Patrick Sin-Chan

Epigenomic alterations define lethal CIMP-positive ependymomas of infancy.

Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland.

Fusion of TTYH1 with the C19MC microRNA cluster drives expression of a brain-specific DNMT3B isoform in the embryonal brain tumor ETMR

Embryonal tumors with multilayered rosettes (ETMRs) are rare, deadly pediatric brain tumors characterized by high-level amplification of the microRNA cluster C19MC. We performed integrated genetic and epigenetic analyses of 12 ETMR samples and identified, in all cases, C19MC fusions to TTYH1 driving expression of the microRNAs. ETMR tumors, cell lines and xenografts showed a specific DNA methylation pattern distinct from those of other tumors and normal tissues. We detected extreme overexpression of a previously uncharacterized isoform of DNMT3B originating at an alternative promoter that is active only in the first weeks of neural tube development. Transcriptional and immunohistochemical analyses suggest that C19MC-dependent DNMT3B deregulation is mediated by RBL2, a known repressor of DNMT3B. Transfection with individual C19MC microRNAs resulted in DNMT3B upregulation and RBL2 downregulation in cultured cells. Our data suggest a potential oncogenic re-engagement of an early developmental program in ETMR via epigenetic alteration mediated by an embryonic, brain-specific DNMT3B isoform.

Molecular subgroups of atypical teratoid rhabdoid tumours in children: an integrated genomic and clinicopathological analysis

BACKGROUND Rhabdoid brain tumours, also called atypical teratoid rhabdoid tumours, are lethal childhood cancers with characteristic genetic alterations of SMARCB1/hSNF5. Lack of biological understanding of the substantial clinical heterogeneity of these tumours restricts therapeutic advances. We integrated genomic and clinicopathological analyses of a cohort of patients with atypical teratoid rhabdoid tumours to find out the molecular basis for clinical heterogeneity in these tumours. METHODS We obtained 259 rhabdoid tumours from 37 international institutions and assessed transcriptional profiles in 43 primary tumours and copy number profiles in 38 primary tumours to discover molecular subgroups of atypical teratoid rhabdoid tumours. We used gene and pathway enrichment analyses to discover group-specific molecular markers and did immunohistochemical analyses on 125 primary tumours to evaluate clinicopathological significance of molecular subgroup and ASCL1-NOTCH signalling. FINDINGS Transcriptional analyses identified two atypical teratoid rhabdoid tumour subgroups with differential enrichment of genetic pathways, and distinct clinicopathological and survival features. Expression of ASCL1, a regulator of NOTCH signalling, correlated with supratentorial location (p=0·004) and superior 5-year overall survival (35%, 95% CI 13-57, and 20%, 6-34, for ASCL1-positive and ASCL1-negative tumours, respectively; p=0·033) in 70 patients who received multimodal treatment. ASCL1 expression also correlated with superior 5-year overall survival (34%, 7-61, and 9%, 0-21, for ASCL1-positive and ASCL1-negative tumours, respectively; p=0·001) in 39 patients who received only chemotherapy without radiation. Cox hazard ratios for overall survival in patients with differential ASCL1 enrichment treated with chemotherapy with or without radiation were 2·02 (95% CI 1·04-3·85; p=0·038) and 3·98 (1·71-9·26; p=0·001). Integrated analyses of molecular subgroupings with clinical prognostic factors showed three distinct clinical risk groups of tumours with different therapeutic outcomes. INTERPRETATION An integration of clinical risk factors and tumour molecular groups can be used to identify patients who are likely to have improved long-term radiation-free survival and might help therapeutic stratification of patients with atypical teratoid rhabdoid tumours. FUNDING C17 Research Network, Genome Canada, b.r.a.i.n.child, Mitchell Duckman, Tal Doron and Suri Boon foundations.

A novel C19MC amplified cell line links Lin28/let-7 to mTOR signaling in embryonal tumor with multilayered rosettes

BACKGROUND Embryonal tumor with multilayered rosettes (ETMR) is an aggressive central nervous system primitive neuroectodermal tumor (CNS-PNET) variant. ETMRs have distinctive histology, amplification of the chromosome 19 microRNA cluster (C19MC) at chr19q13.41-42, expression of the RNA binding protein Lin28, and dismal prognosis. Functional and therapeutic studies of ETMR have been limited by a lack of model systems. METHODS We have established a first cell line, BT183, from a case of ETMR and characterized its molecular and cellular features. LIN28 knockdown was performed in BT183 to examine the potential role of Lin28 in regulating signaling pathway gene expression in ETMR. Cell line findings were corroborated with immunohistochemical studies in ETMR tissues. A drug screen of 73 compounds was performed to identify potential therapeutic targets. RESULTS The BT183 line maintains C19MC amplification, expresses C19MC-encoded microRNAs, and is tumor initiating. ETMRs, including BT183, have high LIN28 expression and low let-7 miRNA expression, and show evidence of mTOR pathway activation. LIN28 knockdown increases let-7 expression and decreases expression of IGF/PI3K/mTOR pathway components. Pharmacologic inhibition of the mTOR pathway reduces BT183 cell viability. CONCLUSIONS BT183 retains key genetic and histologic features of ETMR. In ETMR, Lin28 is not only a diagnostic marker but also a regulator of genes involved in growth and metabolism. Our findings indicate that inhibitors of the IGF/PI3K/mTOR pathway may be promising novel therapies for these fatal embryonal tumors. As the first patient-derived cell line of these rare tumors, BT183 is an important, unique reagent for investigating ETMR biology and therapeutics.

