Surface and global proteome analyses identify ENPP1 and other surface proteins as actionable immunotherapeutic targets in Ewing sarcoma

Clin Cancer Res. 2023 Oct 9. doi: 10.1158/1078-0432.CCR-23-2187. Online ahead of print.


Purpose: Ewing sarcoma (EwS) is the second most common bone sarcoma in children, with 1 case per 1.5 million in the United States. While the survival rate of patients diagnosed with localized disease is ~70%, this decreases to ~30% for patients with metastatic disease and only ~10% for treatment refractory disease, which have not changed for decades. Therefore new therapeutic strategies are urgently needed for metastatic and refractory EwS.

Experimental design: This study analyzed 19 unique EwS patient or cell line-derived xenografts (from 14 primary and 5 metastatic specimens) using proteomics to identify surface proteins for potential immunotherapeutic targeting. Plasma membranes were enriched using density gradient ultracentrifugation and compared to a reference standard of 12 immortalized non-EwS cell lines prepared in a similar manner. In parallel, global proteome analysis was carried out on each model to compliment the surfaceome data. All models were analyzed by Tandem Mass Tags (TMT)-based mass spectrometry to quantify identified proteins.

Results: The surfaceome and global proteome analyses identified 1,131 and 1,030 annotated surface proteins, respectively. Among surface proteins identified, both approaches identified known EwS-associated proteins including IL1RAP, CD99, STEAP1, and ADGRG2, and many new cell surface targets, including ENPP1 and CDH11. Robust staining of ENPP1 was demonstrated in EwS tumors compared to other childhood sarcomas and normal tissues.

Conclusions: Our comprehensive proteomic characterization of the EwS surfaceome provides a rich resource of surface-expressed proteins in EwS. This dataset provides the preclinical justification for exploration of targets such as ENPP1 for potential immunotherapeutic application in EwS.