Hereditary Thrombocytosis Caused By a Novel Germ-Line Mutation In The Gelsolin Gene

Abstract Hereditary thrombocytosis (HT) is a familial myeloproliferative disorder with clinical features resembling sporadic essential thrombocythemia. In some families germline mutations causing HT have been identified in the genes for thrombopoietin (THPO) and its receptor, MPL. However, in many HT families the disease-causing genetic lesion remains unknown. Here we studied a HT pedigree with 15 affected family members in 5 generations. Thrombocytosis is transmitted as an autosomal dominant trait with high penetrance (Figure 1). Genome-wide linkage analysis was performed on DNA from 21 family members using microsatellites and single nucleotide polymorphism arrays. A single co-segregating region with a LOD score of 4.3 was found on chromosome 9. All exons and splice junctions of genes within the co-segregating region were sequenced by classical DNA sequencing and by Next Generation Sequencing (Illumina). We found a candidate mutation in the gelsolin gene (GSN). This C/T transversion was not reported in any SNP database. Computational analysis predicts that the resulting Gly to Cys amino acid change will be damaging to the protein structure. Platelet biogenesis in vitro assays in DAMI cell line stably transfected with the mutant GSN showed that the candidate alteration increased the release of platelets-like particles. To study the in vivo role of the candidate mutation, we generated transgenic mice expressing the mutant GSN gene. These mice developed thrombocytosis and showed increased numbers of megakaryocytes in bone marrow (Figure 2). Thus, our genetic and functional data strongly suggest that GSN mutation is causing thrombocytosis in this family. The exact mechanism of how this newly identified genetic alteration leads to increased megakaryopoiesis needs further investigation. Disclosures: No relevant conflicts of interest to declare.

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New Approaches for Mapping Epigenome Dynamics

Blood ◽

10.1182/blood.v126.23.sci-21.sci-21 ◽

2015 ◽

Vol 126 (23) ◽

pp. SCI-21-SCI-21

Author(s):

Steven Henikoff

Keyword(s):

Transcription Factors ◽

Rna Polymerase Ii ◽

Conflicts Of Interest ◽

Regulation Of Transcription ◽

Base Pairs ◽

Simple Method ◽

Genome Wide ◽

Resolution Mapping

Abstract The protein complexes that package our genomes must be mobilized for active processes to occur, including replication and transcription, but until recently we have only had a static, low resolution view of the "epigenome". Genomes are packaged into nucleosomes, octamers of four core histones wrapped by 147 base pairs of DNA. Nucleosomes present obstacles to transcription, which over genes is the RNA Polymerase II (RNAPII) complex, and one current challenge is to understand what happens to a nucleosome when RNAPII transcribes through the DNA that it occupies. We study this process by developing methods for following nucleosomes as they are evicted and replaced. Among the factors that we have implicated in the process is torsional stress, which we can now measure genome-wide. RNAPII movement can unwrap nucleosomes and thus destabilize them, causing them to be occasionally evicted and replaced. Interestingly, we find that destabilization of nucleosomes during transcription is enhanced by anthracycline compounds, widely used chemotherapeutic drugs that intercalate between DNA base pairs, thus suggesting a new mechanism for cell killing during chemotherapy. We are also interested in what happens to RNAPII during its encounter with a nucleosomes. In vitro, RNAPII cannot transcribe completely through a nucleosome, but rather stalls as it tries to unwrap the DNA from around the core. We have been studying this process in vivo, and have developed a simple method for precisely mapping RNAPII genome-wide. We have used this method to show exactly where RNAPII stalls as it unwraps a nucleosome in vivo, surprisingly in a different place in vivo from where it stalls in vitro. We also have discovered that a variant histone, H2A.Z, which is found in essentially all eukaryotes, helps to reduce the nucleosome barrier to transcription, and in this way may modulate transcription. Other protein components of the epigenome involved in dynamic processes are nucleosome remodelers, which use the energy of ATP to slide or even evict nucleosomes from DNA. Some remodelers help RNAPII get started and others help it overcome the nucleosome barrier to transcription, and by mapping them at base-pair resolution, we can gain insight into how they act. We have also applied our high-resolution mapping tools to transcription factors, which bind DNA at specific sites to regulate transcription and other processes. Our ability to achieve high spatial and temporal resolution mapping of the binding and action of nucleosomes, transcription factors, remodelers and RNAPII provides us with a detailed picture of epigenome dynamics. By using these tools we are beginning to understand how DNA sequence and conformation are recognized for regulation of transcription and other epigenomic processes. Disclosures No relevant conflicts of interest to declare.

