Abstract 2323: Mutant B-Raf-mediated activation of deubiquitinases in melanoma defines Usp5 as a potential therapeutic target.

Abstract

Abstract Melanoma is a lethal form of cutaneous cancer which accounts for 80% of skin cancer deaths owing to its highly metastatic behavior and few clinically effective therapies. More than 60% of melanoma patients present with mutations in B-Raf, a serine/threonine kinase that increases proliferation and survival through the constitutive activation of ERK signaling. Inhibition of mutated B-Raf with specific inhibitors like vemurafenib reduces tumor growth and extends patient survival. However, clinical responsiveness to vemurafenib is limited in most melanoma patients, prompting analysis of additional pathways that could be targeted to extend vemurafenib responsiveness or overcome resistance. Towards this goal, we examined the impact of vemurafenib on substrates or cascades activated in B-Raf mutant and wild-type melanoma cells. We were particularly interested in assessing the impact of mutant B-Raf inhibition on deubiquitinases (DUBs), which are overexpressed or activated in many tumor types. DUBs control the stability, activity and localization of multiple proteins and are emerging as therapeutic targets. We found that vemurafenib treatment or lentiviral-mediated B-Raf knockdown reduced the activity of several DUBs in B-Raf mutant melanoma cells, including Usp9x, Usp14 and Usp5. These DUBs were not affected by vemurafenib in w/t B-Raf-expressing cells. Interestingly lentiviral-mediated knockdown of Usp5, but not Usp9x, reduced melanoma growth and increased vemurafenib sensitivity whereas Usp5 overexpression suppressed vemurafenib activity. Inhibition of Usp5 activity by vemurafenib or by Usp5 shRNA KD alone had little effect on melanoma cell apoptosis. However B-Raf inhibition in Usp5 KD cells resulted in >5-fold increased melanoma cell apoptosis. Further, Usp5 KD increased accumulation of p53 and p53-related proteins as well as their transcriptional targets. Our data clearly shows that mutant B-Raf activates Usp5 to suppress apoptotic responsiveness to vemurafenib and kinase inhibition alone was not sufficient to completely suppress Usp5 activity. Silencing studies demonstrate that Usp5 inhibition will enhance cell killing in response to B-Raf inhibition in melanoma. To address that potential, we compared anti-tumor activity in mice treated with vemurafenib and a small molecule DUB inhibitor, WP1130, with activity against Usp5. Treatment with either agent alone reduced tumor growth while mice receiving both vemurafenib and WP1130 had enhanced anti-tumor responses. Together, these results suggest that mutant B-Raf activates Usp5 activity to suppress p53 induction and apoptotic responsiveness to vemurafenib. These observations also suggest that further testing and refinement of Usp5 inhibitors as an additional approach to targeted therapy for melanoma are warranted. Citation Format: Harish Potu, Luke F. Peterson, Monique Verhaegen, Moshe Talpaz, Nicholas J. Donato. Mutant B-Raf-mediated activation of deubiquitinases in melanoma defines Usp5 as a potential therapeutic target. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2323. doi:10.1158/1538-7445.AM2013-2323