Engineered LINE-1 retrotransposition in nondividing human neurons.

Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80-100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought.

Enhanced hemato-endothelial specification during human embryonic differentiation through developmental cooperation between AF4-MLL and MLL-AF4 fusions.

The t(4;11)(q21;q23) translocation is associated with high-risk infant pro-B-cell acute lymphoblastic leukemia and arises prenatally during embryonic/fetal hematopoiesis. The developmental/pathogenic contribution of the t(4;11)-resulting MLL-AF4 (MA4) and AF4-MLL (A4M) fusions remains unclear; MA4 is always expressed in patients with t(4;11)+ B-cell acute lymphoblastic leukemia, but the reciprocal fusion A4M is expressed in only half of the patients. Because prenatal leukemogenesis manifests as impaired early hematopoietic differentiation, we took advantage of well-established human embryonic stem cell-based hematopoietic differentiation models to study whether the A4M fusion cooperates with MA4 during early human hematopoietic development. Co-expression of A4M and MA4 strongly promoted the emergence of hemato-endothelial precursors, both endothelial- and hemogenic-primed. Double fusion-expressing hemato-endothelial precursors specified into significantly higher numbers of both hematopoietic and endothelial-committed cells, irrespective of the differentiation protocol used and without hijacking survival/proliferation. Functional analysis of differentially expressed genes and differentially enriched H3K79me3 genomic regions by RNA-sequencing and H3K79me3 chromatin immunoprecipitation-sequencing, respectively, confirmed a hematopoietic/endothelial cell differentiation signature in double fusion-expressing hemato-endothelial precursors. Importantly, chromatin immunoprecipitation-sequencing analysis revealed a significant enrichment of H3K79 methylated regions specifically associated with HOX-A cluster genes in double fusion-expressing differentiating hematopoietic cells. Overall, these results establish a functional and molecular cooperation between MA4 and A4M fusions during human hematopoietic development.

RUNX1c Regulates Hematopoietic Differentiation of Human Pluripotent Stem Cells Possibly in Cooperation with Proinflammatory Signaling.

Runt-related transcription factor 1 (Runx1) is a master hematopoietic transcription factor essential for hematopoietic stem cell (HSC) emergence. Runx1-deficient mice die during early embryogenesis due to the inability to establish definitive hematopoiesis. Here, we have used human pluripotent stem cells (hPSCs) as model to study the role of RUNX1 in human embryonic hematopoiesis. Although the three RUNX1 isoforms a, b, and c were induced in CD45+ hematopoietic cells, RUNX1c was the only isoform induced in hematoendothelial progenitors (HEPs)/hemogenic endothelium. Constitutive expression of RUNX1c in human embryonic stem cells enhanced the appearance of HEPs, including hemogenic (CD43+) HEPs and promoted subsequent differentiation into blood cells. Conversely, specific deletion of RUNX1c dramatically reduced the generation of hematopoietic cells from HEPs, indicating that RUNX1c is a master regulator of human hematopoietic development. Gene expression profiling of HEPs revealed a RUNX1c-induced proinflammatory molecular signature, supporting previous studies demonstrating proinflammatory signaling as a regulator of HSC emergence. Collectively, RUNX1c orchestrates hematopoietic specification of hPSCs, possibly in cooperation with proinflammatory signaling. Stem Cells 2017;35:2253-2266.

HOXA9 promotes hematopoietic commitment of human embryonic stem cells.

