Hematopoietic Progenitor Cell Line Research Articles

The Sensitization of Murine Hematopoietic Progenitors and Stem Cells to DNA Damaging Agents By Quizartinib Is Associated with the Transcriptional Downregulation of DNA Damage Repair Genes

Background: The FLT3 inhibitor quizartinib induces quiescence of murine hematopoietic progenitors, and this affords their protection from S phase specific chemotherapies. In this study we investigated whether quizartinib can also protect hematopoietic progenitors from the cytotoxic effects of DNA damaging chemotherapies and radiation. Methods: C57BL/6 mice were primed with 30 mg/kg of quizartinib or vehicle (5% cyclodextrin) by oral gavage, and treated with DNA damaging chemotherapies, either cyclophosphamide, carboplatin, cisplatin, temozolomide or mitoxantrone 6 hours later, or 5.5Gy total body irradiation (TBI) after 9 hours. This TBI dose represents half the 11 Gy split dose used for bone marrow conditioning of C57BL/6 mice prior to HSC transplantation. Bone marrow cells were analyzed by flow cytometry after 3 to 6 days to identify progenitor and stem cell populations. Results: In contrast to quizartinib's protection against the cytotoxic effects of 5-FU and gemcitabine, all 5 DNA-damaging chemotherapies caused an increased loss of total bone marrow cellularity when mice were primed with quizartinib compared to vehicle primed controls. Flow cytometry analysis revealed that the numbers of myeloid and multipotent progenitors, and short-term and long-term hematopoietic stem cells (HSCs), were markedly lower in the quizartinib-treated mice. DNA damage and the resultant cell death caused by ionizing radiation is cell cycle specific, particularly for cells traversing from G 1 to S phases of the cell cycle. The ability of quizartinib to induce quiescence in myeloid and multipotent progenitors suggested that quizartinib may protect these cells from radiation. To test this, mice were dosed with vehicle or 30 mg/kg of quizartinib and 9 hours later exposed mice to 5.5 Gy of TBI. Analysis of bone marrow cells at day 6 revealed that total bone marrow cellularity and the numbers of progenitor and stem cells were markedly reduced in quizartinib-dosed mice compared to vehicle controls. For subsequent experiments mice were treated with 3 Gy TBI because the combination of quizartinib and 5.5 Gy caused severe myeloablation. As part of a series of experiments examining the kinetics of quizartinib's sensitization of bone marrow cells we tested whether the sensitization was maintained when mice were dosed with quizartinib 24 hours prior to 3 Gy TBI. Analysis of bone marrow cells 6 days later revealed that quizartinib dosed mice showed a significantly greater loss of total bone marrow cells, progenitor cells and stem cells compared to vehicle dosed mice (Figure 1). To identify possible mechanisms by which quizartinib sensitizes hematopoietic progenitors to DNA damaging agents we investigated quantitative gene expression profiling of the HPC7 hematopoietic progenitor cell line. These cells provide a suitable in vitro model since quizartinib induces their quiescence and provides protection against 5-FU. HPC7 cells were treated with 100 µM quizartinib, or vehicle, for 18 hr before RNA was harvested and sent for Next-generation sequencing. We found that quiescence induction in HPC7 cells by quizartinib correlated with the transcriptional downregulation of a wide range of genes involved in DNA damage repair pathways (including Brca1/2, Rad51, Palb2, Chek1 and Nbn), thus providing a possible mechanism for the enhanced cytotoxic effects of DNA damaging agents. Conclusions: Quizartinib sensitizes hematopoietic progenitors and stem cells to DNA-damaging agents, an effect that correlates with the transcriptional downregulation of many DNA damage repair genes. The enhanced depletion of hematopoietic progenitors and stem cells by DNA damaging agents in the presence of quizartinib has important clinical implications. Administering concurrent DNA damaging chemotherapy or radiotherapy with quizartinib treatment should be avoided. However, this interaction could facilitate a reduction in radiation/chemotherapy doses administered for bone marrow conditioning prior to HSC transplantation. By reducing these doses, but not compromising the conditioning process, non-hematopoietic side effects could be minimized and may allow currently excluded patients to be considered for HSC transplantation. Disclosure: This study was partly funded by Daiichi Sankyo.

Targeting STAT5 Signaling Overcomes Resistance to IDH Inhibitors in Acute Myeloid Leukemia through Suppression of Stemness.

A CRISPR knockout screen identifies a mechanism of resistance to IDH inhibitors in AML involving activated STAT5 signaling, suggesting a potential strategy to improve the clinical efficacy of IDH inhibitors.

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro.

