages (47.7 ± 14.7% in 0.33 µM SFN versus 93.1 ± 14.6% in DMSO control, p < 0.05). Previously, we have shown that AA enhances hyperoxic macrophage functions by reducing hyperoxia-induced HMGB1 release. Using a mouse model of HALI, we determined the effects of AA on hyperoxia-induced inflammatory lung injury. The i.p. administration of 50 mg/kg of AA to mice exposed to 72 h of ≥98% O2 significantly decreased hyperoxia-induced oxidative and nitrosative stress in mouse lungs. There was a significant decrease in the levels of airway HMGB1 (43.3 ± 12.2% in 50 mg/kg AA versus 96.7 ± 9.39% in hyperoxic control, p < 0.05), leukocyte infiltration (60.39 ± 4.137% leukocytes numbers in 50 mg/kg AA versus 100 ± 5.82% in hyperoxic control, p < 0.05) and improved lung integrity in mice treated with AA. Our study is the first to report that the dietary antioxidants, ascorbic acid and sulforaphane, ameliorate HALI and attenuate hyperoxia-induced macrophage dysfunction through an HMGB1-mediated pathway. Thus, dietary antioxidants could be used as potential treatments for oxidative-stress-induced acute inflammatory lung injury in patients receiving mechanical ventilation.

Effect of nitric oxide on the anticancer activity of the topoisomerase-active drugs etoposide and adriamycin in human melanoma cells.

Nitric oxide (·NO) was originally identified as an innate cytotoxin. However, in tumors it can enhance resistance to chemotherapy and exacerbate cancer progression. Our previous studies indicated that (·NO/·NO-derived species react with etoposide (VP-16) in vitro and form products that show significantly reduced activity toward HL60 cells and lipopolysaccharide (LPS)-induced macrophages. Here, we further confirm the hypothesis that (÷)NO generation contributes to VP-16 resistance by examining interactions of ·NO with VP-16 in inducible nitric-oxide synthase (iNOS)-expressing human melanoma A375 cells. Inhibition of iNOS catalysis by N(6)-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL) in human melanoma A375 cells reversed VP-16 resistance, leading to increased DNA damage and apoptosis. Furthermore, we found that coculturing A375 melanoma cells with LPS-induced macrophage RAW cells also significantly reduced VP-16 cytotoxicity and DNA damage in A375 cells. We also examined the interactions of (·)NO with another topoisomerase active drug, Adriamycin, in A375 cells. In contrast, to VP-16, (·)NO caused no significant modulation of cytotoxicity or Adriamycin-dependent apoptosis, suggesting that (⋅)NO does not interact with Adriamycin. Our studies support the hypothesis that (·)NO oxidative chemistry can detoxify VP-16 through direct nitrogen oxide radical attack. Our results provide insights into the pharmacology and anticancer mechanisms of VP-16 that may ultimately contribute to increased resistance, treatment failure, and induction of secondary leukemia in VP-16-treated patients.

Predictive Radiation Oncology - A New NCI-DOE Scientific Space and Community.

With a widely attended virtual kickoff event on January 29, 2021, the National Cancer Institute (NCI) and the Department of Energy (DOE) launched a series of 4 interactive, interdisciplinary workshops-and a final concluding "World Café" on March 29, 2021-focused on advancing computational approaches for predictive oncology in the clinical and research domains of radiation oncology. These events reflect 3,870 human hours of virtual engagement with representation from 8 DOE national laboratories and the Frederick National Laboratory for Cancer Research (FNL), 4 research institutes, 5 cancer centers, 17 medical schools and teaching hospitals, 5 companies, 5 federal agencies, 3 research centers, and 27 universities. Here we summarize the workshops by first describing the background for the workshops. Participants identified twelve key questions-and collaborative parallel ideas-as the focus of work going forward to advance the field. These were then used to define short-term and longer-term "Blue Sky" goals. In addition, the group determined key success factors for predictive oncology in the context of radiation oncology, if not the future of all of medicine. These are: cross-discipline collaboration, targeted talent development, development of mechanistic mathematical and computational models and tools, and access to high-quality multiscale data that bridges mechanisms to phenotype. The workshop participants reported feeling energized and highly motivated to pursue next steps together to address the unmet needs in radiation oncology specifically and in cancer research generally and that NCI and DOE project goals align at the convergence of radiation therapy and advanced computing.

Overview and Lessons From the Preclinical Chemoradiotherapy Testing Consortium.

