Melanoma is an aggressive skin cancer characterized by gene mutations, high metastatic potential and few clinically effective systemic therapies. BRAF, MEK1 kinase inhibitors and CTLA4 antagonist have improved patient survival, but only a subset of patients respond or derived long-lasting clinical benefit, illustrating the need for discovery of additional targets and pathways that may be inhibited with small molecules. Given the clinical impact of inhibitors of the ubiquitin-proteasome system in other cancers, we explored the potential for aberrant deubiquitinase (DUB) activity to play a role in the tumorigenic properties of melanoma. We also assessed whether specific DUBs play a role in apoptotic responsiveness and resistance to kinase inhibitors. Initially, we noted that the activity of several DUBs, including Usp9x and Usp5, was elevated in melanoma compared to normal melanocytes. Further, Usp9x and Usp5 expression and activity were up-regulated by expression of BRAFV600E in 293T cells and kinase inhibition partially suppressed both Usp9x and Usp5 activity in vemurafenib sensitive, but not resistant cells. Usp9x knockdown (KD) in BRAF mutant or wild-type melanoma did not affect 2-dimensional cell growth or survival but significantly amplified the apoptotic activity of BRAF or MEK1 inhibitors, respectively. Usp5 KD in melanoma suppressed cell growth by reinforced the S/G2-M checkpoint, enhanced extrinsic caspase activation through modulation of p53 and FAS levels and amplified the apoptotic activity of kinase inhibitors (and other chemotherapeutic agents) and overcame vemurafenib resistance in A375R cells. To further assess the clinical relevance of activated DUBs in melanoma, primary human melanoma explants characterized for their metastatic efficiency in patients and NSG mice demonstrated that Usp9x and Usp5 expression and activity were elevated in efficient metastasizers, but only a subset of inefficient metastasizers, suggesting a potential role for DUBs in the regulation of metastatic potential. To investigate that possibly, control and Usp9x KD cells were grown in Matrigel or suspension culture and colony growth and survival, as well as Usp9x activity were examined. Usp9x KD significantly suppressed melanoma colony growth and fully reduced survival of highly metastatic SK-Mel147 cells in 3D culture. Growth in 3D conditions resulted in activation of Usp9x, but also increased their sensitivity (IC50 <400 nM) to our partially selective Usp9x inhibitor, EOAI3402143 (G9). G9 inhibits both Usp9x and Usp5 activity and phenocopied the activities of both Usp9x and Usp5 KD cells. G9 was safely administered to NSG mice, with complete block of melanoma growth. We conclude that targeted inhibition of specific DUBs will suppress metastatic melanoma and amplify existing melanoma therapies.
Citation Format: Harish Potu, Anupama Pal, Hanshi Sun, Luke Peterson, Moshe Talpaz, Monique Verhaegen, Juxiang Cao, Ugur Eskiocak, Sean Morrison, Nicholas J. Donato. Deubiquitinases Usp9x and Usp5 control tumorigenicity and apoptotic responsiveness in malignant melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4435. doi:10.1158/1538-7445.AM2014-4435