The molecular determinants regulating the specification of human embryonic stem cells (hESCs) into hematopoietic cells remain elusive. HOXA9 plays a relevant role in leukemogenesis and hematopoiesis. It is highly expressed in hematopoietic stem and progenitor cells (HSPCs) and is downregulated upon differentiation. Hoxa9-deficient mice display impaired hematopoietic development, and deregulation of HOXA9 expression is frequently associated with acute leukemia. Analysis of the genes differentially expressed in cord blood HSPCs vs hESC-derived HSPCs identified HOXA9 as the most downregulated gene in hESC-derived HSPCs, suggesting that expression levels of HOXA9 may be crucial for hematopoietic differentiation of hESC. Here we show that during hematopoietic differentiation of hESCs, HOXA9 expression parallels hematopoietic development, but is restricted to the hemogenic precursors (HEP) (CD31(+)CD34(+)CD45(-)), and diminishes as HEPs differentiate into blood cells (CD45(+)). Different gain-of-function and loss-of-function studies reveal that HOXA9 enhances hematopoietic differentiation of hESCs by specifically promoting the commitment of HEPs into primitive and total CD45(+) blood cells. Gene expression analysis suggests that nuclear factor-κB signaling could be collaborating with HOXA9 to increase hematopoietic commitment. However, HOXA9 on its own is not sufficient to confer in vivo long-term engraftment potential to hESC-hematopoietic derivatives, reinforcing the idea that additional molecular regulators are needed for the generation of definitive in vivo functional HSPCs from hESC.

SCL/TAL1-mediated transcriptional network enhances megakaryocytic specification of human embryonic stem cells.

Human embryonic stem cells (hESCs) are a unique in vitro model for studying human developmental biology and represent a potential source for cell replacement strategies. Platelets can be generated from cord blood progenitors and hESCs; however, the molecular mechanisms and determinants controlling the in vitro megakaryocytic specification of hESCs remain elusive. We have recently shown that stem cell leukemia (SCL) overexpression accelerates the emergence of hemato-endothelial progenitors from hESCs and promotes their subsequent differentiation into blood cells with higher clonogenic potential. Given that SCL participates in megakaryocytic commitment, we hypothesized that it may potentiate megakaryopoiesis from hESCs. We show that ectopic SCL expression enhances the emergence of megakaryocytic precursors, mature megakaryocytes (MKs), and platelets in vitro. SCL-overexpressing MKs and platelets respond to different activating stimuli similar to their control counterparts. Gene expression profiling of megakaryocytic precursors shows that SCL overexpression renders a megakaryopoietic molecular signature. Connectivity Map analysis reveals that trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), both histone deacetylase (HDAC) inhibitors, functionally mimic SCL-induced effects. Finally, we confirm that both TSA and SAHA treatment promote the emergence of CD34(+) progenitors, whereas valproic acid, another HDAC inhibitor, potentiates MK and platelet production. We demonstrate that SCL and HDAC inhibitors are megakaryopoiesis regulators in hESCs.

FLT3 activation cooperates with MLL-AF4 fusion protein to abrogate the hematopoietic specification of human ESCs.

Mixed-lineage leukemia (MLL)-AF4 fusion arises prenatally in high-risk infant acute pro-B-lymphoblastic leukemia (pro-B-ALL). In human embryonic stem cells (hESCs), MLL-AF4 skewed hematoendothelial specification but was insufficient for transformation, suggesting that additional oncogenic insults seem required for MLL-AF4-mediated transformation. MLL-AF4+ pro-B-ALL expresses enormous levels of FLT3, occasionally because of activating mutations, thus representing a candidate cooperating event in MLL-AF4+ pro-B-ALL. Here, we explored the developmental impact of FLT3 activation alone, or together with MLL-AF4, in the hematopoietic fate of hESCs. FLT3 activation does not affect specification of hemogenic precursors but significantly enhances the formation of CD45(+) blood cells, and CD45(+)CD34(+) blood progenitors with clonogenic potential. However, overexpression of FLT3 mutations or wild-type FLT3 (FLT3-WT) completely abrogates hematopoietic differentiation from MLL-AF4-expressing hESCs, indicating that FLT3 activation cooperates with MLL-AF4 to inhibit human embryonic hematopoiesis. Cell cycle/apoptosis analyses suggest that FLT3 activation directly affects hESC specification rather than proliferation or survival of hESC-emerging hematopoietic derivatives. Transcriptional profiling of hESC-derived CD45(+) cells supports the FLT3-mediated inhibition of hematopoiesis in MLL-AF4-expressing hESCs, which is associated with large transcriptional changes and downregulation of genes involved in hematopoietic system development and function. Importantly, FLT3 activation does not cooperate with MLL-AF4 to immortalize/transform hESC-derived hematopoietic cells, suggesting the need of alternative (epi)-genetic cooperating hits.

