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Recent results from clinical trials with the BRAF inhibitors GSK2118436 (dabrafenib) and PLX4032 (vemurafenib) have shown encouraging response rates; however, the duration of response has been limited. To identify determinants of acquired resistance to GSK2118436 and strategies to overcome the resistance, we isolated GSK2118436 drug-resistant clones from the A375 BRAFV600E and the YUSIT1 BRAFV600K melanoma cell lines. These clones also showed reduced sensitivity to the allosteric mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitor GSK1120212 (trametinib). Genetic characterization of these clones identified an in-frame deletion in MEK1 (MEK1K59del) or NRAS mutation (NRASQ61K and/or NRASA146T) with and without MEK1P387S in the BRAFV600E background and NRASQ61K in the BRAFV600K background. Stable knockdown of NRAS with short hairpin RNA partially restored GSK2118436 sensitivity in mutant NRAS clones, whereas expression of NRASQ61K or NRASA146T in the A375 parental cells decreased sensitivity to GSK2118436. Similarly, expression of MEK1K59del, but not MEK1P387S, decreased sensitivity of A375 cells to GSK2118436. The combination of GSK2118436 and GSK1120212 effectively inhibited cell growth, decreased ERK phosphorylation, decreased cyclin D1 protein, and increased p27kip1 protein in the resistant clones. Moreover, the combination of GSK2118436 or GSK1120212 with the phosphoinositide 3-kinase/mTOR inhibitor GSK2126458 enhanced cell growth inhibition and decreased S6 ribosomal protein phosphorylation in these clones. Our results show that NRAS and/or MEK mutations contribute to BRAF inhibitor resistance in vitro, and the combination of GSK2118436 and GSK1120212 overcomes this resistance. In addition, these resistant clones respond to the combination of GSK2126458 with GSK2118436 or GSK1120212. Clinical trials are ongoing or planned to test these combinations. Mol Cancer Ther; 11(4); 909–20. ©2012 AACR.

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>10μM) and PLX4032 (IC50>10μM). In addition, they displayed greater than 10-fold less sensitivity to an allosteric inhibitor of MEK1/2, GSK1120212 (IC50>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 >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 >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.

Activation of the Ras-Raf-MEK-ERK pathway has been implicated in a large range of human cancers. Growth factor receptor stimulation by extracellular ligands activates Ras, which then sets in motion a signal transduction cascade through the Raf, MEK and ERK serine/threonine kinases. Mutation of the B-Raf kinase constitutively activates MAPK signalling, thus bypassing the need for upstream stimuli. This has been genetically associated with several human cancers, especially occurrence of the B-RafV600E mutant and its high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. The ability to selectively and potently inhibit B-Raf should provide a potential therapy for patients with mutant B-Raf tumors, for which addictive dependency on this pathway is observed. We have identified a novel, potent, and selective Raf kinase inhibitor that is capable of inhibiting the kinase activity of wild-type B-Raf, B-RafV600E and c-Raf with IC50 values of 3.2, 0.8, and 5.0 nM, respectively. Kinase panel screening for over 270 kinases has indicated that this inhibitor is selective for Raf kinase, with ∼400 fold selectivity towards B-Raf over 91% of the other kinases tested. Specific cellular inhibition of B-RafV600E kinase by this inhibitor leads to decreased ERK phosphorylation and inhibition of cell proliferation by an initial arrest in the G1 phase of the cell cycle, followed by cell death. This inhibition is selective for cancer cells that specifically encode the mutation for B-RafV600E. Oral compound administration inhibits the growth of B-RafV600E mutant melanoma (A375P) and colon cancer (Colo205) human tumor xenografts, growing subcutaneously in immuno-compromised mice. This cell-specific B-RafV600E inhibitor is currently being evaluated in a human Phase I clinical trial. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B88.