PURPOSE: In the current molecular-targeted cancer treatment era, many new agents are being developed so that optimizing therapy with a combination of radiation and drugs is complex. The use of emerging laboratory technologies to further biological understanding of drug-radiation mechanisms of action will enhance the efficiency of the progression from preclinical studies to clinical trials. In 2017, the National Cancer Institute (NCI) solicited proposals through PAR 16-111 to conduct preclinical research combining targeted anticancer agents in the Cancer Therapy Evaluation Program's portfolio with chemoradiation. METHODS AND MATERIALS: The Preclinical Chemo-Radiotherapy Testing Consortium (PCRTC) was formed with 4 U01 programs supported to generate validated high-quality preclinical data on the effects of molecular therapeutics when added to standard-of-care therapies with a concentration on cancers of the pancreas, lung, head and neck, gastrointestinal tract, and brain. RESULTS: The PCRTC provides a rational basis for prioritizing NCI-supported investigational new drugs or agents most likely to have clinical activity with chemoradiotherapy and accelerate the pace at which combined modality treatments with greater efficacy are identified and incorporated into standard treatment practices. CONCLUSIONS: Herein, we introduce and summarize the course of the PCRTC to date and report 3 preliminary observations from the consortium's work to date.

Moving Forward in the Next Decade: Radiation Oncology Sciences for Patient-Centered Cancer Care.

In a time of rapid advances in science and technology, the opportunities for radiation oncology are undergoing transformational change. The linkage between and understanding of the physical dose and induced biological perturbations are opening entirely new areas of application. The ability to define anatomic extent of disease and the elucidation of the biology of metastases has brought a key role for radiation oncology for treating metastatic disease. That radiation can stimulate and suppress subpopulations of the immune response makes radiation a key participant in cancer immunotherapy. Targeted radiopharmaceutical therapy delivers radiation systemically with radionuclides and carrier molecules selected for their physical, chemical, and biochemical properties. Radiation oncology usage of "big data" and machine learning and artificial intelligence adds the opportunity to markedly change the workflow for clinical practice while physically targeting and adapting radiation fields in real time. Future precision targeting requires multidimensional understanding of the imaging, underlying biology, and anatomical relationship among tissues for radiation as spatial and temporal "focused biology." Other means of energy delivery are available as are agents that can be activated by radiation with increasing ability to target treatments. With broad applicability of radiation in cancer treatment, radiation therapy is a necessity for effective cancer care, opening a career path for global health serving the medically underserved in geographically isolated populations as a substantial societal contribution addressing health disparities. Understanding risk and mitigation of radiation injury make it an important discipline for and beyond cancer care including energy policy, space exploration, national security, and global partnerships.

Asbestos redirects nitric oxide signaling through rapid catalytic conversion to nitrite.

Asbestos exposure is strongly associated with the development of malignant mesothelioma, yet the mechanistic basis of this observation has not been resolved. Carcinogenic transformation or tumor progression mediated by asbestos may be related to the generation of free radical species and perturbation of cell signaling and transcription factors. We report here that exposure of human mesothelioma or lung carcinoma cells to nitric oxide (NO) in the presence of crocidolite asbestos resulted in a marked decrease in intracellular nitrosation and diminished NO-induced posttranslational modifications of tumor-associated proteins (hypoxia-inducible factor-1alpha and p53). Crocidolite rapidly scavenged NO with concomitant conversion to nitrite (NO(2)(-)). Crocidolite also catalyzed the nitration of cellular proteins in the presence of NO(2)(-) and hydrogen peroxide. Nitrated protein adducts are a prominent feature of asbestos-induced lung injury. These data highlight the ability of asbestos to induce phenotypic cellular changes through two processes: (a) by directly reducing bioactive NO levels and preventing its subsequent interaction with target molecules and (b) by increasing oxidative damage and protein modifications through NO(2) production and 3-nitrotyrosine formation.

Association of an endogenous inhibitor of nitric oxide synthase with cerebral vasospasm in patients with aneurysmal subarachnoid hemorrhage.

