Wnt/?-catenin signaling plays an important role in controlling lung epithelial cell homeostasis and fate commitment and is dysregulated in COPD with reduced Wnt activity. As it controls alveolar epithelial cell self-renewal, we performed a whole genome CRISPR screen to identify negative regulators of Wnt/?-catenin signaling for potential regenerative therapies addressing emphysema. 152 hits were identified in A549 cells (p<0.1 vs. neutral control), which were then screened in small airway epithelial cells for Wnt pathway activation by ?-catenin nuclear translocation and ViewRNA for AXIN2 expression, yielding 34 candidates for further progression.

As Wnt signaling may drive myofibroblast activation and fibrosis, we performed a counter screen on these hits in normal human lung fibroblasts. Our control gene CSNK1A1 robustly induced ?-smooth muscle actin (?SMA) expression, indicating that the assay could be used to identify pro-fibrotic targets. Four of the targets investigated either caused cell toxicity or induced ?10% increased ?SMA expression above the untreated control. Interestingly, some targets attenuated the effects of transforming growth factor ? (TGF-?) induced ?SMA expression, suggesting anti-fibrotic effects and differential roles in mesenchymal and epithelial cell lineages.

We have validated the Wnt activation of one of our top hits using small molecule inhibitor and RNA interference. We are currently establishing a CRISPR-based alveolar epithelial cell organoid assay to investigate the potential of the top 19 targets to promote alveolar type II cell self-renewal, and we are performing a bioinformatics-based hit ranking of the targets.