Dasatinib Is Effective in the Treatment of Mice Models with Immune Thrombocytopenia

Abstract Background: Immune thrombocytopenia (ITP) is an autoimmune disease in which anti-platelet antibody (APA) is produced. APA-coated platelets are captured and phagocytized by macrophages in the spleen. Recent study revealed that spleen tyrosine kinase (Syk) inhibitor is effective in the treatment of ITP, because Syk phosphorylation is the key step of the phagocytosis by macrophages. Activated Fc receptor signal transduction is initiated by phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) tyrosine residues by SRC family kinases. Recruitment of Syk to dually phosphorylated ITAMs triggers the activation of Syk. To prove the hypothesis that the inhibition of SRC family kinase induces the decreased phosphorylation of Syk, resulting in decreased phagocytosis by macrophages, a SRC family kinase inhibitor, dasatinib, was used in the experiments. Methods, Results and Discussion: In vitro study; Murine macrophage cell line, RAW, was incubated with APA coated murine platelets for 30 minutes. Phagocytosis by RAW was significantly decreased with dasatinib (100nM, p<0.01), indicating SRC family kinase activity is required for efficient phagocytosis. Phosphorylated Syk was decreased in RAW, incubated with anti-Fc receptor antibody (rat IgG) and anti-rat IgG antibody with dasatinib (100nM), shown in the Western blot analysis (Figure 1). These results suggest that Syk phosphorylation is the key step in phagocytosis. In vivo study; (1) Three hours before APA intra-peritoneal injection, dasatinib (2.5mg/kg) was oral-administrated. Six hours after APA injection, platelet counts were measured. The platelet counts were 366 ± 164 x109/L with dasatinib (n=4, mean ± SD) and 114 ± 51 x109/L without dasatinib (n=4)(P=0.026)(Figure 2). (2) Osmotic pump, filled with APA, were inserted in murine intra-peritoneal cavity and dasatinib (2.5mg/kg) was oral-administered once daily for 7 days. The platelet counts were 499 ± 98 x109/L with dasatinib (n=4, mean ± SD) and 82 ± 131 x109/L without dasatinib (n=4) at day 7 (p<0.0022) (Figure 3). These results strongly suggest that dasatinib inhibit the phagocytosis in vivo. Conclusion: Dasatinib inhibits phosphorylation of Syk, inducing decreased phagocytosis of APA-coated platelets via decreased SRC family kinase activity. These findings reveal that SRC family kinase controls the efficiency of phagocytosis in part through the regulation of Syk function. Dasatinib might be effective in the treatment of ITP. Disclosures No relevant conflicts of interest to declare.

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Mice Lacking the Inhibitory Collagen Receptor LAIR-1 Exhibit a Mild Thrombocytosis and Hyperactive Platelets

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.117.309253 ◽

2017 ◽

Vol 37 (5) ◽

pp. 823-835 ◽

Cited By ~ 18

Author(s):

Christopher W. Smith ◽

Steven G. Thomas ◽

Zaher Raslan ◽

Pushpa Patel ◽

Maxwell Byrne ◽

...

Keyword(s):

Tyrosine Kinases ◽

Kinase Activity ◽

Thrombus Formation ◽

Physiological Role ◽

Src Family Kinase ◽

Glycoprotein Vi ◽

Collagen Receptor ◽

Deficient Mice

Objective— Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a collagen receptor that belongs to the inhibitory immunoreceptor tyrosine-based inhibition motif–containing receptor family. It is an inhibitor of signaling via the immunoreceptor tyrosine-based activation motif–containing collagen receptor complex, glycoprotein VI-FcRγ-chain. It is expressed on hematopoietic cells, including immature megakaryocytes, but is not detectable on platelets. Although the inhibitory function of LAIR-1 has been described in leukocytes, its physiological role in megakaryocytes and in particular in platelet formation has not been explored. In this study, we investigate the role of LAIR-1 in megakaryocyte development and platelet production by generating LAIR-1–deficient mice. Approach and Results— Mice lacking LAIR-1 exhibit a significant increase in platelet counts, a prolonged platelet half-life in vivo, and increased proplatelet formation in vitro. Interestingly, platelets from LAIR-1–deficient mice exhibit an enhanced reactivity to collagen and the glycoprotein VI–specific agonist collagen-related peptide despite not expressing LAIR-1, and mice showed enhanced thrombus formation in the carotid artery after ferric chloride injury. Targeted deletion of LAIR-1 in mice results in an increase in signaling downstream of the glycoprotein VI–FcRγ-chain and integrin αIIbβ3 in megakaryocytes because of enhanced Src family kinase activity. Conclusions— Findings from this study demonstrate that ablation of LAIR-1 in megakaryocytes leads to increased Src family kinase activity and downstream signaling in response to collagen that is transmitted to platelets, rendering them hyper-reactive specifically to agonists that signal through Syk tyrosine kinases, but not to G-protein–coupled receptors.

