Abstract 4170: A transgenic zebrafish model for gliomas with mutations in isocitrate dehydrogenase 1

Abstract

Abstract The gene encoding Isocitrate dehydrogenase 1 (IDH1) is frequently mutated in gliomas, chondrosarcomas, acute myeloid leukemia and intrahepatic cholangiocarcinomas. As there are few in-vivo model systems for IDH-mutated tumors we have created a transgenic zebrafish (Danio rerio) model expressing mutant IDH1. We have chosen the zebrafish as a model because they are transparent (allowing monitoring of the transgene in-vivo) and drug screening assays are straightforward (they are simply added to the aquarium). IDH1R132H and IDH1R132C, mutations found in tumors that both produce D-2-hydroxyglutarate (D2HG) instead of alpha ketoglutarate, were cloned into an expression construct that is driven either by the Nestin or GFAP promoter. IDH1G70D (a loss of function mutation), IDH1wildtype and GFP were used as control. All IDH1 constructs were fused to GFP for visualization. These constructs were injected into fertilized zebrafish eggs at the one-cell stage. All of our transgenic zebrafish lines remain healthy and produce offspring. Transgene expression was detected in the mid/hindbrain of the central nervous system by immunohistochemistry, Western blot and RT-QPCR. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1R132C or IDH1R132H, but not in any of the lines expressing control constructs (IDH1wildtype, IDH1G70D or GFP). In contrast to reported, we failed to detect any differences in hydroxymethyl cytosine (the first step in DNA-demethylation) and mature collagen IV levels between wildtype and mutant IDH1 transgenic fish. We also performed microinjections on fertilized eggs to screen for early developmental effects of IDH1R132H and IDH1R132C. Despite of the high expression of the transgene, no developmental effects were found. Our observations therefore suggest that elevated levels of D2HG are insufficient to initiate tumorigenesis or other phenotypic effects in our fish. Treatment of the transgenic zebrafish with an IDH1 mutant inhibitor, AGI-5198, resulted in a reduction in the D2HG level in the mutant zebrafish. The L2HG level was not affected by AGI-5198. As no tumors were formed in our transgenic zebrafish lines, we backcrossed them with Tp53 mutant fish. Analysis of these lines is currently being performed. In summary, we have generated a transgenic zebrafish model system that expresses mutated IDH1 that can be used to study effects of mutant IDH1 (or elevated levels of D2HG) in vivo and can be used for drug screening. Citation Format: Ya D. Gao, Maurice de Wit, Eduard A. Struys, Martine L.M. Lamfers, Gajja S. Salomons, Peter A.E. Sillevis Smitt, Pim J. French. A transgenic zebrafish model for gliomas with mutations in isocitrate dehydrogenase 1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4170.