Abstract B36: Enhancement of histone deacetylase inhibitor's efficacy with hydroxychloroquine via autophagy inhibition in pediatric tumor cell lines
Authors: Cruz Abigail Joy , Bansal Hima , Tomlinson Gail , Bansal Sanjay , Weitman Steven 
Published: May 2016

Background: Histone deacetylase (HDAC) inhibitors, such as vorinostat, have been of interest in adult and pediatric cancers. Preclinical in vitro data has shown an effect in many different cancer cell types, but response has not correlated well in xenograft models. A concern is the effect of the HDAC inhibitors being compensated by autophagy. Autophagy is induced by multiple anticancer agents, as a tumor survival-promoting mechanism. Hydroxychloroquine, an autophagy inhibitor, treatment leads to the accumulation of autophagosomes, and accelerates tumor cell death. The hypothesis of this study is that by combining a HDAC inhibitor with hydroxychloroquine, there will be an improved anti-tumor response compared to HDAC inhibitor alone in pediatric cancer cell lines.

Objectives: To determine if there is an improved efficacy of vorinostat when combined with hydroxychloroquine in pediatric solid tumor cell lines.

Methods: Three pediatric cancer cell lines: Hepatoblastoma (Hep293TT), Ewing's Sarcoma (SKES1), and Medulloblastoma (D283) were treated with different concentrations of vorinostat or hydroxychloroquine, and cell viability was measured by CellTiter Glo assay. Dose-response curves were plotted to determine IC50 (inhibitory concentration 50%). The expression of proliferation and autophagy markers was determined by Western blot analysis.

Results: The IC50 of vorinostat and hydroxychloroquine was in the rage of 0.5-1.25μM and 25-50μM, respectively. To evaluate the functional consequences, cells were treated with vorinostat and hydroxychloroquine alone and in combination for 72 hours. The combination treatment resulted in a dramatic decrease in cell viability, with these two drugs cooperating in a dose-dependent manner. Overall, in all three cell lines there was an improved response when vorinostat was combined with hydroxychloroquine. LC3, a mammalian homolog of yeast Atg8, is a reliable marker of autophagosomes, and tracking the conversion of LC3-I to LC3-II is indicative of autophagy blockade. Hydroxychloroquine treatment resulted in profound conversion of LC3-I to LC3-II, and this effect was greater in cells treated with both vorinostat and hydroxychloroquine alone. Therefore, hydroxychloroquine blocked autophagic flux, which was more pronounced in the setting of autophagy induction by vorinostat. Finally, exposure to the combination of vorinostat and hydroxychloroquine resulted in decreased levels of phospho-ERK compared to single-agent treatment, indicating reduced cell proliferation. Taken together, our data demonstrate that when autophagy was inhibited by hydroxychloroquine in the presence of vorinostat, autophagosomes accumulated and cell viability/proliferation reduced.

Conclusion: Preclinical studies in the different pediatric cancer cell lines show a clear and improved response when vorinostat is combined with hydroxychloroquine compared to vorinostat alone. The increased LC3-II/I ratio indicative of efficient autophagy inhibition while the decrease in the phospho-ERK signifies a decrease in proliferation. Furthermore the IC50 concentration of vorinostat, about 200ng/ml, necessary for effect is well within the achievable clinical range of 150-500ng/ml. The next step would be to validate these results in a xenograft model with one of the pediatric cancer tumors, and then hopefully continue forward to build a phase 1 clinical trial in recurrent pediatric solid tumors. This combination of drugs has yet to be explored in pediatrics but has the potential to enhance the effect of an established chemotherapy agent with a well-known and easily accessible non-cancer drug.

Citation Format: Abigail Joy Cruz, Hima Bansal, Gail Tomlinson, Sanjay Bansal, Steven Weitman. Enhancement of histone deacetylase inhibitor's efficacy with hydroxychloroquine via autophagy inhibition in pediatric tumor cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B36.