Project 3

项目3

• 批准号: 10676736
• 负责人: John L. Cleveland
• 金额: $ 33.81万
• 依托单位: H. LEE MOFFITT CANCER CTR & RES INST
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-25 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676736
• 关键词: Alanine; Amino Acids; Binding; Biological Assay; CRISPR screen; Cancer Model; Cancer Patient; Cancer cell line; Carbon; Cell Death; Cell Survival; Chemoresistance; Cholesterol; Cisplatin; Classification; Clinic; Clinical; Collaborations; Combined Modality Therapy; Cysteine; Data Set; Development; Disease; Disease Resistance; Drug Screening; Enzymes; Epidermal Growth Factor Receptor; Etoposide; Fatty Acids; Genetic; Genetic Engineering; Genetic study; Genetically Engineered Mouse; Genomics; Glucose; Glutamates; Glutamine; Glycolysis; Growth; Hexoses; Human; Immunologic Surveillance; Immunosuppression; Impairment; Knock-out; Lactate Transporter; Lead; Maintenance; Malignant Neoplasms; Malignant neoplasm of lung; Mass Spectrum Analysis; Metabolic; Metabolism; Mus; Neoplasm Circulating Cells; Non-Small-Cell Lung Carcinoma; Oleates; Oncogenic; Oxidative Phosphorylation; Patients; Phosphotransferases; Primary Neoplasm; Proteome; Proteomics; Proto-Oncogene Proteins c-myc; Resistance; Retinoblastoma Protein; Role; Structure of parenchyma of lung; Survival Rate; TP53 gene; Testing; Therapeutic; Tumor Tissue; Tumor-infiltrating immune cells; Up-Regulation; Xenobiotics; Xenograft procedure; cancer genomics; cancer therapy; checkpoint therapy; chemotherapy; data integration; druggable target; effective therapy; efficacy study; embryonic stem cell; enzyme activity; experimental study; genetic approach; in vivo; inhibitor; inorganic phosphate; lipid metabolism; lipidomics; loss of function mutation; metabolome; metabolomics; mouse model; mutant; neoplastic cell; new therapeutic target; novel therapeutics; overexpression; patient derived xenograft model; pharmacologic; prevent; purine metabolism; relapse patients; response; retinoblastoma tumor suppressor; safety study; safety testing; small cell lung carcinoma; standard of care; synergism; targeted treatment; transcription factor; transcriptome sequencing; treatment strategy; tumor; tumor metabolism; tumor xenograft; validation studies

PROJECT 3 SUMMARY ROLES AND MECHANISMS OF ACTION OF METABOLIC VULNERABILITIES OF SCLC Small cell lung cancer (SCLC) lacks targeted therapies, and with only 7% overall survival on standard-of-care cisplatin/etoposide chemotherapy, and only 10% survival on immune checkpoint therapy, NCI has classified SCLC as a recalcitrant malignancy. Thus, there is an urgent need to identify new and effective targeted therapies for SCLC. In non-small cell lung cancer (NSCLC) genomic studies led to effective targeted therapies directed at drivers such as mutant EGFR. Unfortunately, drivers of SCLC are undruggable, where there are loss-of-function mutations in the tumor suppressors retinoblastoma protein (RB1), p53 (TP53) and p73, as well as amplification and/or overexpression of MYC oncogenic transcription factors. To identify vulnerabilities for SCLC, we performed unbiased mass spectrometry-based screens for SCLC-specific changes in the ATP-binding proteome via activity-based proteome profiling (ABPP), and in the metabolome (metabolomics and lipidomics) using a large bank of SCLC and NSCLC cell lines, patient-derived xenografts (PDX) and primary tumor tissue. Further, we performed screens with compounds that inhibit different aspects of metabolism. Integrating these data revealed SCLC has highly elevated levels of glycolysis, 1-carbon and purine and lipid metabolism, and that combined treatment with inhibitors of two metabolic regulators – MCT lactate transporters (MCTi) and the glycolytic enzyme PFKFB3 (PFKFB3i) – triggers SCLC cell line metabolic collapse, growth arrest and cell death. Genetic studies validated these findings, and confirmed the paradoxical observation that PFKFB3 inhibition provokes a collapse in oxidative phosphorylation (OxPhos). Given these findings, we will assess the roles of MCTs and PFKFB3 in the metabolism, development and maintenance of SCLC using established (from Project 1) and new (from Core 2) genetically engineered SCLC mouse models (GEMM), SCLC PDX, and circulating SCLC xenografts (CDX) (Aim 1). These SCLC models will also be used to test the safety and efficacy of the MCTi/PFKB3i combination as a therapeutic strategy for SCLC, and we will also assess effects of targeting MCTs and/or PFKB3 on the repertoire and activity of intratumoral immune cells using SCLC GEMM (with Project 4). Further, we will use ABPP, in vivo tracing (with Project 2), metabolic flux, and metabolomics studies (with Core 3) to identify and then target adaptive metabolic changes provoked by the loss or inhibition of MCTs and/or PFKFB3. These studies will define the mechanism by which PFKFB3 inhibition impairs OxPhos, and how combined MCTi/PFKFB3i treatment provokes SCLC metabolic collapse (Aim 2). Importantly, our metabolomic studies of paired sensitive and cisplatin/etoposide-resistant SCLC PDX supports the hypothesis that MCTi/PFKB3i therapy represents an opportunity to treat and prevent emergence of chemoresistant disease, which we will test using these SCLC PDX models and SCLC GEMM (Aim 3). Finally, guided by our ABPP, metabolomic and lipidomic analyses, we will use targeted CRISPR screens to identify new metabolic vulnerabilities in SCLC, which we will validate and characterize using our SCLC models and ABPP, metabolic flux and metabolomics experiments (Aim 4).