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Thrombopoietin Stimulation Leads to Enhanced Polyploidization in p53-/- Bone Marrow Megakaryocytes: In Vivo and in Vitro Studies.

Blood ◽

10.1182/blood.v114.22.2531.2531 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2531-2531

Author(s):

Pani A. Apostolidis ◽

Stephan Lindsey ◽

William M. Miller ◽

Eleftherios T. Papoutsakis

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Platelet Surface ◽

Platelet Counts ◽

Reticulated Platelets ◽

High Ploidy ◽

And Control ◽

Platelet Formation

Abstract Abstract 2531 Poster Board II-508 BACKGROUND AND HYPOTHESIS. We have previously shown that tumor suppressor p53 is activated in differentiating megakaryocytic (Mk) cells and its knock-down (KD) leads to increased polyploidization and delayed apoptosis in CHRF, a human Mk cell line. Furthermore, bone marrow (BM)-derived Mks from p53−/− mice reach higher ploidy classes in culture. Accordingly, we hypothesized that the role of p53 during megakaryopoiesis is to delimit polyploidization and control the transition from endomitosis by inhibiting DNA synthesis and promoting apoptosis. Here, we test this hypothesis by examining the differential effect of mouse thrombopoietin (rmTpo) on the ploidy of p53−/− and p53+/+ mouse Mk cells. METHODS. 8–10 week-old, male p53−/− mice and p53+/+ littermates were injected once with 1.2 μg rmTpo or saline. On days 2 and 5 after Tpo/saline treatment, tail-bleeding assays were performed to measure bleeding times/volumes, mice were bled for platelet counts and sacrificed to harvest BM. We employed flow cytometry to examine baseline ploidy in BM-resident Mks in p53−/− and p53+/+ mice as well as Mk cells generated from BM progenitors after 4 and 6 days of culture with rmTpo. RESULTS. At steady state, ploidy in BM-resident CD41+ Mk cells was similar in p53−/− and p53+/+ mice: 11.8±2.3% and 10.7±1.3% of p53−/− and p53+/+ Mks, respectively, reaching a ploidy of ≥32N (n=3-4). Platelet counts were 1.3×106±1×105/μl (12.5±1.0% reticulated) and 1.1×106±5×104/μl (12.4±1.3% reticulated) in p53−/− and p53+/+ mice, respectively (n=8). Two days following Tpo treatment of the mice, we did not observe significantly increased platelet levels, while ploidy was marginally affected. However, 5 days following Tpo treatment, we found greater ploidy in the BM in the absence of p53: 22±1.6% 16N and 10.1±0.8% ≥32N Mks in the p53−/− versus 18.6±3.3% 16N and 7.1±1.4% ≥32N Mks in the p53+/+ (n=2). This was accompanied by increased platelet formation: 23.6±8.3% reticulated platelets in the p53−/− versus 17.8±2.6% in the p53+/+ (n=2). Culture of BM cells from non-Tpo treated mice with 50ng/ml rmTpo resulted in a 50% increase in total Mks and increased polyploidy by day 6 of culture: 38.6±4.6% of p53−/− versus 19.2±2.3% of p53+/+ Mks reached ploidy classes of ≥32N (n=3-4, p < 0.01). Lack of p53 led to hyperploid Mk cells; by day 6 of culture 10.3±2.2% of p53−/− Mks were in ploidy classes of 128N and higher, while only 0.6±0.1% p53+/+ Mks achieved such high ploidy (n=3-4). In addition, a 6 day culture with Tpo of BM cells derived from p53−/− and p53+/+ mice pre-treated with Tpo 5 days prior to sacrifice led to more profound polyploidization compared to Mks generated from the non-Tpo treated mice but only in the p53−/− Mks: 48.8±1.1% of p53−/− versus only 17.6±0.2% of p53+/+ Mks reached ploidy ≥32N (n=2). Microarray analysis comparing p53KD to control CHRF cells undergoing Mk differentiation revealed down-regulation of genes coding for platelet surface complex CD41/CD61 and CD62P in the p53KD cells. To examine the possibility of altered functionality of platelets in p53−/− mice, we performed tail-bleeding assays on the mice that did not receive Tpo. Bleeding times and volumes were generally prolonged in the absence of p53 (all p53−/− mice exceeded the 10 min duration of the assay; mean p53−/− and p53+/+ blood loss was 17μl and 10μl, respectively, n=3-4). CONCLUSIONS. Our data indicate that in vivo polyploidization and platelet formation from Mks is increased in the p53−/− relative to p53+/+ mice after Tpo administration. These data are in line with our hypothesis that p53 activation decreases the ability of Mks to respond to Tpo and undergo polyploidization. Additionally, our preliminary data on platelet functionality suggest that p53 may have a role in hemostasis. Disclosures: No relevant conflicts of interest to declare.

