Abstract
INTRODUCTION: Children with MYC-amplified medulloblastoma face a dismal prognosis. We have previously found BET-bromodomain inhibition to have preclinical promise as a therapeutic strategy for MYC-amplified medulloblastoma. However, the mechanisms of action of these inhibitors have not been fully elucidated. In addition, the development of resistance is likely to impact their clinical efficacy. To address this, we characterized cancer cell evolution in response to BET-bromodomain inhibition in MYC-amplified medulloblastoma models. METHODS: We applied genome wide over-expression (ORF) and CRISPR-cas9 screens to identify pathways which, when altered, rescue cells from the phenotypic effects of BET-bromodomain inhibition in MYC-amplified medulloblastoma cell lines. Candidate genes were validated as resistance drivers in medulloblastoma cell lines chronically passaged with BET-bromodomain inhibitors until the acquisition of resistance. These lines were also subjected to whole-exome sequencing to uncover genomic alterations that confer resistance. RESULTS: Altered expression of the anti-apoptosis pathway, cell cycle activation and PI3-kinase signaling, in addition to several lineage-specific transcription factors, rescued MYC-amplified cells from BET-bromodomain inhibition. Cell-lines that had acquired resistance to BET-bromodomain inhibitors exhibited altered cell-state and increased expression of candidate resistance proteins. These lines were resistant to structurally distinct BET-bromodomain inhibitors, and whole-exome sequencing did not reveal mutations in BRD2, BRD3 or BRD4. Resistant cell-lines were sensitive to inhibitors targeting candidate resistance drivers. CONCLUSIONS: MYC-amplified medulloblastoma cell-lines evolve to increase expression of candidate resistant pathways to acquire resistance to BET-bromodomain inhibition. These represent potential therapeutic targets for combination therapies to increase the clinical efficacy of BET-bromodomain inhibitors for children with MYC-amplified medulloblastoma.