Interestingly, while sensitivity to single except agents differed among the various cell lines, in all KRAS-mutated cells the combined treatments caused significantly higher growth inhibition compared to single treatments. Synergism was then studied more extensively over a range of inhibitors concentrations, using the method described by Chou and Talalay, based on the mass-action law principle [41]. Combination Index values (CI) were calculated from experimental data along the whole range of fractional effects and are shown as XY plots in figure 2. Specific CI values at ED50, ED75 and ED90 are reported in Table 1. As predicted by oncogene mutation status, all cell lines carrying mutations in both Wnt pathway (APC or ��-catenin) and KRAS showed synergistic interactions (CI<1), except at extreme points of fraction affected, where effects are either negligible or too strong.

In contrast, HT-29 cells (carrying wild-type KRAS and a mutated BRAFV600E allele) showed no synergism. Rather, antagonistic effects (CI>1) were noted. However, combination of PKF115-584 with a BRAF inhibitor (PLX-4032) showed weak synergism in HT-29 cells at high doses (figure S3). Additional data obtained with pyrvinium/FTS in two KRAS- and one BRAF-mutated cell lines are shown in figure S4. Overall, analysis of CI values at ED50 (pyrvinium/FTS) shows correlation between KRAS/BRAF mutational status and synergistic interaction (p=0.0021): all KRAS mutant cells showed synergism, while neither of two BRAF mutant cell lines did (figure S5), although the limited number of BRAF mutant cell lines tested does not allow to draw definitive conclusions.

In contrast, the effects did not correlate with p53 or MSI status (table 1). Altogether, these results suggest that PKF115-584 and pyrvinium can be combined with FTS in CRC cells harbouring concomitant Wnt/KRAS genetic abnormalities, to achieve better therapeutic effects. Figure 2 Synergistic effects of PKF115-584/FTS and pyrvinium/FTS combinations on CRC cells. Table 1 Combination Index values obtained at ED50, ED75 and ED90. The average of the three values is reported in the last row. We then further characterized the synergistic combinations Drug_discovery using additional independent assays (figure 3 and figure S6). Cell growth was monitored during the course of several days, after exposing Ls174T and DLD-1 cells to the three drugs, alone or in combination, showing that FTS potentiated PKF115-584 and pyrvinium toxicity already after 3�C4 days (figure 3A and figure S6A). Growth inhibition was accompanied by induction of apoptosis: the number of early apoptotic cells was significantly higher in the combination samples compared to either drug alone (figure 3B and figure S6B).