During the first half of this project, we focused on aim 1: “Identification of genes that contribute to RT resistance” and their validation in vitro (aim 2.1). Moreover, we have started to work on all other aims (2.2 and 3.1-3.4). Regarding aim1, we successfully carried out functional genetic CRISPR/Cas9 screens under RT selection in mouse and human BRCA1-proficient and -deficient cell lines. This resulted in a unique list of about 500 genes that affect RT response. In aim 2, we successfully validated, functionally characterized, and published selected hits from this screen. Moreover, we generated a CRISPR/Cas9 sublibrary to unambiguously validate all genes that we identified and get a complete essentialome of RT resistance. Regarding aim 3, we established 3D organoid cultures of RT-resistant and -sensitive tumors and have started testing the transduction efficacy of the organoids using the CRISPR/Cas9 sublibrary generated in aim 2. To dissect the intratumoral heterogeneity of residual disease, we combined single-cell RNA sequencing (scRNAseq) with spatial transcriptomics in the KB1P model as planned. We identified specific subpopulations of tumor cells that have a survival benefit after treatment. Moreover, we observed that residual tumor cells form distinct compartments that separate them from immune cells. Using functional CRISPR-Cas9 in vivo screens, we are currently testing which differentially expressed genes are essential for the survival of residual cells and we hope that this may pinpoint specific subpopulations from which tumors regrow.
