PDF - 476K, Figure S1. Establishment of vemurafenib-resistant A375 melanoma cell line A375RF21 from its parental A375 cell line using chronic selection over 3 months with increasing concentrations of vemurafenib; Figure S2. Anti-phospho-histone H3 and PI (propidium iodide) bivariate staining cell cycle analysis on vemurafenib-resistant cells; Figure S3. In vitro dose-response curves (n = 5) of each combination in A375 and A375RF21 cells; Figure S4. Major vemurafenib resistance mechanisms in A375RF21 cells are the over-expression of PDGFβ and the activations of the PI3K-AKT pathway; Figure S5. Effect of single agent and combination treatment on purified-protein based tubulin polymerization assay (n = 3); Figure S6. In vitro growth curve for A375 and vemurafenib-resistant subline A375RF21 cells.
ARTICLE ABSTRACT
Acquired clinical resistance to vemurafenib, a selective BRAFV600E inhibitor, arises frequently after short-term chemotherapy. Because inhibitions of targets in the RAF–MEK–ERK pathway result in G0–G1 cell-cycle arrest, vemurafenib-resistant cancer cells are expected to escape this cell-cycle arrest and progress to the subsequent G2–M phase. We hypothesized that a combined therapy using vemurafenib with a G2–M phase blocking agent will trap resistant cells and overcome vemurafenib resistance. To test this hypothesis, we first determined the combination index (CI) values of our novel tubulin inhibitor ABI-274 and vemurafenib on parental human A375 and MDA-MB-435 melanoma cell lines to be 0.32 and 0.1, respectively, suggesting strong synergy for the combination. We then developed an A375RF21 subline with significant acquired resistance to vemurafenib and confirmed the strong synergistic effect. Next, we studied the potential mechanisms of overcoming vemurafenib resistance. Flow cytometry confirmed that the combination of ABI-274 and vemurafenib synergistically arrested cells in the G1–G2–M phase, and significantly increased apoptosis in both parental A375 and the vemurafenib-resistant A375RF21 cells. Western blot analysis revealed that the combination treatment effectively reduced the level of phosphorylated and total AKT, activated the apoptosis cascade, and increased cleaved caspase-3 and cleaved PARP, but had no significant influence on the level of extracellular signal–regulated kinase (ERK) phosphorylation. Finally, in vivo coadministration of vemurafenib with ABI-274 showed strong synergistic efficacy in the vemurafenib-resistant xenograft model in nude mice. Overall, these results offer a rational combination strategy to significantly enhance the therapeutic benefit in patients with melanoma who inevitably become resistant to current vemurafenib therapy. Mol Cancer Ther; 13(1); 16–26. ©2013 AACR.
