Abstract LB-6: Exploring nicotinamide-N-methyltransferase kinetics through targeted metabolomic profiling for its prognostic value in glioblastoma

Abstract

Abstract Purpose: Glioblastoma (GBM) is the most aggressive form of glioma. With average patient survival of about 14 months, largely due to its resistance to conventional therapies, there is opportunity for significant improvements to care. Nicotinamide-N-methyltransferase (NNMT) is an enzyme responsible for the methylation of Nicotinamide and other xenobiotic compounds. Although NNMT's exact role in the malignant phenotype remains poorly understood, it has been investigated for its potential use as a prognostic marker or therapeutic target in other cancers. It is detected at very low levels in normal brain tissue but at significantly higher levels in GBM. Additionally, Kaplan-Meier plots from publically available data sets suggest that higher NNMT expression is correlated with adverse patient outcome. In addition to gene interference and various functional assays, we employed a targeted metabolomic approach, using LC-MS Quadropole Time of Flight (Q-TOF) and LC-MS Triple Quad (QQQ) instruments to study intracellular levels of Nicotinamide and N-methylnicotinamide to gain a better understanding of NNMT's role in GBM. Methods: We determined the expression profile of NNMT in established (ATCC) and in our panel of patient derived, primary GBM cell lines. By generating an NNMT isogenic model in U87 cells, we were able to study the functional consequences of differential NNMT expression using in vitro assays such as MTS Assay, Clonogenic Survival Assay, Annexin V, ATP Assay, Comet Assay, and Western Blotting. We intracranially injected NOD-SCID mice with U87 NNMT cells to assess differences in tumorgenicity in vivo. We have also used LC-MS Quadropole Time of Flight (Q-TOF) and LC-MS Triple Quad (QQQ) instruments to measure intracellular levels of the metabolites relevant to NNMT enzymatic function. Results and Conclusions: Results indicate that NNMT protein is highly expressed in primary cell lines. Encouragingly, U87 NNMT Knockdown cells are less proliferative, more sensitive to radiation in vitro, and are less tumorgenic in vivo. Recapitulating the clinical observations, these results could justify why clinically patients with lower expression of NNMT enjoyed increased overall survival. These results also suggest that finding ways to decrease NNMT in patient's tumors may improve response to radiation. Additional preliminary results indicate that intracellular N-methylnicotinamide levels correlate directly with NNMT protein expression and could be used a surrogate biomarker for intracellular NNMT levels, potentially predicting not only overall survival but perhaps response to radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-6. doi:1538-7445.AM2012-LB-6