OBJECT: Delayed cerebral vasospasm after subarachnoid hemorrhage (SAH) may be evoked by the decreased availability of nitric oxide (NO). Increased cerebrospinal fluid (CSF) levels of asymmetric dimethyl-L-arginine (ADMA), an endogenous inhibitor of NO synthase (NOS), have been associated with the course and degree of cerebral vasospasm in a primate model of SAH. In this study, the authors sought to determine if similar changes in CSF ADMA levels are observed in patients with SAH, and whether these changes are associated with NO and NOS metabolite levels in the CSF and the presence of cerebral vasospasm. METHODS: Asymmetric dimethyl-L-arginine, L-arginine, L-citrulline, and nitrite levels were measured in CSF and serum samples collected during the 21-day period after a single aneurysmal SAH in 18 consecutive patients. Samples were also obtained in a control group consisting of seven patients with Chiari malformation Type I and five patients with spontaneous intracerebral hemorrhage without SAH. Vasospasm, defined as a greater than 11% reduction in the anterior circulation vessel diameter ratio compared with the ratio calculated from the initial arteriogram, was assessed on cerebral arteriography performed around Day 7. RESULTS: In 13 patients with SAH, arteriographic cerebral vasospasm developed. Cerebrospinal fluid ADMA levels in patients with SAH were higher than in those in the control group (p < 0.001). The CSF ADMA level remained unchanged in the five patients with SAH without vasospasm, but was significantly increased in patients with vasospasm after Day 3 (6.2 +/- 1.7 microM) peaking during Days 7 through 9 (13.3 +/- 6.7 microM; p < 0.001) and then gradually decreasing between Days 12 and 21 (8.8 +/- 3.2 microM; p < 0.05). Nitrite levels in the CSF were lower in patients with vasospasm compared to patients without vasospasm (p < 0.03). Cerebrospinal fluid ADMA levels positively correlated with the degree of vasospasm (correlation coefficient [CC] = 0.88, p = 0.0001; 95% confidence interval [CI] 0.74-0.95) and negatively correlated with CSF nitrite levels (CC = -0.55; p = 0.017; 95% CI -0.81 to -0.12). CONCLUSIONS: These results support the hypothesis that ADMA is involved in the progression of cerebral vasospasm. Asymmetric dimethyl-L-arginine and its metabolizing enzymes may be a future target for treatment of cerebral vasospasm after SAH.

The biphasic nature of nitric oxide responses in tumor biology.

The dual or biphasic responses of cancer to nitric oxide (NO) arise from its concentration dependent ability to regulate tumor growth, migration, invasion, survival, angiogenesis, and metastasis. The outcome of these various NO-dependent processes is dictated by several factors including NO flux, the chemical redox environment, and the duration of NO exposure. Further, it was recently discovered that an NO-induced redox flux in vascular endothelial cells hypersensitizes these cells to the antiangiogenic effects of thrombospondin-1. This suggests a novel treatment paradigm for targeting tumor-driven angiogenesis that combines redox modulation with mimetic derivatives of thrombospondin-1. This article discusses the biphasic nature of NO in cancer biology and the implications of NO-driven redox flux for modulation of tumor-stimulated angiogenesis, growth, and metastasis.

Malignant glioma progression and nitric oxide.

Glioblastoma multiforme, the most common of the malignant gliomas, carries a dismal prognosis in spite of the most aggressive therapy and recent advances in molecular pathways of glioma progression. Although it has received relatively little attention in the setting of malignant gliomas, nitric oxide metabolism may be intimately associated with the disease process. Interestingly, nitric oxide has both physiological roles (e.g., neurotransmitter-like activity, stimulation of cyclic GMP), and pathophysiological roles (e.g., neoplastic transformation, tumor neovascularization, induction of apoptosis, free radical damage). Moreover, whether nitric oxide is neuroprotective or neurotoxic in a given disease state, or whether it enhances or diminishes chemotherapeutic efficacy in malignant neoplasia, is unresolved. This review discusses the multifaceted activity of nitric oxide with particular reference to malignant gliomas.

Neurotoxicity of nitroxyl: insights into HNO and NO biochemical imbalance.

Nitroxyl anion (NO-), and/or its conjugate acid, HNO, may be formed in the cellular milieu by several routes under both physiological and pathophysiological conditions. Since experimental evidence suggests that certain reactive nitrogen oxide species can contribute significantly to cerebral ischemic injury, we investigated the neurotoxic potential of HNO/NO- using Angeli's salt (AS), a spontaneous HNO/NO(-)-generating compound. Exposure to AS resulted in a time- and concentration-dependent increase in neural cell death that progressed markedly following the initial exposure. Coadministration of the donor with Tempol (1 mM), a one-electron oxidant that converts NO- to NO, prevented its toxic effect, as did the concomitant addition of Fe(III)TPPS. Media containing various chelators, catalase, Cu/Zn superoxide dismutase, or carboxy-PTIO did not ameliorate AS-mediated neurotoxicity, ruling out the involvement of transition metal complexes, H2O2, O2-, and NO, respectively. A concentration-dependent increase in supernatant protein 3-nitrotyrosine immunoreactivity was observed when cultures were exposed to AS under aerobic conditions, an effect lost in the absence of oxygen. A bell-shaped curve for augmented AS-mediated nitration was observed with increasing Fe(III)TPPS concentration, which contrasted with its linear effect on abating cytotoxicity. Finally, addition of glutamate receptor antagonists, MK-801 (10 microM) and CNQX (30 microM) to the cultures abrogated toxicity when given during, but not following, AS exposure; as did pretreatment with the exocytosis inhibitor, tetanus toxin (300 ng/ml). Taken together, our data suggest that under aerobic conditions, AS toxicity is initiated via HNO/NO- but progresses via secondary excitotoxicity.

