Lung involvement in systemic sclerosis (SSc) is common, occurs more frequently in SSc than in other connective tissue diseases and is its leading cause of death, manifesting as interstitial lung disease (ILD) or pulmonary arterial hypertension (PAH). Only two Food and Drug Administration (FDA)-approved compounds (nintedanib and tocilizumab) have been shown to slow the decline of lung function in SSc-ILD, but do not stop or reverse the progression of SSc-ILD. This highlights an unmet medical need for improved molecular understanding of SSc- ILD pathogenesis for identification of novel targets for drug development. SSc-ILD is thought to be triggered by immune-mediated microvascular injuries that induce and perpetuate inflammation, autoimmune responses, and fibroblast-to-myofibroblast activation with subsequent collagen deposition ultimately leading to lung fibrosis. We have recently provided the largest known lung proteome in human and mouse, encompassing more than 8000 proteins, uncovering novel druggable mediators and cell types driving lung fibrosis. We have identified several shared mediators of fibrosis (e.g., the SMAD3/TGF-beta pathway) between SSc skin lesions and IPF lung tissue. An accurate quantification and characterization of the SSc lung proteome, however, remains to be performed. Using novel technological advances, we propose to define and quantify the intricate composition of the fibrotic SSc lung and uncover components within the lung proteome that will serve as peripheral biomarkers and/or drug candidates for the monitoring and treatment of SSc-ILD, respectively. The overarching goal of this application is 1) to define, in greatest detail, the molecular composition of the SSc-ILD lung and peripheral blood, 2) to identify novel disease-specific network modules and mechanisms of tissue fibrosis for improved drug development, and 3) to uncover proteins that can serve as diagnostic, prognostic, or predictive biomarkers for SSC-ILD. We hypothesize that the SSc lung proteome identifies SSc-specific druggable cues, produced by resident fibroblasts, that drive persistent lung fibrosis. We will define and quantify changes in the composition of the proteomes of the lung and blood of SSc-ILD patients that correlate with disease severity in cross-sectional and longitudinal cohorts of SSc-ILD patients. We will provide the SSc-ILD lung’s epigenetic landscape and transcriptome at single cell resolution by performing gene expression and open chromatin accessibility assays using Chromium single multiome ATAC + Genome Expression analysis in nuclear preparations of lung cell suspensions. Finally, we will use a novel drug screening platform of TGF-beta- induced Smad translocation in SSc-derived primary fibroblasts, and characterize inhibitors of the class III phosphatidylinositol-3-kinase (PI3K) vacuolar protein sorting 34 (Vps34) as novel targets for the inhibition of lung fibrosis. We will work closely with the other CORT cores and projects to gener...