Dendritic cells (DCs) are important antigen-presenting cells that connect innate and adaptive immune responses. DCs are heterogeneous and can be divided into conventional DCs (cDCs) and plasmacytoid DCs (pDCs). cDCs specializes in presenting antigens to and activate naïve T cells. On the other hand, pDCs can produce large quantities of type I interferons (IFN-I) during viral infection. The specification of DCs occurs at an early stage of DC progenitors in the bone marrow (BM) and is defined by a network of transcription factors (TFs). For example, cDCs highly express ID2, while pDCs highly express E2-2. Since more and more subsets of DCs are being identified, there is a growing interest in understanding specific TFs controlling DC development. Here, we establish a method to screen TFs critical for DCs differentiation in vitro by delivering lentivirus carrying short hairpin RNA (shRNA) into an immortalized hematopoietic stem and progenitor cell (iHSPCs) line. After the selection and in vitro differentiation, cDC and pDC potential of the stable knockdown cell lines are analyzed by flow cytometry. This approach provides a platform to identify genes potentially governing DC fates from progenitors in vitro.

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line <em>In vitro</em>

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line <em>In vitro</em>

A Precise Tool to Edit Human Hematopoietic Stem Cells at High Efficiency and Decipher and Optimize the Different Double Strand Break Repair Outcomes

The CRISPR/Cas9 discovery has revolutionized precise editing of the genome. However, Cas9-induced site-specific double-strand breaks (DSB) are predominantly repaired by the ‘error-prone’ non-homologous end-joining (NHEJ)/alternate NHEJ (aNHEJ) mechanisms, with relatively low precise correction using homology-directed repair (HDR). The HDR-corrected cells can be selected with drugs/surface reporter in cell lines, induced pluripotent cells, mouse embryonic cells. However, selection of gene edited/corrected hematopoietic stem cells (HSC) is not feasible in vitro. HSC remain the most promising targets because of their unique self-renewing, expansion and differentiation potential. Assessment and optimization of editing/correction of the rare human HSC comprising 1-2% of the CD34+ hematopoietic stem and progenitor cell (HSPC) population is particularly challenging, as their selection before/after often results in insufficient cell numbers for detailed molecular analysis; and selection post-editing for clinical translation can reduce recovery/engraftment potential from increased processing. Currently, the readout of the edited HSC is by molecular analysis for NHEJ- and aNHEJ-induced insertions/deletions and HDR detection of total CD34+ cells and assessment of long term repopulating potential (LTRP) in in vivo models. Herein, we report a unique platform that can precisely quantify NHEJ, aNHEJ, HDR and random integration of the HDR donor template in the human HSC using a flow cytometry. We targeted the CD45 surface reporter (PTPRC), present on all leukocytes and HSPC, to induce a DSB in exon 2 of the PTPRC gene using CRISPR/Cas9, and an in-frame promoter-less GFP containing homology template and a downstream tNGFR expression cassette (GFP/tNGFR HDR donor). HSC and HSPC that repaired Cas9-induced DSB via NHEJ on a single allele in diploid cells lost CD45 expression by flow cytometry. Furthermore, the gRNA/Cas9 induced DSB site was designed to have a 3bp micro-homology so that DSB repaired via aNHEJ resulted in cells express CD45 in frame, but a 6bp deletion detectable by TIDE assay. Provision of the GFP/tNGFR HDR donor repair template resulted in GFP and CD45 expressing, but not tNGFR expressing cells, but if the GFP/tNGFR HDR donor integrated randomly, cells were tNGFR+. Using this system, we optimized editing of hematopoietic progenitor cell lines (K562, Kasumi cells) and EBV transformed B-lymphoblasts to >95% efficiency, and molecular analysis of total and CD45+, and CD45- and CD45+GFP+ cell populations confirmed the precision of the flow cytometry based quantification of NHEJ, aNHEJ. In fact, molecular analysis of the CD45+ cells led to the discovery that K562 cells had a high rate of aNHEJ, while this editing via aNHEJ was absent in LCL- and Kasumi- B lymphoid cells. Inhibition of the aNHEJ pathway with small molecule inhibitors/siRNA resulted in loss of the 6bp aNHEJ-mediated deletion in K562 cells. We also found that random integration in LCL, Kasumi and CD34+ cells was a rare event, but occurred with high frequency in K562 cells. Due to the rapid detection of editing events by flow cytometry, we were able to optimize different editing and cell parameters in both cord blood and mobilized peripheral blood derived CD34+ HSPC and CD34+38-90+RA- HSC populations to achieve high viability and 80-87% editing efficiency (CD45-) in HSPC and HSC, and the FACS-based results were similar to molecular analysis. The edited CD34+ HSPC, when injected into NSG mice resulted in robust human cell engraftment, with 45±10% total engraftment, and 39±16% edited cell engraftment (n=12 mice). Using this platform, we improved HDR frequency to 30% and reduced NHEJ events to 60% in hematopoietic cell lines and similar optimization of HDR in HSPC/HSC is underway. In summary, we have developed a novel platform that allows rapid quantification of gene editing by flow cytometry of human HSC at a single cell and a single allele level, in a single step; with concurrent molecular analysis that identifies different DNA repair pathways utilized by different cell types. This platform allows rapid identification of edited events in the rare HSC population, rather than bulk CD34+ HSPC. Furthermore, ability to sort the edited HSC allows precise determination of the genotoxicity of gene editing, and optimization of DSB repair pathway needed, and optimize editing parameters that result in engraftable edited HSC with LTRP. DisclosuresNo relevant conflicts of interest to declare.