Nitric oxide is a signaling molecule that regulates gene expression.

Nitric oxide (NO) is a dynamic and bioreactive molecule that can both participate in and inhibit the genesis of disease. Its ability to have an impact on a wide range of physiological events stems from its capacity to reversibly alter the expression of specific genes and the activities of a wide range of proteins and signaling pathways. Yet, NO* remains an enigmatic molecule. Recently developed technologies, including gene-chips, two-dimensional electrophoresis, RNA interference, matrix-assisted laser desorption ionization (MALDI)-TOF (time-of-flight) mass spectrometry, and protein arrays will allow us to better understand how NO* and associated reactive nitrogen species (RNS) regulate both physiology and disease states, toward the development of treatments using NO* synthase inhibitors or NO* donors.

In vitro and in vivo effects of probucol on hydrolysis of asymmetric dimethyl L-arginine and vasospasm in primates.

OBJECT: Increased cerebrospinal fluid (CSF) levels of asymmetric dimethyl L-arginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase (eNOS), are associated with delayed vasospasm after subarachnoid hemorrhage (SAH); however, the source, cellular mechanisms, and pharmacological inhibition of ADMA production following SAH are unknown. METHODS: In an in vitro experiment involving human umbilical vein endothelial cells (HUVECs), the authors examined mechanisms potentially responsible for increased ADMA levels during vasospasm and investigated whether this increase can be inhibited pharmacologically. In a second study, an in vivo experiment, the authors used probucol, which effectively inhibited ADMA increase in HUVEC cultures in vitro, in a randomized double-blind placebo-controlled experiment in a primate model of delayed cerebral vasospasm after SAH. Oxidized low-density lipids (OxLDLs; positive control; p < 0.02) and bilirubin oxidation products (BOXes; p < 0.01), but not oxyhemoglobin (p = 0.74), increased ADMA levels in HUVECs. Probucol inhibited changes in ADMA levels evoked by either OxLDLs (p < 0.001) or BOXes (p < 0.01). Comparable changes were observed in cell lysates. In vivo probucol (100 mg/kg by mouth daily) did not alter serum ADMA levels on Days 7, 14, and 21 after SAH compared with levels before SAH, and these levels were not different from those observed in the placebo group (p = 0.3). Despite achieving therapeutic levels in plasma and measurable levels in CSF, probucol neither prevented increased CSF ADMA levels nor the development of vasospasm after SAH. Increased CSF ADMA and decreased nitrite levels in both groups were strongly associated with the degree of delayed vasospasm after SAH (correlation coefficient [CC] 0.5, 95% confidence interval [CI] 0.19-0.72, p < 0.002 and CC -0.43, 95% CI -0.7 to -0.05, p < 0.03, respectively). CONCLUSIONS: Bilirubin oxidation products, but not oxyhemoglobin, increased ADMA levels in the HUVEC. Despite its in vitro ability to lower ADMA levels, probucol failed to inhibit increased CSF ADMA and decreased nitrite levels, and it did not prevent delayed vasospasm in a primate SAH model.

The chemical dynamics of NO and reactive nitrogen oxides: a practical guide.

Nitric oxide has emerged as one of the most important and diverse players in physiology. This small diatomic radical stunned researchers because of its existence and unique biological properties in human physiology. Over the last two decades it was found that NO often has fickle behavior in pathophysiological mechanisms. Where benefiting the host in one case yet inducing and augmenting injury in another. This has lead to confusion in is NO good or bad? Much of the answers to this dichotomy lies in the chemistry of NO and its related nitrogen oxide species. To help understand the complex chemistry with perspective to biology, a discussion on the chemical biology of NO is useful. The chemical biology defines the relevant chemical reaction of NO and nitrogen monoxide in the context of the biological conditions. We discuss in this article the chemistry of nitrogen oxide with different types of biological motifs. Reaction of NO with metal complexes and radicals require low concentration, where formation of reactive nitrogen oxide species require considerably higher amounts and generally are isolated to specific microenvironments in vivo. Though many reactive nitrogen oxide species are formed from chemical reactions with NO, there are several which appear to not require NO to be present, HNO and NO(2). These two species have unique physiological effects and represent additional complexity to this biological picture. From this discussion, a picture can be formed concerning the possible chemical dynamics, which can be plausible in different biological mechanisms.

