Identification and characterization of novel regulatory genes of post-embryonic hematopoiesis

Comprehensive integration site analysis for monitoring the clonal dynamics in clinical gene therapy has revealed that the insertion of the therapeutic retroviral vector (RV) can deregulate and even substantially activate neighboring genes leading to selection advantage and clonal outgrowth. Strikingly, 7 out of 10 Wiskott Aldrich Syndrome (WAS) gene therapy patients developed acute leukemia driven by gene corrected cell clones aberrantly expressing LMO2, MDS1 or MN1. This indicates that RV-mediated activation of adjacent regions cannot only influence the fate of hematopoietic stem cells but also cause clonal dominance up to leukemia. To identify novel regulators of benign hematopoiesis we established a systematic selection strategy using the total genomic integration site dataset of normal and highly polyclonal clinical blood and bone marrow samples. In this thesis, the unique integration site (IS) dataset within a cohort of 10 WAS gene therapy patients was systematically analyzed to select for candidate genes involved in the regulation of hematopoiesis. Initially, a total of 12.887 unique IS in vicinity of 3.267 genes were identified. Next, we selected all genes with at least 10 different IS within a 200 kb window around the gene (n=588). To enrich for genes with increased probability of transcriptional activation we then chose those genes with at least 10 IS within a 50kb window around the transcriptional start site (n=424). After stringent exclusion of all genes located within gene clusters 32 candidate genes were identified. To evaluate the hematopoietic activity of gene corrected cell clones, their contribution to blood cell formation within four years post gene therapy was monitored. We observed that these clones were detectable 15 to 93 times in a total of 102 individually analyzed patient samples, demonstrating long term activity of these hematopoietic stem cell clones. Interestingly, 20 out of the 32 highest ranked genes such as EVI1, CCND2 and LMO2 are known hematopoietic key regulators, strongly validating our selection strategy. After identification of 12 novel hematopoietic regulatory candidate genes, the top five ranked genes, ZNF217, LRRC33, PLCB4, EVL and IRF2BPL were chosen for further functional analysis in murine hematopoietic primary cells. To evaluate the endogenous expression of candidate genes in murine hematopoietic stem and progenitor cells global transcriptome datasets from purified populations were evaluated. We observed that all five selected candidate genes were expressed in at least one out of the five analyzed hematopoietic stem and progenitor cell populations which may point to an important role in the respective cell fraction. In order to validate our selection II strategy and to further investigate whether the chosen candidate genes play roles in hematopoiesis, we performed various in vitro and in vivo assays. We tested the effect of the selected candidate genes on proliferation, differentiation, and cytokine independency as well as for their influence on long term multilineage reconstitution and self-renewal after murine bone marrow transplantation. The first candidate gene, ZNF217, is a zinc finger protein known as a transcription factor. To analyze the impact of ZNF217 on transcriptional activity, global gene expression profiling in hematopoietic cells was performed. We observed that 337 out of 422 genes were significantly downregulated and that they are mainly involved in cellular movement indicating that ZNF217 plays a role in hematopoietic cell migration. Since ZNF217 is known as a proto-oncogene in breast and ovarian carcinoma we evaluated its effect on growth factor independency of hematopoietic cells. Interleukin 3 (IL3)-dependent cells overexpressing ZNF217 acquired the capacity to survive and form colonies in the absence of IL3 suggesting a transforming role for ZNF217 in hematopoietic cells. The second candidate LRRC33 resembles the protein structure of Toll-like receptor (TLR) proteins which are involved in innate immunity. The overexpression of LRRC33 and TLR4 decreased the activity of NF-κB in vitro when stimulated with bacterial lipopolysaccharide. This may point to an inhibitory role of LRRC33 in NF-kB signaling, an important pathway for maintaining stem cell integrity. Transplanted LRRC33-overexpressing LSK (Lin-Sca-1+cKit+) cells gave rise to a 1.3-6.7-fold lower ratio of T-cells and in contrast a 1.1-7.2-fold higher amount of donor derived macrophages in secondary recipients compared to control mice. To study the function of the third candidate gene on hematopoiesis constitutive PLCB4 knockout mice were obtained. This phospholipase has been shown to be important for brain development but has not been linked to hematopoiesis so far. Preliminary results indicate that PLCB4 deficient mice have a reduced LSK cell fraction compared to age matched littermates within the first 18 days after birth. These results demonstrate that clinical integration site datasets can be used to identify regulatory genes of hematopoiesis. Here, we identified ZNF217 as a driver of hematopoietic transformation applying the established selection strategy. In total, we could show that four out of five candidate genes play a role in hematopoiesis and they will be further evaluated for their stem cell regulatory potential. Systematic identification of novel regulatory genes in meta-datasets derived from a larger number of gene therapy studies and subsequent validation in vitro and in vivo will allow to gain new insights into the biology of post-embryonic hematopoiesis

