Abstract #DDT02-1: Discovery and characterization of E6201: A potent natural product-inspired synthetic MEK1 inhibitor
Authors: Kenichi Nomoto
Journal: Cancer Research (2009)

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

Background The RAS/RAF/MEK/ERK signal transduction pathway regulates cell proliferation in diverse types of cells. Mutations in this pathway are often observed in transformed cell lines and are frequently linked to human cancer (1). For instance, B-type Raf (BRAF) somatic missense mutations have been identified in 67% of malignant melanomas and 12% of colorectal cancers. Most BRAF mutations are within the kinase domain, with a predominant single substitution, V600E, accounting for 90% of mutations. Mutated BRAF proteins have elevated kinase activity, leading to activation of MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase), which then triggers ERK (extracellular-signal-regulated kinase) phosphorylation, with subsequent activation of downstream proliferation pathways (2). These data suggest that cancers harboring BRAF mutations, such as melanoma, may be attractive therapeutic targets for MEK inhibitors. Overview of E6201 E6201, a synthetic analog of the zearalenone natural product family member LL-Z1640-2 (3), is a novel inhibitor of MEK1 and other kinases, and suppresses cancer cell growth with nmol/L (nM) IC50 values. E6201 inhibited the growth of 5 human cancer cell lines that carry the V600E BRAF kinase activating mutation (SK-MEL-28, LOX, and MDA-MB-435 melanoma; DBTRG-05MG glioblastoma; COLO 205 colon cancer) with IC50 values of 44-464 nM. In contrast, AU-565 human breast cancer cells, which do not carry the V600E BRAF activating mutation, were relatively resistant to E6201 (IC50 >10 \#956;M). E6201 blocked cell cycle progression in G1 in several cell lines, with no evidence of hypodiploid cells (apoptosis), indicating the cytostatic nature of E6201. Consistent with MEK1 inhibition, E6201 decreased protein levels of phosphorylated ERK (phospho-ERK) with an IC50 value of 2.8 nM. Also, E6201 inhibited cyclin D1 protein expression and phosphorylation of retinoblastoma protein (pRb) in SK-MEL-28 cells, leading to G1 cell cycle arrest. Changes in expression levels of phospho-ERK, cyclin D1, and phosphorylated pRb in cancer cells are currently being assessed as pharmacodynamic biomarkers for MEK1 signaling pathway inhibition in E6201 Phase I clinical trials. In addition to changes in these proteins, spontaneous secretion of interleukins (IL) -6 and -8 was also inhibited by E6201 in LOX melanoma cells with IC50 values of 21.8 and 10.5 nM, respectively. Thus, measurements of IL-6 or -8 are also being assessed as pharmacodynamic biomarkers in clinical trials to monitor biological responses to E6201. The therapeutic efficacy of E6201 was tested in vivo against 3 V600E BRAF-mutated human cancer xenograft models (LOX melanoma, COLO 205 colon cancer, DBTRG-05MG glioblastoma), using a Q4D×3 iv administration schedule. Administration of E6201 at doses of 5, 10, 20, and 40 mg/kg resulted in statistically significant anticancer activity in these 3 models. Importantly, a single iv administration of 40 mg/kg E6201 resulted in time-dependent inhibition of phosphorylation of ERK and pRb in LOX tumors as assessed by Western blot and immunohistochemistry. Staining for Ki-67, a marker for proliferating cells, also showed inhibition of LOX tumor proliferation. E6201 penetrates the intact blood brain barrier (BBB), with high drug concentrations seen in brain tissue using 20 mg/kg iv dosing in mice and rats. The drug concentration of E6201 in brain was about 4.8-6.4 times higher than in plasma in both species, indicating that penetration of E6201 into brain may be sufficient for treating tumor metastases in brain. To assess such effects of E6201 in vivo, brain metastases were established by intra-cardiac injection of G464V BRAF-mutated MDA-MB-231 human breast cancer cells into nude mice. Successive rounds of in vivo passaging of isolated brain metastases produced a subline with increased selectivity for brain compared to other metastatic sites. E6201 showed a survival benefit in this in vivo brain metastasis model, suggesting that E6201 may be effective for treating brain metastases in the clinic. Overall, our preclinical results with E6201 suggest that BRAF mutation status could be an important patient stratification tool for human clinical trials, and that brain metastases of BRAF-mutated cancer types could be considered among the potential clinical indications. References 1. Wallace EM. et al. Progress towards therapeutic small molecule MEK Inhibitors for use in cancer therapy. Curr. Top. Med. Chem., 2005, 5: 215-229. 2. Davies H. et al. Mutations of the BRAF gene in human cancer. Nature, 2002, 417: 949-954. 3. Ellestad, GA. et al. New zearalenone related macrolides and isocoumarins from an unidentified fungus. J. Org. Chem., 1978, 43: 2339-2343.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr DDT02-1.