Abstract 7207: Targeting TGFBR2 neddylation suppresses metastatic/immunosuppressive abilities and enhances responsiveness to BRAF inhibitor in melanoma with BRAF V600E mutation

Abstract Background: Approximately 50% of melanomas harbor activating BRAF mutations (over 90% V600E). BRAF V600E mutation promotes melanoma progression, including metastasis and immunosuppression. While targeted therapies for metastatic BRAF V600E melanoma exist, resistance limits their efficacy. Crosstalk between TGF-β and receptor tyrosine kinase (RTK) signaling pathway is considered a possible mechanism for drug resistance to RTK inhibitors in melanoma with BRAF V600E mutation. In addition, the post-translational modification of TGFBR2 through a non-canonical protein neddylation pathway has been shown to prevent its proteolytic degradation by the proteasome system, which reinforces the activation of TGF-β signaling axis. This study explores whether targeting TGFBR2 neddylation suppresses metastasis/immunosuppression and enhances BRAF inhibitor response. Methods: Gene Set Enrichment Analysis (GSEA) was performed to predict the possible pathways suppressed by the treatment with the protein neddylation inhibitor MLN4924 in melanoma cells. Site-directed mutagenesis was employed to generate a TGFBR2 variant with mutation at its neddylation sites Lys556 and Lys567. MTT assays assessed cell survival post-treatment. Transwell cultivation and co-culture with activated T cells were utilized to estimate the cellular migration and immunosuppressive abilities of melanoma cells, respectively. Xenograft implantation was established to estimate tumor growth and metastatic ability of melanoma cells. Results: GSEA results demonstrated that the treatment with neural precursor cell-expressed, developmentally downregulated gene 8 (NEDD8)-activating enzyme inhibitor MLN4924 significantly suppresses epithelial-mesenchymal transition (EMT) progression and the activity of TGF-β signaling pathways in melanoma cells with BRAF V600E mutation. Conversely, TGFBR2 knockdown in the highly metastatic/BRAF V600E mutation melanoma cell lines RPMI7951 and A2058 suppressed migration and immunosuppressive abilities, but its overexpression in the poorly metastatic/BRAF V600E mutation melanoma cell lines A375 and C32 promoted cellular migration/immunosuppressive abilities and drug resistance to the BRAF inhibitor PLX4720 in vitro. The restoration of wild-type, not Lys556Arg/Lys567Arg mutant, rescued the cellular migration/immunosuppressive abilities and drug resistance characteristics of the TGFBR2-silenced RPMI7951 and A2058 cells. Animal experiments revealed that the overexpression of wild-type, not Lys556Arg/Lys567Arg mutant, enhances the metastatic ability and promotes drug resistance to PLX4720. Conclusion: Our data suggest that targeting TGFBR2 neddylation might be a novel strategy to combat the metastatic/immunosuppressive and BRAF inhibitor-resistant melanoma with BRAF V600E mutation. Citation Format: Leon Tsung-Ju Lee, Hui-Wen Chiu, Yuan-Feng Lin. Targeting TGFBR2 neddylation suppresses metastatic/immunosuppressive abilities and enhances responsiveness to BRAF inhibitor in melanoma with BRAF V600E mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7207.