American Association for Cancer Research
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Supplementary Methods from Resistance to Selective BRAF Inhibition Can Be Mediated by Modest Upstream Pathway Activation

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posted on 2023-03-30, 21:15 authored by Fei Su, William D. Bradley, Qiongqing Wang, Hong Yang, Lizhong Xu, Brian Higgins, Kenneth Kolinsky, Kathryn Packman, Min Jung Kim, Kerstin Trunzer, Richard J. Lee, Kathleen Schostack, Jade Carter, Thomas Albert, Soren Germer, Jim Rosinski, Mitchell Martin, Mary Ellen Simcox, Brian Lestini, David Heimbrook, Gideon Bollag

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A high percentage of patients with BRAFV600E mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAFV600E melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAFV600E inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance. Cancer Res; 72(4); 969–78. ©2011 AACR.

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