Genetic deletion of 5’ methylthioadenosine phosphorylase (MTAP) is the most common homozygous deletion (~15%) in human cancers due to its close proximity to tumor suppressor gene locus CDKN2A/B. MTAP deletions are synthetically lethal with co-deletions in the gene encoding the metabolic enzymes methionine adenosyltransferase 2 alpha (MAT2a) and protein arginine methyltransferase 5 (PRMT5). MTAP functions in the methionine salvage pathways to recycle methylthioadenosine (MTA) to methionine, which MAT2a uses to synthesize s-adenosylmethionine (SAM). SAM is a ubiquitous cofactor for polyamination and methylation reactions in the cell. Current inhibitors of MAT2a are in clinical trials for cancers with MTAP deletions, however, this strategy only covers a small fraction of all cancers. We propose using MTAP inhibitor methylthio-DADMe-Immucillin A (MTDIA), transition state analogue developed by Schramm laboratory in combination with MAT2a inhibitor AG-270 will phenocopy the therapeutic effects seen with AG-270 in MTAP-/- cancers. We hypothesize that our novel drug combination will perturb methionine metabolism and have specific downstream effect on the activity of oncotarget protein arginine methyltransferase 5, a known regulator of gene splicing and transcription factor localization. To test this hypothesis, two cell lines representing cancers with rare natural MTAP deletions were selected. MTDIA and AG-270 displayed synergistic cell growth inhibition in MTAP+ FaDu and HT-29 cells by colorimetric WST-1 assay. Metabolic disturbances were determined through tandem LC-MS/MS analysis of extracts from cells treated over time. Western blotting of the PRMT5-specific product symmetric dimethylarginine was performed to demonstrate the inhibition of PRMT5. We report that MTDIA and AG-270 together, but not alone, have growth-inhibiting properties. MTDIA phenocopies the metabolic signature of MTAP deletion including increased MTA, and combination with AG-270 further increases the ratio of MTA:SAM by depleting the SAM pool. PRMT5 is inhibited only under high MTA:SAM ratio conditions, such as with combination therapy. The data suggest that MTDIA will expand the anti-cancer efficacy of MAT2a inhibitors through specific inhibition of PRMT5 and may extend therapeutic potential to the other 85% of MTAP+ cancers. Experiments on gene expression alterations, proteomic quantification of global and specific methylation changes, and safety and efficacy in animal models are underway.

MTAP inhibitor MTDIA expands anti-cancer efficacy of MAT2a inhibitors to MTAP+/+ cancers and inhibits PRMT5

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