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When detectable (in HT29 cells), GPC levels decreased post-belinostat treatment. induction of choline kinase (ChoK) expression. Furthermore, metabolic labeling experiments with13C-glucose showed that differential glucose routing favored alanine formation at the expense of lactate production. Additional analysis revealed increases in the choline/water and phosphomonoester (including PC)/total phosphate ratiosin vivo. Together, our findings provide mechanistic insights into the impact of HDAC inhibition on cancer cell metabolism and highlight PC as a candidate non-invasive imaging biomarker for monitoring the action of HDAC inhibitors. Keywords:HDAC, targeted cancer therapy, mechanism of action, non-invasive metabolic biomarkers, choline == INTRODUCTION == Histone acetylation is a key regulator of eukaryotic gene expression which controls DNA accessibility to transcription factors and mRNA transcription. The histone acetylation/deacetylation balance is maintained by the opposing activities of rac-Rotigotine Hydrochloride histone acetyl transferases and histone deacetylases (HDACs) resulting in cell-specific gene expression patterns (1). Deregulation of histone acetylation results in abnormal gene expression profiles involved in controlling cell proliferation, differentiation and apoptosis, and is associated with malignancy (2-5). HDACs also act on other non-histone proteins that are subject to regulation by acetylation including some transcription factors (e.g. E2F) and the heat shock protein 90 (HSP90) molecular chaperone, which maintains the conformational stability of several rac-Rotigotine Hydrochloride oncogenic proteins (e.g. ErbB2) (6). HDAC inhibition is a promising anti-tumor approach for simultaneously targeting multiple oncogenic players and pathways. Several HDAC inhibitors have been described that induce potent anti-tumor effects in cells and tumor xenografts (6-8). The HDAC inhibitors SAHA (vorinostat) and depsipeptide FK228 (romidepsin) have gained FDA approval for cutaneous T-cell lymphoma treatment and many more are currently under clinical evaluation (6;8-11). One example is belinostat which has shown promising activity in pre-clinical cancer models and in patients (8;12). The development and evaluation of novel HDAC inhibitors requires the identification and validation of pharmacodynamic (PD) biomarkers of drug activity. These are important because they inform on the inhibition of the intended biochemical target, help assess response dynamics, aid treatment schedule and dose planning, and subsequently allow therapeutic efficacy assessment (13-15). In contemporary drug development, non-invasive endpoints of target modulation are highly desirable as they do not Rabbit Polyclonal to FGFR1 Oncogene Partner involve surgical intervention and allow longitudinal studies in the same patient to be performed (15-17). Non-invasive imaging of cancer metabolism is a valuable approach for PD biomarker discovery that exploits the altered metabolic features of tumors relative to normal tissues, including increased lipid synthesis and aerobic glycolysis (18;19). These metabolic changes are increasingly being investigated as diagnostic as well as treatment response biomarkers, with techniques such as magnetic resonance spectroscopy (MRS) being of particular value for translating findings from pre-clinical models to humans (16;17;20-22). MRS allows the detection of many metabolites (e.g. those related to glucose, protein and lipid metabolism), and in pre-clinical studies has shown that rac-Rotigotine Hydrochloride response to molecularly-targeted therapeutics is often associated with altered metabolism (17;20). For example, inhibitors of HSP90 (23;24), phospholipase C1 (25), mitogen activated protein kinase MEK (26) or phosphoinositide 3-kinase rac-Rotigotine Hydrochloride (27;28) have all been shown to alter choline phospholipid metabolism in human cancer cells. In the case of the HDAC inhibitor LAQ824,in vitroandin vivoMRS showed increased phosphocholine (PC) levels both in human colon cancer cells and tumors post-treatment (29). A similar effect was also observed in human colon and prostate cancer cells treated with the HDAC inhibitor SAHA (29) or its fluoro-analogue (30), respectively. Furthermore, LAQ824 treatment caused a substantial reduction in tumor bioenergy-related metabolites (e.g. NTP, glucose) that was observedin vivobut notin vitro(29). This effect was attributed to the anti-angiogenic action of LAQ824, while the rise in PC was likely to relate to the effect of HDAC inhibition on tumor cell metabolism (29) although the molecular and biochemical mechanisms behind this change remain unclear. Here we assess i) whether similar metabolic effects would be observed with the alternative chemotype (chemical scaffold) rac-Rotigotine Hydrochloride HDAC inhibitor and probe compound belinostat, and ii) the molecular and biochemical processes underlying the observed metabolic alterations. We show that HDAC inhibition with belinostat in human cancer cells leads to increased alanine and branched-chain amino acid.