Use of Physiologic Media to Study Metabolic Regulation and Requirements in Cancer

使用生理介质研究癌症的代谢调节和要求

• 批准号: 10132249
• 负责人: Jason Robert Cantor
• 金额: $ 18.98万
• 依托单位: MORGRIDGE INSTITUTE FOR RESEARCH, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10132249
• 关键词: Adult; Affect; Alanine; Amino Acids; Antineoplastic Agents; Area; Bar Codes; Biochemical; Biological; Biological Models; CRISPR screen; Cancer cell line; Cell Culture Techniques; Cell Line; Cell Proliferation; Cell model; Cells; Cellular Metabolic Process; Characteristics; Citric Acid Cycle; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Consumption; Cultured Cells; Data; Defect; Diphosphates; Doxorubicin; Drug Interactions; Engineering; Environmental Impact; Environmental Risk Factor; Excision; Fluorouracil; Genes; Goals; Growth; Hematopoietic Neoplasms; Homeostasis; Human; Human body; Isotope Labeling; Libraries; Malignant Neoplasms; Metabolic; Metabolism; Mitochondria; Mitochondrial Proteins; Non-Essential Amino Acid; Nutrient; Oncology; Oxidation-Reduction; Patients; Pharmaceutical Preparations; Phenotype; Phosphotransferases; Physiological; Plasma; Proliferating; Property; Recipe; Regulation; Respiration; Role; Salts; Technology; Testing; Therapeutic Intervention; Uric Acid; Validation; Work; base; cancer cell; cancer therapy; cell growth; cell type; comparative; design; fitness; high throughput screening; improved; in vitro Model; in vivo; insight; interest; loss of function; metabolic abnormality assessment; mitochondrial metabolism; new therapeutic target; small molecule; targeted treatment; tool; tumor metabolism; Adult; Affect; Alanine; Amino Acids; Antineoplastic Agents; Area; Bar Codes; Biochemical; Biological; Biological Models; CRISPR screen; Cancer cell line; Cell Culture Techniques; Cell Line; Cell Proliferation; Cell model; Cells; Cellular Metabolic Process; Characteristics; Citric Acid Cycle; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Consumption; Cultured Cells; Data; Defect; Diphosphates; Doxorubicin; Drug Interactions; Engineering; Environmental Impact; Environmental Risk Factor; Excision; Fluorouracil; Genes; Goals; Growth; Hematopoietic Neoplasms; Homeostasis; Human; Human body; Isotope Labeling; Libraries; Malignant Neoplasms; Metabolic; Metabolism; Mitochondria; Mitochondrial Proteins; Non-Essential Amino Acid; Nutrient; Oncology; Oxidation-Reduction; Patients; Pharmaceutical Preparations; Phenotype; Phosphotransferases; Physiological; Plasma; Proliferating; Property; Recipe; Regulation; Respiration; Role; Salts; Technology; Testing; Therapeutic Intervention; Uric Acid; Validation; Work; base; cancer cell; cancer therapy; cell growth; cell type; comparative; design; fitness; high throughput screening; improved; in vitro Model; in vivo; insight; interest; loss of function; metabolic abnormality assessment; mitochondrial metabolism; new therapeutic target; small molecule; targeted treatment; tool; tumor metabolism

