Abstract 7295: The role of PHD2 inhibition in melanoma progression, metabolic adaptation, and therapy resistance

Abstract Therapy-resistant melanoma poses significant challenges due to its diverse phenotypes and limited treatment options. A strong dependency of melanoma cells on EGLN1, which encodes prolyl hydroxylase domain protein 2 (PHD2)—a key regulator of HIF-α degradation— is evident in large-scale dependency datasets, prompting us to investigate EGLN1/PHD2 as a potential therapeutic vulnerability. Melanoma cell lines were engineered using CRISPR-Cas9 to generate knockout (KO) models of EGLN1, HIF1A, and HIF1AN. Pharmacologic assays were performed using the PHD inhibitor roxadustat and the FIH inhibitor tool compound DM-NOFD. Proliferation was assessed in short- and long-term assays. Protein expression was analyzed by immunoblotting, and transcriptional changes were evaluated by RT-qPCR. Metabolic consequences were assessed by measuring NADH/NAD+ ratios. To model resistance to BRAF + MEK inhibition (BRAFi+MEKi), two systems were generated: (1) TGFβ1-induced adaptive resistance and (2) drug-acquired resistance following continuous BRAFi+MEKi exposure. Resistance was confirmed by phosphorylated ERK immunoblotting and IC50 analysis.EGLN1 KO reduced melanoma proliferation by 70–80% relative to control cells (p < 0.05). Roxadustat inhibited growth in a HIF-1α–dependent manner, with IC50 values of 40–80 µM in wild-type cells versus >100 µM in HIF1A KO cells. Long-term treatment with 10–20 µM roxadustat reduced wild-type proliferation by 30–70%, but only 0–10% in HIF1A KO cells. Disruption of HIF1AN or treatment with DM-NOFD enhanced roxadustat sensitivity, decreasing proliferation by 40–60%, an effect absent in HIF1A KO models. Roxadustat increased PHD2 protein levels and induced EGLN1 transcription in wild-type cells, accompanied by upregulation of canonical HIF-1 targets (LDHA, PDK1, BNIP3). A 20–50% increase in the NADH/NAD+ ratio after 4 h of roxadustat treatment indicated a metabolic shift toward reductive stress. In BRAFi+MEKi-resistant models, roxadustat enhanced BRAFi+MEKi potency and inhibited ERK phosphorylation, reversing MAPK pathway reactivation in A375 acquired-resistant cells, but had no effect in A375 HIF1A KO resistant models, demonstrating a requirement for HIF-1α. In TGFβ1-induced adaptive resistance, HIF1A was necessary for the development and maintenance of resistance phenotypes, indicating that HIF-1α signaling contributes to early adaptive BRAFi+MEKi tolerance. These studies demonstrate that PHD2 inhibition suppresses melanoma growth through HIF-1α stabilization and can be enhanced by concurrent FIH inhibition. The requirement of HIF-1α for both adaptive and acquired BRAFi+MEKi resistance and for roxadustat-mediated re-sensitization highlights a functional link between hypoxia signaling and therapeutic tolerance. These findings support continued evaluation of PHD2-directed strategies, including combination approaches, in melanoma. Citation Format: Claire Erdaje Palma, Stephen M. F. Jamieson, Tet-Woo Lee, Dean Singleton. The role of PHD2 inhibition in melanoma progression, metabolic adaptation, and therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7295.