Tumor-barcoding coupled with high-throughput sequencing for quantitative radiogenomics of the abscopal response in NSCLC

癌症免疫治疗(IMT)在某些情况下可以产生强大而持久的抗肿瘤免疫反应。 然而，许多癌症对IMT没有反应，联合治疗需要积极 研究，特别是在致命肿瘤，如IMT不敏感的非小细胞肺癌(NSCLC)。临床前 总的来说，研究发现IMT制剂和放化疗的成功与否并不是很好的预测因素。 临床上的组合，可能是由于糟糕的构思和执行的治疗方案，过时的疾病模型 缺乏现有的交叉验证临床前结果的框架。有必要发展 针对现有IMT制剂结合放化疗的严格的临床前测试计划。非小细胞肺癌遗传 主要分子NSCLC亚型的工程化小鼠模型(GEMM)已经建立。然而， 据我们所知，没有NSCLC GEMM对IMT表现出可靠的“非范围”反应， 这是目前这项提议的一个独特优势。此外，现有GEMM的一个主要限制是相对 可以产生的不同的基因类型数量很少，而且缺乏定量精度。这项建议 利用一种新技术，肿瘤条形码和条形码深度测序(Tuba-Seq)和体内CRE-lox和 CRISPR/CRE介导的GEMMS以前所未有的方式模拟肿瘤发生和放射药物反应 精确度和基因组全面性。我们在本提案中通过以下两种方式使用R01机制 下面陈述的具体目标是为了测试以下中心假设：(I)我们假设同时使用两种方法 抗PD-L1和新型口服生物可用ATR抑制剂AZD6738联合放化疗 将改善免疫原性差的非小细胞肺癌转基因生物的持久抗肿瘤免疫反应；以及， (Ii)Tuba-seq我们将允许以前所未有的方式看到非小细胞肺癌反应的放射-药物遗传图景 在活体内。特定目标1-建立癌基因驱动的非小细胞肺的放射药物遗传学图谱 肿瘤对放化疗和放化疗/抗PD-L1联合治疗的影响我们建议使用新的 CRISPR/Cre介导的非小细胞肺癌GEMMs与抗PD-L1的最佳放化疗组合 IMT.然后，我们将用多参数研究IMT的放化疗作用机制。 免疫学方法和一种创新技术，使血统追踪和直接量化 体内对这些不同遗传背景的治疗效果。特异性AIM#2-确定肿瘤细胞 联合抗PD-L1免疫治疗和ATRI联合放化疗对癌基因的影响 导致非小细胞肺癌。这一目标利用我们的新型CRISPR/CRE介导的非小细胞肺癌GEMM来 检测基因型对抗PD-L1和ATRI联合放化疗的影响。通过使用所有这些工具， 我们将能够破译化学辐射和联合的分子基础的主要方面。 非小细胞肺癌患者的IMT抵抗，有助于改进治疗并积极影响患者的预后。

Cancer immunotherapy (IMT) can produce robust and durable anti-tumor immune responses in some cases. However, many cancers are non-responsive to IMT and combination approaches need to be actively investigated, particularly in lethal tumors such as IMT-insensitive non-small cell lung cancer (NSCLC). Preclinical studies in general have been found to be poor predictors of success for IMT agents and chemoradiotherapy combinations in the clinic, likely due to poorly conceived and executed treatment protocols, dated disease model systems and lack of an existing framework for cross-validation of preclinical results. There is a need to develop a rigorous preclinical testing program for existing IMT agents combined with chemoradiation. NSCLC genetically engineered mouse models (GEMMs) of the major molecular NSCLC subtypes have been created. However, there are no NSCLC GEMMs that to our knowledge has demonstrated “abscopal” responses reliably to IMT, which is one unique strength of this current proposal. Also, a major limitation of existing GEMMs is the relatively small number of different genotypes that can be generated and their lack of quantitative precision. This proposal leverages a new technique, tumor barcoding with barcode deep-sequencing (Tuba-seq) and in vivo Cre-lox and CRISPR/Cre-mediated GEMMs to model oncogenesis and radiation-drug response with unprecedented precision and genomic-comprehensiveness. We are using this R01 mechanism in the present proposal via two Specific Aims stated below to test the following central hypotheses: (i) we hypothesize that treating with both anti-PD-L1 and a novel orally bioavailable ATR inhibitor (ATRi), AZD6738, in combination with chemoradiation will result in an improved and durable anti-tumor immune response in poorly immunogenic NSCLC GEMMs; and, (ii) Tuba-seq we will allow an unprecedented view of the radio-pharmacogenetic landscape of NSCLC responses in vivo. SPECIFIC AIM #1 – Establish a radio-pharmacogenetic map of oncogene-driven non-small cell lung cancer to both chemoradiation and combined chemoradiation/anti-PD-L1 therapy We propose to use novel CRISPR/Cre-mediated GEMMs of NSCLC to test the optimal combinations of chemoradiation with anti-PD-L1 IMT. We will then examine the mechanism of action of chemoradiation with IMT using multiparametric immunologic approaches and an innovative technique enabling lineage tracing and direct quantification of treatment effects on these different genetic backgrounds in vivo. SPECIFIC AIM #2 – Determine tumor cell genotype effects on combination anti-PD-L1 immunotherapy and ATRi with chemoradiotherapy in oncogene- driven non-small cell lung cancer. This Aim leverages our novel CRISPR/Cre-mediated GEMMs of NSCLC to test genotype effects on the combination of anti-PD-L1 and ATRi with chemoradiation. By using all these tools, we will be able to decode the major aspects of the molecular underpinnings of chemoradiation and combination IMT resistance in NSCLC, contributing to improved therapies and positively impacting patient outcomes.