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Unique Thrombopoietic Activity of Novel PKC Agonist Ingenol 3,20 Dibenzoate.

Blood ◽

10.1182/blood.v114.22.3622.3622 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3622-3622

Author(s):

Frederick Karl Racke ◽

Maureen E Baird ◽

Rolf Barth ◽

Tianyao Huo ◽

Weilian Yang ◽

...

Keyword(s):

Conflicts Of Interest ◽

Leukemic Cell ◽

The Novel ◽

Drug Induced ◽

Platelet Recovery ◽

Platelet Production ◽

Platelet Counts ◽

Pkc Isoforms

Abstract Abstract 3622 Poster Board III-558 Despite recent advances in our understanding of megakaryocytic growth and platelet production, thrombocytopenia remains a difficult problem in the clinical management of patients with hematologic malignancies. Thrombopoietin (TPO) is the major cytokine involved in the normal production of platelets. However, the use of TPO has been relatively unsuccessful for the treatment of these patients and platelet transfusions remain the primary treatment for thrombocytopenia despite their significant cost and relatively short-lived responses. Thus, there remains an important clinical need for the development of novel approaches to generate platelets. Despite numerous reports on protein kinase C (PKC) agonists as promoters of megakaryocytic differentiation in leukemic cell lines and primary cells, little is known about their in vitro effects on primary CD34-selected progenitors or when administered in vivo. In the present study, we examine that effects of the novel PKC isoform agonist ingenol 3,20 dibenzoate (IDB) on megakaryocyte differentiation from CD34+ cells cultured in TPO and stem cell factor (SCF) or erythropoietin/SCF and its effects on platelet production in BALB/c mice. IDB potently stimulates early megakaryopoiesis and redirects the specificity of EPO to favor megakaryopoiesis over erythropoiesis. In contrast, broad spectrum PKC agonists such as phorbol myristate acetate, mezerein, and indolactam V fail to promote megakaryopoiesis. In vitro, IDB stimulates early expression of the promegakaryopoietic transcription factors egr1 and fli-1 and downregulates the proerythropoietic factors KLF1 and c-myb. Induction of the early megakaryocytic marker, CD9, was observed within the first 24 hrs of treatment with IDB and CD9 induction was blocked by the PKC inhibitor bisindolylmaleimide, which inhibits both novel and conventional PKC isoforms. In contrast, an inhibitor of conventional PKC isoforms, Gö6976, failed to block CD9 induction. In vivo, single intraperitoneal injections of IDB selectively increased platelet counts in BALB/c mice by 50% (plt= 630,000 vs. 985,000/μl; p<.005) at day 7 without affecting hemoglobin (Hgb) concentration or white counts (WBC). Mice treated with low dose radiation (2-4 Gy) had a transient drop in both platelet and WBC counts. Pretreatment with IDB 3 hrs prior to irradiation increased the platelet counts without improving WBC. More severe radiation exposure (6-8 Gy) causes pancytopenia. IDB treatment 3 hrs prior to 6 Gy irradiation significantly reduced the thrombocytopenia (plt=192,000 vs 594,000/μl; p<0.005) and anemia (hemoglobin=11.9 vs. 13.5gm/dl); p<0.005) without affecting the drop in WBC (WBC=1,200 vs. 1,300/μl; p=NS) at 14 days following irradiation. For mice treated with 8 Gy radiation, IDB pretreatment resulted in similar improvements in platelet counts (plt=111,000 vs. 443,000/μl; p<0.005) and hemoglobin (hgb=8.2 vs. 12.7 gm/dl; p<0.005) at 21 days following irradiation. The mitigation of thrombocytopenia is accompanied by marked increases in the megakaryocyte content in both the spleens and bone marrows of IDB-treated mice. Most importantly, IDB mitigated radiation-induced thrombocytopenia, even when administered 24 hrs after irradiation (plt=80,000 vs. 241,000/μl at 14 days following 6 Gy irradiation; p<0.01). Finally, IDB improved the survival of lethally irradiated mice. Our data suggest that the novel PKC isoform agonist IDB promotes the early differentiation of megakaryocytes from hematopoietic progenitors at the resulting in a significant improvement in platelet recovery following irradiation. IDB also improved Hgb levels following higher radiation doses. This may be due to improved hemostasis secondary to increased platelet numbers; however, an additional radioprotective effect on erythroid precursors cannot be excluded. These results strongly support our hypothesis that the novel PKC agonist IDB may be useful for the treatment of radiation and possibly drug-induced thrombocytopenia. Disclosures: No relevant conflicts of interest to declare.