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Targeting Myeloid-Cell Specific Integrin α9β1 Inhibits Arterial Thrombosis in Mice

Blood ◽

10.1182/blood-2019-127006 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3581-3581

Author(s):

Nirav Dhanesha ◽

Manasa K Nayak ◽

Prakash Doddapattar ◽

Anil K Chauhan

Keyword(s):

Platelet Aggregation ◽

Arterial Thrombosis ◽

Conflicts Of Interest ◽

Myeloid Cell ◽

Cathepsin G ◽

In Vitro Assays ◽

Laser Injury

Background: Coordinated interactions between neutrophils, platelets and endothelial cells contribute towards the development of arterial thrombosis. Neutrophils along with platelets are the first immune cells that are recruited at the site of endothelial activation/injury or infection. Recent studies have suggested that neutrophils modulate thrombosis via several mechanisms, including NETosis (formation of neutrophil extracellular traps). The integrin α9 is highly expressed on neutrophils while platelets do not express it. The integrin α9 up-regulated upon neutrophil activation and is implicated in stable adhesion and transmigration. The mechanisms underlying the role of integrin α9 towards the progression of arterial thrombosis has not been explored yet. Objective: To elucidate the mechanistic insights into the role of myeloid-cell specific integrin α9 in neutrophil adhesion and arterial thrombosis. Methods: We generated novel myeloid-specific α9-/- mice (α9fl/fl LysMcre+l-) by crossing α9fl/fl with LysMcr+/+mice. Littermates α9fl/flLysMcre-l-mice were used as controls. Standardized in vitro assays were used to evaluate the role of integrin α9 in neutrophil mediated platelet aggregation, NETosis and Cathepsin-G release. Susceptibility to arterial thrombosis and hemostasis was evaluated in vivo (FeCl3-induced carotid and laser-injury induced mesenteric artery thrombosis models) by utilizing intravital microscopy and tail bleeding assay respectively. Results: α9fl/flLysMCre+/-mice developed smaller thrombi (~40% occlusion), when compared with α9fl/flmice (~80% occlusion, 10 minutes post-FeCl3 induced injury). The mean time to complete occlusion was significantly prolonged in α9fl/flLysMCre+/-mice (P<0.05 vs α9fl/fl mice). Consistent with this, α9fl/flLysMCre+/-mice displayed significantly decreased platelet mean fluorescence intensity (MFI) and reduced rate of thrombus growth in laser injury-induced thrombosis model (P<0.05 vs. α9fl/fl mice). Together, these results suggest that myeloid cell-specific integrin α9 contributes to the experimental thrombosis at arterial shear rates. Monocytes depletion experiments demonstrated a minimal role for monocyte in progression of arterial thrombosis. In vitro mechanistic studies demonstrated a reduction in neutrophil-mediated platelet aggregation and cathepsin-G secretion in myeloid cell-specific integrin α9-/- mice, when compared with litter-mates control wild-type mice. Notably, the percentage of cells releasing NETs was markedly reduced in myeloid cell-specific integrin α9-/- mice that was concomitant with reduced MPO levels in carotid thrombus of α9fl/flLysMCre+/-mice. Together, these results suggest most likely integrin α9 expressed on neutrophils, but not monocytes, promotes arterial thrombosis. Comparable tail bleeding time between α9fl/flLysMcreand littermate α9fl/fl mice suggested that myeloid-cell specific deficiency of integrin α9 does not alter hemostasis. Conclusion: These findings reveal a novel role for integrin α9 in modulation of arterial thrombosis. While the clinical implications of these findings remains to be explored, we suggest that targeting integrin α9 may reduce post reperfusion thrombo-inflammatory injury, following acute myocardial infarction or stroke. Disclosures No relevant conflicts of interest to declare.

