Mixed lineage kinase 3 inhibition induces T cell activation and cytotoxicity

Significance The agents that inhibit mitogen-activated protein kinases (MAPKs) are reported to have antineoplastic efficacies; however, their impact on immune cells is not clearly defined. We identified that genetic loss/inhibition of a MAP3K member, MLK3, increases CD8+ T cell cytotoxicity via inhibition of a prolyl isomerase, Ppia, and nuclear translocation of NFATc1. The MLK3 inhibitor increased the tumor infiltration of cytotoxic T cells in an immune-competent mouse model of breast cancer. Similarly, the MLK3 inhibitor increased the cytotoxic T cell population in pan T cells isolated from breast cancer patients with metastatic disease. These results suggest that small-molecule inhibitor of MLK3 might have clinical usage even in the advanced stage of the disease, where tumor-induced immunosuppression is frequent. Mixed lineage kinase 3 (MLK3), also known as MAP3K11, was initially identified in a megakaryocytic cell line and is an emerging therapeutic target in cancer, yet its role in immune cells is not known. Here, we report that loss or pharmacological inhibition of MLK3 promotes activation and cytotoxicity of T cells. MLK3 is abundantly expressed in T cells, and its loss alters serum chemokines, cytokines, and CD28 protein expression on T cells and its subsets. MLK3 loss or pharmacological inhibition induces activation of T cells in in vitro, ex vivo, and in vivo conditions, irrespective of T cell activating agents. Conversely, overexpression of MLK3 decreases T cell activation. Mechanistically, loss or inhibition of MLK3 down-regulates expression of a prolyl-isomerase, Ppia, which is directly phosphorylated by MLK3 to increase its isomerase activity. Moreover, MLK3 also phosphorylates nuclear factor of activated T cells 1 (NFATc1) and regulates its nuclear translocation via interaction with Ppia, and this regulates T cell effector function. In an immune-competent mouse model of breast cancer, MLK3 inhibitor increases Granzyme B-positive CD8+ T cells and decreases MLK3 and Ppia gene expression in tumor-infiltrating T cells. Likewise, the MLK3 inhibitor in pan T cells, isolated from breast cancer patients, also increases cytotoxic CD8+ T cells. These results collectively demonstrate that MLK3 plays an important role in T cell biology, and targeting MLK3 could serve as a potential therapeutic intervention via increasing T cell cytotoxicity in cancer.