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The CD47 Pathway Is Deregulated In Human Immune Thrombocytopenia (ITP).

Blood ◽

10.1182/blood.v116.21.3776.3776 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3776-3776

Author(s):

Lucia Catani ◽

Daria Sollazzo ◽

Francesca Ricci ◽

Francesca Palandri ◽

Nicola Polverelli ◽

...

Keyword(s):

Cell Death ◽

Immune Thrombocytopenia ◽

Conflicts Of Interest ◽

Regulatory Protein ◽

Immunoglobulin Superfamily ◽

Phagocytic Capacity ◽

Platelet Apoptosis ◽

Cell Death Pathway

Abstract Abstract 3776 The CD47 antigen is a transmembrane glycoprotein ubiquitously expressed on hematopoietic and non-hematopoietic cells. It serves as a receptor for Thrombospondin (TSP) and a ligand for signal regulatory protein-alpha (SIRP-alpha) receptor, acting, respectively, as a regulator of apoptosis and as antagonistic to phagocyte activity. Ligation of CD47 with antibodies, its natural physiological ligand TSP or the specific CD47-binding peptide 41NK induces apoptosis in nucleated blood cells. This apoptosis is characterized by mitochondrial damage and the exposure of phosphatydilserine on the outerleaflet of the plasma membrane. Interaction of SIRP-alpha with CD47 is important also for the regulation of phagocytosis. SIRP-alpha is an immunoglobulin superfamily member and is predominantly expressed in neurons, dendritic cells (DCs) and monocytes/macrophages. Phagocytes engulf foreign cells but not “self” in part because “self” cells express CD47 as a ligand for SIRP-alpha, which inhibits phagocytosis. Thus CD47 functions as a “don't eat me” signal. Based on studies in mice, a novel mechanism of platelet destruction involving the CD47/SIRP-alpha system has been recently suggested in Immune Thrombocytopenia (ITP). Specifically, it has been demonstrated that: 1) platelet homeostasis is regulated by platelet expression of CD47 under normal conditions and in immune thrombocytopenia in a mouse model; 2) interaction between platelet CD47 and macrophage SIRP-alpha is important in regulating normal platelet turnover and FcgammaR-mediated clearance of IgG-sensitized platelets; 3) CD47-deficient platelets have a shortened half-life in the circulation of CD47 wild-type mice and are also more sensitive to Fcgamma receptor-mediated clearance, both in vivo and in vitro. However, the role of CD47 pathway in the pathogenesis of human ITP has not yet been studied. Therefore, the main purpose of the present study was to evaluate whether alterations of this system (platelets/phagocytes) might play a pathogenetic role in human ITP. In particular, we investigated whether in ITP: i) platelets are more susceptible to CD47-induced cell death; ii) expression of CD47 on fresh and in vitro aged platelets is reduced; iii) the platelet phagocytic capacity of CD14-derived DCs and macrophages is differentially modulated in the presence or absence of antibodies against CD47 and SIRP-alpha. Phenotypical and functional analysis of the expression of CD47 on platelets and SIRP-αlpha on CD14-derived/circulating DCs and on CD14-derived macrophages was performed in 32 ITP patients. Patients were newly diagnosed (14 cases) or with persistent (15 cases) or chronic (3 cases) ITP. At the time of the study, patients with persistent or chronic ITP were off therapy by at least two months. None of the patients were splenectomized. The median platelet count at the time of the study was 49×109/L (range 14–98). We found that in healthy subjects CD47 expression increased in in vitro aged platelets and ligation of CD47 with anti-CD47 antibody induced a dose-dependent increase of platelet apoptosis. Immature and mature CD14-derived DCs and circulating myeloid DCs were strongly positive for SIRP-α. Conversely, we demonstrated that in ITP: 1) CD47 expression was unchanged in freshly isolated and in vitro aged platelets; 2) increased platelet apoptosis was not due to the activation of the CD47-induced cell death pathway, which instead was shown to be blocked; 3) the blockage of SIRP-αlpha on immature CD14-derived DCs or CD47 on platelets by specific antibodies failed to modify platelet uptake/phagocytosis of DCs; in contrast, targeting platelet CD47 with specific antibody significantly increases platelet phagocytosis of CD14-derived macrophages. In conclusion, our data demonstrate that in ITP the increased platelet clearance is not due to reduced CD47 expression on platelets. However, platelets from ITP patients are not healthy because 1) apoptosis is increased; 2) platelet apoptosis is independent from CD47 death signal; 3) CD47 expression is not modified by in vitro ageing/apoptosis. In addition, we show that the CD47 pathway plays a role in platelet phagocytosis of macrophages, but not in DCs. We conclude that in ITP patients platelet homeostasis is differentially modulated by the CD47 pathway. Disclosures: No relevant conflicts of interest to declare.

