22 42 inactivation of the signaling mechanism can translate into specific consequences in DNA repair 43 capacity. The heterogeneity of the responses reported herein could help define subtypes of 44 melanoma that are associated with resistance to targeted therapies. 3 45 131 132 Cell Pellet Preparation 133 The concentrations of the inhibitors used in the DNA repair experiments were selected on the 134 basis of cytotoxicity experiments. Specifically, the doses were selected because they induced 135 about 20-30% toxicity (Table 1 and S1 Fig). Cells were treated for 24h and rinsed two times in 136 PBS. Pellets containing 3-5 million cells were prepared and frozen at -80°C in albumin/DMSO

About 50% of melanomas carry activating mutation in BRAF or NRAS genes. BRAF/MEK inhibitors elicit a transient effective response but resistance rapidly develops through various MAPK/PI3K/AKT pathway activating mechanisms. DNA Repair mechanisms are regulated by these signaling pathways. We hypothesized that effective inhibition of the MAPK pathway should translate into decrease of DNA Repair capacities. To gain insights into this hypothesis we measured the DNA Repair capacities of 12 melanoma cell lines treated or not by BRAF and MEK inhibitors (respectively Vemurafenib V and Cobimetinib C, Roche laboratories), alone and in combination. We evaluated various excision synthesis repair mechanisms using a multiplexed functional DNA Repair assay using cell extracts. We thus obtained a comprehensive overview of the cell lines DNA Repair capacities. Significant qualitative and quantitative differences were observed between the DNA Repair profiles of the 3 mutation groups in non-treated cells. Globally DNA Repair capacities of BRAFm cells significantly decreased following treatment by V and V+C confirming that excision synthesis repair mechanisms are under the control of the MAPK pathway. When the cell lines were examined individually, interesting features were observed. 3/7 showed drastic decrease of DNA repair with V and V+C treatment. On the contrary, C alone activated repair in 3/7, possibly reflecting a paradoxical activation of some pathways. In addition, among the 3 NRASm cell lines, only 1 exhibited a marked decreased of DNA Repair after C treatment. Surprisingly V and V+C activated some repair activities in one WT cell line, possibly reflecting some paradoxical activation of the complex kinase network. Our results suggest that mutations in signaling kinase pathways impact the so-called DNA Damage Response and translate into specific DNA Repair Enzyme Signatures. Crosstalk of tyrosine kinases with the DNA damage signaling pathway is well known. It is possible to take advantage of their intricate regulation through characterization of DNA Repair Enzymatic Signature to gain information on the inhibition efficacy of targeted drugs. Interestingly this approach could be conducted on clinical samples and we propose to use the DNA Repair Enzyme Signature as a new tool to investigate effectiveness of targeted therapies in metastatic melanoma. This study was sponsored by Institut Roche. Citation Format: Sylvie Sauvaigo, Manel Benkhiat, Florian Braisaz, Florence de Fraipont, Caroline Aspord, Stéphane Mouret, Joël Plumas, Fanny Bouquet, Marie-Thérèse Leccia. DNA Repair Enzyme Signature as a biomarker of MAPK pathway inhibition by targeted therapies in melanoma cell lines [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 2888.