Membrane Protein-focused CRISPR Screen Identifies ATP2A2 as a Druggable Transcriptional Co-regulator of CCND1 (Cyclin D1) in Lung Adenocarcinoma

Lung adenocarcinoma (LUAD) is one of the most commonly seen non-small cell lung cancer (NSCLC). Recent progress has highlighted cell cycle-oriented medications such as cyclin-dependent kinase (CDK) inhibitors for treating diverse cancers including NSCLC. However, in clinical practice limitations associated with CDK inhibitors have been reported such as acquired resistance. This prompted the exploration of druggable regulatory or compensatory pathways in cell cycle as alternative or combination therapies of CDK inhibitors. Here, we conducted a membrane protein-focused CRISPR screen in A549 cells to discover upstream regulatory proteins in LUAD. An endoplasmic reticulum (ER)-localized protein ATP2A2 was identified as a top hit and was subsequently verified to be critical for LUAD cell proliferation, migration and invasion. Transcriptomic analyses illustrated that ATP2A2 affected LUAD by involving in cell cycle regulation. Mechanistic studies revealed that ATP2A2 interacted with another ER protein HACD3 and co-regulated the transcription of CCND1 gene (cyclin D1), which forms complex with CDK4/6. It was found that ATP2A2 and HACD3 promoted CCND1 expression by activating NF-kB signaling and nuclear translocation of p65 protein, which is known to be the transcription factor of CCND1. Most importantly, curcumin, an ATP2A2-targeting natural product, was found to inhibit LUAD both in vitro and in xenografted mouse model. Collectively, our results revealed ATP2A2-HACD3-NF-kB as a regulatory axis of cyclin D1 expression and shed light on developing ATP2A2-targeted medications as cell cycle-oriented treatment for LUAD.