A robust approach for the generation of functional hematopoietic progenitor cell lines to model leukemic transformation.

Studies of molecular mechanisms of hematopoiesis and leukemogenesis are hampered by the unavailability of progenitor cell lines that accurately mimic the situation in vivo. We now report a robust method to generate and maintain LSK (Lin-, Sca-1+, c-Kit+) cells, which closely resemble MPP1 cells. HPCLSKs reconstitute hematopoiesis in lethally irradiated recipient mice over >8 months. Upon transformation with different oncogenes including BCR/ABL, FLT3-ITD, or MLL-AF9, their leukemic counterparts maintain stem cell properties in vitro and recapitulate leukemia formation in vivo. The method to generate HPCLSKs can be applied to transgenic mice, and we illustrate it for CDK6-deficient animals. Upon BCR/ABLp210 transformation, HPCLSKsCdk6-/- induce disease with a significantly enhanced latency and reduced incidence, showing the importance of CDK6 in leukemia formation. Studies of the CDK6 transcriptome in murine HPCLSK and human BCR/ABL+ cells have verified that certain pathways depend on CDK6 and have uncovered a novel CDK6-dependent signature, suggesting a role for CDK6 in leukemic progenitor cell homing. Loss of CDK6 may thus lead to a defect in homing. The HPCLSK system represents a unique tool for combined in vitro and in vivo studies and enables the production of large quantities of genetically modifiable hematopoietic or leukemic stem/progenitor cells.

Characterization of p190-Bcr-Abl chronic myeloid leukemia reveals specific signaling pathways and therapeutic targets

The oncogenic protein Bcr-Abl has two major isoforms, p190Bcr-Abl and p210Bcr-Abl. While p210Bcr-Abl is the hallmark of chronic myeloid leukemia (CML), p190Bcr-Abl occurs in the majority of Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) patients. In CML, p190Bcr-Abl occurs in a minority of patients associating with distinct hematological features and inferior outcomes, yet the pathogenic role of p190Bcr-Abl and potential targeting therapies are largely uncharacterized. We employed next generation sequencing, phospho-proteomic profiling, and drug sensitivity testing to characterize p190Bcr-Abl in CML and hematopoietic progenitor cell line models (Ba/f3 and HPC-LSK). p190Bcr-Abl CML patients demonstrated poor response to imatinib and frequent mutations in epigenetic modifiers genes. In contrast with p210Bcr-Abl, p190Bcr-Abl exhibited specific transcriptional upregulation of interferon, interleukin-1 receptor, and P53 signaling pathways, associated with hyperphosphorylation of relevant signaling molecules including JAK1/STAT1 and PAK1 in addition to Src hyperphosphorylation. Comparable to p190Bcr-Abl CML patients, p190Bcr-Abl cell lines demonstrated similar transcriptional and phospho-signaling signatures. With the drug sensitivity screening we identified targeted drugs with specific activity in p190Bcr-Abl cell lines including IAP-, PAK1-, and Src inhibitors and glucocorticoids. Our results provide novel insights into the mechanisms underlying the distinct features of p190Bcr-Abl CML and promising therapeutic targets for this high-risk patient group.

HMGA1 Is Upregulated in Myelodysplastic Syndromes with Mutation in Pre-mRNA Splicing Genes and Inhibits Leukemia Cell Differentiation

