RhoA Is Essential for Maintaining Normal Megakaryocyte Ploidy Distribution and Platelet Generation

Abstract Abstract 385 The small GTPase, RhoA orchestrates actin cytoskeletal dynamics, which plays a role in platelet development and function. After platelet activation, RhoA rearranges the cytoskeleton by facilitating shape change, granule release, and clot retraction. In addition, RhoA is involved in platelet development. It does this by presumably regulating cytokinesis during megakaryocyte-erythroid progenitor cell expansion and megakaryopoiesis. RhoA also regulates thrombopoiesis by coordinating end bifurcation of the proplatelet extensions to amplify preplatelet numbers and the regulation of platelet release. Previously, most studies utilized pharmacological Rho inhibitors, such as C3 exotoxin. This could potentially obfuscate the data because of the incomplete knockdown of RhoA, the non-specific disruption of closely related Rho family members, or the long incubation times that could alter platelet biology. Therefore, we developed a transgenic mouse model that knocked out RhoA only in megakaryocytes and in platelets by using a CRE-LOX strategy to further investigate the role of RhoA in platelet biology. First, mice were generated that had loxP sites flanking the 3rd exon of RhoA (RhoAfl/fl). These mice were then crossed with mice expressing CRE recombinase driven by the platelet factor 4 promoter (PF4 CRE+), thus limiting CRE expression only in megakaryocytes and in platelets. The offspring, RhoAfl/fl PF4CRE+ mice were phenotypically normal, and had normal complete blood counts, except for macrothrombocytopenia. Their platelet counts were 25 ±3% of that observed in their littermate controls, (RhoAfl/fl PF4CRE−) and their platelet size was 130 ±10% of their littermate controls. The lack of RhoA only disrupted aggregation, granule release, and clot retraction when stimulated at the lowest dosage of agonists. To determine the causes of macrothrombocytopenia in RhoAfl/fl PF4CRE+ mice, histological examination of the spleen showed that 26.0±13.3% of the megakaryocytes had pyknotic nuclei as compared to 1.0±0.5% of the controls. In the bone marrow, apoptosis was present in 6.8±3.3% of the RhoA null megakaryocytes, but only in 1.2 ±0.3% of the control megakaryocytes. Furthermore, flow cytometry revealed that megakaryocyte counts in the bone marrow were 51.5 ±4.2% lower than in that of the controls. To determine if lacking RhoA impairs normal megakaryocyte maturation, we measured DNA ploidy using propidium iodide and flow cytometry. In megakaryocytes derived from adult bone marrow or from cultured fetal livers (E13.5, 8 days culture), the RhoAfl/fl PF4CRE+ cells had higher ploidy, a lower number of 2N cells, and an increased number of 16N cells than megakaryocytes derived from control animals. Together these data show that loss of RhoA causes deficiency of megakaryocytes probably due to increased apoptosis, and also causes aberrant maturation of the surviving megakaryocyte. To analyze whether RhoA was also required for thrombopoiesis, cultured RhoA-null megakaryocytes derived from fetal livers were infused into recipient mice. The megakaryocytes lacking RhoA more rapidly release platelets during the first 3 hours post infusion than controls. However, unlike control platelets, the Rho-null platelets were essentially gone within 24 hours. We analyzed whether the increased release of knockout platelets could be due to the up-regulation of proplatelet generation since the deletion of RhoA might impair myosin activity and impair cortical tension. However, micropipette aspiration analysis, which mimics the shear forces found in the bone marrow sinusoid capillaries, showed that the RhoA-null megakaryocytes were less compliant than the controls, primarily due to their large size. These data suggest that the higher ploidy and larger sizes of RhoA knockout megakaryocytes causes them to lodge in the pulmonary capillary bed more quickly, and to rapidly release defective macrothrombocytes. In contrast, the smaller (and thereby more compliant) wild type-cell megakaryocytes fragmented more slowly into platelets through proplatelet extension. Together, our findings demonstrate that RhoA is essential for normal megakaryocyte survival, maturation, and thrombopoiesis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

CongenitalSTAT3mutation Mediates Primary Persistent Polymorphic Inflammatory Activation and T Cell Leukemogenesis

Blood ◽

10.1182/blood-2019-132138 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1054-1054 ◽

Cited By ~ 1

Author(s):

