Uncovering vulnerabilities prompted by dysregulated transcription through Cas13-based genetic interaction profiling

Carbonell Adames, Andrés Alejandro (2025): Uncovering vulnerabilities prompted by dysregulated transcription through Cas13-based genetic interaction profiling. Dissertation, LMU München: Faculty of Medicine

Abstract

Novel technological advances in high-throughput sequencing have paved the way for understanding the mutational landscape of human cancer. Some of these mutations occur in genes encoding druggable proteins, but the functional consequences of such aberrations can hardly be inferred by “passive” sequencing data alone. Functional screens using technologies such as RNA interference and CRISPR aim to fill this gap by assigning functions to mutated genes. This functional knowledge facilitates the development of molecular cancer therapeutics. Direct inhibition of a mutated proto-oncogene such as BRAF with a small molecule inhibitor is the simplest application of a molecular cancer therapy. However, studies such as the INFORM trial have shown that in high-risk and recurrent pediatric cancers, only about one in ten tumors has a clear molecular target. This underscores the need to discover and therapeutically exploit vulnerabilities beyond driving oncogenes. One example of “indirect” cancer vulnerabilities is negative genetic interactions in the context of cellular fitness. The most extreme form of a negative genetic interaction is synthetic lethality, which refers to situations in which one mutation does not result in major fitness defects but renders cells hyperdependent on secondary genes or processes. This increased functional dependence can then be exploited therapeutically. A well-known example of this concept is the dependence of BRCA-mutant cancers on PARP1, which is exploited clinically by treating certain ovarian and breast cancer patients with PARP inhibitors such as olaparib. Since genetic interactions cannot be easily inferred from passive sequencing studies, there is a strong need for functional screens to foster their discovery. Cancer cells rely on increased transcription to support survival and tumor progression, rendering them "transcriptionally addicted". Many small molecules are currently in clinical trials for their ability to target different aspects of transcription, splicing, nuclear RNA export and cytoplasmic RNA turnover. Perturbations in transcriptional fidelity caused by mutations or pharmacological interference can increase the dependence of cells on secondary processes, leading to synthetic lethality. Here, we developed a CRISPR-Cas13d mediated RNA knockdown platform and performed a combinatorial massively parallel Cas13d screen with 47,727 crRNA-crRNA combinations to investigate molecular vulnerabilities that arise after transcriptional dysregulation. We recapitulated known genetic interactions and discovered novel context-dependent synthetic lethalities. These findings were validated by distinct methods. In addition, we investigated the underlying biochemical mechanisms and predicted rationales for pre-stratification of patients.

• Item Type: Theses (Dissertation, LMU Munich)
• Subjects: 600 Technology, Medicine; 600 Technology, Medicine > 610 Medical sciences and medicine
• Faculties: Faculty of Medicine
• Language: English
• Date of oral examination: 23. April 2025
• 1. Referee: Braun, Christian
• Signature of the printed copy: 0700/UMD 22748