Tank Binding Kinase (Tbk1) Inhibitor Bx795 Impairs Anti-Tumor Efficacy Of T Cells By Abrogating Granzyme And Perforin Mobilization

TANK binding kinase 1 (TBK1) is a non-canonical IκB kinase that regulates the innate immune response and is highly expressed in lung, breast, pancreatic and colon cancers. It phosphorylates and activates downstream targets such as IRF3 and c-Rel and mediates NF-κB activation, facilitating the expression of pro-inflammatory genes and interferons. In addition to its role in regulating innate immunity, TBK1 also promotes oncogenesis downstream of K-Ras by phosphorylating Akt and promoting cell survival, proliferation, autophagy and mitophagy. Therefore, TBK1 inhibitors have been proposed to be potentially efficacious against inflammatory diseases and cancer. Unfortunately, TBK1 inhibitors have not been found to be highly efficacious anti-cancer agents and we made efforts to elucidate the underlying reasons. Earlier findings from our lab revealed a novel role for TBK1 in regulating mitosis. Specifically, we found that active phospho-TBK1 levels were increased during mitosis and TBK1 localized to the centrosomes, mitotic spindles and midbody; selective inhibition or silencing of TBK1 triggered defects in spindle assembly and prevented mitotic progression (Pillai et al., Nature Communications, 2015). TBK1 was found to facilitate the binding of the kinesis Kif2b to CEP170 and dynein to NuMA; depletion or inhibition of TBK1 significantly altered microtubule stability and dynamics, probably due to its regulation of dyenein-NuMA interaction. Since dynein function and appropriate microtubule dynamics is necessary for proper trafficking of vesicles for secretion, hypothesized that inhibition of TBK1 might adversely affect the tumor microenvironment by interfering with the mobilizing of cytotoxic molecules like granzyme and perforin by T cells. Hence we tested if inhibition of TBK1 would impair the anti-tumor activity of T-cells. Pilot immunofluorescence experiments conducted on JURKAT T-cells treated with the TBK1 inhibitor BX795 showed higher percent of cells retaining granzyme-b and perforin as compared to control cells. Further, we carried out in vitro cytotoxicity assay using normal human peripheral blood mononuclear cells (PBMC9s) treated with BX795 (2.5uM for 24 hrs), activated with T cell mitogen concanavalin A (1.25ug/ml) and co-cultured with KRAS mutant A549 lung cancer cells for 72 hrs. Our results showed that BX795 treatment of PBMC9s significantly inhibited the ability of the T cells to induce apoptosis in A549 cells, as measured by Annexin-V / PI +ve signal. These findings suggest that inhibiting TBK1 may be causing dysfunction of immune mediated T cell functions and interference with the anti-tumor immunity. Further experiments are in progress to study the regulatory role of TBK1 on different T cell populations and the associated cytokine signaling. Citation Format: Fatema Khambati, Jaya Padmanabhan, SriKumar Chellappan. Tank binding kinase (TBK1) inhibitor BX795 impairs anti-tumor efficacy of T cells by abrogating granzyme and perforin mobilization [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1011.