Identification of chromatin modifiers regulating vincristine resistance in medulloblastoma

Medulloblastoma (MB) is the most prevalent brain cancer in children, typically occurring between the ages of six and eight. While primary MB treatments have a success rate of over 50%, relapse affects more than 30% of the patients, resulting in a poor prognosis with survival rates dropping below 25%. Furthermore, acquired-drug resistance is widely observed in patients with relapse, posing a significant challenge for the treatment of MB. Recent cohorts revealed mutations and differential regulation of chromatin modifiers in distinct subgroups of MB, emphasizing the importance of epigenetic regulations in MB tumorigenesis. In this study, we aimed to identify the epigenetic vulnerabilities of MB by employing loss-of-function screening approaches. Using paired parental and vincristine-resistant MB cells previously generated in our lab, we first conducted a chemical screen with an epigenetic probe library. Second, we performed genetic perturbation screens using our CRISPR/Cas9-based epigenome-wide knockout library (EPIKOL). The ultimate goal was to discover new epigenetic mechanisms of drug response in MB. The established vincristine-resistant MB cells showed up to 100-fold resistance to vincristine, displaying Multi-Drug Resistance (MDR) characteristics primarily attributed to a significant upregulation of ABCB1. With chemical screens, we demonstrated that inhibition of the bromodomain of CBP/p300 histone acetyltransferases leads to sensitization of vincristine-resistant MB by regulating the expression of ABC and SLC transporters. With genetic screens using EPIKOL, we further validated the sensitizing effect of CBP inhibition on drug resistance. Additionally, we identified KEAP1, the regulator of NRF2, as a novel target. Accordingly, KEAP1 was identified from 3 different screens on vincristine-resistant MB, where the negative selection was applied with increasing doses of vincristine, but not on parental MB cells. KEAP1 loss by CRISPR/Cas9 using multiple sgRNAs also confirmed the sensitizing effect through viability assays. Moreover, upon exposure to vincristine, a significant increase in apoptosis was also observed in KEAP1-KO vincristine-resistant cells. Additionally, the transcriptomic analysis revealed the downregulation of ABCB1, the main efflux pump of vincristine, on KEAP1-KO vincristine-resistant cells, further supporting the sensitizing effect of KEAP1 loss. This thesis identified two major regulators of acquired vincristine resistance in MB, which have the potential to serve as therapeutic intervention points in the future.