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Forming Cytoplasmic Fragment Is Implicated In the Final Step of Platelet Production

Blood ◽

10.1182/blood.v116.21.4793.4793 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4793-4793

Author(s):

Shugo Kowata ◽

Kazunori Murai ◽

Kenichi Nomura ◽

Tatsuo Oyake ◽

Shigeki Ito ◽

...

Keyword(s):

Conflicts Of Interest ◽

Time Lapse ◽

In Vitro Assays ◽

Spatial Continuity ◽

Platelet Production ◽

Microscopic Evaluation ◽

Large Particles ◽

Time Lapse Imaging

Abstract Abstract 4793 Introduction: Conventional microscopic evaluation of bone marrow (BM) and in vitro assays have suggested that platelets arise from the proplatelet (PP) that extend from the mature megakaryocytes (MKs) in BM. On the other hand, recently, a study with in vivo imaging showed that MKs routinely release heterogeneous substantial large particles into BM sinusoids (Tobias Junt et. al., Science 317,1767, 2007). They noted that large particles may represent multiple intertwined or single immature proplatelets. However, it has been unclear whether the heterogeneous large particles consist of intertwined strings PPs or “cytoplasmic fragment (CF)”. Thus, in our study, to resolve this riddle, we planed to observe the dynamics of MKs with a modified imaging technique and we have cleared the presence and role of CF in platelet production. Materials and methods: Our study was approved by the Iwate Medical University Institutional Animal Care and Use Committee. 1) Mice: Six- to 8-week-old transgenic C57/BL6 (actin promoter driven EGFP) was used. 2) In vitro time lapse imaging of MK and PPF study: Primary mature MKs from femur BM were cultured for 12 hrs. Time lapse images were taken using Zeiss LSM510 meta confocal microscope (CLM). 3) 3-D reconstitution imaging of fixed BM study: the BM core was removed and immediately fixed and stained with PE-conjugated anti-CD61 antibody. Images were taken using CLM and reconstituted to 3-D images to keep the continuity between MK cell body and PP. 4) BM imaging by Multiphoton intravital microscopy (MP-IVM): Mice were anesthetized, and the frontoparietal skull was exposed. To trace individual MK over time in BM of living mice, time lapse images were taken. Results: By in vitro time lapse imaging of MK study, it become clear that primary cultured MK formed CF in which morphology was distinctly different from PP (Fig.1). Reversible interconversion between CF and PP was observed also. We observed that CF formation was more augmented in the presence of other BM cells. Because 3-D reconstitution imaging of fixed BM study has a benefit to observe amorphous structure without breaking of spatial continuity, we successfully proved the presence of CF and PP in BM sinusoid clearly (Fig.2). BM imaging by MP-IVM demonstrated that MK formed CF and extended protrusions into sinusoids. We have proved that MK formed and extended CF and PP coincidentally into sinusoids (Fig.3). Discussion: We had taken an evidence of presence of CF by in vitro time lapse imaging and 3-D reconstitution imaging. The meaning of reversible interconversion between CF and PP remains unclear in our study. This fact may closely associate with the efficacy of platelet production and avoiding precocious platelet activation in BM. The result that MK produced and extended CF and PP coincidentally suggests that both PP and CF formation may be essential for platelet production process. In conclusion, MK forms PP and CF in living BM. Both PP and CF have critical roles in platelet production mechanism. Disclosures: No relevant conflicts of interest to declare.

