Targeting aspartate biosynthesis in pancreatic tumors

胰腺导管腺癌（PDAC）是胰腺癌死亡的第二大原因。 美国，一旦扩散到远处器官，存活率为3%。肿瘤内的PDAC细胞 由于它们的高增殖率和血管系统不足，经常缺氧。这种程度的 肿瘤中的缺氧引发癌细胞的强烈代谢适应，使其存活， 增殖在这些适应中，改变了对主要营养素（如谷氨酰胺）的吸收和利用。 然而，胰腺癌代谢适应缺氧的确切机制，以及是否 这可能被用于治疗，仍然未知。 在初步研究中，我们发现，当胰腺癌和肺癌暴露于低水平的 蛋白质和核苷酸合成所需的氨基酸天冬氨酸氧变得有限。简单 通过表达质膜天冬氨酸转运蛋白增加外源天冬氨酸的摄取 强烈促进癌细胞在缺氧和肿瘤中的生长速度，以及增强其 转移潜能这些发现为天冬氨酸可能是癌症生长限制代谢物提供了证据 in vivo.此外，使用靶向限速代谢酶的sgRNA文库进行CRISPR/Cas9筛选， 酶显示，GOT 2是从头合成细胞天冬氨酸的两种酶之一， 谷氨酰胺对于KRAS/TP 53突变PDAC细胞系在缺氧条件下的体外增殖至关重要。建筑 基于这些结果，我建议检验这样一个假设，即靶向从头天冬氨酸合成可能具有 在胰腺肿瘤原发性肿瘤生长和转移水平的治疗潜力。 在该奖项的整个初始阶段，我们将定义GOT 2介导的天冬氨酸的重要性， 当暴露于低氧张力时，重要的是，当暴露于低氧张力时， 在远处器官的定殖过程中作为肿瘤生长。此外，我们将确定捐款额， 谷氨酰胺转化为天冬氨酸的两种不同代谢途径对癌症增殖的影响： 氧化和还原代谢在所获得的结果的基础上，我们将在 临床前患者来源的模型和KRAS/TP 53突变小鼠模型，并通过使用同位素标记 通过代谢组学分析，我们将显示哪种天冬氨酸合成途径在体内被胰腺肿瘤使用。 最后，通过在原发性和转移性肿瘤中使用体内代谢组学和线粒体下拉， 建议确定缺氧触发的代谢重新连接是否是转移潜力的决定因素， PDAC细胞。这些分析将确定PDAC细胞在转移期间经历哪些代谢变化， 揭示了潜在的责任，可以有针对性的，以减少传播PDAC到遥远的器官。 总之，所提出的实验将确定天冬氨酸合成在PDAC肿瘤生长中的作用。 和转移，并将测试PDAC细胞转移是否需要任何其他代谢变化。

Pancreatic ductal adenocarcinomas (PDACs) are the second leading cause of cancer death in the United States, with a survival rate of 3% once it spreads to distant organs. PDAC cells within a tumor are frequently starved for oxygen due to their high proliferation rate and insufficient vasculature. This degree of hypoxia in the tumor triggers strong metabolic adaptations on cancer cells that allow their survival and proliferation. Among these adaptations are altered uptake and utilization of major nutrients, such as glutamine. However, the precise mechanisms through which pancreas cancer metabolism adapts to hypoxia, and whether this could be exploited for therapy, remain unknown. In preliminary studies, we found that, when pancreatic and lung cancers are exposed to low levels of oxygen, the amino acid aspartate, required for protein and nucleotide synthesis, becomes limiting. Simply increasing the uptake of exogenous aspartate by expression of a plasma membrane aspartate transporter strongly promoted the growth rate of cancer cells under hypoxia and in tumors, as well as enhanced their metastatic potential. These findings provide evidence that aspartate can be a cancer growth-limiting metabolite in vivo. Furthermore, a CRISPR/Cas9 screen using a library of sgRNAs targeting rate-limiting metabolic enzymes revealed that GOT2, one of the two enzymes that de novo synthesizes cellular aspartate from glutamine, is essential for in vitro proliferation under hypoxia of a KRAS/TP53 mutant PDAC cell line. Building upon these results, I propose to test the hypothesis that targeting de novo aspartate synthesis may have therapeutic potential in pancreatic tumors at the level of primary tumor growth and metastasis. Throughout the initial phase of this award, we will define the essentiality of GOT2-mediated aspartate synthesis in a panel of PDAC cell lines upon being exposed to low tensions of oxygen and, importantly, when grown as tumors and during colonization of distant organs. Additionally, we will determine the contribution and impact on cancer proliferation of the two divergent metabolic routes of glutamine conversion into aspartate: oxidative and reductive metabolism. Building upon the obtained results, we will target aspartate synthesis in pre-clinical patient-derived models and KRAS/TP53 mutant mouse models and, by using isotope-labeling metabolomic analysis, we will show which aspartate synthesis route is used by pancreatic tumors in vivo. Finally, by using in vivo metabolomics and mitochondrial pull-downs in primary and metastatic tumors, I propose to define whether hypoxia-triggered metabolic rewiring is a determinant of the metastatic potential of PDAC cells. These analysis will identify which metabolic changes PDAC cells undergo during metastasis, unveiling potential liabilities that could be targeted in order to decrease spread of PDAC to distant organs. Altogether, the proposed experiments will define the role of aspartate synthesis in PDAC tumor growth and metastasis, and will test whether any other metabolic changes are required for PDAC cells to metastasize.