Lung cancer is one of the problematic cancers due to the low 5-year survival rate (< 15%). Mutations and dysregulated proteins contribute to chemoresistance in non-small (85%) and in small cell lung cancers (15%). Increased RTK signaling (e.g. EGFR family, AKT, mTOR) and overexpressed epigenetic erasers such as histone deacetylases (HDACs) lead to dysregulation of metabolism, cell cycle, cell death and signal pathways. Whereas inhibitors of RTK signaling are established therapies based on the mutation status of a patient, HDAC inhibitors are experimental in solid cancers so far. HDAC inhibitors have the potential to increase chemosensitivity of cancers against classical cytostatics and targeted therapies as e.g. kinase or KRAS inhibitors. This project investigates drug combinations of HDAC and KRAS inhibitors for synergy in drug-resistant and KRAS wildtype or mutated non-small and small cell lung cancers.

Non-small cell lung cancers A427/97 and DV90 and small cell lung cancers DMS114 and SHP77 were investigated. Cytotoxic effect of compounds were analysed by MTT assay. Caspase activation was determined by CellEvent Caspase-3/7 Green Detection Reagent from Thermo Scientific. Drug synergy analysis was performed by Chou-Talalay using Combenefit. HDACs, kinases, KRAS and phosphorylated proteins were analyzed by western blotting.

HDAC1/2/3, EGFR, pEGFR, HER2, pHER2, and KRAS are highly expressed in lung cancer cells. KRAS mutations G12D, G13D and G12V were identified in A427/97, DV90 and SHP77, respectively. Incubation with 0.5 µM of the class I HDAC inhibitor entinostat for 48h prior to addition of the pan-KRAS inhibitor BI2865 decreased the IC50 of BI2865 by a factor of 5- to 50-fold in A427/97, DV90, and SHP77. This chemosensitization of lung cancer cells against the KRAS inhibitor BI2865 by entinostat was shown to be synergistic according to Chou-Talalay. Furthermore, the combination of entinostat and BI2865 increased caspase 3/7 mediated apoptosis in a synergistic manner. Observed synergistic effects between entinostat and BI2865 are likely mediated by increased p21 expression and changes in the expression of pro- and antiapoptotic proteins.

Pretreatment with the HDAC inhibitor entinostat and subsequent treatment with the pan-KRAS inhibitor BI2865 is better than treatment with entinostat or BI2865 alone. The combination not only led to an increase in cytotoxicity, but also to an increased activation of caspase 3/7 with subsequent apoptosis. A proteome analysis before and after treatment provides mechanistic evidence. p21 was upregulated and various pro- and anti-apoptotic proteins were differentially regulated. This combination of epigenetic and targeted therapy represents a new clinical option for the treatment of chemoresistant lung cancers.