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 虽然许多有希望的候选放射增敏剂已经被追求，但临床批准的一种 放射增敏剂仍然是临床放射生物学的“圣杯”。我们的提案代表了一个令人兴奋的联盟 传统翻译基础科学与尖端技术的结合：使用CRISPR筛选文库 寻找新的分子靶点，使体内的癌细胞辐射增敏，既有内在的，也有通过 免疫系统的参与(目标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)。Weichselbaum实验室是辐射领域的先驱 40多年来的治疗突破，以及以下一代研究人员为特色的芒古索实验室 他们是体内CRISPR筛查这一新兴领域的世界领先者，使我们唯一有资格使用 尖端技术解决了一个长期存在的问题，将立即改善临床放射治疗。 具体地说，我们计划使用体内CRISPR-筛选新的癌细胞内在遗传靶点 增加或降低与检查站封锁或不与检查站封锁相结合的放射效果，直接或通过 免疫系统的参与。通过确定哪些sgRNA在接受治疗的小鼠和未接受治疗的小鼠中被耗尽， 这表明引入了致敏的功能丧失，我们可以识别RT的假定靶点 单独或在组合策略中。SgRNA将根据枯竭程度进行排序。候选目标将是 评选依据是(I)具有最高的累积分数，(Ii)新颖性，以及(Iii)MC38和 LLC肿瘤模型。我们将使用类似SBRT和分段IR方案+/-检查点封锁(反 PD1/anti-CTLA-4)在体内测试三个直接增加放射治疗效果的靶点，即在 免疫缺陷小鼠和提高IR联合检查点治疗效果的三个靶点 在免疫功能正常的小鼠体内阻断。最后，我们将检查外显子组测序数据中的靶扩增。 来自我们机构接受RT或放射免疫治疗的患者。此外，尽管不是主要的 这项提案的焦点是，对辐射小鼠中丰富的sgRNA而不是耗尽的sgRNA的检查将指向 靶向基因在癌症放射抗性中可能起到未知的作用。 我们提出的支持我们的建议的研究结果突出了地方之间相互作用的关键作用 肿瘤细胞和免疫细胞在照射后肿瘤微环境中决定治疗结果 单纯放射治疗或联合免疫治疗。发现新奇需要不偏不倚的方法 并更好地确定组合战略的优先次序，以改善治疗。顺利完成我们的学业 将解决长期寻求的未得到满足的放射增敏剂需求，以改善癌症患者的预后。这 这将是第一次基于CRISPR的体内辐射增敏肿瘤基因靶点的筛选。一个活体内 对小鼠的筛选将首次使发现潜在的间接放射增敏靶点成为可能 除了直接的(内在的)放射增敏靶标外，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.