PROJECT SUMMARY Globally, colorectal cancer (CRC) ranks third in incidence and second in mortality. Immune checkpoint blockade (ICB) has demonstrated impressive efficacy in microsatellite instability (MSI)-positive CRC, however, there remains a substantial unmet need for 96% of patients with microsatellite stable (MSS) advanced CRC who don’t respond. A growing literature supports an immunomodulatory role of glycogen synthase kinase-3 (GSK-3) in the context of anti-tumor immunity. The proposed research focuses on GSK-3 inhibitor 9-ING-41. We hypothesize that GSK-3 inhibition can improve efficacy of ICB by (1) activating immune cells via increased inflammatory mediators and decreased checkpoint receptor expression and by (2) sensitizing tumor cells to immune cell-mediated tumor cell killing via increased death receptor expression and signaling, increased checkpoint ligand expression, and decreased survival pathway signaling. In year one, Aim 1 experiments will examine the potential of 9-ING-41 to promote anti-tumor immunity, modify NF-κB signaling, and regulate immune checkpoint expression in vitro. In our preliminary evaluation of 9-ING-41 in a co-culture assay we observed increased immune cell-mediated tumor cell killing with 9-ING-41, compared to control. We will analyze cytokine profiles of tumor and immune cells treated with 9-ING-41 to make predictions about therapeutic impact on anti-tumor immunity. We plan to evaluate checkpoint ligand expression and downstream targets of NF-κB signaling using western blots, RNA-seq, and flow cytometry in 9-ING-41-treated cells. In year two, Aim 2 experiments will utilize an immunocompetent, syngeneic murine CRC model to monitor immune cell invasion and activation, and tumor cell killing in response to 9-ING-41 monotherapy or combination ICB therapy with αPD- 1 or αPD-L1. This work will be carried out in Dr. Wafik El-Deiry’s lab, which has expertise in CRC, cytokine profiling, in vivo experimental design, biomarkers, and clinical translation. Because the El-Deiry Lab is located at Brown University, we have access to state-of-the-art bioimaging, genomics, microscopy, and flow cytometry facilities. The Cancer Center at Brown, located within the same building as the lab, offers the applicant access to invited speaker seminars, research program meetings, and translational research disease group meetings, while the Pathobiology Program offers invited speaker seminars and journal clubs. This environment enables the training plan by facilitating the applicant’s new technical skills, cancer biology and immunology expertise, and through individualized mentorship, teaching, and scientific communication opportunities. The project will lead to the applicant’s mastery of new technical and research design skills in addition to career development included within the fellowship training plan. This novel research will offer mechanistic insights into the immunomodulatory mechanisms of GSK-3 inhibitors and will address the significant unmet need of effective immunotherapy combinations for the vast majority of patients with CRC who don’t respond to ICB therapy.

PROJECT NARRATIVE Immune checkpoint blockade has demonstrated impressive efficacy in microsatellite instability positive colorectal cancer, however, there remains a substantial unmet need in the approximately 96% of patients with microsatellite stable advanced colorectal cancer who do not respond. This project will investigate the immunomodulatory mechanisms of glycogen synthase kinase-3 (GSK-3) inhibitor 9-ING-41 and the potential for GSK-3 inhibitors in combination with immune checkpoint blockade in the context of colorectal cancer. This novel research will offer mechanistic insights into the immunomodulatory mechanisms of GSK-3 inhibitors and will address the significant unmet need of effective immunotherapy combinations for the vast majority of patients with CRC who do not respond to immune checkpoint blockade therapy.