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Enhancing Functional Platelet Release In Vivo from in Vitro-Grown Megakaryocytes Using the Protein Kinase Inhibitor SU6656

Blood ◽

10.1182/blood.v128.22.1029.1029 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1029-1029

Author(s):

Danuta Jadwiga Jarocha ◽

Karen K Vo ◽

Randolph B Lyde ◽

Vincent M Hayes ◽

Mortimer Poncz

Keyword(s):

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Specific Inhibitor ◽

Annexin V ◽

Rock Inhibitor ◽

Medicine Research ◽

Nsg Mice ◽

Platelet Release

Abstract The clinical demand for platelet transfusions is increasing, threatening the ability to obtain sufficient healthy donors to provide these platelets. Advances in regenerative medicine research have opened the possibility of generating sufficient in vitro-grown megakaryocytes and consequent platelets to supply a portion of the clinical platelet transfusion demand. We have shown that infusing megakaryocytes for obtaining released, functional platelets is a viable alternative strategy than trying to release platelets in vitro. However, for both approaches, in vitro-cultured megakaryocytes have lower ploidy and release fewer platelets than likely occurs in vivo by primary cells. SU6656 inhibitor, a Src kinase inhibitor, has been shown to influence ploidization in several megakaryocyte-like line with purported increase in proplatelets release. However, in our hands, other agents - such as the ROCK inhibitor Y27632 - while increasing polyploidization markedly, inhibited platelet release per infused megakaryocyte in vivo. We grew megakaryocytes from CD34+ cells for 12 days with or without SU6656 (2.5 µM) supplementation during the last 4 days. We found that the SU6656 inhibitor only increased the number of CD34+-derived megakaryocytes by ~15% at the end of the 12 day growth, but more markedly increase the percent of large megakaryocytes measured by FSC parameter in flow cytometry evaluation from 28 up to 41% and percent of high granular megakaryocytes from 27 to 45%. These changes were accompanied with a shift in average ploidy from 4.9 to 6.9 (p<0.0003, N=6). Notably, SU6656-treated megakaryocytes released ~4-fold more platelets per infused megakaryocytes in immunocompromized NSG mice than untreated similarly in vitro-grown megakaryocytes. By 24 hrs, there were 6.5-fold platelets from the infused SU6656-treated megakaryocytes than control untreated (p<0.037, N=6). Released platelets from the drug-treated and untreated megakaryocytes had similar levels of percent thiazole orange positivity as an indication that they were young platelets. Importantly, baseline annexin V, CD62p and PAC1 binding prior to agonist exposure were also similarly and increased to the same extent after thrombin (1U/ml) stimulation. Additionally, incorporation into a growing cremaster laser injury-induced thrombus in vivo was similar further indicating retained function by the platelets released from the drug-treated megakaryocytes. A number of strategies such as modifying the level of transcription factors have been proposed to increase the size, ploidy or proplatelets release from in vitro-grown megakaryocytes. In none of these cases have these released platelets in vivo biology been examined and demonstrated to replicate high release number per megakaryocyte and retained functionality. We show that terminal exposure of in vitro-grown megakaryocytes to the non-specific inhibitor SU6656 significantly increases in vivo yield while leaving in vivo half-life and functionality intact. The exact pathway affected by SU6656 that leads to these results is now being pursued. Disclosures No relevant conflicts of interest to declare.

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Combined Targeting of BCR-ABL and JAK2 with ABL and JAK2 Inhibitors Is Effective Against CML Patients' Leukemic Stem/Progenitor Cells.

Blood ◽

10.1182/blood.v116.21.3404.3404 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3404-3404

Author(s):

Donna DeGeer ◽

Paolo Gallipoli ◽

Min Chen ◽

Ivan Sloma ◽

Heather Jorgensen ◽

...

