VEGF-C prophylaxis favors lymphatic drainage and modulates neuroinflammation in a stroke model.

Meningeal lymphatic vessels (MLVs) promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelial growth factor-C (VEGF-C) regulates MLV development and maintenance and has therapeutic potential for treating neurological disorders. Herein, we investigated the effects of VEGF-C overexpression on brain fluid drainage and ischemic stroke outcomes in mice. Intracerebrospinal administration of an adeno-associated virus expressing mouse full-length VEGF-C (AAV-mVEGF-C) increased CSF drainage to the deep cervical lymph nodes (dCLNs) by enhancing lymphatic growth and upregulated neuroprotective signaling pathways identified by single nuclei RNA sequencing of brain cells. In a mouse model of ischemic stroke, AAV-mVEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage, associated with mitigated microglia-mediated inflammation and increased BDNF signaling in brain cells. Neuroprotective effects of VEGF-C were lost upon cauterization of the dCLN afferent lymphatics and not mimicked by acute post-stroke VEGF-C injection. We conclude that VEGF-C prophylaxis promotes multiple vascular, immune, and neural responses that culminate in a protection against neurological damage in acute ischemic stroke.

Hippocampal and neocortical BRAF mutant non-expansive lesions in focal epilepsies.

OBJECTIVE: Mesial Temporal Lobe Epilepsy-associated Hippocampal Sclerosis (MTLE-HS) is a syndrome associated with various aetiologies. We previously identified CD34-positive extravascular stellate cells (CD34+ cells) possibly related to BRAFV600E oncogenic variant in a subset of MTLE-HS. We aimed to identify the BRAFV600E oncogenic variants and characterise the CD34+ cells. METHODS: We analysed BRAFV600E oncogenic variant by digital droplet Polymerase Chain Reaction in 53 MTLE-HS samples (25 with CD34+ cells) and nine non-expansive neocortical lesions resected during epilepsy surgery (five with CD34+ cells). Ex vivo multi-electrode array recording, immunolabelling, methylation microarray and single nuclei RNAseq were performed on BRAFwildtype MTLE-HS and BRAFV600E mutant non-expansive lesion of hippocampus and/or neocortex. RESULTS: We identified a BRAFV600E oncogenic variant in five MTLE-HS samples with CD34+ cells (19%) and in five neocortical samples with CD34+ cells (100%). Single nuclei RNAseq of resected samples revealed two unique clusters of abnormal cells (including CD34+ cells) associated with senescence and oligodendrocyte development in both hippocampal and neocortical BRAFV600E mutant samples. The co-expression of the oncogene-induced senescence marker p16INK4A and the outer subventricular zone radial glia progenitor marker HOPX in CD34+ cells was confirmed by multiplex immunostaining. Pseudotime analysis showed that abnormal cells share a common lineage from progenitors to myelinating oligodendrocytes. Epilepsy surgery led to seizure freedom in eight of the 10 patients with BRAF mutant lesions. INTERPRETATION: BRAFV600E underlies a subset of MTLE-HS and epileptogenic non-expansive neocortical focal lesions. Detection of the oncogenic variant may help diagnosis and open perspectives for targeted therapies.

VEGF-C promotes brain-derived fluid drainage, confers neuroprotection, and improves stroke outcomes.

Meningeal lymphatic vessels promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelium growth factor-C (VEGF-C) is essential for meningeal lymphatic development and maintenance and has therapeutic potential for treating neurological disorders, including ischemic stroke. We have investigated the effects of VEGF-C overexpression on brain fluid drainage, single cell transcriptome in the brain, and stroke outcomes in adult mice. Intra-cerebrospinal fluid administration of an adeno-associated virus expressing VEGF-C (AAV-VEGF-C) increases the CNS lymphatic network. Post-contrast T1 mapping of the head and neck showed that deep cervical lymph node size and drainage of CNS-derived fluids were increased. Single nuclei RNA sequencing revealed a neuro-supportive role of VEGF-C via upregulation of calcium and brain-derived neurotrophic factor (BDNF) signaling pathways in brain cells. In a mouse model of ischemic stroke, AAV-VEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage. AAV-VEGF-C thus promotes CNS-derived fluid and solute drainage, confers neuroprotection, and reduces ischemic stroke damage. Short abstract: Intrathecal delivery of VEGF-C increases the lymphatic drainage of brain-derived fluids confers neuroprotection, and improves neurological outcomes after ischemic stroke.

