Identifying Combinatorial Drug Targets for Ras Pathway-Driven Melanomas

Abstract

Cutaneous melanoma is a highly metastatic skin cancer, with ~100,000 new cases estimated to occur in 2020 in the US. Melanomas are defined by oncogenic “driver” mutations that constitutively activate the Ras/Raf/MEK/ERK signaling (henceforth called Ras/ERK) pathway. Clinical agents that target various components of this pathway have been developed, although they are not typically curative. For BRAF-mutant melanomas, combined BRAF and MEK inhibitors prolong survival, although patients still relapse within ~11 months. Unfortunately, Ras/ERK pathway inhibitors are even less effective in NRAS-mutant tumors, with MEK inhibitors stimulating only partial responses in 15-20% of patients. Thus, NRAS mutations are associated with poor overall survival, and there is a great need for improved therapies. The goal of my thesis has been to identify agents that potentiate the effects of Ras/ERK pathway inhibitors as a means of developing more effective therapies for NRAS and other Ras pathway-driven melanomas. This dissertation describes three promising therapeutic combinations. In one approach, we discovered a new combinatorial therapy for NRAS-mutant melanomas by performing an unbiased negative selection CRISPR-Cas9 screen. Specifically, we identified the de-ubiquitinase USP7 as a target that when suppressed, sensitizes NRAS-mutant melanomas to MEK inhibitors. Further genetic and mechanistic analysis revealed USP7 interacts with two other hits from our screen and together form a complex that regulates H2B ubiquitination, a marker of transcriptional elongation. Through epigenetic and transcriptional studies, we found that this complex regulates the expression of CABLES1, which is required for triggering cell death and tumor regression. Using candidate-based approaches, we also identified two additional combination therapies that target BRAF-, NRAS-, and NF1-mutant melanomas. Specifically, we discovered that histone de-acetylase (HDAC) inhibitors potentiate responses to Ras/ERK signaling pathway inhibitors in BRAF-, NRAS-, and NF1-mutant melanomas by suppressing DNA repair pathways (Maertens et al. 2019). Additionally, we demonstrated that the combination of Ras/ERK signaling pathway and bromodomain and extraterminal domain (BET) inhibitors represents a promising new therapeutic approach to reduce acquired and intrinsic resistance in BRAF-mutant melanomas (Katherine R. Singleton 2017). In summary, this dissertation details three promising combination therapies for Ras/ERK pathway-driven melanomas and describes the distinct mechanisms by which they function.