P27 The combined use of anti-EGFr and anti-JAK/STAT-3 agents as radiosensitizers in HPV negative and positive head and neck cancer cell lines

Abstract

Human Papilloma Virus (HPV) is a common etiological factor in human head and neck squamous cell cancer (HHNSCC). Our group has reported that HPV-infected HHNSCC cell lines are more sensitive to radiation and deficient in DNA repair compared to uninfected lines (Cooper T, et al. Cancer Research, 73(8 Supplement 1), AACR, 2013). EGFr inhibitors (primarily cetuximab) have evolved as an important therapeutic modality for head and neck cancer since it was found that cetuximab sensitizes HHNSCC’s to radiation in laboratory studies, as well as in phase III clinical studies (Bonner JA, et al. Lancet Oncology; 11(1):21–28, Epub 2009, November 2010). Early on, it was recognized that cetuximab not only inhibits EGFr, but the downstream inhibitor of apoptosis STAT-3 (Bonner JA, et al. J Clin Oncol 2001;19:3234–3243). Therefore, we have previously assessed cetuximab in combination with the inhibition of JAK/STAT as a means of accentuating the targeting of this pathway and increasing radiosensitization (Bonner JA, et al. Radiother Oncol 2011;99:339–343). Herein, we report the radiosensitizing properties of dual EGFr and JAK/STAT inhibition in three HPV negative HHNSCC’s and two HPV positive HHNSCC’s. Three HPV negative HHNSCC’s (UM-SCC-1, UM-SCC-6, UM-SCC-5) and two HPV positive HHNSCC’s (SCC-47, SCC-90) cell lines were used for these studies. All five cell lines showed greater anti-proliferative effects for the combination of cetuximab (0.25 μg/ML) followed 1hr by the JAK2/STAT-3 inhibitor, BMS911543 (10 μM) than for either agent alone. This enhanced anti-proliferative effect, for the combination of inhibitors (EGFr and JAK1i/STAT3), correlated with greater inhibition of phosphorylated STAT-3 as measured by immunoblot. Additionally, there was a suggestion that greater radiosensitization (proliferation and colony formation) occurred when both agents were used prior to radiation compared to the use of either single agent in all five cell lines. The enhanced anti-proliferative effects of combining the agents (JAK2/STAT-3 and cetuximab) showed correlation with enhanced apoptosis. Additionally, there was a suggestion of enhanced apoptosis when both agents were given prior to radiation compared to either single agent treatment given prior to radiation. However, the apoptotic events were markedly pronounced (2-fold increase) in the HPV negative cell lines compared to the HPV positive cell lines even though the HPV positive cell lines showed 5–10-fold greater radiosensitivity compared to the HPV negative lines (colony formation). Additional studies are being performed to assess mechanisms associated with the regulation of cellular proliferation that may account for the discordant findings with respect to apoptosis and radiosensitivity relative to HPV status. The combined treatment of Cetuximab (anti-EGFr) and BMS911543 (anti-JAK-STAT-3) with or without radiation was studied in four HHNSCC cell lines. The combination of these two inhibitors increased the anti-proliferative effects of all cell lines compared to either individual treatment in HPV positive and negative human head and neck squamous cell cancers. The combination treatment also led to marked radiosensitization which correlated with apoptotic events. However, the treatment-induced apoptotic events were muted in the HPV positive cell lines compared to the HPV negative cell lines even though the HPV positive cell lines showed the greatest radiosensitivity. Further study of the effects of these inhibitors on the regulation of cellular proliferation in HPV positive and HPV negative tumors is warranted.