PDF file - 386K, A, Cell viability relative to day 0 is shown following the indicated treatments in the indicated cell lines. Error bars represent the mean plus-minus SD of three replicates per condition. B, qPCR assessment of MYC mRNA expression in three MYC-amplified patient derived medulloblastoma cell lines treated with JQ1R or JQ1S at doses shown. Values represent mean plus-minus SD of six replicate measurements.

PDF file - 291K, Densitometry quantification of Western Immunoblots shown in Figures 4 and 5.

PDF file - 63K, MYC gene sets enriched in medulloblastoma samples with high expression of MYC isoforms.

PDF file - 567K, A, Gene expression sub-group profiling of MB002 and MB004 patient-derived medulloblastoma cell lines. B, Western blot (left) and densitometry of western blot (right) depicting MYC expression relative to β actin loading control in patient derived medulloblastoma cell lines. ** depict cell lines which are known to harbor amplification of MYC.

Medulloblastoma models which harbor amplifications of myc family members are sensitive to BET-Bromodomain inhibition

Cancers dependent on hedgehog pathway signaling are susceptible to the BET bromodomain inhibitor JQ1. Hedgehog signaling drives oncogenesis in several cancers, and strategies targeting this pathway have been developed, most notably through inhibition of Smoothened (SMO). However, resistance to Smoothened inhibitors occurs by genetic changes of Smoothened or other downstream Hedgehog components. Here we overcome these resistance mechanisms by modulating GLI transcription through inhibition of bromo and extra C-terminal (BET) bromodomain proteins. We show that BRD4 and other BET bromodomain proteins regulate GLI transcription downstream of SMO and suppressor of fused (SUFU), and chromatin immunoprecipitation studies reveal that BRD4 directly occupies GLI1 and GLI2 promoters, with a substantial decrease in engagement of these sites after treatment with JQ1, a small-molecule inhibitor targeting BRD4. Globally, genes associated with medulloblastoma-specific GLI1 binding sites are downregulated in response to JQ1 treatment, supporting direct regulation of GLI activity by BRD4. Notably, patient- and GEMM (genetically engineered mouse model)-derived Hedgehog-driven tumors (basal cell carcinoma, medulloblastoma and atypical teratoid rhabdoid tumor) respond to JQ1 even when harboring genetic lesions rendering them resistant to Smoothened antagonists. Altogether, our results reveal BET proteins as critical regulators of Hedgehog pathway transcriptional output and nominate BET bromodomain inhibitors as a strategy for treating Hedgehog-driven tumors with emerged or a priori resistance to Smoothened antagonists.

Abstract Aberrant activation of Hedgehog signaling drives oncogenesis in several types of cancer. As a result, there has been significant interest in developing therapeutic strategies targeting this pathway, most notably through inhibition of Smoothened. Though Smoothened inhibitors have shown efficacy in several cancer clinical trials, the initial enthusiasm for these inhibitors has been tempered by emergence of resistance and a priori resistance, often via mutation of Smoothened itself or through dysregulation of downstream components of the Hedgehog signaling axis. Here we reveal a strategy that overcomes these resistance mechanisms by targeting the far downstream transcriptional mediators of Hedgehog signaling through inhibition of the BET bromodomain protein, BRD4. We show that knockdown of BRD4 or treatment with the BET bromodomain inhibitor, JQ1, dramatically inhibits transcription of GLI1 and other Hedgehog target genes upon ligand-mediated or genetic activation of the Hedgehog pathway. We confirm the inhibitory effect of JQ1 occurs downstream of SMO and SUFU and verify by chromatin immunoprecipitation that Brd4 directly occupies the GLI1 and GLI2 promoters, with a substantial decrease in the engagement of these genomic sites upon treatment with JQ1. We observe a corresponding downregulation of genes associated with medulloblastoma-specific GLI1 binding sites upon exposure to JQ1, confirming the direct regulation of GLI1 by BET bromodomain proteins. Finally, using patient- and GEMM-derived cell lines of Hedgehog-driven cancer (basal cell carcinoma, medulloblastoma and ATRT), we show that JQ1 decreases Hh pathway output and proliferation, even in cell lines resistant to Smoothened inhibitors. These results expand the role of BET bromodomain inhibitors to targeting Hedgehog-driven cancers and highlight a strategy that overcomes the limitation of Hedgehog pathway inhibitors currently in clinical use. Citation Format: Yujie Tang, Simone Schubert, Jun Qi, Brian Nguyen, Sabran Masoud, Nujsaunusi Vue, Brianna Balansay, Furong Yu, Scott X. Atwood, Ramon J. Whitson, Anitha Ponnuswami, Spencer Chen, Sharareh Gholamin, Woo J. Pamelyn, Michelle Monje-Diesseroth, Sekyung Oh, Alex Lee, Jean Y. Tang, Rob Wechsler-Reya, Anthony E. Oro, James E. Bradner, Yoon-Jae Cho. Epigenetic regulation of Hedgehog pathway transcriptional output by BET bromodomain proteins. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B6.

