Genotype-Informed Drosophila Kinome Screening Prioritizes PRKX as a Candidate Vulnerability in Bladder Cancer

Muscle-invasive bladder cancer is molecularly heterogeneous, and the basal/squamous subtype frequently exhibits elevated EGFR expression and TP53 and RB1 alterations. Functional vulnerabilities associated with these alterations remain incompletely defined, while large-scale in vivo screening in mammalian models is experimentally demanding. Therefore, we developed a genotype-defined Drosophila melanogaster model combining Egfr overexpression, mutant p53, and Rbf suppression in Malpighian tubules. The resulting 3-hit model showed increased Malpighian tubule volume and reduced adult eclosion rate. A dominant modifier screen targeting 216 kinase genes identified 72 genes whose perturbation significantly increased the eclosion rate, and a complementary RNA interference screen identified eight kinases whose knockdown increased the eclosion rate. Among these hits, Pka-C3, which is a Drosophila ortholog of human PRKX, emerged as one of the strongest suppressors of the 3-hit phenotype. PRKX was prioritized for further study because public transcriptomic datasets showed higher PRKX expression in bladder tumors than in normal bladder tissue. PRKX knockdown reduced spheroid volume in four human bladder cancer cell lines with distinct EGFR, TP53, and RB1 profiles. Both small interfering RNAs produced significant reductions in two cell lines, whereas only one siRNA reached significance in the other two. These findings nominate PRKX as a candidate vulnerability in bladder cancer and highlight a cross-species approach that combines genotype-informed Drosophila screening with functional testing in human cancer cells to prioritize targets for further investigation.