Abstract Background: Expression of programmed death-ligand 1 (PD-L1) and down-regulation of MHC class I are well known tumor immune evasion mechanisms. The purpose of this study is to investigate the regulation of PD-L1 and MHC class I expression in head and neck squamous cell carcinoma (HNSCC) cell lines by MEK inhibitor trametinib. Methods: Six HNSCC cell lines (SNU-1041, SNU-1066, SNU-1076, PCI-13, Detroit 562, and FaDu) were used. Trametinib was purchased from Selleckchem. Growth inhibition of trametinib was measured using MTT assay. Trametinib was dosed at 10 - 640 nM for investigating their effect on PD-L1 and MHC class I molecule expression. Changes of PD-L1 and MHC class I (including HLA-A2) expression levels were analyzed by flow cytometry after treatment with trametinib (20 nM) and/or IFN-γ (1 - 10 ng/ml). Total and phosphorylated STAT1 and STAT3 were analyzed by western blot. Results: Overall IC50 values of trametinib were over 10 μM in cell lines except SNU-1066 and Detroit 562 with IC50 values still higher than 100 nM. All the six cell lines have no upstream RAS or RAF mutation. Although trametinib treatment did not inhibit growth of these cells, it up-regulated MHC class I expression level in all cells. Furthermore, trametinib enhanced exogenous IFN-γ-induced MHC class I up-regulation in four out of six cell lines. Mechanistically, trametinib treatment increased phosphorylated STAT1 levels by IFN-γ in all cells, and subsequent total STAT1 levels in two out of six cell lines. Interestingly, trametinib treatment directly phosphorylated STAT3 although total STAT3 level was not changed. Either trametinib alone or with IFN-γ induce PD-L1 expression, but the effect was various in these cells. These results imply that blockade of MAPK pathway may alternatively activate JAK/STAT pathway in HNSCC cells, and MHC class I and PD-L1 expression levels were changed as the results Conclusion: Our results suggest that MEK pathway is closely related with regulation of PD-L1 and MHC class I expression. Up-regulated levels of PD-L1 and MHC class I by inhibiting MEK pathway suggest a possible synergistic role of MEK inhibitor with anti-PD-1/PD-L1 immunotherapy in HNSCC. Citation Format: Seong-Ho Kang, Bhumsuk Keam, Soyeon Kim, Miso Kim, Yong-Oon Ahn, Tae Min Kim, Dong-Wan Kim, Dae Seog Heo. MEK pathway inhibition upregulates PD-L1 and MHC class I expression levels in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4029.