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Alcoholic fatty liver disease (AFLD) is characterized by lipid accumulation and inflammation and can progress to cirrhosis and cancer in the liver. AFLD diagnosis currently relies on histological analysis of liver biopsies. Early detection permits interventions that would prevent progression to cirrhosis or later stages of the disease. Herein, we have conducted the first comprehensive time-course study of lipids using novel state-of-the art lipidomics methods in plasma and liver in the early stages of a mouse model of AFLD, i.e., Lieber-DeCarli diet model. In ethanol-treated mice, changes in liver tissue included up-regulation of triglycerides (TGs) and oxidized TGs and down-regulation of phosphatidylcholine, lysophosphatidylcholine, and 20-22-carbon-containing lipid-mediator precursors. An increase in oxidized TGs preceded histological signs of early AFLD, i.e., steatosis, with these changes observed in both the liver and plasma. The major lipid classes dysregulated by ethanol play important roles in hepatic inflammation, steatosis, and oxidative damage. Conclusion: Alcohol consumption alters the liver lipidome before overt histological markers of early AFLD. This introduces the exciting possibility that specific lipids may serve as earlier biomarkers of AFLD than those currently being used.

Purpose Oxidative stress has been shown to be intimately involved in the pathogenesis of fatty liver disease (FLD). A major factor contributing to oxidative stress is the depletion of glutathione (GSH). Our previous studies in glutamate cysteine ligase modifier subunit (Gclm)-null mice demonstrate that ~85% deficiency in hepatic GSH renders mice protected from fatty liver injuries induced by varieties of hepatic insults, including alcohol exposure. It is well-known that epigenetic mechanisms play active roles in modulating gene expression and disease outcome in FLD. The purpose of the current study is to utilize the Gclm-null mouse model to examine in vivo interplay between chronic oxidative stress and DNA methylation at the epigenome level and whether these epigenetic changes are functionally involved in FLD. Methods we performed DNA methylation profiling in the liver and whole blood using the Mouse Promoter Methylation Array. Genes with CpG sites that were significantly affected by GSH deficiency were further investigated for network and functional enrichment. Results Among 22,327 gene promoter regions (from -1300bp to +500bp of the Transcription Start Sites) that were analyzed, in the liver, only 52 and 6 gene promoters were hypo- and hyper-methylated, respectively, in Gclm-null mice relative to wild-type mice, indicating an overall hypo-methylated genome associated with low GSH. Interestingly, the DNA methylation profiles in the liver and blood appeared to be comparable irrespective of the genotype, which leads to the possibility of potential circulating biomarker(s) for hepatic epigenetic fingerprints related to chronic oxidative stress. Ingenuity Pathway Analysis (IPA) revealed that hypo-methylated genes in the KO liver are enriched in canonical pathways of protein phosphorylation, RNA transcription, cytoplasm organization and organismal death. Conclusion our preliminary data indicate that chronic GSH deficiency induces global DNA hypomethylation. The functional significance of observed epigenomic changes is under investigation.

ALDH1A1 is expressed at high levels in normal HSCs and we previously reported that its function might involve metabolism of compounds termed reactive aldehydes. We also reported that loss of ALDH1A1 led to a compensatory increase in a related isoform, ALDH3A1, which also metabolizes reactive aldehydes. Double knockouts for both ALDH1A1/3A1 accumulate reactive aldehydes, which appear to impact a number of cellular processes including signal transduction and gene expression. As reactive aldehydes also cause DNA damage, we hypothesized that excess accumulation of reactive aldehydes may predispose to leukemic transformation of HSCs. In support of this, we found that ALDH1A1/3A1 double knockout HSCs readily form acute leukemia following transduction with a NUP98-HOXA10 fusion gene, which rarely causes leukemic transformation in wild type HSCs. Furthermore, in human AML, frequent absence of ALDH1A1 and the universal absence of ALDH3A1 was observed. A human AML cell line, Kasumi-1, was found to be ALDH1A1/3A1 deficient and to have high levels of intracellular reactive aldehydes. In addition, Kasumi-1 was highly sensitive to DNA damage and cell death following exposure to exogenous 4-HNE, a prototypic reactive aldehyde. In contrast, normal CD34+ HSCs were relatively resistant to 4-HNE. Based on these observations, we further hypothesized that treatment of ALDH1A1/3A1 deficient AMLs with clinically relevant compounds that further increase intracellular 4-HNE levels would selectively eliminate AML while sparing normal CD34+ HSCs. To test this, Kasumi-1 were exposed to a series of compounds including the pro-oxidant Arsenic tri-oxide (ATO), the sesquiterpene lactone parthenolide (PTL) and 4-HC, the active metabolite of cyclophosphamide (Cy) and a substrate of ALDH1A1. All increased intracellular 4-HNE levels and DNA damage. Exposure to combinations of 4-HC, ATO and PTL induced high levels of cell death in Kasumi-1. In contrast, Kasumi-1 cells engineered to express ALDH1A1 through lentiviral gene transfer and normal CD34+ HSCs were relatively resistant to several of these treatments. Primary ALDH1A1/3A1- AMLs were also relatively sensitive to treatment with these same compounds. In conclusion, ALDHs and reactive aldehydes may play important roles in HSCs and leukemia and exploitation of their biology may lead to novel therapies for AML and possibly other cancers. As an initial application of this treatment strategy, we are developing a clinical trial to treat patients with relapsed/refractory ALDH1A1/3A1 deficient AML with Cy/ATO. Disclosures: No relevant conflicts of interest to declare.