Inflammatory Signaling Pathways in Preleukemic and Leukemic Stem Cells

Hematopoietic stem cells (HSCs) are a rare subset of bone marrow cells that usually exist in a quiescent state, only entering the cell cycle to replenish the blood compartment, thereby limiting the potential for errors in replication. Inflammatory signals that are released in response to environmental stressors, such as infection, trigger active cycling of HSCs. These inflammatory signals can also directly induce HSCs to release cytokines into the bone marrow environment, promoting myeloid differentiation. After stress myelopoiesis is triggered, HSCs require intracellular signaling programs to deactivate this response and return to steady state. Prolonged or excessive exposure to inflammatory cytokines, such as in prolonged infection or in chronic rheumatologic conditions, can lead to continued HSC cycling and eventual HSC loss. This promotes bone marrow failure, and can precipitate preleukemic states or leukemia through the acquisition of genetic and epigenetic changes in HSCs. This can occur through the initiation of clonal hematopoiesis, followed by the emergence preleukemic stem cells (pre-LSCs). In this review, we describe the roles of multiple inflammatory signaling pathways in the generation of pre-LSCs and in progression to myelodysplastic syndrome (MDS), myeloproliferative neoplasms, and acute myeloid leukemia (AML). In AML, activation of some inflammatory signaling pathways can promote the cycling and differentiation of LSCs, and this can be exploited therapeutically. We also discuss the therapeutic potential of modulating inflammatory signaling for the treatment of myeloid malignancies

The balance between the intronic miR-342 and its host gene Evl determines hematopoietic cell fate decision

Protein-coding and non-coding genes like miRNAs tightly control hematopoietic differentiation programs. Although miRNAs are frequently located within introns of protein-coding genes, the molecular interplay between intronic miRNAs and their host genes is unclear. By genomic integration site mapping of gamma-retroviral vectors in genetically corrected peripheral blood from gene therapy patients, we identified the EVL/MIR342 gene locus as a hotspot for therapeutic vector insertions indicating its accessibility and expression in human hematopoietic stem and progenitor cells. We therefore asked if and how EVL and its intronic miRNA-342 regulate hematopoiesis. Here we demonstrate that overexpression (OE) of Evl in murine primary Lin- Sca1+ cKit+ cells drives lymphopoiesis whereas miR-342 OE increases myeloid colony formation in vitro and in vivo, going along with a profound upregulation of canonical pathways essential for B-cell development or myelopoietic functions upon Evl or miR-342 OE, respectively. Strikingly, miR-342 counteracts its host gene by targeting lymphoid signaling pathways, resulting in reduced pre-B-cell output. Moreover, EVL overexpression is associated with lymphoid leukemia in patients. In summary, our data show that one common gene locus regulates distinct hematopoietic differentiation programs depending on the gene product expressed, and that the balance between both may determine hematopoietic cell fate decision

EVI1 inhibits apoptosis induced by antileukemic drugs via upregulation of CDKN1A/p21/WAF in human myeloid cells.

