Electronic cigarettes (E-cigs) have experienced sharp increases in popularity over the past five years due to many factors, including aggressive marketing, increased restrictions on conventional cigarettes, and a perception that E-cigs are healthy alternatives to cigarettes. Despite this perception, studies on health effects in humans are extremely limited and in vivo animal models have not been generated. Presently, we determined that E-cig vapor contains 7x1011 free radicals per puff. To determine whether E-cig exposure impacts pulmonary responses in mice, we developed an inhalation chamber for E-cig exposure. Mice that were exposed to E-cig vapor contained serum cotinine concentrations that are comparable to human E-cig users. E-cig exposure for 2 weeks produced a significant increase in oxidative stress and moderate macrophage-mediated inflammation. Since, COPD patients are susceptible to bacterial and viral infections, we tested effects of E-cigs on immune response. Mice that were exposed to E-cig vapor showed significantly impaired pulmonary bacterial clearance, compared to air-exposed mice, following an intranasal infection with Streptococcus pneumonia. This defective bacterial clearance was partially due to reduced phagocytosis by alveolar macrophages from E-cig exposed mice. In response to Influenza A virus infection, E-cig exposed mice displayed increased lung viral titers and enhanced virus-induced illness and mortality. In summary, this study reports a murine model of E-cig exposure and demonstrates that E-cig exposure elicits impaired pulmonary anti-microbial defenses. Hence, E-cig exposure as an alternative to cigarette smoking must be rigorously tested in users for their effects on immune response and susceptibility to bacterial and viral infections.

Loss of function mutations in Kelch Like ECH Associated Protein 1 (KEAP1) or gain-of-function mutations in nuclear factor erythroid 2-related factor 2 (NRF2) are common in non-small cell lung cancer (NSCLC) and is associated with therapeutic resistance. To discover novel NRF2 inhibitors for targeted therapy, we conducted a quantitative high-throughput screen using a diverse set of ~400,000 small molecules (Molecular Libraries Small Molecule Repository Library, MLSMR) at the National Center for Advancing Translational Sciences. We identified ML385 as a probe molecule that binds to NRF2 and inhibits its downstream target gene expression. Specifically, ML385 binds to the Neh1, the Cap ‘N’ Collar Basic Leucine Zipper (CNC-bZIP) domain of NRF2, and interferes with the binding of the V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homolog G (MAFG)-NRF2 protein complex to regulatory DNA binding sequences. In clonogenic assays, when used in combination with platinum-based drugs such as doxorubicin or taxol, ML385 substantially enhances cytotoxicity in NSCLC cells compared to single agents alone. ML385 shows specificity and selectivity for NSCLC cells with KEAP1 mutation leading to gain of NRF2 function. In preclinical models of NSCLC with gain of NRF2 function, ML385 in combination with carboplatin showed significant anti-tumor activity. We demonstrate the discovery and validation of ML385 as a novel and specific NRF2 inhibitor and conclude that targeting NRF2 may represent a promising strategy for the treatment of advanced NSCLC.

BackgroundCOPD patients have high pulmonary and systemic oxidative stress that correlates with severity of disease. Sulforaphane has been shown to induce expression of antioxidant genes via activation of a transcription factor, nuclear factor erythroid-2 related factor 2 (Nrf2).MethodsThis parallel, placebo-controlled, phase 2, randomized trial was conducted at three US academic medical centers. Patients who met GOLD criteria for COPD and were able to tolerate bronchoscopies were randomly assigned (1:1:1) to receive placebo, 25 μmoles, or 150 μmoles sulforaphane daily by mouth for four weeks. The primary outcomes were changes in Nrf2 target gene expression (NQ01, HO1, AKR1C1 and AKR1C3) in alveolar macrophages and bronchial epithelial cells. Secondary outcomes included measures of oxidative stress and airway inflammation, and pulmonary function tests.ResultsBetween July 2011 and May 2013, 89 patients were enrolled and randomized. Sulforaphane was absorbed in the patients as evident from their plasma metabolite levels. Changes in Nrf2 target gene expression relative to baseline ranged from 0.79 to 1.45 and there was no consistent pattern among the three groups; the changes were not statistically significantly different from baseline. Changes in measures of inflammation and pulmonary function tests were not different among the groups. Sulforaphane was well tolerated at both dose levels.ConclusionSulforaphane administered for four weeks at doses of 25 μmoles and 150 μmoles to patients with COPD did not stimulate the expression of Nrf2 target genes or have an effect on levels of other anti-oxidants or markers of inflammation.Trial RegistrationClinicaltrials.gov: NCT01335971.

