Abstract PR08: Inhibition of ATR as a therapeutic strategy to overcome cisplatin resistance in HNSCC

Abstract

Abstract Background: Standard treatment for head and neck squamous cell carcinoma (HNSCC) includes platinum-based therapy given in combination with radiation. Although platinum has been a key drug of HNSCC treatment for decades, many patients develop resistance and the prognosis is unsatisfactory, and therefore development of novel treatment strategies is needed. Targeting ATR has been shown to be effective as a monotherapy in various type of cancers. Thus, inhibition of ATR could be a new approach when combined with DNA damaging agents to kill cancer cells. Here, we hypothesize that addition of ATR inhibitor AZD6738 can enhance the antitumor effects of cisplatin in HNSCC cells and result in better survival in preclinical models of HNSCC. Materials and Methods: Clonogenic survival assays, reverse phase protein array (RPPA), Western blotting, cell cycle analysis, and apoptosis assay were used to evaluate the in vitro sensitivity of HNSCC cells to AZD6738 and cisplatin and the molecular mechanisms associated with antitumor response. An orthotopic mouse model of oral cancer was used to explore in vivo efficacy of the combination treatment. Results: HNSCC cells treated with various concentrations of AZD6738 showed an IC50 value of 0.24-3.00 μmol/L and no differential sensitivity was observed among cells with different TP53 mutational status. Treatment with constant drug concentration ratios of AZD6738 and cisplatin (2:1) resulted in a combination index of <1, indicating synergistic effects. RPPA pointed out proteins involved in DNA repair, DNA replication, and cell cycle to be predominantly changed in the combination groups compared to single treatments. Among these, the replication factors Cdc6 and Cdt1 were both reduced with the combination treatment. Furthermore, Western blot results revealed that combination treatments lead to enhanced DNA damage indicated by increased phosphorylation of H2AX and induction of replication stress marker (decrease of phospho-CDK1 and phospho-CHK1). Moreover, levels of RRM2 and Rad51 were diminished, suggesting insufficient nucleotide supply and impaired homologous replication. Combining both drugs also resulted in a time-dependent increase of cleaved PARP. This apoptotic death was further confirmed by Annexin V/PI assay. Cell cycle analysis and live cell imaging revealed that an accumulation of cells in the S phase was more than doubled after 12 and 48 hours, indicating replicative stress. In a pilot in vivo study, cisplatin combined with AZD6738 reduced tumor growth in mice bearing oral tumors. Conclusions: Addition of AZD6738 synergizes with cisplatin in vitro and potentiates cisplatin response in vivo. Mechanistically, combination of cisplatin and AZD6738 induces DNA damage and replication stress, resulting in apoptotic cell death. Interestingly, proteins essential for the origin licensing as part of the DNA replication initiation machinery were decreased following combination treatment, offering a potential mechanistic insight and warranting further investigation. Citation Format: Antje Lindemann, Hideaki Takahashi, Ameeta A. Patel, Walter N. Hittelman, Abdullah A. Osman, Jeffrey N. Myers. Inhibition of ATR as a therapeutic strategy to overcome cisplatin resistance in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr PR08.