Tenley C. Archer

MEDULLOBLASTOMA EXOME SEQUENCING UNCOVERS SUBTYPE-SPECIFIC SOMATIC MUTATIONS

Medulloblastomas are the most common malignant brain tumours in children. 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 profiles. 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.

Pleiotropic effects of miR-183~96~182 converge to regulate cell survival, proliferation and migration in medulloblastoma

Medulloblastomas are the most common malignant brain tumors in children. Several large-scale genomic studies have detailed their heterogeneity, defining multiple subtypes with unique molecular profiles and clinical behavior. Increased expression of the miR-183~96~182 cluster of microRNAs has been noted in several subgroups, including the most clinically aggressive subgroup associated with genetic amplification of MYC. To understand the contribution of miR-183~96~182 to the pathogenesis of this aggressive subtype of medulloblastoma, we analyzed global gene expression and proteomic changes that occur upon modulation of miRNAs in this cluster individually and as a group in MYC-amplified medulloblastoma cells. Knockdown of the full miR-183~96~182 cluster results in enrichment of genes associated with apoptosis and dysregulation of the PI3K/AKT/mTOR signaling axis. Conversely, there is a relative enrichment of pathways associated with migration, metastasis and epithelial to mesenchymal transition, as well as pathways associated with dysfunction of DNA repair in cells with preserved miR-183 cluster expression. Immunocytochemistry and FACS analysis confirm induction of apoptosis upon knockdown of the miR-183 cluster. Importantly, cell-based migration and invasion assays verify the positive regulation of cell motility/migration by the miR-183 cluster, which is largely mediated by miR-182. We show that the effects on cell migration induced by the miR-183 cluster are coupled to the PI3K/AKT/mTOR pathway through differential regulation of AKT1 and AKT2 isoforms. Furthermore, we show that rapamycin inhibits cell motility/migration in medulloblastoma cells and phenocopies miR-183 cluster knockdown. Thus, the miR-183 cluster regulates multiple biological programs that converge to support the maintenance and metastatic potential of medulloblastoma.

Interaction of Sox1, Sox2, Sox3 and Oct4 during primary neurogenesis

Sox1, Sox2 and Sox3, the three members of the SoxB1 subgroup of transcription factors, have similar sequences, expression patterns and overexpression phenotypes. Thus, it has been suggested that they have redundant roles in the maintenance of neural stem cells in development. However, the long-term effect of overexpression or their function in combination with their putative co-factor Oct4 has not been tested. Here, we show that overexpression of sox1, sox2, sox3 or oct91, the Xenopus homologue of Oct4, results in the same phenotype: an expanded neural plate at the expense of epidermis and delayed neurogenesis. However, each of these proteins induced a unique profile of neural markers and the combination of Oct91 with each SoxB1 protein had different effects, as did continuous misexpression of the proteins. Overexpression studies indicate that Oct91 preferentially cooperates with Sox2 to maintain neural progenitor marker expression, while knockdown of Oct91 inhibits neural induction driven by either Sox2 or Sox3. Continuous expression of Sox1 and Sox2 in transgenic embryos represses neuron differentiation and inhibits anterior development while increasing cell proliferation. Constitutively active Sox3, however, leads to increased apoptosis suggesting that it functions as a tumor suppressor. While the SoxB1s have overlapping functions, they are not strictly redundant as they induce different sets of genes and are likely to partner with different proteins to maintain progenitor identity.