Hongxing Liu

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cell ◽

Lymph Nodes ◽

Conflicts Of Interest ◽

Sh2 Domain ◽

Janus Kinase ◽

Gingival Hyperplasia ◽

Hematopoietic Stem ◽

T Cell Leukemogenesis

Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways play a pivotal role in inflammation and immunity, among which, JAK/STAT3 pathway is the most potent and leads the crosstalk of immunity and oncogenesis. Somatic STAT3 activatingmutations have been found in about 40% of T cell large granular lymphocytic leukemia (T-LGLL) patients, most of which are located in exon 21 which encodes Src homology 2 (SH2) domain leading to the increased activity of aberrant STAT3 protein and the upregulation of its transcriptional targets. While germline STAT3activatingmutations represent a newly defined entity of immune dysregulations named infantile-onset multisystem autoimmune disease-1 (ADMIO1, #MIM 615952). Both the two diseases are rare and poorly understood. Here, we report a pedigree including a proband, a six-year-old girl, primarily manifesting as thrombocytopenia and lymphadenopathy and her father diagnosed as T-LGLL with pure red cell aplastic anemia without autoimmune disorders preceding or during his disease course. Morphology of the bone marrow smears of the proband indicated normal hyperplasia without evident dyspepsia or increased blast cells. However, the vacuoles in monocytes and the density and size of granules in neutrophils increased, and megaloblast transformation was observed in some neutrophils. (Fig. 1A, 1B) Biopsy of an enlarged lymph node showed the reactive follicular hyperplasia. (Fig. 1C) Whole exon sequencing and pedigree analysis of the family revealed the germline STAT3 c.833G>A/p.R278Hmutation harbored by the proband which originated de novo from her father who additionally carried a germline TAL1G62Rmutation and somatically accumulated an FLT3-ITD mutation. (Fig. 2) Through single-cell RNA sequencing, we also found the increase of circulating CD8+ T cells and the decrease of NK cells of the proband. (Fig. 3) The STAT3 target genes were generally overactivated, and the expression of cytokines decreased in transcription level. In the genes participating in JAK/STATs pathways, the expression of JAK3, STAT1, and STAT3was up-regulated significantly. (data not shown) Immunophenotype of the proband by flow cytometry confirmed change in immunocyte compartments, (Fig. 4) but the serum cytokine concentrations measured by flow cytometry yielded controversial results, that most of cytokines were moderately elevated, and IL-1β, IL-5, TNF-α, and IFN-γ were of the most evident. (data not shown) During the treatment and follow-up, Cyclosporin A (CsA) was efficient in maintaining her circulating platelets in the range of 166×109/L to 302×109/L, but the enlarged lymph nodes and hepatosplenomegaly had no response. Eleven months later, CsA was replaced by tacrolimusfor the severe gingival hyperplasia, which has efficiently stabilized her platelets count and normalized the enlarged lymph nodes, liver, and spleen. On the contrary, in the three and a half years' span of illness, the father was refractory to CsA and methotrexate (MTX), moreover, lethal bone marrow suppression was induced by one course of fludarabine. For the high level of HLA-I and HLA-II antibodies in the circulation, plantlets transfusions were only efficient after plasmapheresis. The father eventually died from pulmonary and gastrointestinal infection due to the failure of maternal HLA-haploidentical hematopoietic stem cell transplantation (HSCT). We comprehensively elaborated the immunophenotype of the proband and thoroughly elucidated the genetic alternations of the father which led to the T cell leukemogenesis, which brought new insight on these two rare diseases and highlighted a more scrupulous therapeutic strategy in T-LGLL with congenital mutations. Figure 1 Disclosures No relevant conflicts of interest to declare.

Download Full-text

Abnormal Distribution of Erythroid Progenitors Populations in Mice Lacking p45 NF-E2.

Blood ◽

10.1182/blood.v114.22.1988.1988 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1988-1988

Author(s):

Jadwiga Gasiorek ◽

Gregory Chevillard ◽

Zaynab Nouhi ◽

Volker Blank

Keyword(s):

Bone Marrow ◽

Red Blood Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Globin Genes ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Adult Mice ◽

Deficient Mice

Abstract Abstract 1988 Poster Board I-1010 The NF-E2 transcription factor is a heterodimer composed of a large hematopoietic-specific subunit called p45 and widely expressed 18 to 20-kDa small Maf subunits. In MEL (mouse erythroleukemia) cells, a model of erythroid differentiatin, the absence of p45 is inhibiting chemically induced differentiation, including induction of globin genes. In vivo, p45 knockout mice were reported to show splenomegaly, severe thrompocytopenia and mild erythroid abnormalities. Most of the mice die shortly after birth due to haemorrhages. The animals that survive display increased bone, especially in bony sites of hematopoiesis. We confirmed that femurs of p45 deficient mice are filled with bone, thus limiting the space for cells. Hence, we observed a decrease in the number of hematopoietic cells in the bone marrow of 3 months old mice. In order to analyze erythroid progenitor populations we performed flow cytometry using the markers Ter119 and CD71. We found that p45 deficient mice have an increased proportion of early erythroid progenitors (proerythroblasts) and a decreased proportion of late stage differentiated red blood cells (orthochromatic erythroblasts and reticulocytes) in the spleen, when compared to wild-type mice. We showed that the liver of p45 knockout adult mice is also becoming a site of red blood cell production. The use of secondary sites, such as the spleen and liver, suggests stress erythropoiesis, likely compensating for the decreased production of red blood cells in bone marrow. In accordance with those observations, we observed about 2 fold increased levels of erythropoietin in the serum of p45 knockout mice.Overall, our data suggest that p45 NF-E2 is required for proper functioning of the erythroid compartment in vivo. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Genetic and Pharmacologic Evidence Shows That Cdc42 GTPase Plays a Central Role in the Regulation of Both GPVI- and Non-GPVI-Dependent Activation of Platelets.

