Good afternoon, everyone. My name is Cody Nesvick. I'm a PGY-5 neurosurgery resident at Mayo Clinic. I work in the lab of Dave Daniels, and I'll be discussing the epigenetic and phenotypic impact of SMARCB1 restoration in ATRT. This work was funded by an NREF Research Fellowship grant sponsored by the Academy of Neurological Surgeons, to whom I am very grateful. ATRT is a rare CNS cancer of young children. Despite surgical resection, high-dose chemotherapy, and radiotherapy, the median overall survival remains less than a year, and long-term survivors suffer significant treatment-related morbidity. ATRT is very unique amongst cancers in its genomic stability. The only recurring genomic event in 98% of cases is loss of SMARCB1, which is a subunit of the SWE-SNF or BAF chromatin remodeling complex, which plays a key role in regulation of enhancers. In ATRT, typical enhancers involved in cell differentiation are lost, and residual super-enhancer activity likely contributes to tumorigenesis in specific patterns, yielding at least three known molecular subtypes. The epigenetic mechanisms by which SMARCB1 loss leads to tumorigenesis are not yet well-defined. To determine the cellular and molecular features lost and gained in ATRT, our study question is, how does SMARCB1 restoration impact the epigenetic landscape and phenotype of ATRT? To answer that question, we have a library of ATRT cell lines in which SMARCB1 is re-expressed to be a lentiviral vector. These cells are then analyzed in a battery of assays that assess phenotypic, epigenomic, and transcriptomic changes. We then integrate all these data to make new hypotheses about tumorigenesis and cell differentiation. This figure demonstrates the phenotypic impact of SMARCB1 restoration in ATRT cell lines. Panel B shows data from an incusite proliferation assay showing that SMARCB1 restoration significantly abrogates the proliferative capacity of adherent ATRT cell lines. In panel C, we see that SMARCB1 expressing ATRT cells of both MYC and sonic hedgehog subtypes have dramatically diminished clonogenic capacity as well, indicating that SMARCB1 loss continues to be a requirement for cancer-like features in vitro and that SMARCB1 restoration significantly abrogates these features. Given that the BATH complex is necessary for normal chromatin remodeling, we next performed ATAC-Seq, which is a transposase-based assay that defines open regions of chromatin. On the left are landscape tracings. The blue peaks that rise from the horizon of each tracing represent areas of open chromatin, and a flat line indicates regions of chromatin that are closed and therefore not accessible. We see that open regions specific to ATRT MYC and tyrosinase tumors are conserved in ATRT MYC cell lines, and that subtype specificity of open regions in human tumors is retained in vitro, indicating these regions serve an important pathophysiologic function in ATRT and are conserved in our model. When we look at all differentially accessible regions, we see many regions are opened and closed following SMARCB1 restoration. The opened regions are expectedly enriched at regions far from transcriptional start sites, consistent with the creation of new regulatory elements, whereas closed regions are highly enriched near transcriptional start sites. Interestingly, these regions do not overlap with subtype specific regions of chromatin openness, indicating they are generally not sites of differential regulation. So what regions are being regulated? This is a really busy slide, but the message is pretty simple. There is a lot of overlap between cellular programs that are predicted by ATAC-seq data and observed in the transcriptome after SMARCB1 restoration. Specifically, pathways involved in cell morphology, differentiation, and cellular identity are enriched in ATRT cells expressing SMARCB1, and these might be predicted by differentially accessible regions. In conclusion, SMARCB1 loss remains necessary for the display of cancer-like features of ATRT cells, and SMARCB1 restoration significantly reduces their proliferative and clonogenic capacity. Re-expression of SMARCB1 makes regions of chromatin involved in cell morphology and identity more accessible, but the mechanisms by which this occurs are unclear. Our current work is defining the role of specific factors that initiate cellular programs after SMARCB1 restoration. Thank you all very much for listening, and I'd be happy to take any questions in the Q&A session.

SMARCB1 restoration

ATRT

CNS cancer

epigenetic impact

phenotypic impact