Xenopus Sox3 activates sox2 and geminin and indirectly represses Xvent2 expression to induce neural progenitor formation at the expense of non-neural ectodermal derivatives

The SRY-related, HMG box SoxB1 transcription factors are highly homologous, evolutionarily conserved proteins that are expressed in neuroepithelial cells throughout neural development. SoxB1 genes are down-regulated as cells exit the cell-cycle to differentiate and are considered functionally redundant in maintaining neural precursor populations. However, little is known about Sox3 function and its mode of action during primary neurogenesis. Using gain and loss-of-function studies, we analyzed Sox3 function in detail in Xenopus early neural development and compared it to that of Sox2. Through these studies we identified the first targets of a SoxB1 protein during primary neurogenesis. Sox3 functions as an activator to induce expression of the early neural genes, sox2 and geminin in the absence of protein synthesis and to indirectly inhibit the Bmp target Xvent2. As a result, Sox3 increases cell proliferation, delays neurogenesis and inhibits epidermal and neural crest formation to expand the neural plate. Our studies indicate that Sox3 and 2 have many similar functions in this process including the ability to activate expression of geminin in naïve ectodermal explants. However, there are some differences; Sox3 activates the expression of sox2, while Sox2 does not activate expression of sox3 and sox3 is uniquely expressed throughout the ectoderm prior to neural induction suggesting a role in neural competence. With morpholino-mediated knockdown of Sox3, we demonstrate that it is required for induction of neural tissue by BMP inhibition. Together these data indicate that Sox3 has multiple roles in early neural development including as a factor required for nogginmediated neural induction.

SMARCB1-mediated SWI/SNF complex function is essential for enhancer regulation

SMARCB1 (also known as SNF5, INI1, and BAF47), a core subunit of the SWI/SNF (BAF) chromatin-remodeling complex, is inactivated in nearly all pediatric rhabdoid tumors. These aggressive cancers are among the most genomically stable, suggesting an epigenetic mechanism by which SMARCB1 loss drives transformation. Here we show that, despite having indistinguishable mutational landscapes, human rhabdoid tumors exhibit distinct enhancer H3K27ac signatures, which identify remnants of differentiation programs. We show that SMARCB1 is required for the integrity of SWI/SNF complexes and that its loss alters enhancer targeting—markedly impairing SWI/SNF binding to typical enhancers, particularly those required for differentiation, while maintaining SWI/SNF binding at super-enhancers. We show that these retained super-enhancers are essential for rhabdoid tumor survival, including some that are shared by all subtypes, such as SPRY1, and other lineage-specific super-enhancers, such as SOX2 in brain-derived rhabdoid tumors. Taken together, our findings identify a new chromatin-based epigenetic mechanism underlying the tumor-suppressive activity of SMARCB1.

Hedgehog-GLI Pathway in Medulloblastoma

Medulloblastoma (MB), the most common malignant brain tumor in children, arises in the cerebellum. Aberrant activation of the sonic hedgehog (SHH) pathway has been unambiguously tied to the etiology of one subtype of the disease. Although the majority of tumors arise through somatic mutations, familial predisposition to MB is well known to occur in association with germline mutations of the SHH receptor patched (PTCH) in patients with Gorlin’s syndrome. In Journal of Clinical Oncology, Brugieres et al report their investigation of the incidence of germline mutations in suppressor of fused (SUFU), a repressor in the SHH pathway, in a cohort of 131 patients with MB. They discovered germline SUFU mutations in eight of these patients, most commonly in infants. Developmentally important signaling pathways are frequently recapitulated in cancer, and SHH-driven MB is one such classic example. As germline mutations in SUFU deregulate SHH signaling and potentially other signaling pathways, understanding how the SUFU mutations lead to oncogenic transformation is a useful tool to gain insight into all SHH-driven MB. The cerebellum begins to form early on in embryonic development but does not fully mature until a few months after birth. This prolonged maturation makes the cerebellum especially vulnerable to developmental abnormalities and cancer. SHH is secreted by the Purkinje neurons that are present underneath the external granular layer of the developing cerebellum. SHH drives proliferation of granule neuron precursors to orchestrate cerebellar patterning and dictate the size of the organ. It is not surprising that deregulated hyperactive SHH signaling in granule neuron precursors during cerebellar development is a primary event during MB pathogenesis. The ability of SHH to control cerebellar patterning originated from its anterior/posterior patterning, induction of polarity, and

DiSCoVERing Innovative Therapies for Rare Tumors: Combining Genetically Accurate Disease Models with In Silico Analysis to Identify Novel Therapeutic Targets.

