The Efficacy of Anti-Cancer Drugs Targeting Nicotinamide N-Methyltransferase (NNMT) in Cultured Human Oral Squamous Cell Carcinoma (OSCC).

Objectives: Oral squamous cell carcinoma (OSCC) accounts for 90% of oral malignancies. We are interested in a targeted approach to complement the established cancer therapeutics via the enzyme, N-Nicotinamide Methyltransferase (NNMT). NNMT has been demonstrated to alter protein methylation profiles promoting oncogenesis. Our laboratory identified two inhibitors of NNMT (AG-670 and AO-022) based on a pharmacophore of the in-silico nicotinamide binding site. The effect of these modulators of NNMT on OSCC cell viability and energy metabolism was evaluated.
Methods: All cells were cultured as per ATCC recommendations. Detection of NNMT was assessed by immunoblot (Novus Biologicals NBP2-00537) from cell lysates. Cell viability was determined by alamar blue assay using a range of inhibitor concentrations. Cellular respiration and extracellular acidification rate were determined by Seahorse XF analyser. NADH-dependent oxygen consumption in the presence/absence of NNMT inhibitors on rat liver mitochondria was determined using a Rank Oxygen Electrode. An Inhibitor Screening Assay kit was used to determine the potency of inhibitors on the isolated enzyme.
Results: SCC-4 and DOK cells express NNMT. AG-670 and AO-022 caused the death of SCC-4 cells over the aforementioned concentration range and their respective IC₅₀ were calculated (45.7µM for AG-670 and 52.1µM for AO-022). Significantly, our data has shown that both inhibitors reduce oxidative phosphorylation and increase glycolysis. Neither inhibitor significantly affected NADH-dependant oxygen consumption in the coupled/uncoupled rat mitochondria.
Conclusions: NNMT is expressed in oral squamous cell carcinoma (SCC-4) cells. The NNMT inhibitors are potent to SCC-4 cells. Provisional data confirms that the compounds do inhibit the enzymic function of isolated NNMT. It was observed that AG-670 and AO-022 inhibit mitochondrial oxygen consumption by intact SCC4 and DOK cells commensurate with increased glycolytic flux. However, we demonstrate that inhibitors do not directly inhibit mitochondrial function, suggesting indirect inhibition of mitochondrial function via NNMT via a mechanism yet to be determined.