Abstract 4676: Metabolic reprogramming evoked by nitrosative stress enhances the effect of radiation in brain metastases.
Authors: Cerchietti Leandro C. , Navigante Alfredo , Yang Shaoning , Bonomi Marcelo , Castro Monica , Roth Berta , Martinez Manuel 
Published: August 2014

Abstract
Previous findings from our lab and others showed that brain metastases (BM) overexpress one or more nitric oxide (NO) synthetase isoforms. NO formation represents a growth advantage but also an oxidative load for these tumor cells. Proliferation during NO stress (NS) requires multiple homeostatic mechanisms, including metabolic adaptation. We wished to determine the metabolic reprogramming of BM cells under NS and whether this effect could be therapeutically capitalized. We performed cellular metabolomic studies (GC/UPLC-MS) in 5 human breast cancer cell lines including the brain metastatic subclone BrM2 under NS. Cell lines were exposed to L-arginine (or control) 60 min to induce NS. We found that NS induced profound changes in several metabolic pathways affecting glucose and glutamine metabolism rewiring with decreasing pyruvate and lactate production. This is relevant for BM pts, since virtually all of them will receive radiotherapy as part of their treatment. Accordingly, upon treatment with radiation (3Gy), cancer cells pre-exposed to L-Arg 50 mM exhibited more DNA damage (by p-H2AX) and apoptosis (caspase 7/3 activation) than untreated controls. We therefore hypothesized that L-Arg, by decreasing TLC, could behave as randiosensitizer for pts with BM.
Since oral L-Arg is approved as nutritional supplement for human use, we conducted a clinical trial with this formulation to determine its pharmacological properties and characterize its metabolic effects. We found that the maximum tolerable dose of L-Arg was 10 g, whith a plasma peak at 60 min (88 ± 12 nmol/ml, p=0.03 to basal, n=5). Accordingly, 60 min after L-Arg 10 g, we found an increase in tumor blood flow by PW-MRI (15% ± 2, p=0.02, n=5) and a decrease in TLC by Magnetic Resonance Spectroscopy (from 432 ± 23 pg to 213 ± 20 pg, p&lt;0.001, n=10). These results suggest that L-Arg induces metabolic reprogramming with decreasing TLC in BM pts. Consequently, we conducted a second study to assess toxicity and effect of L-arg administered with radiation. A second cohort of 53 pts with BM was irradiated to 54.4 Gy fractionated in 1.6 Gy bid and randomized to oral L-Arg 10 g (n=22) or placebo (PLA) 10 g (n=31), 60 min before each fraction. We found a significant difference for PLA vs L-Arg in radiological responses (CR+PR) (23 vs 86% respectively, p&lt;0.001) and symptomatic responses (CR + PR) (51 vs 95% respectively, p=0.002). The overall responses for PLA vs L-Arg were: CR (10 vs 27%, p=0.09), PR (13 vs 59%, p&lt;0.001), SD (7 vs 9%) and PD (70 vs 5%, p&lt;0.001). Median progression-free and OS in months (Kaplan-Meier) for PLA were 2 (0-3) and 3 (2-5), whereas for L-Arg were 6 (4-17) and 8 (5-19) (p&lt;0.001 and p=0.02, Cox-Mantel). No relevant adverse events attributed to L-Arg were noted.
In conclusion, oral L-Arg induced a transient metabolic reprogramming characterized by a reduction in TLC. This effect was capitalized to increase the efficacy of radiation for pts with BM.
Citation Format: Leandro C. Cerchietti, Alfredo Navigante, Shaoning Yang, Marcelo Bonomi, Monica Castro, Berta Roth, Manuel Martinez. Metabolic reprogramming evoked by nitrosative stress enhances the effect of radiation in brain metastases. [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 4676. doi:10.1158/1538-7445.AM2013-4676