Background: The loss of normal cell cycle regulation via disruption of CDKN2A in HPV-negative (-) head and neck squamous cell carcinoma (HNSCC) is universal. Targeting CDK4/6 with a specific inhibitor, such as palbociclib, is therefore a logical strategy in this disease. Previous work has demonstrated that CDK4/6 inhibition leads to cell cycle arrest and senescence in cancer cells, and specifically HPV(-) HNSCC. Recent studies have also shown that the BCL-xL inhibitor, ABT-263 (navitoclax), effectively targets senescent cells. This study aimed to evaluate the efficacy and mechanism of a combination approach utilizing palbociclib and ABT-263 in HNSCC.

Methods: Three HPV (-) HNSCC cell lines (CAL27, HN31, PCI15B) were treated with palbociclib. Cresyl violet staining and annexin V apoptosis assays were used to assess cell viability in response to palbociclib, navitoclax, or the combination of the two drugs. Senescence following treatment with palbociclib was measured using β-galactosidase expression on western blot analysis, as well as via immunofluorescence staining assessed and quantified using confocal microscopy. Levels of BCL-xL were also measured using these methods.

Results: Following treatment with palbociclib, all cell lines exhibited phenotypic evidence of senescence, and protein expression of β-galactosidase was significantly increased compared to untreated controls (mean fold change ± SD: CAL27 2.67±0.26; HN31 1.79±0.41; PCI15B 1.26±0.15). Protein expression of BCL-xL was concurrently increased under these conditions (mean fold change ± SD: CAL27 1.57±0.04; HN31 1.58±0.39; PCI15B 1.33±0.17). Immunofluorescent staining was also used to determine if cells exhibiting senescence specifically demonstrated higher BCL-xL expression. Co-expression of β-galactosidase and BCL-xL showed a strong positive correlation (CAL27 r = 0.94, p < 0.0001; HN31 r = 0.94, p < 0.0001; PCI15B r = 0.71, p < 0.0001). Treatment with the combination of palbociclib and navitoclax resulted in significantly decreased survival compared to each agent alone in all HNSCC cell lines tested (data summarized for the cell line panel: mean percent survival, navitoclax 76.40%; palbociclib 38.76%; combination 16.74%). Annexin V apoptosis assay also revealed that there was a significant increase in apoptosis observed in all cell lines tested comparing each agent to combination treatment (data summarized for the cell line panel: mean percent apoptotic cells, navitoclax 35.53%; palbociclib 29.95%; combination 60.98%). Apoptosis with combination of palbociclib and navitoclax approached or exceeded levels achieved with staurosporine treated controls.

Conclusions: Palbociclib treatment leads to consistent induction of senescence in HPV (-) HNSCC. Adding a senolytic agent, such as the BCL-xL inhibitor ABT-263 (navitoclax) appears to be an effective strategy which holds promise in this disease.

Citation Format: Nicholas Gadsden, Carlos Thomas, Daniel Li, Nitisha Shrivastava, Nicolas Schlecht, Michael Prystowsky, Jeffrey Segall, Thomas Ow. Inducing and targeting senescence in HPV-negative head and neck squamous cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 90.