In myelodysplastic syndrome (MDS), genes associated with pre-mRNA splicing such as SRSF2 (Serine and Arginine Rich Splicing Factor 2) and U2AF1 (U2 small nuclear RNA auxiliary factor 1) are frequently mutated. While several reports demonstrated that MDS patients with these mutations had worse prognosis, few treatments specific to these mutations are available, partly due to the dearth of material on functions of these mutated genes. To assess the role of pre-mRNA splicing gene mutations in the pathogenesis of MDS, we performed RNA-seq analysis using CD34-postitive fractions of bone marrow samples of MDS patients. Patients with mutations in either SRSF2 or U2AF1 tended to have shorter 1-year leukemia free survival compared to patients without these mutations (66.7% vs 73.7%, respectively; p=0.067). Gene set enrichment analysis revealed that MYC target genes were enriched in patients with mutations in either SRSF2 or U2AF1 compared to patients (n=4) without these mutations (n=3). Among them, we found that HMGA1 (High Mobility Group A1) was distinctly upregulated in these patients. Expression levels of HMGA1 are known to be higher in hematopoietic stem cells. HMGA1 is involved in various cellular processes such as transcriptional regulation, DNA repair, cell differentiation and regulated cell death. We confirmed that expression levels of HMGA1 in murine bone marrow cells were higher in Lineage marker-negative, Sca1-positive, and c-Kit-positive (LSK) fractions than other more differentiated fractions. To determine whether upregulation of HMGA1 contributed to pathogenesis of MDS, we retrovirally transduced HMGA1 to murine hematopoietic progenitor cell line 32D-cl3. We found that proportions of Gr-1 positive cells after treatment with G-CSF were lower in HMGA1-transduced 32D-cl3 cells. Further, expression levels of myeloid-associated genes were also suppressed in these 32D-cl3 cells. These results suggested that 32D-cl3 with extrinsic expression of HMGA1 were more resistant to G-CSF induced myeloid differentiation. Similarly, primary murine hematopoietic progenitor cells retrovirally transduced with HMGA1 showed increased colony-forming capacities. Considering that differentiation block plays a major role in the onset of MDS, these results supported our hypothesis that upregulation of HMGA1 would contribute to pathogenesis of MDS through blocking normal myeloid differentiation. To investigate whether inhibition of HMGA1 could affect the pathogenesis of myeloid malignancies, we silenced expression of HMGA1 via short-hairpin RNA (shRNA) in various cell lines of acute myeloid leukemia (AML). Human acute monocytic leukemia cell line THP-1 became more easily induced to differentiate upon phorbol12-myristate13-acetate treatment when HMGA1 was silenced. Similarly, we silenced expression of HMGA1 in HL60, which is a human acute promyelocytic leukemia (APL) cell line. We treated HL60 cells with all-trance retinoic acid (ATRA) to induce myeloid differentiation. As expected, we found that expression levels of CEBP-β and Gr-1 in ATRA-treated HL60 cells were significantly higher when HMGA1 was silenced, which meant that HL60 cells became more sensitive to ATRA-induced myeloid differentiation by inhibition of HMGA1. Considering that differentiation induction therapy has improved clinical outcome not only in APL but also in AML, these results suggested that HMGA1 would be a potential therapeutic target for myeloid malignancies. In summary, our experiments demonstrated that expression levels of HMGA1 were higher in patients with splicing mutations. Considering its inhibitory effects on differentiation demonstrated by our experiments, upregulation of HMGA1 could be involved in the pathogenesis of MDS. Further, therapeutic intervention for HMGA1 had a potential to improve clinical outcome of myeloid malignancies. Disclosures Kurokawa: Nippon Shinyaku: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Teijin: Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bioverativ Japan: Consultancy; Shire Plc: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Eisai: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau; Pfizer: Research Funding; Sanwa-Kagaku: Consultancy; Chugai: Consultancy, Research Funding, Speakers Bureau; MSD: Consultancy, Research Funding, Speakers Bureau.

Hematopoietic Stem Cells Culture, Expansion and Differentiation: An Insight into Variable and Available Media.

Owing to differentiation and self-renewal capacity, hematopoietic stem cells clasp potentiality to engender all blood cell types, leading to their immense competence to play a diverse role in therapeutic applications. Although these stem cells are the most investigated and exploited until now, further research is still essential to comprehend their nature, fate, and potential. Enhanced usage of hematopoietic stem cells in research and therapeutics intensified the requirement of expansion and differentiation of hematopoietic stem cells under in vitro conditions. Since these cells remain in senescence for a prolonged period before isolation, selection of appropriate growth medium along with supplements and culture conditions are crucial to initiate their cell division and to designate their destiny. The precise equilibrium between self-renewal and differentiation of stem cells sustained by exclusive medium along with special growth or differentiation factors is accountable for generating diverse cell lineages. Maintenance of hematopoietic stem and progenitor cell lines along with the advancement of research work generate an inexorable demand for production and commercialization of specialized stem cell culture media, with or without serum along with specific growth factors and supplements. Media commercialization for precise stem cell types, culturing and differentiation is a cost-effective developing field. Here in this review, we are assembling various types of hematopoietic stem cell self-renewal, expansion and differentiation media along with supplements and culture conditions, either developed and used by various scientists or are available commercially.

