Human Hematological Disorders Research Articles

Znf687 recruits Brd4-Smrt complex to regulate gfi1aa during neutrophil development.

Neutrophils are key component of the innate immune system in vertebrates. Diverse transcription factors and cofactors act in a well-coordinated manner to ensure proper neutrophil development. Dysregulation of the transcriptional program triggering neutrophil differentiation is associated with various human hematologic disorders such as neutropenia, neutrophilia, and leukemia. In the current study we show the zinc finger protein Znf687 is a lineage-preferential transcription factor, whose deficiency leads to an impaired neutrophil development in zebrafish. Mechanistically, Znf687 functions as a negative regulator of gfi1aa, a pivotal modulator in terminal granulopoiesis, to regulate neutrophil maturation. Moreover, we found BRD4, an important epigenetic regulator, directly interacts with ZNF687 in neutrophils. Deficiency of brd4 results in similar defective neutrophil development as observed in znf687 mutant zebrafish. Biochemical and genetic analyses further reveal that instead of serving as a canonical transcriptional coactivator, Brd4 directly interacts and bridges Znf687 and Smrt nuclear corepressor on gfi1aa gene's promoter to exert transcription repression. In addition, the ZNF687-BRD4-SMRT-GFI1 transcriptional regulatory network is evolutionary conserved in higher vertebrate. Overall, our work indicates Znf687 and Brd4 are two novel master regulators in promoting terminal granulopoiesis.

Label-free photoacoustic microscopy: a potential tool for the live imaging of blood disorders in zebrafish

The zebrafish has emerged as a useful model for human hematological disorders. Transgenic zebrafish that express green fluorescence protein (GFP) in red blood cells (RBCs) visualized by fluorescence microscopy (FLM) is a fundamental approach in such studies to understand the cellular processes and biological functions. However, additional and cumbersome efforts are required to breed a transgenic zebrafish line with reliable GFP expression. Further, the yolk autofluorescence and finite GFP fluorescence lifetimes also have an adverse impact on the observation of target signals. Here, we investigate the identification of intracerebral hemorrhage (ICH) and hemolytic anemia (HA) in zebrafish embryos using label-free photoacoustic microscopy (PAM) for imaging. First, ICH and HA in transgenic LCR-EGFP zebrafish are mainly studied by PAM and FLM. The results show that PAM is comparable to FLM in good identification of ICH and HA. Besides, PAM is more advantageous in circumventing the issue of autofluorescence. Secondly, ICH and HA in the transparent casper zebrafish without fluorescent labeling are imaged by PAM and bright-field microscopy (BFM). Because of the high contrast to reveal RBCs, PAM obviously outperforms BFM in the identification of both ICH and HA. Note that FLM cannot observe casper zebrafish due to its lack of fluorescent labeling. Our work proves that PAM can be a useful tool to study blood disorders in zebrafish, which has advantages: (i) Reliable results enabled by intrinsic absorption of RBCs; (ii) wide applicability to zebrafish strains (no requirement of a transgene); (iii) high sensitivity in identification of ICH and HA compared with BFM.

Hematologic disorder–associated Cxcr4 gain-of-function mutation leads to uncontrolled extrafollicular immune response

The extrafollicular immune response is essential to generate a rapid but transient wave of protective antibodies during infection. Despite its importance, the molecular mechanisms controlling this first response are poorly understood. Here, we demonstrate that enhanced Cxcr4 signaling caused by defective receptor desensitization leads to exacerbated extrafollicular B-cell response. Using a mouse model bearing a gain-of-function mutation of Cxcr4 described in 2 human hematologic disorders, warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome and Waldenström macroglobulinemia, we demonstrated that mutant B cells exhibited enhanced mechanistic target of rapamycin signaling, cycled more, and differentiated more potently into plasma cells than wild-type B cells after Toll-like receptor (TLR) stimulation. Moreover, Cxcr4 gain of function promoted enhanced homing and persistence of immature plasma cells in the bone marrow, a phenomenon recapitulated in WHIM syndrome patient samples. This translated in increased and more sustained production of antibodies after T-independent immunization in Cxcr4 mutant mice. Thus, our results establish that fine-tuning of Cxcr4 signaling is essential to limit the strength and length of the extrafollicular immune response.

