Towards the mechanism of early-life programming:Consequences of a maternal western diet

Maternal diet is associated with the development of metabolism-related and other diseases in the offspring. Underlying mechanisms and molecular markers are only starting to be revealed. In this research project, we explored the physiological and molecular impact of maternal western-style diet on the development of the offspring. Mice were exposed to either a low fat/low cholesterol diet (LFD) or a western-style high fat/high cholesterol diet (WSD) before mating, as well as during gestation and lactation. Dams and offspring were sacrificed at different developmental stages. In offspring of both sexes of WSD dams, liver and blood parameters, as well as hepatic gene expression profiles were changed. Exclusively male offspring had significantly higher body weight and a non-alcoholic fatty liver-phenotype upon maternal WSD. We conclude that maternal WSD affects physiological parameters and induces vital changes in the molecular profile of the liver in offspring. Remarkably, the observed biological responses of the offspring reveal pronounced sex-specificity, which in part might be related to a differential development of the placenta. We describe that the labyrinth diameter is larger in male placentas compared to the female placentas, which let us speculate that the male placenta supports transport of excess maternal nutrients to the male fetus. In addition we studied DNA methylation changes that might facilitate programming effects to persist into adulthood. In our mice we found a core set of specific methylation patterns corresponding to human steatohepatitis

HIF1/2-exerted control over glycolytic gene expression is not functionally relevant for glycolysis in human leukemic stem/progenitor cells

Background Hypoxia-inducible factors (HIF)1 and 2 are transcription factors that regulate the homeostatic response to low oxygen conditions. Since data related to the importance of HIF1 and 2 in hematopoietic stem and progenitors is conflicting, we investigated the chromatin binding profiles of HIF1 and HIF2 and linked that to transcriptional networks and the cellular metabolic state. Methods Genome-wide ChIPseq and ChIP-PCR experiments were performed to identify HIF1 and HIF2 binding sites in human acute myeloid leukemia (AML) cells and healthy CD34(+) hematopoietic stem/progenitor cells. Transcriptome studies were performed to identify gene expression changes induced by hypoxia or by overexpression of oxygen-insensitive HIF1 and HIF2 mutants. Metabolism studies were performed by 1D-NMR, and glucose consumption and lactate production levels were determined by spectrophotometric enzyme assays. CRISPR-CAS9-mediated HIF1, HIF2, and ARNT(-/-) lines were generated to study the functional consequences upon loss of HIF signaling, in vitro and in vivo upon transplantation of knockout lines in xenograft mice. Results Genome-wide ChIP-seq and transcriptome studies revealed that overlapping HIF1- and HIF2-controlled loci were highly enriched for various processes including metabolism, particularly glucose metabolism, but also for chromatin organization, cellular response to stress and G protein-coupled receptor signaling. ChIP-qPCR validation studies confirmed that glycolysis-related genes but not genes related to the TCA cycle or glutaminolysis were controlled by both HIF1 and HIF2 in leukemic cell lines and primary AMLs, while in healthy human CD34(+) cells these loci were predominantly controlled by HIF1 and not HIF2. However, and in contrast to our initial hypotheses, CRISPR/Cas9-mediated knockout of HIF signaling did not affect growth, internal metabolite concentrations, glucose consumption or lactate production under hypoxia, not even in vivo upon transplantation of knockout cells into xenograft mice. Conclusion These data indicate that, while HIFs exert control over glycolysis but not OxPHOS gene expression in human leukemic cells, this is not critically important for their metabolic state. In contrast, inhibition of BCR-ABL did impact on glucose consumption and lactate production regardless of the presence of HIFs. These data indicate that oncogene-mediated control over glycolysis can occur independently of hypoxic signaling modules.</p

Inhibition of the succinyl dehydrogenase complex in acute myeloid leukemia leads to a lactate-fuelled respiratory metabolic vulnerability

