Cancer Cell Intrinsic Interferon-I pathway Activation by Fractionated Radiation

 描述（由申请人提供）：最近的证据表明，最强大的抗肿瘤T细胞识别源自个体肿瘤表达的独特突变蛋白的新抗原，这表明需要精确接种疫苗以诱导癌症患者的有效抗肿瘤免疫。重要的是，这种抗肿瘤T细胞在治疗性免疫检查点阻断后在晚期疾病阶段引起肿瘤消退。我们开创了探索使用局部肿瘤放疗（RT）作为产生原位个体化疫苗的手段的研究。我们是第一个在临床前模型中证明RT通过诱导对内源性肿瘤抗原特异性的T细胞使对CTLA-4阻断无反应的肿瘤敏感的人。我们和其他人的新证据表明，RT在临床上也有类似的效果。然而，缺乏有关机制的知识，阻碍了快速进展，有效利用RT作为免疫佐剂。一个关键的未回答的问题是剂量和分割是否影响RT引发抗肿瘤免疫应答的能力。我们先前已经在两种小鼠癌中发现，通过以8戈伊的3个部分给予RT而不是通过单次20戈伊剂量，在诱导免疫介导的辐射和同步非辐射肿瘤消退（远位效应）中与抗CTLA-4治疗协同的原位疫苗的产生得以实现，这表明所采用的RT方案是关键的。我们现在有数据支持这一假设，即癌细胞内在激活的I型干扰素（IFN-I）途径的RT需要产生原位疫苗，这表明RT触发典型的防御途径在肿瘤上皮细胞，模仿病毒感染。我们的数据还表明，分次（FRT），而不是单剂量（SDRT）辐射可以通过激活细胞溶质DNA传感器环GMP-AMP合酶（cGAS）和下游刺激因子IFN基因（STING）在辐射的癌细胞内实现这一点。为了测试上述假设，将采用几种小鼠和人癌细胞来确定哪种RT剂量/分级激活cGAS/STING。接下来，将使用植入同基因免疫活性野生型、cGAS缺陷型和STING缺陷型小鼠中的具有cGAS或STING的选择性敲低的癌细胞进行体内实验，以确定该途径在RT介导的介导肿瘤消退和远位应答的抗肿瘤T细胞的诱导中的作用。为了确定由辐射的癌细胞产生的IFN-I的作用是癌细胞自主的还是需要宿主DC中的信号传导，IFNAR 1缺陷型小鼠将被用作肿瘤受体。将建立癌细胞衍生的IFN-I和DC募集至肿瘤之间的关系。最后，我们将研究RT激活癌细胞中cGAS/STING通路的机制。总的来说，获得的数据将有重要的意义，一个新的使用放射治疗作为一个相对简单和广泛使用的方式为个性化的肿瘤疫苗。

 DESCRIPTION (provided by applicant): Recent evidence indicates that the most powerful anti-tumor T cells recognize neoantigens derived from unique mutated proteins expressed by an individual tumor, suggesting that precision vaccination is required to induce effective anti-tumor immunity in cancer patients. Importantly, such anti-tumor T cells cause tumor regression at advanced disease stages upon therapeutic immune checkpoints blockade. We have pioneered studies exploring the use of local tumor radiotherapy (RT) as a means to generate an in situ individualized vaccine. We were the first to demonstrate in a pre-clinical model that RT sensitizes unresponsive tumors to CTLA-4 blockade by inducing T cells specific for endogenous tumor antigens. Emerging evidence by us and others suggests a similar effect of RT in the clinic. However, lack of knowledge about the mechanisms involved precludes rapid progress towards the effective use of RT as an immune adjuvant. One critical unanswered question is whether dose and fractionation affect RT ability to elicit anti-tumor immune responses. We have previously found in two mouse carcinomas that generation of an in situ vaccine synergistic with anti-CTLA-4 treatment in inducing immune-mediated regression of irradiated and synchronous non-irradiated tumors (abscopal effect) was achieved by RT given in 3 fractions of 8 Gy but not by a single 20 Gy dose, suggesting that the RT regimen employed is critical. We now have data supporting the hypothesis that carcinoma cell-intrinsic activation of type I interferon (IFN-I) pathway by RT is required to generate an in situ vaccine, suggesting that RT triggers canonical defense pathways in neoplastic epithelial cells that mimic a viral infection. Our data also indicat that fractionated (FRT) but not single dose (SDRT) radiation can accomplish this via activation of the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) and downstream stimulator of IFN genes (STING) within the irradiated cancer cells. To test the above hypothesis several mouse and human carcinoma cells will be employed to determine which RT dose/fractionation activates cGAS/STING. Next, in vivo experiments using cancer cells with selective knockdown of cGAS or STING implanted in syngeneic immunocompetent wild type, cGAS-deficient, and STING-deficient mice will be performed to determine the role of this pathway in RT-mediated induction of anti-tumor T cells that mediate tumor regression and abscopal responses. To determine whether the effects of IFN-I produced by irradiated cancer cells are cancer cell autonomous or require signaling in host DC, IFNAR1-deficient mice will be used as tumor recipient. The relationship between cancer cell-derived IFN-I and DC recruitment to tumors will be established. Finally, we will investigate the mechanisms whereby RT activates the cGAS/STING pathway in cancer cells. Overall, data obtained will have important implications for a novel use of radiotherapy as a relatively simple and widely available modality for individualized tumor vaccination.