Multiplex organoid mutagenesis reveals gene combinations driving transformation, pathology, and chemosensitivity in ovarian carcinoma.

High-grade serous ovarian carcinoma (HGSC) is the sixth leading cause of cancer-related death among women. Most tumors arise from the Fallopian tubal epithelium (TE), exhibit numerous mutations, and present heterogenous pathological features. However, the contribution of specific mutation combinations to cellular transformation, pathological phenotype, and chemotherapeutic responses remains unclear. We used a mouse TE organoid platform for combinatorial CRISPR mutagenesis of 20 candidate HGSC driver genes. Besides Trp53, mutations in Nf1, Cdkn2a, and Map2k4 were the most prevalent in phenotypically transformed organoids. Map2k4 mutant organoids transplanted into mice predominantly yielded papillary-glandular carcinomas, whereas those with Nf1 mutations were more mesenchymal-like. Map2k4 mutant cells were particularly sensitive to paclitaxel, and Rho kinase inhibitor (ROCKi) increased trametinib sensitivity in both Map2k4- and Nf1-mutant organoids. This organoid mutagenesis strategy is powerful for unraveling the genetic and phenotypic complexity of HGSC, and identified Map2k4 as a potential therapeutic target in select HGSC cases.