eEF2K promotes PD-L1 stabilization through inactivating GSK3β in melanoma

Abstract

BackgroundImmune checkpoint blockade (ICB) targeting programmed death ligand-1 (PD-L1)/programmed cell death protein-1 (PD-1) pathway has become an attractive strategy for cancer treatment; however, unsatisfactory efficacy has limited its clinical benefits. Therefore, a more comprehensive understanding of the regulation of PD-L1 expression is essential for developing more effective cancer immunotherapy. Recent studies have revealed the important roles of eukaryotic elongation factor 2 kinase (eEF2K) in promoting epithelial-mesenchymal transition (EMT), angiogenesis, tumor cell migration and invasion; nevertheless, the exact role of eEF2K in the regulation of tumor immune microenvironment (TIME) remains largely unknown.MethodsIn this study, we used a cohort of 38 patients with melanoma who received anti-PD-1 treatment to explore the association between eEF2K expression and immunotherapy efficacy against melanoma. Immunoprecipitation-mass spectrometry analysis and in vitro assays were used to examine the role and molecular mechanism of eEF2K in regulating PD-L1 expression. We also determined the effects of eEF2K on tumor growth and cytotoxicity of CD8+ T cells in TIME in a mouse melanoma model. We further investigated the efficacy of the eEF2K inhibition in combination with anti-PD-1 treatment in vivo.ResultsHigh eEF2K expression is correlated with better therapeutic response and longer survival in patients with melanoma treated with PD-1 monoclonal antibody (mAb). Moreover, eEF2K protein expression is positively correlated with PD-L1 protein expression. Mechanistically, eEF2K directly bound to and inactivated glycogen synthase kinase 3 beta (GSK3β) by phosphorylating it at serine 9 (S9), leading to PD-L1 protein stabilization and upregulation, and subsequently tumor immune evasion. Knockdown of eEF2K decreased PD-L1 expression and enhanced CD8+ T cell activity, thus dramatically attenuating murine B16F10 melanoma growth in vivo. Clinically, p-GSK3β/S9 expression is positively correlated with the expressions of eEF2K and PD-L1, and the response to anti-PD-1 immunotherapy. Furthermore, eEF2K inhibitor, NH125 treatment or eEF2K knockdown enhanced the efficacy of PD-1 mAb therapy in a melanoma mouse model.ConclusionsOur results suggest that eEF2K may serve as a biomarker for predicting therapeutic response and prognosis in patients receiving anti-PD-1 therapy, reveal a vital role of eEF2K in regulating TIME by controlling PD-L1 expression and provide a potential combination therapeutic strategy of eEF2K inhibition with ICB therapy.

Reduction of eEF2 kinase alleviates the learning and memory impairment caused by acrylamide

“…The primary role of eukaryotic elongation factor 2 (eEF2) is to participate in the elongation phase of protein synthesis, with the phosphorylation of eEF2 representing a key mechanism in this phase. Eukaryotic elongation factor 2 kinase (eEF2K) is the exclusive protein kinase known to phosphorylate eEF2 at the 56th threonine residue (Thr-56, p-eEF2) (Chen et al, 2022). Studies indicate that eEF2K is expressed in neurons and may contribute to processes such as learning, memory, and depression (Klupt and Jia, 2023).…”

The role of ubiquitin pathway‐mediated regulation of immune checkpoints in cancer immunotherapy

“…Conversely, inhibition of β‐TrCP or certain special molecules that inactivate GSK3β could in turn block PD‐L1 ubiquitination, promote its stability, and eventually, induce cancer immunosuppression. Recently, we found that eukaryotic elongation factor 2 kinase (eEF2K) promotes immunosuppression through GSK3β inactivation‐mediated PD‐L1 stabilization in melanoma, providing a potential combinational therapeutic strategy to enhance the efficacy of immunotherapy 83 . The CKLF‐like MARVEL transmembrane domain‐containing protein 6 (CMTM6), which is expressed on the cell surface, colocalized with PD‐L1 at the plasma membrane to interrupt PD‐L1 ubiquitination and prolong its half‐life, and as a result, enhanced the capacity of cancer cells to escape immune surveillance.…”

“…Recently, we found that eukaryotic elongation factor 2 kinase (eEF2K) promotes immunosuppression through GSK3β inactivation-mediated PD-L1 stabilization in melanoma, providing a potential combinational therapeutic strategy to enhance the efficacy of immunotherapy. 83 The CKLF-like MARVEL transmembrane domain-containing protein 6 (CMTM6), which is expressed on the cell surface, colocalized with PD-L1 at the plasma membrane to interrupt PD-L1 ubiquitination and prolong its half-life, and as a result, enhanced the capacity of cancer cells to escape immune surveillance. CMTM6 depletion improved PD-L1 mediated Tcell suppression, highlighting potential values of CMTM6 as a therapeutic target or in enhancing the efficacy of the present PD-L1/PD-1 blocking therapies.…”

Combination Approaches to Target PD-1 Signaling in Cancer

“…More importantly, anti-PD-1 and GSK-3 inhibitor combinations may be effective to treat solid tumors that are otherwise unresponsive to immune checkpoint blockade. This is likely because, GSK-3β phosphorylates PD-L1 in tumor cells to induce its degradation, and GSK-3β inactivation can be seen in some cancers to stabilize PD-L1 expression ( 116 , 117 ). Similarly, inhibiting GSK-3β with the chemotherapy-sensitization combination disulfiram and copper stabilizes PD-L1 expression in a hepatocellular carcinoma model ( 118 ).…”