In vivo CRISPR-screening of novel cancer cell-intrinsic targets that sensitize to local ionizing radiation, and possible combination with systemic checkpoint blockade.

项目摘要 虽然已经追求了许多有希望的候选放射增敏剂，但临床上批准的放射增敏剂的开发仍然是一个难题。 放射增敏剂仍然是临床放射生物学的“圣杯”。我们的提议代表了一个令人兴奋的联盟， 传统的转化基础科学与最先进的技术：使用CRISPR筛选库， 确定新的分子靶点，在体内对癌细胞进行放射增敏，包括内在的和通过 免疫系统的参与（目的1），并在体内小鼠肿瘤模型中进行测试，并使用独特的 临床数据集（目标2）。这是Weichselbaum实验室与辐射实验室的合作， 治疗突破超过40年，和Manguso实验室，具有下一代的研究人员 他们是体内CRISPR筛选这一新兴领域的世界领导者， 尖端技术，以解决一个长期存在的问题，将立即改善临床放射治疗。 具体来说，我们计划使用体内CRISPR筛选新的癌细胞内在遗传靶点， 直接或通过检查点阻断增加或降低放射联合或不联合的疗效 免疫系统的参与。通过确定哪些sgRNA在治疗的小鼠与未治疗的小鼠中被耗尽， 这将表明引入了致敏性功能丧失，我们可以确定RT的假定靶点 单独或组合策略。sgRNA将通过消耗程度进行排序。候选目标将是 选择基于（i）具有最高累积分数，（ii）新奇，以及（iii）在MC 38和MC 38中均被耗尽 LLC肿瘤模型。我们将使用类SBRT和分级IR方案+/-检查点阻断（抗- PD 1/抗CTLA-4）来体内测试直接增加放射治疗效果的三种靶点，即，在 免疫缺陷小鼠，以及三个增加IR与检查点组合的治疗效果的靶点 在免疫活性小鼠中的阻断。最后，我们将检查外显子组测序数据中的靶扩增 在我们机构接受RT或放射免疫治疗的患者。此外，虽然不是主要的 对于这一建议的焦点，在辐照小鼠中富集而不是耗尽的sgRNA的检查将指向 这些基因的靶向作用可能在癌症的辐射抗性中发挥未知的作用。 我们提出的支持我们的建议的研究结果强调了当地相互作用的关键作用， 癌细胞和免疫细胞在照射后的肿瘤微环境中，以确定治疗结果。 单独的放射疗法或与免疫疗法组合。需要无偏见的方法来发现新的 目标和更好地优先考虑组合策略，以改善治疗。顺利完成学业 将解决长期寻求的未满足的放射增敏剂需求，以改善癌症患者的预后。这 这将是第一个基于CRISPR的体内筛选肿瘤放射敏感性遗传靶点。体内 在小鼠中的筛选将首次能够发现潜在的间接放射增敏靶点， 除了直接的（内在的）放射增敏剂靶点外，还需要免疫系统的参与。

Project Summary While many promising candidate radiosensitizers have been pursued, the development of a clinically approved radiosensitizer remains a “holy grail” of clinical radiobiology. Our proposal represents an exciting union of traditional translational basic science with state-of-the-art technology: the use of a CRISPR screening library to identify new molecular targets that would radiosensitize cancer cells in vivo, both intrinsically and through the involvement of the immune system (Aim 1), and testing in both in vivo mouse tumor models and using a unique clinical dataset (Aim 2). This collaboration between the Weichselbaum lab, which has pioneered radiation therapy breakthroughs for over 40 years, and the Manguso lab, featuring the next generation of investigators who are world leaders in the nascent field of in vivo CRISPR screenings, makes us uniquely qualified to use cutting-edge technology to solve a long-standing problem that would immediately improve clinical radiotherapy. Specifically, we plan to use in vivo CRISPR-screening for novel cancer cell-intrinsic genetic targets that increase or decrease the efficacy of radiation combined or not with checkpoint blockade, directly or through the participation of the immune system. By determining which sgRNAs are depleted in treated vs. untreated mice, which would indicate that a sensitizing loss-of-function was introduced, we can identify putative targets for RT alone or in combinational strategies. sgRNAs will be ranked by degree of depletion. Candidate targets will be selected based on (i) having highest cumulative scores, (ii) novelty, and (iii) being depleted in both MC38 and LLC tumor models. We will use SBRT-like and fractionated IR schemes +/- checkpoint blockade (anti- PD1/anti-CTLA-4) to test in vivo three targets that increase the therapeutic effect of radiation directly, i.e., in immunodeficient mice, and three targets that increase the therapeutic effect of IR combined with checkpoint blockade in immunocompetent mice. Finally, we will examine target amplification in exome sequencing data from patients treated in our institution with RT or radio-immunotherapy. Additionally, although not the main focus for this proposal, an inspection of sgRNAs enriched instead of depleted in irradiated mice will point at genes whose targeting could play an unknown role in cancer radio-resistance. The findings we present supporting our proposal highlight the key role of the local interaction between cancer cells and immune cells in the irradiated tumor microenvironment to determine treatment outcome after radiotherapy alone, or combined with immunotherapy. Unbiased approaches are required to discover novel targets and better prioritize combination strategies to improve treatment. Successful completion of our studies will address the long sought unmet need of a radiosensitizer to improve outcomes for cancer patients. This would be the first in vivo CRISPR-based screening of genetic targets that radiosensitize tumors. An in vivo screening in mice will for the first time enable the discovery of potential indirect radio-sensitizing targets that would require the involvement of the immune system, in addition to direct (intrinsic) radio-sensitizer targets.