PIKfyve regulation of IL-12 signaling in dendritic cells and cancer immunity

项目摘要/摘要 自从IL-12细胞因子被发现以来，它一直被认为是免疫的“主调节器” 系统。IL-12主要由树突状细胞产生，是连接先天适应性的关键信号分子 免疫以驱动干扰素𝛾𝛾/Th1介导的T细胞对病原体和癌症的反应。临床前研究已经 Long表明，加强IL-12信号转导可以改进疫苗。最近，树突状细胞中的IL-12被发现 在小鼠癌症模型中抗PD1疗效所需的药物。尽管有数据表明IL-12途径 作为癌症的治疗靶点，以前在临床试验中直接给药IL-12的尝试导致了 患者死于细胞毒性反应或因IL-10介导的负反馈而疗效不佳。后续 治疗癌症的努力主要集中在改进传递和释放IL-12的方法上，但收效甚微。 因此，有必要更好地了解树突状细胞中IL-12调节的生物学特性 确定新的、安全和有效的靶向该途径的策略作为抗癌策略。 本课题组先前发现PIKfyve脂蛋白激酶是一种新的高效、高效、高效的靶点。 耐受性和口服多激酶抑制剂ESK981。我们证明了这种药物有能力 减少多种同基因小鼠癌症模型中的肿瘤生长，并加强抗PD1治疗。 有趣的是，药物的疗效取决于CD8T细胞的存在，以及IL-12和干扰素𝛾𝛾和 体内的信号通路。此外，ESK981可直接增强原发树突状细胞IL-12信号转导 通过转录调控IL12B在体外和体内的细胞。 因此，本项目的总体目标是明确PIKfyve和PIKfyve对IL-12的调节机制 证明PIKfyve抑制的性质，作为一种抗癌疗法，在抗肿瘤免疫反应中。这个 最重要的假设是，抑制PIKfyve诱导树突状细胞中的IL-12信号，以增强抗原- 癌症中CD8T细胞的特异性反应。目标1中的实验将调查PIKfyve的效果 小鼠树突状细胞对IL-12基因和蛋白亚基的敲除和条件性敲除 体外和体内的信号通路。为了检测树突状细胞亚群，如CD8CD8和CD103CDC， 带有BATF3基因敲除的小鼠将被纳入体外和体内研究。AIM 2中的实验将 PIKfyve抑制剂ESK981和Apilimod对树突状细胞的潜在抗肿瘤作用 PIKfyve条件性基因敲除、BATF3基因敲除和野生型小鼠。我们将监测肿瘤的进展和 检测肿瘤引流淋巴结和肿瘤微环境中的T细胞反应，包括OVA 模型抗原和新抗原四聚体对MC38肿瘤特异性CD8 T细胞的激活作用 模特。这项拟议的研究将为树突状细胞中IL-12信号的生物学和 可以为选择PIKfyve抑制剂作为单独增强IL-12的安全、有效的方法提供依据 并与依赖IL-12的治疗相结合，如检查点抑制剂和癌症疫苗。

PROJECT SUMMARY/ABSTRACT Since the discovery of the IL-12 cytokine, it has been considered a “master regulator” of the immune system. Primarily produced by dendritic cells, IL-12 is the key signaling molecule linking innate to adaptive immunity to drive IFN𝛾𝛾/Th1-mediated T cell responses against pathogens and cancer. Preclinical studies have long shown that enhancing IL-12 signaling improves vaccines. Recently, IL-12 in dendritic cells was shown to be required for anti-PD1 efficacy in mouse models of cancer. Despite the data nominating the IL-12 pathway as a therapeutic target in cancer, previous attempts to directly administer IL-12 in clinical trials have resulted in patient death from cytotoxic response or poor efficacy due to IL-10-mediated negative feedback. Subsequent efforts in cancer have largely focused on improving methods to deliver and release IL-12 with limited success. Therefore, there is a significant need to better understand the biology of IL-12 regulation in dendritic cells to identify novel, safe and effective strategies of targeting this pathway as an anti-cancer strategy. Our group previously identified PIKfyve lipid kinase as a novel target of the highly efficacious, well- tolerated and orally administered multi-kinase inhibitor, ESK981. We demonstrated the ability of this drug to reduce tumor growth in multiple syngeneic mouse models of cancer and enhance anti-PD1 therapy. Interestingly, drug efficacy was dependent on the presence of CD8+ T cells, and the IL-12 and IFN𝛾𝛾 and signaling pathways in vivo. Furthermore, ESK981 could directly enhance IL-12 signaling in primary dendritic cells in vitro and in vivo through transcriptional regulation of IL12B. Therefore, the overall goal of this project is to define the mechanism of IL-12 regulation by PIKfyve and demonstrate the nature of PIKfyve inhibition, as an anti-cancer therapy, in anti-tumor immune responses. The overarching hypothesis is that PIKfyve inhibition induces IL-12 signaling in dendritic cells to enhance antigen- specific, CD8+ T cell responses in cancer. Experiments in Aim 1 will investigate the effect of Pikfyve knockdown and conditional knock-out in mouse dendritic cells on the genes and protein subunits in the IL-12 signaling pathway in vitro and in vivo. To examine dendritic cell subsets, such as CD8α+ and CD103+ cDCs, mice with genetic knock-out of Batf3 will be included for in vitro and in vivo studies. Experiments in Aim 2 will study the potential anti-tumor effect of PIKfyve inhibitors, including ESK981and Apilimod, on dendritic cell Pikfyve conditional knock-out, Batf3 knock-out, and wild type mice. We will monitor tumor progression and examine T cell responses in the tumor draining lymph nodes and tumor microenvironment, including OVA model antigen and neoantigen specific CD8+ T cell activation using tetramers available for the MC38 tumor model. The proposed study will provide greater insight into the biology of IL-12 signaling in dendritic cells and could provide the rationale for selecting PIKfyve inhibitors as safe, effective methods of enhancing IL-12 alone and in combination with IL-12-dependent therapies, such as checkpoint inhibitors and vaccines in cancer.