DRES-05. IDENTIFYING DRIVERS OF CHEMORESISTANCE IN GROUP 3 MEDULLOBLASTOMA THROUGH A GENOME-WIDE CRISPR/Cas9 ACTIVATION SCREEN

Abstract

Medulloblastoma (MB) is the most common malignant brain tumor in children, accounting for nearly 20% of all pediatric brain cancers. Based on transcriptional profiling studies, MB has been characterized into four molecular subgroups-WNT, SHH, Group 3, and Group 4-that have distinct genetics, patient demographics, histology, and clinical outcomes. Group 3 MB carries the worst prognosis, with 5-year survival rates of 50%. Current therapy for MB consists of maximal safe surgical resection, radiation, and chemotherapy, with cisplatin, vincristine, and cyclophosphamide comprising a standard chemotherapy regimen. However, Group 3 MB commonly develops resistance and becomes refractory to standard therapy. As these mechanisms of resistance remain poorly understood, I am using the CRISPR/ Cas9-based synergistic activation mediator (SAM) system to conduct a genome-wide, gain-of-function, positive selection screen to identify the specific drivers of chemoresistance in Group 3 MB. I cloned and sequenced a library consisting of 70,290 single-guide RNAs (sgRNAs) targeting each of the 23,430 coding isoforms from the human RefSeq database. I have transduced this library into a Group 3 MB patient-derived model stably expressing the other SAM components (dCas9, VP64, MS2, p65, and HSF1) and then selected with standard-of-care chemotherapy conditions: (1) cisplatin; (2) vincristine; (3) 4-hydroperoxycyclophosphamide; and (4) combination of cisplatin, vincristine, and 4-hydroperoxycyclophosphamide. Modulators of chemotherapy sensitivity will be prioritized by sgRNAs that have activated genes conferring chemoresistance, and I will perform next-generation sequencing to identify those sgRNAs. I will then validate the top hits from our screen and also cross-reference them with recently published whole-genome sequencing data on six pairs of human diagnostic and post-therapy Group 3 MBs. These findings will provide insights into the mechanisms of resistance in Group 3 MB and inform novel therapeutic targets that may sensitize the tumor to chemotherapy and improve future treatment response.