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-? 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.
Current standard of care for glioblastoma (GBM) patients offers a dismal 15-month median survival. This application looks to define a novel connection between intrinsic tumoral tryptophan metabolism via the kynurenine pathway and p53-mediated cell death. Using kynurenine pathway inhibitors in primary patient- derived GBM cell lines, as well as primary patient-derived GBM xenograft mouse models, the proposed study will elucidate novel therapeutic targets for a patient population that is in desperate need.
|Guastella, Anthony R; Michelhaugh, Sharon K; Klinger, Neil V et al. (2018) Investigation of the aryl hydrocarbon receptor and the intrinsic tumoral component of the kynurenine pathway of tryptophan metabolism in primary brain tumors. J Neurooncol 139:239-249|