Project Summary Cervical cancer remains a significant global cause of death in women, and 40% of women with cervical cancer will relapse and die despite chemoradiation treatment. There is an urgent need to identify prognostic and predictive biomarkers for chemoradiation response. We have developed and optimized a novel, non-invasive swab-based biopsy approach to collect tissue before, during, and after chemoradiation without patient discomfort or risks of serial biopsies such as bleeding or fistula. Despite low tumor purity, we have also developed a custom computational pipeline to optimize mutation calling and identify clonally expanded mutations during chemoradiation. Developing a deep understanding of genomic alterations during and after CRT to these findings will help fast-track clinical translation of targeted therapies. To this end, we have used our pipeline in a pilot study of 70 patients to reconstruct the evolution of mutations during CRT and identify proliferatively advantageous driver mutations and pathways. This proposal aims to validate the candidate driver mutation lists in multiple dimensions and develop a patient-derived organoid platform to test potential targeted therapies. First, we will validate the preliminary identified clonally expanded genes and pathways in a larger population of already collected but yet-to-be-sequenced samples. Second, we will perform a CRISPR/Cas9 library screen of these preliminarily identified drivers in available cervical cancer cell lines. Simultaneously, we will develop a cervical cancer organoid biobank to perform single-cell RNA sequencing before and after CRT and validate these preliminarily identified genes. This platform will be used in the future to test targeted agents. When we complete the aims of this R21, we will have validated ideal targets, developed testing platforms, and set the stage for testing clinically impactful therapies in a future R01.