Functional validation and targeting of ENPP1 in Ewing sarcoma
Ewing sarcoma (EwS) is the second most common bone and soft tissue tumor of children and young adults. Despite being one of the most aggressive pediatric cancers, outcomes for EwS patients have not changed in decades, with limited treatment options for patients with metastatic or relapsed disease. Thus, there is an urgent need to identify new and effective therapeutic candidates suitable for the development of alternative treatments to improve disease prognosis. Immunotherapy is one such option, but EwS is characterized by a very immunosuppressive tumor microenvironment (i.e., the immune system is not very active in EwS tumors). We performed a comprehensive analysis of EwS expressed proteins located on the plasma membrane to identify new promising candidates for immunotherapy. Among the newly identified proteins, we discovered ectonucleotide pyrophosphate/phosphodiesterase 1 (ENPP1) as one of the most abundantly expressed proteins on EwS cells but with limited expression in normal tissues. ENPP1 is a surface enzyme that is known to play an immunomodulatory role in several cancer types by dampening proinflammatory antitumor immunity and promoting disease relapse and metastasis growth. Therefore, we postulate that overexpression of ENPP1 on EwS cells may be one of the strategies for securing an immunosuppressive tumor milieu. In this proposal, we will perform functional characterization of ENPP1 in EwS (Aim 1) and develop and evaluate antibody-based therapies targeting ENPP1 in EwS (Aim 2). We will use conventional molecular biology tools to describe the role of ENPP1 in EwS disease development and progression, by evaluating the impact of ENPP1 genetic loss on cellular phenotypes (proliferation, migration, invasion, and resistance to common stressors), as well as coculturing assays with immune cells to evaluate the immunomodulatory role of ENPP1. Moreover, to identify the cellular pathways in which ENPP1 is involved, we will perform so-called whole proteome analysis to determine the changes in cellular proteins when ENPP1 is deleted from cells and will also evaluate which other proteins that ENPP1 binds to in EwS cells, to help determine its function. Furthermore, we will test the ability of ENPP1 depleted EwS cells to form tumors and develop distant metastasis when they are injected into mice. This data will be supported by direct visualization and characterization of metastatic lung colonization using an assay we established to visualize lungs in mice injected with EwS cells. We will leverage the high ENPP1 expression in EwS for potential therapeutic use by identifying ENPP1-specific antibodies that can also block the enzyme properties of ENPP1, combined them with cytotoxic payloads, and evaluate their ability to induce antibody-dependent cellular cytotoxicity. Together, this data will provide an entirely novel and more comprehensive resource for the understanding of mechanisms of immune evasion by EwS cells.