Small-molecule Inhibition of Glycogen Synthase Kinase-3 Sensitizes Colorectal Cancer Cells and Stimulates Immune Cells to Bolster Immune Cell-mediated Tumor Cytotoxicity

项目摘要 在全球范围内，结直肠癌（CRC）的发病率排名第三，死亡率排名第二。免疫检查点阻断 (ICB)在微卫星不稳定性（MSI）阳性CRC中表现出令人印象深刻的疗效，然而， 对于96%的微卫星稳定（MSS）晚期CRC患者， 回答。越来越多的文献支持糖原合成酶激酶-3（GSK-3）在免疫系统中的免疫调节作用。 抗肿瘤免疫的背景。该研究的重点是GSK-3抑制剂9-ING-41。我们假设 GSK-3抑制可以通过以下方式改善ICB的功效：（1）通过增加免疫细胞的活性来激活免疫细胞， 炎症介质和降低检查点受体表达，以及（2）致敏肿瘤 细胞通过增加的死亡受体表达和信号传导对免疫细胞介导的肿瘤细胞杀伤， 增加的检查点配体表达和减少的存活途径信号传导。第一年，目标1 实验将检测9-ING-41促进抗肿瘤免疫、修饰NF-κB信号传导和 在体外调节免疫检查点表达。在我们对9-ING-41在共培养试验中的初步评价中， 与对照相比，我们观察到用9-ING-41增加的免疫细胞介导的肿瘤细胞杀伤。我们将 分析用9-ING-41处理的肿瘤和免疫细胞的细胞因子谱， 对抗肿瘤免疫的影响。我们计划评估检查点配体表达和下游靶点， 在9-ING-41处理的细胞中使用蛋白质印迹、RNA-seq和流式细胞术的NF-κB信号传导。第二年，Aim 2 实验将利用具有免疫活性的同基因小鼠CRC模型来监测免疫细胞侵袭 9-ING-41单药治疗或ICB联合αPD治疗的肿瘤细胞杀伤 1或αPD-L1。这项工作将在Wafik El-Deiry博士的实验室进行，该实验室具有CRC，细胞因子和细胞因子的专业知识。 分析、体内实验设计、生物标志物和临床翻译。因为El-Deiry实验室位于 在布朗大学，我们可以使用最先进的生物成像、基因组学、显微镜和流式细胞术 设施布朗大学的癌症中心与实验室位于同一栋大楼内， 受邀演讲者研讨会、研究计划会议和转化研究疾病组会议， 而病理生物学项目提供特邀演讲者研讨会和期刊俱乐部。这种环境使 培训计划通过促进申请人的新技术技能，癌症生物学和免疫学专业知识， 并通过个性化的指导，教学和科学交流的机会。该项目将 导致申请人掌握新的技术和研究设计技能，除了职业发展 列入研究金培训计划。这项新的研究将提供机械的见解， GSK-3抑制剂的免疫调节机制，并将解决有效的 免疫治疗组合用于绝大多数对ICB治疗无反应的CRC患者。

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.