Abstract A16: The in vitro and in vivo activity of the pyrrolomycin marinopyrrole RL71 alone and combined with temsirolimus (TEM) in MYCN-amplified neuroblastoma (NB)
Authors: McGuire Timothy R. , Coulter Donald W. , Liu Yan , Chhonker Yashpal S. , Murry Daryl J. , Alexander Gracey R. , McIntyre Erin M. , Sughroue Jason A. , Sharp Graham J. , Li Rongshi 
Published: February 2019

Abstract
Introduction: We previously reported a series of novel marinopyrroles as Mcl-1/Bcl-2 inhibitors for potential anticancer activity. The marinopyrrole agents have not been studied in pediatric tumors including neuroblastoma (NB). Temsirolimus (TEM) is an mTOR inhibitor that has been used clinically in renal cell carcinoma and breast cancer and more recently has been used in relapse/refractory pediatric tumors, including NB. In the following study we describe the in vitro and in vivo activity of the most active of the marinopyrroles (RL71) alone or with the mTOR inhibitor TEM in a chemoresistant NB cell line.
Methods: RL71, which was determined to be the most active of 39 pyrrolomycin derivatives, was tested in a MYCN amplified BE-2c NB cell line using an MTT assay and was selected for study in experiments to determine potential mechanism of action and for evaluation of in vivo activity. RL71 was tested in a series of in vitro assays to evaluate effects on apoptosis, metabolism, and cell ultrastructure. Apoptosis was evaluated using propidium iodide cell cycle analysis by flow cytometry and measurement of the sub-G1 fraction. Apoptosis was also evaluated by the measurement of cleaved PARP by Western blot. The effect of RL71 alone and combined with TEM on metabolism was determined using Seahorse Xp Metabolic Analyzer. The effect of agent(s) on OXPHOS metabolism was measured by oxygen consumption rate and effect on glycolysis by extracellular acidification rate (ECAR). Electron microscopy (EM) was used to determine changes in cell ultrastructure, particularly of mitochondria, in treated and nontreated cells. Using a mouse xenograft model, the antitumor effect for RL71 and the combination of RL71 plus TEM was tested. RL71 concentrations were measured in blood, tumor, and brain.
Results: RL71 at low concentrations (500 nM) caused disruption of ATP production and increased proton leak in mitochondria after injection. This effect was associated with sharp drop in ATP cellular levels. There was also a modest decline in glycolytic capacity and a lack of compensation for loss of mitochondrial ATP production. These effects were not seen with TEM alone, nor did TEM combined with RL71 show potentiation. Measures of apoptosis, which included cleaved PARP and sub-G1 peaks on flow cytometry, were not consistently elevated in either RL71 or TEM alone or when the two were combined. EM showed mitochondrial and cell membrane disruption with RL71 treatment alone but not with TEM alone; drugs combined appeared to produce greater disruption of cell membranes. There was no clear effect on nuclear morphology on EM, further supporting that apoptosis was unlikely to be the mechanism of cell death. The cell membrane disruption was consistent with necrosis, perhaps as a result of sharp decline in ATP. Three of 5 mice dosed with the combination of RL71 plus TEM had complete responses while none of the mice in the control group, TEM-alone group, or PL71-alone group responded (p = 0.009). Concentrations of RL71 in blood, tumor, and brain generally achieved concentrations exceeding the IC50 when it was combined with TEM (IC50=25 nM).
Conclusion: The combination of RL71 and TEM may be active in chemoresistant, MYCN-amplified NB. The mechanism of action of the combination continues to be evaluated; it is unlikely to be completely explained by disrupted metabolism or induction of apoptosis but likely results from nonapoptotic mechanisms of cell death such as necrosis or autophagy.
Citation Format: Timothy R. McGuire, Donald W. Coulter, Yan Liu, Yashpal S. Chhonker, Daryl J. Murry, Gracey R. Alexander, Erin M. McIntyre, Jason A. Sughroue, Graham J. Sharp, Rongshi Li. The in vitro and in vivo activity of the pyrrolomycin marinopyrrole RL71 alone and combined with temsirolimus (TEM) in MYCN-amplified neuroblastoma (NB) [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A16.