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

体内 CRISPR 筛选对局部电离辐射敏感的新型癌细胞内在靶标，并可能与全身检查点封锁相结合。

• 批准号: 10684850
• 负责人: RALPH R WEICHSELBAUM
• 金额: $ 18.48万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-16 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10684850
• 关键词: Algorithms; Basic Science; Bioinformatics; Biological; CRISPR library; CRISPR screen; CRISPR/Cas technology; Cancer Patient; Cell Line; Cells; Chemicals; Chicago; Clinic; Clinical; Clinical Data; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Colon Carcinoma; DNA Damage; DNA Repair; Data; Data Set; Development; Gene Targeting; Genes; Genetic; Genetic Screening; Genomic DNA; Goals; Human; Immune; Immune system; Immunocompetent; Immunodeficient Mouse; Immunotherapy; Institution; Ionizing radiation; Irradiated tumor; Libraries; MC38; Malignant Neoplasms; Measures; Mediating; Modality; Modeling; Molecular Target; Mus; Patients; Phase; Play; Public Health; Qualifying; Radiation; Radiation Toxicity; Radiation therapy; Radiation-Sensitizing Agents; Radio; Radiobiology; Radioimmunotherapy; Radiosensitization; Research; Research Personnel; Role; Scheme; System; Technology; Testing; Therapeutic; Therapeutic Effect; Time; Toxic effect; Treatment outcome; Universities; Validation; anti-CTLA4; anti-PD-1; anti-PD-L1; anti-PD-L1 antibodies; cancer cell; cancer therapy; exome sequencing; experience; experimental study; gene discovery; genome editing; immune checkpoint blockade; improved; improved outcome; in vivo; in vivo evaluation; innovation; irradiation; loss of function; lung Carcinoma; next generation; novel; pharmacologic; pre-clinical; radiation effect; radiation resistance; resistance mechanism; screening; small molecule; small molecule inhibitor; treatment response; tumor; tumor growth; tumor microenvironment; vector

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.

项目概要 虽然人们一直在寻找许多有前途的候选放射增敏剂，但临床批准的放射增敏剂的开发 放射增敏剂仍然是临床放射生物学的“圣杯”，我们的建议代表了令人兴奋的结合。 传统转化基础科学与最先进技术的结合：利用 CRISPR 筛选文库 确定新的分子靶标，这些靶标可以内在地或通过 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）是 Weichselbaum 实验室之间的合作，该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 Manguso 实验室 他们是体内 CRISPR 筛选新兴领域的世界领导者，使我们具有独特的使用资格 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。 具体来说，我们计划使用体内 CRISPR 筛选新的癌细胞内在遗传靶标， 直接或通过检查点封锁来增加或减少放射治疗的效果 通过确定治疗组小鼠和未治疗组小鼠中哪些 sgRNA 被耗尽， 这表明引入了敏化功能丧失，我们可以确定 RT 的假定目标 单独或组合策略中的 sgRNA 将按耗尽程度进行排序。 选择基于 (i) 具有最高累积分数，(ii) 新颖性，以及 (iii) MC38 和 LLC 肿瘤模型。我们将使用类似 SBRT 和分次 IR 方案 +/- 检查点阻断（抗 PD1/抗CTLA-4）在体内测试直接增加放射治疗效果的三个靶点，即 免疫缺陷小鼠，以及增强IR联合检查点治疗效果的三个靶点 最后，我们将检查外显子组测序数据中的目标扩增。 来自在我们机构接受放疗或放射免疫疗法的患者，尽管不是主要疗法。 该提案的重点是，对受辐射小鼠中富集而不是耗尽的 sgRNA 进行检查将指出 其靶向可能在癌症放射抗性中发挥未知作用的基因。 我们提出的支持我们提案的调查结果强调了当地之间互动的关键作用 受辐射的肿瘤微环境中的癌细胞和免疫细胞，以确定治疗后的结果 需要单独的放射治疗或与免疫治疗相结合来发现新的治疗方法。 目标和更好地优先考虑组合策略以改善我们的研究。 将解决放射增敏剂长期以来未得到满足的需求，以改善癌症患者的治疗结果。 这将是第一个基于 CRISPR 的体内放射敏感性肿瘤遗传靶点筛选。 对小鼠进行筛选将首次能够发现潜在的间接放射增敏靶标 除了直接（内在）放射增敏剂目标之外，还需要免疫系统的参与。