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

项目概要/摘要 自从发现IL-12细胞因子以来，它一直被认为是免疫调节的“主调节剂”。 系统由树突状细胞产生的IL-12是连接先天性和适应性的关键信号分子。 免疫力驱动IFNγ/Th 1介导的T细胞应答以对抗病原体和癌症。临床前研究已经 长期以来，增强IL-12信号传导可以改善疫苗。最近，树突状细胞中的IL-12被证明是 在小鼠癌症模型中的抗PD 1功效所需要的。尽管有数据表明IL-12通路 作为癌症的治疗靶点，先前在临床试验中直接施用IL-12的尝试已经导致 患者死于细胞毒性反应或因IL-10介导的负反馈而导致疗效不佳。后续 在癌症方面的努力主要集中在改进递送和释放IL-12的方法，但成功有限。 因此，非常需要更好地理解树突状细胞中IL-12调节的生物学， 确定靶向该途径的新型、安全和有效的策略作为抗癌策略。 我们的研究小组先前将PIKfyve脂质激酶鉴定为高效、良好- 耐受和口服多激酶抑制剂ESK 981。我们证明了这种药物能够 在多种同基因小鼠癌症模型中减少肿瘤生长并增强抗PD 1治疗。 有趣的是，药物疗效依赖于CD 8 + T细胞的存在，以及IL-12和IFNγ的表达。 体内的信号通路。此外，ESK 981可直接增强原代树突状细胞中IL-12信号转导。 通过IL 12 B的转录调节在体外和体内对细胞进行研究。 因此，本项目的总体目标是确定PIKfyve调节IL-12的机制， 证明PIKfyve抑制作为抗癌疗法在抗肿瘤免疫应答中的性质。的 总体假设是PIKfyve抑制诱导树突状细胞中的IL-12信号传导以增强抗原- 特异性，CD 8 + T细胞反应。目标1中的实验将研究Pikfyve的效果 小鼠树突状细胞中IL-12基因和蛋白亚基的敲除和条件性敲除 在体外和体内的信号通路。为了检查树突状细胞亚群，如CD 8 α+和CD 103 + cDC， 体外和体内研究将包括基因敲除Batf 3的小鼠。目标2的实验将 研究PIKfyve抑制剂，包括ESK 981和阿吡莫德，对树突状细胞的潜在抗肿瘤作用 Pikfyve条件性敲除、Batf 3敲除和野生型小鼠。我们将监测肿瘤进展， 检查肿瘤引流淋巴结和肿瘤微环境中的T细胞反应，包括OVA 使用可用于MC 38肿瘤的四聚体的模型抗原和新抗原特异性CD 8 + T细胞活化 模型这项拟议的研究将为树突状细胞中IL-12信号传导的生物学提供更深入的了解， 可以为选择PIKfyve抑制剂作为单独增强IL-12的安全、有效方法提供依据 以及与IL-12依赖性疗法（例如癌症中的检查点抑制剂和疫苗）组合。

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.