Keyword(s):

Progenitor Cells ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Integration Site ◽

Single Agent ◽

K562 Cells ◽

High Rate ◽

Jak2 Inhibitors

Abstract Abstract 3404 Imatinib mesylate (IM) is a tyrosine kinase inhibitor (TKI) that induces clinical responses in most chronic myeloid leukemia (CML) patients. Nevertheless, early relapses and later emergence of IM-resistant disease pose serious concerns for many. The inadequacies of IM therapy are due, at least in part, to the unique properties of CML stem/progenitor cells that make them generally less responsive to IM and, indeed, other TKIs, and also confer on them a genetic instability that leads to a high rate of formation of BCR-ABL mutants. Improved treatment approaches to prevent the development of resistant subclones by targeting other key molecular elements active in CML stem/progenitor cells are thus clearly needed. One candidate is a complex that forms in CML stem/progenitor cells between the oncoproteins encoded by AHI-1 (Abelson helper integration site 1), BCR-ABL and the JAK2 kinase. This complex contributes to the transforming activity of BCR-ABL both in vitro and in vivo and also plays a role in the IM response/resistance of primary CML stem/progenitor cells. We now describe the results of experiments designed to test the ability of ABL and JAK2 inhibitors to block the activity of this protein complex in CML cells. K562 cells engineered to stably overexpress AHI-1 showed a significantly reduced sensitivity to both IM (at 1 and 5 μM) and TG101209, a JAK2 inhibitor, (at 0.5 and 1 μM), as determined by assays for cell viability, apopotosis, and colony-forming activity. K562 cells engineered to suppression AHI-1 showed an opposite effect, with a heightened sensitivity to IM at concentrations as low as 1 μM. In addition, IM together with TG101209 was more effective at killing AHI-1-overexpressing K562 cells, IM-resistant K562 cells and IM-resistant T315I-mutant cells than either treatment alone. Western blot and co-IP experiments demonstrated a significant reduction of p-BCR-ABL, p-JAK2 and p-STAT5 in cells treated with IM plus TG101209 compared to cells treated with IM or TG101209 alone. Importantly, treatment with 5 μM IM, 150 nM dasatinib (DA) or 5 μM nilotinib (NL) in combination with 100 nM TG101209 caused a significantly greater reduction in the viability of primary CD34+CD38− and CD34+CD38+ CML cells when these responses were compared to any of the TKIs or TG101209 alone (~2-4 fold, n=3). Apoptotic cells at 72 hours were also significantly increased for all drug combinations compared to single agent treatments (40%-52% for the combinations vs 15%-18% for the single agents). CFSE tracking analysis of cell division in these cells further demonstrated additive anti-proliferative activity from the TKI plus TG101219 combinations, although some rare undivided cells were not eliminated. Nevertheless, exposure of CD34+ CML cells from IM-nonresponders (n=4) to TG101209 plus IM or DA did cause a greater inhibition (81% and 85%) of patients' colony-forming cells as compared to the same cells treated with the combination of IM plus DA only, or IM or DA only (60%, 41% and 50% inhibition, p<0.05). Long-term culture-initiating cell assays were undertaken to compare the effect of these combination treatments versus the effects of TKIs or TG101209 alone on very primitive CML cells. The results again showed a more significant reduction of these cells treated with the combination (n=3). Intracellular staining revealed a greater reduction in the levels of p-CrKL and p-STAT5 in CD34+ CML cells treated for 24 hours with the combination of TKIs plus TG101219 as compared to single TKI-treated cells (~44% vs 65% for p-CrKL and 36% vs 57% for p-STAT5, n=3). Strikingly, the combination treatment produced an even greater inhibition of both p-CrKL and p-STAT5 after 72 hours while p-CrKL was almost fully reactivated with TKIs alone (~29% vs 89% for p-CrKL and 23% vs 50% for p-STAT5). These results point to the possibility of achieving improved therapeutic outcomes in CML patients by simultaneously targeting both BCR-ABL and JAK2 activities in the critical TKI-insensitive CML stem/progenitor reservoir. Disclosures: No relevant conflicts of interest to declare.

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Lycorine Modulates the Expression of p21 Via a p53-Independent Pathway in HL-60 Cells

Blood ◽

10.1182/blood.v118.21.4297.4297 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4297-4297

Author(s):

Jing Liu ◽

Shu-Ling Wang ◽

Lin Fang ◽

Mao Ye ◽

Zhi-Wei Sun ◽

...

