TRAF6 modulates PD-L1 expression through YAP1-TFCP2 signaling in melanoma

Background Immunotherapy represented by the programmed death-1 (PD-1)/ligand 1 (PD-L1) monoclonal antibodies has led tumor treatment into a new era. However, the low overall response rate and high incidence of drug resistance largely damage the clinical benefits of existing immune checkpoint therapies. Recent studies correlate the response to PD-1/PD-L1 blockade with PD-L1 expression levels in tumor cells. Hence, identifying molecular targets and pathways controlling PD-L1 protein expression and stability in tumor cells is a major priority. Methods In this study, we first performed a Stress and Proteostasis CRISPR interference library-based screening to identify PD-L1 positive modulators. We then used in vitro and in vivo assays to investigate the biological function and mechanism of TRAF6 and its downstream YAP1/TFCP2 signaling in malignant melanoma. Results Here, we identified TRAF6 as a critical regulator of PD-L1 in melanoma cells. Suppression of TRAF6 expression down-regulates PD-L1 expression on the membrane surface of melanoma cells. We also found that PD-L1 protein abundance is regulated by YAP1/TFCP2 transcriptional complex. TRAF6 stabilizes YAP1 by K63 poly-ubiquitination modification, subsequently promoting the formation of YAP1/TFCP2 and PD-L1 transcription. Furthermore, inhibition of TRAF6 by Bortezomib enhanced cytolytic activity of CD8+ T cells by reduction of endogenous PD-L1. Notably, Bortezomib enhances anti-tumor immunity to an extent that is comparable to anti-PD-1 mAb therapies with no obvious toxicity. Conclusions These findings uncover a novel molecular mechanism for regulating PD-L1 protein abundance by a E3 ligase in cancer cells and reveal the potential of using TRAF6 inhibitors to stimulate internal anti-tumor immunological effect for TRAF6-PD-L1 overexpressing cancers. ### Competing Interest Statement The authors have declared no competing interest. * CRISPR : Clustered Regularly Interspaced Short Palindromic Repeats TRAF6 : Tumor Necrosis Factor Receptor Associated Factor 6 PD-L1 : Programmed death ligand 1 PD-L2 : Programmed death ligand 2 YAP1 : Yes-associated protein 1 TFCP2 : Transcription Factor CP2 JAK2 : Janus Kinase 2 PTEN : Phosphatase and Tensin Homolog PI3K : Phosphoinositide 3-kinase EGFR : Epidermal Growth Factor Receptor CDK5 : Cyclin Dependent Kinase 5 IFN-γ : IFN-gamma mAb : monoclonal antibody