Cancer Cell Intrinsic Interferon-I pathway Activation by Fractionated Radiation

 描述(申请人提供)：最近的证据表明，最强大的抗肿瘤T细胞识别来自单个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才能在癌症患者中诱导有效的抗肿瘤免疫。重要的是，当治疗性免疫检查点被阻断时，这种抗肿瘤T细胞会在疾病晚期导致肿瘤消退。我们已率先开展研究，探索使用局部肿瘤放射治疗(RT)作为一种产生就地个体化疫苗的方法。我们是第一个在临床前模型中证明RT通过诱导针对内源性肿瘤抗原的T细胞而使对CTLA-4阻断无反应的肿瘤变得敏感。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，缺乏对相关机制的了解，阻碍了RT作为免疫佐剂的有效使用的快速进展。一个关键的悬而未决的问题是，剂量和分割是否会影响RT诱导抗肿瘤免疫反应的能力。我们此前在两个小鼠肿瘤中发现，在诱导放射和同步非辐射肿瘤的免疫介导消退(非内窥镜效应)方面，与抗CTLA-4治疗有协同作用的原位疫苗的产生是通过放射治疗(RT)实现的，剂量为8Gy3次，而不是单一的20Gy剂量，这表明所采用的放射治疗方案是关键的。我们现在有数据支持这样的假设，即需要通过RT激活I型干扰素(IFN-I)途径来产生原位疫苗，这表明RT在模拟病毒感染的肿瘤上皮细胞中触发了典型的防御途径。我们的数据还表明，分次(FRT)而不是单次剂量(SDRT)照射可以通过激活受照射癌细胞内胞浆DNA传感器环GMP-AMP合成酶(CGAS)和干扰素基因下游刺激物(STING)来实现这一点。为了验证上述假设，将使用几个小鼠和人类癌细胞来确定哪个RT剂量/分割激活cGAS/STIN。接下来，将进行体内实验，使用选择性击倒cGAS或STING的癌细胞植入同基因免疫活性野生型、cGAS缺陷型和STING缺陷型小鼠，以确定该途径在RT介导的抗肿瘤T细胞诱导中的作用，该T细胞介导肿瘤消退和异常反应。为了确定受辐射的癌细胞产生的干扰素-I的作用是癌细胞自主的还是需要宿主DC的信号，将使用IFNAR1缺陷小鼠作为肿瘤受体。肿瘤细胞来源的干扰素-I和DC向肿瘤的募集之间的关系将被建立。最后，我们将研究RT激活癌细胞中cGAS/STIN通路的机制。总体而言，所获得的数据将对放射治疗作为一种相对简单和广泛可用的个体化肿瘤疫苗接种方式的新用途具有重要意义。

 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.