Nicotinamide Inhibits Self-renewal and Induces Granulocyte Differentiation of Multipotent Progenitor Cells.

Nicotinamide (NAM) a form of vitamin B3, is an essential precursor of NAD. This dinucleotide (pyridine nucleotide) participates in the regulation of fundamental processes including transcription, cell cycle progression and DNA repair. Here we assessed the effect of NAM on myeloid differentiation of the IL-3 dependent, multipotent hematopoietic progenitor cell line FDCP-Mix. We found that NAM reduces the pSTAT5 signaling response, cell cycling and self-renewal potential. It initiates an atypical program of myeloid differentiation that results in the emergence of granulocytic cells in the absence of added myeloid differentiation factors. NAM did not affect the expression the of cell surface granulocyte marker GR1 but led to a strong downregulation of MHC-II molecules. Taken together our data show that NAM induces a differentiation program in hematopoietic progenitors prompting them to undergo differentiation along the granulocyte path without reaching the status of fully developed granulocytes. Graphical abstract.

CRISPR Gene Editing of Murine Blood Stem and Progenitor Cells Induces MLL-AF9 Chromosomal Translocation and MLL-AF9 Leukaemogenesis.

Chromosomal rearrangements of the mixed lineage leukaemia (MLL, also known as KMT2A) gene on chromosome 11q23 are amongst the most common genetic abnormalities observed in human acute leukaemias. MLL rearrangements (MLLr) are the most common cytogenetic abnormalities in infant and childhood acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL) and do not normally acquire secondary mutations compared to other leukaemias. To model these leukaemias, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL-AF9 (MA9) chromosomal rearrangements in murine hematopoietic stem and progenitor cell lines and primary cells. By utilizing a dual-single guide RNA (sgRNA) approach targeting the breakpoint cluster region of murine Mll and Af9 equivalent to that in human MA9 rearrangements, we show efficient de novo generation of MA9 fusion product at the DNA and RNA levels in the bulk population. The leukaemic features of MA9-induced disease were observed including increased clonogenicity, enrichment of c-Kit-positive leukaemic stem cells and increased MA9 target gene expression. This approach provided a rapid and reliable means of de novo generation of Mll-Af9 genetic rearrangements in murine haematopoietic stem and progenitor cells (HSPCs), using CRISPR/Cas9 technology to produce a cellular model of MA9 leukaemias which faithfully reproduces many features of the human disease in vitro.

Radioresistance of Serpinb3a-/- Mice and Derived Hematopoietic and Marrow Stromal Cell Lines.

Serpins are a group of serine-proteases involved in multiple signal transduction pathways in mammalian cells. In particular, Serpinb3a is involved in the lysosomal necrosis cell death pathway with components that overlap with radiation-induced apoptosis. We investigated the radiation response of Serpinb3a-/- mice compared to Serpinb3a+/+ mice on the Balb/c background. Serpinb3a-/- mice showed significant radioresistance to a dose of 8.0 Gy total-body irradiation, compared to Serpinb3a+/+ Balb/c mice. Long-term bone marrow cultures from Serpinb3a-/- mice showed increased longevity. In clonogenic survival assays, fresh bone marrow hematopoietic progenitors, as well as clonal interleukin-3 (IL-3)-dependent hematopoietic progenitor and bone marrow stromal cell lines from Serpinb3a-/- mice were radioresistant. Serpinb3a-/- mouse bone marrow-derived stromal cell lines had increased baseline and postirradiation antioxidant capacity. Serpinb3a-/- bone marrow stromal cells showed increased radiation-induced RNA transcripts for MnSOD and p21, and decreased levels of p53 and TGF-b. Both irradiated Serpinb3a-/- mouse bone marrow stromal cell lines and plasma removed from total-body irradiated mice had decreased levels of expression of stress response and inflammation-associated proteins. Abrogation of Serpinb3a may be a potential new target for mitigation of radiation effects.

Malignant Transformation of Fanconi Anemia Complementation Group D2-deficient (Fancd2 -/-) Hematopoietic Progenitor Cells by a Single HPV16 Oncogene.

To demonstrate that Fanconi anemia complementation group D2-deficient (Fancd2-/-) hematopoietic progenitor cell lines can be transformed by transfection with a plasmid containing either the E6 or E7 oncogene of human papillomavirus (HPV) to generate malignant plasmacytoma-inducing cell lines. In order to determine whether a single HPV type 16 (HPV16) oncogene induced malignant transformation, Fancd2-/- and Fancd2+/+ interleukin 3 (IL3)-dependent hematopoietic progenitor cell lines were transfected with plasmids containing E6 or E7 oncogene, or control empty plasmid. Fancd2-/- but not Fancd2+/+ cells were transformed into malignant IL3-independent cells by both E6, and E7 oncogenes, but not by empty plasmid. Hematopoietic cell lines and tumors induced by Fancd2-/- E6 and Fancd2-/- E7 cell lines were positive for each respective HPV RNA and protein. A single HPV16 oncogene is adequate to produce malignant transformation of Fancd2-/- hematopoietic cells.

