Our recent work has demonstrated that HNSCC is specifically dependent on the upregulation of the pro-survival anti-apoptosis molecules Bcl-xL and Mcl1. Nevertheless, the mechanism of HNSCC to evade apoptosis is still not well understood. BH3 profiling, a high throughput in vitro method of evaluating apoptosis signaling, measures mitochondrial depolarization in response to the introduction of BH3 peptides, which are responsible for intrinsic apoptosis signaling. In addition, a series of small molecules targeting BH3 proteins, such as ABT-263 (navitoclax), an inhibitor of Bcl-2/Bcl-xL/Bcl-w and S63845, inhibitor of Mcl-1, have been recently developed. We hypothesize that HNSCC cells can be functionally categorized based on apoptosis potential as determined by BH3 profiling, which can then be utilized to tailor drug combinations that will optimally induce apoptosis in HNSCC cells.
10 HNSCC cell lines (8 HPV- and 2 HPV+) were investigated. Functional BH3 profiling was carried out as previously described to evaluate apoptosis signaling and to determine the level of dependency on BCL-family pro-survival molecules. Response to ABT-263 Navitoclax and S63845 both as single agents and in combination was measured using cell viability assays (via ATP quantification using Cell Titre Glo®) after exposure to a matrix of dose ranges for each drug. Bliss Independence analysis was utilized to determine whether drug combinations yielded synergistic activity.
BH3 signaling in 8 HPV- cell lines and two HPV+ cell lines was profiled, and lines were categorized into three classes of apoptosis potential (incompetent, unprimed, and primed). In 1/8 HPV- and 0/2 HPV+ lines, BH3 profiling demonstrated intact apoptosis signaling, with a lack of response to apoptosis sensitizers (Class A - unprimed). 0/8 HPV- and 1/2 HPV+ were characterized by apparent loss of BAX and BAK activity (Class B – low apoptosis competence). In 7/8 HPV- lines and 1/2 HPV+ lines, apoptosis signaling is fully intact, and inhibited via dependence on expression of antiapoptotic proteins (Class C - primed). Treatment with ABT-263 combined with S63845 showed synergistic activity in 7/8 HPV- lines and 2/2 HPV+ cell lines tested based on Bliss Analysis.
Apoptosis signaling appears to be intact in the majority of HNSCC cancer cells, either in a primed or unprimed state. In both settings, inhibition of BCL-family pro-survival molecules remains a treatment approach with potential efficacy. Our data suggest that co-dependency on Bcl-xL and Mcl-1 is common, and co-inhibition of these molecules appears is synergistic in most HNSCC cells.