Enforced expression of MLL-AF4 fusion in cord blood CD34+ cells enhances the hematopoietic repopulating cell function and clonogenic potential but is not sufficient to initiate leukemia.

Infant acute lymphoblastic leukemia harboring the fusion mixed-lineage leukemia (MLL)-AF4 is associated with a dismal prognosis and very brief latency. Our limited understanding of transformation by MLL-AF4 is reflected in murine models, which do not accurately recapitulate the human disease. Human models for MLL-AF4 disease do not exist. Hematopoietic stem or progenitor cells (HSPCs) represent probable targets for transformation. Here, we explored in vitro and in vivo the impact of the enforced expression of MLL-AF4 in human cord blood-derived CD34(+) HSPCs. Intrabone marrow transplantation into NOD/SCID-IL2Rγ(-/-) mice revealed an enhanced multilineage hematopoietic engraftment, efficiency, and homing to other hematopoietic sites on enforced expression of MLL-AF4. Lentiviral transduction of MLL-AF4 into CD34(+) HSPCs increased the in vitro clonogenic potential of CD34(+) progenitors and promoted their proliferation. Consequently, cell cycle and apoptosis analyses suggest that MLL-AF4 conveys a selective proliferation coupled to a survival advantage, which correlates with changes in the expression of genes involved in apoptosis, sensing DNA damage and DNA repair. However, MLL-AF4 expression was insufficient to initiate leukemogenesis on its own, indicating that either additional hits (or reciprocal AF4-MLL product) may be required to initiate ALL or that cord blood-derived CD34(+) HSPCs are not the appropriate cellular target for MLL-AF4-mediated ALL.

SCL/TAL1 regulates hematopoietic specification from human embryonic stem cells.

Determining the molecular regulators/pathways responsible for the specification of human embryonic stem cells (hESCs) into hematopoietic precursors has far-reaching implications for potential cell therapies and disease modeling. Mouse models lacking SCL/TAL1 (stem cell leukemia/T-cell acute lymphocytic leukemia 1) do not survive beyond early embryogenesis because of complete absence of hematopoiesis, indicating that SCL is a master early hematopoietic regulator. SCL is commonly found rearranged in human leukemias. However, there is barely information on the role of SCL on human embryonic hematopoietic development. Differentiation and sorting assays show that endogenous SCL expression parallels hematopoietic specification of hESCs and that SCL is specifically expressed in hematoendothelial progenitors (CD45(-)CD31(+)CD34(+)) and, to a lesser extent, on CD45(+) hematopoietic cells. Enforced expression of SCL in hESCs accelerates the emergence of hematoendothelial progenitors and robustly promotes subsequent differentiation into primitive (CD34(+)CD45(+)) and total (CD45(+)) blood cells with higher clonogenic potential. Short-hairpin RNA-based silencing of endogenous SCL abrogates hematopoietic specification of hESCs, confirming the early hematopoiesis-promoting effect of SCL. Unfortunately, SCL expression on its own is not sufficient to confer in vivo engraftment to hESC-derived hematopoietic cells, suggesting that additional yet undefined master regulators are required to orchestrate the stepwise hematopoietic developmental process leading to the generation of definitive in vivo functional hematopoiesis from hESCs.

Abrogation of stemness in osteosarcoma by the mithramycin analog EC-8042 is mediated by its ability to inhibit NOTCH-1 signaling.

