(a) Combination of cobimetinib and GDC-0994 demonstrates significantly greater anti-tumor activity in multiple KRAS mutant tumor models A549 and NCI-H2122 (cobimetinib at 5 mg/kg, PO, QD + GDC-0994 at 60 mg/kg, PO, QD) compared to single agent (upper panels). Mean tumor volume is plotted ± SEM (n = 10 mice per group). Study was terminated on day 20. All treatments were tolerated with minimal body weight loss (lower panels), One way ANOVA, * p<0.05, ** p<0.01, *** p<0.005, ****p<0.001. (b) A549 (NSCLC, KRAS^G12S) tumor-bearing mice (n = 3 per time point) were treated with GDC-0994 (60 mg/kg, PO, QDx4), cobimetinib (GDC-0994; 5 mg/kg, PO, QDx4) or the combination and then MAPK target genes expression was assessed in tumor samples (Nanostring^®) and the quantified results are plotted for each individual gene over time. The combination results in deeper, more prolonged suppression of multiple MAPK target genes, including DUSP4, DUSP6, SPRY2, SPRY4, ETV4, and ETV5. Student’s t test at the 24 hr time point, * p<0.05, ** p<0.01, *** p<0.005, ****p<0.001. (c) The combination of cobimetinib and GDC-0994 results in stronger and more prolonged suppression of p-p90RSK/total p90RSK phosphorylation (as determined by quantitative western blot), cyclin D1 and Ki-67, as well as increased induction of cleaved caspase 3 (CC3) (as determined by IHC) in A549 xenograft tumors treated for 4 days (values were quantified from n = 4 mice/time point). Student’s t test at the 24 hr time point, * p<0.05, ** p<0.01, *** p<0.005.

https://doi.org/10.1371/journal.pone.0192059.g001