Therapeutic targeting of ependymoma as informed by oncogenic enhancer profiling

Genomic sequencing has driven precision-based oncology therapy; however, the genetic drivers of many malignancies remain unknown or non-targetable, so alternative approaches to the identification of therapeutic leads are necessary. Ependymomas are chemotherapy-resistant brain tumours, which, despite genomic sequencing, lack effective molecular targets. Intracranial ependymomas are segregated on the basis of anatomical location (supratentorial region or posterior fossa) and further divided into distinct molecular subgroups that reflect differences in the age of onset, gender predominance and response to therapy. The most common and aggressive subgroup, posterior fossa ependymoma group A (PF-EPN-A), occurs in young children and appears to lack recurrent somatic mutations. Conversely, posterior fossa ependymoma group B (PF-EPN-B) tumours display frequent large-scale copy number gains and losses but have favourable clinical outcomes. More than 70% of supratentorial ependymomas are defined by highly recurrent gene fusions in the NF-κB subunit gene RELA (ST-EPN-RELA), and a smaller number involve fusion of the gene encoding the transcriptional activator YAP1 (ST-EPN-YAP1). Subependymomas, a distinct histologic variant, can also be found within the supratetorial and posterior fossa compartments, and account for the majority of tumours in the molecular subgroups ST-EPN-SE and PF-EPN-SE. Here we describe mapping of active chromatin landscapes in 42 primary ependymomas in two non-overlapping primary ependymoma cohorts, with the goal of identifying essential super-enhancer-associated genes on which tumour cells depend. Enhancer regions revealed putative oncogenes, molecular targets and pathways; inhibition of these targets with small molecule inhibitors or short hairpin RNA diminished the proliferation of patient-derived neurospheres and increased survival in mouse models of ependymomas. Through profiling of transcriptional enhancers, our study provides a framework for target and drug discovery in other cancers that lack known genetic drivers and are therefore difficult to treat.

Basic Science of Pediatric Brain Tumors

Recent advances in genomic and transcriptomic technologies have revolutionized our knowledge of the genetic and molecular basis of pediatric brain tumors. These discoveries have pinpointed novel genes and pathways, identified distinct molecular subgroups, and have led to developments of new mouse models. This chapter details our current understanding of the basic science of pediatric brain tumors, providing an outline of disease mechanisms and potential targets for molecular therapy.

Abstract LB-179: Integrated (epi)genomic analyses identify subgroup-specific therapeutic targets in CNS rhabdoid tumors

Atypical Teratoid Rhabdoid Tumors (ATRTs) are the most common malignant embryonal brain tumors arising in younger children that are distinctly lethal cancers for which effective therapies are lacking. Although ATRTs exhibit substantial clinical heterogeneity, exome studies reveal a relatively bland coding genome with only recurrent alterations of SMARCB1. Despite apparent genomic/genetic homogeneity, we recently reported that ATRTs comprise at least two transcriptional subclasses that correlate with different clinical features and treatment outcomes. However, the biological mechanisms and basis for molecular and therapeutic heterogeneity in ATRTs remained unclear. In this study, we integrated whole genome, exome, RNAseq as well as genome wide methylation and nucleosomal profiling analyses to comprehensively define the genomic and epigenomic landscape of ATRT sub-groups and identify sub-group specific therapeutic targets. Integration of multiplatform genomic analyses revealed novel recurrent genetic alterations in upto 20% of ATRTs. We observed predominantly structural coding events that targeted genes with functions in neural development and epigenetic regulation including BCR, MKL1 and EP300, thus suggesting greater complexity to the ATRT genome than previously appreciated. Global methylation (162) and gene expression analyses (90) of primary tumors indicated further segregation of ATRTs into three epigenetic sub-groups (group 1, 2A and 2B) that correlated with distinct lineage enriched gene expression profiles, global and SMARCB1 specific genotypes and different anatomic tumor locations and age at diagnosis. Group 1 ATRT exhibited enrichment of neurogenic/NOTCH signaling loci (ASCL1, FABP7, MYCN, C1ORF61, HES5/6, DLL1) and were predominantly supra-tentorial tumors arising in children at a median age of 24 months. In contrast Group 2A tumors arose predominantly in infra-tentorial locations in the youngest patients, while group 2B tumors were characteristically spinal in location. BMP signaling and mesenchymal differentiation genes (BMP4, BAMBI, PDGFRB) were commonly enriched in group 2A and B tumors; Group 2B tumors were additionally characterized by enrichment of MYCC, HOXB & C gene clusters. Remarkably, ATAC-seq analyses revealed distinct chromatin landscape associated with each ATRT sub-group, that correlated strikingly with sub-group specific therapeutic response in ATRT cell lines to a panel of signaling (NOTCH, BMP, Dasatinib) and epigenetic (EZH2, G9a, BRD4) inhibitors. Significantly, we discovered that differential methylation of a novel, PDGFRβ associated enhancer element confers robust sensitivity to tyrosine kinase inhibitors Dasatinib and Nilotinib in group 2 ATRTs, and suggest these as novel agents for this highly lethal ATRT sub-type. Citation Format: Jonathon Torchia, Shengrui Feng, King Ching Ho, Louis Letourneau, Daniel Picard, Tiffany S. Chan, Alexandre Vasiljevic, Dong Anh Khuong Quang, Brian Golbourn, Dalia Barsyte-Lovejoy, Constanze Zeller, Patrick Sin-Chan, Natalia R. Agamez, Mei Lu, Lucie Lafay-Cousin, Joseph D. Norman, Maryam Fouladi, Lindsey M. Hoffman, Stefan Rutkowski, Torsten Pietsch, Alexander R. Judkins, Eric Bouffet, James T. Rutka, Cynthia E. Hawkins, Cheryl H. Arrowsmith, Daniel De Carvalho, Nada Jabado, Annie Huang. Integrated (epi)genomic analyses identify subgroup-specific therapeutic targets in CNS rhabdoid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-179.