Beytagh, Mary C.

Abstract Medulloblastoma (MB) is one of the most prevalent malignant brain tumors in children, with tremendous cognitive and neuroendocrine disability among survivors. Group 3 (G3) MBs have poor overall survival at < 50%, higher frequency of metastasis, and no targeted therapies. Amplification of MYC and activation of TGFβ signaling are frequent in G3MB. Remarkably, some tumors have no reported mutations, suggesting roles for epigenetic mechanisms in driving disease. We have generated novel humanized models for G3MB by transducing neuroepithelial stem cells (NESC) derived from human induced pluripotent stem (iPSC) cells with TGFβ effectors alone and/or in combination with MYC, prioritizing combinations observed in patients. We determined that both MYC and TGFβ effectors drove tumor formation in vivo with the combination driving more aggressive tumors and higher frequency of metastasis. NESC-derived tumors clustered with human G3/G4MB, indicating our models recapitulated human disease. To decipher mechanisms of resistance, we integrated CUT&RUN for MYC genomic localization and histone PTMs with RNA-sequencing and discovered MYC-bound neural developmental genes repressed in MYC and TGFβR1-driven lines. Further interrogation identified JARID1B, a MYC binding partner, as a regulator of many of these repressed neural genes. We postulate that deregulation of MYC and context-specific targeting to the chromatin template culminates in transcriptional alterations that promote aggressive G3MB. To further investigating therapeutic vulnerabilities, we have performed a in vivo whole genome CRISPRi screen to reveal important drivers of malignancy, one of the first of it’s kind in brain tumors. We are functionally validating key candidate sgRNAs that were differentially significant in primary tumors and may also have key roles in driving metastasis. Excitingly, many sgRNAs identified from the screen were chromatin modifiers and may be important co-factors of MYC-mediated regulation on the epigenome. Collectively, our studies provide a critical framework for understanding mechanisms of resistance and the epigenetic landscape of MYC- and TGFβ-driven MB.

Abstract Medulloblastoma (MB) is one of the most prevalent malignant brain tumors in children, with tremendous cognitive and neuroendocrine disability among survivors. Group 3 (G3) MBs have poor overall survival at <50%, higher frequency of metastasis, and no targeted therapies. Amplification of MYC and activation of TGFβ signaling are frequent in G3MB. Remarkably, some tumors have no reported mutations, suggesting roles for chromatin-driven mechanisms in this disease. We have generated novel humanized models for G3MB by transducing neuroepithelial stem cells (NESC) derived from human induced pluripotent stem cells with TGFβ effectors alone and/or in combination with MYC, prioritizing combinations observed in patients. We determined that both MYC and TGFβ effectors drove tumor formation in vivo with the combination driving more aggressive tumors and higher frequency of metastasis. Importantly, NESC-derived tumors clustered with human G3MB, indicating our models recapitulated human disease. To decipher mechanisms of resistance, we integrated CUT&RUN for MYC genomic localization and histone PTMs with RNA-sequencing and discovered MYC-bound neural developmental genes repressed in MYC and TGFβR1-driven lines. These gene signatures were associated with the Polycomb Repressive Complex (PRC) and demarcated with H3K27me3; the histone mark directly regulated by PRC. Further interrogation identified JARID1B, a MYC binding partner and H3K4me3 demethylase, as a regulator of these repressed neural genes. This is further reinforced by the observation of higher H3K27me3 levels concurrent with increased JARID1B expression in human G3MB patient samples, compared to other subtypes. We postulate that deregulation of MYC and context-specific targeting to the chromatin template culminates in transcriptional alterations that promote aggressive G3MB. These studies support a critical role of MYC in repressing target genes to drive tumor formation and JARID1B as a new target for therapy. Collectively, we present a critical framework for understanding mechanisms of resistance and the chromatin landscape of MYC- and TGFβ-driven G3MB.