Blood ◽

10.1182/blood.v114.22.2997.2997 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2997-2997 ◽

Cited By ~ 1

Author(s):

Huzoor Akbar ◽

Kevin Funk ◽

Mark Berryman ◽

Joshua Raines ◽

Rehana Perveen ◽

...

Keyword(s):

Platelet Aggregation ◽

G Protein ◽

Conflicts Of Interest ◽

Therapeutic Potential ◽

Shape Change ◽

Specific Inhibitor ◽

Small Gtpases ◽

Clot Retraction ◽

Coated Surfaces ◽

Induced Aggregation

Abstract Abstract 2997 Poster Board II-975 Cdc42 and Rac1, members of the Rho family of small GTPases, play critical roles in reorganization of actin cytoskeleton in platelets. Previously we have shown that Rac1 GTPase is involved in regulation of platelet secretion and aggregation by diverse signaling pathways (J Thromb Haemost 2007; 5: 1747-55). Others have reported that Rac1 is essential for GPVI-, but not G protein-dependent platelet aggregation (Pflugers Arch. 2009; 457:1173-85). Cdc42 was recently reported to be involved in collagen, but not collagen related peptide (CRP), a GPVI specific agonist, induced platelet aggregation (Platelets 2008; 19: 199-210). In this study we have investigated the role of Cdc42 in regulation of platelet function by using complementary approaches of (a) mouse gene targeting of Cdc42, and (b) specific inhibition of Cdc42 activity by a newly identified chemical inhibitor of Cdc42, CASIN (Cdc42 activity-specific inhibitor). Platelets from Cdc42−/− mice exhibited a complete lack of filopodia formation and spreading on collagen coated surfaces. Threshold concentrations of collagen, CRP or thrombin failed to induce shape change or aggregation in platelets from Cdc42−/− mice compared with induction of shape change and maximal aggregation in platelets from Cdc42+/+ mice. Platelets from Cdc42−/− mice, as compared to Cdc42+/+ mice, exhibited a significant inhibition of CRP- or thrombin-induced secretion of ATP and release of P-selectin from the dense- and alpha-granules respectively. Increasing concentrations of the agonists only partially corrected the defective aggregation and secretion responses in Cdc42−/− platelets. These data provide the genetic evidence that Cdc42 is required for collagen, CRP and thrombin mediated platelet signaling and activation. Treatment of platelets with CASIN, but not a pharmacologically inactive analog, blocked collagen induced activation of Cdc42 without detectably affecting the Rac1 activity. Human platelets pre-incubated with CASIN (10 micro-M) exhibited a complete lack of filopodia formation and spreading on collagen coated surfaces. Further, treatment of platelets with CASIN (1-10 micro-M) inhibited: (a) aggregation induced by collagen, CRP, thrombin, ADP or U46619; (b) release of P-selectin and secretion of ATP induced by U46619; and (c) collagen induced phosphorylation of Akt. Addition of CASIN to platelets also blocked collagen or CRP induced aggregation in aspirinated platelets in the presence of apyrase. In other experiments, addition of CASIN to citrated platelet-rich plasma inhibited thrombin induced clot retraction. Significantly, removal of CASIN from the platelet samples by washing reversed inhibition of aggregation as well as clot retraction, reflecting a reversible suppression of Cdc42 activity by CASIN. Administration of CASIN into C57Bl/6 mice inhibited ex vivo platelet aggregation induced by collagen or ADP as well as significantly prolonged tail bleeding times. These data suggest that: (a) Cdc42 plays an essential, non-redundant role in platelet filopodia formation, spreading, secretion, aggregation and clot retraction; (b) Cdc42 is involved in GPVI, non-GPVI- and G protein-dependent signaling in platelets; (c) the pharmacologic inhibitor CASIN is capable of specifically and reversibly inhibiting Cdc42 activity in platelets, mimicking Cdc42 genetic knockout in mice. Altogether, our studies strongly implicate Cdc42 as a novel anti-platelet target, and present evidence that the Cdc42 specific small molecule inhibitor, CASIN, may have therapeutic potential. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

DISTINCT ROLES for Rap1b In PLATELET SECRETION and INTEGRIN aIIBb3 OUTSIDE-In SIGNALING

Blood ◽

10.1182/blood.v118.21.2200.2200 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2200-2200

Author(s):

Guoying Zhang ◽

Binggang Xiang ◽

Ye Shaojing ◽

Magdalena Chrzanowska-Wodnicka ◽

Andrew J. Morris ◽

...

