PROJECT SUMMARY/ABSTRACT The rapid proliferation of cancer cells is often fueled by a significant elevation in glucose consumption. A consequence of sustained elevation in glycolytic flux is the generation of reactive carbonyl species such as methylglyoxal (MG). MG can form advanced glycation end- products (AGEs) with amino acids, nucleic acids and lipids. The DNA AGE is of particular interest because it serves as a molecular link between dysregulated glucose metabolism and genome instability. In order to prevent the formation of AGEs, cells deploy the glutathione- dependent Glyoxalase 1/2 (Glo1/2) pathway. Glo1 is over-expressed in breast and prostate tumors and has been correlated with poor prognosis. Separately, BRCA1/2 defective breast, ovarian and metastatic prostate cancers are being treated effectively in the clinic with inhibitors of poly(ADP-ribose) polymerase (PARP). Here, we propose to investigate the inhibition of Glo1 using a glutathione analogue, S-p-bromobenzyl glutathione cyclopentyl diester [p- BrBzGSH(Cp)2]. A novel stable isotope dilution liquid chromatography tandem mass spectrometry (SID-LC/MS/MS) method will be used to quantify the DNA AGE, CEdG in Glo1 inhibitor treated cancer cells. Our long-term goal is to see if we can leverage Glo1 to target aggressive glycolytic tumors that lack targeted therapy. We propose that effective inhibition of MG detoxification pathways via Glo1 or aldo-keto reductase (AKR) inhibition will lead to the formation of CEdG and downstream mutations and single strand breaks and potentiate cell death. (Aim 1) To determine the biological effects of Glo1 inhibition in breast, prostate, ovarian, and pancreatic cancer cells. (Aim 2) To interrogate the synergy of Glo1 inhibition with the inhibition of DNA repair pathways. Progress towards these Specific Aims will contribute towards establishing a strategy of pairing a novel metabolic target with a PARP inhibitor to selectively kill glycolytic cancer cells with diminished DNA repair capacity. We do anticipate that there may be compensatory responses to Glo1 inhibition, we are aware of the aldo-keto reductase superfamily of NADPH-dependent oxido-reductases that may detoxify MG in the absence of Glo1 activity. We are also aware of the potential over-expression of the glutathione biosynthesis pathway that may respond in the event of effective Glo1 inhibition. This proposal will work towards illuminating a potential novel strategy for targeting glycolytic tumors for which there are no targeted therapy such as triple negative breast cancer and metastatic castration-resistant prostate cancer.