An integrated global regulatory network of hematopoietic precursor cell self-renewal and differentiation.

Systematic study of the regulatory mechanisms of Hematopoietic Stem Cell and Progenitor Cell (HSPC) self-renewal is fundamentally important for understanding hematopoiesis and for manipulating HSPCs for therapeutic purposes. Previously, we have characterized gene expression and identified important transcription factors (TFs) regulating the switch between self-renewal and differentiation in a multipotent Hematopoietic Progenitor Cell (HPC) line, EML (Erythroid, Myeloid, and Lymphoid) cells. Herein, we report binding maps for additional TFs (SOX4 and STAT3) by using chromatin immunoprecipitation (ChIP)-Sequencing, to address the underlying mechanisms regulating self-renewal properties of lineage-CD34+ subpopulation (Lin-CD34+ EML cells). Furthermore, we applied the Assay for Transposase Accessible Chromatin (ATAC)-Sequencing to globally identify the open chromatin regions associated with TF binding in the self-renewing Lin-CD34+ EML cells. Mass spectrometry (MS) was also used to quantify protein relative expression levels. Finally, by integrating the protein-protein interaction database, we built an expanded transcriptional regulatory and interaction network. We found that MAPK (Mitogen-activated protein kinase) pathway and TGF-β/SMAD signaling pathway components were highly enriched among the binding targets of these TFs in Lin-CD34+ EML cells. The present study integrates regulatory information at multiple levels to paint a more comprehensive picture of the HSPC self-renewal mechanisms.

A DNA Contact Map for the Mouse Runx1 Gene Identifies Novel Haematopoietic Enhancers

The transcription factor Runx1 is essential for definitive haematopoiesis, and the RUNX1 gene is frequently translocated or mutated in leukaemia. Runx1 is transcribed from two promoters, P1 and P2, to give rise to different protein isoforms. Although the expression of Runx1 must be tightly regulated for normal blood development, the mechanisms that regulate Runx1 isoform expression during haematopoiesis remain poorly understood. Gene regulatory elements located in non-coding DNA are likely to be important for Runx1 transcription. Here we use circular chromosome conformation capture sequencing to identify DNA interactions with the P1 and P2 promoters of Runx1, and the previously identified +24 enhancer, in the mouse multipotent haematopoietic progenitor cell line HPC-7. The active promoter, P1, interacts with nine non-coding regions that are occupied by transcription factors within a 1 Mb topologically associated domain. Eight of nine regions function as blood-specific enhancers in zebrafish, of which two were previously shown to harbour blood-specific enhancer activity in mice. Interestingly, the +24 enhancer interacted with multiple distant regions on chromosome 16, suggesting it may regulate the expression of additional genes. The Runx1 DNA contact map identifies connections with multiple novel and known haematopoietic enhancers that are likely to be involved in regulating Runx1 expression in haematopoietic progenitor cells.

The EMT Modulator SNAI1 Drives AML Development Via Its Interaction with the Chromatin Modulator LSD1

