Identification of genes regulated by ID2 in Ewing sarcoma cells.

The ID family of proteins (ID1-4), which bind to basic helix-loop-helix (bHLH) transcription factors and prevent bHLH-directed transcription, are critical regulators of the differentiation and chemoresistance of cancer cells derived from multiple cellular lineages. ID2 was previously shown to impair the in vitro differentiation of human mesenchymal stem cells. However, the functional role, if any, of ID2 in regulating differentiation, developmental pathways, and the oncogenic phenotype of Ewing sarcoma tumors is unknown. We used CRISPR/Cas9 to knockout ID2 in Ewing sarcoma cell lines and identified that the loss of ID2 significantly decreases cell growth in vitro and in vivo in a xenograft experiment. Using RNA-seq and gene set enrichment analysis with ID2-knockout and ID2-Knockout-Rescue cell lines we identified that ID2 regulates genes related to differentiation and development in Ewing sarcoma cell lines. We performed RNA-seq with wildtype, ID2-knockout, and ID2-Knockout-Rescue Ewing sarcoma TC71 cell lines.