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Supplementary Figures S1-S6.

Purpose: Despite their preclinical promise, previous MEK inhibitors have shown little benefit for patients. This likely reflects the narrow therapeutic window for MEK inhibitors due to the essential role of the P42/44 MAPK pathway in many nontumor tissues. GSK1120212 is a potent and selective allosteric inhibitor of the MEK1 and MEK2 (MEK1/2) enzymes with promising antitumor activity in a phase I clinical trial (ASCO 2010). Our studies characterize GSK1120212' enzymatic, cellular, and in vivo activities, describing its unusually long circulating half-life.Experimental Design: Enzymatic studies were conducted to determine GSK1120212 inhibition of recombinant MEK, following or preceding RAF kinase activation. Cellular studies examined GSK1120212 inhibition of ERK1 and 2 phosphorylation (p-ERK1/2) as well as MEK1/2 phosphorylation and activation. Further studies explored the sensitivity of cancer cell lines, and drug pharmacokinetics and efficacy in multiple tumor xenograft models.Results: In enzymatic and cellular studies, GSK1120212 inhibits MEK1/2 kinase activity and prevents Raf-dependent MEK phosphorylation (S217 for MEK1), producing prolonged p-ERK1/2 inhibition. Potent cell growth inhibition was evident in most tumor lines with mutant BRAF or Ras. In xenografted tumor models, GSK1120212 orally dosed once daily had a long circulating half-life and sustained suppression of p-ERK1/2 for more than 24 hours; GSK1120212 also reduced tumor Ki67, increased p27Kip1/CDKN1B, and caused tumor growth inhibition in multiple tumor models. The largest antitumor effect was among tumors harboring mutant BRAF or Ras.Conclusions: GSK1120212 combines high potency, selectivity, and long circulating half-life, offering promise for successfully targeting the narrow therapeutic window anticipated for clinical MEK inhibitors. Clin Cancer Res; 17(5); 989–1000. ©2011 AACR.

Purpose: Despite their preclinical promise, previous MEK inhibitors have shown little benefit for patients. This likely reflects the narrow therapeutic window for MEK inhibitors due to the essential role of the P42/44 MAPK pathway in many nontumor tissues. GSK1120212 is a potent and selective allosteric inhibitor of the MEK1 and MEK2 (MEK1/2) enzymes with promising antitumor activity in a phase I clinical trial (ASCO 2010). Our studies characterize GSK1120212' enzymatic, cellular, and in vivo activities, describing its unusually long circulating half-life. Experimental Design: Enzymatic studies were conducted to determine GSK1120212 inhibition of recombinant MEK, following or preceding RAF kinase activation. Cellular studies examined GSK1120212 inhibition of ERK1 and 2 phosphorylation (p-ERK1/2) as well as MEK1/2 phosphorylation and activation. Further studies explored the sensitivity of cancer cell lines, and drug pharmacokinetics and efficacy in multiple tumor xenograft models. Results: In enzymatic and cellular studies, GSK1120212 inhibits MEK1/2 kinase activity and prevents Raf-dependent MEK phosphorylation (S217 for MEK1), producing prolonged p-ERK1/2 inhibition. Potent cell growth inhibition was evident in most tumor lines with mutant BRAF or Ras. In xenografted tumor models, GSK1120212 orally dosed once daily had a long circulating half-life and sustained suppression of p-ERK1/2 for more than 24 hours; GSK1120212 also reduced tumor Ki67, increased p27Kip1/CDKN1B, and caused tumor growth inhibition in multiple tumor models. The largest antitumor effect was among tumors harboring mutant BRAF or Ras. Conclusions: GSK1120212 combines high potency, selectivity, and long circulating half-life, offering promise for successfully targeting the narrow therapeutic window anticipated for clinical MEK inhibitors. Clin Cancer Res; 17(5); 989–1000. ©2011 AACR.

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 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

Background: We recently reported a 49% RECIST response rate in conjunction with a phase 2 clinical trial of the multi-targeted kinase inhibitor pazopanib in differentiated thyroid cancers (DTC; Lancet Oncology 2010, 11:962-72), prompting a search for synergistic pazopanib-containing doublets in thyroid cancer cell lines. Methods and Results: Among all explored pazopanib-containing combinations, paclitaxel and pazopanib produced the greatest combined cytotoxic effects (continuous exposure pazopanib, 24 h paclitaxel; Combination Index, CI, at IC90=0.3). Interestingly, analogous synergy resulted when combining pazopanib with other antimicrotubule agents including other taxanes (docetaxel), Vinca alkyloids (vincristine) and epothilones (ixabepilone) – suggesting a class effect. Further, we found this combination to be synergistic in multiple cancer cell lines including DTC, medullary and anaplastic thyroid cancer lines, and in the A549 lung cancer line. In pursuit of underlying mechanism(s), we noted that the combination produced a greater fraction of cells in G2/M phase of the cell cycle and increased apoptosis in comparison to paclitaxel alone, prompting us to examine whether pazopanib might potentiate the effects of paclitaxel on mitosis. Analysis of cell fate resulting from pazopanib and paclitaxel alone and in combination using time-lapsed video microscopy demonstrated that as low as 2.5 μM pazopanib augmented paclitaxel-induce mitotic catastrophe by 2.4 fold, while inducing no appreciable cell death alone. As pazopanib is somewhat promiscuous in inhibition of kinases, we hypothesized that observed combined effects with antimicrotubule agents might be attributed to its inhibition of cell cycle by targeting CDKs or mitosis by targeting aurora kinases. Inhibition of CDKs 1 and 2 required pazopanib concentrations 1000-times those required to inhibit its primary kinase targets, VEGF-Rs; whereas aurora kinases A and B were inhibited at much lower concentrations (IC50s, 500-1000 nM). Hence, we pursued shRNA aurora A knockdown experiments: potentiation of pazopanib effects were observed in knockdown clones, suggesting that pazopanib single-agent effects are at least in part mediated via aurora A inhibition. Moreover, augmentation of paclitaxel effects were achieved in knockdown clones that rivaled those observed for the pazopanib/paclitaxel combination. Additionally, pazopanib/paclitaxel synergy was reduced in aurora A knockdown clones (CI at IC80: vector1.0), indicating that aurora A inhibition by pazopanib is contributory to paclitaxel/pazopanib synergy. Conclusions: Pazopanib combines with antimicrotubule agents to produce cytotoxic synergy associated with augmentation of mitotic catastrophe that appears at least in part mediated by its ability to inhibit aurora kinases. Supported in part by CA125750. 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 3597. doi:10.1158/1538-7445.AM2011-3597

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.