Cellular senescence in malignant cells promotes tumor progression in mouse and patient Glioblastoma.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, yet it remains refractory to systemic therapy. Elimination of senescent cells has emerged as a promising new treatment approach against cancer. Here, we investigated the contribution of senescent cells to GBM progression. Senescent cells are identified in patient and mouse GBMs. Partial removal of p16Ink4a-expressing malignant senescent cells, which make up less than 7 % of the tumor, modifies the tumor ecosystem and improves the survival of GBM-bearing female mice. By combining single cell and bulk RNA sequencing, immunohistochemistry and genetic knockdowns, we identify the NRF2 transcription factor as a determinant of the senescent phenotype. Remarkably, our mouse senescent transcriptional signature and underlying mechanisms of senescence are conserved in patient GBMs, in whom higher senescence scores correlate with shorter survival times. These findings suggest that senolytic drug therapy may be a beneficial adjuvant therapy for patients with GBM.

Portal fibroblasts with mesenchymal stem cell features form a reservoir of proliferative myofibroblasts in liver fibrosis.

BACKGROUND AND AIMS: In liver fibrosis, myofibroblasts derive from HSCs and as yet undefined mesenchymal cells. We aimed to identify portal mesenchymal progenitors of myofibroblasts. APPROACH AND RESULTS: Portal mesenchymal cells were isolated from mouse bilio-vascular tree and analyzed by single-cell RNA-sequencing. Thereby, we uncovered the landscape of portal mesenchymal cells in homeostatic mouse liver. Trajectory analysis enabled inferring a small cell population further defined by surface markers used to isolate it. This population consisted of portal fibroblasts with mesenchymal stem cell features (PMSCs), i.e., high clonogenicity and trilineage differentiation potential, that generated proliferative myofibroblasts, contrasting with nonproliferative HSC-derived myofibroblasts (-MF). Using bulk RNA-sequencing, we built oligogene signatures of the two cell populations that remained discriminant across myofibroblastic differentiation. SLIT2, a prototypical gene of PMSC/PMSC-MF signature, mediated profibrotic and angiogenic effects of these cells, which conditioned medium promoted HSC survival and endothelial cell tubulogenesis. Using PMSC/PMSC-MF 7-gene signature and slit guidance ligand 2 fluorescent in situ hybridization, we showed that PMSCs display a perivascular portal distribution in homeostatic liver and largely expand with fibrosis progression, contributing to the myofibroblast populations that form fibrotic septa, preferentially along neovessels, in murine and human liver disorders, irrespective of etiology. We also unraveled a 6-gene expression signature of HSCs/HSC-MFs that did not vary in these disorders, consistent with their low proliferation rate. CONCLUSIONS: PMSCs form a small reservoir of expansive myofibroblasts, which, in interaction with neovessels and HSC-MFs that mainly arise through differentiation from a preexisting pool, underlie the formation of fibrotic septa in all types of liver diseases.

Molecular Profiling Reclassifies Adult Astroblastoma into Known and Clinically Distinct Tumor Entities with Frequent Mitogen-Activated Protein Kinase Pathway Alterations.

