Background/Aim:
Resistance to BRAF and MEK inhibitors (BRAFi+MEKi) remains a major challenge in melanoma and is associated with substantial phenotypic plasticity. EGLN1, encoding prolyl hydroxylase domain protein 2 (PHD2), a key regulator of HIF-α stability, is a strong melanoma dependency evident in large-scale functional genomic datasets. We investigated the PHD2–HIF-1α axis as a therapeutic vulnerability in melanoma and examined the role of HIF-1α in the response to PHD inhibition in adaptive and acquired BRAFi+MEKi resistance.
Methods:
CRISPR–Cas9 knockout models of EGLN1, HIF1A and HIF1AN were generated in melanoma cell lines. PHD/HIF signalling was pharmacologically modulated using the PHD inhibitor roxadustat and the factor inhibiting HIF (FIH) inhibitor DM-NOFD. Proliferation, signalling and transcriptional responses were assessed using short- and long-term proliferation assays, immunoblotting and RT-qPCR. BRAFi+MEKi resistance was investigated using TGFβ1-induced adaptive resistance and acquired-resistant populations generated through prolonged dabrafenib/trametinib exposure.
Results:
EGLN1 deletion reduced melanoma proliferation by 70–80% relative to controls (p<0.05), while PHD inhibition suppressed growth in a HIF-1α-dependent manner. Genetic disruption of HIF1AN or pharmacological FIH inhibition further enhanced the antiproliferative response to PHD inhibition, with effects dependent on HIF-1α. Roxadustat induced canonical HIF-1 target genes (LDHA, PDK1, BNIP3) in a time-dependent manner, while increased EGLN1 transcription and PHD2 protein were consistent with HIF-mediated feedback regulation. In A375 cells with acquired dabrafenib/trametinib resistance, roxadustat produced a 15 ± 2-fold increase in BRAFi+MEKi sensitivity and reduced ERK phosphorylation, whereas this response was lost following HIF1A deletion. HIF-1α-dependent responses were also observed in TGFβ1-induced adaptive resistance. In contrast, acquired-resistant C32 and WM266-4 populations lost HIF-1α expression and no longer responded to roxadustat, accompanied by reduced MITF and increased AXL expression, consistent with dedifferentiated melanoma phenotype.
Conclusions:
These findings identify the PHD2–HIF-1α axis as a therapeutic vulnerability in melanoma but demonstrate that its relevance varies across BRAFi+MEKi-resistant states. Loss of HIF-1α in a subset of acquired-resistant melanomas limit responsiveness to PHD inhibition, supporting HIF-1α status as a potential biomarker for PHD-directed therapeutic strategies.
Financial support: Cancer Society Auckland Northland (CSAN).
Conflicts of interest: None.