Investigating the roles of extracellular cGAMP and harnessing it for cancer treatment

项目总结 癌症免疫治疗的目标是唤醒人体的抗肿瘤免疫反应，以对抗 所有患者的所有癌症。我们的实验室最近发现了一种关键机制，通过这种机制，我们的 先天免疫系统。我们现在知道，具有内在染色体不稳定性的癌细胞不断地隐藏着 可溶性小分子2‘3’-环-GMP-AMP(CGAMP)。作为免疫递质，细胞外 CGAMP被周围的组织和免疫细胞摄取，以旁分泌的方式激活其受体刺痛， 从而产生下游的抗癌免疫反应。细胞外cGAMP的重要性被证明 根据我们的发现，在乳腺癌小鼠模型中，它是电离辐射疗效所必需的。 此外，我们还鉴定了胞外酶ENPP1是唯一可检测到的胞外水解酶 CGAMP阻断其信号通路。因此，我们假设ENPP1是一种先天免疫 检查站，可以成为目标，以扩大癌症免疫治疗的范围。 虽然ENPP1是cGAMP唯一的水解酶，但它以类似的效力水解胞外ATP。 因此，基于完全消除ENPP1活性的遗传和药理学工具不能 用于区分胞外cGAMP和ATP的生理作用。在这里，我们建议选择性地 遗传和化学生物学方法：我们将描述选择性取消ENPP1的遗传工具的特征‘S 水解酶活性朝向胞外cGAMP而不是ATP(DENPP1cGAMP)；同时，我们将开发底物- 特定的ENPP1抑制剂作为工具化合物和潜在的免疫疗法。 在目标1中，我们将充分描述突变体的动力学、选择性和作用机制。 DENPP1cGAMP，以及dEnpp1cGAMP小鼠的病理生理。在目标2中，我们将评估多种肿瘤 在dEnpp1cGAMP小鼠模型中确定细胞外cGAMP在 控制肿瘤生长和与检查点阻滞剂的协同作用。在目标3中，我们将首先描述机制 我们的先导cGAMP选择性ENPP1抑制剂的底物选择性，然后使用该抑制剂来驾驭 胞外cGAMP的抗肿瘤作用这项建议将仔细的生化分析与MICE相结合 遗传学和工具化合物开发以解决细胞外cGAMP在癌症中的作用，目标是 提高癌症免疫治疗水平。

PROJECT SUMMARY The goal of cancer immunotherapy is to awaken the body’s anti-tumoral immune response to fight against all cancers in all patients. Our lab recently identified a key mechanism by which cancer cells are detected by our innate immune system. We now know that cancer cells, with intrinsic chromosomal instability, constantly secret the soluble small molecule 2’3’-cyclic-GMP-AMP (cGAMP). Acting as an immunotransmitter, extracellular cGAMP is taken up by surrounding tissues and immune cells to activate its receptor STING in a paracrine fashion, resulting in downstream anti-cancer immune responses. The importance of extracellular cGAMP is demonstrated by our finding that it is required for the curative effect of ionizing radiation in a murine model of breast cancer. Furthermore, we identified the extracellular enzyme ENPP1 as the only detectable hydrolase of extracellular cGAMP that blocks its signaling pathway. Therefore, we hypothesize that ENPP1 is an innate immune checkpoint that could be targeted to expand the reach of cancer immunotherapy. Although ENPP1 is cGAMP’s only hydrolase, it hydrolyzes extracellular ATP at comparable potency. Therefore, genetic and pharmacological tools that are based on complete ablation of ENPP1 activity cannot be used to distinguish the physiological role between extracellular cGAMP and ATP. Here, we propose selective genetic and chemical biology approaches: we will characterize genetic tools that selectively abolish ENPP1’s hydrolase activity toward extracellular cGAMP but not ATP (dENPP1cGAMP); in parallel we will develop substrate- specific ENPP1 inhibitors as tool compounds and potential immunotherapeutics. In Aim 1, we will fully characterize the kinetics, selectivity, and mechanisms of action of mutant dENPP1cGAMP, as well as the pathophysiology of dEnpp1cGAMP mice. In Aim 2, we will evaluate multiple tumor models in the dEnpp1cGAMP mouse strain to determine the physiological roles of extracellular cGAMP in controlling tumor growth and synergism with checkpoint blockers. In Aim 3, we will first characterize mechanism of substrate selectivity of our lead cGAMP-selective ENPP1 inhibitor and then use this inhibitor to harness the anti-tumoral effects of extracellular cGAMP. This proposal combines careful biochemical analyses with mouse genetics and tool compound development to address the role of extracellular cGAMP in cancer, with the goal of improving cancer immunotherapy.