BACKGROUND: Astroblastoma (ABM) is a rare glial brain tumor. Recurrent meningioma 1 (MN1) alterations have been recently identified in most pediatric cases. Adolescent and adult cases, however, remain molecularly poorly defined. MATERIALS AND METHODS: We performed clinical and molecular characterization of a retrospective cohort of 14 adult and 1 adolescent ABM. RESULTS: Strikingly, we found that MN1 fusions are a rare event in this age group (1/15). Using methylation profiling and targeted sequencing, most cases were reclassified as either pleomorphic xanthoastrocytomas (PXA)-like or high-grade glioma (HGG)-like. PXA-like ABM show BRAF mutation (6/7 with V600E mutation and 1/7 with G466E mutation) and CD34 expression. Conversely, HGG-like ABM harbored specific alterations of diffuse midline glioma (2/5) or glioblastoma (GBM; 3/5). These latter patients showed an unfavorable clinical course with significantly shorter overall survival (p = .021). Mitogen-activated protein kinase pathway alterations (including FGFR fusion, BRAF and NF1 mutations) were present in 10 of 15 patients and overrepresented in the HGG-like group (3/5) compared with previously reported prevalence of these alterations in GBM and diffuse midline glioma. CONCLUSION: We suggest that gliomas with astroblastic features include a variety of molecularly sharply defined entities. Adult ABM harboring molecular features of PXA and HGG should be reclassified. Central nervous system high-grade neuroepithelial tumors with MN1 alterations and histology of ABM appear to be uncommon in adults. Astroblastic morphology in adults should thus prompt thorough molecular investigation aiming at a clear histomolecular diagnosis and identifying actionable drug targets, especially in the mitogen-activated protein kinase pathway. IMPLICATIONS FOR PRACTICE: Astroblastoma (ABM) remains a poorly defined and controversial entity. Although meningioma 1 alterations seem to define a large subset of pediatric cases, adult cases remain molecularly poorly defined. This comprehensive molecular characterization of 1 adolescent and 14 adult ABM revealed that adult ABM histology comprises several molecularly defined entities, which explains clinical diversity and identifies actionable targets. Namely, pleomorphic xanthoastrocytoma-like ABM cases show a favorable prognosis whereas high-grade glioma (glioblastoma and diffuse midline gliome)-like ABM show significantly worse clinical courses. These results call for in-depth molecular analysis of adult gliomas with astroblastic features for diagnostic and therapeutic purposes.

Primary CNS lymphoma patient-derived orthotopic xenograft model capture the biological and molecular characteristics of the disease.

Primary CNS lymphomas (PCNSL) are rare and poor prognosis diffuse large B-cell lymphomas. Because of the brain tumor environment and the restricted distribution of drugs in the CNS, specific PCNSL patient-derived orthotopic xenograft (PDOX) models are needed for preclinical research to improve the prognosis of PCNSL patients. PCNSL patient specimens (n = 6) were grafted in the caudate nucleus of immunodeficient nude mice with a 83% rate of success, while subcutaneous implantation in nude mice of human PCNSL sample did not generate lymphoma, supporting the role of the brain microenvironment in the PCNSL physiopathology. PDOXs showed diffuse infiltration of B-cell lymphoma cells in the brain parenchyma. Each model had a unique mutational signature for genes in the BCR and NF-κB pathways and retained the mutational profile of the primary tumor. The models can be stored as cryopreserved biobank. Human IL-10 levels measured in the plasma of PCNSL-PDOX mice showed to be a reliable tool to monitor the tumor burden. Treatment response could be measured after a short treatment with the targeted therapy ibrutinib. In summary, we established a panel of human PCNSL-PDOX models that capture the histological and molecular characteristics of the disease and that proved suitable for preclinical experiments. Our methods of generation and characterization will enable the generation of additional PDOX-PCNSL models, essential tools for cognitive and preclinical drug discovery.

Identification of novel recurrent ETV6-IgH fusions in primary central nervous system lymphoma.