The contribution of fish models in understanding of blood disorders

Human and fish peripheral blood contains erythrocytes or red blood cells, leukocytes or white blood cells and platelets or thrombocytes. Although the fish present the same types of blood cells as humans, studies have highlighted some morphological, ultrastructural and cytochemical differences in blood cells of the two groups of vertebrates. In both human and fish hematological tests are indispensable tools which provide a considerable amount of useful information for evaluation of the general state of health. Although there are limited amount of studies on blood cells in fish, over the years especially Danio rerio, has become an powerful model organism for studying human hematological diseases, as anemia, leukemia, myeloproliferative diseases and others human hematological diseases. Thereby, fish are ideal model organisms for studying hematological diseases due to external fertilization, brief development time, large number of embryos and the results can be extrapolated to mammals, including human. Furthermore, ikaros, rag-1, rag-2 and lck genes that are found in human were observed in Danio rerio revealing the spatio-temporal proximity between these groups of vertebrates. Therefore, studies realized on fish can offer essential information about molecular mechanism which causes the appearance of numerous human hematological diseases and discovery of possible therapeutic methods. This review provides a comparative overview of blood cells in fish and humans and describes zebrafish mutants as models of human hematologic disorders.

GATA2 mutations in myeloid malignancies: Two zinc fingers in many pies

The GATA family of transcription factors are zinc finger (ZF) DNA-binding proteins that regulate transcription during development and cell differentiation. GATA2 plays an essential role in the regulation of hematopoiesis. As a result, mutations in this gene or alterations in its expression level or function have been linked to a variety of human hematologic disorders. In this review, we summarize the findings and developments over the recent years regarding the clinical correlations and functional properties of distinct GATA2 mutations in hematopoietic malignancies, with particular focus on the mutational hotspots in the ZF domains.

The Engraftment of Lentiviral Vector-Transduced Human CD34+ Cells into Humanized Mice.

Humanized mouse models have been developed to study human hematopoiesis and therapeutic application of hematopoietic stem cell transplantation. To evaluate the safety and efficacy of lentiviral vectors for gene therapy, human CD34+ cells have been transduced with lentiviral vectors and transplanted into the humanized mice. Recipient mice are monitored over time and sacrificed for bone marrow analyses with regard to human cell engraftment, lineage distribution, and vector transduction. This chapter details the procedure for lentiviral transduction and transplantation of human hematopoietic stem/progenitor cells into humanized mice to study inherited human hematological disorders.

Clonal expansion and myeloid leukemia progression modeled by multiplex gene editing of murine hematopoietic progenitor cells

Recent advances in next-generation sequencing have identified novel mutations and revealed complex genetic architectures in human hematological malignancies. Moving forward, new methods to quickly generate animal models that recapitulate the complex genetics of human hematological disorders are needed to transform the genetic information to new therapies. Here, we used a ribonucleoprotein-based CRISPR/Cas9 system to model human clonal hematopoiesis of indeterminate potential and acute myeloid leukemia (AML). We edited multiple genes recurrently mutated in hematological disorders, including those encoding epigenetic regulators, transcriptional regulators, and signaling components in murine hematopoietic stem/progenitor cells. Tracking the clonal dynamics by sequencing the indels induced by CRISPR/Cas9 revealed clonal expansion in some recipient mice that progressed to AML initiated by leukemia-initiating cells. Our results establish that the CRISPR/Cas9-mediated multiplex mutagenesis can be used to engineer a variety of murine models of hematological malignancies with complex genetic architectures seen in human disease.

GATA family transcriptional factors: emerging suspects in hematologic disorders.

GATA transcription factors are zinc finger DNA binding proteins that regulate transcription during development and cell differentiation. The three important GATA transcription factors GATA1, GATA2 and GATA3 play essential roles in the development and maintenance of hematopoietic systems. GATA1 is required for the erythroid and megakaryocytic commitment during hematopoiesis. GATA2 is crucial for the proliferation and survival of early hematopoietic cells, and is also involved in lineage specific transcriptional regulation as the dynamic partner of GATA1. GATA3 plays an essential role in T lymphoid cell development and immune regulation. As a result, mutations in genes encoding the GATA transcription factors or alteration in the protein expression level or their function have been linked to a variety of human hematologic disorders. In this review, we summarized the current knowledge regarding the disrupted biologic function of GATA in various hematologic disorders.