Keyword(s):

Conflicts Of Interest ◽

Critical Role ◽

Wild Type ◽

Clot Retraction ◽

Platelet Factor ◽

Dense Granules ◽

Activation Mechanisms ◽

Platelet Spreading ◽

Inside Out ◽

Platelet Secretion

Abstract Abstract 2200 Rap1b is activated by platelet agonists, and plays a critical role in integrin aIIbb3 inside-out signaling and platelet aggregation. In this study, we identify two novel functions of Rap1b in platelets. We show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that aIIbb3 outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Defect in secretion of Rap1b deficient platelets was not due to reduced granule contents, because the amount of serotonin (5HT) and platelet factor 4 (PF4) in Rap1b deficient platelets is similar to that in wild type platelets. Data from transmission electron microscopy indicate that under resting conditions, wild type and Rap1b deficient platelets had normal discoid shapes with similar numbers of granules. When stimulated with thrombin, wild type platelets showed an irregular appearance with protruding filopodia and lack of granules. In contrast, thrombin-stimulated Rap1b−/– platelets showed that more Rap1b−/– platelets contained visible a granules than wild type platelets. Dense granules were also more obvious in thrombin-stimulated Rap1b−/– platelets than wild type platelets. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y12 or TP deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-BAPTA. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b deficient platelets compared with wild type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b deficient platelets were not rescued by addition of MnCl2, which elicits aIIbb3 outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin aIIbb3 outside-in signaling. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Impaired Precursor B-Cell Maturation/Production In The Bone Marrow: Association With Molecular and Immunophenotypic Markers Of Myeloid Dysplasia In Suspected Low-Grade MDS

Blood ◽

10.1182/blood.v122.21.4943.4943 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4943-4943

Author(s):

Charles Repetti ◽

Hsueh-Hua Chen ◽

Yongbao Wang ◽

Vanessa A Jones ◽

Albert K Ho ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Somatic Mutation ◽

Conflicts Of Interest ◽

Statistical Significance ◽

Sequence Variant ◽

Low Grade ◽

Precursor B Cells

Abstract Rationale Myelodysplastic syndromes (MDS) are clonal stem cell disorders that disrupt orderly maturation of multiple hematopoietic lineages. Several studies have suggested that maturation of precursor B cells (hematogones) is also abnormal in MDS. As a result, the presence of normal numbers or increased precursor B cells in bone marrow (BM) is frequently used as a diagnostic feature arguing against a diagnosis of MDS. We compared the presence of myeloid-associated gene mutations and myeloid maturation abnormalities with qualitative and quantitative precursor B cell findings in BM samples submitted for workup of cytopenias or MDS. Methods Seventeen BM aspirate samples with <5% blasts submitted for cytopenia or MDS evaluation were compared with 10 samples having 5% or more blasts and changes diagnostic of MDS or AML. Mutation analysis was performed on genomic DNA using a targeted exome sequencing assay. This assay employs a TruSeq custom amplicon design on the MiSeq platform (Illumina, San Diego, CA). The assay covers the commonly mutated areas of 19 myeloid-associated genes. Somatic mutation status was assigned based on mutation levels, previous association with myeloid neoplasia, and no prior identification in public or internal databases as a normal sequence variant. Flow cytometry using 6-color (CD19/CD34) and 8-color (CD19/10) formats was used to assess lymphoblasts; CD34/13 was used to assess myeloblasts; and CD11b, CD13, CD16, and CD38 were used to assess abnormalities in myelopoiesis. Results Among the 17 BM samples submitted for cytopenia or MDS evaluation that had <5% blasts, 7 (41%) had immunophenotypic myeloid maturation abnormalities. Ten (59%) of the 17 cases had at least one myeloid-associated somatic mutation, with TET2 and ASXL1being the most commonly mutated genes. The ratio of myeloblasts to B-lymphoblasts, calculated using either CD10 or CD19, was >10:1 in 10/17 (59%) cases. Nine of the 17 (53%) cases had virtually no precursor B cells detected. Discrete abnormalities in more mature myeloid forms were seen in 7/10 (70%) cases with low numbers of B-lymphoblasts but in none of the 7 cases with significant numbers of B-lymphoblasts. MDS-associated mutations were more common in cases with rare B-lymphoblasts (7/9) than in those with higher percentages of precursor B cells (3/8), but the difference did not reach statistical significance (P = 0.15). Genes mutated in the group with B-lymphoblasts present included ASXL1 (3 cases), DNMT3A (2), TET2 (1) and TP53 (2). Two of these mutated cases presented with isolated thrombocytopenia. By comparison, myeloblast/lymphoblast ratios were >50:1 in all 10 unequivocal MDS/AML samples (>5% blasts); 8 (80%) of these cases had MDS-associated mutations, and 4 (50%) had mutations in multiple genes. Conclusions Decreases in BM precursor B cells in cases of possible low-grade MDS were usually, but not always, associated with the presence of MDS-associated mutations. However, cases with normal or increased precursor B cell numbers also showed MDS-associated mutations although immunophenotypic evidence of myeloid maturation abnormalities was not seen in this group. The identification of a subgroup of cytopenic patients with likely pathogenic mutations in bone marrow precursors but minimal phenotypic evidence of myeloid dysplasia may indicate clonal abnormalities primarily located outside the granulocyte or common stem precursor populations, e.g. restricted to the megakaryocytic lineage. Therefore, the presence of intact precursor lymphoblast and myeloid maturation by higher-dimensional flow cytometry as a primary criterion to argue against a diagnosis of low-grade MDS needs further evaluation, especially when granulocytopenia is absent. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Erythro-Myeloblastic Island (EMBI): A Hematopoietic Niche Balancing Erythropoiesis and Myelopoiesis