Background: Primary central nervous system lymphoma (PCNSL) represents a particular entity within non-Hodgkin lymphomas and is associated with poor outcome. The present study addresses the potential clinical relevance of chimeric transcripts in PCNSL discovered by using RNA sequencing (RNA-seq). Methods: Seventy-two immunocompetent and newly diagnosed PCNSL cases were included in the present study. Among them, 6 were analyzed by RNA-seq to detect new potential fusion transcripts. We confirmed the results in the remaining 66 PCNSL. The gene fusion was validated by fluorescence in situ hybridization (FISH) using formalin-fixed paraffin-embedded (FFPE) samples. We assessed the biological and clinical impact of one new gene fusion. Results: We identified a novel recurrent gene fusion, E26 transformation-specific translocation variant 6-immunoglobulin heavy chain (ETV6-IgH). Overall, ETV6-IgH was found in 13 out of 72 PCNSL (18%). No fusion conserved an intact functional domain of ETV6, and ETV6 was significantly underexpressed at gene level, suggesting an ETV6 haploinsufficiency mechanism. The presence of the gene fusion was also validated by FISH in FFPE samples. Finally, PCNSL samples harboring ETV6-IgH showed a better prognosis in multivariate analysis, P = 0.03, hazard ratio = 0.33, 95% CI = 0.12-0.88. The overall survival at 5 years was 69% for PCNSL harboring ETV6-IgH versus 29% for samples without this gene fusion. Conclusions: ETV6-IgH is a new potential surrogate marker of PCNSL with favorable prognosis with ETV6 haploinsufficiency as a possible mechanism. The potential clinical impact of ETV6-IgH should be validated in larger prospective studies.

Mutations in DCC cause isolated agenesis of the corpus callosum with incomplete penetrance.

Brain malformations involving the corpus callosum are common in children with developmental disabilities. We identified DCC mutations in four families and five sporadic individuals with isolated agenesis of the corpus callosum (ACC) without intellectual disability. DCC mutations result in variable dominant phenotypes with decreased penetrance, including mirror movements and ACC associated with a favorable developmental prognosis. Possible phenotypic modifiers include the type and location of mutation and the sex of the individual.

Multi-omics analysis of primary glioblastoma cell lines shows recapitulation of pivotal molecular features of parental tumors.

Background: Glioblastoma (GBM) is the deadliest primary brain cancer in adults. Emerging innovative therapies hold promise for personalized cancer treatment. Improving therapeutic options depends on research relying on relevant preclinical models. In this line we have established in the setting of the GlioTex project (GBM and Experimental Therapeutics) a GBM patient-derived cell line (GBM-PDCL) library. A multi-omic approach was used to determine the molecular landscape of PDCL and the extent to which they represent GBM tumors. Methods: Single nucleotide polymorphism array, expression arrays, exome sequencing, and RNA sequencing were used to measure and compare the molecular landscapes of 20 samples representing 10 human GBM tumors and paired GBM-PDCLs. Results: Copy number variations were similar for a median of 85% of the genome and for 59% of the major focal events. Somatic point mutations were similar in a median of 41%. Mutations in GBM driver and "druggable" genes were maintained in 67% of events. Mutations that were not conserved in the PDCL were mainly low allelic fraction and/or non-driver mutations. Based on RNA expression profiling, PDCLs cluster closely to their parental tumor with overexpression of pathways associated with cancer progression in PDCL. Conclusions: Overall, PDCLs recapitulate pivotal molecular alterations of paired-parental tumors supporting their use as a preclinical model of GBM. However, some driver aberrations are lost or gained in the passage from tumor to PDCL. Our results support using PDCL as a relevant preclinical model of GBM. Further investigations of changes between PDCLs and their parental tumor may provide insights into GBM biology.

Identifying and Assessing Interesting Subgroups in a Heterogeneous Population.

Biological heterogeneity is common in many diseases and it is often the reason for therapeutic failures. Thus, there is great interest in classifying a disease into subtypes that have clinical significance in terms of prognosis or therapy response. One of the most popular methods to uncover unrecognized subtypes is cluster analysis. However, classical clustering methods such as k-means clustering or hierarchical clustering are not guaranteed to produce clinically interesting subtypes. This could be because the main statistical variability--the basis of cluster generation--is dominated by genes not associated with the clinical phenotype of interest. Furthermore, a strong prognostic factor might be relevant for a certain subgroup but not for the whole population; thus an analysis of the whole sample may not reveal this prognostic factor. To address these problems we investigate methods to identify and assess clinically interesting subgroups in a heterogeneous population. The identification step uses a clustering algorithm and to assess significance we use a false discovery rate- (FDR-) based measure. Under the heterogeneity condition the standard FDR estimate is shown to overestimate the true FDR value, but this is remedied by an improved FDR estimation procedure. As illustrations, two real data examples from gene expression studies of lung cancer are provided.

