Patient-derived glioblastoma in vitro and in vivo studies of tryptophan metabolism via the kynurenine pathway

患者来源的胶质母细胞瘤通过犬尿氨酸途径进行色氨酸代谢的体外和体内研究

• 批准号: 9332670
• 负责人: Anthony Guastella
• 金额: $ 4.05万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9332670
• 关键词: AHR gene; Aftercare; Apoptosis; Aryl Hydrocarbon Receptor; Biological Assay; Brain Neoplasms; Cell Death; Cell Line; Cell Proliferation; Cell Survival; Cells; Cerebrospinal Fluid; Clinical; Data; Development; Dioxygenases; Disease; Environment; Enzymes; FDA approved; Functional disorder; Future; Gene Targeting; Glioblastoma; Glioma; Human; Immunohistochemistry; Immunosuppressive Agents; Implant; In Vitro; Individual; Knowledge; Kynurenine; Laboratories; Ligands; Link; Literature; Luciferases; Malignant Neoplasms; Measurement; Measures; Mediating; Molecular; Mus; Nature; Neuraxis; Nicotinamide adenine dinucleotide; Pathway interactions; Patient-Focused Outcomes; Patients; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Plasma; Play; Positron-Emission Tomography; Process; Quantitative Reverse Transcriptase PCR; Receptor Activation; Reporter; Research Personnel; Role; Serum; Signal Transduction; Staining method; Stains; Survival Rate; TP53 gene; Testing; Therapeutic Intervention; Training; Transforming Growth Factor beta; Tryptophan 2,3 Dioxygenase; Tryptophan Metabolism Pathway; Tryptophanase; Tumor Biology; Tumor Suppressor Proteins; Tumor Tissue; U251; Ubiquitin; Ubiquitin-Conjugating Enzymes; Western Blotting; Work; Xenograft procedure; aryl hydrocarbon receptor ligand; carcinogenesis; chemotherapy; clinically relevant; design; extracellular; immortalized cell; improved; in vivo; inhibitor/antagonist; innovation; malignant phenotype; metabolomics; methyl tryptophan; mouse model; neoplastic cell; neurotoxic; new therapeutic target; novel; novel therapeutics; patient population; standard of care; subcutaneous; temozolomide; transcription factor; tumor; tumor growth; tumor heterogeneity; tumor microenvironment; tumorigenesis

Glioblastomas (GBMs) are the most common primary malignancy of the central nervous system, and currently poses a significant clinical problem. Even with aggressive standard of care treatment, GBM patients have an average survival of only ~15 months, and a 5-year survival of less than 5%. Currently, there are ~160 FDA-approved cancer chemotherapeutics; however, only three are approved for treatment of GBM patients. Therefore, it is critical that novel therapeutics are developed to improve the dismal clinical outcome of these patients. Recently, Adams, et al., discovered that tryptophan (TRP) metabolism via the kynurenine pathway (KP) plays a role in the pathophysiology of gliomas. The KP is responsible for majority of the TRP metabolism in the central nervous system, and in brain tumor patients, this pathway becomes highly dysregulated. Opitz et al., discovered that one metabolite of the KP, kynurenine, is an endogenous ligand of the aryl hydrocarbon receptor (AHR). AHR is a transcription factor commonly associated with carcinogenesis, and its signaling generates a malignant phenotype in gliomas via control of cell proliferation, clonogenicity, invasiveness, and the TGF-β pathway. AHR has implications in the degradation of p53, as one of AHR’s gene targets is Ube2l3, an E2 ubiquitin- conjugating enzyme that degrades p53. Therefore, I hypothesize that suppression of the KP in GBM cells will decrease AHR activation, producing increased p53 activity and will inhibit tumor growth. This hypothesis will be tested in two specific aims using my laboratory’s unique primary patient-derived GBM cell lines and patient-derived xenograft mouse models. By using patient-derived cell lines over conventional immortalized cell lines (e.g. U87 and U251), we will encompass the tumoral heterogeneity observed within GBMs. I plan to elucidate the relationship between the endogenous AHR ligand KYN and p53 degradation. Initial in vitro drug treatments with selective inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1; epacadostat), IDO2 (tenatoprazole), tryptophan 2,3-dioxygenase (TDO2; 680C91), and AHR (CH223191), show that tenatoprazole and CH223191 have the greatest effect on cells, as measured by MTT assay. Further studies will measure the effect of these drugs on p53 degradation, as well as intra/extra- cellular KYN levels. Immunohistochemical staining, western blots, and qRT-PCR will be used to study the effect of the drugs on the KP in the cells. A luciferase reporter for AHR will be transduced into cells to quantitatively measure the AHR activity in treated and untreated cells. I will select the two most responsive cell lines from my in vitro work to perform in vivo studies in GBM primary patient-derived xenograft (PDX) mouse models. Both subcutaneous and orthotopic PDX mouse models will be used for in vivo drug treatments. PDX mice will have the luciferase reporter-expressing cells implanted, thereby allowing for weekly in vivo measurements of AHR activity. The two most effective inhibitors from my Aim 1 studies will be selected and tested against temozolomide (current standard-of-care first-line chemotherapy for GBM). To measure in vivo TRP metabolism, alpha-[11C]-methyl-tryptophan (AMT) positron emission tomography (PET) scans will beconducted one-day pre- and one-day post-treatment. My work will likely identify a novel connection linking the KP and p53, thereby expanding the knowledge of GBM tumor biology and revealing new targets for therapeutic intervention.