Blood ◽

10.1182/blood-2018-99-118728 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 842-842

Author(s):

Katie Giger Seu ◽

Laurel Romano ◽

Julien Papoin ◽

Edward David Muench ◽

Diamantis Konstantinidis ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Multispectral Imaging ◽

Conflicts Of Interest ◽

Myeloid Cells ◽

Mouse Bone Marrow ◽

Erythroid Cells ◽

Murine Models ◽

Native Bone ◽

Hematopoietic Niche

Abstract Mammalian erythropoiesis has long been established to occur within erythroblastic islands (EBIs), niches where erythroblasts differentiate in close contact with a central macrophage. While it is generally accepted that EBI macrophages play an important role in regulation of erythropoiesis, very little is known about the specific macrophage populations involved in EBI formation, the regulation that occurs within EBIs, or how this niche fits into the broader context of hematopoiesis. We analyzed native EBIs isolated from mouse bone marrow using multispectral imaging flow cytometry (Seu et. al. Front Immunol 2017). Consistent with historical observations, the EBIs were heterogeneous and many contained a number of closely CD11b+ cells in addition to erythroblasts and a central F4/80+ macrophage. Flow cytometry analysis of cells dissociated from native bone marrow EBIs indicated these niches are also enriched 2-3 fold in myeloblasts and granulocytic precursors up to metamyelocytes relative to the total bone marrow while they are depleted of mature granulocytes (bands and segmented cells). Bulk RNAseq of the CD11b+ population isolated from EBIs showed high expression of genes characteristic of the granulocytic lineage (e.g. Elane, Mpo, Gfi1, Cebpe, Camp, and Mmp9), indicating the EBI macrophages may regulate myelopoiesis along with erythropoiesis and that EBIs should really be considered as erythro-myeloblastic islands (EMBIs). To critically document the various hematopoietic cell populations that constitute EMBIs, we used the 10x Genomics Chromium system to obtain single cell gene expression data on ~3,500 total cells from isolated EMBIs along with at least 1,000 sorted cells from each of the 3 major EMBI-associated populations (F4/80+, CD71+, and CD11b+) (Fig 1a, b). The data were analyzed using 10x Genomics' Loupe cell Browser and Iterative Clustering and Guide-gene Selection (ICGS, http://www.AltAnalyze.org, Olsson et. al. Nature 2016). From the ICGS analysis, ~30% of the total EMBI-associated cells were myeloid cells that segregated into at least 3 transcriptionally distinct clusters representing granulocytic progenitors and precursors. As expected, erythroblasts with a progressive maturation pattern made up the bulk (60%) of the EMBI-associated cells, while up to 10% were a heterogeneous population of cells that exhibited expression of macrophage markers such as Csf1R and Irf8, along with genes previously described to characterize resident macrophages, such as Fn1and Fsp1/S100A4 (Fig 1c). In order to investigate the balance of myeloid cells with erythroid cells within the EMBIs, we examined the ratio of CD71+ cells to CD11b+ and how this ratio changes in models of altered granulopoiesis. While the number of myeloid cells at any island varied, the overall ratio of CD11b+ area to CD71+ within the EMBIs was relatively constant at steady state. In three different murine models of anemia of inflammation (AoI), we found that this ratio of CD11b+ to CD71+ cells within the EMBI increases dramatically indicating that the increased granulopoiesis and suppression of erythropoiesis noted in AoI is a result of altered balance of the hematopoiesis within the EMBI unit. Similarly, stimulation of granulopoiesis with GCSF also results in a shift within the EMBIs to CD11b+ myeloid cells and suppression of erythroid cells. Alternatively, in gfi1 KO mice, a model of congenital neutropenia in which granulopoiesis fails at an early stage, the ratio shifts toward CD71+ erythroid cells with paucity of the granulocytic precursors that are typically found at the EMBIs. Taken together, these data indicate that granulocyte progenitors and precursors are specifically associated with EMBI macrophages in the mouse bone marrow. The preferential localization of myeloid precursors within EMBIs suggests this niche is a site for granulopoiesis as well as erythropoiesis and production of these lineages is dynamically regulated within this niche. Our work with multiple murine models of altered granulopoiesis demonstrates that pathological expansion of one of the lineages within this niche may suppress the other and that the interactions within the EMBI could be a useful therapeutic target for AoI. These novel findings significantly broaden our understanding of the role of this hematopoietic niche in the regulated development of lineage committed erythroid and myeloid cells. Disclosures No relevant conflicts of interest to declare.

