Periodic Reporting for period 2 - TETHER (Targeting the essentialome of radiotherapy-resistant cancer)
Berichtszeitraum: 2023-04-01 bis 2024-09-30
More than 50% of cancer patients undergo irradiation as part of their cancer treatment. Although radiotherapy (RT) significantly contributes to cancer cure, local therapy resistance and the subsequent emergence of distant metastasis remain major obstacles for its success. The molecular mechanisms underlying tumor cell-intrinsic RT resistance are ill-defined. It is therefore crucial to better define these mechanisms and identify new vulnerabilities of RT-resistant tumors in order to decrease the current annual cancer mortality of >1.3 million persons in EU Member States alone. The overall goal of this project is to understand the molecular mechanisms that affect radiotherapy (RT) response, and to dissect the genetic essentialome of RT-resistant tumors.To reach this overall goal, the following specific aims will be addressed:
Aim1: Identification of genes that are essential for RT resistance
Aim 2: Functional in vitro analysis and mapping the interactome of genes that contribute to RT resistance
Aim 3: Functional in vivo validation and targeting RT escape
Aim1: Identification of genes that are essential for RT resistance
Aim 2: Functional in vitro analysis and mapping the interactome of genes that contribute to RT resistance
Aim 3: Functional in vivo validation and targeting RT escape
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.
The results we have obtained indicate that we have discovered thus far unknown radiotherapy resistance mechanisms, which we expect them to be major breakthroughs that significantly advance the research field beyond the state of the art. We have not yet published our latest findings, as we are still working on further validating our hypothesis mechanistically. Our results are somewhat unexpected as there is no direct link to DNA repair. Instead, based on our current analysis, there seems to be an indirect effect that reduces the IR-induced DNA damage of the tumor cell nucleus by 50%. A 50% reduction in DNA damage makes a major difference on the RT response of the tumors.