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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.

Multiple summer events, including large indoor gatherings, in Provincetown, Massachusetts (MA), in July 2021 contributed to an outbreak of over one thousand COVID-19 cases among residents and visitors. Most cases were fully vaccinated, many of whom were also symptomatic, prompting a comprehensive public health response, motivating changes to national masking recommendations, and raising questions about infection and transmission among vaccinated individuals. To characterize the outbreak and the viral population underlying it, we combined genomic and epidemiological data from 467 individuals, including 40% of known outbreak-associated cases. The Delta variant accounted for 99% of sequenced outbreak-associated cases. Phylogenetic analysis suggests over 40 sources of Delta in the dataset, with one responsible for a single cluster containing 83% of outbreak-associated genomes. This cluster was likely not the result of extensive spread at a single site, but rather transmission from a common source across multiple settings over a short time. Genomic and epidemiological data combined provide strong support for 25 transmission events from, including many between, fully vaccinated individuals; genomic data alone provides evidence for an additional 64. Together, genomic epidemiology provides a high-resolution picture of the Provincetown outbreak, revealing multiple cases of transmission of Delta from fully vaccinated individuals. However, despite its magnitude, the outbreak was restricted in its onward impact in MA and the US, likely due to high vaccination rates and a robust public health response.

BET-bromodomain inhibition (BETi) has shown pre-clinical promise for MYC-amplified medulloblastoma. However, the mechanisms for its action, and ultimately for resistance, have not been fully defined. Here, 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 identify bHLH/homeobox transcription factors and cell-cycle regulators as key genes mediating BETi’s response and resistance. Cells that acquire drug tolerance exhibit a more neuronally differentiated cell-state and expression of lineage-specific bHLH/homeobox transcription factors. However, they do not terminally differentiate, maintain expression of CCND2, and continue to cycle through S-phase. Moreover, CDK4/CDK6 inhibition delays acquisition of resistance. Therefore, our data provide insights about the mechanisms underlying BETi effects and the appearance of resistance and support the therapeutic use of combined cell-cycle inhibitors with BETi in MYC-amplified medulloblastoma., BET-bromodomain inhibitors could be used to treat medulloblastoma tumors with Myc amplifications. Here, the authors show that both the response and resistance to BET inhibitors in mice is mediated by bHLH/homeobox transcription factors.

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 to 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 metabolic vulnerabilities to overcome BETi resistance and provide a more efficacious upfront therapy.

BET-bromodomain inhibition (BETi) is a promising therapeutic strategy to target MYC-driven cancers, including Group 3 medulloblastoma, a deadly childhood brain tumor. We have shown that BET inhibitors exhibit preclinical efficacy against MYC¬-amplified medulloblastoma, providing motivation to evaluate this drug class in early phase clinical trials. However, our work has also found that MYC-amplified medulloblastoma cells can acquire resistance to BETi, suggesting that curative responses for this disease will require combination therapy. To guide the development of such combination therapies, we have focused our efforts on elucidating the mechanisms through which medulloblastoma cells acquire resistance to BETi. We found that medulloblastoma cells can develop tolerance to BETi by reinstating the expression of cell-essential “rescue genes,” which include bHLH transcription factors, cell-cycle regulators, and anti-apoptosis genes. This transition to the resistant cell state is mediated through changes in chromatin structure including the upregulation of H3K4me3 promoters. Our preliminary results suggest that BETi-resistant cells maintain mRNA transcription and protein translation of important mediators of resistance. Importantly, we observe that BETi-resistant medulloblastoma cells are more dependent on specific protein complexes involved in transcriptional regulation. This project explores the mechanisms through which these transcriptional regulators help maintain transcription of rescue genes that drive BETi resistance and evaluates the potential of targeting these drivers of BETi resistance. These results will help guide the development of combination approaches to improve the efficacy of BETi for the treatment of MYC-driven medulloblastoma.

While investigating a signal of adaptive evolution in humans at the gene LARGE, we encountered an intriguing finding by Dr. Stefan Kunz that the gene plays a critical role in Lassa virus binding and entry. This led us to pursue field work to test our hypothesis that natural selection acting on LARGE—detected in the Yoruba population of Nigeria—conferred resistance to Lassa Fever in some West African populations. As we delved further, we conjectured that the “emerging” nature of recently discovered diseases like Lassa fever is related to a newfound capacity for detection, rather than a novel viral presence, and that humans have in fact been exposed to the viruses that cause such diseases for much longer than previously suspected. Dr. Stefan Kunz’s critical efforts not only laid the groundwork for this discovery, but also inspired and catalyzed a series of events that birthed Sentinel, an ambitious and large-scale pandemic prevention effort in West Africa. Sentinel aims to detect and characterize deadly pathogens before they spread across the globe, through implementation of its three fundamental pillars: Detect, Connect, and Empower. More specifically, Sentinel is designed to detect known and novel infections rapidly, connect and share information in real time to identify emerging threats, and empower the public health community to improve pandemic preparedness and response anywhere in the world. We are proud to dedicate this work to Stefan Kunz, and eagerly invite new collaborators, experts, and others to join us in our efforts.

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.

INTRODUCTION We have previously found that up to 15% of all DIPGs harbor mutations in PPM1D, resulting in the expression of an activated and truncated PPM1D (PPM1Dtr). Here we evaluate the mechanisms through which PPM1Dtr enhances glioma formation and identify its associated therapeutic vulnerabilities. METHODS We have developed multiple in vitro and in vivo models of PPM1D-mutant DIPGs and applied quantitative proteomic and functional genomic approaches to identify pathways altered by PPM1Dtr and associated dependencies. RESULTS PPM1D mutations are clonal events that are anti-correlated to TP53 mutations. We find ectopic expression of PPM1Dtr to be sufficient to enhance glioma formation and to be necessary in PPM1D-mutant DIPG cells. In addition, endogenous truncation of PPM1D is sufficient to enhance glioma formation in the presence of mutant H3F3A and PDGFRA. PPM1Dtr overexpression attenuates g-H2AX formation and suppresses apoptosis and cell-cycle arrest in response to radiation treatment. Deep scale phosphoproteomics analyses reveal DNA-damage and cell cycle pathways to be most significantly associated with PPM1Dtr. Furthermore, preliminary analysis of genome-wide loss-of-function CRISPR/Cas9 screens in isogenic GFP and PPM1Dtr overexpressing mouse neural stem cells reveal differential dependency on DNA-damage response genes in the PPM1Dtr overexpressing cells. Consistent with PPM1D’s role in stabilizing MDM2, PPM1D-mutant DIPG models are sensitive to a panel of MDM2 inhibitors (Nutlin-3a, RG7388, and AMG232). CONCLUSION Our study shows that PPM1Dtr is both an oncogene and a dependency in PPM1D- mutant DIPG, and there are novel therapeutic vulnerabilities associated with PPM1D that may be exploited.