Download Full-text

C-CBL Is a Critical Negative Regulator of Megakaryopoesis

Blood ◽

10.1182/blood.v124.21.1581.1581 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1581-1581

Author(s):

Sebastian J. Saur ◽

Melanie Märklin ◽

Alexandra Poljak ◽

Manuela Ganser ◽

David E. James ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Negative Regulator ◽

Cytokine Signaling ◽

Wild Type ◽

Platelet Factor ◽

Physiological Level ◽

Hematopoietic Stem ◽

Primary Mouse

Abstract Megakaryopoiesis is controlled by a variety of hematopoietic growth factors in order to maintain a physiological level of circulating platelets. Thrombopoietin (TPO) is the main regulator of megakaryopoiesis modulating megakaryocyte differentiation, promoting endomitosis and proplatelet formation and as such supports the self-renewal and survival of hematopoietic stem cells. To allow proper proliferation and differentiation of different hematopoetic lineages, TPO signal transduction must be tightly regulated. Several mechanisms negatively modulating hematopoiesis and differentiation of the megakaryocytic lineage have previously been identified. Among those are suppressors cytokine signaling, protein phosphatases as well as a multitude of negative regulatory signaling pathways. However, one of the most effective mechanisms to permanently disable activated signaling proteins is by targeted degradation via lysosomes or proteasomes. In this study, we investigated the mechanisms that regulate TPO-mediated MPL degradation in primary mouse cells. Previous studies have identified CBL as an E3 ligase responsible for the ubiquitination of MPL in cell lines. In order to determine the potential role of c-CBL in murine thrombopoiesis, we used Cre/loxP technology to specifically delete c-CBL in the megakaryocytic lineage. Mice expressing two floxed c-CBL alleles were crossed to mice expressing Cre recombinase under the control of the platelet factor 4 (PF4) promoter. This yielded progeny with the desired genotype of c-CBLfl/fl PF4-Cre (CBL ko) after two generations of breeding. The desired cohort exhibited a quantitative absence of c-CBL in megakaryocytes and platelets as assessed by western blotting compared with wild type C57/BL6 mice. The expression of CBL in other hematopoietic cells such as B cells, T cells, neutrophils, monocytes and dendritic cells remained unaffected in this conditional ko strain. The experimental cohort showed significantly higher numbers of megakaryocytes in the bone marrow and of platelets in the peripheral blood as compared to wild type mice (1.2 mio vs. 1.8 mio cells/µl, p<0.0001). In addition, the platelets from the mutant mouse strain were of significantly smaller size (43 vs. 38 fL, p=0.0022). To evaluate the role of c-CBL in mature megakaryocytes, total bone marrow was collected from 12 wk old CBL ko mice and grown in TPO-containing culture medium for 72 h. Megakaryocytes derived from the bone marrow of wild type mice served as controls. Mature megakaryocytes were eventually isolated on a BSA-density gradient. Subsequent Western Blot analysis revealed a significant reduction of MPL ubiquitination in the CBL ko mice as compared to wild type mice, thereby identifying c-CBL as a critical negative regulator of megakaryopoesis. Taken together, we have successfully ablated c-CBL specifically from the megakaryocyte lineage and could demonstrate that this has profound effects on platelet counts and platelet size. In addition, we were able to show that c-CBL ablation leads to reduced ubiquitination of MPL and a consecutively longer half life of this protein culminating in substantially increased megakaryopoiesis in the c-CBL ko cohort. In summary, these data enhance our understanding of the regulation of TPO signaling and the physiological role of CBL in the megakaryocytic lineage. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Immunophenotypic Response After Allografting In Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.3371.3371 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3371-3371 ◽

Cited By ~ 1

Author(s):

Luisa Giaccone ◽

Lucia Brunello ◽

Roberto Passera ◽

Moreno Festuccia ◽

Milena Gilestro ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Conflicts Of Interest ◽

