Comparing genome-scale metabolic models of the non-resistant Enterococcus faecalis ATCC 19433 and the multi-resistant Enterococcus faecalis V583.

Enterococcus faecalis is a versatile lactic acid bacterium with a large variety of implications for humans. While some strains of this species are pathobionts being resistant against most of the common antibiotics, other strains are regarded as biological protectants or even probiotics. Accordingly, E. faecalis strains largely differ in the size and content of their accessory genome. In this study, we describe the genome-scale metabolic network reconstruction of E. faecalis ATCC 19433, a non-resistant human-associated strain. A comparison of the genome-scale metabolic model (GSM) of E. faecalis ATCC 19433 with a previously published GSM of the multi-resistant pathobiontic E. faecalis V583 reveals high similarities in the central metabolic abilities of these two human associated strains. This is reflected, e.g., in the identical amino acid auxotrophies. The ATCC 19433 strain, however, has a 14.1 % smaller genome than V583 and lacks the multiple antibiotic resistance genes and genes involved in capsule formation. Based on the measured metabolic fluxes at different growth rates, the energy demand at zero growth was calculated to be about 40 % lower for the ATCC 19433 strain compared to V583. Furthermore, the ATCC 19433 strain seems less prone to the depletion of amino acids utilizable for energy metabolism. This might hint at a lower overall energy demand of the ATCC 19433 strain as compared to V583.

Clonal heterogeneity in ER+ breast cancer reveals the proteasome and PKC as potential therapeutic targets.

Intratumoral heterogeneity impacts the success or failure of anti-cancer therapies. Here, we investigated the evolution and mechanistic heterogeneity in clonal populations of cell models for estrogen receptor positive breast cancer. To this end, we established barcoded models of luminal breast cancer and rendered them resistant to commonly applied first line endocrine therapies. By isolating single clones from the resistant cell pools and characterizing replicates of individual clones we observed inter- (between cell lines) and intra-tumor (between different clones from the same cell line) heterogeneity. Molecular characterization at RNA and phospho-proteomic levels revealed private clonal activation of the unfolded protein response and respective sensitivity to inhibition of the proteasome, and potentially shared sensitivities for repression of protein kinase C. Our in vitro findings are consistent with tumor-heterogeneity that is observed in breast cancer patients thus highlighting the need to uncover heterogeneity at an individual patient level and to adjust therapies accordingly.

Erythropoietin-driven dynamic proteome adaptations during erythropoiesis prevent iron overload in the developing embryo.

Erythropoietin (Epo) ensures survival and proliferation of colony-forming unit erythroid (CFU-E) progenitor cells and their differentiation to hemoglobin-containing mature erythrocytes. A lack of Epo-induced responses causes embryonic lethality, but mechanisms regulating the dynamic communication of cellular alterations to the organismal level remain unresolved. By time-resolved transcriptomics and proteomics, we show that Epo induces in CFU-E cells a gradual transition from proliferation signature proteins to proteins indicative for differentiation, including heme-synthesis enzymes. In the absence of the Epo receptor (EpoR) in embryos, we observe a lack of hemoglobin in CFU-E cells and massive iron overload of the fetal liver pointing to a miscommunication between liver and placenta. A reduction of iron-sulfur cluster-containing proteins involved in oxidative phosphorylation in these embryos leads to a metabolic shift toward glycolysis. This link connecting erythropoiesis with the regulation of iron homeostasis and metabolic reprogramming suggests that balancing these interactions is crucial for protection from iron intoxication and for survival.

Cell-to-cell variability in JAK2/STAT5 pathway components and cytoplasmic volumes defines survival threshold in erythroid progenitor cells.

Survival or apoptosis is a binary decision in individual cells. However, at the cell-population level, a graded increase in survival of colony-forming unit-erythroid (CFU-E) cells is observed upon stimulation with erythropoietin (Epo). To identify components of Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5) signal transduction that contribute to the graded population response, we extended a cell-population-level model calibrated with experimental data to study the behavior in single cells. The single-cell model shows that the high cell-to-cell variability in nuclear phosphorylated STAT5 is caused by variability in the amount of Epo receptor (EpoR):JAK2 complexes and of SHP1, as well as the extent of nuclear import because of the large variance in the cytoplasmic volume of CFU-E cells. 24-118 pSTAT5 molecules in the nucleus for 120 min are sufficient to ensure cell survival. Thus, variability in membrane-associated processes is sufficient to convert a switch-like behavior at the single-cell level to a graded population-level response.

The ParaHox gene Cdx4 induces acute erythroid leukemia in mice.

Acute erythroid leukemia (AEL) is a rare and aggressive form of acute leukemia, the biology of which remains poorly understood. Here we demonstrate that the ParaHox gene CDX4 is expressed in patients with acute erythroid leukemia, and that aberrant expression of Cdx4 induced homogenously a transplantable acute erythroid leukemia in mice. Gene expression analyses demonstrated upregulation of genes involved in stemness and leukemogenesis, with parallel downregulation of target genes of Gata1 and Gata2 responsible for erythroid differentiation. Cdx4 induced a proteomic profile that overlapped with a cluster of proteins previously defined to represent the most primitive human erythroid progenitors. Whole-exome sequencing of diseased mice identified recurrent mutations significantly enriched for transcription factors involved in erythroid lineage specification, as well as TP53 target genes partly identical to the ones reported in patients with AEL. In summary, our data indicate that Cdx4 is able to induce stemness and inhibit terminal erythroid differentiation, leading to the development of AEL in association with co-occurring mutations.