HIF-2α inhibition rescues zebrafish VHL model phenotypes

Patients with VHL develop a range of hypervascular tumours including renal cell carcinoma (RCC), retinal and central nervous system hemangioblastomas (HB). The loss of VHL protein (pVHL) reduces HIF protein degradation increasing HIF-1α and HIF-2α signalling and the expression of target genes involved in angiogenesis, erythropoiesis, metabolism and cell proliferation. Increased HIF signalling has also been linked to tumourigenesis in sporadic RCC, BHD, TSC and HLRCC (Kim et al., 2006, Preston et al., 2011). HIF-2α is reported to be more important than HIF-1α in renal tumourigenesis – specific inhibition of HIF-2α has been shown to be sufficient to block aberrant growth in VHL-null cell lines (Kondo et al., 2003, Zimmer et al., 2004). As such the development of treatments that target HIF-2α may be more therapeutically beneficial that current downstream targeted therapies which show little efficacy in VHL patients.

Metelo et al., (2015) report that HIF-2α-specific inhibition can reverse vhl-associated pathologies in a zebrafish model – a good model system for VHL as the hypoxia, angiogenesis and erythropoiesis constitute pathways are conserved between mammals and fish. The authors assessed the ability of compound 76, originally identified in a cell line (786-O) screen (Zimmer et al., 2008), to reduce HIF-2α signalling both in the vhl-/- model zebrafish and wildtype zebrafish that were hypoxia-challenged using the hypoxia-mimetic DMOG. In both groups, when untreated, there was an increase in vascular branching – most notable in the trunk and head – and erythrocytosis. The vhl-/- zebrafish also have cardiomegaly with decreased cardiac contractility believed to be the major cause of death during early development.

Compound 76 inhibits HIF-2α by promoting iron regulatory protein 1 (IRP1) binding to 5’-UTR of HIF2a-mRNA thereby blocking translation (Zimmer et al., 2008). As HIF-1α mRNA does not have a IRP1-binding loop inhibition is specific to HIF-2α. Metelo et al. used qRT-PCR to determine levels of HIF1α and HIF-2α target genes in hypoxia-challenged zebrafish to confirm inhibitor efficacy and specificity was retained.

Incubation of hypoxia-challenged or vhl-/- zebrafish with compound 76 reduced vascular sprouting in the trunk and brain. As the complex networks formed in the nervous system are reminiscent of the highly vascularised HBs in patients this suggests the HIF-2α inhibition treatment could be effective for multiple VHL pathologies. The compound 76 treated zebrafish also showed decreased erythrocytosis potentially through increased terminal differentiation to mature erythrocytes. Compound 76 also increased cardiac contractility in vhl-/- zebrafish, which is most likely responsible for the reported increase in early larval survival.

The efficacy of compound 76, a non-optimised compound, in a vertebrate VHL model raises hope for its eventual use in patients, although further refinement is required before use in clinical trials. PT2385, an inhibitor which binds to the HIF-2α protein blocking the heterodimerisation required for target gene transcription (Scheuermann et al., 2013), is currently in phase I clinical trials for advanced clear cell RCC (NCT02293980). Preclinical trial data suggests that PT2385 is a potent and selective inhibitor that can readily be absorbed from oral preparations (Peloton Therapeutics).

In BHD increased HIF signalling is the result of aberrant activation of AMPK and increased PCG1α activity initiating mitochondrial biogenesis and ROS production (Yan et al., 2014). Increased expression of HIF-2α was reported in BHD pulmonary cyst samples (Nishii et al., 2013) and although the authors did not assess levels of HIF-2a the increase in VEGF in these samples would suggest increased HIF-2α expression. If HIF signalling is associated with multiple aspects of BHD pathology then HIF-2α inhibitors could prove effective treatments for multiple currently untreatable rare diseases.


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