Investigating transcriptional programs in hyperploid Ewing Sarcoma cells

Ava Stephens

Mentor: Dr. Markus Hoffmann, Assistant Professor, Oncology, Georgetown University.

Date/Time: August 25th, 2026 at 8:15 PM.

Abstract: Ewing Sarcoma is an aggressive bone and soft tissue cancer that primarily affects children and young adults. It is often associated with poor outcomes when metastasis occurs, particularly to the bone. Previous studies indicated that Ewing sarcoma bone metastasis is initiated by a population of polyploid tumor cells arising in hypoxia. However, the mechanisms driving their osseous dissemination are not fully understood. To investigate transcriptional differences that may contribute to this metastatic behavior, I analyzed RNA-seq data from the SKES1 Ewing Sarcoma cell line under two conditions: a normal diploid state (NOR, 2N) and a hyperploid state (HYP, 4N), each with three biological replicates.

Using DESeq2, I identified substantial differences in gene expression between HYP and NOR cells. Principal component analysis showed clear separation between the two conditions, with PC1 accounting for 99% of the variance, while heatmaps and differential expression analysis further demonstrated distinct transcriptional profiles. GAP43, a gene of interest in this project, was strongly upregulated in HYP cells. Gene ontology over-representation analysis across multiple log2 fold change thresholds consistently identified enrichment of developmental and signaling processes, including pattern specification, axogenesis, axon guidance, neuron projection guidance, and embryonic organ development. MSigDB Gene Set Enrichment Analysis also identified changes in metabolic and signaling programs, including oxidative phosphorylation, fatty acid metabolism, inflammatory response, and NF-κB related signaling. Overall, these results show that hyperploid SKES1 cells undergo broad transcriptional changes involving developmental, neuronal guidance, metabolic, and stress response pathways. These changes may contribute to the increased plasticity and metastatic behavior of hyperploid Ewing Sarcoma cells and provide candidate pathways for further investigation.