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PSC-Derived Hematopoietic System to Elucidate the Cooperation Between Gene Alterations and Cell Lineages in Leukemogenesis

Blood ◽

10.1182/blood.v128.22.1522.1522 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1522-1522

Author(s):

Akira Niwa ◽

Megumu K Saito ◽

Tatsutoshi Nakahata

Keyword(s):

Chromosomal Abnormalities ◽

Conflicts Of Interest ◽

Disease Model ◽

In Vitro Assays ◽

Cell Lineages ◽

Common Causes ◽

Pediatric Aml ◽

Gene Alterations

Abstract Onset of acute myeloid leukemia (AML) has been accounted for by accumulated genetic mutations including chromosomal abnormalities. For example, MLL fusion genes, which have been proven to impair cell differentiation, proliferation and epigenetic regulations, are among the common causes of pediatric AML. However, although those alterations are thought to occur in very immature stages, leukemia cells often show phenotypes similar to specific later-stage progenitors such as myeloid and monocytic cells. Until today, it remains unclear how those lineage specifications and mutations are cooperatively involved in disease pathogenesis. To address this issue in reproducible manner, we generated the lines of human pluripotent stem cells (hPSCs) harbouring doxycycline (Dox)-inducible leukemic gene cassettes, applied them for hematopoietic differentiations in a step-wise manner, and sought to identify the mechanisms behind the phenomena. Using our system, we first evaluated the in vitro and in vivo phenotypes compatible with AML symptoms. When MLL-AF9 (MF9) transgene expression was induced in combination with cKIT (822K) or FLT3-ITD mutations, PSC-derived hematopoietic cells showed reinforced growth in liquid culture and prolonged colony forming efficacies in serial replanting assays in methylcellulose-containing semisolid media. In addition to in vitro assays, in vivo transplantations also showed the prolonged detection of graft cells as long as eight months, while those sets of "leukemia-like" phenotypes were not observed under the condition of any single transgene induction. In order to find what lineages were most responsible for those phenomena, we next induced the combination of MF9 and FLT3-ITD in various sorted subpopulations of cells. As a result, CD34+/-CD43+CD13+ myeloid precursors showed the strongest tendencies to emerge highly proliferative clones followed by CD34+CD43+CD13- immature progenitors. In the contrast, CD34-CD71+CD41+ erythro-megakaryocytic cells hardly emerged those kinds of long-term expanding clones. Those results together indicated the bias of cell lineages and stages in our disease model, and encouraged us to explore a key pathway. Interestingly, we found that a set of NFkB pathway-associating genes were significantly activated when the transgenes were induced not in erythroid but in myeloid cells, which indicated the myeloid specific mechanisms forming a bridge between this pathway and leukemic gene alterations. In conclusion, we succeeded in establishing the way to dissect the leukemogenesis from the view of relationships between cell stages and gene alterations using PSCs. We believe that our model will enable us to better understand the pathogenesis of leukemia. Disclosures No relevant conflicts of interest to declare.

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Redundancy of primary RNA-binding functions of the bacterial transcription terminator Rho

Nucleic Acids Research ◽

10.1093/nar/gku690 ◽

2014 ◽

Vol 42 (15) ◽

pp. 9677-9690 ◽

Cited By ~ 20

Author(s):

Rajesh Shashni ◽

M. Zuhaib Qayyum ◽

V. Vishalini ◽

Debashish Dey ◽

Ranjan Sen

Keyword(s):

Rna Binding ◽

Classical Model ◽

In Vitro Assays ◽

Elongation Complex ◽

Transcription Terminator ◽

Bacterial Transcription ◽

Genome Wide ◽

Termination Function

Abstract The bacterial transcription terminator, Rho, terminates transcription at half of the operons. According to the classical model derived from in vitro assays on a few terminators, Rho is recruited to the transcription elongation complex (EC) by recognizing specific sites (rut) on the nascent RNA. Here, we explored the mode of in vivo recruitment process of Rho. We show that sequence specific recognition of the rut site, in majority of the Rho-dependent terminators, can be compromised to a great extent without seriously affecting the genome-wide termination function as well as the viability of Escherichia coli. These terminators function optimally only through a NusG-assisted recruitment and activation of Rho. Our data also indicate that at these terminators, Rho-EC-bound NusG interaction facilitates the isomerization of Rho into a translocase-competent form by stabilizing the interactions of mRNA with the secondary RNA binding site, thereby overcoming the defects of the primary RNA binding functions.