7. Project Summary/Abstract Altered metabolism is a nearly universal characteristic of cancer cells, which require metabolic adaptations to support proliferation and survival. However, mechanisms that dictate the diverse metabolic liabilities and phenotypes of cancer remain poorly understood. Thus, the resurgence of interest to target cancer metabolism for patient benefit will require an improved understanding of metabolic regulation and requirements in malignant cell types. Cells in culture are commonly used to study cancer metabolism and to develop drugs that exploit metabolic vulnerabilities. However, while it is increasingly appreciated that environmental factors impact cell metabolism, our current understanding of metabolic rewiring in cancer is largely based on findings from cells cultured in media that poorly reflect the metabolic composition of human plasma. Therefore, our overarching hypothesis is that many important aspects of cancer cell metabolism have been overlooked or misconstrued as a consequence of utilizing model systems that inadequately resemble physiologic conditions. To begin to test this, we developed a new culture medium (human plasma-like medium; HPLM) that contains polar metabolites and salts at concentrations comparable to those of adult human plasma. We then showed that, relative to traditional media, HPLM has widespread and largely unexplained effects on cell metabolism. Our preliminary data reveal that, when cultured in traditional media, certain cell lines secrete alanine, but that in HPLM, those cells instead consume alanine at rates exceeding those for most other amino acids. Therefore, we will test the hypothesis that alanine has a key role for cells cultured in physiologic conditions (Aim 1). Using a chemostat technology that we developed, we will also determine how the removal of alanine from HPLM affects the growth of over 40 barcoded blood cancer cell lines in a pooled fashion. In addition, through the use of CRISPR-based loss-of-function screens, we identified NME6, a gene that encodes a poorly studied mitochondrial protein, as conditional lethal with HPLM. Thus, we will also test the hypothesis that NME6 serves a critical and unforeseen role in mitochondrial homeostasis for cells cultured in physiologic conditions (Aim 2). Lastly, we also found that HPLM dramatically influences cell sensitivity to the cancer drug 5-fluorouracil without affecting the potency of doxorubicin, another common chemotherapeutic. Therefore, using a library of > 1,900 diverse oncology-related small molecules, we will perform a high-throughput screen to test the hypothesis that, relative to traditional media, HPLM alters the potency of additional compounds. We will then pursue validation and mechanistic insights for differential drug phenotypes in cell line and primary cell models (Aim 3). Our proposed work uses distinct approaches to better understand how environmental factors that more closely reflect physiologic conditions impact the metabolism of blood cancer cells, and has the potential to not only identify unforeseen biological insights, but to also uncover new therapeutic targets and approaches that may have greater in vivo relevance for cancer therapy.

7。项目摘要/摘要 改变新陈代谢是癌细胞几乎普遍的特征，需要代谢 适应以支持增殖和生存。但是，决定了多种代谢的机制 癌症的责任和表型仍然了解不足。因此，目标癌的兴趣复兴 用于患者利益的代谢将需要提高对代谢法规和要求的了解 在恶性细胞类型中。培养细胞通常用于研究癌症代谢和开发药物 这种利用代谢脆弱性。但是，尽管越来越多地赞赏环境因素 影响细胞代谢，我们目前对癌症代谢重新布线的理解主要基于发现 来自在培养基中培养的细胞，这些细胞反映了人血浆的代谢组成。因此，我们的 总体假设是，癌细胞代谢的许多重要方面已被忽略或 由于利用模型系统不足的生理条件而被误解。 为了开始测试这一点，我们开发了一种新的培养基（类似人等离子体的培养基； HPLM） 极性代谢物和盐的浓度与成年人类血浆的浓度相当。然后我们展示了 相对于传统培养基，HPLM对细胞代谢具有广泛的且在很大程度上无法解释的影响。 我们的初步数据表明，当在传统媒体中培养时，某些细胞系会分泌丙氨酸， 但是在HPLM中，这些细胞以超过大多数其他氨基酸的速率消耗丙氨酸。 因此，我们将检验以下假设：丙氨酸在生理条件下培养的细胞具有关键作用 （目标1）。使用我们开发的化学稳定技术，我们还将确定如何去除丙氨酸 来自HPLM以合并的方式影响40多个条形的血液癌细胞系的生长。此外， 通过使用基于CRISPR的功能丢失屏幕，我们确定了NME6，该基因编码较差 研究线粒体蛋白，作为HPLM的条件致命。因此，我们还将检验以下假设 NME6在生理学中培养的细胞的线粒体稳态中起着至关重要且不可预见的作用 条件（目标2）。最后，我们还发现HPLM极大地影响细胞对癌症药物的敏感性 5-氟尿嘧啶不影响阿霉素的效力，阿霉素是另一种常见的化学治疗性。所以， 使用> 1,900种不同肿瘤相关的小分子的库，我们将执行高通量屏幕 为了检验以下假设：相对于传统培养基，HPLM改变了其他化合物的效力。我们 然后，将寻求验证和机械见解，以对细胞系和原代细胞中的差异药物表型进行验证 模型（目标3）。我们提出的工作使用不同的方法来更好地了解环境因素如何 更紧密地反映生理状况会影响血液癌细胞的代谢，并具有 不仅可以识别不可预见的生物学见解，还可以发现新的治疗靶标和 可能与癌症治疗具有更大相关性的方法。