Introduction: Regulation of the epithelial to mesenchymal transition (EMT) is an emerging theme in acute leukemia biology (1, 2). EMT is required for many aspects of development, including gastrulation and mesoderm formation. It has also been associated with malignant processes such as epithelial tumor cell invasion and metastasis, and the development, function and chemo-resistance of cancer stem cells. Master regulators of EMT belong to the Snail (Snai1/Snai2/Snai3) and Zeb (Zeb1/Zeb2) families of transcription factors. While they have primarily been studied in relation to solid organ development and embryogenesis, recent work has begun to uncover novel roles for these proteins in both normal and malignant hematopoiesis.A mounting body of evidence implicates the founding member of the Snail family, SNAI1, in Acute Myeloid Leukaemia (AML) development. Transgenic Snai1 expression induces myeloid leukaemia in mice (3), and human AML cells show increased SNAI1 expression compared to haematopoietic stem and progenitor cells (HSPCs). We have also found that shRNA knockdown of SNAI1 in AML cells induces differentiation, as measured by increased CD11b expression. In this paper we elucidate the mechanisms by which elevated levels of Snai1 impair normal hematopoiesis and promote leukemogenesis.Results: We employed murine retroviral and transgenic models to drive high levels of Snai1 in hematopoietic cells in vivo. Homozygous expression of a Snai1 transgene, using the endothelial and hematopoietic restricted Tie2-Cre, resulted in embryonic lethality at E15.5-17.5. Embryos were highly anemic, exhibiting normal primitive erythropoiesis, but severely impaired definitive erythropoiesis. Retroviral mediated overexpression of Snai1 during adult hematopoiesis resulted in a significant expansion of the Granulocyte-Macrophage progenitor (GMP) compartment, with a concomitant reduction in Megakaryocyte-Erythroid progenitor (MEP) numbers. Downstream myeloid development was also perturbed, with a strong differentiation bias towards macrophage production, at the expense of granulocyte development. Mice (n=4) have also begun to develop an aggressive AML beginning at 12 months of age, consistent with the previously published transgenic model (3).The effects of Snai1 expression on myeloid development are reminiscent of those observed in mice lacking Gfi1 or Gfi1b, suggesting possible competition for, or altered function of, their common co-repressor: the chromatin modulator Lsd1. To test this hypothesis, we generated a mutant form of Snai1 that does not bind Lsd1 and retrovirally expressed it in HSPCs. Unlike wild-type Snai1, the mutant form was unable to induce hematopoietic defects or AML development in mice.We next conducted gene expression profiling on the HPC7 hematopoietic progenitor cell line, with or without ectopic Snai1 expression. We observed a significant up-regulation of GMP-associated genes and more mature monocyte/macrophage related genes. These data, along with morphological analysis, suggested that Snai1 expressing HPC7 cells take on a macrophage-biased GMP-like phenotype, but do not fully differentiate into mature myeloid cells. Moreover, there was a significant correlation to gene expression programs from Gfi1/1b knockout HSCs, further implicating loss of Gfi1/1b activity in driving these myeloid phenotypes.Strikingly, we also observed evidence of inflammatory cytokine production and activation of key downstream signaling pathways that contribute to AML cell survival and proliferation. Whether this effect is due to altered regulation of Lsd1 targets, or instead represents a distinct Lsd1-independent mechanism, remains to be determined.Conclusion: We have shown that Snai1 expression perturbs myeloid cell development and induces AML in an Lsd1-dependent manner. Snai1 expression likely contributes to increased survival and proliferation of leukemic cells via induction of key inflammatory signaling pathways that support tumor cell growth. These data confirm the importance of Snai1 in AML development and pathogenesis.

A Runx1 Interactome Identifies Novel Hematopoietic Enhancers

The transcription factor Runx1 is essential for definitive hematopoiesis, and human RUNX1 is frequently translocated or mutated in leukaemia. Runx1 expression from its two promoters, the proximal P1 and distal P2, must be tightly regulated for normal hematopoiesis to occur. The mechanism(s) of promoter-specific Runx1 expression during development and differentiation of hematopoietic progenitors is poorly understood. Gene regulatory elements located in non-coding DNA, such as the previously identified +24 enhancer (Ng et al, Stem Cells 2010), are likely to be important for Runx1 transcription. Here, we identify novel cis-regulatory DNA elements that are competent to drive hematopoietic expression in zebrafish, and that interact long-range with the Runx1 gene.Circular chromosome conformation capture sequencing (4C-seq) was used to identify long-range interactions of DNA elements with the promoters of Runx1 in the mouse hematopoietic progenitor cell line, HPC7. We used the P1 and P2 promoters and the +24 enhancer (located 24 kb downstream from the P1 promoter) as anchor points from which to view cis-interactions on mouse chromosome 16. 4C-seq reveals that high levels of chromatin interactions at Runx1 are contained within a 1 megabase domain, spanning from the upstream Setd4 gene to the downstream Clic6 gene. A comparison with HiC data in K562 cells shows that the same domain is conserved in humans.HPC7 cells express Runx1 almost exclusively from the P1 promoter, such that DNA elements interacting with P1 are likely to activate gene expression. The active P1 promoter interacts with multiple non-coding regions both upstream and downstream of Runx1-P1. Interacting regions were overlapped with DNAse hypersensitivity peaks and transcription factor binding sites (available in the USCS genome browser) to identify putative enhancers for Runx1. The majority of putative enhancer regions function as blood specific enhancers in zebrafish (Table). Interestingly, the previously identified +24 enhancer interacted promiscuously within and outside of the 1 megabase Runx1 domain, indicating it may regulate the expression of many additional genes. Overall, our study of the Runx1 interactome identifies multiple novel hematopoietic enhancers that directly interact with the P1 promoter of Runx1, suggesting they are likely involved in regulating Runx1 expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Anti-apoptotic quinolinate phosphoribosyltransferase (QPRT) is a target gene of Wilms' tumor gene 1 (WT1) protein in leukemic cells

