23 3330 Hospital Drive NW 24 Calgary, AB T2N 4N1 25 Canada 26 jawchan@ucalgary.ca 27 28 ABSTRACT 32Capicua (Cic) is a transcriptional repressor mutated in the brain cancer 33 oligodendroglioma. Despite its cancer link, little is known of Cic's function in the 34 brain. Here, we investigated the relationship between Cic expression and cell type 35 specification in the brain. Cic is strongly expressed in astrocytic and neuronal 36 lineage cells but is more weakly expressed in stem cells and oligodendroglial lineage 37 cells. Using a new conditional Cic knockout mouse, we show that forebrain-specific 38 Cic deletion increases proliferation and self-renewal of neural stem cells. 39 Furthermore, Cic loss biases neural stem cells toward glial lineage selection, 40 expanding the pool of oligodendrocyte precursor cells (OPCs). These proliferation 41 and lineage selection effects in the developing brain are dependent on de-repression 42 of Ets transcription factors. In patient-derived oligodendroglioma cells, CIC re-43 expression or ETV5 blockade decreases lineage bias, proliferation, self-renewal and 44 tumorigenicity. Our results identify Cic is an important regulator of cell fate in 45 neurodevelopment and oligodendroglioma, and suggest that its loss contributes to 46 oligodendroglioma by promoting proliferation and an OPC-like identity via Ets 47 overactivity. 48 49 INTRODUCTION 50 51The identification of genes recurrently mutated in cancer often presents opportunities to uncover 52 previously unappreciated mechanisms regulating normal development, and vice versa. The 53 transcriptional repressor Capicua (CIC) has been identified as a likely tumour suppressor gene, as 54 recurrent mutations in CIC and/or reduced expression of CIC are found in several cancer types. In the 55 brain, CIC mutations are nearly exclusively found in oligodendrogliomas (ODGs) -glial tumors that 56 are composed of cells resembling oligodendrocyte precursor cells (1, 2). Indeed, concurrent IDH1/2 57 mutation, single-copy whole-arm losses of 1p and 19q, and mutation of the remaining copy of CIC on 58 chr 19q13 are highly characteristic of ODG and are not found in other cancer types (3-5). These 59 associations suggest a unique relationship between CIC and glial biology. 61Prior work in Drosophila and in mammalian cultured cells has shown that Cic is a transcriptional 62 repressor downstream of receptor tyrosine kinase (RTK) signalling (6). Binding of Cic to the sequence 63 T(G/C)AATG(G/A)A in enhancers and promoters leads to transcriptional repression of its target genes 64 (7, 8). This default repression is relieved upon RTK signalling (6,(9)(10)(11), permitting transcription of 65 targets -among which are PEA3/ETS transcription factors ETV1/4/5 (12). To date, there is limited 66 knowledge of Cic's function in mammalian development or in the brain. Recently, Yang et al. using Cic 67 conditional knockout mice, reported that Cic loss increases a population of proliferating Olig2+ cells in 68 the brain, and that ) loss of Cic potentiates glial tum...

Pineoblastoma (PB), a rare and aggressive brain tumor affecting children, presents with a highly variable age distribution and treatment outcome. Our recent bulk tumor analyses of DNA methylation and mutational landscapes uncovered four discrete PB molecular subgroups (PB-miRNA1, PB-miRNA2, PB-MYC/FOXR2, and PB-RB), providing a major advance in our understanding of biological and clinical heterogeneity. However, developmental origins of PB subgroup heterogeneity and mechanisms governing how specific genetic alterations promote malignancy remain unknown. We conducted single-nucleus RNA sequencing of 38 primary tumors, including cases from all subgroups, to uncover intra-tumoral heterogeneity, developmental origins and expose selective dependencies. Transcriptional programs driving intra-tumoral heterogeneity showed subgroup-specific patterns such as mRNA splicing associated with PB-miRNA1/2 and phototransduction in PB-RB and PB-MYC/FOXR2. Next, we created a single-cell transcriptional atlas of the murine pineal gland across 11 developmental stages and found significant associations between PB subgroups and specific differentiation states of the pinealocyte lineage, suggesting subgroup-specific developmental origins. Characterization of pineal development informed generation of biologically faithful disease models, including a novel genetically engineered mouse model of the PB-RB subgroup. PB-Rb1 mouse tumors were histologically and molecularly validated for their fidelity to human tumor counterparts, exhibiting up-regulation of key pinealocyte lineage markers that are diagnostic in patients. Using cell lines derived from our PB-Rb1 mouse model we identified several transcription factor dependencies we believe are high-jacked during normal pinealocyte development. Our findings inform the developmental origins of pineoblastoma as well as provides candidates for potential therapeutic targets.

Pineoblastoma (PB) is a rare and aggressive childhood brain tumor with highly variable age and treatment-associated outcomes. Our recent bulk tumor analyses of DNA methylation and mutational landscapes uncovered four discrete PB molecular subgroups (PB-miRNA1, PB-miRNA2, PB-MYC/FOXR2, and PB-RB), providing a major advance in our understanding of biological and clinical heterogeneity. However, developmental origins of PB subgroup heterogeneity and mechanisms governing how specific genetic alterations promote malignancy remain unknown. To resolve the cellular origins of PB, we assembled a large single-nucleus RNA-sequencing cohort (n=32) of primary PB tumors, including representatives from each subgroup. Transcriptomic analysis identified subgroup-specific gene expression programs driving intra-tumoral heterogeneity. In addition, we discovered substantial differences in the expression of miRNA biogenesis genes between the PB-miRNA1 and PB-miRNA2 subgroups, providing mechanistic support for their distinct subgroup identities despite overlapping driver events. The MYC/FOXR2 subgroup was characterized by over-expression of the FOXR2 proto-oncogene in bulk RNA-seq, which we validated in single-nuclei and identified co-expressed downstream target genes. To map PB subgroups to their putative developmental beginnings, we created a single-cell transcriptional atlas of the murine pineal gland across 11 developmental stages (E11-P21). Trajectory inference within the developing pineal gland revealed a differentiation continuum of early, mid, and mature alpha-/beta pinealocytes. Cross-species correlation and deconvolution identified significant associations between multiple PB subgroups and specific differentiation states of the pinealocyte lineage, suggestive of developmental origins. Characterization of pinealocyte development informed generation of biologically faithful disease models, including a novel genetically engineered mouse model of the PB-RB subgroup. PB-Rb1 mouse tumors were histologically and molecularly validated for their fidelity to human tumor counterparts, exhibiting up-regulation of key pinealocyte lineage markers that are diagnostic in patients. Finally, high-throughput drug screening identified several promising pharmacological candidates that may attenuate consequences of Rb1 deficiency in affected children.