Osteosarcomas are frequently associated to a poor prognosis and a modest response to current treatments. EC-8042 is a well-tolerated mithramycin analog that has demonstrated an efficient ability to eliminate tumor cells, including cancer stem cell subpopulations (CSC), in sarcomas. In transcriptomic and protein expression analyses, we identified NOTCH1 signaling as one of the main pro-stemness pathways repressed by EC-8042 in osteosarcomas. Overexpression of NOTCH-1 resulted in a reduced anti-tumor effect of EC-8042 in CSC-enriched 3D tumorspheres cultures. On the other hand, the depletion of the NOTCH-1 downstream target HES-1 was able to enhance the action of EC-8042 on CSCs. Moreover, HES1 depleted cells failed to recover after treatment withdrawal and showed reduced tumor growth potential in vivo. In contrast, mice xenografted with NOTCH1-overexpressing cells responded worse than parental cells to EC-8042. Finally, we found that active NOTCH1 levels in sarcoma patients was associated to advanced disease and lower survival. Overall, these data highlight the relevant role that NOTCH1 signaling plays in mediating stemness in osteosarcoma. Moreover, we demonstrate that EC-8042 is powerful inhibitor of NOTCH signaling and that the anti-CSC activity of this mithramycin analog highly rely on its ability to repress this pathway.

Expression profile of telomere-associated genes in multiple myeloma.

To further contribute to the understanding of multiple myeloma, we have focused our research interests on the mechanisms by which tumour plasma cells have a higher survival rate than normal plasma cells. In this article, we study the expression profile of genes involved in the regulation and protection of telomere length, telomerase activity and apoptosis in samples from patients with monoclonal gammopathy of undetermined significance, smouldering multiple myeloma, multiple myeloma (MM) and plasma cell leukaemia (PCL), as well as several human myeloma cell lines (HMCLs). Using conventional cytogenetic and fluorescence in situ hybridization studies, we identified a high number of telomeric associations (TAs). Moreover, telomere length measurements by terminal restriction fragment (TRF) assay showed a shorter mean TRF peak value, with a consistent correlation with the number of TAs. Using gene expression arrays and quantitative PCR we identified the hTERT gene together with 16 other genes directly involved in telomere length maintenance: HSPA9, KRAS, RB1, members of the Small nucleolar ribonucleoproteins family, A/B subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins, and 14-3-3 family. The expression levels of these genes were even higher than those in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), which have unlimited proliferation capacity. In conclusion, the gene signature suggests that MM tumour cells are able to maintain stable short telomere lengths without exceeding the short critical length, allowing cell divisions to continue. We propose that this could be a mechanism contributing to MM tumour cells expansion in the bone marrow (BM).

Ribosomal 18 S RNA processing by the IGF-I-responsive WDR3 protein is integrated with p53 function in cancer cell proliferation.

Insulin-like growth factor-I (IGF-I) signaling is strongly associated with cell growth and regulates the rate of synthesis of the rRNA precursor, the first and the key stage of ribosome biogenesis. In a screen for mediators of IGF-I signaling in cancer, we recently identified several ribosome-related proteins, including NEP1 (nucleolar essential protein 1) and WDR3 (WD repeat 3), whose homologues in yeast function in ribosome processing. The WDR3 gene and its locus on chromosome 1p12-13 have previously been linked with malignancy. Here we show that IGF-I induces expression of WDR3 in transformed cells. WDR3 depletion causes defects in ribosome biogenesis by affecting 18 S rRNA processing and also causes a transient down-regulation of precursor rRNA levels with moderate repression of RNA polymerase I activity. Suppression of WDR3 in cells expressing functional p53 reduced proliferation and arrested cells in the G(1) phase of the cell cycle. This was associated with activation of p53 and sequestration of MDM2 by ribosomal protein L11. Cells lacking functional p53 did not undergo cell cycle arrest upon suppression of WDR3. Overall, the data indicate that WDR3 has an essential function in 40 S ribosomal subunit synthesis and in ribosomal stress signaling to p53-mediated regulation of cell cycle progression in cancer cells.

Heme-binding protein HRG-1 is induced by insulin-like growth factor I and associates with the vacuolar H+-ATPase to control endosomal pH and receptor trafficking.