A chemical perspective on the interplay between NO, reactive oxygen species, and reactive nitrogen oxide species.

Nitric oxide (nitrogen monoxide, NO) plays a veritable cornucopia of regulatory roles in normal physiology. In contrast, NO has also been implicated in the etiology and sequela of numerous neurodegenerative diseases that involve reactive oxygen species (ROS) and nitrogen oxide species (RNOS). In this setting, NO is often viewed solely as pathogenic; however, the chemistry of NO can also be a significant factor in lessening injury mediated by both ROS and RNOS. The relationship between NO and oxidation, nitrosation, and nitration reactions is summarized. The salient factors that determine whether NO promotes, abates, or interconnects these chemistries are emphasized. From this perspective of NO chemistry, the type, magnitude, location, and duration of either ROS or RNOS reactions may be predicted.

Association between cerebrospinal fluid levels of asymmetric dimethyl-L-arginine, an endogenous inhibitor of endothelial nitric oxide synthase, and cerebral vasospasm in a primate model of subarachnoid hemorrhage.

OBJECT: Decreased availability of nitric oxide (NO) has been proposed to evoke delayed cerebral vasospasm after sub-arachnoid hemorrhage (SAH). Asymmetric dimethyl-L-arginine (ADMA) inhibits endothelial NO synthase (eNOS) and, therefore, may be responsible for decreased NO availability in cases of cerebral vasospasm. The goal of this study was to determine whether ADMA levels are associated with cerebral vasospasm in a primate model of SAH. METHODS: Twenty-two cynomolgus monkeys (six control animals and 16 with SAH) were used in this study. The levels of ADMA, L-arginine, L-citrulline, nitrites, and nitrates in cerebrospinal fluid (CSF) and serum were determined on Days 0, 7, 14, and 21 following onset of SAH. Cerebral arteriography was performed to assess the degree of vasospasm. Western blot analyses of the right and left middle cerebral arteries (MCAs) were performed to assess the expression of eNOS, type I protein-arginine methyl transferase (PRMT1) and dimethylarginine dimethylaminohydrolase (DDAH2). Cerebrospinal fluid levels of ADMA remained unchanged in the control group (six animals) and in animals with SAH that did not have vasospasm (five animals; p = 0.17), but the levels increased in animals with vasospasm (11 animals) on Day 7 post-SAH (p < 0.01) and decreased on Days 14 through 21 (p < 0.05). Cerebrospinal fluid levels of ADMA correlated directly with the degree of vasospasm (correlation coefficient = 0.7, p = 0.0001; 95% confidence interval: 0.43-0.83). Levels of nitrite and nitrate as well as those of L-citrulline in CSF were decreased in animals with vasospasm. Furthermore, DDAH2 expression was attenuated in the right spastic MCA on Day 7 post-SAH, whereas eNOS and PRMT1 expression remained unchanged. CONCLUSIONS: Changes in the CSF levels of ADMA are associated with the development and resolution of vasospasm found on arteriograms after SAH. The results indicate that endogenous inhibition of eNOS by ADMA may be involved in the development of delayed cerebral vasospasm. Inhibition of ADMA production may provide a new therapeutic approach for cerebral vasospasm after SAH.

Comparison of the reactivity of nitric oxide and nitroxyl with heme proteins. A chemical discussion of the differential biological effects of these redox related products of NOS.

Investigations on the biological effects of nitric oxide (NO) derived from nitric oxide synthase (NOS) have led to an explosion in biomedical research over the last decade. The chemistry of this diatomic radical is key to its biological effects. Recently, nitroxyl (HNO/NO(-)) has been proposed to be another important constituent of NO biology. However, these redox siblings often exhibit orthogonal behavior in physiological and cellular responses. We therefore explored the chemistry of NO and HNO with heme proteins in different redox states and observed that HNO favors reaction with ferric heme while NO favors ferrous, consistent with previous reports. Further results show that HNO and NO were equally effective in inhibiting cytochrome P450 activity, which involves ferric and ferrous complexes. The differential chemical behavior of NO and HNO toward heme proteins provides insight into mechanisms of activity that not only helps explain some of the opposing effects observed in NOS-mediated events, but offers a unique control mechanism for the biological action of NO.