Neural tumors often express neurotransmitter receptors as markers of their developmental lineage. Although these receptors have been well characterized in electrophysiological, developmental and pharmacological settings, their importance in the maintenance and progression of brain tumors and, importantly, the effect of their targeting in brain cancers remains obscure. Here, we demonstrate high levels of GABRA5, which encodes the α5-subunit of the GABAA receptor complex, in aggressive MYC-driven, "Group 3" medulloblastomas. We hypothesized that modulation of α5-GABAA receptors alters medulloblastoma cell survival and monitored biological and electrophysiological responses of GABRA5-expressing medulloblastoma cells upon pharmacological targeting of the GABAA receptor. While antagonists, inverse agonists and non-specific positive allosteric modulators had limited effects on medulloblastoma cells, a highly specific and potent α5-GABAA receptor agonist, QHii066, resulted in marked membrane depolarization and a significant decrease in cell survival. This effect was GABRA5 dependent and mediated through the induction of apoptosis as well as accumulation of cells in S and G2 phases of the cell cycle. Chemical genomic profiling of QHii066-treated medulloblastoma cells confirmed inhibition of MYC-related transcriptional activity and revealed an enrichment of HOXA5 target gene expression. siRNA-mediated knockdown of HOXA5 markedly blunted the response of medulloblastoma cells to QHii066. Furthermore, QHii066 sensitized GABRA5 positive medulloblastoma cells to radiation and chemotherapy consistent with the role of HOXA5 in directly regulating p53 expression and inducing apoptosis. Thus, our results provide novel insights into the synthetic lethal nature of α5-GABAA receptor activation in MYC-driven/Group 3 medulloblastomas and propose its targeting as a novel strategy for the management of this highly aggressive tumor.

Purpose: MYC-amplified medulloblastomas are highly lethal tumors. Bromodomain and extraterminal (BET) bromodomain inhibition has recently been shown to suppress MYC-associated transcriptional activity in other cancers. The compound JQ1 inhibits BET bromodomain-containing proteins, including BRD4. Here, we investigate BET bromodomain targeting for the treatment of MYC-amplified medulloblastoma. Experimental Design: We evaluated the effects of genetic and pharmacologic inhibition of BET bromodomains on proliferation, cell cycle, and apoptosis in established and newly generated patient- and genetically engineered mouse model (GEMM)-derived medulloblastoma cell lines and xenografts that harbored amplifications of MYC or MYCN. We also assessed the effect of JQ1 on MYC expression and global MYC-associated transcriptional activity. We assessed the in vivo efficacy of JQ1 in orthotopic xenografts established in immunocompromised mice. Results: Treatment of MYC-amplified medulloblastoma cells with JQ1 decreased cell viability associated with arrest at G1 and apoptosis. We observed downregulation of MYC expression and confirmed the inhibition of MYC-associated transcriptional targets. The exogenous expression of MYC from a retroviral promoter reduced the effect of JQ1 on cell viability, suggesting that attenuated levels of MYC contribute to the functional effects of JQ1. JQ1 significantly prolonged the survival of orthotopic xenograft models of MYC-amplified medulloblastoma (P < 0.001). Xenografts harvested from mice after five doses of JQ1 had reduced the expression of MYC mRNA and a reduced proliferative index. Conclusion: JQ1 suppresses MYC expression and MYC-associated transcriptional activity in medulloblastomas, resulting in an overall decrease in medulloblastoma cell viability. These preclinical findings highlight the promise of BET bromodomain inhibitors as novel agents for MYC-amplified medulloblastoma. Clin Cancer Res; 20(4); 912–25. ©2013 AACR.

Abstract DDX3X is an ATP-dependent RNA helicase that belongs to an extended family of D-E-A-D motif (DEAD-box) containing proteins whose functions have been implicated in key biological processes ranging from RNA transcription and splicing to the regulation of translation initiation. Through whole exome sequencing of 94 primary medulloblasotmas, we have identified DDX3X as one of the most common recurrently mutated genes in medulloblastoma. These mutations predominantly occur in WNT pathway medulloblastomas which concurrently harbor stabilized forms of mutant beta-catenin. Interestingly, we have also identified SMARCA4 mutations in WNT pathway tumors that are mutually exclusive of DDX3X mutations. In addition, two WNT pathway tumors that have neither DDX3X nor SMARCA4 gene mutations have mutations in genes known to encode for direct binding partners of DDX3X and SMARCA4 (EIF3E and ACTL6A, respectively). This mutual exclusivity of DDX3X and SMARCA4 mutations suggests either a direct interaction between the DDX3X and SMARCA4 gene products or an overlap of the biological processes regulated by each. Here we reveal the direct interaction between DDX3X and SMARCA4 in normal physiological conditions and further investigate the impact of somatic mutations in DDX3X and SMARCA4 on this interaction in WNT pathway associated medulloblastomas. Our results point to a critical link between RNA helicases and the SWI/SNF chromatin remodeling complex in WNT/beta-catenin signaling and disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1313. doi:1538-7445.AM2012-1313

Medulloblastoma is the most common brain tumour in children; using exome sequencing of tumour samples the authors show that these cancers have low mutation rates and identify 12 significantly mutated genes, among them the gene encoding RNA helicase DDX3X. Medulloblastoma is the most common malignant brain tumour in children. Four papers published in the 2 August 2012 issue of Nature use whole-genome and other sequencing techniques to produce a detailed picture of the genetics and genomics of this condition. Notable findings include the identification of recurrent mutations in genes not previously implicated in medulloblastoma, with significant genetic differences associated with the four biologically distinct subgroups and clinical outcomes in each. Potential avenues for therapy are suggested by the identification of targetable somatic copy-number alterations, including recurrent events targeting TGFβ signalling in Group 3, and NF-κB signalling in Group 4 medulloblastomas. Medulloblastomas are the most common malignant brain tumours in children1. Identifying and understanding the genetic events that drive these tumours is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma on the basis of transcriptional and copy number profiles2,3,4,5. Here we use whole-exome hybrid capture and deep sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas have low mutation rates consistent with other paediatric tumours, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were newly identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR and LDB1. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant, but not wild-type, β-catenin. Together, our study reveals the alteration of WNT, hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and within specific subtypes of this disease, and nominates the RNA helicase DDX3X as a component of pathogenic β-catenin signalling in medulloblastoma.