Disrupting glutathione dependency in pancreatic cancer

项目摘要/摘要 胰腺导管癌(PDAC)是胰腺癌中最常见的一种，具有高度的致命性和 抗拒治疗。有必要探索新的、有效的治疗PDAC的策略，因为只有~10%的 这些患者可以存活五年以上。PDAC过度利用细胞外营养来维持其生长。这 营养依赖，加上血液供应不足，限制了PDAC微环境中的营养供应。 为了实现治疗和改善患者的生存结果，了解PDAC是如何在 营养受限的条件和维持其生存的肿瘤内在或微环境因素。在这 建议，我们表明，PDAC细胞对半胱氨酸的依赖程度远远超过其他氨基酸。代谢组学 分析显示，PDAC细胞几乎完全使用半胱氨酸来维持细胞内谷胱甘肽(GSH)。 虽然一些PDAC细胞在半胱氨酸饥饿时迅速产生GSH，但另一些细胞在以下情况下维持其GSH池 同时缺乏半胱氨酸和精氨酸，这表明使用了各种机制来维持GSH和存活 PDAC细胞。此外，我们还发现，在同样的半胱氨酸饥饿下，巨噬细胞产生GSH，这是 是一个重要的发现，因为在PDAC微环境中巨噬细胞的高丰度，它们的精氨酸 巨噬细胞来源的GSH对PDAC有支持作用。在多种基因表达中 在患者肿瘤的数据集上，我们观察到PDAC表达高水平的GSH途径基因。基于这些 数据，我们假设GSH是PDAC生长所需的核心营养物质，潜在地通过 肿瘤相关的巨噬细胞，而干扰GSH的利用可以改善PDAC的治疗。这个 本研究的目的有：1)。为了确定驱动PDAC对GSH依赖的分子机制- 包括GSH途径的表观遗传调控；2)。确定肿瘤相关基因的作用 巨噬细胞是PDAC中GSH的来源和调节者。首要目标是探索阻止 单独使用GSH或与巨噬细胞活动一起使用可能是改善PDAC治疗的一种方法。目标1将 目标2的大部分目标将在R00阶段实现。方法将包括基因 干扰(例如，CRISPR/Cas9、shRNA、siRNA)、GSH途径的药理抑制剂(包括 磷酸戊糖途径)、细胞培养分析、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、流式细胞术和质量细胞术。该项目将收到来自5个人的意见 在肿瘤免疫学、新陈代谢、生物信息学和表观遗传学方面拥有专长的导师团队。这个 预期结果可能a)为阻断GSH途径以抑制PDAC生长提供新的见解，b)揭示 新的微环境机制使肿瘤在营养受限状态下能够适应，以及c)揭示了新的 在PDAC中克服化疗或免疫治疗耐药性的机会。

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.