Keyword(s):

Molecular Mechanisms ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Fold Increase ◽

Alternative Pathways ◽

Cyclin Dependent Kinase Inhibitor ◽

M Phase ◽

Induced Apoptosis

Abstract Abstract 4297 Leukemia is one of the most life-threatening cancers today, and acute promyelogenous leukemia is a common type of leukemia. We have previously shown that lycorine, a natural alkaloid extract from Amaryllidaceae, exhibited anti-leukemia effects in vitro and in vivo. Lycorine treatment of HL-60 cell arrested cell cycle at G2/M phase and induced apoptosis. In the present study, we sought to explore the molecular mechanisms for the anti-leukemia action of lycorine. Gene chip analysis revealed that lycorine treatment of HL-60 cells induced more than 9 fold increase of p21, a cyclin-dependent kinase inhibitor, whose expression is mainly regulated by p53. Since HL-60 cells are p53 null, the above findings suggest that lycorine activates p21 expression through p53-independent pathway. To further explore the alternative pathways for the activation of p21 induced by lycorine, we examined the effect of lycorine on the expression of Rb, pRb, E2F, c-Myc and HDACs which have shown to regulate p21 expression. We show that expression of pRb (ser780) and c-Myc was down-regulated, Rb and E2F were up-regulated, while the expression of HDAC1 and HDAC3 was not changed. Together these findings suggest that lycorine exerts its anti-leukemia effect by activating p21 expression via pRb/E2F and c-Myc pathways. Disclosures: No relevant conflicts of interest to declare.

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Homosexual men with thrombocytopenia have impaired reticuloendothelial system Fc receptor-specific clearance

Blood ◽

10.1182/blood.v70.2.392.bloodjournal702392 ◽

1987 ◽

Vol 70 (2) ◽

pp. 392-395 ◽

Cited By ~ 1

Author(s):

BS Bender ◽

TC Quinn ◽

JL Spivak

Keyword(s):

Immune Complexes ◽

Fc Receptor ◽

Red Cells ◽

Circulating Immune Complexes ◽

Homosexual Men ◽

Severe Thrombocytopenia ◽

Half Time ◽

Igg Antibody ◽

Platelet Counts

Classic immune thrombocytopenia purpura (ITP) occurs predominantly in women and is associated with either normal or impaired Fc receptor- mediated clearance of antibody-coated cells. Recently, an increasing incidence of thrombocytopenia has been observed in homosexual men, but whether Fc receptor-mediated clearance of antibody-coated cells is normal or impaired in these men is unknown. To study this question, we measured the in vivo clearance of anti-Rho(D) IgG antibody-sensitized 51Cr-labeled autologous red cells in five homosexual men with thrombocytopenia without an evident cause. All five had antibodies to human immunodeficiency virus, and four had circulating immune complexes as determined by a Clq-binding assay. Two of the men tested also had an increase in platelet-associated IgG. In the four homosexual men with platelet counts of 20,000/microL or less, the clearance half-time of IgG-sensitized red cells was prolonged (mean, 106 minutes; range, 72 to 140 minutes) as compared with the clearance of such cells in five hematologically normal men (mean, 39 minutes; range 30 to 50 minutes; P less than .005). One homosexual man with a platelet count of 81,000/microL had a normal clearance half-time (30 minutes). Three patients whose platelet counts increased after corticosteroid therapy were restudied. In all three, the clearance of antibody-coated cells was shortened and returned to normal in the one patient who had achieved a complete remission. No correlation was observed between the presence of platelet-associated IgG or circulating immune complexes and the clearance half-time. These data indicate that severe thrombocytopenia occurring in homosexual men as in some patients with classic ITP is associated with defective in vivo Fc receptor-mediated clearance of antibody-coated cells.

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Red Cell-Derived Microparticles (RMP) As Hemostatic Agent: Evaluation in a Rabbit Ear Bleeding Model.

Blood ◽

10.1182/blood.v120.21.2238.2238 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2238-2238

Author(s):

Wenche Jy ◽

Carlos J Bidot ◽

Sandra Nunez ◽

Max E Johansen ◽

Lawrence L Horstman ◽

...

Keyword(s):

