Tunable-Combinatorial Mechanisms of Acquired Resistance Limit the Efficacy of BRAF/MEK Cotargeting but Result in Melanoma Drug Addiction

• BRAFi+MEKi-resistant melanomas augment mutational mechanisms of BRAFi resistance
• Extreme gene dosage changes or concurrent mutations drive double-drug resistance
• Neophysiologic V600EBRAF-WTCRAF or -MUTMEK interactions can drive MAPK reactivation
• Acute double-drug withdrawal elicits ERK rebound and loss of melanoma viability

SummaryCombined BRAF- and MEK-targeted therapy improves upon BRAF inhibitor (BRAFi) therapy but is still beset by acquired resistance. We show that melanomas acquire resistance to combined BRAF and MEK inhibition by augmenting or combining mechanisms of single-agent BRAFi resistance. These double-drug resistance-associated genetic configurations significantly altered molecular interactions underlying MAPK pathway reactivation. V600EBRAF, expressed at supraphysiological levels because of V600EBRAF ultra-amplification, dimerized with and activated CRAF. In addition, MEK mutants enhanced interaction with overexpressed V600EBRAF via a regulatory interface at R662 of V600EBRAF. Importantly, melanoma cell lines selected for resistance to BRAFi+MEKi, but not those to BRAFi alone, displayed robust drug addiction, providing a potentially exploitable therapeutic opportunity.