Genome-wide microarray expression and genomic alterations by array-CGH analysis in neuroblastoma stem-like cells.

Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-ß) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-ß and Hh signalling pathways, confirming the inhibition of repressors of TGF-ß pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells.

Genetic evidence of a precisely tuned dysregulation in the hypoxia signaling pathway during oncogenesis.

The classic model of tumor suppression implies that malignant transformation requires full "two-hit" inactivation of a tumor-suppressor gene. However, more recent work in mice has led to the proposal of a "continuum" model that involves more fluid concepts such as gene dosage-sensitivity and tissue specificity. Mutations in the tumor-suppressor gene von Hippel-Lindau (VHL) are associated with a complex spectrum of conditions. Homozygotes or compound heterozygotes for the R200W germline mutation in VHL have Chuvash polycythemia, whereas heterozygous carriers are free of disease. Individuals with classic, heterozygous VHL mutations have VHL disease and are at high risk of multiple tumors (e.g., CNS hemangioblastomas, pheochromocytoma, and renal cell carcinoma). We report here an atypical family bearing two VHL gene mutations in cis (R200W and R161Q), together with phenotypic analysis, structural modeling, functional, and transcriptomic studies of these mutants in comparison with classical mutants involved in the different VHL phenotypes. We demonstrate that the complex pattern of disease manifestations observed in VHL syndrome is perfectly correlated with a gradient of VHL protein (pVHL) dysfunction in hypoxia signaling pathways. Thus, by studying naturally occurring familial mutations, our work validates in humans the "continuum" model of tumor suppression.

MET is a potential target across all papillary renal cell carcinomas: result from a large molecular study of pRCC with CGH array and matching gene expression array.

PURPOSE: Papillary renal cell carcinomas (pRCC) are the most common nonclear cell RCC subtype. Germline mutations of the MET oncogene at 7q31 have been detected in patients with hereditary type I pRCC and in 13% of sporadic type I pRCC. Recent report of MET inhibition strengthened the role of c-Met inhibition across pRCC. EXPERIMENTAL DESIGN: We collected 220 frozen samples of sporadic pRCC through the French RCC Network and quality controlled for percentage of malignant cells >70%. Gene expression was assessed on 98 pRCC using human whole-genome Agilent 8 × 60K arrays. Copy number alterations were analyzed using Agilent Human 2 × 400K and 4× 180K array for type II pRCC and comparative genomic microarray analysis method for type I pRCC. MET gene sequencing was performed on type I pRCC. RESULTS: MET expression level was high across all pRCC. We identified copy number alterations (gain) in 46% of type II pRCC and in 81% of type I pRCC. Correlation between DNA copy number alterations and mRNA expression level was highly significant. Eleven somatic mutations of MET gene were identified amongst 51 type I pRCC (21.6%), including 4 new mutations. We validated LRRK2 cokinase as highly correlated to MET expression. CONCLUSION: The present report expands the role of MET activation as a potential target across all pRCC subtypes. These data support investigating MET inhibitors in pRCC in correlation with MET activation status.

Integrated molecular portrait of non-small cell lung cancers.

