Glycine N-methyltransferase (GNMT) is the main enzyme responsible for catabolism of excess hepatic S-adenosylmethionine (SAMe). GNMT is absent in hepatocellular carcinoma (HCC), messenger RNA (mRNA) levels are significantly lower in livers of patients at risk of developing HCC, and GNMT has been proposed to be a tumor-susceptibility gene for liver cancer. The identification of several children with liver disease as having mutations of the GNMT gene further suggests that this enzyme plays an important role in liver function. In the current study we studied development of liver pathologies including HCC in GNMTknockout (GNMT-KO) mice. GNMT-KO mice have elevated serum aminotransferase, methionine, and SAMe levels and develop liver steatosis, fibrosis, and HCC. We found that activation of the Ras and Janus kinase ( T he first steps in mammalian methionine metabolism are conversion to S-adenosylmethionine (SAMe) and transfer of the methyl group of SAMe to a large variety of substrates (including DNA, RNA, histones, and small molecules such as glycine, guanidinoacetate, and phosphatidylethanolamine) with the formation of S-adenosylhomocysteine (SAH), an inhibitor of many SAMe-dependent methyltransferases. 1 Although there are a large number of SAMe-dependent methyltransferases, 2 methylation of glycine by glycine Nmethyltransferase (GNMT) to form sarcosine (N-methylglycine) is one of the reactions that contribute most to total transmethylation flux. 3 The importance of GNMT is to remove excess SAMe and maintain a constant hepatic SAMe/SAH ratio to avoid aberrant methylation. 2 Consistent with this function, the activation of GNMT in rats by the administration of retinoic acid causes a reduction in plasma methionine and homocysteine levels, as well as in liver DNA methylation. 4,5 In GNMT-knockout (GNMT-KO) mice, liver SAMe content is elevated 35-fold, and the SAMe/SAH ratio increases about 100-fold, 6 and individuals with GNMT mutations, which leads to inactive forms of the enzyme, have elevated plasma levels of methionine and SAMe but a normal concentration of homocysteine. 7,8 GNMT is expressed in the liver, pancreas, and prostate 9 and is absent in hepatocellular carcinoma (HCC) 10 and down-regulated in the livers of patients at risk of Abbreviations: GNMT, HCC, hepatocellular carcinoma; H3K27me3, trimethylated Received September 10, 2007; accepted November 26, 2007. Supported by NIH grants AA12677, AA13847, and AT-1576 (to S.C.L. and J.M.M.); DK15289 (to C.W.), PN IϩD SAF 2005-00855, HEPADIP-EULSHM-CT-205, and ETORTEK 2005 (to J.M.M. and M.L.M.-C.); Program Ramón y Cajal (to M.L.M.-C.); and Fundación "La Caixa" (to M.L.M.-C., R.M., and A.M.A.).

DNA-dependent protein kinase (DNA-PK) is a critical player in the DNA damage response (DDR) and instrumental in the non-homologous end-joining pathway (NHEJ) used to detect and repair DNA double-strand breaks (DSBs). We demonstrate that the potent and highly selective DNA-PK inhibitor, AZD7648, is an efficient sensitizer of radiation- and doxorubicin-induced DNA damage, with combinations in xenograft and patient-derived xenograft (PDX) models inducing sustained regressions. Using ATM-deficient cells, we demonstrate that AZD7648, in combination with the PARP inhibitor olaparib, increases genomic instability, resulting in cell growth inhibition and apoptosis. AZD7648 enhanced olaparib efficacy across a range of doses and schedules in xenograft and PDX models, enabling sustained tumour regression and providing a clear rationale for its clinical investigation. Through its differentiated mechanism of action as an NHEJ inhibitor, AZD7648 complements the current armamentarium of DDR-targeted agents and has potential in combination with these agents to achieve deeper responses to current therapies.

Our understanding of the mechanisms by which nonalcoholic fatty liver disease (NAFLD) progresses from simple steatosis to steatohepatitis (NASH) is still very limited. Despite the growing number of studies linking the disease with altered serum metabolite levels, an obstacle to the development of metabolome-based NAFLD predictors has been the lack of large cohort data from biopsy-proven patients matched for key metabolic features such as obesity. We studied 467 biopsied individuals with normal liver histology (n=90) or diagnosed with NAFLD (steatosis, n=246; NASH, n=131), randomly divided into estimation (80% of all patients) and validation (20% of all patients) groups. Qualitative determinations of 540 serum metabolite variables were performed using ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS). The metabolic profile was dependent on patient body-mass index (BMI), suggesting that the NAFLD pathogenesis mechanism may be quite different depending on an individual’s level of obesity. A BMI-stratified multivariate model based on the NAFLD serum metabolic profile was used to separate patients with and without NASH. The area under the receiver operating characteristic curve was 0.87 in the estimation and 0.85 in the validation group. The cutoff (0.54) corresponding to maximum average diagnostic accuracy (0.82) predicted NASH with a sensitivity of 0.71 and a specificity of 0.92 (negative/positive predictive values = 0.82/0.84). The present data, indicating that a BMI-dependent serum metabolic profile may be able to reliably distinguish NASH from steatosis patients, have significant implications for the development of NASH biomarkers and potential novel targets for therapeutic intervention.

Background & Aims-Hepatic de-differentiation, liver development, and malignant transformation are processes in which the levels of hepatic S-adenosylmethionine (SAMe) are tightly regulated by two genes, MAT1A and MAT2A. MAT1A is expressed in the adult liver, whereas MAT2A expression is primarily extra-hepatic and is strongly associated with liver proliferation. The mechanisms that regulate these expression patterns are not completely understood. In silico analysis of the 3′ untranslated region of MAT1A and MAT2A revealed putative binding sites for the RNA-binding proteins AUF1 and HuR, respectively. We investigated the post-transcriptional regulation of MAT1A and MAT2A by AUF1, HuR and methyl-HuR in the aforementioned biological processes.