Metastatic melanoma is a lethal malignancy leading to an estimated 65,000 deaths annually worldwide. The serine threonine kinase BRAF is mutated in 40-60% of all melanoma patients frequently at a single hotspot BRAF V600E, which results in hyperactivation of MAPK pathway. RAF kinase inhibitors are clinically active in patients with BRAF (V600E) mutant melanoma. However, rarely do tumours regress completely with the majority of responses being partial and short-lived. Several mechanisms of resistance to RAF inhibitors have been suggested in melanoma. In addition to somatic genomic alterations recent studies have revealed the importance of ubiquitination in the role of MAPK signaling. Yet very little is known about the deubiquitinating enzymes that counteract ubiquitination mediated functions. Our study identified loss of deubiquitinating enzyme, USP28 as a novel mechanism of resistance to vemurafenib in BRAF mutant melanoma.
A genome wide shRNA screen targeting all known deubiquitinating enzymes was performed and level of phosphorylated ERK as the representative of MAPK pathway was assessed by western blotting. Significant hits of the screen were validated and mechanism of action in regulation of MAPK pathway and resistance to MAPK inhibitors in melanoma has been investigated.
Using a functional RNAi screen targeting all known human deubiquitinating enzymes, we identified USP28 as a critical regulator of MAPK pathway. It is known that USP28 binds to and stabilizes the E3 ligase substrate recognition subunit, FBW7 to regulate stability of various proteins. We showed that the USP28/FBW7 complex directly ubiquitinates BRAF and targets BRAF for ubiquitin mediated degradation. Importantly, TCGA datasets indicates that USP28 is deleted in 9% of melanoma patients. Using Kaplan-Meier analysis, we showed that loss of USP28 confers poorer overall survival in melanoma patients. We showed that loss of USP28 enhances MAPK activity through the stabilization of BRAF. Our results revealed the Loss of USP28 drives resistance to RAF inhibitor therapy in BRAF(V600E) tumors both in vitro and in vivo.
Taken together we showed loss of USP28 as a potential biomarker for MAPK activation and vemurafenib resistance in BRAF 600E mutant melanoma.
Clinical trial identification
Legal entity responsible for the study
National University of Singapore
Cancer Science Institute of Singapore, National University of Singapore
All authors have declared no conflicts of interest.