Overcoming Acquired Cetuximab Resistance In Head And Neck Squamous Cell Carcinoma: An In Vitro Study On The Potential Of Pi3k Inhibition.

Abstract Resistance to therapies targeting the epidermal growth factor receptor (EGFR), such as cetuximab, remains a major roadblock in the search for effective therapeutic strategies in head and neck squamous cell carcinoma (HNSCC). Increasing evidence suggests that aberrant signalling of the PI3K/Akt pathway is involved in resistance to cetuximab treatment. Therefore, this study aims to investigate whether combined inhibition of EGFR and PI3K might be a novel therapeutic strategy to overcome acquired cetuximab resistance in HNSCC. Acquired cetuximab resistant HNSCC cell lines (SC263-R and SCC22b-R) were generated by chronically exposing initially sensitive cell lines to cetuximab. In parallel, control cell lines (SC263-PBS and SCC22b-PBS) were established by exposing these cells to the vehicle control (PBS). All cell lines were HPV-negative. Cytotoxicity of cetuximab (72h, 25 and 50 nM) plus the PI3K inhibitor buparlisib (72h, 0-1μM) was assessed using the Tecan Spark Cyto. Cells were stained with Hoechst 33342 and Cytotox Green (Sartorius) after 72h of treatment. Possible synergism between cetuximab and buparlisib was determined by calculating the combination index (CI) using the additive model. CI<0.8, CI = 1.0±0.2, and CI>1.2 indicated synergism, additivity, and antagonism, respectively. The effects of the combination treatment on cell cycle arrest and apoptosis were analysed flow cytometrically using the Vindelov and Annexin V-FITC/PI method, respectively. Simultaneous exposure to cetuximab and buparlisib revealed a synergistic interaction in the SC263-PBS and SC263-R cell lines (CI≤0.797). Furthermore, the percentage of cell death was the highest in the combination treatments, with a maximum of 30.33±4.18% for 50 nM cetuximab plus 1 μM buparlisib. Cetuximab monotherapy induced a small cell cycle arrest, while buparlisib monotherapy had little to no effect on cell cycle distribution. The addition of buparlisib to cetuximab resulted in a small increase in G0/G1 phase cells. Therapy-induced apoptosis was limited when cells were treated with cetuximab monotherapy. Buparlisib monotherapy induced a small percentage of apoptosis in a concentration-dependent manner. The addition of buparlisib to cetuximab resulted in an increase in the percentage of apoptotic cells with a maximum of 22.30±7.48%. These results are currently being validated in other acquired resistant cell lines. Preliminary results also show an additive to synergistic interaction between both drugs in the SCC22b-PBS and SCC22b-R cell lines. In conclusion, these results can serve as a preclinical rationale for a novel therapeutic strategy combining buparlisib with cetuximab in HNSCC patients who experience acquired cetuximab resistance. Further research is necessary to obtain a more profound understanding of the molecular mechanisms underlying this targeted combination therapy. Citation Format: Hannah Zaryouh, Hasan Baysal, Patrick Pauwels, Marc Peeters, Jan B. Vermorken, Filip Lardon, An Wouters, Ines De Pauw. Overcoming acquired cetuximab resistance in head and neck squamous cell carcinoma: An in vitro study on the potential of PI3K inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1095.