Abstract 2676: GSK3β inhibition by small molecule 9-ING-41 decreases VEGF and other cytokines, and boosts NK and T cell-mediated killing of colorectal tumor cells
Authors: Huntington Kelsey E. , Zhang Shengliang , Carneiro Benedito A. , El-Deiry Wafik S. 
Published: July 2021

The GSK3β/beta-catenin axis activates vascular endothelial growth factor (VEGF) signaling in endothelial cells to promote angiogenesis. VEGF has been noted as immunosuppressive, it dampens the immune cell response by promoting recruitment of tumor associated macrophages (TAMs), and has been proposed as a target to modulate antitumor immunity. A panel of human colorectal cell lines was chosen to provide a varied mutational background (TP53, KRAS, BRAF, TRK, APC, PIK3CA). Cells were treated with 9-ING-41, a selective and potent small molecule inhibitor of GSK3β, at doses up to 50 µM for 72 hours to determine IC10, IC30, IC50, and IC70. Cytokine, chemokine, and growth factor levels were analyzed in the tumor cell culture supernatants after treatment at IC10, IC30, IC50, and IC70 for 48 hours using a Luminex 200 multiplexing instrument. Co-culture experiments were conducted with GFP+ SW480 colorectal cancer cells and either NK-92 natural killer cells or TALL-104 T cells at various effector/target ratios in a 48-well plate, in the presence or absence of 9-ING-41. Overall cytokine levels showed a decreasing trend in response to increasing doses of 9-ING-41. Among the most prominently decreased growth factors in the profile was VEGF. In an immune cell-killing co-culture assay we observed a significant increase in natural killer (NK-92) cell and T cell (TALL-104) killing of the colorectal cancer cells in response to treatment with 9-ING-41 as compared to controls without drug treatment. Follow-up experiments compared the effect of pre-treating either the effector or the target cell population with 9-ING-41 before the co-culture experiment was started. Pre-treatment of the target tumor cells, but not the effector immune cells, bolstered cell-killing, implying that the drug is sensitizing the tumor cells to killing by the immune cells. Moreover, we saw an increase in the expression of chemokine CXCL14 (BRAK) in the tumor cell culture supernatant with increasing doses of 9-ING-41. BRAK is known to stimulate activated NK cell migration and could have a beneficial therapeutic effect by increasing NK cell migration into the tumor microenvironment. Furthermore, we saw a decrease in macrophage colony-stimulating factor (M-CSF) which, along with VEGF, has been associated with recruitment of TAMs. We hypothesize that a 9-ING-41-mediated decrease of VEGF in conjunction with a 9-ING-41-mediated increase of BRAK secreted by the tumor cells may increase the capacity of NK- and T cell-mediated killing of the tumor cells. Utilizing a compound such as 9-ING-41 could be a way to increase the host's anti-tumor immune response to decrease tumor burden in conjunction with other therapeutic agents.

Citation Format: Kelsey E. Huntington, Shengliang Zhang, Benedito A. Carneiro, Wafik S. El-Deiry. GSK3β inhibition by small molecule 9-ING-41 decreases VEGF and other cytokines, and boosts NK and T cell-mediated killing of colorectal tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2676.