Abstract 3717: New therapies for the treatment of BRAF/NRAS wild type melanoma
Authors: Ranzani Marco , Kemper Kristel , Michaut Magali , Krijgsman Oscar , Iyer Vivek , Speak Anneliese , Nsengimana Jeremie , Wong Kim , Grinkevich Vera , Aben Nanne , Velasco-Herrera Martin Del Castillo , Alsinet Clara , Sjoberg Marcela , Rashid Mamunur , Turner Gemma , Behan Fiona , Supper Emmanuelle , Thompson Nicola , Bignell Graham , Dutton-Regester Ken , Pritchard Antonia , Wong Chi , McDermott Ultan , Hayward Nicholas K. , Yusa Kosuke , Newton-Bishop Julia , Wessels Lodewyk , Garnett Mathew , Peeper Daniel , Adams David 
Published: July 2017

Abstract
Melanoma represents the common tumor whose incidence has increased the most in the last 30 years and causes more than one death every hour in the US alone. Despite significant advances in targeted and immunotherapies, most patients cannot still be cured. Our aim is to identify new drug combinations that are synergistic in BRAF/NRAS wild type melanoma, a sub-type representing 30% of cases for which targeted therapies are not currently available. We high-throughput screened a collection of 20 BRAF/NRAS wild type melanoma cell lines with 180 drug combinations (60 library drugs used at 5 different concentrations combined with 3 clinically relevant anchor drugs) and generated over 8000 survival curves . We found that 25% of cell lines are highly sensitive to a combination of nilotinib plus trametinib and confirmed this finding with 2 independent assays. We further validated the drug synergy firstly using an independent collection of BRAF/NRAS wild type melanoma cell lines (n=7), then a collection of BRAF/NRAS wild type patient derived xenotransplant cultures (n=3), and finally with a collection of BRAFV600E and NRASQ61 melanoma cell lines (n=12). Further, we generated a gene expression signature of cell lines that display synergy for the nilotinib/trametinib combination, and used it to classify human melanomas from Leeds Melanoma Project (N=171) and TCGA (n=470) cohorts. Tumors classified as “synergistic-like” (27.9 and 36.7%, respectively) are associated to decreased overall and recurrence free survival (P&lt;0.05), suggesting that our combination might be effective in a relevant fraction of aggressive tumors. In order to identify drug resistance mechanisms we deployed a genome-wide CRISPR/Cas9 screen. We found that loss of the tuberous sclerosis complex can confer resistance to nilotinib/trametinib, and validated this mechanism using clonal engineered lines. Since tuberous sclerosis complex genes are mutated in 10% of melanomas, this approach can help to identify patients potentially refractory to the treatment. We also investigated the molecular mechanism of nilotinib/trametinib synergy by analysing the level of several phosphoproteins upon treatment. We discovered that the nilotinib/trametinib combination synergistically reduce the level of P-ERK in synergistic cell lines but not in cell lines resistant to the drug combination, thus pointing out the MAPK pathway dependence of the synergy. This finding provides a putative marker to identify tumors responsive to the treatment. Finally, we tested in vivo the nilotinib/trametinib combination in a patient derived xenotransplant mouse model and showed that the combination is well tolerated and significantly more effective than the 2 drugs alone (P&lt;0.01). These data suggest a strong clinical translation potential for nilotinib/trametinib combination and pave the way to the development of clinical trials for BRAF/NRAS wild type melanoma.
Citation Format: Marco Ranzani, Kristel Kemper, Magali Michaut, Oscar Krijgsman, Vivek Iyer, Anneliese Speak, Jeremie Nsengimana, Kim Wong, Vera Grinkevich, Nanne Aben, Martin Del Castillo Velasco-Herrera, Clara Alsinet, Marcela Sjoberg, Mamunur Rashid, Gemma Turner, Fiona Behan, Emmanuelle Supper, Nicola Thompson, Graham Bignell, Ken Dutton-Regester, Antonia Pritchard, Chi Wong, Ultan McDermott, Nicholas K. Hayward, Kosuke Yusa, Julia Newton-Bishop, Lodewyk Wessels, Mathew Garnett, Daniel Peeper, David Adams. New therapies for the treatment of BRAF/NRAS wild type melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3717. doi:10.1158/1538-7445.AM2017-3717