Endocytosis and trafficking of receptors and nutrient transporters are dependent on an acidic intra-endosomal pH that is maintained by the vacuolar H(+)-ATPase (V-ATPase) proton pump. V-ATPase activity has also been associated with cancer invasiveness. Here, we report on a new V-ATPase-associated protein, which we identified in insulin-like growth factor I (IGF-I) receptor-transformed cells, and which was separately identified in Caenorhabditis elegans as HRG-1, a member of a family of heme-regulated genes. We found that HRG-1 is present in endosomes but not in lysosomes, and it is trafficked to the plasma membrane upon nutrient withdrawal in mammalian cells. Suppression of HRG-1 with small interfering RNA causes impaired endocytosis of transferrin receptor, decreased cell motility, and decreased viability of HeLa cells. HRG-1 interacts with the c subunit of the V-ATPase and enhances V-ATPase activity in isolated yeast vacuoles. Endosomal acidity and V-ATPase assembly are decreased in cells with suppressed HRG-1, whereas transferrin receptor endocytosis is enhanced in cells that overexpress HRG-1. Cellular uptake of a fluorescent heme analogue is enhanced by HRG-1 in a V-ATPase-dependent manner. Our findings indicate that HRG-1 regulates V-ATPase activity, which is essential for endosomal acidification, heme binding, and receptor trafficking in mammalian cells. Thus, HRG-1 may facilitate tumor growth and cancer progression.

GARP is a key molecule for mesenchymal stromal cell responses to TGF-ß and fundamental to control mitochondrial ROS levels.

Multipotent mesenchymal stromal cells (MSCs) have emerged as a promising cell therapy in regenerative medicine and for autoimmune/inflammatory diseases. However, a main hurdle for MSCs-based therapies is the loss of their proliferative potential in vitro. Here we report that glycoprotein A repetitions predominant (GARP) is required for the proliferation and survival of adipose-derived MSCs (ASCs) via its regulation of transforming growth factor-ß (TGF-ß) activation. Silencing of GARP in human ASCs increased their activation of TGF-ß which augmented the levels of mitochondrial reactive oxygen species (mtROS), resulting in DNA damage, a block in proliferation and apoptosis. Inhibition of TGF-ß signaling reduced the levels of mtROS and DNA damage and restored the ability of GARP-/low ASCs to proliferate. In contrast, overexpression of GARP in ASCs increased their proliferative capacity and rendered them more resistant to etoposide-induced DNA damage and apoptosis, in a TGF-ß-dependent manner. In summary, our data show that the presence or absence of GARP on ASCs gives rise to distinct TGF-ß responses with diametrically opposing effects on ASC proliferation and survival.

Establishment of 2 control and 2 hPSC cell lines constitutively expressing the Notch ligand DLL4.

The Notch ligand DLL4 has key roles during embryonic development of different tissues, but most of the data comes from animal models. Here we describe the generation and characterization of 2 human Pluripotent Stem Cell (hPSC) lines that overexpress DLL4, as well as the two corresponding control hPSC lines. DLL4 expression can be detected at the mRNA and protein level, and does not affect the pluripotency of the cells. These hPSC lines can be used to study the role of DLL4 during human embryonic development.

New hPSC-based human models to study pediatric Acute Megakaryoblastic Leukemia harboring the fusion oncogene RBM15-MKL1.

Pediatric Acute Megakaryoblastic Leukemia not associated to Down Syndrome (non-DS AMKL) is a rare disease with a dismal prognosis. Around 15% of patients carry the chromosomal translocation t(1;22) that originates the fusion oncogene RBM15-MKL1, which is linked to an earlier disease onset (median of 6months of age) and arises in utero. Here we report the generation of two hPSC cell lines constitutively expressing the oncogene RBM15-MKL1, resulting in an increased expression of known RBM15-MKL1 gene targets. These cell lines represent new disease models of pediatric AMKL to study the impact of the RBM15-MKL1 oncogene on human embryonic hematopoietic development.

