Abstract B71: Acquired resistance to the BRAF inhibitor GSK2118436, mediated by NRAS or MEK mutation, can be overcome with combinations that inhibit BRAF and MEK, BRAF and PI3K/mTOR, or MEK and PI3K/mTOR.
Authors: Greger James G. , Eastman Stephen D. , Zhang Vivian , Bleam Maureen R. , Hughes Ashley , Smitheman Kimberly N. , Laquerre Sylvie , Liu Li , Gilmer Tona M. 
Published: June 2012

Abstract
Recent results from clinical trials with the BRAF inhibitors PLX4032 and GSK2118436 have shown encouraging clinical response rates; however, acquired resistance has limited response duration. In an effort to identify determinants of acquired resistance to GSK2118436 and strategies to overcome the resistance, we isolated nine GSK2118436 resistant clones derived from A375 BRAFV600E melanoma cell line (IC50=0.028μM). In three day cell growth assays, the resistant clones were insensitive to the BRAF inhibitors GSK2118436 (IC50&gt;10μM) and PLX4032 (IC50&gt;10μM). In addition, they displayed greater than 10-fold less sensitivity to an allosteric inhibitor of MEK1/2, GSK1120212 (IC50&gt;0.06μM), as compared to the parental A375 cells (IC50=0.005μM). Genetic characterization of these clones by Sanger sequencing identified an in-frame deletion in MEK1 (MEK1K59del), or NRAS mutations (NRASQ61K and/or NRASA146T) with and without a MEK1P387S mutation. In addition, all clones retained the BRAFV600E mutation and no additional mutations were identified in BRAF, KRAS, HRAS, ARAF, CRAF or PTEN. Stable knockdown of NRAS by shRNA partially restored GSK2118436 sensitivity in the NRAS mutant clones. Expression of mutant NRAS in the parental cells decreased sensitivity to GSK2118436 similar to that observed in the NRAS mutant clones. Similarly, expression of MEK1K59del, but not MEK1P387S, decreased sensitivity of the parental cells to GSK2118436. In three day growth assays, treatment of the resistant clones with GSK2118436 in combination with GSK1120212 enhanced cell growth inhibition 19 to 50% at the IC50 of the combination and was ≥3-fold more potent than the most active single agent, GSK1120212. Sustained treatment of greater than ten days with this combination inhibited &gt;90% of cell growth compared to 30–70% with GSK1120212 alone. By western blot analysis, the combination of GSK2118436 and GSK1120212 effectively decreased ERK phosphorylation and cyclin D1 protein, and increased p27kip1 protein levels in all resistant clones. S6 ribosomal protein phosphorylation was not completely inhibited in all clones with this combination. In contrast, the combination of GSK2118436 or GSK1120212 with the PI3K/mTOR inhibitor GSK2126458 effectively blocked S6 ribosomal protein phosphorylation in the resistant clones. Decreases in ERK phosphorylation and cyclin D1 protein were similar to the combination of GSK2118436 and GSK1120212. The addition of GSK2118436 to GSK2126458 was slightly more potent (∼2-fold) than GSK2126458 alone and enhanced cell growth inhibition 12 to 35% at the IC50 for the combination in 5/7 clones with NRAS mutation and both MEK1K59del clones. The combination of GSK1120212 and GSK2126458 was &gt;2-fold more potent than the most active single agent and enhanced cell growth inhibition by 23 to 33% in all the resistant clones. This combination was synergistic (combination index = 0.24–0.75) in 8/9 clones.
Taken together these results demonstrate that mutation within NRAS or MEK contribute to resistance to BRAF inhibitors in BRAFV600E mutant melanoma. These resistant cells are responsive to the combination of BRAF and MEK inhibitors, as well as BRAF or MEK inhibitors with PI3K/mTOR inhibitors. Clinical trials are ongoing or planned to test these combinations.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B71.