USP43 downregulation as driver of acquired resistance to dabrafenib and trametinib in melanoma via the HIF1A-PDK1 axis-mediated oxidative phosphorylation.

Abstract

e21517 Background: Acquired resistance to combined BRAF and MEK inhibition (Dabrafenib+Trametinib) limits long-term survival in BRAF-mutant melanoma. Metabolic reprogramming toward oxidative phosphorylation (OXPHOS) is a key driver of this resistance, yet the post-translational mechanisms triggering this switch remain elusive. We investigated the role of the deubiquitinase USP43 in regulating metabolic plasticity and drug sensitivity. Methods: Dabrafenib+Trametinib-resistant melanoma cell lines were established. Transcriptomic profiling and paired clinical sample analysis were performed to identify key regulators. Metabolic phenotypes were assessed using Seahorse extracellular flux analysis. Mechanistic studies included Co-IP, K48-linkage specific ubiquitination assays, cycloheximide (CHX) chase assays, and nucleocytoplasmic fractionation. Therapeutic potential was validated in xenograft models with rescue arms using HIF1A inhibitors. Results: 1) We successfully generated resistant melanoma cell lines with significantly elevated IC50 values. RNA-sequencing and immunohistochemistry of paired clinical samples revealed a specific downregulation of USP43 in resistant tumors compared to pre-treatment baselines. 2) Metabolic profiling and Seahorse assays demonstrated that resistant cells underwent a metabolic switch characterized by elevated oxygen consumption rates (OCR) and mitochondrial ATP production, distinct from the glycolytic profile of sensitive cells. 3) Mechanistically, USP43 was identified to directly interact with HIF1A. USP43 specifically cleaved K48-linked poly-ubiquitin chains from HIF1A, thereby extending its protein half-life and preventing proteasomal degradation. 4) In resistant cells, USP43 deficiency led to rapid HIF1A degradation, resulting in impaired nuclear translocation and reduced transcriptional activation of PDK1. This downregulation of PDK1 facilitated pyruvate entry into mitochondria, fueling hyper-active oxidative phosphorylation. 5) Overexpression of USP43 in resistant cells restored the HIF1A-PDK1 axis, suppressed mitochondrial respiration, and re-sensitized cells to treatment both in vitro and in xenograft models. Crucially, this re-sensitization was abrogated by pharmacological inhibition of HIF1A or PDK1, confirming that USP43 functions strictly through this metabolic axis. Conclusions: Our study elucidates a novel USP43-HIF1A-PDK1 signaling axis that safeguards against metabolic drug resistance. The loss of USP43 unleashes oxidative phosphorylation by destabilizing HIF1A, allowing melanoma cells to survive targeted therapy. These findings highlight USP43 as a potential predictive biomarker and therapeutic target for overcoming resistance in BRAF-mutant melanoma.