Abstract A162: A comprehensive platform for unraveling the molecular mechanisms and vulnerabilities of colorectal cancer: A step forward in target discovery

Abstract

Abstract Colorectal cancer (CRC) is a heterogeneous malignancy that originates from the colonic and rectal epithelium. It ranks among the most prevalent and deadly cancers globally, representing about 10% of cancer incidence and 9% of cancer mortality in 2018. The oncogenesis and evolution of CRC are driven by the accumulation of genetic and epigenetic alterations that dysregulate key signaling pathways, such as Wnt, RAS, TGF-β, and DNA mismatch repair. However, the molecular mechanisms and vulnerabilities of CRC remain poorly understood, and many patients exhibit poor response or resistance to the current therapies. Therefore, there is an urgent need for novel and effective therapies that can target the specific features and dependencies of different CRC subtypes. To better understand the molecular mechanisms and vulnerabilities of CRC, we established a comprehensive platform based on the CRC model cells derived from human intestinal stem cells (hISCs), patient-derived xenografts (PDXs), and clinical samples, on which we conducted integrative genomic and functional analyses. We employed CRISPR/Cas9 to introduce common CRC mutations, such as APC truncation, KRAS G12D, TP53 and SMAD4 knockouts into hISCs and generated isogenic CRC model cells with different mutational backgrounds. We also developed PDXs from CRC patients and collected biopsy samples from primary and metastatic tumors. We characterized the transcriptomic and genomic profiles of all the samples using RNA-seq and whole-exome sequencing (WES). Then, we conducted high-throughput drug screening (drug HTS) with a collection of standard-of-care drugs and CRISPR/Cas9 dropout screening with a genome-wide library on the CRC model cells. RNA-seq and WES analyses identified distinct molecular signatures and pathways associated with different mutational variants. We validated the presence of driver mutations in the patient-derived cultures, such as APC, KRAS, and TP53, and compared them with the model cells. Our primary cancer cell culture platform was proven suitable for high-throughput identification of new molecular targets through CRISPR/Cas9 dropout screening with a genome-wide library performed on CRC model cells. Additionally, our in-house drug HTS, utilizing a collection of standard-of-care drugs, met industrial standards of QC values such as signal-to-noise ratio, Z-factor, and robustness.. The QC values, such as read depth, library complexity, and alignment rate met the established criteria and we were able to identify essential and tumor suppressor gene sets. Moreover, by utilizing normal hIPCs, we were able to identify among the essential genes those that are critical only for the transformed cells. This innovative platform provides a powerful tool for target discovery and validation in CRC, with potential applicability to other cancer types and personalized medicine approaches. Our findings underscore the importance of using primary material-derived cells in the process of understanding the molecular intricacies of CRC and pave the way for the development of novel, effective therapies. Citation Format: Marcin Duleba, Joanna Szuszkiewicz, Marcin Serocki, Joanna Szklarczyk, Olga Dracz, Anna Ledwon, Michał Combik, Oleksii Bryzghalov, Agata Stachowicz, Michal Mikula, Krzysztof Brzozka, Rafal Dziadziuszko, Tomasz Rzymski, Eliza Zimolag, Andrzej Mazan. A comprehensive platform for unraveling the molecular mechanisms and vulnerabilities of colorectal cancer: A step forward in target discovery [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A162.