Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies.

Azad Saei,Paolo Nuciforo,Prasanna Vasudevan Iyengar,Javier Cortés,Violeta Serra,Eva Muñoz-Couselo,Marta Palafox,John Lalith Charles Richard,Christopher Nötzel,Touati Benoukraf,Nishi Kumari,Brendan Pang,Henry Yang,Wai Leong Tam,Nesaretnam Barr Kumarakulasinghe,Zul Fazreen Bin Adam Isa,Marta Guzmán ,Shiwei Zhou ,Patrick Jaynes ,Pieter J.a Eichhorn

doi:10.1084/jem.20171960

Abstract

RAF kinase inhibitors are clinically active in patients with BRAF (V600E) mutant melanoma. However, rarely do tumors regress completely, with the majority of responses being short-lived. This is partially mediated through the loss of negative feedback loops after MAPK inhibition and reactivation of upstream signaling. Here, we demonstrate that the deubiquitinating enzyme USP28 functions through a feedback loop to destabilize RAF family members. Loss of USP28 stabilizes BRAF enhancing downstream MAPK activation and promotes resistance to RAF inhibitor therapy in culture and in vivo models. Importantly, we demonstrate that USP28 is deleted in a proportion of melanoma patients and may act as a biomarker for response to BRAF inhibitor therapy in patients. Furthermore, we identify Rigosertib as a possible therapeutic strategy for USP28-depleted tumors. Our results show that loss of USP28 enhances MAPK activity through the stabilization of RAF family members and is a key factor in BRAF inhibitor resistance.

Highlights

Activating mutations in the BRAF oncogene are found in 40–60% of patients with advanced melanoma (Davies et al, 2002)
Pools of shRNAs targeting 94 known or predicted deubiquitinating enzymes (DUBs) were introduced into 293T cells and the abundance of phosphorylated ERK to total ERK was quantified (Fig. S1 A and Table S1; Brummelkamp et al, 2003)
To determine whether expression of any of these genes are regulated by MAPK signaling in melanoma, we analyzed their expression by quantitative real-time PCR after treatment with the BRAF inhibitor vemurafenib (PLX4032) in the BRAF (V600E) mutant melanoma cell line WM164

Summary

Introduction

Activating mutations in the BRAF oncogene are found in 40–60% of patients with advanced melanoma (Davies et al, 2002). Several functional genomic and next-generation sequencing–based approaches probing resistant models have identified COT/TPL2, STAG family members, loss of RNF125, and YAP overexpression as mechanisms of BRAF inhibitor resistance (Johannessen et al, 2010; Kim et al, 2015; Lin et al, 2015; Shen et al, 2016). These mechanisms are not prevalent enough to justify the high frequency of primary and acquired resistance to BRAF inhibitors

Methods

Results

Conclusion