Abstract 961: GSK1120212 inhibits both MEK kinase activity and activation
Authors: Fisher Kelly , Yang Jingsong , Zappacosta Francesca , Groy Arthur , Rominger Cynthia , Gilmartin Aidan G. 
Published: June 2012

Negative feedback loops are commonplace in signal transduction pathways, restoring homeostatic equilibrium. Inhibitory drugs to these pathways, by suppressing the negative feedback, can result in hyperactivation of upstream pathway components. MEK inhibitors have been reported to suppress ERK1/2 mediated activation of DUSPs and Sprouty, resulting in increased phosphorylation and activation of both Raf and Mek. This feedback response may negatively impact an inhibitor's efficacy both by increasing pathway activation and by weakening the compound binding site.

Here we report on studies characterizing the effect of the MEK1/2 inhibitor GSK1120212 in preventing activation of MEK by Raf kinases, a complementary effect to its inhibition of MEK kinase activity. We conducted in vitro MEK activation assays with Raf1 or BRAF and U-MEK in the presence or absence of GSK1120212, and analyzed MEK phosphorylation by ftMSMS. We observed that GSK1120212 completely prevented Raf-dependent phosphorylation of S217 on MEK1, resulting in mono-phosphorylated (S221) MEK1 (p-MEK1). We then showed that although p-MEK1(pS221) is more active than U-MEK, it is 83-fold less active than the fully-activated diphospho-MEK1 (pp-MEK1). Our inhibition study indicated that the affinity of GSK1120212 for MEK1 was reduced by S217 phosphorylation since the IC50 for pp-MEK1 was 15.3 nM, but &lt;2.5nM for p-MEK1 and &lt;0.4 nM for U-MEK1. Taken together, these results indicate that GSK1120212, in addition to potently inhibiting activated MEK, also partially blocks MEK activation by avidly binding U-MEK and preventing S217 phosphorylation by Raf kinases.

We then assessed whether the effect of GSK1120212 on MEK1 activating phosphorylation occurs in cells. In Sk-MEL-28, A375P, and HCT116 cells, treatment with GSK1120212 results in a transient decrease in MEK phosphorylation detected by immunoblotting; however the levels increase over time for both HCT116 and A375P. Since the phospho-MEK antibodies may not distinguish the mono from diphospho MEK, we immunoprecipitated MEK1 from lysates and analyzed MEK phosphorylation by MS. As with the in vitro reaction, GSK1120212 prevented S217 phosphorylation, but not phosphorylation of S221. As suggested by immunblotting, S221 phosphorylation increased over time, presumably reflecting the block of negative feedback mechanisms.

Since GSK1120212 does not directly inhibit either RAF1 or BRAF kinase activity, we believe that GSK1120212 binds to MEK in a way that specifically blocks the accessibility of S217 to Raf kinases. This is further supported by the observation that once S217 is phosphorylated, as in pp-MEK, the affinity for GSK1120212 is reduced. S217 phosphorylation likely alters the adjacent activation loop that partially defines the compound binding site. These results suggest that in Ras and Raf mutant tumors, GSK1120212 may suppress both MEK kinase activity and partially abrogate the activating effects due to negative feedback.

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 961. doi:10.1158/1538-7445.AM2011-961