Overexpression of ecotropic viral integration site 1 (EVI1) is associated with aggressive disease in acute myeloid leukemia (AML). Despite of its clinical importance, little is known about the mechanism through which EVI1 confers resistance to antileukemic drugs. Here, we show that a human myeloid cell line constitutively overexpressing EVI1 after infection with a retroviral vector (U937_EVI1) was partially resistant to etoposide and daunorubicin as compared to empty vector infected control cells (U937_vec). Similarly, inducible expression of EVI1 in HL-60 cells decreased their sensitivity to daunorubicin. Gene expression microarray analyses of U937_EVI1 and U937_vec cells cultured in the absence or presence of etoposide showed that 77 and 419 genes were regulated by EVI1 and etoposide, respectively. Notably, mRNA levels of 26 of these genes were altered by both stimuli, indicating that EVI1 regulated genes were strongly enriched among etoposide regulated genes and vice versa. One of the genes that were induced by both EVI1 and etoposide was CDKN1A/p21/WAF, which in addition to its function as a cell cycle regulator plays an important role in conferring chemotherapy resistance in various tumor types. Indeed, overexpression of CDKN1A in U937 cells mimicked the phenotype of EVI1 overexpression, similarly conferring partial resistance to antileukemic drugs

The balance between the intronic miR-342 and its host gene Evl determines hematopoietic cell fate decision

Protein-coding and non-coding genes like miRNAs tightly control hematopoietic differentiation programs. Although miRNAs are frequently located within introns of protein-coding genes, the molecular interplay between intronic miRNAs and their host genes is unclear. By genomic integration site mapping of gamma-retroviral vectors in genetically corrected peripheral blood from gene therapy patients, we identified the EVL/MIR342 gene locus as a hotspot for therapeutic vector insertions indicating its accessibility and expression in human hematopoietic stem and progenitor cells. We therefore asked if and how EVL and its intronic miRNA-342 regulate hematopoiesis. Here we demonstrate that overexpression (OE) of Evl in murine primary Li

Overexpression of <i>EVI1</i> decreases the sensitivity of U937 cells to drugs used in AML therapy.

<p>A–D) U937_vec and U937_EVI1 cells were treated with the indicated concentrations of etoposide (A, C) or daunorubicin (B, D) for 48 h. Cellular viability/metabolic activity was determined based on ATP content (A, B), and apoptosis was measured via caspase 3/7 activity (C, D). Data points represent the mean +/− SEM from at least three independent experiments. *, p<0.05 (paired Student’s t-test). E) Nuclear morphology of U937_vec and U937_EVI1 cells after treatment with or without 400 nM etoposide for 48 h. Apoptotic nuclei are marked by arrows. Please note difference in scale between etoposide and control treated cells. F) Quantitative assessment of nuclear morphology. Nuclei prepared as in E were counted as ‘intact’ or ‘apoptotic’ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056308#s2" target="_blank">Methods</a>) by an observer blinded to the identity of the samples. Data points represent the mean+SEM from 3 independent experiments. **, p<0.01; n.s., not significant (paired Student’s t-test).</p

Establishment and characterization of a human myeloid cell line constitutively overexpressing <i>EVI1</i>.

<p>A) Immunoblot analysis for the detection of EVI1 in U937_vec and U937_EVI1 cells. HNT-34 cells, which express EVI1 due to a rearrangement of chromosome band 3q26 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056308#pone.0056308-Hamaguchi1" target="_blank">[43]</a>, were included for comparison. Hybridization with a β-tubulin antibody was used as a loading control. B) Intracellular FACS staining for detection of EVI1 in U937_vec, U937_EVI1, and HNT-34 cells. Dark grey histogram curves, EVI1 antibody; light grey histogram curves, isotype control. C) High-resolution gel analysis of LAM-PCR amplicons obtained from Tsp509I digested genomic DNA from U937_vec and U937_EVI1 cells. DNA was isolated shortly after infection and sorting (t1) as well as after another 12–15 weeks in culture (t2).</p