Abstract 5475: Functional profiling of germline EPAS1 variants associated with pheochromocytoma and paraganglioma
Authors: Kim Edward , Benn Diana E. , Clifton-Bligh Roderick J. , Dwight Trisha 
Published: July 2017

Abstract
Background: Endothelial PAS domain-containing protein 1 (EPAS1), encoding hypoxia-inducible transcription factor 2-alpha (HIF2α), is involved in regulation of cellular hypoxia responses and has been shown to play a role in several cancers including renal cell cancer and pheochromocytoma/paraganglioma (PPGL). HIF2 levels are tightly regulated, being low in normoxia but increased in hypoxia. Studying heritable PPGL syndromes has uncovered diverse genetic events that lead to oxygen-independent HIF stabilization. Using massively parallel sequencing, we noted several germline EPAS1 variants that occurred in subjects also carrying pathogenic mutations in either VHL or SDHB. In addition, two distinct EPAS1 variants were identified in separate individuals who did not harbor germline mutation in other known susceptibility genes. We therefore investigated whether these variants affected EPAS1 function.
Method: Six different EPAS1 variants were identified in the germline of patients with PPGL; p.His194Arg, p.Arg247Ser, p.Phe374Tyr, p.Thr766Pro, p.Pro785Thr and p.Ile789Val. GFP-tagged HIF2α expressing constructs - expressing either empty vector, wildtype or mutant HIF2α- were generated (including positive control p.Pro531Thr) and transfected into HEK293 cells. HEK293 cells transfected with wildtype HIF2α were also subjected to hypoxic conditions for use as a positive control. Co-immunoprecipitation followed by Western blotting analyses were performed to determine whether: a) HIF2α mutants are still able to interact with VHL and ARNT; and b) HIF2α mutants are stabilized under normoxia. Candidate target gene expression (CCND1 and SLC2A) was measured by RT-qPCR.
Results: Western blot analyses showed that p.Arg247Ser, p.Phe374Tyr, p.Pro531Thr and p.Pro785Thr amino acid changes in HIF2α were all more stable than wild-type HIF2α under normoxia. Co-IP of HIF2α with VHL showed significantly reduced interaction with p.Arg247Ser and p.Pro531Thr (p&lt;0.05). Co-IP of HIF2α with ARNT failed to show any effect on interaction occurring as a result of the HIF2α variants. Interestingly only p.Pro531Thr was able to induce CCND1 and SLC2A gene expression (p&lt;0.05).
Conclusion: Germline EPAS1 variants p.Arg247Ser and p.Phe374Tyr share some functional features in common with the known oncogenic somatic variant p.Pro531Thr. These variants may therefore be functionally relevant to developing cancers that are dependent upon pseudo-hypoxia. Whether any of these EPAS1 variants will affect response to HIF2α antagonist therapies is yet unknown and may be of importance.
Citation Format: Edward Kim, Diana E. Benn, Roderick J. Clifton-Bligh, Trisha Dwight. Functional profiling of germline EPAS1 variants associated with pheochromocytoma and paraganglioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5475. doi:10.1158/1538-7445.AM2017-5475