BACKGROUND: Non-small cell lung cancer (NSCLC), a leading cause of cancer deaths, represents a heterogeneous group of neoplasms, mostly comprising squamous cell carcinoma (SCC), adenocarcinoma (AC) and large-cell carcinoma (LCC). The objectives of this study were to utilize integrated genomic data including copy-number alteration, mRNA, microRNA expression and candidate-gene full sequencing data to characterize the molecular distinctions between AC and SCC. METHODS: Comparative genomic hybridization followed by mutational analysis, gene expression and miRNA microarray profiling were performed on 123 paired tumor and non-tumor tissue samples from patients with NSCLC. RESULTS: At DNA, mRNA and miRNA levels we could identify molecular markers that discriminated significantly between the various histopathological entities of NSCLC. We identified 34 genomic clusters using aCGH data; several genes exhibited a different profile of aberrations between AC and SCC, including PIK3CA, SOX2, THPO, TP63, PDGFB genes. Gene expression profiling analysis identified SPP1, CTHRC1 and GREM1 as potential biomarkers for early diagnosis of the cancer, and SPINK1 and BMP7 to distinguish between AC and SCC in small biopsies or in blood samples. Using integrated genomics approach we found in recurrently altered regions a list of three potential driver genes, MRPS22, NDRG1 and RNF7, which were consistently over-expressed in amplified regions, had wide-spread correlation with an average of ~800 genes throughout the genome and highly associated with histological types. Using a network enrichment analysis, the targets of these potential drivers were seen to be involved in DNA replication, cell cycle, mismatch repair, p53 signalling pathway and other lung cancer related signalling pathways, and many immunological pathways. Furthermore, we also identified one potential driver miRNA hsa-miR-944. CONCLUSIONS: Integrated molecular characterization of AC and SCC helped identify clinically relevant markers and potential drivers, which are recurrent and stable changes at DNA level that have functional implications at RNA level and have strong association with histological subtypes.

Retinoic acid receptor alpha amplifications and retinoic acid sensitivity in breast cancers.

BACKGROUND: Molecular segmentation of breast cancer allows identification of small groups of patients who present high sensitivity to targeted agents. A patient, with chemo- and trastuzumab-resistant HER2-overexpressing breast cancer, who presented concomitant acute promyelocytic leukemia, showed a response in her breast lesions to retinoic acid, arsenic, and aracytin. We therefore investigated whether RARA gene amplification could be associated with sensitivity to retinoic acid derivatives in breast cancers. MATERIALS AND METHODS: Array comparative genomic hybridization and gene expression arrays were used to characterize RARA amplifications and expression in 103 breast cancer samples. In vitro activity of ATRA was characterized in T47D, SKBR3, and BT474 cell lines. RESULTS: Retinoic acid receptor alpha was gained or amplified in 27% of HER2-positive and 13% of HER2-negative breast cancer samples. Retinoic acid receptor alpha can be coamplified with HER2. Retinoic acid receptor alpha copy number changes could be correlated with messenger RNA expression. All-trans-retinoic acid reduced cell viability of RARA-amplified, but not RARA-normal, cell lines through apoptosis. Gene expression arrays showed that ATRA-induced apoptosis in RARA-amplified cell lines was related to an increase in CASP1 and IRF1. CONCLUSION: The results of this study suggest that breast cancers exhibiting RARA amplifications could be sensitive to retinoic acid. A phase II trial will evaluate this hypothesis in the clinical setting.

Cisplatin resistance associated with PARP hyperactivation.

Non-small cell lung carcinoma patients are frequently treated with cisplatin (CDDP), most often yielding temporary clinical responses. Here, we show that PARP1 is highly expressed and constitutively hyperactivated in a majority of human CDDP-resistant cancer cells of distinct histologic origin. Cells manifesting elevated intracellular levels of poly(ADP-ribosyl)ated proteins (PAR(high)) responded to pharmacologic PARP inhibitors as well as to PARP1-targeting siRNAs by initiating a DNA damage response that translated into cell death following the activation of the intrinsic pathway of apoptosis. Moreover, PARP1-overexpressing tumor cells and xenografts displayed elevated levels of PAR, which predicted the response to PARP inhibitors in vitro and in vivo more accurately than PARP1 expression itself. Thus, a majority of CDDP-resistant cancer cells appear to develop a dependency to PARP1, becoming susceptible to PARP inhibitor-induced apoptosis.

