Astegolimab, an anti-ST2, in chronic obstructive pulmonary disease (COPD-ST2OP): a phase 2a, placebo-controlled trial.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a heterogeneous inflammatory airway disease. The epithelial-derived IL-33 and its receptor ST2 have been implicated in airway inflammation and infection. We aimed to determine whether astegolimab, a selective ST2 IgG2 monoclonal antibody, reduces exacerbations in COPD. METHODS: COPD-ST2OP was a single-centre, randomised, double-blinded, placebo-controlled phase 2a trial in moderate-to-very severe COPD. Participants were randomly assigned (1:1) with a web-based system to received 490 mg subcutaneous astegolimab or subcutaneous placebo, every 4 weeks for 44 weeks. The primary endpoint was exacerbation rate assessed for 48 weeks assessed with a negative binomial count model in the intention-to-treat population, with prespecified subgroup analysis by baseline blood eosinophil count. The model was the number of exacerbations over the 48-week treatment period, with treatment group as a covariate. Safety was assessed in the whole study population until week 60. Secondary endpoints included Saint George's Respiratory Questionnaire for COPD (SGRQ-C), FEV1, and blood and sputum cell counts. The trial was registered with ClinicalTrials.gov, NCT03615040. FINDINGS: The exacerbation rate at 48 weeks in the intention-to-treat analysis was not significantly different between the astegolimab group (2·18 [95% CI 1·59 to 2·78]) and the placebo group (2·81 [2·05 to 3·58]; rate ratio 0·78 [95% CI 0·53 to 1·14]; p=0·19]). In the prespecified analysis stratifying patients by blood eosinophil count, patients with 170 or fewer cells per µL had 0·69 exacerbations (0·39 to 1·21), whereas those with more than 170 cells per µL had 0·83 exacerbations (0·49 to 1·40). For the secondary outcomes, the mean difference between the SGRQ-C in the astegolimab group versus placebo group was -3·3 (95% CI -6·4 to -0·2; p=0·039), and mean difference in FEV1 between the two groups was 40 mL (-10 to 90; p=0·094). The difference in geometric mean ratios between the two groups for blood eosinophil counts was 0·59 (95% CI 0·51 to 0·69; p<0·001) and 0·25 (0·19 to 0·33; p<0·001) for sputum eosinophil counts. Incidence of treatment-emergent adverse events was similar between groups. INTERPRETATION: In patients with moderate-to-very severe COPD, astegolimab did not significantly reduce exacerbation rate, but did improve health status compared with placebo. FUNDING: Funded by Genentech and National Institute for Health Research Biomedical Research Centres.

Effects of indoor particulate matter exposure on daily asthma control.

BACKGROUND: Household dust often contains aeroallergens, such as the house dust mite antigen Der p 1. It has been proposed that overnight exposure to particulate matter from bedding and other sources may be an important driver of atopic asthma. Whether variability in overnight particulate matter exposure is a significant determinant of asthma control is unknown. OBJECTIVE: To test the hypothesis that overnight particulate matter exposure is associated with day-to-day symptoms, lung function, and airway inflammation in patients with asthma who are sensitized to house dust mite. METHODS: We undertook a prospective, single-center panel study in 28 adults with asthma and house dust mite sensitization. Overnight exposure to particulate matter was measured using a commercially available indoor air quality monitor. Symptom scores, peak expiratory flow, and exhaled nitric oxide were measured and electronically recorded daily. Participants were followed up for 12 weeks and attended study visits every 4 weeks, at which they underwent spirometry and completed the Asthma Control Questionnaire and Asthma Quality of Life Questionnaire. Data were analyzed using cross-correlation and linear mixed-effects models. RESULTS: No significant associations were observed between overnight particulate matter exposure and clinical outcomes measured daily or at study visits. CONCLUSION: Natural variability in overnight particulate matter exposure does not appear to be a major determinant of daily asthma control in patients with asthma and house dust mite sensitization.

Loss of catalase increases malignant mouse keratinocyte cell growth through activation of the stress activated JNK pathway.

A cell line that produces mouse squamous cell carcinoma (6M90) was modified to develop a cell line with an introduced Tet-responsive catalase transgene (MTOC2). We have previously reported that the overexpressed catalase in the MTOC2 cells reverses the malignant phenotype in part by decreasing epidermal growth factor receptor (EGFR) signaling. With this work we expanded the investigation into the differences between these two cell lines. We found that the decreased EGFR pathway activity of the MTOC2 cells is not because of reduced autocrine secretion of an epidermal growth factor (EGF) ligand but rather because of lower basal receptor activity. Phosphorylated levels of the mitogen-activated protein kinase (MAPK) members JNK and p38 were both higher in the 6M90 cells with low catalase when compared with the MTOC2 cell line. Although treatment with an EGFR inhibitor, AG1478, blocked the increased activity of JNK in the 6M90 cells, a similar effect was not observed for p38. Basal levels of downstream c-jun transcription were also found to be higher in the 6M90 cells versus MTOC2 cells. Activated p38 was found to down-regulate the JNK MAPK pathway in the 6M90 cells. However, the 6M90 cells contain constitutively high levels of phosphorylated JNK, generating higher levels of phosphorylated c-jun and total c-jun than those in the MTOC2 cells. Inhibition of JNK activity in the 6M90 cells reduced AP-1 transcription and cell proliferation. The data confirm the inhibitory effects of catalase on tumor cell growth, specifically through a ligand-independent decrease in the stress activated JNK pathway.

Inhibition of UVA-induced c-Jun N-terminal kinase activity results in caspase-dependent apoptosis in human keratinocytes.

