Abstract
Abstract Uveal melanoma (UM) is the most common primary intraocular malignancy in human, and is associated with fatal metastases in up to 50% of patients despite effective primary tumor treatment. We previously showed that UMs can be divided into two prognostic subgroups based on gene expression profile (GEP), where class 1 GEP tumors have a low risk of metastasis, and those with the class 2 GEP have a high risk. Recently, we identified PRAME (Preferentially Expressed Antigen in Melanoma) as an independent predictor of metastasis in both class 1 and class 2 UM. In order to gain insight into the mechanisms by which PRAME drives tumor progression, we developed Tet-inducible cell lines allowing for the inducible expression (MEL290 Tet-On PRAME) and knockdown (FP41 Tet-shPRAME) of PRAME. PRAME expression correlates strongly with invasion in cell culture experiments. Further, we developed a Nod-Scid Gamma (NSG) mouse xenograft model using a specialized suprachoroidal injection technique that results in a pattern of metastasis that mimics human UM. In this model, UM cells that express high levels of PRAME developed significantly more and larger liver metastasis and had decreased survival rates compared to the same cell line in which PRAME was depleted using shRNA (p<0.028). These results indicate that PRAME is not only a biomarker of poor outcome, but a functional mediator of metastasis in UM. Furthermore, we performed mass spectrometry after immunoprecipitation and found that components of the Cul2 ubiquitin ligase complex were the most abundant PRAME binding proteins, which is similar to previous findings in leukemia, where PRAME was found to function as a substrate specificity motif for the Cul2 complex. To identify novel PRAME interacting partners and potential substrates of the PRAME-Cul2 complex, we then used a biotin ligase proximity labelling approach (BioID2), followed by biotin pulldown and mass spectrometry. This approach revealed multiple interacting partners from pathways known to modulate proliferation, cell cycle, and invasion. To quantify ubiquitination changes on these newly discovered interacting partners, we performed mass spectrometry after enriching for ubiquitinated peptides (PTMScan®). We observed that PRAME expression significantly changes the ubiquitome of the cells, including several of the interacting partners found by the BioID2 approach. In addition, global mass spectrometry was used to quantify changes in protein levels, allowing to correlate altered ubiquitination of proteins with their degradation. Taken together, these findings show that PRAME misexpression in UM promotes invasion and metastasis by effecting ubiquitomic and proteomic alterations. These findings could serve as a basis for novel drug discovery in the treatment of advanced UM. Citation Format: Margaret I. Sanchez, Stefan Kurtenbach, Jeffim N. Kuznetsov, Daniel A. Rodriguez, Dien G. Pham, Henry Barreras, J. William Harbour. PRAME misexpression alters protein ubiquitination and leads to increased invasion and metastasis in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2737.