Purpose: Stabilization of the transcription factor NRF2 through genomic alterations in KEAP1 and NFE2L2 occurs in a quarter of patients with lung adenocarcinoma and a third of patients with lung squamous cell carcinoma. In lung adenocarcinoma, KEAP1 loss often co-occurs with STK11 loss and KRAS-activating alterations. Despite its prevalence, the impact of NRF2 activation on tumor progression and patient outcomes is not fully defined. Experimental Design: We model NRF2 activation, STK11 loss, and KRAS activation in vivo using novel genetically engineered mouse models. Furthermore, we derive a NRF2 activation signature from human non–small cell lung tumors that we use to dissect how these genomic events impact outcomes and immune contexture of participants in the OAK and IMpower131 immunotherapy trials. Results: Our in vivo data reveal roles for NRF2 activation in (i) promoting rapid-onset, multifocal intrabronchiolar carcinomas, leading to lethal pulmonary dysfunction, and (ii) decreasing elevated redox stress in KRAS-mutant, STK11-null tumors. In patients with nonsquamous tumors, the NRF2 signature is negatively prognostic independently of STK11 loss. Patients with lung squamous cell carcinoma with low NRF2 signature survive longer when receiving anti–PD-L1 treatment. Conclusions: Our in vivo modeling establishes NRF2 activation as a critical oncogenic driver, cooperating with STK11 loss and KRAS activation to promote aggressive lung adenocarcinoma. In patients, oncogenic events alter the tumor immune contexture, possibly having an impact on treatment responses. Importantly, patients with NRF2-activated nonsquamous or squamous tumors have poor prognosis and show limited response to anti–PD-L1 treatment.

Sussan TE, Gajghate S, Chatterjee S, Mandke P, McCormick S, Sudini K, Kumar S, Breysse PN, Diette GB, Sidhaye VK, Biswal S. Nrf2 reduces allergic asthma in mice through enhanced airway epithelial cytoprotective function.

Oncogenic Kras mutations are one of the most common alterations in non-small cell lung cancer and are associated with poor response to treatment and reduced survival. Driver oncogenes, such as Kras are now appreciated for their ability to promote tumor growth via up-regulation of anabolic pathways. Therefore, we wanted to identify metabolic vulnerabilities in Kras-mutant lung cancer. Using the Kras lung cancer model, we show that mutant Kras drives a lipogenic gene-expression program. Stable-isotope analysis reveals that mutant Kras promotes de novo fatty acid synthesis in vitro and in vivo. The importance of fatty acid synthesis in Kras-induced tumorigenesis was evident by decreased tumor formation in Kras mice after treatment with a fatty acid synthesis inhibitor. Importantly, with gain and loss of function models of mutant Kras, we demonstrate that mutant Kras potentiates the growth inhibitory effects of several fatty acid synthesis inhibitors. These studies highlight the potential to target mutant Kras tumors by taking advantage of the lipogenic phenotype induced by mutant Kras.-Singh, A., Ruiz, C., Bhalla, K., Haley, J. A., Li, Q. K., Acquaah-Mensah, G., Montal, E., Sudini, K. R., Skoulidis, F., Wistuba, I. I., Papadimitrakopoulou, V., Heymach, J. V., Boros, L. G., Gabrielson, E., Carretero, J., Wong, K.-k., Haley, J. D., Biswal, S., Girnun, G. D. De novo lipogenesis represents a therapeutic target in mutant Kras non-small cell lung cancer.

Background Broccoli sprouts (BS) are the richest source of sulforaphane (SFN), which is a potent inducer of Phase II enzymes, which play a critical role in preventing oxidative stress (OS) and inflammation. Objectives To determine if ingestion of whole BS improves airway inflammatory and physiologic outcomes, and OS in adults with asthma and allergic sensitization to an indoor allergen. Methods The study is a double-blind, placebo-controlled, randomized trial to compare the effects of BS to placebo (alfalfa sprouts (AS)) on airway inflammation and markers of OS. Forty adults (age 18–50 years) were randomized to eat either: (a) 100g of BS daily or (b) 100g of AS daily for three days. Fractional exhaled nitric oxide (FENO), FEV1, nasal epithelial and PBMC gene expression, inflammatory and OS biomarkers, and symptoms were assessed both before and after ingestion of the sprouts. The primary outcome variable was the change in FENO. Secondary outcome measures included rhinitis and asthma symptoms, lung function, OS and inflammatory biomarkers. Results BS ingestion for 3 consecutive days did not reduce FENO despite resulting in a marked increase in serum SFN concentrations (21 vs 22 ppb, p=0.76). Furthermore, BS consumption did not induce cytoprotective antioxidant genes in either PBMCs or nasal epithelial cells, reduce OS and inflammatory markers, or improve lung function. Conclusions Ingestion of whole BS for 3 days does not appear to improve eosinophilic pulmonary inflammation, inflammatory and OS biomarkers, or clinical features of asthma among atopic adults with asthma despite resulting in a marked increase in serum SFN levels.

The risk model developed may help radiologists suggest the possibility of TON and prioritize ophthalmology consults. However, future external validation of this prediction model is necessary.