Abstract 4457: The HDAC inhibitor romidepsin prevents the emergence of drug-tolerant cancer cells.
Authors: Norng Manith , Nguyen Aaron N. , DiMartino Jorge F. , MacBeth Kyle J. 
Published: August 2014

Abstract
Background: Drug resistance represents a major obstacle to successful cancer treatment. Various resistance mechanisms have been described (e.g., drug efflux pumps). Recent data points to the pre-existence of a subpopulation of cancer cells termed drug-tolerant persisters (DTPs) that exhibit an epigenetically-mediated tolerance to high concentrations of chemotherapeutic drugs (Sharma, 2010). Although DTPs were largely quiescent, a small fraction resumed growth even in the presence of high drug concentrations, giving rise to drug-tolerant expanded persisters (DTEPs). Levels of histone H3 lysine 4 (H3K4) methylation and H3K14 acetylation were decreased in DTPs and DTEPs, and co-treatment (Q3D) of cancer cells with the anticancer drug in combination with histone deacetylase inhibitors (HDACi) inhibited DTP/DTEP growth, implicating an epigenetic mechanism.
Purpose: To assess whether the HDACi romidepsin (ROMI) can prevent the emergence of drug-tolerant cancer cells, to determine the stage at which ROMI acts (killing DTPs or preventing DTEP expansion), and to establish the dose-schedule requirements for the effect.
Methods: The M14 melanoma and HCC827 NSCLC cell lines were treated in 24-well plates with AZ628 (B-Raf inhibitor) or Erlotinib (ERL; EGFR inhibitor), respectively, alone or in combination with ROMI, TSA, or azacitidine (AZA). ROMI, TSA, AZ628, and ERL were added every 3 days, while AZA was added daily. Cell viability was monitored periodically over 45 days using CellTiter-Glo. In subsequent experiments, reduced schedules from the “continuous” (Q3D for 45 days) schedule were tested, including Q3D schedules given for 1-8 times. To evaluate minimal daily exposure, cancer cells were exposed to ROMI for only 6 hours.
Results: Massive cell death was observed after 9 days of treatment of M14 and HCC827 cells with 100X IC50 concentrations of AZ628 and ERL, respectively. With continued AZ628/ERL treatments, however, the few remaining cells (DTPs) began to proliferate, ultimately yielding a large population of AZ628/ERL-resistant cells (DTEPs). “Continuous” (Q3D for 45 days) co-treatment of cancer cells with AZ628/ERL in combination with sub-lethal concentrations of ROMI and TSA, but not AZA, prevented the emergence of DTEPs. Short (6-hour) exposures to higher ROMI and TSA concentrations on the “continuous” schedule were also sufficient to prevent DTEP growth. Interestingly, “continuous” dosing of HDACi was not required to prevent DTEPs from forming, as reducing the schedule to 2 weeks Q3D had the same effect, suggesting that there is a period of susceptibility to the DTP or early DTEP population.
Conclusions: ROMI prevents DTEP emergence. Dosing ROMI for 2 weeks Q3D and 6-hour exposures are sufficient for this effect. These results show that co-treatment with ROMI can prevent drug resistance and provide guidance to the dose and schedule requirements to consider for clinical evaluation in this setting.
Citation Format: Manith Norng, Aaron N. Nguyen, Jorge F. DiMartino, Kyle J. MacBeth. The HDAC inhibitor romidepsin prevents the emergence of drug-tolerant cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4457. doi:10.1158/1538-7445.AM2013-4457