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

项目摘要 癌症免疫治疗的目标是唤醒人体的抗肿瘤免疫反应， 所有癌症患者。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，癌细胞，固有的染色体不稳定性，不断秘密 可溶性小分子2 '3'-环-GMP-AMP（cGAMP）。作为一种免疫递质，细胞外 cGAMP被周围组织和免疫细胞摄取以旁分泌方式激活其受体STING， 导致下游抗癌免疫应答。证明了细胞外cGAMP的重要性 我们发现，电离辐射在乳腺癌小鼠模型中的疗效需要它。 此外，我们鉴定了胞外酶ENPP 1作为唯一可检测的胞外蛋白水解酶。 cGAMP阻断其信号通路。因此，我们假设ENPP 1是一种先天性免疫， 检查点，可以有针对性地扩大癌症免疫治疗的范围。 虽然ENPP 1是cGAMP的唯一水解酶，但它以相当的效力水解细胞外ATP。 因此，基于ENPP 1活性的完全消除的遗传和药理学工具不能被应用于临床。 用于区分细胞外cGAMP和ATP之间的生理作用。在这里，我们建议选择性地 遗传和化学生物学方法：我们将描述选择性消除ENPP 1的遗传工具 水解酶对细胞外cGAMP的活性，但不对ATP（dENPP 1cGAMP）的活性;同时，我们将开发底物- 作为工具化合物和潜在的免疫治疗剂的特异性ENPP 1抑制剂。 在目标1中，我们将充分表征突变体的动力学、选择性和作用机制。 dENPP 1cGAMP，以及dEnpp 1cGAMP小鼠的病理生理学。在目标2中，我们将评估多个肿瘤 在dEnpp 1cGAMP小鼠品系中建立模型，以确定细胞外cGAMP在 控制肿瘤生长并与检查点阻断剂协同作用。在目标3中，我们将首先描述机制 我们的cGAMP选择性ENPP 1抑制剂的底物选择性，然后使用这种抑制剂来利用 细胞外cGAMP的抗肿瘤作用。这项建议结合了细致的生化分析与小鼠 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 改善癌症免疫疗法。

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.