Purpose: We used human stem and progenitor cells to develop a genetically accurate novel model of MYC-driven Group 3 medulloblastoma. We also developed a new informatics method, Disease-model Signature versus Compound-Variety Enriched Response (“DiSCoVER”), to identify novel therapeutics that target this specific disease subtype. Experimental Design: Human neural stem and progenitor cells derived from the cerebellar anlage were transduced with oncogenic elements associated with aggressive medulloblastoma. An in silico analysis method for screening drug sensitivity databases (DiSCoVER) was used in multiple drug sensitivity datasets. We validated the top hits from this analysis in vitro and in vivo. Results: Human neural stem and progenitor cells transformed with c-MYC, dominant-negative p53, constitutively active AKT and hTERT formed tumors in mice that recapitulated Group 3 medulloblastoma in terms of pathology and expression profile. DiSCoVER analysis predicted that aggressive MYC-driven Group 3 medulloblastoma would be sensitive to cyclin-dependent kinase (CDK) inhibitors. The CDK 4/6 inhibitor palbociclib decreased proliferation, increased apoptosis, and significantly extended the survival of mice with orthotopic medulloblastoma xenografts. Conclusions: We present a new method to generate genetically accurate models of rare tumors, and a companion computational methodology to find therapeutic interventions that target them. We validated our human neural stem cell model of MYC-driven Group 3 medulloblastoma and showed that CDK 4/6 inhibitors are active against this subgroup. Our results suggest that palbociclib is a potential effective treatment for poor prognosis MYC-driven Group 3 medulloblastoma tumors in carefully selected patients. Clin Cancer Res; 22(15); 3903–14. ©2016 AACR.

α5-GABAA receptors negatively regulate MYC-amplified medulloblastoma growth

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.

OTX2 Activity at Distal Regulatory Elements Shapes the Chromatin Landscape of Group 3 Medulloblastoma

Medulloblastoma is the most frequent malignant pediatric brain tumor and is divided into at least four subgroups known as WNT, SHH, Group 3, and Group 4. Here, we characterized gene regulation mechanisms in the most aggressive subtype, Group 3 tumors, through genome-wide chromatin and expression profiling. Our results show that most active distal sites in these tumors are occupied by the transcription factor OTX2. Highly active OTX2-bound enhancers are often arranged as clusters of adjacent peaks and are also bound by the transcription factor NEUROD1. These sites are responsive to OTX2 and NEUROD1 knockdown and could also be generated de novo upon ectopic OTX2 expression in primary cells, showing that OTX2 cooperates with NEUROD1 and plays a major role in maintaining and possibly establishing regulatory elements as a pioneer factor. Among OTX2 target genes, we identified the kinase NEK2, whose knockdown and pharmacologic inhibition decreased cell viability. Our studies thus show that OTX2 controls the regulatory landscape of Group 3 medulloblastoma through cooperative activity at enhancer elements and contributes to the expression of critical target genes.Significance: The gene regulation mechanisms that drive medulloblastoma are not well understood. Using chromatin profiling, we find that the transcription factor OTX2 acts as a pioneer factor and, in cooperation with NEUROD1, controls the Group 3 medulloblastoma active enhancer landscape. OTX2 itself or its target genes, including the mitotic kinase NEK2, represent attractive targets for future therapies. Cancer Discov; 7(3); 288-301. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 235.

Defining the Molecular Landscape of Ependymomas

Ependymomas have a variable prognosis. In this issue of Cancer Cell, Pajtler and colleagues identify nine subgroups of ependymoma using DNA methylation profiles. Two subgroups, predominately pediatric, are responsible for most of the mortality, with all others having nearly 100% overall survival after 5 years.