Abstract 4108: Longitudinal diffusion-weighted MRI assessment of NRAS mutant melanoma response to dual RAF-MEK inhibition reveals differences associated with collagen deposition
Authors: Delgado-Goni Teresa , Wantuch Slawomir , Casals-Galobart Teresa , Banerji Udai , Tunariu Nina , Robinson Simon P. , Leach Martin O. , Beloueche-Babari Mounia 
Published: August 2018

Abstract
Background and Purpose: Oncogenic NRAS, which drives the constitutive activation of the RAS-RAF-MEK-ERK pathway, is expressed in 15–20% of melanomas and is the second most common driver mutation in this disease. Inhibition of oncogenic RAS has proven extremely challenging with no agents clinically approved to date. RO5126766 is a dual RAF/MEK inhibitor with activity in mutant RAS driven cancers currently in early phase clinical trials.
Here the potential of non-invasive diffusion-weighted MRI (DW-MRI) to detect response to acute and long-term treatment with RO5126766 was investigated in NRAS mutant human melanoma xenografts.
Experimental procedures: Female NCr/nu mice with established D04 (NRAS(Q61L) melanoma) subcutaneous tumors (~300mm3) were treated with either vehicle or RO5126766 (5mg/kg once daily) for 3 days (acute protocol, n=4 /group) or 10 days (long-term protocol, n=5/group). DW-MRI was performed prior to and at the end of each treatment protocol to determine tumor apparent diffusion coefficient (ADC), a parameter proportional to tissue water diffusivity. MRI data were correlated with immunohistochemical analysis of tumor tissue stained with H&E (cellularity and necrosis), Masson's trichrome (MTC, collagen) and P-ERK (target inhibition). In addition, D04 cells were treated in vitro with RO5126766 (1µM, 10xGI50) for 24h and mRNA expression of genes involved in collagen synthesis (COL1A1 and COL1A2) assessed with qRT-PCR.
Results: RO5126766 induced a significant reduction in D04 tumor volume, relative to pre-treatment values, after both acute (76 ± 14%, p=0.04) and long-term treatments (63 ± 20%, p=0.01). Baseline tumor ADC equaled ca. 700x10-6mm2/s and showed no changes in the 3-day treatment cohort relative to pre-treatment values (95 ± 10%). In contrast, a significant decrease in ADC was observed in the long-term RO5126766 treatment cohort (to 81 ± 16% of day 0 value, p=0.04). Whilst H&E staining revealed no change in tumor necrosis following either treatment protocol, a significant increase in collagen deposition, shown by MTC staining, was detected in long-term RO5126766-treated tumors compared to vehicle controls (p=0.01). qRT-PCR analysis in D04 in vitro cell cultures following exposure to RO5126766 revealed a significant increase in COL1A1 expression (up to 2.6-fold, p=0.006), indicating that the increase in tumor collagen observed in vivo was, at least in part, due to increased synthesis by melanoma cells.
Conclusions: Our data show that response to the dual RAF-MEK inhibitor RO5126766 in mutant NRAS melanoma xenografts is associated with extracellular matrix remodeling via collagen deposition following long-term treatment. This process can be monitored non-invasively using DW-MRI indicating potential for clinical translation.
Citation Format: Teresa Delgado-Goni, Slawomir Wantuch, Teresa Casals-Galobart, Udai Banerji, Nina Tunariu, Simon P. Robinson, Martin O. Leach, Mounia Beloueche-Babari. Longitudinal diffusion-weighted MRI assessment of NRAS mutant melanoma response to dual RAF-MEK inhibition reveals differences associated with collagen deposition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4108.