Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specifi c associations between age and phenotype. We observed that fusion oncogenes, such as ETO2-GLIS2 , are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that ETO2-GLIS2 expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed in vivo leukemogenic potential. Chromatin accessibility and singlecell transcriptome analyses indicate ontogeny-dependent intrinsic and ETO2-GLIS2 -induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. SIGNIFICANCE:This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state.

Acute erythroleukemia (AML-M6 or AEL) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing three genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (e.g. DNMT3A, TET2 or IDH2), and undefined cases with low mutational burden. We established an erythroid vs. myeloid transcriptomics-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, more than 25% of AEL patients, including in the genetically-undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1 binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicates that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.

Dystrophin is essential to skeletal muscle function and confers resistance to the sarcolemma by interacting with cytoskeleton and membrane. In the present work, we characterized the behavior of dystrophin 11-15 (DYS R11-15), five spectrin-like repeats from the central domain of human dystrophin, with lipids. DYS R11-15 displays an amphiphilic character at the liquid/ air interface while maintaining its secondary ␣-helical structure. The interaction of DYS R11-15 with small unilamellar vesicles (SUVs) depends on the lipid nature, which is not the case with large unilamellar vesicles (LUVs). In addition, switching from anionic SUVs to anionic LUVs suggests the lipid packing as a crucial factor for the interaction of protein and lipid. The monolayer model and the modulation of surface pressure aim to mimic the muscle at work (i.e. dynamic changes of muscle membrane during contraction and relaxation) (high and low surface pressure). Strikingly, the lateral pressure modifies the protein organization. Increasing the lateral pressure leads the proteins to be organized in a regular network. Nevertheless, a different protein conformation after its binding to monolayer is revealed by trypsin proteolysis. Label-free quantification by nano-LC/ MS/MS allowed identification of the helices in repeats 12 and 13 involved in the interaction with anionic SUVs. These results, combined with our previous studies, indicate that DYS R11-15 constitutes the only part of dystrophin that interacts with anionic as well as zwitterionic lipids and adapts its interaction and organization depending on lipid packing and lipid nature. We provide strong experimental evidence for a physiological role of the central domain of dystrophin in sarcolemma scaffolding through modulation of lipid-protein interactions.Dystrophin is a rod-shaped cytoplasmic protein that constitutes a vital part of a protein complex that connects the cytoskeleton of muscle fibers to the surrounding extracellular matrix through the cell membrane. This long, filamentous protein (Fig. 1A) is essential to skeletal muscle function, which is demonstrated by the lethal pathophysiology associated with its deficiency, namely Duchenne muscular dystrophy (1). Several membrane and cytoskeletal binding partners of dystrophin have been identified, including ␤-dystroglycan from the dystrophin-glycoprotein complex (2, 3). ␤-Dystroglycan interacts with the cysteine-rich region of dystrophin that is located between the stabilizing central domain, which consists of 24 spectrin-like repeats and is known as the rod domain, and the C-terminal end of the molecule. Cytoskeletal actin interacts with the dystrophin molecule through two actin-binding domains, ABD1 and ABD2, which are situated at the N-terminal end and at the center of the dystrophin rod domain (repeats 11-15), respectively (4). The subsarcolemmal location of dystrophin and its association with both the cytoskeleton and membrane suggest a role in the mechanical regulation of membrane stress during contraction and elongation of muscle fi...

The susceptibility to development of hepatic steatosis is known to differ between Muscovy and Pekin ducks. Although some experiments were conducted to decipher these differences, few data have been produced to analyse the role of specific genes in this process. For this purpose, expression levels of genes involved in lipid (ATP citrate lyase, malic enzyme 1, fatty acid synthase, stearoyl-CoA desaturase 1, diacylglycerol O-acyl transferase 2, microsomal triglyceride transfer protein, apolipoprotein A1, apolipoprotein B, sterol regulatory element binding factor 1, hepatocyte nuclear factor 4, choline/ethanolamine phosphotransferase 1, carnitine palmitoyl transferase 1A, peroxisome proliferator-activated receptor alpha and sterol O-acyltransferase) and carbohydrate (activating transcription factor 4 or cAMP-response element binding protein, mitochondrial malate dehydrogenase 2 and carbohydrate responsive element binding protein) metabolism and in other functions were analysed in the liver of Pekin and Muscovy ducks fed ad libitum or overfed. A specific positive effect of feeding was observed on the expression of genes involved mainly in fatty acids and TG synthesis and glycolysis, and negative effect on genes involved in beta-oxidation. Interestingly, a strong species effect was also observed on stearoyl-CoA desaturase 1 and to a lesser extent on diacylglycerol O-acyl transferase 2 expression, leading to large differences in expression levels between Pekin and Muscovy overfed ducks, which could explain the difference in lipid metabolism and steatosis ability observed between the two duck species. These results should shed light on gene expression that might underlie susceptibility to hepatic steatosis in humans.

