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

基本信息

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
项目类别：
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

项目摘要

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（GBMS）是中枢神经系统中最常见的主要恶性肿瘤，目前提出了一个重大的临床问题。即使采用积极的护理水平治疗， GBM患者的平均存活率仅约15个月，5年生存率少于5％。目前，约有160种FDA批准的癌症化学治疗药。但是，只有三个被批准用于治疗GBM患者。因此，至关重要的是开发新的疗法改善这些患者的临床结果令人沮丧。最近，亚当斯等人发现色氨酸（TRP）通过Kynurenine途径（KP）代谢在病理生理中起作用神经胶质瘤。 KP负责中枢神经系统中的大多数TRP代谢，在脑肿瘤患者中，该途径高度失调。 Opitz等人发现 KP的一种代谢产物Kynurenine是芳基烃接收器的内源性配体（啊）。 AHR是通常与癌变有关的转录因子及其信号传导通过控制细胞增殖，克隆性，在神经胶质瘤中产生恶性表型侵入性和TGF-β途径。 AHR对p53的退化有影响，作为之一 AHR的基因靶标是ube2l3，这是一种降解p53的E2泛素偶联酶。因此，我假设在GBM细胞中KP的抑制作用将降低AHR激活，从而产生 p53活性增加并会抑制肿瘤的生长。该假设将使用我的实验室独特的主要主要目标进行两个特定目标测试患者衍生的GBM细胞系和患者衍生的Xenographic小鼠模型。通过使用患者来源传统永生的细胞系（例如U87和U251）上的细胞系，我们将包括在GBM中观察到的肿瘤异质性。我计划阐明内源性AHR配体Kyn和p53降解。有选择性的初始体外药物治疗吲哚胺2,3-二氧酶1（IDO1; epacadostat），IDO2（Tenatoprapopophan），色氨酸的抑制剂 2,3-二氧酶（TDO2; 680C91）和AHR（CH223191），表明tenatoraprozole和CH223191具有通过MTT分析测量，对细胞的影响最大。进一步的研究将衡量这些药物在p53降解以及细胞内KYN水平上。免疫组织化学染色，蛋白质印迹和QRT-PCR将用于研究药物对KP的影响细胞。 AHR的荧光素酶报告基因将被翻译成细胞，以定量测量 AHR活性在未经处理的细胞和未处理细胞中。我将从我在GBM初级患者衍生型（PDX）小鼠中进行体内研究的体外工作型号。皮下和原位PDX小鼠模型都将用于体内药物治疗。 PDX小鼠将植入荧光素酶报告的细胞，从而每周 AHR活性的体内测量。我的目标1研究中的两个最有效的抑制剂将选择并针对替莫唑胺进行测试（当前的现行标准一线化学疗法 GBM）。为了测量体内TRP代谢，α-[11C] - 甲基 - 色氨酸（AMT）极性发射断层扫描（PET）扫描将进行一日和一日的治疗。我的工作可能会确定连接KP和P53的新型联系，从而扩大了GBM肿瘤生物学的知识并揭示了新的知识治疗干预的目标。