项目 3 摘要 SCLC 代谢脆弱性的作用和作用机制 小细胞肺癌 (SCLC) 缺乏靶向治疗，标准治疗下的总生存率仅为 7% NCI 已将顺铂/依托泊苷化疗，以及免疫检查点治疗的存活率仅为 10% SCLC 是一种顽固性恶性肿瘤。因此，迫切需要寻找新的有效的靶向治疗方法 对于小细胞肺癌。在非小细胞肺癌 (NSCLC) 基因组研究中，针对以下疾病开发出有效的靶向治疗： 驱动因素，例如突变型 EGFR。不幸的是，SCLC 的驱动程序是不可药物的，其中存在功能丧失 肿瘤抑制因子视网膜母细胞瘤蛋白 (RB1)、p53 (TP53) 和 p73 的突变以及扩增 和/或 MYC 致癌转录因子的过度表达。为了识别 SCLC 的漏洞，我们执行了 基于无偏质谱法筛选 ATP 结合蛋白质组中 SCLC 特异性变化 基于活动的蛋白质组分析（ABPP），以及在代谢组学（代谢组学和脂质组学）中使用大量 SCLC 和 NSCLC 细胞系、患者来源的异种移植物 (PDX) 和原发性肿瘤组织库。此外，我们 对抑制新陈代谢不同方面的化合物进行了筛选。整合这些数据揭示 SCLC 的糖酵解、1-碳、嘌呤和脂质代谢水平高度升高，并且这些因素结合在一起 使用两种代谢调节剂抑制剂进行治疗 - MCT 乳酸转运蛋白 (MCTi) 和糖酵解酶 PFKFB3 (PFKFB3i) – 触发 SCLC 细胞系代谢崩溃、生长停滞和细胞死亡。遗传学研究 验证了这些发现，并证实了 PFKFB3 抑制引起崩溃的矛盾观察 氧化磷酸化（OxPhos）。鉴于这些发现，我们将评估 MCT 和 PFKFB3 在 使用已建立的（来自项目 1）和新的（来自核心 2) 基因工程 SCLC 小鼠模型 (GEMM)、SCLC PDX 和循环 SCLC 异种移植物 (CDX) （目标 1）。这些 SCLC 模型还将用于测试 MCTi/PFKB3i 组合的安全性和有效性 作为 SCLC 的治疗策略，我们还将评估靶向 MCT 和/或 PFKB3 对 使用 SCLC GEMM（项目 4）研究瘤内免疫细胞的库和活性。此外，我们将使用 ABPP、体内追踪（通过项目 2）、代谢流和代谢组学研究（通过核心 3）来识别并随后 目标是由 MCT 和/或 PFKFB3 的丢失或抑制引起的适应性代谢变化。这些研究 将定义 PFKFB3 抑制损害 OxPhos 的机制，以及如何结合 MCTi/PFKFB3i 治疗会引发 SCLC 代谢崩溃（目标 2）。重要的是，我们对配对敏感的代谢组学研究 顺铂/依托泊苷耐药 SCLC PDX 支持以下假设：MCTi/PFKB3i 疗法代表了一种 治疗和预防耐药疾病出现的机会，我们将使用这些 SCLC 进行测试 PDX 模型和 SCLC GEMM（目标 3）。最后，在 ABPP、代谢组学和脂质组学分析的指导下，我们 将使用有针对性的 CRISPR 筛选来识别 SCLC 中新的代谢漏洞，我们将对其进行验证和 使用我们的 SCLC 模型和 ABPP、代谢流和代谢组学实验进行表征（目标 4）。