PDX tumor-TME acquired resistance

项目假设和具体目标：我们假设EGFR和KRAS的分子亚型 具有获得性突变靶向耐药性的突变型NSCLC肿瘤与 免疫抑制TME包括免疫和非免疫基质细胞，可用于治疗 靶向的方式将提高针对内在耐药途径的药物的疗效。 具体目标1：开发和表征现有的和新的突变型EGFR和KRAS抑制剂模型 获得性抗性，包括PDX和PDX衍生的有机物及其机制 分子亚型与TME的关系，包括免疫和非免疫间质。 鉴定将包括组织学、免疫组织化学(IHC)、RNAseq和MS蛋白质组学。我们会 确定可以进行功能测试的特定分子变化，并确定它们对TME的影响。 目前正在测试的例子包括3-磷脂酰肌醇依赖的激酶1(PDK1，PDPK1)的表达， SETD1B失活突变和YAP(YES相关蛋白)表达。我们将测试针对目标的药物 TME中的特定细胞可增强免疫反应。我们目前的目标是与M2肿瘤相关的 巨噬细胞()。将对Osimertinib和G12C Krasi抗性PDX和有机类化合物进行讯问，以确定 利用MS蛋白质组学、scRNAseq和CRISPR筛选获得性耐药分子途径。 具体目标2：确定突变的EGFR和KRAS抑制剂获得性耐药的治疗策略 当前和未来研究中涉及的基于靶向通路(例如，PDK1、YAP、NF-B)的模型 肿瘤-TME细胞网络(如CSFR1、CD74)引起的获得性耐药。潜在的药物靶点 克服获得性耐药的奥西美替尼(见初步数据)包括PDK1、YAP、NFkB和。我们 将开发基于TME特征的治疗方法。H1975R具有更强的免疫刺激作用 TME比PC9 YAP更好，从而为测试检查点封锁与PDK1抑制在 H1975R。在PC9中过表达wtYAP的M2极化巨噬细胞增加，从而为 用CSFR 1抑制剂检测PC9YAP对的抑制作用。奥西美替尼和KRAS G12Ci治疗后的肿瘤残留 将在使用MS蛋白质组学、scRNAseq和CRISPR屏幕进行治疗后进行表征，以确定新的 获得性抗性途径。特定靶点在获得性耐药性中的功能作用将得到验证 使用CRISPR KO.我们将开发新的模型，以包括未来针对这些和其他目标开发的新药 小路。

Project hypothesis and Specific Aims: We hypothesize that the molecular subtypes of EGFR and KRAS mutant NSCLC tumors with acquired mutation targeted drug resistance are associated with an immunosuppressive TME including both immune and non-immune stromal cells that can be therapeutically targeted in a way that will enhance the efficacy of drugs targeted to intrinsic resistance pathways. Specific Aim 1: Develop and characterize existing and new models of mutant EGFR and KRAS inhibitor acquired resistance including PDXs and PDX-derived organoids and characterize mechanistic relationships between molecular subtypes and the TME, including immune and non-immune stroma. Characterization will include histology, immunohistochemisry (IHC), RNAseq, and MS proteomics. We will identify specific molecular alterations that can be functionally tested and determine their effect on the TME. Examples being tested currently include 3-phosphoinositide-dependent kinase 1 (PDK1, PDPK1) expression, SETD1B inactivating mutations, and YAP (yes-associated protein) expression. We will test drugs that target specific cells in the TME to enhance the immune response. We are currently targeting M2 tumor associated macrophages (TAM). Osimertinib and G12C KRASi resistant PDXs and organoids will be interrogated to identify acquired drug resistance molecular pathways using MS proteomics, scRNAseq, and CRISPR screens. Specific Aim 2: Define treatment strategies in mutant EGFR and KRAS inhibitor acquired resistance models based on targeting pathways (e.g., PDK1, YAP, NF-B) implicated in current and future studies of acquired resistance caused by tumor-TME cell networks (e.g., CSFR1, CD74). Potential drug targets for overcoming acquired resistance to osimertinib (see preliminary data) include PDK1, YAP, NFkB, and TAM. We will develop therapeutic approaches based on TME characterization. H1975R has a more immune stimulatory TME than PC9 YAP thus providing a rationale for testing checkpoint blockade combined with PDK1 inhibition in H1975R. M2 polarized macrophages are increased in PC9 overexpressing wtYAP thus providing a rationale for testing TAM inhibition in PC9 YAP with a CSFR1 inhibitor. Residual tumors after osimertinib and KRAS G12Ci will be characterized following treatment using MS proteomics, scRNAseq, and CRISPR screens to identify novel acquired resistance pathways. The functional role of specific targets in acquired drug resistance will be validated using CRISPR KO. We will develop new models to include future novel drugs developed to target these and other pathways.