Univariate Analysis ◽

Conditioning Regimen ◽

First Line ◽

Significant Difference ◽

The Impact

Abstract Background Minimal residual disease (MRD) by multiparameter flow-cytometry recently showed a promising role in predicting outcomes in patients with multiple myeloma. However, data on immunophenotypic response (IR) after allografting are lacking. Aim To evaluate the impact of IR and compare it to conventional complete remission (CR) following allografting in myeloma patients. Methods Sixty-six consecutive patients, median age 54 years (35-66), who underwent an allograft between January 2000 and December 2011 with a follow-up of at least 3 months were included. Disease response was evaluated by serum and urine electrophoresis, and bone marrow aspirate at baseline, 3, 6, 12, 18, 24 months after transplant and yearly thereafter. Skeletal survey or MRI were performed yearly or as clinically indicated (overt relapse or complaints of bone pain). Bone marrow aspirates had to contain at least 13000 cells/µL for flow-cytometry studies and IR was defined as absence of monoclonal plasma-cells detected by 4 or 6-colour staining with the following antibodies: CD38, CD138, CD56, CD19, CD45, cyKappa, cyLambda. CR was defined according to standard criteria (Durie et al, Leukemia 2006; 20:1467-73). Results Conditioning regimen was non-myeloablative 2Gy TBI-based in 55 patients, reduced intensity (fludarabine-melphalan-based) in 10 and myeloablative in 1 patient. Post-grafting immunosuppression consisted of cyclosporine with mycophenolate mofetil or methotrexate. Donors were HLA identical siblings in 58 patients and unrelated in 8. Only 1 patient received bone marrow as source of stem cells. Thirty-five/66 (53%) received the allograft as part of the first line treatment, whereas the remaining 31/66, (47%) were transplanted at relapse. At the time of transplant, 5/66 were both in IR and CR, 16 were only in IR and 4 patients were only in clinical CR. All 21 patients in IR at the time of transplant maintained it, while 26/45 (58%) entered IR after the allograft. Among patients surviving at least 3 months, overall treatment related mortality was 10.6% at 3 years. After a median follow-up of 69 months (range 19-147), the incidence of acute and chronic graft-versus-host disease was 45.6% and 49.3% without significant difference between responsive and non-responsive patients. At follow-up, overall, 24 patients achieved CR and IR (CR/IR group), 21 achieved IR but not CR because of persistence of urine/serum M-component (noCR/IR group), and 21 did not achieve either CR or IR (noCR/noIR group). Interestingly, none achieved CR without IR. Median overall survival (OS) and event-free survival (EFS) in patients who achieved IR were 96 and 55 months versus 36 and 7 months in those who did not (p<0.001). Median OS and EFS were not reached and 59 months in the CR/IR group, 77 and 15 months in the noCR/IR, and 30 and 5 months in the noCR/noIR respectively (p<0.001 for both EFS and OS-fig.1). In univariate analysis, being in the CR/IR group was the only significant predictor for prolonged OS and EFS (p<0.001). Of note, cumulative incidence of extra-medullary disease at first relapse after the allograft was 4% in the CR/IR, 32% in the noCR/IR and 15% in the noCR/noIR groups respectively (p<0.001). Receiving the allograft as first line therapy or later during the disease course did not significantly impact on OS and EFS. Conclusion The achievement of IR confers a favorable impact on OS and EFS after allografting. A higher incidence of extra-medullary in the noCR/IR group (some 30% of our patient cohort) may suggest that myeloma cells escape immune control outside the bone marrow. In this group, imaging studies such as positron emission tomography may clinically be indicated during follow-up to detect early relapse. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Sustained Growth of Primary Myeloma Cells in Coculture with Whole Donor Bone Marrow Is Associated with Induced Secretion of the Microenvironmental Mediator of Cytokinesis, Hemicentin-1

Blood ◽

10.1182/blood.v124.21.3403.3403 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3403-3403

Author(s):

Rakesh Bam ◽

Ricky D Edmondson ◽

Caleb K Stein ◽

Xin Li ◽

Wen Ling ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Extracellular Matrix ◽

Matrix Protein ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Extracellular Matrix Protein ◽

