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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）是中枢神经系统最常见的原发性恶性肿瘤