P0168 Cotargeting MAPK and CDK4 signaling with concurrent radiotherapy as a strategy for the treatment of non-small-cell lung cancer

Abstract

<h3>Background</h3> Non-small-cell lung cancer frequently exhibits mutational activation of the Ras/MAPK pathway, which is implicated in resistance to ionising radiation and chemotherapy. There is an urgent need for the development of novel therapies to treat KRAS-mutant lung cancer. Activation through Mek-Erk-dependent signaling occurs through cyclin D1 induction and subsequent assembly of the cyclin D1-CDK4/CDK6 complex, which results in Rb phosphorylation and Rb-E2F dissociation and G1-S cell cycle. MEK inhibitors do not engage the cell-cycle checkpoint, resulting in tumour stasis but not regression, despite robust apoptosis. The aim of the current study was to test the hypothesis that MAP kinase inhibitors and CDK4 inhibitors will offer clear therapeutic benefit when integrated into radiotherapy for treatment of KRAS-mutant lung cancer. <h3>Methods</h3> Clonogenic survival assays were done with the H358, A549, and H460 cell lines using the MEK inhibitor trametinib and the CDK4 inhibitor PD033291. Cell viability was assessed by tetrazolium-based CellTiter 96 aqueous non-radioactive cell proliferation assay (MTS). DNA repair was analysed using immunocytochemistry. Western blots were used to investigate the effects on downstream signaling. Flow cytometry was used to analyse cell cycle and apoptosis. Growth delay was used to determine the effects of trametinib and PD0332991 on in vivo tumour radiosensitivity. <h3>Findings</h3> Preradiation treatment with trametinib and PD0332991 significantly decreased clonogenic survival compared with either agent alone. Dose modifying factors at a surviving fraction for trametinib and PD0332991 were 2.38, 1.62, and 1.77 for H358, A549, and H460, respectively. Cell proliferation was decreased by treatment with trametinib and PD0332991 as compared with either agent alone. Flow cytometric analysis indicated that dual inhibition combined with irradiation significantly induced G0/G1 phase arrest and enhanced apoptosis in these cell lines. The expression of phospho-Rb and phospho-Erk1/2 were fully attenuated by simultaneous treatment with both inhibitors in combination with irradiation. In addition, dual inhibition combined with irradiation induced dramatic tumour growth delay in A549 xenografts. <h3>Interpretation</h3> These data suggest that trametinib can be used with CDK inhibitors to augment the radiation response in KRAS-mutant non-small-cell lung cancer.