Directional dependencies of chemo-resistant lung cancer.

• Applicants: Professor Dr. Manuel Kaulich, Ph.D.; Professor Dr. Mario Looso
• Subject Area: General Genetics and Functional Genome Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 531902760

CRISPR-based functional genomics was applied to large panels of cancer cell lines to identify genetic co-dependencies. Some of these are tested in clinical trials, emphasizing the potential of unbiased genotype-to-phenotype associations. However, actionable vulnerabilities associated with cancer drug resistance remain sparse. This is, in part, attributed to the fact that resistance-causing mutations do not co-emerge but rather occur during iterative rounds of cellular adaptation. A logical consequence is that multiple mechanisms contribute to resistance and that a temporal order of vulnerabilities exists that must be understood to overcome resistance. On a genetic level, this suggests the existence of directional dependencies (DDs) in which the order of mutations dictates the phenotypic outcome (phenotypes of A→B and B→A differ). Exploiting our experimental and computational expertise, we propose to investigate DDs and characterize their potential as drug targets in chemo-resistant non- small cell lung cancer (NSCLC). To do so, we will address the following questions: (i) what is the nature of DDs and how do they relate to classical genetic interactions, (ii) can iterative drug exposure force cell adaptation and recapitulate DDs, and (iii) to what degree can DDs inform about higher-order genetic dependencies? In more detail, WP1 will investigate the relationship between DDs and genetic interactions, develop a pipeline to aggregate, rank, and extract gene pairs based on pathway-level information, and mechanistically explore our observation of genes having several DDs (A→B; A→C; A→D), a phenomenon we coined directional hubs. The establishment of combinatorial CRISPR Switch reagents that circumvent the need for time-consuming cell engineering to perform time-resolved CRISPR genomics is the main goal of WP2. Building on this knowledge, WP3 will perform time-resolved combinatorial CRISPR screens among cancer-druggable genes in a panel of parental and gemcitabine- resistant NSCLC cells and probe for >200.000 pairwise and iterative gene combinations. Based on these data sets, WP4 will establish algorithms to extract and analyze DDs at scale, derive networks and extract directional hubs, and identify DDs that form trajectories (A→B→C). Experimentally, WP4 will mechanistically explore clinically relevant DDs, use iterative drug regimens to chemically mimic genetic relationships, and investigate their context robustness in a larger panel of gemcitabine-resistant cancer models. To the best of our knowledge, our proposed project represents the first joint experimental and computational approach to investigate the biological phenomenon of directional phenotypes. While our focus is on cancer- druggable genes and their significance for chemo-resistant NSCLC, our work packages promise to produce innovative and multifaceted biological insights into cellular adaptation and inform about directional vulnerabilities associated with chemo-resistance.

• DFG Programme: Research Grants