In vitro Kinase-to-Phosphosite database (iKiP-DB) predicts kinase activity in phosphoproteomic datasets

Phosphoproteomics routinely quantifies changes in the levels of thousands of phosphorylation sites, but functional analysis of such data remains a major challenge. While databases like PhosphoSitePlus contain information about many phosphorylation sites, the vast majority of known sites are not assigned to any protein kinase. Assigning changes in the phosphoproteome to the activity of individual kinases therefore remains a key challenge.. A recent large-scale study systematically identified in vitro substrates for most human protein kinases. Here, we reprocessed and filtered these data to generate an in vitro Kinase-to-Phosphosite database (iKiP-DB). We show that iKiP-DB can accurately predict changes in kinase activity in published phosphoproteomic datasets for both well-studied and poorly characterized kinases. We apply iKiP-DB to a newly generated phosphoproteomic analysis of SARS-CoV-2 infected human lung epithelial cells and provide evidence for coronavirus-induced changes in host cell kinase activity. In summary, we show that iKiP-DB is widely applicable to facilitate the functional analysis of phosphoproteomic datasets.

Evaluation of SARS-CoV-2 entry, inflammation and new therapeutics in human lung tissue cells

The development of physiological models that reproduce SARS-CoV-2 infection in primary human cells will be instrumental to identify host-pathogen interactions and potential therapeutics. Here, using cell suspensions directly from primary human lung tissues (HLT), we have developed a rapid platform for the identification of viral targets and the expression of viral entry factors, as well as for the screening of viral entry inhibitors and anti-inflammatory compounds. The direct use of HLT cells, without long-term cell culture and in vitro differentiation approaches, preserves main immune and structural cell populations, including the most susceptible cell targets for SARS-CoV-2; alveolar type II (AT-II) cells, while maintaining the expression of proteins involved in viral infection, such as ACE2, TMPRSS2, CD147 and AXL. Further, antiviral testing of 39 drug candidates reveals a highly reproducible method, suitable for different SARS-CoV-2 variants, and provides the identification of new compounds missed by conventional systems, such as VeroE6. Using this method, we also show that interferons do not modulate ACE2 expression, and that stimulation of local inflammatory responses can be modulated by different compounds with antiviral activity. Overall, we present a relevant and rapid method for the study of SARS-CoV-2.

A validated hypoxia-inducible factor (HIF) signature across tissue compartments predicts clinical outcome in human lung fibrosis

We previously reported that oxidative stress drives pseudohypoxic hypoxia-inducible factor (HIF) pathway activation to promote pathogenetic collagen structure-function in human lung fibrosis (Brereton et al., 2022). Here, through bioinformatic studies we investigate HIF pathway activation status in patients with idiopathic pulmonary fibrosis (IPF) and whether this has prognostic significance. Applying a well-established HIF gene expression signature, we classified publicly available datasets into HIF score-high and score-low groups across multiple tissue compartments. TheHIF scores in lung tissue, bronchoalveolar lavage (BAL) and peripheral blood mononuclear cells (PBMC) were increased in IPF patients and significantly correlated with an oxidative stress signature consistent with pseudohypoxic HIF pathway activation. A high HIF score in BAL and in PBMC was a strong independent predictor of mortality in multivariate analysis. Thus, a validated HIF gene signature predicts survival across tissue compartments in IPF and merits prospective study as a non-invasive biomarker of lung fibrosis progression.

Developmental origins of cell heterogeneity in the human lung

The lung contains numerous specialized cell-types with distinct roles in tissue function and integrity. To clarify the origins and mechanisms generating cell heterogeneity, we created a first comprehensive topographic atlas of early human lung development. We report 83 cell states, several spatially-resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated scRNA-Seq and spatial transcriptomics into a web-based, open platform for interactive exploration. To illustrate the utility of our approach we show distinct states of secretory and neuroendocrine cells, largely overlapping with the programs activated either during lung fibrosis or small cell lung cancer progression. We define the origin of uncharacterized airway fibroblasts associated with airway smooth muscle in bronchovascular bundles, and describe a trajectory of Schwann cell progenitors to intrinsic parasympathetic neurons controlling bronchoconstriction. Our atlas provides a rich resource for further research and a reference for defining deviations from homeostatic and repair mechanisms leading to pulmonary diseases.

A human fetal lung cell atlas uncovers proximal-distal gradients of differentiation and key regulators of epithelial fates

We present a multiomic cell atlas of human lung development that combines single cell RNA and ATAC sequencing, high throughput spatial transcriptomics and single cell imaging. Coupling single cell methods with spatial analysis has allowed a comprehensive cellular survey of the epithelial, mesenchymal, endothelial and erythrocyte/leukocyte compartments from 5-22 post conception weeks. We identify new cell states in all compartments. These include developmental-specific secretory progenitors that resemble cells in adult fibrotic lungs and a new subtype of neuroendocrine cell related to human small cell lung cancer; observations which strengthen the connections between development and disease/regeneration. Our datasets are available for the community to download and interact with through our web interface (https://fetal-lung.cellgeni.sanger.ac.uk). Finally, to illustrate its general utility, we use our cell atlas to generate predictions about cell-cell signalling and transcription factor hierarchies which we test using organoid models.