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Evaluation of Novel S-Allyl Derivatives of 6-Mercapto-Purine against B-CLL: Combining the Effects of Different Compounds in A Single Molecule.

Blood ◽

10.1182/blood.v114.22.3437.3437 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3437-3437

Author(s):

Fabian David Arditti ◽

Mordechai Shtalrid ◽

Lucette Bassous ◽

Lev Shvidel ◽

Alain Berrebi ◽

...

Keyword(s):

Single Molecule ◽

Conflicts Of Interest ◽

Annexin V ◽

Daily Basis ◽

High Reactivity ◽

In Vitro Assays ◽

Dependent Manner ◽

Purine Analogs

Abstract Abstract 3437 Poster Board III-325 Previously, we have shown that Allicin, the highly active compound of freshly crushed garlic, produced by the reaction of the enzyme Alliinase with its substrate Alliin, induced the apoptotic killing of B-CLL cells in vitro. In addition, we also reported that generation of Allicin in situ on the surface of B-CLL cells by targeting Alliinase to the cell surface of the CD20+ cells by Rituximab, resulted in the eradication of primary B-CLL in a human-mouse chimeric model, denoting the marked anti-CLL potential of combining these two different molecules, with different mechanism of action, into a single drug entity (Arditti et al., Mol Cancer Ther 2005;4(2)325-331). Indeed, monotherapeutic approaches, even if effective, are usually not sufficient to fully eradicate B-CLL and the most effective therapeutic protocols require the utilization of more than one agent. With this in mind, we took advantage of the high reactivity of Allicin, with SH-containing compounds, and created novel chimeric compounds by the combination of Allicin with 6-Mercapto-Purine (6MP) and 6MP-riboside (6MPR), both SH-containing purine analogs used for decades for the treatment of hematologic malignancies. The resulting novel compounds, S-Allyl-6MP (SA-6MP) and S-Allyl-6MPR (SA-6MPR), were examined against primary B-CLL cells obtained from the peripheral blood of patients at Binnet stage C. In our in vitro assays, Annexin-V staining indicated that SA-6MP acted in a dose dependent manner, inducing the apoptotic death of 37.9% and 95.2% of plated CD19+CD5+ B-CLL cells (10.9% in untreated cells) incubated for 16 h at 37 °C in the presence of 50 uM or 100 uM, respectively. As expected, the original 6MP compound had no impact on the viability of plated B-CLL cells (9.7% and 8.7%) at doses of up to 150 uM. In preliminary in vivo experiments, we compared the anti-BCLL activity of SA-6MP with that of SA-6MPR and the original 6MP compound on primary B-CLL cells from 5 different patients (Binnet stage C) in a human-SCID/Beige mouse model. Following the engraftment of the human B-CLL cells, mice were treated with i.p. injections of 2.5 mg/kg body weight of SA-6MP, SA-6MPR, or 6MP on a daily basis throughout 7 consecutive days, after which, the engraftment of primary B-CLL cells was examined by the recovery of CD45+CD19+CD5+ from injected mice. An additional group of mice injected with vehicle (1% DMSO) was also examined as a control. In close similarity to our in vitro results, engraftment of primary B-CLL cells was considerably reduced following treatment with SA-6MP (>90% reduction), as compared with treatment with the original 6MP drug. In addition, the chimeric riboside 6MP derivative, SA-6MPR, induced a potent anti-BCLL effect comparable to that of SA-6MP. In summary, our results in vitro and in vivo suggests that combining the pro-apoptotic effects of Allicin with the antiproliferative effects of 6MP or 6MPR is superior to the effect of either of the purine analogs alone. This approach may be evaluated at first instance in B-CLL patients with refractory disease. Disclosures No relevant conflicts of interest to declare.