Abstract 5531: OTX015, a novel BET inhibitor, is a promising anticancer agent for multiple myeloma

Abstract Introduction. The activity of BET inhibitors in several models human hematologic disorders, including multiple myeloma (MM) has recently been demonstrated. Exposure of cancer cells to BET inhibitors results in a significant down-regulation of c-Myc expression, and suppression of transcriptional programs associated with proliferation and survival. OTX015 is a new selective orally bioavailable inhibitor of the BET family proteins in clinical development. We evaluated its activity in a panel of MM models with known upregulation of the c-MYC pathway. Materials and Methods. Human cell lines derived from MM (RPMI 8226, KMM1, JJN3, OPM2, KMS27, U266, KMS18) were treated with increasing doses of OTX015 (OncoEthix SA, Switzerland). Cell proliferation was evaluated by ATPlite and MTT assays over time, and cell cycle analysis by FACScan flow cytometer. RNA was extracted using TRIzol and reverse-transcribed with the Superscript First-Strand Synthesis System kit. RT-qPCR was performed using SYBR Green Master Mix on a Bio-RAD Real-Time PCR System. For Western blot analysis, lysates were fractionated on 8% polyacrilamide gels and membranes incubated with commercial antibodies. OPM2 cells were inoculated (1*10^6 cells both flanks) in NSG mice and 72h after treatments started (vehicle; OTX015 50 mg/kg bid, po, 5 days on-2 days off).Tumor growth was measured over time. Results. OTX105 treatment resulted in G1 cell cycle arrest in a dose-dependent manner in all MM cell lines. Increased percentages of G1 cells (∼20%) were found after 24h exposure, reaching a maximum after 72h. OTX015 treatment for 24h followed by drug washout induced an initial cell cycle arrest, which was then overcome at 72-96h. RPMI 8226 and KMS27 cell lines also displayed increased apoptosis after prolonged exposure (> 48h) at 500 nM of OTX015. In 7 out of 9 MM cell lines, OTX015 treatment resulted in rapid downregulation of c-MYC mRNA and protein, while two cell lines (U266 and KMM1) displayed dose and time dependent c-MYC upregulation. Equivalent activity was observed with OTX015 (250 nM) and the OTX015 analog and bona fide BET-inhibitor, JQ1. In OPM2-bearing tumor mice, OTX015 treatment showed impaired tumor growth respect to controls after 4 weeks of treatment. Conclusions. OTX015 is a novel potent BRD-inhibitor with cytostatic anti-proliferative or pro-apoptotic activity in MM demonstrated by in vitro and in vivo experiments. The different changes of c-MYC expression in MM cell lines after OTX015 exposure suggest that the biological effects could be due to multiple and/or alternative mechanisms, which are not strictly linked to down-regulation of this transcription factor. These findings support the ongoing clinical development of OTX015 in MM patients. Citation Format: Maria Todaro, Michela Boi, Valentina Vurchio, Elisabetta Ercole, Rodolfo Machiorlatti, Katia Messana, Indira Landra, Susanna Urigu, Sabrina Aliberti, Eugenia Riveiro, Francesco Bertoni, Giorgio Inghirami. OTX015, a novel BET inhibitor, is a promising anticancer agent for multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5531. doi:10.1158/1538-7445.AM2014-5531

Engraftment Of Human Polycythemia Vera CD34+ Cells In hSIRPα-Transgenic-Human-TPO-Expressing RAG2-/-, IL2Rγ-/- Immunodeficient Mice

