Disrupting glutathione dependency in pancreatic cancer

项目总结/摘要 胰腺导管癌（PDAC）是胰腺癌的最常见形式，并且是高度致命的， 对治疗有抵抗力有必要探索新的，有效的，治疗PDAC的策略，因为只有~10%的PDAC患者， 患者存活超过5年。PDAC过度利用细胞外营养物质来维持其生长。这 营养依赖性加上低血液供应限制了PDAC微环境中的营养可用性。 为了实现治疗和改善患者生存结局，重要的是要了解PDAC如何在 营养有限的条件和维持其生存的肿瘤内在或微环境因素。在这 根据这一提议，我们表明PDAC细胞对半胱氨酸的依赖程度远远大于其他氨基酸。代谢组 分析显示PDAC细胞几乎专门使用半胱氨酸来维持细胞内谷胱甘肽（GSH）。 虽然一些PDAC细胞在缺乏半胱氨酸时快速产生GSH，但其他细胞在缺乏半胱氨酸时维持其GSH库。 饥饿的半胱氨酸和精氨酸，表明使用各种机制来维持GSH和生存， PDAC细胞。此外，我们发现在相同的半胱氨酸饥饿下，巨噬细胞产生GSH， 这是一个重要的发现，因为PDAC微环境中巨噬细胞的丰度很高，它们的精氨酸 巨噬细胞源性GSH可维持PDAC。在多基因表达 在患者肿瘤的数据集中，我们观察到PDAC表达高水平的GSH途径基因。基于这些 根据数据，我们假设GSH是PDAC生长所需的核心营养素， 肿瘤相关巨噬细胞，破坏GSH利用可以改善PDAC的治疗。的 本研究的目的是（1）。以确定驱动PDAC对GSH依赖性的分子机制- 包括GSH途径的表观遗传调控; 2）.以确定肿瘤相关的 巨噬细胞作为PDAC中GSH的来源和调节剂。首要目标是探索是否阻止 GSH单独使用或与巨噬细胞活性一起使用可能是改善PDAC治疗的一种方法。目标1将 在K99阶段进行，而目标2的大部分将在R 00阶段进行。方法将包括基因 干扰（例如，CRISPR/Cas9、shRNA、siRNA）、GSH途径的药理学抑制剂（包括 戊糖磷酸途径），细胞培养测定，代谢组学（包括稳定同位素示踪），饮食小鼠 模型，生物信息学，启动子分析/表观遗传学方法，RNA测序（单细胞和批量）， 免疫组织化学、流式细胞术和质谱细胞术。该项目将收到一个5人的输入 在肿瘤免疫学、代谢、生物信息学和表观遗传学方面具有专长的导师团队。的 预期的结果可以a）提供关于破坏GSH途径以抑制PDAC生长的新见解，B）揭示 新的微环境机制，使肿瘤适应营养有限的状态，和c）揭示新的 有机会克服PDAC对化疗或免疫治疗的耐药性。

PROJECT SUMMARY/ABSTRACT Pancreatic ductal carcinoma (PDAC) is the most common form of pancreatic cancer and is highly lethal and resistant to therapy. There is a need to explore new, effective, strategies to treat PDAC, given that only ~10% of the patients survive beyond five years. PDAC overutilize extracellular nutrients to sustain their growth. This nutrient dependency, coupled with a low blood supply, limits nutrient availability in the PDAC microenvironment. To achieve therapy and improve patient survival outcome, it is important to understand how PDAC survive in the nutrient-limited condition and the tumor-intrinsic or microenvironmental factors that sustain their survival. In this proposal, we show that PDAC cells rely on cysteine at a far greater extent than other amino acids. Metabolomics profiling revealed that the PDAC cells almost exclusively use cysteine to sustain intracellular glutathione (GSH). While some PDAC cells rapidly generate GSH when starved of cysteine, others maintain their GSH pool when starved of both cysteine and arginine, indicating the use of various mechanisms to sustain GSH and survival in PDAC cells. In addition, we found that under the same cysteine starvation, macrophages produce GSH, which is an important discovery given the high abundance of macrophages in PDAC microenvironment, their arginine catabolic function, and that the macrophage-derived GSH could sustain PDAC. In multiple gene expression datasets of patient tumors, we observed that PDAC express a high level of GSH pathway genes. Based on these data, we hypothesize that GSH is a core nutrient required for PDAC growth, is potentially sustained by tumor-associated macrophages, and that disrupting GSH utilization could improve therapy in PDAC. The aims of this study are 1). to determine the molecular mechanisms driving the dependency of PDAC on GSH – including the epigenetic regulation of GSH pathway, and 2). to determine the role of tumor-associated macrophages as a source and modulator of GSH in PDAC. The overarching goal is to explore whether blocking GSH utilization alone or alongside macrophage activities could be a way to improve PDAC therapy. Aim 1 will be pursued at the K99 phase, while most of Aim 2 will be pursued at the R00 phase. Methods will include gene interference (e.g., CRISPR/Cas9, shRNA, siRNA), pharmacological inhibitors of GSH pathways (including the pentose phosphate pathway), cell culture assays, metabolomics (including stable isotope tracing), dietary mouse models, bioinformatics, promoter analysis/epigenetic methods, RNA sequencing (single cell and bulk), immunohistochemistry, flow cytometry and mass cytometry. The project will receive input from a 5-person mentorship team that have expertise in tumor immunology, metabolism, bioinformatics, and epigenetics. The expected results could a) offer new insights on disrupting GSH pathway to suppress PDAC growth, b) reveal new microenvironmental mechanisms that enable tumor adaptation in nutrient-limited state, and c) reveal new opportunities to overcome resistance to chemotherapy or immunotherapy in PDAC.