Focal Lesion ◽

Adherent Cells ◽

Bone Biopsies

Abstract Primary human myeloma (MM) cells do not survive in culture while current systems for growing these cells are limited to coculture with specific bone marrow (BM) cell type or growth in immunodeficient animals. The aim of the study was to establish a coculture system for studying long-term growth of primary MM and their interaction with whole BM microenvironment, namely normal bone marrow (NBM) system . Whole BM cells from healthy donors (n=20) were cultured in medium supplemented with serum (10% v/v) pooled from MM patients for 7 days followed by coculture with CD138-selected primary MM cells (4:1 NBM:MM ratio) or MM cell lines (10:1 NBM:MM ratio) for ≥7 days. This NBM system is composed of adherent and non-adherent compartments. Adherent cells were mainly macrophages and mesenchymal stem cells (MSCs) whereas non-adherent cells contained typical hematopoietic cells including CD19+, CD3+, CD11b+ and CD33+ cells. Growth of MM cells was determined by CD45/CD38 flow cytometry and by bioluminescence of luciferase-expressing MM cells. MM cells or subset of MM cells from all patients (n=60) survived and grew in this system regardless of molecular risk or subtype, and MM growth was comparable to coculture with the supportive osteoclasts or MSCs. Adherent and non-adherent compartments supported MM cells which required patient’s serum for optimal growth. In 14 of 20 experiments, number of MM plasma cells, quantified by flow cytometry or bioluminescence analysis was increased by 58±12% (p<0.0005) in the NBM system and cell proliferation was evident by the loss of cell membrane PKH26 dye or by BudR uptake in dividing cocultured MM cells. Growth of OPM2, H929 and ARP1 lines was also stimulated in the NBM system which protected these cells from dexamethasone (1-2.5µM) but not bortezomib (0.01-5nM), while the effect of lenalidomide varied (0.1-5µM). For identifying secreted proteins that may mediate MM growth in the NBM system, supernatant were collected from serum-free culture of NBM, MM cells and NBM/MM coculture (18 hrs, n=3). Proteomics analysis performed on supernatant samples identified 1843 proteins. The clinical markers B2M and LDHA were present at high levels and were significantly higher by 2-2.4 folds in NBM/MM coculture compared to cultured NBM (p<0.04). Further filtration revealed 89 proteins that were significantly changed upon NBM/MM coculture but minimally detected in the MM cells culture: 14 were significantly lower and 75 were higher in NBM/MM cocultures compared to cultured NBM. These factors include mediators of extracellular matrix, immunity, and inflammation. A microenvironmental secreted factor that was not detected in the supernatant from MM cells or NBM but was secreted in cocultures was hemicentin-1 (HMCN1), a unique extracellular matrix protein directly involved in cytokinesis (Xu and Vogel, Curr Biol 2011) but has yet not been implicated in MM. Hemicentin-1 gene expression was detected in cultured NBM and MSCs but not in primary MM cells, MM lines or CD11b+ NBM cells. Induction of hemicentin-1 expression in MSCs after coculture with MM cells was validated by immunohistochemistry. Hemicentin-1 expression is higher in random bone biopsies from newly diagnosed MM patients (n=406) compared to donor biopsies (n=25, p<0.008) and highest in MM focal lesion biopsies (n=49, q<0.0005 vs. paired random bone biopsies). Higher baseline HMCN1 expression in biopsies was associated with inferior overall survival in TT3b clinical trial (p<0.027). The NBM system demonstrates the ability of primary MM plasma cells to interact with and to survive in coculture with healthy allogeneic adult BM through secretion of factors involved in immune evasion and extracellular matrix modification. Ongoing work is underway to unravel the role of hemicentin-1 in MM growth. Disclosures No relevant conflicts of interest to declare.

Download Full-text

THE ROLE OF GPIIb-IIIa IN MODULATION OF ADHESION REACTIONS

10.1055/s-0038-1643516 ◽

1987 ◽

Author(s):

J C Mattson ◽

D W Estry ◽

D Peterson ◽

R LaFevre ◽

J Chirco

Keyword(s):

Shape Change ◽

Atp Release ◽

Dense Granule ◽

Clot Retraction ◽

Virtual Absence ◽

Fibrinogen Binding ◽

Related Defect ◽

Rice University ◽

Granule Release

We have previously reported that patients with Glanzmann’s Thrombasthenia (GT) fail to adhere to a carbon-formvar surface and undergo contact-induced shape change in a non-flow system. The ability of ADP to reverse this adhesion defect suggested that it may be secondary to defective dense granule release rather that a direct requirement for GPIIb-IIIa. To further assess the role of GPIIb-IIIa in adhesion, we examined the effect of two mouse monoclonal antibodies to the GPIIb-IIIa complex, AP2 (IgG, kappa) from T. Kunicki, Milwaukee Blood Center and MAb36 (IgM, lambda) from D. Peterson, Rice University. AP2 (1:50 dil) and MAb36 (1:200 dil) both completely abolished aggregation by ADP, collagen and epinephrine and prevented clot retraction. In a transmission EM (TEM) whole mount assay of adhesion and contact-induced shape change, both antibodies inhibited platelet attachment to the substrate and impaired spreading in those few platelets that did attach. This antibody-induced adhesion defect was reversed by the addition of 2×10−6 m ADP just prior to exposure of platelets to the activating surface. In parallel studies, antibody treated platelets demonstrated a dose-related defect in ATP release as measured in a Lumiaggregometer with total absence of release at antibody dilutions that abolished aggregation. Using a colloidal gold-fibrinogen probe, virtual absence of binding of exogenous fibrinogen was demonstrated in antibody treated platelets induced to. spread by ADP stimulation. These studies suggest that while GPIIb-IIIa may play a role in adhesion in non-flow systems, as suggested by the altered adhesion seen in GT platelets, adhesion and adhesion-induced shape change can be supported by ADP stimulation in the absence of fibrinogen binding to GPIIb-IIIa.

Download Full-text