Results Sensitivity to E6201 in a melanoma cell line panel Sensitivity to E6201 was assessed in a panel of 31 cell lines for which the mutation status of common mel anoma genes was known. These lines were chosen to represent different mutational profiles from a larger panel of more than one hundred melanoma cell lines. Western blots in Additional file 1 Figure S1 trichostatin a clinical trials confirm that E6201 efficiently inhibits MEK1/2 ac tivity by virtue of its ability to abrogate phosphoryl ation of ERK1/2 in our entire panel of melanoma cell lines. The majority of the melanoma cell lines were sensitive to E6201. MAPK activation due to mutations Inhibitors,Modulators,Libraries in BRAF and NRAS was not significantly associated with increased sensitivity to E6201. In the 26 cell lines carrying mutations in BRAF, NRAS, or HRAS, sensitivity to E6201 was statistically associated with wildtype PTEN status.

Specific ally, of the 18 cell lines with Inhibitors,Modulators,Libraries wildtype PTEN, 17 were sensitive whereas in the 8 cell lines with mutant PTEN, only 4 were sensitive. Moreover, even if PTEN status alone is examined, E6201 sensitivity is associated, albeit non significantly, with wildtype PTEN status. 23/31 cell lines are wildtype for PTEN and of these 20 are sensitive. Interestingly, Inhibitors,Modulators,Libraries 18 of the 24 sensitive cell lines also demonstrated hypersensitivity to E6201, with an IC50 100 nM. Using this criterion, BRAF mutation status correlated with E6201 hypersensitivity, with 15 out of the 18 hypersensitive cell lines possessing a BRAF mutation. In contrast, of the 11 cell lines with wildtype BRAF, only 3 were hypersensitive.

In those cell lines carrying mutations in BRAF, sensitivity to E6201 was not statistically associated with wildtype PTEN status. NRAS/HRAS mutation status correlated with E6201 resistance, where none of the 5 NRAS/HRAS mutant cell lines were hypersensitive to E6201 Inhibitors,Modulators,Libraries and 18 of the 26 NRAS/HRAS Inhibitors,Modulators,Libraries wildtype cell lines were hypersensitive. Neither CDKN2A, CDK4 or TP53 mutational status in our panel of melanoma cell lines, irrespective of their BRAF and RAS mutational status, reference 4 was associated with E6201 sensitivity. E6201 sensitivity and downstream pathway activation To determine whether E6201 responsiveness correlated with direct Akt or ERK1/2 activation, the phosphoryl ation status of Akt and ERK1/2 proteins was evaluated following serum starvation. Phosphorylated Akt was detectable in 7/7 cell lines with mutant PTEN. In addition, pAkt was present in 5/23 cell lines with wildtype PTEN although the mechanism re sponsible for phosphorylation of Akt in these cell lines is unknown. Phosphorylated ERK1/2 was detected in all cell lines with mutant BRAF. Consistent with previous reports, elevated pERK1/2 was detected in 3/5 cell lines with mutant NRAS or HRAS.