Metabolic programs can differ substantially across genetically distinct subtypes of acute myeloid leukemia (AML). These programs are not static entities but can change swiftly as a consequence of extracellular changes or in response to pathway-inhibiting drugs. Here, we uncover that AML patients with FLT3 internal tandem duplications (FLT3-ITD+) are characterized by a high expression of succinate-CoA ligases and high activity of mitochondrial electron transport chain (ETC) complex II, thereby driving high mitochondrial respiration activity linked to the Krebs cycle. While inhibition of ETC complex II enhances apoptosis in FLT3-ITD+ AML, cells also quickly adapt by importing lactate from the extracellular microenvironment. 13C3-labelled lactate metabolic flux analyses reveal that AML cells use lactate as a fuel for mitochondrial respiration. Inhibition of lactate transport by blocking Monocarboxylic Acid Transporter 1 (MCT1) strongly enhances sensitivity to ETC complex II inhibition in vitro as well as in vivo. Our study highlights a metabolic adaptability of cancer cells that can be exploited therapeutically.</p

Oxidative Phosphorylation Fueled by Fatty Acid Oxidation Sensitizes Leukemic Stem Cells to Cold

Dependency on mitochondrial oxidative phosphorylation (OxPhos) is a potential weakness for leukemic stem cells (LSC) that can be exploited for therapeutic purposes. Fatty acid oxidation (FAO) is a crucial OxPhos-fueling catabolic pathway for some acute myeloid leukemia (AML) cells, particularly chemotherapy-resistant AML cells. Here, we identified cold sensitivity at 4◦C (cold killing challenge; CKC4), commonly used for sample storage, as a novel vulnerability that selectively kills AML LSCs with active FAO-supported OxPhos while sparing normal hematopoietic stem cells. Cell death of OxPhos-positive leukemic cells was induced by membrane permeabilization at 4◦C; by sharp contrast, leukemic cells relying on glycolysis were resistant. Forcing glycolytic cells to activate OxPhos metabolism sensitized them to CKC4. Lipidomic and proteomic analyses showed that OxPhos shapes the composition of the plasma membrane and introduces variation of 22 lipid subfamilies between cold-sensitive and cold-resistant cells. Together, these findings indicate that steady-state energy metabolism at body temperature predetermines the sensitivity of AML LSCs to cold temperature, suggesting that cold sensitivity could be a potential OxPhos biomarker. These results could have important implications for designing experiments for AML research to avoid cell storage at 4◦C.</p

The Glycolytic Gatekeeper PDK1 defines different metabolic states between genetically distinct subtypes of human acute myeloid leukemia

Acute myeloid leukemia remains difficult to treat due to strong genetic heterogeneity between and within individual patients. Here, we show that Pyruvate dehydrogenase kinase 1 (PDK1) acts as a targetable determinant of different metabolic states in acute myeloid leukemia (AML). PDK1(low) AMLs are OXPHOS-driven, are enriched for leukemic granulocyte-monocyte progenitor (L-GMP) signatures, and are associated with FLT3-ITD and NPM1cyt mutations. PDK1(high) AMLs however are OXPHOS(low), wild type for FLT3 and NPM1, and are enriched for stemness signatures. Metabolic states can even differ between genetically distinct subclones within individual patients. Loss of PDK1 activity releases glycolytic cells into an OXPHOS state associated with increased ROS levels resulting in enhanced apoptosis in leukemic but not in healthy stem/progenitor cells. This coincides with an enhanced dependency on glutamine uptake and reduced proliferation in vitro and in vivo in humanized xenograft mouse models. We show that human leukemias display distinct metabolic states and adaptation mechanisms that can serve as targets for treatment

Depletion of SAM50 Specifically Targets BCR-ABL-Expressing Leukemic Stem and Progenitor Cells by Interfering with Mitochondrial Functions

