Abstract 4688: HPV+ cervical cancer cells are selectively killed by the combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the cyclin-dependent kinase-9 inhibitor (CDK9i) dinaciclib
Authors: Kotlyar David S. , Greer Yoshimi E. , Voeller Donna , Hernandez Lidia , Annunziata Christina M. , Lipkowitz Stanley 
Published: July 2016

Abstract
Background: Cervical cancer affects more than 520,000 women worldwide yearly, kills over 250,000 and disproportionately affects younger women. With optimal systemic treatment, the median overall survival of patients with metastatic disease is approximately 1.5 years. Previously, a cyclin dependent kinase-9 inhibitor and TRAIL have shown synergistic efficacy in a variety of cancer cell lines in vitro and in vivo. We investigated whether TRAIL combined with dinaciclib (also a CDK9 inhibitor) was selective for HPV+ tumor cells and also explored the mechanisms by which this combination killed HPV+ killed these cells.
Methods: We used a colorimetric MTS assay to assess viability after 24 h of treatment with CDK9i and TRAIL on 3 HPV+ cervical cancer lines (HeLa, SiHa, CaSki) and one HPV- cervical cancer line. Synergy was assessed by the CompuSyn program. The pan-caspase inhibitor ZVAD-FMK was used to evaluate caspase-dependence. Morphological changes were evaluated by light microscopy.
Results: The combination of TRAIL and dinaciclib resulted in greater growth inhibition than either alone in the HPV+ cell lines HeLa, Caski, and Siha. By contrast, TRAIL + dinaciclib did not inhibit the growth of the HPV - cervical cancer cell line C33a more than dinaciclib alone. The growth inhibition by TRAIL + dinaciclib was synergistic in the HPV+ lines but not the HPV- line as calculated by the Combination Index (CompuSyn). Treatment with the pan-caspase inhibitor ZVAD-FMK completely blocked TRAIL-mediated loss of viability and blocked the increased toxicity when TRAIL was added to dinaciclib, but only partially rescued dinaciclib induced loss of viability. When TRAIL and dinaciclib were combined, microscopic analysis was consistent with the synergistic killing of the HPV+ cell lines, but not the HPV- cell line. Morphologically, most cells showed signs of apoptotic cell death due to TRAIL + dincaciclib. However, in 2 HPV+ cell lines (CaSki and Hela) in addition to evidence of shrinkage of some cells with dense nuclei, seen in apoptosis, there was evidence of other cells with cell swelling with heterogeneous nuclei, which can be seen in necroptosis.
Conclusions: Our results demonstrate synergistic killing in HPV+ cells by the combination of TRAIL and dinaciclib. As one HPV- line did not show any effect of TRAIL nor synergistic killing, this is also suggestive of potential HPV selectivity of killing. Dinaciclib induces both caspase-dependent apoptosis, and a non-caspase dependent mechanism of cell death. Direct examination of the cells by light microscopy suggests dinaciclib being associated with apoptosis and necroptosis. TRAIL alone appears to solely cause apoptosis. Future experiments will investigate the mechanisms of cell death and will test these drugs in mouse xenografts.
Citation Format: David S. Kotlyar, Yoshimi E. Greer, Donna Voeller, Lidia Hernandez, Christina M. Annunziata, Stanley Lipkowitz. HPV+ cervical cancer cells are selectively killed by the combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the cyclin-dependent kinase-9 inhibitor (CDK9i) dinaciclib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4688.