Project 3

项目3

• 批准号: 10438715
• 负责人: John L. Cleveland
• 金额: $ 33.98万
• 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/10438715
• 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; Clinic; Clinical; Collaborations; Combined Modality Therapy; Cysteine; Data; Data Set; Development; Disease; Disease Resistance; Drug Screening; Enzymes; Epidermal Growth Factor Receptor; Etoposide; Fatty Acids; Genetic; Genetic Engineering; Genetic study; Genetically Engineered Mouse; 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; Pharmacology; Phosphotransferases; Primary Neoplasm; Proteome; Proteomics; Proto-Oncogene Proteins c-myc; Resistance; Role; Structure of parenchyma of lung; Survival Rate; TP53 gene; Testing; Therapeutic; Tumor Tissue; Tumor-infiltrating immune cells; Up-Regulation; Xenobiotics; Xenograft procedure; base; cancer genomics; cancer therapy; checkpoint therapy; chemotherapy; 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; lung small cell carcinoma; metabolome; metabolomics; mouse model; mutant; neoplastic cell; new therapeutic target; novel therapeutics; overexpression; patient derived xenograft model; prevent; purine metabolism; relapse patients; response; retinoblastoma tumor suppressor; safety study; safety testing; standard of care; synergism; targeted treatment; transcription factor; transcriptome sequencing; treatment strategy; tumor; tumor metabolism; tumor xenograft

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代谢脆弱性的作用的角色和机制 小细胞肺癌（SCLC）缺乏靶向疗法，在护理标准方面只有7％的总生存率 顺铂/依托泊苷化学疗法，免疫检查点疗法中只有10％的生存率，NCI已分类 SCLC作为顽固性恶性肿瘤。那迫切需要确定新的有效的目标疗法 对于SCLC。在非小细胞肺癌（NSCLC）基因组研究中，导致了针对的有效靶向疗法 驾驶员，例如突变EGFR。不幸的是，SCLC的驱动程序不得不难，有功能丧失 肿瘤补充的突变视网膜细胞瘤蛋白（RB1），p53（TP53）和p73以及放大 和/或MYC致癌转录因子的过表达。为了确定SCLC的漏洞，我们执行了 基于质谱的公正质谱屏幕，用于通过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（代谢组学和脂质组学）中使用较大的 SCLC和NSCLC细胞系，患者衍生的Xenographictics（PDX）和原发性肿瘤组织。此外，我们 进行了具有抑制新陈代谢不同方面的化合物的筛选。集成这些数据显示 SCLC具有高度升高的糖酵解水平，1-碳和嘌呤和脂质代谢，并且合并 用两个代谢调节剂的抑制剂进行治疗 - MCT刀片转运蛋白（MCTI）和糖酵解酶 PFKFB3（PFKFB3I） - 触发SCLC细胞系代谢崩溃，停滞和细胞死亡。遗传研究 验证了这些发现，并证实了PFKFB3抑制作用造成崩溃的悖论观察结果 在氧化磷酸化（OXPHOS）中。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）的新陈代谢，开发和维护 2）基因设计的SCLC小鼠模型（GEMM），SCLC PDX和循环SCLC Xenographictics（CDX） （目标1）。这些SCLC模型还将用于测试MCTI/PFKB3I组合的安全性和效率 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（与项目4）使用肿瘤内免疫细胞的曲目和活性。此外，我们将使用 ABPP，体内跟踪（带有项目2），代谢通量和代谢组学研究（带有核心3），以识别然后 目标自适应代谢变化因MCT和/或PFKFB3的损失或抑制而引起。这些研究 将定义PFKFB3抑制损害OXPHOS的机制，以及MCTI/PFKFB3I的组合 治疗会促进SCLC代谢崩溃（AIM 2）。重要的是，我们对配对敏感的代谢组学研究 和顺铂/依托泊苷耐药的SCLC PDX支持MCTI/PFKB3I治疗代表的假设 治疗和预防化学抗性疾病的机会，我们将使用这些SCLC进行测试 PDX型号和SCLC GEMM（AIM 3）。最后，在我们的ABPP，代谢组学和脂肪组分析的指导下，我们 将使用有针对性的CRISPR屏幕来识别SCLC中的新代谢漏洞，我们将验证并 使用我们的SCLC模型和ABPP，代谢通量和代谢组学实验表征（AIM 4）。