Hoxa9 and EGFP reporter expression in human Embryonic Stem Cells (hESC) as useful tools for studying human development.

HoxA9 is an evolutionarily conserved homeobox gene implicated in embryo development. To study the roles of Hoxa9 during human development we generated a transgenic H9 (hESC) line that overexpresses HoxA9 and the Enhanced Green Fluorescent Protein (EGFP), and a control H9 with a stable expression of the EGFP. The resulting H9-HoxA9-EGFP and H9-EGFP cell lines allow an efficient visualization of hESCs by fluorescent microscopy, quantification by flow cytometry and cell differentiation tracking. Both transgenic cell lines maintained the pluripotent phenotype, the ability to differentiate into all three germ layers and a normal karyotype.

Gene expression profiles in cells transformed by overexpression of the IGF-I receptor.

To identify genes associated with insulin-like growth factor-I receptor (IGF-IR)-mediated cellular transformation, we isolated genes that are differentially expressed in R- cells (derived from the IGF-IR knockout mouse) and R+ cells (R- cells that overexpress the IGF-IR). From these, 45 genes of known function were expressed at higher levels in R+ cells and 22 were expressed at higher levels in R- cells. Differential expression was confirmed by Northern blot analysis of R+ and R- cells. Genes expressed more abundantly in R+ cells are associated with (1) tumour growth and metastasis including, betaigH3, mts1, igfbp5 protease, and mystique; (2) cell division, including cyclin A1 and cdk1; (3) signal transduction, including pkcdeltabp and lmw-ptp; and (4) metabolism including ATPase H+ transporter and ferritin. In MCF-7 cells IGF-I induced expression of two genes, lasp-1 and mystique, which could contribute to metastasis. Lasp-1 expression required activity of the PI3-kinase signalling pathway. Mystique was highly expressed in metastatic but not in androgen-dependent prostate cancer cell lines and Mystique overexpression in MCF-7 cells promoted cell migration and invasion. We conclude that genes identified in this screen may mediate IGF-IR function in cancer progression.

Generation of induced pluripotent stem cells (iPSCs) from a Bernard-Soulier syndrome patient carrying a W71R mutation in the GPIX gene.

We generated an induced pluripotent stem cell (iPSC) line from a Bernard-Soulier Syndrome (BSS) patient carrying the mutation p.Trp71Arg in the GPIX locus (BSS1-PBMC-iPS4F4). Peripheral blood mononuclear cells (PBMCs) were reprogrammed using heat sensitive non-integrative Sendai viruses containing the reprogramming factors Oct3/4, SOX2, KLF4 and c-MYC. Successful silencing of the exogenous reprogramming factors was checked by RT-PCR. Characterization of BSS1-PBMC-iPS4F4 included mutation analysis of GPIX locus, Short Tandem Repeats (STR) profiling, alkaline phosphatase enzymatic activity, analysis of conventional pluripotency-associated factors at mRNA and protein level and in vivo differentiation studies. BSS1-PBMC-iPS4F4 will provide a powerful tool to study BSS.

Proapoptotic activity of ITM2B(s), a BH3-only protein induced upon IL-2-deprivation which interacts with Bcl-2.

Growth factor deprivation is a physiological mechanism to induce apoptosis. We used an IL-2-dependent murine T cell line to identify proteins that trigger apoptosis. Here we report the identification, the cloning and characterization of ITM2B(s), a protein induced upon IL-2-deprivation. ITM2B(s), which shares the BH3 domain of Bcl-2 family members, is a cytoplasmic and mitochondrial protein. Expression of ITM2B(s) induces apoptosis in IL-2-stimulated cells, but not in IL-4-stimulated cells, while overexpression of the long form of the protein is not able to induce apoptosis. In IL-2-stimulated cells, ITM2B(s) interacts with the antiapoptotic protein Bcl-2, and does not interact with the proapoptotic Bad. Mutation of the critical L and D residues within the BH3 domain abolished the ability of ITM2B(s) to promote apoptosis.