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A Subset of MicroRNAs and Genes Involved in AML Has a Pivotal Role in the in Vitro differentiation of Hematopoietic Stem Cell Precursors

Blood ◽

10.1182/blood.v118.21.1290.1290 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1290-1290

Author(s):

Julian Pulecio ◽

Leopoldo Laricchia-Robbio ◽

Juan Carlos Izpisua ◽

Montserrat Barragan ◽

Marianna Vitaloni ◽

...

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Embryonic Stem ◽

In Vitro Assays ◽

Main Role ◽

In Vitro Differentiation ◽

Related Factors ◽

Hematopoietic Stem

Abstract Abstract 1290 After the finding of a set of transcription factors capable of reprogram any somatic cell into an embryonic stem-like cell by Yamanaka's group a lot of effort has been put to differentiate and produce in-vitro engraftable cells that could replace and fix damaged tissues. One of the most attractive and promising fields is the differentiation towards blood, considering it is a tissue without a complex tridimensional structure and that the phenotypes of the different sublineages are already well characterized. Nonetheless, so far there are no reports of successful differentiation into blood progenitors which are able to completely recover functionally in vivo blood-depleted mice. We previously reported the differentiation from induced pluripotent stem cells (iPS) towards hematopoietic cells capable of distinguish into sub lineages in in vitro assays, while another group obtained blood precursors by transdifferentiation of fibroblasts; however a complete recovery of the hematopoietic lineages in vivo was not seen. Our hypothesis is that the gap missing in the current protocols to obtain repopulating blood stem cells can be filled by the microRNA profiling of Cord Blood (CB) progenitors, in order to find the key players in the maintenance of blood stemness. In particular, it has been shown that population with the highest capacity to be engrafted in mice is the CD34+/CD90+ from CB. Our preliminary results depict a set of miRNAs that are specifically overexpressed in the CD34+/CD90+ population from CB cells when compared against a less specific CD34+ population. These miRNAs are currently being tested as a tool to improve the efficiency of iPS differentiation and fibroblasts conversion towards blood progenitors by means of lentiviral infection of the miRNA precursors. Interestingly, we have found that these miRNAs have been previously reported to have a main role in the occurrence of Acute Myeloid Leukemia in humans and mice. These results led us to look for genes that are highly expressed in blood progenitors but also have been shown to be correlated with AML.As a safety study, we are currently evaluating the effect of overexpressing AML related factors (miRNAs and genes) when added to the established protocols to obtain blood progenitors from iPS and fibroblasts. Surprisingly, our initial results show that the overxpression of the above mentioned genes and miRNAs have an intrinsic potential to induce in vitro differentiation or conversion from iPS and fibroblasts towards blood progenitors. Disclosures: No relevant conflicts of interest to declare.

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Dissection of BET Protein Function in a Hematopoietic Differentiation Model

Blood ◽

10.1182/blood.v124.21.1574.1574 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1574-1574

Author(s):

Aaron J Stonestrom ◽

Sarah C Hsu ◽

Kristen S Jahn ◽

Chris CS Hsuing ◽

Peng Huang ◽

...

Keyword(s):

