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

项目总结 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。免疫检查点封锁 (ICB)在微卫星不稳定(MSI)阳性的结直肠癌中表现出令人印象深刻的疗效，然而，有 96%的微卫星稳定(MSS)晚期结直肠癌患者的需求仍未得到满足 请回答。越来越多的文献支持糖原合成酶激酶-3(GSK-3)在心脏疾病中的免疫调节作用。 抗肿瘤免疫的背景。拟议的研究重点是GSK-3抑制剂9-ING-41。我们假设 抑制GSK-3可通过以下途径提高ICB的疗效：(1)通过增加 炎症介质和Checkpoint受体表达减少以及(2)致敏肿瘤 通过增加死亡受体的表达和信号转导免疫细胞介导的肿瘤细胞杀伤， 增加检查点配体的表达，减少生存通路信号。在第一年，目标1 实验将检测9-ING-41促进抗肿瘤免疫，修改NF-κB信号转导的潜力，以及 在体外调节免疫检查点的表达。在我们对9-ING-41共培养实验的初步评价中 我们观察到，与对照组相比，9-ING-41增加了免疫细胞介导的肿瘤细胞杀伤率。我们会 分析9-ING-41处理的肿瘤和免疫细胞的细胞因子谱以预测治疗 对抗肿瘤免疫的影响。我们计划评估检查点配体的表达和下游靶点 在9-κ-41处理的细胞中使用免疫印迹、rna-seq和流式细胞术检测核因子-ING-B信号。在第二年，目标2 实验将利用免疫活性的同基因小鼠CRC模型来监测免疫细胞的入侵 和9-ING-41单独治疗或联合αPD治疗对肿瘤细胞的激活和杀伤作用 1或αPD-L1。这项工作将在Wafik El-Deary博士的实验室进行，该实验室拥有CRC、细胞因子等方面的专业知识 简介、活体实验设计、生物标记物和临床翻译。因为艾尔-戴利实验室位于 在布朗大学，我们可以获得最先进的生物成像、基因组学、显微镜和流式细胞术 设施。布朗癌症中心与实验室位于同一栋建筑内，为申请者提供 到受邀的演讲者研讨会、研究项目会议和转化研究疾病小组会议， 而病理生物学项目则提供受邀的演讲者研讨会和杂志俱乐部。此环境可实现 培训计划通过促进申请者的新技术技能、癌症生物学和免疫学专业知识， 并通过个性化的指导、教学和科学交流机会。该项目将 使申请人在职业发展之外，还能掌握新的技术和研究设计技能 包括在研究金培训计划中。这项新颖的研究将提供对 GSK-3抑制剂的免疫调节机制，并将解决有效的显著未满足的需求 免疫联合疗法适用于绝大多数对ICB治疗无效的结直肠癌患者。

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.