Fig 2: In vivo therapeutic efficacy. a MDA-MB-231 is a cell line with high levels of p-ERK and CDK6, but no visible levels of c-Kit. It can be observed that, although imatinib-based doublets significantly prolonged mice overall survival as compared with the singlets or vehicle, the magnitude of the improvement is much lower than that seen with the p-ERK + CDK6-targeting doublet (GDC-0994 and palbociclib, mid chart; >5-fold increase in overall survival compared with ~2-fold). Below the Kaplan-Meier curves, the median overall survival (days) for each combination (or single-agent) in addition to the Log-Rank P-values are shown. Finally, representative tumor burdens of each treatment group are depicted. b MDA-MB-231 shows relatively high p-PNKP levels; CRISPR PNKP MDA-MB-231 transfectant xenografts treated with GDC-0994 also showed statistically significantly longer overall survival as compared with GDC-0994 administered to wild-type MDA-MB-231 or untreated CRISPR PNKP transfectants. Mice treated with: vehicle (n = 5), imatinib (n = 6), GDC-0994 (n = 4), palbociclib (n = 5), GDC-0994 + palbociclib (n = 5), palbociclib + imatinib (n = 5), sgPNKP (n = 11) and sgPNKP + GDC-0994 (n = 11). c Compared to MDA-MB-231, the levels of p-PNKP in MDA-MB-468 are almost undetectable. The levels of p-ERK and CDK6 are high, and those of c-Kit are low. Matching the observations in the other models, when a targeted doublet includes a target with low or absent expression (namely, p-PNKP in MDA-MB-468), no synergy is observed. In the three Kaplan-Meier curves it can be observed than the doublet is not better than any of the monotherapies (or sgPNKP alone). Representative tumor burden charts are shown below the survival curves. Mice treated with: vehicle (n = 6), sgPNKP and sgPNKP + imatinib (n = 5), imatinib (n = 8), GDC-0994 (n = 11), GDC-0994 + imatinib (n = 13), palbociclib (n = 12), GDC-0994 + palbociclib (n = 9), sgPNKP + palbociclib (n = 4) and sgPNKP + GDC-0994 (n = 7). d Finally, in PDX156 (c-Kit and pERK higher than CDK6), imatinib plus GDC-0994, and GDC-0994 plus palbociclib significantly prolonged median overall survival compared to the monotherapies. Mice treated with: vehicle (n = 5), imatinib (n = 4), GDC-0994 (n = 4), palbociclib (n = 4), GDC-0994 + palbociclib (n = 5) and GDC-0994 + imatinib (n = 9). In tumor burden graph, each point represents a tumor. The data are represented as mean±SEM and Student ´s t test was performed. *p < 0.05, **p < 0.01, ***p < 0.001