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

胶质母细胞瘤是中枢神经系统最常见的原发恶性肿瘤， 目前构成了一个重大的临床问题。即使有积极的护理标准治疗， GBM患者平均生存期仅为15个月，5年生存率不到5%。 目前，大约有160种FDA批准的癌症化疗药物；然而，只有3种获得批准 用于治疗GBM患者。因此，开发新的治疗方法以 改善这些患者令人沮丧的临床结果。最近，亚当斯等人发现 色氨酸(Trp)通过犬尿氨酸途径(KP)代谢在脑血管疾病的病理生理过程中起重要作用。 神经胶质瘤。KP负责中枢神经系统中色氨酸的大部分代谢， 而在脑肿瘤患者中，这一途径变得高度失调。Opitz等人发现 KP的代谢物Kynurenine是芳烃受体的内源性配体 (AHR)。AHR是一种转录因子，通常与癌症的发生有关，其信号转导 通过控制细胞增殖，克隆性， 侵袭性和转化生长因子-β途径。AHR在P53的降解中有意义，作为一种 AHR的基因靶标是Ube213，一种能降解P53的E2泛素结合酶。因此，我 假设抑制GBM细胞中的KP会减少AHR的激活，产生 P53活性升高，并会抑制肿瘤生长。 这一假设将在两个特定的目标上进行验证，使用我的实验室唯一的初级 患者来源的GBM细胞系和患者来源的异种移植小鼠模型。通过使用患者派生的 与传统的永生化细胞系(如U87和U251)相比，我们将包括 在基底节内观察到肿瘤的异质性。我打算澄清两人之间的关系 内源性AHR配体Kyn和P53的降解。选择性体外药物治疗的初步研究 吲哚胺2，3-双加氧酶1(IDO1；epacadostat)、IDO2(替托拉唑)、色氨酸的抑制剂 2，3-双加氧酶(TDO2；680C91)和AHR(CH223191)，表明泰托拉唑和CH223191具有 四甲基偶氮唑盐比色法测定对细胞的影响最大。进一步的研究将衡量 这些药物对P53的降解以及细胞内/细胞外KYN水平的影响。免疫组织化学 采用染色、Western blotting和qRT-PCR等方法研究药物对小鼠肾小管上皮细胞Kp的影响。 细胞。AHR的荧光素酶报告将被转导到细胞内，以定量测量 处理细胞和未处理细胞中的AHR活性。我将选择两个反应最灵敏的细胞系 我的体外工作是在GBM患者来源的异种移植(PDX)小鼠体内进行研究 模特们。皮下和原位PDX小鼠模型都将用于体内药物治疗。 PDX小鼠将被植入荧光素酶报告表达细胞，从而允许每周 AHR活性的活体测量。我在Aim 1研究中发现的两种最有效的抑制剂将 选择替莫唑胺并进行测试(目前的标准护理一线化疗 GBM)。为了测量体内色氨酸代谢，α-[11C]-甲基色氨酸(AMT)正电子发射 治疗前一天和治疗后一天进行体层摄影术(PET)扫描。我的工作可能会发现一种新的连接KP和P53的连接，从而扩大对GBM肿瘤生物学的知识，并揭示新的 治疗干预的靶点。

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.