Shyamal Dilhan Weeraratne

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.

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

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.

Could α5-GABA-A receptor activation be used as a target for managing medulloblastomas?

‘Medulloblastoma cerebelli’ was initially described by Cushing and Bailey in 1925 [1]. Medulloblastomas are the most common malignant brain tumors in children and are a significant cause of cancer-related deaths in children [2]. Since their initial description, enormous headway has been made in the molecular characterization of this highly malignant predominantly pediatric brain tumor [3–6]. Recent genomic studies have delineated the molecular heterogeneity of these tumors. They are now classified into subtypes, each with a unique molecular signature and clinical outcome: SHH, WNT, group 3 and group 4 [3–6]. Interestingly, patients with group 3 medulloblastomas have a worse prognosis compared with the other medulloblastoma subtypes [7]. Indeed, 5-year progression-free survival is less than 20% for group 3 medulloblastomas after maximal therapy with externalbeam radiation and multidrug chemotherapy, as compared with 70% 5-year progression-free survival for all other medulloblastoma subtypes. Ten years after disease onset, almost all group 3 medulloblastoma patients will have succumbed to the disease. Therefore, it is of paramount importance to develop effective targeted therapy to manage this subtype of patients. Group 3 medulloblastomas are characterized by oncogenic MYC signaling, often through a high level of MYC amplification. In terms of the current arsenal of chemotherapeutic regimens and radiotherapy, this makes this subtype of medulloblastoma particularly resistant to standard chemotherapy. Unfortunately, attempts at developing drugs that target the MYC protein in group 3 medulloblastomas have so far been unsuccessful. Gene-expression profiling revealed an enrichment of genes associated with GABA pathway signaling in MYC-driven/group 3 medulloblastomas, largely from increased GABRA5 expression [3]. GABRA5 encodes the α5 subunit of the GABA-A receptor complex. This appears to be unique to the group 3 medulloblastomas. This receptor

Identification of very rare clinically actionable KRAS variants in colorectal cancer patients using a comprehensive large gene panel.

e15632Background: While commercially available hotspot testing detects over 99% of the activating mutations in KRAS located at codons 12, 13, 61, and 146, rare oncogenic variants beyond these regio...

Abstract 1313: Mutually exclusive somatic mutations in WNT pathway medulloblastomas reveal a critical interaction between DDX3X and SMARCA4

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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