Abstract 5159: Assessing the importance of the kinase domain through kinome-focused CRISPR screening

Abstract

Abstract The identification of new therapeutic targets is critical in the fight against cancer. Over the past few decades, the inhibition of kinase cancer drivers has dramatically changed the cancer treatment landscape. Kinases play an important role in modulating the cellular signaling cascades that are often altered in cancer. This role is mainly carried out by the kinase domain(s) which control catalytic activity through binding ATP. Consequently, small molecule inhibition of catalytic activity can disrupt cancer signaling and halt tumor growth. Although examples of kinase drivers in cancer development are well established, there are still numerous understudied kinases with limited information about their biological function. Therefore, we designed a comprehensive kinase-focused CRISPR library specifically aimed at interrogating the contribution of kinase catalytic activity. Our library contains a high density of guides targeting the kinase domain, allowing for quick assessment of the importance of the active site. Our library aims to create opportunities for identifying novel therapeutic targets and for further understanding the cancer signaling mechanisms. We utilized our newly designed library in a high throughput, kinome-wide CRISPR pooled screen applied to multiple cellular models. CRISPR Pooled Screening is a powerful tool for conducting large-scale studies involving hundreds or thousands of genes. However, due to a general preference to increase throughput, most screening efforts include a limited number of guides per gene. This increases chances of detecting false positives and reduces the screen's power to differentiate between the activity of different gene domains. In our library, however, we used a minimum of 24 guides per gene strategically distributed both inside and outside the catalytic domains of each kinase. Additionally, our library allows us to rule out likely false-positive hits due to genomic copy number amplifications through the addition of guides targeting the nearest neighbor gene. As a reference, we used the Broad Institute's Achilles Avana CRISPR Screen. Our library was able to reproduce 100% of Avana's significant kinase hits. Furthermore, we identified novel hits that did not show dependency in Avana. These hits produced a phenotype only when their kinase domain was targeted and did not show any phenotype for the guides outside the kinase domain. Most importantly, our screen highlighted the value of domain-centric CRISPR libraries and how they could be used for identifying kinase targets that have so far eluded previous screens. Citation Format: Ahmad Al Kawam, Alexandra R. Grassian, Fabien Llambi, Erica Evans, Klaus Hoeflich, Chaoyang Ye. Assessing the importance of the kinase domain through kinome-focused CRISPR screening [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5159.