Upregulation of BRAF and MAPK signaling as a mechanism of acquired resistance to the BRAF inhibitor, AZ628 Free

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The Ras/Raf/MEK/ERK pathway is activated in approximately 30% of all human tumors. Activating mutations of BRAF have been reported in multiple tumor types, including melanoma, papillary thyroid and colon cancer. MEK inhibitors are already in development for the treatment of MAPK-dependent tumors and BRAF inhibitors are currently entering clinical trials. Acquired resistance is a major obstacle to successful cancer treatment and secondary resistance mutations have been observed in target kinases in patients and cell lines with resistance to EGFR inhibitors Gefitinib or Erlotinib and Imitanib. To determine potential resistance mechanism/s to BRAF inhibitors in the clinic, V600E BRAF-containing colon cancer cell line Colo205 with acquired resistance was generated through continuous exposure to increasing concentration of the BRAF inhibitor AZ628 in vitro, up to a final concentration of 500nM (Colo-628R). Altered morphology was observed in Colo-628R, with increased colony formation compared to the parental line. Colo-628R cultured in the presence of AZ628 had the same growth rate as the parental cell line in the absence of compound, whereas lower concentrations of AZ628 appeared to promote growth of Colo-628R line. AZ628 inhibited growth of Colo205 and Colo-628R in a 72h MTS assay with GI50 values of 43nM and 2.8μM, respectively, indicating a 64-fold reduction in sensitivity of the resistant versus sensitive line. Colo-628R was also found to be more resistant to other structurally diverse BRAF inhibitors as well as the MEK inhibitor U0126, but equally sensitive to taxol compared to Colo205. Colo-628R had increased BRAF mRNA and protein and pMEK and pERK levels, but equivalent kRas levels, compared to Colo205. No additional BRAF mutations were detected as assessed by full length sequencing of the BRAF gene. AZ628 reduced pMEK and pERK levels in both Colo205 and Colo-628R cell lines, although higher concentrations of compound were required in the resistant versus sensitive line to reduce pMEK and pERK to similar levels in the two cell lines. In summary, a mutant BRAF colon cancer cell line was demonstrated to acquire resistance to a BRAF inhibitor in vitro through upregulation of activated BRAF levels and enhanced MAPK signaling. Tumors that therefore acquire resistance to BRAF inhibition in the clinic may therefore not be subsequently responsive to MEK inhibitors or second-generation BRAF inhibitors.