HIF2 Activation Derails Alveolar Differentiation Through Metabolic, Biosynthetic, and Transcriptional Changes

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Abstract Rationale: The current paradigm of Idiopathic Pulmonary Fibrosis (IPF) pathogenesis implicates recurrent injury and dysfunctional alveolar epithelial repair as a key driving upstream disease mechanism, yet available IPF treatments do not address key barriers to epithelial regeneration. Recent work has demonstrated that Hypoxia-inducible Factor (HIF) activation, specifically HIF2 [EPAS1], impairs adaptive alveolar repair, drives disease-emergent maladaptive cell-states which accumulate in IPF, and is a potential therapeutic target. The mechanism through which HIF2 activity controls facultative stem cell populations required for adaptive alveolar repair following injury is not well understood. Methods: Human alveolar organoids were generated from declined donor or IPF derived explant lungs via CD326+ column-based enrichment and cultured in domed Matrigel droplets under serum-free feeder free conditions for single cell RNA sequencing (scRNA-seq), biochemical experimentation, fixation for immunofluorescence, and other downstream experiments. HIF-form biased activation was generated with Roxadustat, a prolyl-hydroxylase inhibitor to activate HIF-driven signaling, along with PT-2385 (HIF2 inhibitor) or M1002 (HIF2 allosteric activator). Quantitative lipidomics/metabolomics/label free proteomics were performed. Stable isotopic enrichment analysis on fatty acids was performed using D2O and analyzed by GC/MS. scRNA data was analyzed using Scanpy. Cell Painting was performed with whole mount staining and imaged on an ImageXpress.AI and analyzed using InCarta/StratomineR (molecular devices). Results: Pharmacologic HIF-biased activation with Roxadustat in alveolar organoids suppressed AT2-like characteristics including Surfactant Protein C (SPC) expression and increased aberrant intermediate marker Vimentin. Integrative analysis of IPF and control epithelial scRNA-seq and proteomics/lipidomics/metabolomics of patient-derived organoids demonstrated HIF-mediated suppression of TCA-cycle rate-limiting components (p=2.37x10-26; and quantitative reduction in cytosolic citrate) as well as de novo fatty acid biosynthesis pathways (p=9.78x10-6; paired reduction on global surfactant-related phosphatidyl choline lipids) essential in surfactant biosynthesis. Stable isotope measurements showed nearly complete cessation of de novo lipid synthesis upon HIF2-biased activation (p<0.0001). Finally, using HIF2-biased pharmacologic activation, with a combination of scRNA-seq and high-content imaging with Cell Painting (organelle level staining with high-dimensional image analysis), we demonstrated that HIF2-biased activation strongly promoted KRT5- /KRT17+ transitions and drove significant morphologic changes (phenotypic distance for separate donors of 2.43x10-11 and 5.68x10-16) at the organoid level. Conclusion: Persistent HIF2 activation in the alveolar epithelium prevents maturation and terminal differentiation through suppression of essential AT2 biosynthetic components and metabolic machinery, which results in disease-relevant changes in cellular identity and function. This constellation of findings points toward HIF2 inhibition as a novel therapeutic target to enhance alveolar repair in the treatment of fibrotic lung diseases.

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