Abstract 4735: Constitutively active RAS and MEK confer resistance to PLX4032 in BRAF-V600E human melanoma cells
Authors: Somers-Edgar Tiffany J. , Ferrao Petranel , Callahan Jason , Bollag Gideon , Hicks Rod , Johnstone Ricky , McArthur Grant A. 
Published: June 2012

Mutation of the BRAF gene occurs in 50-70% of melanomas. The most common of these encodes a V600E amino acid substitution that confers constitutive kinase activity. BRAF-V600E can act as a transforming oncogene and siRNAs to BRAF inhibit the proliferation and survival of melanoma cells. Therefore, BRAF-V600E represents an important novel drug target in these tumors. PLX4032 is a selective inhibitor of BRAF-V600E with a favorable safety profile and significant single agent response rate in clinical melanoma. However, acquired resistance to BRAF inhibitors represents a significant challenge in the development of these agents.

The current study shows that retrovirus-mediated expression of constitutively active MEK1-DD, KRAS-12D, HRAS-12V, or NRAS-61K in a panel of BRAF-V600E human melanoma cells promotes resistance to PLX4032. For example, expression of MEK1-DD in A375 cells resulted in a 13-fold increase in the PLX4032 IC50 concentration, while expression of any of the three RAS isoforms in these cells resulted in &gt;15-fold increases in the PLX4032 IC50 concentration (determined by SRB assays). While PLX4032 potently inhibits ERK phosphorylation in A375 vector control cells, HRAS-12V and MEK1DD cells maintain ERK phosphorylation in the presence of PLX4032. Inhibition of clonogenicity, induction of G0/G1-phase arrest and apoptosis induction mediated by PLX4032 are also reversed by expression of MEK1-DD or RAS isoforms. For example, after treatment of A375 BRAF-V600E melanoma cells with 10 µM PLX4032 for 96 h, 30.2 ± 2.9% of vector control cells versus 9.3 ± 2.2% of MEK1-DD- and 8.3 ± 4.9% of HRAS-12V-expressing cells stained positive for Annexin-V-APC compared to 0.3 ± 0.2% of untreated cells. These findings indicate that activation of RAS or MEK could confer a proliferative and survival advantage in BRAF-V600E cells in the presence of PLX4032 and therefore, could contribute to acquired resistance to this drug.

Preliminary data also suggest that PLX4032 suppresses glycolysis in BRAF-V600E melanoma cells prior to changes in proliferation and viability. This corroborates recent clinical data from a cohort of patients with advanced metastatic melanoma participating in a phase I study of PLX4032. Baseline and day 15 FDG-PET data (indicating metabolic disease burden) from 27 patients treated with ≥ 320mg BID PLX4032 demonstrated a 100% partial metabolic response rate while 3 patients achieved a complete metabolic response after PLX4032 therapy. The current study demonstrates a 53.7% decrease in the rate of glucose uptake in A375 vector control cells treated with 10 µM PLX4032 for 24 h compared to a 4.0% decrease and 11.9% increase in the rate of glucose uptake in MEK1-DD and HRAS-12V expressing cells, respectively. These findings suggest that inhibition of glycolysis may be an important early response to PLX4032 therapy and that activation of RAS or MEK may reverse this effect.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4735. doi:10.1158/1538-7445.AM2011-4735