Identification of Cancer Stem Cell Susceptibility to Targeted Therapies in a Unique Subset of Colorectal Cancer

Abstract

The colonic epithelium has a fascinating renewal capacity and turns over every 3-4 days. Colorectal cancer develops when the same epithelial cells collect mutations over time which leads to carcinoma. CRC is one of the leading causes of morbidity in the United States and other parts of the world. Classical adenoma formation in the epithelium follow the Vogelstein model of progression, where the driver mutations are sequential. Consensus Molecular Subtypes suggests CRC can be divided into 4 subtypes based on their genetic signature. This has been widely accepted in the field and researchers are finding new categories to understand, diagnose and treat CRC. Driving mutations in CRC usually involve the Wnt Pathway and MAPK Pathway and a lot of the treatments target these pathways to improve patient outcome. CRC is inherently heterogenous in nature and treatment often fails to improve overall survivalin patients with chances of remission and chemoresistance. One unique subset of tumours which have been understudied are BRCA/BRAF tumours. These tumours have driver mutations B-RAF which induce survival and hyperproliferation along with BRCA mutations that protect the tumours from apoptosis. These tumours are said to have high immune infiltration and thus benefit from checkpoint inhibitor therapies. They are treated with B-RAF inhibitors along with EGFR and VEGFR inhibitors. But several studies have shown increased resistance to these therapies and high probability of relapse in patient outcomes. Chemoresistance is a big bottleneck which has been studied over the years to overcome. Studies suggest that resistant populations may either lay dormant or not respond to the treatment and thus increase the probability of relapse. This study aims to understand this unique understudied set of tumours and their response to conventionally used chemotherapeutics. We utilized patient-derived organoid models to characterize chemotherapeutic responses in BRCA/BRAF mutant CRC. We evaluated the efficacy of FDA-approved RAF and PARP inhibitors, as well as DYR895, a novel CLK/DYRK kinase inhibitor targeting the Wnt and AKT pathways. High content single-cell imaging and phenotypic profiling revealed that while RAF inhibitors were largely ineffective, PARP inhibitors and DYR895 significantly reduced viable tumor cell populations and induced DNA damage. Subpopulation analysis highlighted dynamic shifts, particularly the depletion of cancer stem-like (CD44v9+) cells, suggesting potential avenues to overcome chemoresistance. Our findings underscore the importance of targeting both DNA repair deficiencies and survival signaling pathways in BRCA/BRAF-mutant CRC and demonstrate the utility of organoid models for preclinical drug evaluation and personalized therapy development.