The Carcinogenic Properties of Overlooked yet Prevalent Polycyclic Aromatic Hydrocarbons in Human Lung Epithelial Cells

The WHO classified air pollution as a human lung carcinogen and polycyclic aromatic hydrocarbons (PAHs) are components of both indoor (e.g., tobacco smoke and cookstoves) and outdoor (e.g., wildfires and industrial and vehicle emissions) air pollution, thus a human health concern. However, few studies have evaluated the adverse effects of low molecular weight (LMW) PAHs, the most abundant PAHs in the environment. We hypothesized that LMW PAHs combined with the carcinogenic PAH benzo[a]pyrene (B[a]P) act as co-carcinogens in human lung epithelial cell lines (BEAS-2B and A549). Therefore, in this paper, we evaluate several endpoints, such as micronuclei, gap junctional intercellular communication (GJIC) activity, cell cycle analysis, anti-BPDE-DNA adduct formation, and cytotoxicity after mixed exposures of LMW PAHs with B[a]P. The individual PAH doses used for each endpoint did not elicit cytotoxicity nor cell death and were relevant to human exposures. The addition of a binary mixture of LMW PAHs (fluoranthene and 1-methylanthracene) to B[a]P treated cells resulted in significant increases in micronuclei formation, dysregulation of GJIC, and changes in cell cycle as compared to cells treated with either B[a]P or the binary mixture alone. In addition, anti-BPDE-DNA adducts were significantly increased in human lung cells treated with B[a]P combined with the binary mixture of LMW PAHs as compared to cells treated with B[a]P alone, further supporting the increased co-carcinogenic potential by LMW PAHs. Collectively, these novel studies using LMW PAHs provide evidence of adverse pulmonary effects that should warrant further investigation.

Serum deprivation enhanced ethanol-induced toxic responses in A549, lung carcinoma cells

Aim: The current study explores the toxic consequences of ethanol on human lung carcinoma cell, A549 in serum-deprived condition. Methodology: Human lung carcinoma cells, A549, were cultured in complete and serum-deprived medium for 6 hr. Subsequently, they were exposed to 50 mM and 100 mM concentrations of ethanol. Cytotoxicity studies linked with cell viability, oxidative stress, cell cycle arrest and micronuclei formation were performed using various toxicological parameters, namely MTT assay, DCFDA based ROS generation, cell cycle analysis and micronuclei formation assay. The cytotoxicity of ethanol in complete and serum deprived medium were compared at similar doses and time duration. Results: The metabolic viability assay demonstrated that 50 mM and 100 mM concentration of ethanol did not induce significant levels of cytotoxic alteration in A549 lung carcinoma cells in complete medium. However, in serum-deprived conditions, 50 mM and 100 mM ethanol concentration significantly altered cell viability. Further, exposure of 50 mM and 100 mM concentration of ethanol enhanced reactive oxygen species levels in A549 cells more significantly in serum-deprived conditions than in complete medium. In addition to cytotoxicity and oxidative stress, 50 mM and 100 mM ethanol also arrested the cells at G0 phase more significantly in serum deprived conditions compared to complete medium. Interpretation: Both 50 mM and 100 mM ethanol concentration enhanced the cell cytotoxicity and reactive oxygen species, cell cycle arrest and micronuclei formation more severely in serum-deprived medium than in complete medium (containing 10% FBS) under similar treatment conditions.

BAP1 Downregulates NRF2 Target Genes and Exerts Anti-Tumorigenic Effects by Deubiquitinating KEAP1 in Lung Adenocarcinoma

KELCH-ECH-associated protein 1 (KEAP1) is an adaptor protein of Cullin 3 (CUL3) E3 ubiquitin ligase that targets a redox sensitive transcription factor, NF-E2-related factor 2 (NRF2). BRCA1-associated protein 1 (BAP1) is a tumor suppressor and deubiquitinase whose mutations increase the risk of several types of familial cancers. In the present study, we have identified that BAP1 deubiquitinates KEAP1 by binding to the BTB domain. Lentiviral transduction of BAP1 decreased the expression of NRF2 target genes, suppressed the migration and invasion, and sensitized cisplatin-induced apoptosis in human lung adenocarcinoma (LUAD) A549 cells. Examination of the lung tissues in KrasG12D/+ mice demonstrated that the level of Bap1 and Keap1 mRNAs progressively decreases during lung tumor progression, and it is correlated with NRF2 activation and the inhibition of oxidative stress. Supporting this observation, lentiviral transduction of BAP1 decreased the growth of A549 xenografts in athymic nude mice. Transcriptome analysis of human lung tissues showed that the levels of Bap1 mRNA are significantly higher in normal samples than LUAD samples. Moreover, the expression of Bap1 mRNA is associated with a better survival of LUAD patients. Together, our study demonstrates that KEAP1 deubiquitination by BAP1 is novel tumor suppressive mechanism of LUAD.