IntroductionXenotransplantation of human hematopoietic malignancies into immunodeficient mice represents the most appropriate in vivo model system for human malignant hematopoiesis. While a diversity of immunodeficient mouse strains on the NOD/SCID and RAG2-/-/IL2Rγ-/--BALB/C background are described for the most aggressive human malignancies like acute leukemias, xenotransplantation models of less aggressive human hematologic disorders like myeloproliferative neoplasms and myelodysplastic syndromes show only limited engraftment levels. We recently developed next generation mouse strains expressing human cytokines and key factors of xenogeneic cell acceptance (e.g. hSIRPα to inactivate mouse macrophage activation by human cells) and hypothesized that these would represent suitable models for the assessment of human less aggressive hematologic disorders in vivo by providing an optimized “humanized” microenvironment. MethodsPeripheral blood (PB) and bone marrow (BM) samples of polycythemia vera (PV) patients were collected after informed consent at the Division of Hematology, Zurich University Hospital. Human CD34+ cells were isolated by density gradient centrifugation followed by immunomagnetical selection using anti-CD34 coupled beads. Purity of magnetical selection process was confirmed by FACS analysis. Newborn (24h-48h old) hSIRPα-tg-hTPO-knockin mice on the RAG2-/-/IL2Rγ-/--BALB/C background received sublethal irradiation (split dose of 2x1.5 Gy) and were transplanted intra-hepatically 24 hours later. Transplanted cell dose was dependent on availability of CD34+ stem and progenitor cells isolated from one phlebotomy sample (∼400ml of PB) of the respective patient. Mice were bled 4 weeks after transplantation and chimerism in peripheral blood was analyzed by flow cytometry using a panel of antigens (mCD45.2, hCD45, hCD33, hCD34, hCD3, hCD19). Mice showing positive chimerism in PB (i.e.>0.1% hCD45+ of total MNCs) at week 4 were sacrificed between week 8-16 and engraftment in BM, spleen and PB was analyzed by flow cytometry. To verify engraftment of human malignant hematopoiesis we quantified allele-burden of JAK2V617F point mutation in mouse BM using allele specific (AS)-PCR for the pathognomonic point mutation of the JAK2 gene. ResultsBy transplantation of 4-10x105 CD34+ cells into newborn hSIRPα-tg-hTPO-RAG2-/-/IL2Rγ-/- mice we could detect engraftment of hCD45+ cells in PB at week 4 (median 0.68%, range 0.12-23.8%). At week 8, BM engraftment of hCD45+ cells ranged 0.88-54.1% (median 4.43%) with a high proportion of human myeloid cells detected by hCD45/hCD33 co-staining (median 3.07 %, range 0.6-17.6% of total MNCs). We could detect engraftment until week 16, the latest timepoint assessed. Since all transplanted PV patient samples were positive for the common point mutation JAK2V617F, AS-PCR was used to quantify human malignant hematopoiesis. In tested BM samples of engrafted mice we found JAK2V617F positive alleles with a frequency of 2-12% (median 8%). To further assess the clonal composition of the engrafted population we established single cell sorting of primary and engrafted human PV-CD34+ cells in a 96 well format followed by liquid culture expansion and AS-PCR. In pilot studies we could show the clonal composition of a BM engrafted CD34+ population that split into 80% JAK2 WT expressing, 10% JAK2V617F heterozygous and 10 % JAK2V617F homozygous clones. We are currently extending these findings by side by side comparison of the clonal composition of primary vs. xenografted human PV-CD34+ cells of the same patient to test for the influence of a xenogeneic humanized microenvironment on maintenance of malignant cells in vivo. ConclusionsBy using hSIRPα-tg-hTPO-RAG2-/-/IL2Rγ-/- mice we could show engraftment of PV-CD34+ cells that extends previous reported engraftment levels in other model organisms. To our knowledge this is the first study assessing the clonal composition of human PV engrafted cells in the xenogeneic environment aiming at identifying components that are critical for the maintenance of human malignant hematopoiesis in vivo. This model will thus be a useful tool to test targeted therapies in vivo. Disclosures:No relevant conflicts of interest to declare.

Evaluation of tyrosine-kinase receptor c-KIT (c-KIT) mutations, mRNA and protein expression in canine leukemia: Might c-KIT represent a therapeutic target?