胶质母细胞瘤（GBM）是中枢神经系统最常见的原发性恶性肿瘤， 目前，即使采用积极的标准护理治疗，也构成了重大的临床问题。 GBM患者的平均生存期仅为~15个月，5年生存率低于5%。 目前，FDA 批准了约 160 种癌症化疗药物，但只有 3 种获得批准； 因此，开发新的治疗方法对于治疗 GBM 患者至关重要。 最近，Adams 等人发现， 通过犬尿氨酸途径 (KP) 的色氨酸 (TRP) 代谢在病理生理学中发挥作用 KP 负责中枢神经系统中的大部分 TRP 代谢， Opitz 等人发现，在脑肿瘤患者中，这条通路变得高度失调。 KP 的一种代谢物犬尿氨酸是芳基烃受体的内源配体 (AHR) 是一种通常与癌发生及其信号传导相关的转录因子。 通过控制细胞增殖、克隆形成，在神经胶质瘤中产生恶性表型， 侵袭性和 TGF-β 途径对 p53 的降解有影响，作为其中之一。 AHR 的基因目标是 Ube2l3，一种降解 p53 的 E2 泛素结合酶。 随后抑制 GBM 细胞中的 KP 将减少 AHR 激活，产生 增加p53活性并会抑制肿瘤生长。 该假设将使用我的实验室独特的主要测试在两个特定目标上进行测试 患者来源的 GBM 细胞系和患者来源的异种移植小鼠模型。 与传统永生化细胞系（例如 U87 和 U251）相比，我们将涵盖 我计划阐明 GBM 内观察到的肿瘤异质性之间的关系。 内源性 AHR 配体 KYN 和 p53 选择性降解的初始体外药物治疗。 吲哚胺 2,3-双加氧酶 1（IDO1；epacadostat）、IDO2（替那拉唑）、色氨酸抑制剂 2,3-双加氧酶 (TDO2; 680C91) 和 AHR (CH223191) 表明替那拉唑和 CH223191 具有 通过 MTT 测定来测量对细胞的最大影响。 这些药物影响 p53 降解以及细胞内/外 KYN 水平。 染色、蛋白质印迹和 qRT-PCR 将用于研究药物对 KP 的影响 AHR 的荧光素酶报告基因将被转导到细胞中以定量测量 AHR 的荧光素酶报告基因。 处理和未处理细胞中的 AHR 活性我将从中选择两个反应最灵敏的细胞系。 我的体外工作是在 GBM 原发性患者来源的异种移植 (PDX) 小鼠中进行体内研究 皮下和原位 PDX 小鼠模型都将用于体内药物治疗。 PDX 小鼠将植入表达荧光素酶报告基因的细胞，从而每周进行一次 AHR 活性的体内测量将是我的 Aim 1 研究中的两种最有效的抑制剂。 选择并测试替莫唑胺（当前治疗的标准一线化疗） GBM) 测量体内 TRP 代谢、α-[11C]-甲基色氨酸 (AMT) 正电子发射 我的工作可能会在治疗前一天和治疗后一天进行断层扫描 (PET) 扫描，从而确定连接 KP 和 p53 的新连接，从而扩展 GBM 肿瘤生物学知识并揭示新的知识。 治疗干预的目标。