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MYC-driven medulloblastomas are a particularly devastating group of pediatric brain tumors that exhibit resistance and continued progression despite standard of care treatments. Our preclinical work identified BET-bromodomain inhibitors as a potentially promising new class of drugs for children with medulloblastoma and other MYC-driven cancers, providing rationale to evaluate these agents in clinical trials. However, treatment with BET inhibitor (BETi) alone is unlikely to be sufficient for a cure, with most tumors evolving to acquire resistance to single-agent targeted therapies. We applied an integrative genomics approach to identify genes and pathways mediating BETi response in medulloblastoma. These studies revealed that MYC-driven medulloblastoma cells with acquired resistance to BETi reinstate transcription of essential genes suppressed by drug, and exhibit changes in cell state and new vulnerabilities not present in drug-sensitive cells. We now have a growing body of evidence showing that BET inhibition downregulates the expression of key lipid metabolism genes and metabolism-related signaling pathways, and that medulloblastoma cells with adaptive resistance to drug differentially express and exhibit preferential dependency on specific lipid metabolic genes and transcriptional regulators. Our studies explore the possibility of exploiting these novel metabolic vulnerabilities in order to overcome BETi resistance and provide a more efficacious upfront therapy.

BACKGROUND: BET-bromodomain proteins bind to H3K27ac enhancers and recruit transcriptional proteins to facilitate the expression of genes. Inhibition of BET-bromodomain proteins shows preclinical promise for MYC-driven tumors such as medulloblastoma (MB), however the mechanism of action and means of resistance are not well described. We hypothesized that genes required for MB cell growth and were sufficient to rescue the effects of BET-bromodomain inhibition (BETi) would signify key downstream effectors of this class of inhibitors. METHODS: We applied an integrative genomics approach: expression profiling of genes suppressed following BETi with JQ1, genome-scale CRISPR/Cas9 screens to determine which of the suppressed genes are also essential for cell viability, and a near-genome scale open reading frame (ORF) rescue screen to determine which of the suppressed genes are also sufficient to drive resistance to BETi. Genes that were nominated by all three data sets were considered BETi transcriptional targets responsible for BET-induced reduced cell viability. RESULTS: BETi in MYC-driven MB cell lines generated widespread changes in gene expression. Genes suppressed by BETi had the tendency to be essential to cell survival. The most frequently included pathways were cell-cycle, DNA replication, and RNA processing/transcription. In each cell line, we identified ORF constructs that significantly rescued cells from BETi which included BCL2 family proteins, which also represented genetic dependencies in treatment naïve cell. Overexpression of BCL2 family member BCL2L1 attenuated both apoptosis and JQ1 mediated cell cycle arrest and expression of the pro-apoptotic genes BAX and BAK were required for BETi response. BCL2 family genes were also validated in drug-tolerant cell lines as having been suppressed by BETi and re-expressed in drug-tolerant cells thus demonstrating their relevance in resistance. CONCLUSION: Integrative genomic analysis identifies BCL2 family members as key mediators of response of MB cells to BET-bromodomain inhibition and also mediate resistance to BETi.

BET-bromodomain inhibition (BETi) has shown pre-clinical promise for MYC-amplified medulloblastoma. However, the mechanisms for its action, and ultimately of resistance, have not been fully defined. Using a combination of expression profiling, genome-scale CRISPR/Cas9-mediated loss of function and ORF/cDNA-driven rescue screens, and cell-based models of spontaneous resistance, we identified bHLH/homeobox transcription factors including NEUROD1, NEUROG1 and NEUROG3, and cell-cycle regulators CCND2 and CCND3 as key gene mediators of BETi’s response. Furthermore, these genes also mediated resistance, and were re-expressed in cells that acquire resistance to BETi through chronic dosing. Cells that acquired drug tolerance exhibit altered cell state, with an increase in neuronally differentiated cell-state and expression of lineage-specific bHLH/homeobox transcription factors. However, they did not terminally differentiate, maintaining the expression of SOX2 and the capacity to cycle through S-phase, re-instating expression of CCND2. Analysis of human medulloblastomas revealed heterogeneous populations of cell states, including those that expressed both neuronal and stem markers, and would be predicted to be less sensitive to BETi. Barcoding techniques to track evolution of cells through treatment indicated the existence of cells that are predetermined to acquire resistance to BETi. Finally, we found inhibition of cell-cycling with CDK4/CKD6 inhibition to delay acquisition of resistance, providing a rationale for combination treatments. These findings provide insights into the mechanisms of action of BETi as a therapeutic strategy for MYC-driven medulloblastoma that can be leveraged to increase efficacy in the clinical setting.