Transcriptional Activation ◽

Conflicts Of Interest ◽

Gene Activation ◽

Family Members ◽

Model Organisms ◽

Dependent Manner ◽

Genome Wide ◽

Bet Inhibitors ◽

Bet Protein

Abstract The mammalian Bromodomain and Extra-Terminal motif (BET) proteins associate with acetylated histones and transcription factors including the master erythroid regulator GATA1. Pharmacologic inhibitors that broadly target BET family proteins are being evaluated in clinical trials for hematologic malignancies. Additionally, a rapidly growing number of studies in model organisms and in vitro systems suggests BET modulation is a therapeutic avenue in diverse disorders. However the mechanisms through which BET proteins act as well as the contributions of individual BET family members to biological processes remain mostly uncharacterized. Previously we showed that BET family members Brd3 and Brd4 can bind to GATA1 in an acetylation-dependent manner and that pharmacologic BET inhibition impairs erythroid maturation (Lamonica et al PNAS 2011). Using a combination of genome-wide occupancy analysis, pharmacologic inhibition, genome editing and knockdown we characterized the mechanism and function of BET proteins in the context of GATA1-driven erythroid differentiation. BET inhibitors prevented GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, GATA1 required BETs both for initial chromatin association and for transcriptional activation following the establishment of GATA1 occupancy. As BET proteins associate with chromatin during mitosis when transcription is globally disrupted, they have been implicated as mitotic “bookmarks”. BET inhibition specifically during mitosis failed to elicit a measurable impact on post-mitotic gene activation, calling into question a role of BET proteins as mitotic bookmarks in this system. To determine the BET protein(s) most relevant to GATA1-activated transcription, we genomically disrupted BET family members Brd2, Brd3 and Brd4 using the CRISPR/Cas9 tool. Despite being present at nearly all GATA1-occupied sites genome-wide, Brd3 disruption had little impact on GATA1-dependent transcription. In contrast, both Brd2 and Brd4 were individually required for efficient gene activation by GATA1. A Brd3 requirement was only revealed in the setting of Brd2 deficiency, indicating functional compensation among select BET proteins. These results provide a comprehensive definition of the functions of BET proteins in a model of cellular differentiation. They further suggest that pharmacologic BET inhibition should be viewed in the context of distinct steps in transcriptional activation and overlapping functions among BET family members. Disclosures No relevant conflicts of interest to declare.

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CD38 Regulates Homing and Engraftment of CLL Cells,

Blood ◽

10.1182/blood.v118.21.3873.3873 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3873-3873

Author(s):

Tiziana Vaisitti ◽

Sara Serra ◽

Valentina Audrito ◽

Chris Pepper ◽

Davide Rossi ◽

...

Keyword(s):

Cell Line ◽

De Novo ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

In Vitro Assays ◽

Wild Type ◽

Proliferating Cells

Abstract Abstract 3873 Chronic lymphocytic leukemia (CLL) is considered the result of a dynamic balance between proliferating cells in lymphoid organs and circulating cells resisting apoptosis. Re-circulation of leukemic cells from blood to growth-permissive niches represents an essential step in the maintenance and progression of the disease. This equilibrium is finely tuned by a set of surface molecules expressed by CLL cells and modulated in response to environmental conditions. We previously reported that CD38, an enzyme and a receptor, functionally cooperates with the CXCL12/CXCR4 axis, enhancing the ability of CLL cells to home to bone marrow and lymph nodes. In addition, the use of anti-CD38 mAbs can enhance or impair the chemotactic behavior of the neoplastic cells. New evidence also indicates that CD38 synergizes with the CD49d integrin, increasing adhesion of CLL cells to VCAM-1 or the CS-1 fibronectin fragment, two known ligands of CD49d. To complete the picture, CD38 expression denotes a CLL subset with increased activity of the matrix metalloproteinases MMP-9. Ligation of CD38 with specific antibodies increases MMP-9 secretion and the invasive properties of CLL cells, using in vitro assays. The effects on chemotaxis, adhesion and invasion are obtained through modulation of a ERK1/2-dependent pathway. To further confirm the involvement of CD38 in CLL homing to specific niches, in vivo experiments have been set using NOD/SCID/γ chain−/− (NSG) mice. The CLL-like cell line Mec-1, constitutively CD38−/CD49d+, was adopted as a model and compared to transfectants stably expressing wild-type (wt) CD38, as well a mutant lacking enzyme activities. Results after i.v. injections of tumor cells indicate that de novo expression of CD38 by Mec-1 cells increases growth kinetics in vivo with a higher proliferation rate and metastatic potential, as compared to the Mec-1 mock-trasfected cells. Both these features are lost when the animals are injected with the enzyme-deficient variant of CD38, suggesting that the enzymatic activity is critical for in vivo growth and re-circulation of Mec-1 cells. Microarray data confirm that the genetic signature of the CD38-enzyme mutant overlaps with the wild-type cell line, clearly distinct from cells transfected with CD38. The latter cell line shows up-modulation of several genes involved in chemotaxis and adhesion. All together, these results support the notion that CD38 is part of a complex network of molecules and signals, that regulate homing of CLL cells to growth-permissive niches, suggesting a relationship between the expression of CD38, the ability to migrate and invade and the poor clinical outcome of the CD38+ subset of patients. Disclosures: No relevant conflicts of interest to declare.