The tyrosine-kinase receptor c-KIT (c-KIT) plays an important role in proliferation, survival and differentiation of progenitor cells in normal hematopoietic cells. In human hematological malignancies, c-KIT is mostly expressed by progenitor cell neoplasia and seldom by those involving mature cells. Tyrosine kinase inhibitors (TKIs) are actually licensed for the first- and second-line treatment of human hematologic disorders. Aim of the present study was to evaluate c-KIT mRNA and protein expression and complementary DNA (cDNA) mutations in canine leukemia. Eleven acute lymphoblastic leukemia (ALL) and acute undifferentiated leukemia (AUL) and 12 chronic lymphocytic leukemia (CLL) were enrolled in this study. The amounts of c-KIT mRNA and protein were determined, in peripheral blood samples, by using quantitative real time RT-PCR, flow cytometry and immunocytochemistry, respectively. The presence of mutations on c-KIT exons 8–11 and 17 were investigated by cDNA sequencing. Higher amounts of c-KIT mRNA were found in ALL/AUL compared to CLL, and this latter showed a lower pattern of gene expression. Transcriptional data were confirmed at the protein level. No significant gain-of-function mutations were ever observed in both ALL/AUL and CLL. Among canine hematological malignancies, ALL/AUL typically show a very aggressive biological behavior, partly being attributable to the lack of efficacious therapeutic options. The high level of c-KIT expression found in canine ALL/AUL might represent the rationale for using TKIs in future clinical trials.

Increased Expression of Bcl11b Leads to Chemoresistance Accompanied by G1 Accumulation

BackgroundThe expression of BCL11B was reported in T-cells, neurons and keratinocytes. Aberrations of BCL11B locus leading to abnormal gene transcription were identified in human hematological disorders and corresponding animal models. Recently, the elevated levels of Bcl11b protein have been described in a subset of squameous cell carcinoma cases. Despite the rapidly accumulating knowledge concerning Bcl11b biology, the contribution of this protein to normal or transformed cell homeostasis remains open.Methodology/Principal FindingsHere, by employing an overexpression strategy we revealed formerly unidentified features of Bcl11b. Two different T-cell lines were forced to express BCL11B at levels similar to those observed in primary T-cell leukemias. This resulted in markedly increased resistance to radiomimetic drugs while no influence on death-receptor apoptotic pathway was observed. Apoptosis resistance triggered by BCL11B overexpression was accompanied by a cell cycle delay caused by accumulation of cells at G1. This cell cycle restriction was associated with upregulation of CDKN1C (p57) and CDKN2C (p18) cyclin dependent kinase inhibitors. Moreover, p27 and p130 proteins accumulated and the SKP2 gene encoding a protein of the ubiquitin-binding complex responsible for their degradation was repressed. Furthermore, the expression of the MYCN oncogene was silenced which resulted in significant depletion of the protein in cells expressing high BCL11B levels. Both cell cycle restriction and resistance to DNA-damage-induced apoptosis coincided and required the histone deacetylase binding N-terminal domain of Bcl11b. The sensitivity to genotoxic stress could be restored by the histone deacetylase inhibitor trichostatine A.ConclusionsThe data presented here suggest a potential role of BCL11B in tumor survival and encourage developing Bcl11b-inhibitory approaches as a potential tool to specifically target chemoresistant tumor cells.

GATA Transcription Factors in Hematologic Disease

GATA family transcription factors play essential roles in broad developmental settings. GATA-1, one of the hematopoietically expressed members, is required for normal erythroid and megakaryocytic differentiation. Over the past few years, mutations in the gene encoding GATA-1 have been linked to several human hematologic disorders, including X-linked dyserythropoietic anemia and thrombocytopenia, X-linked thrombocytopenia and beta-thalassemia, and Down syndrome acute megakaryoblastic leukemia. This review summarizes the role of GATA-1 during normal hematopoiesis and discusses how disease-associated mutations may affect its function.

Zebrafish as a model of human hematologic disorders.

This review summarizes the status of zebrafish as a genetic model to study human hematological disorders. Much of our current understanding of the function of genes modulating the process of hematopoietic stem cell generation, specification, and differentiation has come from mutant analysis. Because of the transparency of zebrafish embryos that allows for direct visualization of circulating erythroid cells, mutations affecting zebrafish erythropoiesis were among the first characterized mutants through positional cloning and candidate gene strategies. New technologies have evolved that allow for generation, detection, and characterization of lineage specific alterations in the hematopoietic system. We will also briefly discuss the applications of several of these technologies such as targeted gene knockdown using antisense morpholinos, small molecule screen, transgenesis, and cell transplantation as related to blood disorders and hematopoietic development. The combination of phenotype-driven forward genetic analyses and innovative technical advances has conferred zebrafish as a powerful genetic model to further dissect the function of hematopoietic genes. Through the use of available resources, the identification of novel genes or novel function for known hematopoietic genes will have important implications for our understanding of human disease pathogenesis, treatment, and prevention.