Acute megakaryoblastic leukemia of Down syndrome (DS-AMKL) is a model of clonal evolution from a preleukemic transient myeloproliferative disorder requiring both a trisomy 21 (T21) and a GATA1 s mutation to a leukemia driven by additional driver mutations. We modeled the megakaryocyte differentiation defect through stepwise gene editing of GATA1s , SMC3 +/– , and MPL W515K , providing 20 different T21 or disomy 21 (D21) induced pluripotent stem cell (iPSC) clones. GATA1s profoundly reshaped iPSC-derived hematopoietic architecture with gradual myeloid-to-megakaryocyte shift and megakaryocyte differentiation alteration upon addition of SMC3 and MPL mutations. Transcriptional, chromatin accessibility, and GATA1-binding data showed alteration of essential megakaryocyte differentiation genes, including NFE2 downregulation that was associated with loss of GATA1s binding and functionally involved in megakaryocyte differentiation blockage. T21 enhanced the proliferative phenotype, reproducing the cellular and molecular abnormalities of DS-AMKL. Our study provides an array of human cell–based models revealing individual contributions of different mutations to DS-AMKL differentiation blockage, a major determinant of leukemic progression.

A-PCR screening for the endogenous or ETO2-GLIS2-targeted AAVS1 locus. The expected sizes for the PCR products obtained from amplification of the wild-type allele and the ETO2-GLIS2 recombined allele (represented Figure 1A) are indicated. B-Karyotype for control and #10 iPSC clones. C-Quantitative RT-PCR on RNA from embryoid body (EB) formed after 24 days of differentiation. Expression is represented as a ratio between day 24 EBs and undifferentiated iPSC cells. Genes characteristic of the undifferentiated state (NANOG, OCT4) and of the three embryonic layers, mesoderm (GATA4), endoderm (HAND1) and ectoderm (NeuroD1) were used. D-Histological section of teratoma formed after injection of iPSC into NSG mice. E-Confocal fluorescence microscopy images obtained at day 15 of differentiation (left panel: control, right panel: ETO2-GLIS2). F-Quantitative RT-PCR at day 15 of differentiation for GATA3 expression. Histograms represent means+/-SD (n=3 obtained from one differentiation experiment). Statistical significance is indicated as p values (Student's t-test): **p-value<0.01 Supplemental Figure 2 A-Flow cytometry analysis of CD43 and CD34 hematopoietic markers in control and clones expressing ETO2-GLIS2 fusion gene (clone 10 & 19) at day 13, 15 and 18 of differentiation. B-Mean fluorescent intensity of CD41 and CD61 surface markers in the indicated populations. Mean+/-SD (n=3 independent differentiation experiments) are represented. Statistical significance is indicated as p-value (Student's t-test): ***p<0.001 **p<0.01. C-Intensity of the CD42 marker gating on the CD41 + 42 + populations from control, clone 10 & 19 at day 18 of differentiation (up-left panel) or on the CD41 low 42 low populations from clone 10 & 19 at day 18 of differentiation (up-right panel). Mean fluorescent intensity in the indicated populations. Mean+/-SD (n=3 independent differentiation experiments) are represented. Statistical significance is indicated as p-value (Student's t-test): ***p<0.001 **p<0.01. D-Flow cytometry analysis of CD43 and GPA markers for control and ETO2-GLIS2 cells at different stages of methylcellulose culture serial replating. P1: first plates; P2: 2 ary plates; P3: 3 ary plates.

Few genomic tools are available in ducks. To produce some new resources, we have designed Pekin (Anas platyrhynchos) and Muscovy (Cairina moschata) duck-specific primers for 22 genes involved mainly in lipid metabolism, and to a lesser extent in carbohydrate metabolism and other functions. Primers were designed according to duck sequences when available and otherwise from the corresponding conserved regions in chicken and human sequences. These primers allowed quantitative RT-PCR amplification of RNA from Pekin and Muscovy ducks. Amplified cDNA products from both species were sequenced and were found to be very similar to chicken sequences (about 94%). This work provides additional genomic resources and polymorphism information for some genes in duck species and represents a first step towards gene expression analyses in Pekin and Muscovy ducks.

International audienceWe have studied single curved films stabilized by globular proteins, using small angle scattering. By combining both the use of in-house X-ray and synchrotron radiation, we have measured the structural properties of films (thickness, electronic density) by controlling the physicochemical properties of protein (ovalbumin, pH 7, bulk concentration 10 g L−1). For each experiment, solutions of highly purified protein were freshly prepared to eliminate any problem of aging. The observation of Kiessig fringes shows that the films are thin with an average thickness of 60 nm. Benefiting from the fine angular resolution and the short acquisition time of a synchrotron source, we have highlighted a stratification formation inside the films. This phenomenon suggests protein structural reorganization under confinement, possibly driven by high osmotic pressure