ARNT-dependent HIF-2 transcriptional activity is not sufficient to regulate downstream target genes in neuroblastoma

Persson C.U., von Stedingk K., Fredlund E., Bexell D., Påhlman S., Wigerup C. Mohlin S.Experimental cell research, 2020

Hypoxia-inducible factor (HIF)-2α associates with poor outcomes in neuroblastoma and glioblastoma, and gain-of-function mutations in the EPAS1gene (encoding HIF-2α) have been reported in paragangliomas and pheochromocytomas. Specific targeting of a druggable hydrophobic pocket in the HIF-2αPAS-B domain with PT2385 has demonstrated promising clinical results for clear cell renal cell carcinoma (ccRCC). Here, the authors investigated the effect of PT2385-mediated inhibition of ARNT dependent HIF-2 activity. Neuroblastoma patient-derived xenograft (PDX) cells cultured on Biolaminin 521 were treated with PT2385 and analyzed for HIF-2-dependent gene expression, HIF activity, HIF-2αprotein localization, response to chemotherapy and orthotopic tumor growth in vivo. The authors detected high levels of HIF-2α protein in perivascular niches in neuroblastoma PDXs in vivo and at oxygenated conditions in PDX-derived cell cultures in vitro, particularly in the cytoplasmic fraction. Nuclear HIF-2αexpression was reduced following PT2385 treatment, but surprisingly, virtually no effects on tumor growth in vivo or expression of canonical HIF downstream target genes in vitro were observed. RNA sequencing of PT2385-treated PDX cells revealed a virtually unaffected transcriptome. Treatment with PT2385 did not affect cellular response to chemotherapy. In contrast, HIF-2αprotein knockdown resulted in profound downregulation of target genes. The lack of effect from PT2385 treatment in combination with high cytoplasmic HIF-2αexpressionat normoxia suggests that HIF-2α has additional roles than acting as an ARNT dependent transcription factor. It is important to further unravel the conditions at which HIF-2αhas transcriptional and non-transcriptional roles in neuroblastoma.

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