HIF-independent actions of HIF-prolyl hydroxylase inhibitors reveal off-target pathways with therapeutic implications

HIF-prolyl hydroxylase inhibitors (HIF-PHIs) are used to treat anemia in chronic kidney disease. These drugs stabilize hypoxia-inducible factors HIF-1α and HIF-2α, which activate erythropoiesis and iron metabolism pathways. Clinically approved HIF-PHIs such as roxadustat and molidustat exhibit distinct molecular structures and selectivity profiles, yet their HIF-independent effects remain poorly understood. Here we generated HIF1A/EPAS1 double-knockout cells to systematically dissect non-canonical HIF-PHI actions. Roxadustat and molidustat induced distinct phenotypic alterations and transcriptional reprogramming in HIF-null cells, demonstrating substantial HIF-independent effects. Notably, roxadustat exhibited anti-angiogenic activity in HIF-null cells, contradicting expectations of VEGF-driven angiogenesis via HIF stabilization. RNA sequencing and pathway analysis revealed compound-specific off-target gene regulation affecting cellular processes beyond canonical hypoxia responses including mitochondrial function, lipid metabolism, and immune signaling. These findings illuminate mechanisms underlying potential adverse effects-such as thrombosis- and identify alternative therapeutic pathways, providing a framework for optimizing HIF-PHI safety profiles and expanding their clinical applications in oncology and metabolic disorders.