Differences in transcription patterns between induced pluripotent stem cells produced from the same germ layer are erased upon differentiation.

Little is known about differences between induced pluripotent stem cells produced from tissues originating from the same germ layer. We have generated human myoblast-derived iPS cells by retroviral transduction of human primary myoblasts with the OCT3/4, SOX2, KLF4 and MYC coding sequences and compared them to iPS produced from human primary fibroblasts. When cultivated in vitro, these iPS cells proved similar to human embryonic stem cells in terms of morphology, expression of embryonic stemness markers and gene promoter methylation patterns. Embryonic bodies were derived that expressed endodermal, mesodermal as well as ectodermal markers. A comparative analysis of transcription patterns revealed significant differences in the gene expression pattern between myoblast- and fibroblast-derived iPS cells. However, these differences were reduced in the mesenchymal stem cells derived from the two iPS cell types were compared.

Characterization of novel genomic alterations and therapeutic approaches using acute megakaryoblastic leukemia xenograft models.

Acute megakaryoblastic leukemia (AMKL) is a heterogeneous disease generally associated with poor prognosis. Gene expression profiles indicate the existence of distinct molecular subgroups, and several genetic alterations have been characterized in the past years, including the t(1;22)(p13;q13) and the trisomy 21 associated with GATA1 mutations. However, the majority of patients do not present with known mutations, and the limited access to primary patient leukemic cells impedes the efficient development of novel therapeutic strategies. In this study, using a xenotransplantation approach, we have modeled human pediatric AMKL in immunodeficient mice. Analysis of high-throughput RNA sequencing identified recurrent fusion genes defining new molecular subgroups. One subgroup of patients presented with MLL or NUP98 fusion genes leading to up-regulation of the HOX A cluster genes. A novel CBFA2T3-GLIS2 fusion gene resulting from a cryptic inversion of chromosome 16 was identified in another subgroup of 31% of non-Down syndrome AMKL and strongly associated with a gene expression signature of Hedgehog pathway activation. These molecular data provide useful markers for the diagnosis and follow up of patients. Finally, we show that AMKL xenograft models constitute a relevant in vivo preclinical screening platform to validate the efficacy of novel therapies such as Aurora A kinase inhibitors.

A pair of co-opted retroviral envelope syncytin genes is required for formation of the two-layered murine placental syncytiotrophoblast.

In most mammalian species, a critical step of placenta development is the fusion of trophoblast cells into a multinucleated syncytiotrophoblast layer fulfilling essential fetomaternal exchange functions. Key insights into this process came from the discovery of envelope genes of retroviral origin, the syncytins, independently acquired by the human (syncytin-1 and -2), mouse (syncytin-A and -B), and rabbit (syncytin-Ory1) genomes, with fusogenic properties and placenta-specific expression. We previously showed that mouse syncytin-A is essential for the formation of one of the two syncytiotrophoblast layers and for embryo survival. Here, we have generated syncytin-B KO mice and demonstrate that syncytin-B null placenta displays impaired formation of syncytiotrophoblast layer II (ST-II), with evidence of unfused apposed cells, and enlargement of maternal lacunae disrupting the placenta architecture. Unexpectedly, syncytin-B null embryos are viable, with only limited late-onset growth retardation and reduced neonate number. Microarray analyses identified up-regulation of the connexin 30 gene in mutant placentae, with the protein localized at the fetomaternal interface, suggesting gap junction-mediated compensatory mechanisms. Finally, double-KO mice demonstrate premature death of syncytin-A null embryos if syncytin-B is deleted, indicating cooperation between ST-I and ST-II. These findings establish that both endogenous retrovirus-derived syncytin genes contribute independently to the formation of the two syncytiotrophoblast layers during placenta formation, demonstrating a major role of retroviral gene capture, through convergent evolution, to generate multiple placental structures. Although some are absolutely required for completion of pregnancy, others are still amenable to "epigenetic" compensations, thus illustrating the complexity of the molecular machinery that developed during placental evolution.