Inhibition of c-Jun N-terminal kinase (JNK) with the pharmacologic inhibitor SP600125 in UVA-irradiated HaCaT cells and human primary keratinocytes resulted in dramatic phenotypic changes indicative of cell death. These phenotypic changes correlated with caspase 8, 9 and 3 activations as well as cleavage of the caspase substrate polyADP-ribose polymerase (PARP). Morphologic analysis and analysis of sub-G0 DNA content confirmed apoptotic cell death in these keratinocytes after combination treatment. Addition of the general caspase inhibitor zVAD-fmk to combination-treated HaCaT cells was able to completely block caspase activation, PARP cleavage, the increase in sub-G0 DNA content and the classic morphologic features of apoptosis, indicating that this combination treatment resulted in caspase-dependent apoptotic cell death. zVAD-fmk treatment of primary keratinocytes was able to completely inhibit caspase activation and PARP cleavage, reduce morphologic apoptosis at lower concentrations of SP600125 and decrease the sub-G(0) DNA content detected after UVA + SP600125 treatment. However, cell death and a significant amount of debris was still detected after caspase inhibitor treatment, particularly with 125 nM SP600125. At subconfluent conditions and low passage, primary keratinocytes were more sensitive to UVA irradiation alone than HaCaT cells. In conclusion, we have observed that inhibition of UVA-induced JNK activity with the pharmacologic inhibitor SP600125 resulted in caspase-dependent apoptotic cell death in both the immortalized keratinocyte cell line HaCaT and primary keratinocytes. However, the increased sensitivity of primary keratinocytes to experimental stress may have also resulted in direct cellular injury and caspase-independent cell death.

Catalase reverses tumorigenicity in a malignant cell line by an epidermal growth factor receptor pathway.

We have used a keratinocyte in vivo/in vitro cell model to test the hypothesis that hydrogen peroxide acts as a signaling molecule, contributing to proliferation and tumorigenesis. A cell line, 6M90, that produces squamous cell carcinoma (SCC), has high levels of ROS and low levels of catalase. A new cell line, MTOC2, generated from parental 6M90 cells by introduction of a Tet-responsive catalase transgene, effectively expressed higher peroxisomal catalase. Increased catalase expression diminished constitutive ROS and enhanced viability after treatment with hydrogen peroxide. Protein tyrosine phosphatase activity was higher in the MTOC2 cells with high catalase, consistent with detection of a lower level of phosphorylation at tyrosine 1068 of the epidermal growth factor receptor (EGF-R). Transcription of downstream c-fos, AP-1 transactivation and cell proliferation were higher in the low catalase cells. An EGF-R inhibitor, AG1478, blocks the higher AP-1 transactivation and cell proliferation of the low catalase 6M90 cells. Tumorigenesis in SCID mice was greatly diminished in the high catalase cells. Our data suggest that hydrogen peroxide functions as a signaling molecule that can modulate activity of a protein tyrosine phosphatase/(s) resulting in phosphorylation of tryrosine/(s) on the EGF-R. Therefore, catalase acts as a tumor-suppressor gene in part by decreasing EGF-R signaling.

The role of Rac1 in maintaining malignant phenotype of mouse skin tumor cells.

We have previously developed an in vitro tumor progression model with mouse skin keratinocytes to study the molecular targets that mediate the tumor cell's progression from a benign to a malignant phenotype. The malignantly transformed cells were found to have elevated MAP kinase signaling and increases in AP-1, NFkappaB and cAMP response element (CRE) transcription factors activities compared to their benign counter-part. In this study, we showed that Rac1, a member of the Rho superfamily of small GTPases, functions as a key signaling molecule that mediates these malignant phenotypes. We used a doxycycline inducible system to express dominant negative Rac1 (N17 Rac1) in the squamous cell carcinomas producing 6M90 cell line. Conditional expression of the N17 Rac1 was able to decrease multiple markers of malignancy including: growth rate, colony formation, migration, invasion and most importantly, in vivo tumor growth. In addition, these phenotypic changes were accompanied by decreases in mitogenic signals, which include ERK1/2, JNK, and PI-3 kinase/Akt activation. Transactivation mediated by AP-1, NFkappaB, and CRE were also attenuated by expression of dominant negative Rac1. These observations led us to conclude that Rac1 signaling is required for the malignant phenotypes of the squamous cell carcinoma cells.

Transcriptional repression of catalase in mouse skin tumor progression.

Previous studies in our laboratory have shown that the elevation of reactive oxygen species levels and the repression of the antioxidant enzyme, catalase, played a critical role in the in vitro progression of benign papilloma cells to malignant carcinoma cells. Catalase message, protein levels, and activity levels were found to be downregulated in the malignantly progressed cells. The goal of this study is to further characterize the repression of catalase in malignant progression of mouse skin tumors. To validate the in vitro observations, we examined catalase expression in tumor samples generated by the multistep chemical carcinogenesis protocol. Higher levels of catalase mRNA and protein were observed in benign papillomas versus malignant carcinomas. Nuclear run-on analysis showed that catalase repression in the cultured malignant cells was transcription-dependent. Results from luciferase reporter assays indicated that malignant cells have lower catalase promoter activities than benign papilloma cells, in part through the Wilm's tumor suppressor 1 (WT1) binding site within the proximal promoter region. The WT1 protein levels were found to be inversely correlated with the observed catalase promoter activities, with higher levels observed in the malignant cells versus the benign cells. These results led us to conclude that WT1 is acting as a transcription repressor in catalase gene regulation during tumor progression.