Wilms' tumor gene 1 (WT1) is a zinc finger transcription factor that has been implicated as an oncogene in leukemia and several other malignancies. When investigating possible gene expression network partners of WT1 in a large acute myeloid leukemia (AML) patient cohort, one of the genes with the highest correlation to WT1 was quinolinate phosphoribosyltransferase (QPRT), a key enzyme in the de novo nicotinamide adenine dinucleotide (NAD+) synthesis pathway. To investigate the possible relationship between WT1 and QPRT, we overexpressed WT1 in hematopoietic progenitor cells and cell lines, resulting in an increase of QPRT expression. WT1 knock-down gave a corresponding decrease in QPRT gene and protein expression. Chromatin-immunoprecipitation revealed WT1 binding to a conserved site in the first intron of the QPRT gene. Upon overexpression in leukemic K562 cells, QPRT conferred partial resistance to the anti-leukemic drug imatinib, indicating possible anti-apoptotic functions, consistent with previous reports on glioma cells. Interestingly, the rescue effect of QPRT overexpression was not correlated to increased NAD + levels, suggesting NAD + independent mechanisms. We conclude that QPRT, encoding a protein with anti-apoptotic properties, is a novel and direct target gene of WT1 in leukemic cells.

Evolution of malignant plasmacytoma cell lines from K14E7 Fancd2-/- mouse long-term bone marrow cultures.

We tested the effect of expression of the Human Papilloma Virus (HPV E7) oncogene on hematopoiesis in long-term bone marrow cultures (LTBMCs) derived from K14E7 (FVB) Fancd2−/− (129/Sv), K14E7 Fancd2+/+, Fancd2−/−, and control (FVB X 129/Sv) Fl mice. K14E7 Fancd2−/− and Fancd2−/− LTBMCs showed decreased duration of production of total nonadherent hematopoietic cells and progenitors forming day 7 and day 14 multilineage CFU-GEMM colonies in secondary cultures (7 wks and 8 wks respectively) compared to cultures from K14E7 Fancd2+/+ (17 wks) or control mice (18 wks) p < 0.0001. Marrow stromal cell lines derived from both K14E7 Fancd2−/− and Fancd2−/− cultures were radiosensitive, as were IL-3 dependent hematopoietic progenitor cell lines derived from K14E7 Fancd2−/− cultures. In contrast, Fancd2−/− mouse hematopoietic progenitor cell lines and fresh marrow were radioresistant. K14E7 Fancd2−/− mouse freshly explanted bone marrow expressed no detectable K14 or E7; however, LTBMCs produced K14 positive factor-independent (FI) clonal malignant plasmacytoma forming cell lines in which E7 was detected in the nucleus with p53 and Rb. Transfection of an E6/E7 plasmid into Fancd2−/−, but not control Fancd2+/+ IL-3 dependent hematopoietic progenitor cell lines, increased cloning efficiency, cell growth, and induced malignant cell lines. Therefore, the altered radiobiology of hematopoietic progenitor cells and malignant transformation in vitro by K14E7 expression in cells of the Fancd2−/− genotype suggests a potential role of HPV in hematopoietic malignancies in FA patients.

EVI1 Interferes with Myeloid Maturation via Transcriptional Repression of Cebpa, via Binding to Two Far Downstream Regulatory Elements

One mechanism by which oncoproteins work is through perturbation of cellular maturation; understanding the mechanisms by which this occurs can lead to the development of targeted therapies. EVI1 is a zinc finger oncoprotein involved in the development of acute myeloid leukemia; previous work has shown it to interfere with the maturation of granulocytes from immature precursors. Here we investigate the mechanism by which that occurs, using an immortalized hematopoietic progenitor cell line, EML-C1, as a model system. We document that overexpression of EVI1 abrogates retinoic acid-induced maturation of EML cells into committed myeloid cells, a process that can be documented by the down-regulation of stem cell antigen-1 and acquisition of responsiveness to granulocyte-macrophage colony-stimulating factor. We show that this requires DNA binding capacity of EVI1, suggesting that downstream target genes are involved. We identify the myeloid regulator Cebpa as a target gene and identify two EVI1 binding regions within evolutionarily conserved enhancer elements at +35 and +37 kb relative to the gene. EVI1 can strongly suppress Cebpa transcription, and add-back of Cebpa into EVI1-expressing EML cells partially corrects the block in maturation. We identify the DNA sequences to which EVI1 binds at +35 and +37 kb and show that mutation of one of these releases Cebpa from EVI1-induced suppression. We observe a more complex picture in primary bone marrow cells, where EVI1 suppresses Cebpa in stem cells but not in more committed progenitors. Our data thus identify a regulatory node by which EVI1 contributes to leukemia, and this represents a possible therapeutic target for treatment of EVI1-expressing leukemia.