Pharmacological HIF2α inhibition improves VHL disease–associated phenotypes in zebrafish model
Ana Martins Metelo, … , Randall T. Peterson, Othon Iliopoulos
Ana Martins Metelo, … , Randall T. Peterson, Othon Iliopoulos
Published April 13, 2015
Citation Information: J Clin Invest. 2015;125(5):1987-1997. https://doi.org/10.1172/JCI73665.
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Research Article Oncology

Pharmacological HIF2α inhibition improves VHL disease–associated phenotypes in zebrafish model

Abstract

Patients with a germline mutation in von Hippel-Lindau (VHL) develop renal cell cancers and hypervascular tumors of the brain, adrenal glands, and pancreas as well as erythrocytosis. These phenotypes are driven by aberrant expression of HIF2α, which induces expression of genes involved in cell proliferation, angiogenesis, and red blood cell production. Currently, there are no effective treatments available for VHL disease. Here, using an animal model of VHL, we report a marked improvement of VHL-associated phenotypes following treatment with HIF2α inhibitors. Inactivation of vhl in zebrafish led to constitutive activation of HIF2α orthologs and modeled several aspects of the human disease, including erythrocytosis, pathologic angiogenesis in the brain and retina, and aberrant kidney and liver proliferation. Treatment of vhl–/– mutant embryos with HIF2α-specific inhibitors downregulated Hif target gene expression in a dose-dependent manner, improved abnormal hematopoiesis, and substantially suppressed erythrocytosis and angiogenic sprouting. Moreover, pharmacologic inhibition of HIF2α reversed the compromised cardiac contractility of vhl–/– embryos and partially rescued early lethality. This study demonstrates that small-molecule targeting of HIF2α improves VHL-related phenotypes in a vertebrate animal model and supports further exploration of this strategy for treating VHL disease.

Authors

Ana Martins Metelo, Haley R. Noonan, Xiang Li, Youngnam Jin, Rania Baker, Lee Kamentsky, Yiyun Zhang, Ellen van Rooijen, Jordan Shin, Anne E. Carpenter, Jing-Ruey Yeh, Randall T. Peterson, Othon Iliopoulos

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Figure 1

Small-molecule compound 76 suppresses DMOG-induced expression of HIF2α target genes in WT zebrafish embryos.

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Small-molecule compound 76 suppresses DMOG-induced expression of HIF2α t...
(A) Sequence comparison among IREs present in the 5′-UTR of human HIF2a, zebrafish epas1a, and zebrafish epas1b. The IRE consensus loop and the mandatory 5′ cytosine are circled. (B) IRP1 binds to HIF2a IRE, suppressing the translation of HIF2a mRNA. Compound 76 promotes binding of IRP1 to HIF2a IRE and inhibits HIF2α translation. (C) Luciferase expression in U2OS cells transfected with pTol2 vectors containing the 5′-UTR sequences of hif1aa, hif1ab, epas1a, or epas1b upstream of the luciferase ORF. The cells were treated with 5 μM of compound 76 or DMSO control for 30 hours. Luciferase expression levels were normalized by protein levels. (D) Treatment protocol. WT embryos were treated with compound 76 or DMSO vehicle control from 3 to 7 dpf. Embryos were challenged with the hypoxia chemical mimetic DMOG from 5 to 7 dpf. (E) mRNA expression of the HIF target gene phd3 in WT embryos challenged with 100 μM DMOG and treated with compound 76 (as indicated) or vehicle-only control. Gene expression levels were normalized by 18S gene expression. (F) Relative mRNA levels of HIF target genes, phd3, epo, and vegfab, normalized to DMOG-challenged samples treated with vehicle-only control. All experiments were performed in biological triplicate. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, paired, 2-tailed t test.