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

PIKfyve 对树突状细胞中 IL-12 信号传导和癌症免疫的调节

基本信息

批准号：
10451543
负责人：
Jae Eun Choi
金额：
$ 2.02万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10451543
关键词：
Ablation Affect Antigens Antiviral Agents Autoimmune Automobile Driving Biological Biological Response Modifiers Biology Bone Marrow CD8-Positive T-Lymphocytes Cancer Model Cancer Patient Cells Cessation of life Clinic Clinical Clinical Trials Communicable Diseases Complex Data Dendritic Cells Disease Endosomes Enzyme-Linked Immunosorbent Assay Feedback Flow Cytometry Gene Proteins Genetic Genetic Transcription Goals IL12B gene Immune checkpoint inhibitor Immune system Immunotherapy In Vitro Interleukin-10 Interleukin-12 Knock-out Knowledge Link Lipid III Lipids MC38 Malignant Neoplasms Measures Mediating Methods Modeling Monitor Mus Nature Oral Pathway interactions Patients Pharmaceutical Preparations Pharmacology Phase II Clinical Trials Phosphatidylinositols Phosphotransferases Protein Subunits Proteins Regulation Research Proposals Role Signal Pathway Signal Transduction Signaling Molecule T cell response T-Cell Activation T-Lymphocyte Transcriptional Regulation Translating Tumor Immunity Vaccines Vacuole Viral Wild Type Mouse Work adaptive immunity anti-PD-1 anti-PD1 therapy anti-cancer anti-tumor immune response antitumor effect cancer clinical trial cancer immunotherapy cancer therapy conditional knockout cytokine cytotoxic draining lymph node drug efficacy effector T cell enzyme linked immunospot assay experimental study improved in vivo inhibition of autophagy inhibitor inorganic phosphate insight kinase inhibitor knock-down late endosome mouse model neoantigens novel pathogen phosphatidylinositol 3-phosphate pre-clinical preclinical study response success targeted treatment therapeutic target transcriptome sequencing tumor tumor growth tumor immunology tumor microenvironment tumor progression

项目摘要

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.

项目概要/摘要自IL-12细胞因子被发现以来，它一直被认为是免疫系统的“主调节剂” 系统。 IL-12 主要由树突状细胞产生，是连接先天性和适应性的关键信号分子免疫驱动 IFN𝛾𝛾/Th1 介导的 T 细胞针对病原体和癌症的反应。临床前研究有长期研究表明，增强 IL-12 信号传导可以改善疫苗效果。最近，树突状细胞中的 IL-12 被证明可以需要在小鼠癌症模型中发挥抗 PD1 功效。尽管数据提名 IL-12 途径作为癌症的治疗靶点，之前在临床试验中直接施用 IL-12 的尝试已取得成果患者因细胞毒性反应而死亡，或因 IL-10 介导的负反馈而导致疗效不佳。随后的癌症领域的努力主要集中在改进递送和释放 IL-12 的方法，但成效有限。因此，迫切需要更好地了解树突状细胞中 IL-12 调节的生物学特性，以确定针对该途径作为抗癌策略的新颖、安全且有效的策略。我们的团队之前将 PIKfyve 脂质激酶确定为高效、良好的新靶标。耐受且口服的多激酶抑制剂 ESK981。我们证明了这种药物的能力减少多个同基因小鼠癌症模型中的肿瘤生长并增强抗 PD1 治疗。有趣的是，药物疗效取决于 CD8+ T 细胞、IL-12 和 IFN𝛾𝛾 的存在以及体内信号通路。此外，ESK981 可以直接增强初级树突状细胞中的 IL-12 信号传导通过 IL12B 的转录调节在体外和体内的细胞中发挥作用。因此，该项目的总体目标是明确PIKfyve对IL-12的调节机制以及证明 PIKfyve 抑制作为抗癌疗法在抗肿瘤免疫反应中的本质。这总体假设是 PIKfyve 抑制会诱导树突状细胞中的 IL-12 信号传导，从而增强抗原- 癌症中的特异性 CD8+ 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 基因和蛋白质亚基的敲低和条件敲除体外和体内信号通路。为了检查树突状细胞亚群，例如 CD8α+ 和 CD103+ cDC， Batf3 基因敲除的小鼠将被纳入体外和体内研究。目标 2 中的实验将研究 PIKfyve 抑制剂（包括 ESK981 和 Apilimod）对树突状细胞的潜在抗肿瘤作用 Pikfyve 条件敲除、Batf3 敲除和野生型小鼠。我们将监测肿瘤进展并检查肿瘤引流淋巴结和肿瘤微环境（包括 OVA）中的 T 细胞反应使用可用于 MC38 肿瘤的四聚体激活模型抗原和新抗原特异性 CD8+ T 细胞模型。拟议的研究将更深入地了解树突状细胞和 IL-12 信号转导的生物学可以为选择 PIKfyve 抑制剂作为单独增强 IL-12 的安全、有效方法提供依据并与 IL-12 依赖性疗法相结合，例如癌症检查点抑制剂和疫苗。