Renal cell carcinoma (RCC) is characterized by dysregulated lipid metabolism and frequent resistance to targeted therapies. Mitophagy plays a critical role in cancer progression and drug resistance, but its precise regulatory mechanisms in RCC remain unclear.

We conducted an in vivo genome-wide CRISPR/Cas9 knockout screen to identify key regulators of RCC progression. Functional assays, including in vitro proliferation, migration, invasion, and 3D cultures, were performed, along with in vivo xenograft models. Mechanistic investigations involved immunoprecipitation, mass spectrometry, lipidomics, and bioinformatics analyses, complemented by AlphaFold structural modeling.

Genome-wide CRISPR screening identified PRKAB2 as a critical tumor suppressor in RCC. Low PRKAB2 expression correlated with poor prognosis and aggressive tumor features. Overexpression of PRKAB2 significantly inhibited RCC cell proliferation, migration, invasion, and tumor growth in vitro and in vivo. Mechanistically, PRKAB2 suppressed mitophagy through two distinct pathways: (1) PRKAB2 enhanced the binding between LRPPRC and Parkin, thereby reducing Parkin’s interaction with PINK1 and inhibiting ubiquitin-dependent mitophagy; (2) PRKAB2 downregulated cardiolipin synthesis, a key mitochondrial lipid required for mitophagy initiation. Restoring CLS1 expression, a key enzyme in cardiolipin biosynthesis, rescued PRKAB2-induced mitophagy suppression. Functionally, PRKAB2 overexpression significantly increased RCC sensitivity to tyrosine kinase inhibitors (TKIs), whereas Parkin overexpression counteracted this effect, promoting drug resistance. DepMap analysis further confirmed the association between mitophagy activation and TKI resistance.

PRKAB2 functions as a crucial tumor suppressor in RCC by regulating both protein-protein interactions and lipid metabolism to suppress mitophagy. Targeting PRKAB2-associated pathways may provide a promising therapeutic strategy to enhance treatment efficacy and overcome drug resistance in RCC.