A high proliferation rate of malignant cells requires an increased energy production, both by anaerobic glucose metabolism and mitochondrial respiration. Moreover, increased levels of mitochondria-produced reactive oxygen species (ROS) promote survival of transformed cells and contribute to the disease progression both in solid tumors and leukemia. Consequently, interfering with mitochondrial metabolism has been used as a strategy to specifically target leukemic cells. SAM50 is a mitochondrial outer membrane protein involved in the formation of mitochondrial intermembrane space bridging (MIB) complex. Although the importance of SAM50 in maintaining MIB integrity and in the assembly of mitochondrial respiratory chain complexes has been described, its specific role in the normal and leukemic hematopoietic cells remains unknown. We observed that human leukemic cells display a specific dependency on SAM50 expression, as downregulation of SAM50 in BCR-ABL-expressing, but not normal CD34(+) human hematopoietic stem and progenitor cells (HSPCs) caused a significant decrease in growth, colony formation, and replating capacity. Mitochondrial functions of BCR-ABL-expressing HSPCs were compromised, as seen by a decreased mitochondrial membrane potential and respiration. This effect of SAM50 downregulation was recapitulated in normal HSPCs exposed to cytokine-rich culture conditions that stimulate proliferation. Both oncogene-transduced and cytokine-stimulated HSPCs showed increased mitochondrial membrane potential and increased ROS levels compared to their normal counterparts. Therefore, we postulate that human leukemic HSPCs are highly dependent on the proper functioning of mitochondria and that disruption of mitochondrial integrity may aid in targeting leukemic cells

DNA Methylation and Expression Patterns of Selected Genes in First-Trimester Placental Tissue from Pregnancies with Small-for-Gestational-Age Infants at Birth

Variations in DNA methylation levels in the placenta are thought to influence gene expression and are associated with complications of pregnancy, like fetal growth restriction (FGR). The most important cause for FGR is placental dysfunction. Here, we examined whether changes in DNA methylation, followed by gene expression changes, are mechanistically involved in the etiology of FGR. In this retrospective case-control study, we examined the association between small-for-gestational-age (SGA) children and both DNA methylation and gene expression levels of the genes WNT2, IGF2/H19, SERPINA3, HERVWE1, and PPARG in first-trimester placental tissue. We also examined the repetitive element LINE-1. These candidate genes have been reported in the literature to be associated with SGA. We used first-trimester placental tissue from chorionic villus biopsies. A total of 35 SGA children (with a birth weight below the 10th percentile) were matched to 70 controls based on their gestational age. DNA methylation levels were analyzed by pyrosequencing and mRNA levels were analyzed by real-time PCR. None of the average DNA methylation levels, measured for each gene, showed a significant difference between SGA placental tissue compared to control tissue. However, hypermethylation of WNT2 was detected on two CpG positions in SGA. This was not associated with changes in gene expression. Apart from two CpG positions of the WNT2 gene, in early placenta samples, no evident changes in DNA methylation or expression were found. This indicates that the already reported changes in term placenta are not present in the early placenta, and therefore must arise after the first trimester

Inihibition of Glutaminase impairs proliferation of BCR-ABL⁺ CB cells.

<p><b>A,</b> Enhanced expression of SLC1A5 in K562 and CB BCR-ABL cells as compared to empty vector transduced MiNR1 CB cells. <b>B,</b> Schematic representation of glutaminolysis to participate as an extra anaplerotic carbon. <b>C,</b> Normal CB cells showed no significant effect with 20 μM of BPTES however higher dose of 40 μM seemed to be toxic. <b>D,</b> Dose dependent impaired proliferation observed selectively in BCR-ABL cells as compared to normal CB cells at 20 μM and 40 μM BPTES. <b>E,</b> The clonogenic capacity of BCR-ABL cells was compromised in the presence of BPTES, p value: BCR-ABL untreated vs BCR-ABL 20 μM p = 0.0032; BCR-ABL untreated vs BCR-ABL 40 μM p = 0.0117. <b>F,</b> OCR measurements in K562 cells treated with BPTES using Seahorse. <b>G</b>, OCR measurements in CB CD34⁺ cells transduced with BCR-ABL and treated with BPTES using Seahorse. <b>H,</b> Two primary leukemia CD34⁺ patient samples (#1 in open bars, BC-CML sample; and #2 in striped bars, IDH1 mutant AML) were grown on MS5 stroma in the absence or presence of BPTES and cumulative expansion is shown. #1: 0 μM vs 20 μM p = 0.0085; and #2: 0 μM vs 20 μM p = 0.0017. <b>I</b>, Six CD34⁺-sorted chronic-phase (CP) CML samples were grown on MS5 stroma in the absence or presence of BPTES. p-values: *<0.05, **<0.01, n.s. = not significant.</p