Conflicts Of Interest ◽

In Vivo Studies ◽

Hemostatic Agent ◽

Large Animal ◽

Platelet Activity ◽

Platelet Counts ◽

Rabbit Ear ◽

Hemostatic Efficacy

Abstract Abstract 2238 Introduction: We are developing RMP for use as an infusible hemostatic agent to treat bleeding conditions. Preliminary evaluations in vitro, such as by thromboelesatography (TEG), were very promising, and were supported by in vivo studies in a small animal (rat) [Haemophilia 2011; 17: 44]. It was shown that RMP not only accelerate coagulation and correct hemostatic aberration of factor-deficient plasma but also enhance platelet aggregation and adhesion, suggesting its efficacy in both primary and secondary hemostasis [Blood 2011; 118:3263]. Here we report results of initial studies in a large animal, the rabbit ear bleeding model. Methods: RMP were produced by high-pressure extrusion of human RBC, converting >99% of RBC to RMP. Hemostatic efficacy was evaluated in a rabbit ear bleeding model. New Zealand male white rabbits of 2.5–3.5 kg were used. After sedation, the ear was prepared by chlorhexidime scrub, then a standard incision 6 mm wide was made to penetrate the ear (#11 scalpel blade), in a region of the ear devoid of macroscopic (visible) vessels. The ear was maintained at 37°C with a heating pad. Bleeding time (BT) was defined as the time required for the disappearance of blood stains on a filter paper blot. To reliably measure reduction of BT by the RMP product, bleeding was prolonged by administration of busulfan (20 mg/kg SC) twice each at days 1 and 3, to suppress bone marrow, resulting in thrombocytopenia after 7–12 days. BT was significantly prolonged in animals when platelet counts fell to <60,000/μL. This regimen allowed all animals to survive, but higher doses of busulfan was toxic and caused severe pancytopenia, unacceptable for experimentation. Results: In normal (untreated) rabbits, the BT was 90 ±20 seconds (n = 12). Injection of RMP (3×109/kg, IV) in untreated rabbits resulted in no significant changes in BT, and no major adverse events were seen (n = 6). In thrombocytopenic rabbits, the BT was prolonged to 620 ±130 sec., and prolongation of BT correlated with decrease in platelet counts, 30,000 – 60,000/μL (n = 12). In this range of platelet counts, injection of RMP (at either 1 × 109 or 3 × 109/kg, IV) induced significant shortening of BT, to 440 ±90 sec (n = 8) at the lower dose (p<0.01), and to 280 ±65 sec (n = 8), at the higher dose (p < 0.001). When platelet counts dropped below 10,000/μL and BT >15 min, RMP had no significant effect. The hemostatic effects disappeared if >30 min elapsed between RMP infusion and making the incision, indicating a short half-life of RMP in circulation. Conclusions: These data confirm efficacy of RMP in thrombocytopenic rabbits. Hemostatic efficacy of RMP was evident in moderate thrombocytopenia, and was dose-dependent. However, efficacy fell off if platelets <10,000/μL, indicating that RMP augment platelet activity but do not replace platelets. In both thrombocytopenic and non-thrombocytopenic rabbits, no major adverse reactions were observed with infusion of RMP. Disclosures: No relevant conflicts of interest to declare.

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Homosexual men with thrombocytopenia have impaired reticuloendothelial system Fc receptor-specific clearance

Blood ◽

10.1182/blood.v70.2.392.392 ◽

1987 ◽

Vol 70 (2) ◽

pp. 392-395 ◽

Cited By ~ 16

Author(s):

BS Bender ◽

TC Quinn ◽

JL Spivak

Keyword(s):

Immune Complexes ◽

Fc Receptor ◽

Red Cells ◽

Circulating Immune Complexes ◽

Homosexual Men ◽

Severe Thrombocytopenia ◽

Half Time ◽

Igg Antibody ◽

Platelet Counts

Abstract Classic immune thrombocytopenia purpura (ITP) occurs predominantly in women and is associated with either normal or impaired Fc receptor- mediated clearance of antibody-coated cells. Recently, an increasing incidence of thrombocytopenia has been observed in homosexual men, but whether Fc receptor-mediated clearance of antibody-coated cells is normal or impaired in these men is unknown. To study this question, we measured the in vivo clearance of anti-Rho(D) IgG antibody-sensitized 51Cr-labeled autologous red cells in five homosexual men with thrombocytopenia without an evident cause. All five had antibodies to human immunodeficiency virus, and four had circulating immune complexes as determined by a Clq-binding assay. Two of the men tested also had an increase in platelet-associated IgG. In the four homosexual men with platelet counts of 20,000/microL or less, the clearance half-time of IgG-sensitized red cells was prolonged (mean, 106 minutes; range, 72 to 140 minutes) as compared with the clearance of such cells in five hematologically normal men (mean, 39 minutes; range 30 to 50 minutes; P less than .005). One homosexual man with a platelet count of 81,000/microL had a normal clearance half-time (30 minutes). Three patients whose platelet counts increased after corticosteroid therapy were restudied. In all three, the clearance of antibody-coated cells was shortened and returned to normal in the one patient who had achieved a complete remission. No correlation was observed between the presence of platelet-associated IgG or circulating immune complexes and the clearance half-time. These data indicate that severe thrombocytopenia occurring in homosexual men as in some patients with classic ITP is associated with defective in vivo Fc receptor-mediated clearance of antibody-coated cells.

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