EPCO-54. MULTIOMIC ANALYSIS OF SPINAL EPENDYMOMAS REVEALS MECHANISMS OF TUMOR AGGRESSIVENESS WITH MYCN AMPLIFICATION

Abstract Grade-3 spinal ependymomas, particularly those harboring MYCN amplification, have dismal prognoses despite optimal treatment regimens. MYCN functions as a transcription factor (TF) to drive the expression of key tumor driver genes. Our aim is to identify mechanisms underlying tumor aggressiveness and treatment resistance in MYCN-amplified ependymomas. MYCN amplification in spinal ependymomas was identified with custom next generation sequencing panels and bulk methylation analysis. We then conducted paired single-nucleus RNA (snRNA-seq) and chromatin accessibility (snATAC-seq) sequencing of grade-2 (n=2, 1 patient) and grade-3 spinal ependymomas (n=6, 6 patients, 4 MYCN-amplified). Comparator snRNA-seq datasets (grade-2/grade-3; 8/2) were included from a publicly available dataset (GSE163686; all non-MYCN-amplified). Canonical cell classes were identified with a combination of cluster- and cell-based strategies. Downstream analysis was conducted using R 4.3 (Seurat, Signac, clusterProfiler, CellChat), and Python 3.11 (scanpy) packages. We identified ependymoma tumor cells distinct from canonical immune cell classes with gene expression analysis and confirmed a distinct copy-number-variation pattern in these cells with chromosome 1 and 2p gain, and chromosome 6p and 22q loss. MYCN-amplified tumor cells (compared to non-MYCN-amplified grade2/3 tumor cells) showed increased differential accessibility at genes including VEGFA. Increased MYCN TF activity was confirmed with gene overexpression of downstream targets including NOTCH3, CREB5, PLK1, and VEGFA. Other genes that were highly accessible and overexpressed in MYCN-amplified tumor cells were related to oxidative stress, differentiation, and anti-apoptotic pathways. MYCN-amplified tumor cells modified the tumor microenvironment with increased VISTA signaling between MYCN-amplified tumor cells and tumor-associated macrophages, creating an immunosuppressive environment. We confirmed these effects by observing a higher proportion of M2-like macrophages in MYCN-amplified tumors (29.6% vs 18.2%). In conclusion, MYCN amplification in spinal ependymomas upregulates tumor cell proliferation, angiogenesis, and M2-like macrophage recruitment/programming.