Characterization of RAS Alterations in Myeloma: Why Direct Targeting of RAS May be the Most Appropriate Therapeutic Approach
Authors: Aronson Lauren I , Walker Brian A , Boyle Eileen M , Wardell Christopher P , Morgan Gareth J , Davies Faith E 
Published: October 2019

Abstract
Recent studies have shown that the RAS pathway is a major therapeutic target with which to develop personalized treatment strategies for myeloma patients. In order to translate this knowledge into patient benefit, we have taken a systematic approach by characterizing the mutational profile of the RAS pathway and examining its role in disease progression and patient outcome. Finally we investigated a targeted treatment approach using small molecules to inhibit the RAS MAPK signalling cascade at different levels.
In order to gain a deeper understanding of RAS pathway alterations in myeloma, next-generation sequencing data was analyzed from 463 patients entered into the MRC Myeloma XI trial. Using published papers and online databases, a panel of 72 genes were identified as RAS MAPK family genes and were grouped into 5 categories: RAS GTPases, guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), downstream pathway or RAS processing genes. Thirty seven of these genes were mutated in 50% of patients, with novel mutations described for genes in each of these categories. The mutation frequency of KRAS (22.5%), NRAS (19.7%) and BRAF (7.8%) was in-line with other reports and KRAS was found mutated at more codons compared to NRAS. Additionally, recurrent mutations at the same codon were identified only in KRAS, NRAS and BRAF, attesting to their importance in this disease. A quarter of patients (n=59) had more than one RAS MAPK mutation. When more than one mutation was present, the majority were in two RAS GTPases (n=19) (most commonly KRAS and NRAS (n=8) or both in KRAS (n=6)) or in a RAS GTPase and a downstream pathway gene (n=12). One patient had mutations in KRAS, NRAS and BRAF but at sub-clonal levels, indicating they may be present in different cells.
We also assessed the role of this pathway in myeloma by examining gene expression profiling data from plasma cells at various myeloma disease stages (GSE2113, GSE5900 and GSE6477). This analysis highlighted roles for KRAS, RAF1, MAPK1, MAP2K1, NF1, RASGRP1 and FNTA in disease progression from MGUS to PCL. Additionally, survival analysis suggested important roles for MAPK1 in determining outcome in newly diagnosed patients in the HOVON65/GMMG-HD4 (GSE19784) trial and FNTA, KRAS, MAP2K1 and RAF1 in determining outcome in relapsed patients in the APEX (GSE9782) trial. These results suggest the highlighted genes are important in mediating clinical outcome and could be therapeutic targets.
Given the biological importance of this pathway, we have explored its therapeutic potential by using inhibitors that target the pathway at different levels. Inhibitors included those targeting RAS (n=2), RAF1 (n=2), RAF1/BRAF (n=2), MEK (n=2) and ERK (n=1). The effect of these inhibitors was explored in a panel of 8 myeloma cell lines chosen based on their differing RAS mutational status. Using Western blot analysis, all cell lines showed evidence of activation of the RAS MAPK pathway. No consistent pattern of expression was observed in terms of the levels of activated pathway proteins in KRAS mutant cells compared to NRAS or non-mutated cells. In contrast NRAS and BRAF mutant cells contained elevated levels of activated MEK compared to the other cell lines and were the most sensitive to a MEK inhibitor. Interestingly all cell lines, regardless of mutational status, were similarly sensitive to a novel KRAS inhibitor which prevents KRAS reaching its correct localization. It was shown to be effective at inducing apoptosis, even in the presence of the bone marrow stromal cells. Importantly, protein analysis demonstrated these effects were target-driven with Western blot analysis indicating inhibition of RAS signalling within two to six hours of treatment. Thus, the direct targeting of RAS early in the signalling cascade may be effective against myeloma, irrespective of the RAS mutation status.

Disclosures
Walker: Onyx Pharmaceuticals: Consultancy, Honoraria.