Fig. 6. Combination treatment delays HNSCC progression in a GEM model.

a. Schema of drug treatment strategy in GEM model (left panel). Ten days after Cre activation, KKP mice (K14-CreER^tam;LSL-Kras^G12D;Trp53^R172H) treated with palbociclib (25 mg/kg/day), afatinib (10 mg/kg/day), combination or vehicle control for 21 days. Mice sacrificed and oral tissues analyzed by H&E. b. Immunohistochemistry of p-EGFR (Tyr-1068), p-S6 (Ser-235/236), cyclin D1, and Ki67 in KKP tumors after treatments (n=3). Image scale = 100 μm. The positive staining (OD) of the respective proteins is provided (bottom panel). c. Current proposed model of how combination therapy block tumor progression. Targeting CDK4/6 with palbociclib activates Rb to induce cell cycle arrest. However, palbociclib also activates EGFR to increases cyclin D1 and E1 levels. And afatinib inhibits EGFR signaling and decreases cyclin D1, cyclin E1, and p-S6 (Ser-235/236) expression. In contrast, combination treatment reduces the glucose uptake and inhibits both glycolysis and TCA cycle with no change in OXPHOS. Furthermore, decreased SOD3, NQO1, NRF2, and catalase levels by combination lead to increased ROS levels, mitochondrial dysfunction and metabolic alteration, resulting in cellular senescence with an overall decrease in tumor growth and development. Data represent mean ± SEM. ANOVA followed by Tukey’s multiple comparisons test was used between groups (b). **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns=non-significant. OD=optical density.