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A Small Molecular Agonist of Histone Demethylase KDM3B Selectively Represses MLL-Rearranged Acute Leukemia

Blood ◽

10.1182/blood-2018-99-112018 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4682-4682 ◽

Cited By ~ 1

Author(s):

Xin Xu ◽

Wilhelm G Dirks ◽

Hans G. Drexler ◽

Zhenbo Hu

Keyword(s):

Dna Methylation ◽

Acute Leukemia ◽

Histone Acetylation ◽

Conflicts Of Interest ◽

Family Members ◽

Cell Lineage ◽

Hematopoietic Stem ◽

Acute Myeloid

Abstract Background: Acute leukemia (AL) originates from both genetic and epigenetic changes that can be targeted to cure AL. Dysregulated DNA methylation has been shown to be associated with AL and demethylating agents 5-azacytidine and decitabine show favored improvement in secondary leukemia. Deficient histone acetylation has also been reported in AL and can be corrected to relieve leukemia. Histone methylation harbors more structural complexities compared to DNA methylation and histone acetylation and is broadly involved in AL. In particular, histone H3 lysine 9 (H3K9) methylation has been associated with AL. Di-methylation of H3K9 is reportedly involved in human hematopoietic stem cell lineage commitment. Moreover, tri-methylation of H3K9 predicts AML survival. H3K9 demethylation is catalyzed by exclusive KDM3 family members (KDM3A, KDM3B, and JMJD1C) that catalyze mono- and di-demethylation of H3K9, non-exclusive KDM4 family members (KDM4A, KDM4B, KDM4C, and KDM4D) that catalyze both H3K9 and H3K36 di- and tri-demethylation, KDM1A (LSD1) that catalyzes H3K4 and H3K9 mono- and di-demethylation, and PHF8 that catalyzes H3K9 mono- and di-demethylation and H4K20 demethylation. Among these, KDM3B, JMJD1C, KDM4C, LSD1, and PHF8 have been reported to be associated with AL in an enzymatic activity-dependent way. Furthermore, small molecular inhibitors of KDM4C and LSD1 have been developed for treatment of AML. H3K9 demethylase KDM3B is located at chromosome 5 band 31, a region frequently deleted or lost in acute myeloid leukemias (AML) and myelodysplasias (MDS). Different from other H3K9 demethylases that are usually responsible for leukemia maintenance, KDM3B harbors potential tumor-suppressive activities in acute myeloid leukemia and myelodysplastic syndromes. However, small molecular antagonists and agonists are lacking for KDM3B. Results: We aim to identify small molecular modulators of KDM3B. We focused on crystal structure of KDM3B Jumonji domain that catalyzes histone demethylation for virtual screening. From approximately 200,000 natural products and Chinese medicine components, we identified a potential KDM3B modulator, namely compound #7. Surface plasmon resonance technology showed that compound #7 binds to KDM3B with favorable affinity. In vitro and in vivo demethylation assay showed that compound #7 is able to increase H3K9 demethylating activity of KDM3B. We thus named compound #7 as KA-7 (KDM3B agonist #7). Interestingly, the identified KDM3B agonist KA-7 is able to selectively repress MLL-rearranged AL in cell proliferation and colony formation assays. Considering that KA-7 targets KDM3B that is located at chromosome 5q, a frequently deleted region in AML and MDS, we explored if KA-7 collaborates with Lenalidomide, an FDA approved drug for treating MDS with deletion at 5q where KDM3B is located. KA-7 was found to be able to synergistically increase the selective killing of AL cells by Lenalidomide. Conclusion: To sum up, physiologic H3K9 demethylase activity of KDM3B can be enhanced by a small molecular modulator KA-7 and causes selective killing against MLL-arranged AL cells. Disclosures. No relevant conflicts of interest to declare. Disclosures No relevant conflicts of interest to declare.

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