Novel Preclinical Testing Strategies for Treatment of Metastatic Pheochromocytoma

There is currently no effective treatment for metastatic pheochromocytomas and paragangliomas. A deficiency in currentchemotherapy regimens is that the metastases usually grow very slowly. Drugs that target dividing tumor cells havetherefore had limited success. To improve treatment, new strategies and valid experimental models are required for pre-clinical testing. However, development of models has itself been hampered by the absence of human pheochromocytoma/paraganglioma cell lines for cultures or xenografts. Topoisomerase 1 (TOP1) inhibitors are drugs that interfere withmechanisms that maintain DNA integrity during transcription in both quiescent and dividing cells. We used primary culturesof representative human tumors to establish the cytotoxicity of camptothecin, a prototypical TOP1 inhibitor, against non-dividing pheochromocytoma/paraganglioma cells, and then employed a mouse pheochromocytoma model (MPC) to showthat efficacy of low concentrations of camptothecin and other TOP1 inhibitors is increased by intermittent coadministrationof sub-toxic concentrations of 5-azacytidine, a DNA methylation inhibitor that modulates transcription. We then tested thesame drugs against a clonal MPC derivative that expresses CMV reporter-driven luciferase and GFP, intended for in vivodrug testing. Unexpectedly, luciferase expression, bioluminescence and GFP expression were paradoxically increased byboth camptothecin and SN38, the active metabolite of irinotecan, thereby masking cell death. Expression of chromograninA, a marker for neuroendocrine secretory granules, was not increased, indicating that the drug effects on levels of luciferaseand GFP are specific to the GFP-luciferase construct rather than generalized cellular responses. Our findings provide proof ofprinciple for use of TOP1 inhibitors against pheochromocytoma/paraganglioma and suggest novel strategies for enhancingefficacy and reducing toxicity by optimizing the combination and timing of their use in conjunction with other drugs. Theparadoxical effects of TOP1 inhibitors on luciferase and GFP dictate a need for caution in the use of CMV promoter-regulated constructs for cancer-related imaging studies. Citation: Powers JF, Korgaonkar PG, Fliedner S, Giubellino A, Pacak K, et al. (2014) Cytocidal Activities of Topoisomerase 1 Inhibitors and 5-Azacytidine againstPheochromocytoma/Paraganglioma Cells in Primary Human Tumor Cultures and Mouse Cell Lines. PLoS ONE 9(1): e87807. doi:10.1371/journal.pone.0087807 Editor: Michal Hetman, University of Louisville, United States of America Received August 16, 2013; Accepted December 30, 2013; Published January 31, 2014 Copyright: 2014 Powers et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: JF Powers and AS Tischler were supported by grant PR100171 from the Department of Defense and by a grant from the Pheo Para Alliance (http://www.pheo-para-alliance.org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]