Normal Megakaryocytes Research Articles

Dynamin 2–dependent endocytosis is required for normal megakaryocyte development in mice

AbstractDynamins are highly conserved large GTPases (enzymes that hydrolyze guanosine triphosphate) involved in endocytosis and vesicle transport, and mutations in the ubiquitous and housekeeping dynamin 2 (DNM2) have been associated with thrombocytopenia in humans. To determine the role of DNM2 in thrombopoiesis, we generated Dnm2fl/flPf4-Cre mice specifically lacking DNM2 in the megakaryocyte (MK) lineage. Dnm2fl/flPf4-Cre mice had severe macrothrombocytopenia with moderately accelerated platelet clearance. Dnm2-null bone marrow MKs had altered demarcation membrane system formation in vivo due to defective endocytic pathway, and fetal liver–derived Dnm2-null MKs formed proplatelets poorly in vitro, showing that DNM2-dependent endocytosis plays a major role in MK membrane formation and thrombopoiesis. Endocytosis of the thrombopoietin receptor Mpl was impaired in Dnm2-null platelets, causing constitutive phosphorylation of the tyrosine kinase JAK2 and elevated circulating thrombopoietin levels. MK-specific DNM2 deletion severely disrupted bone marrow homeostasis, as reflected by marked expansion of hematopoietic stem and progenitor cells, MK hyperplasia, myelofibrosis, and consequent extramedullary hematopoiesis and splenomegaly. Taken together, our data demonstrate that unrestrained MK growth and proliferation results in rapid myelofibrosis and establishes a previously unrecognized role for DNM2-dependent endocytosis in megakaryopoiesis, thrombopoiesis, and bone marrow homeostasis.

Bone marrow pathology predicts mortality in chronic hemodialysis patients.

Introduction. A bone marrow biopsy is a useful procedure for the diagnosis and staging of various hematologic and systemic diseases. The objective of this study was to investigate whether the findings of bone marrow studies can predict mortality in chronic hemodialysis patients. Methods. Seventy-eight end-stage renal disease patients on maintenance hemodialysis underwent bone marrow biopsies between 2000 and 2011, with the most common indication being unexplained anemia followed by unexplained leukocytosis and leukopenia. Results. The survivors had a higher incidence of abnormal megakaryocyte distribution (P = 0.001), band and segmented cells (P = 0.021), and lymphoid cells (P = 0.029) than the nonsurvivors. The overall mortality rate was 38.5% (30/78), and the most common cause of mortality was sepsis (83.3%) followed by respiratory failure (10%). In multivariate Cox regression analysis, both decreased (OR 3.714, 95% CI 1.671–8.253, P = 0.001) and absent (OR 9.751, 95% CI 2.030–45.115, P = 0.004) megakaryocyte distribution (normal megakaryocyte distribution as the reference group), as well as myeloid/erythroid ratio (OR 1.054, CI 1.012–1.098, P = 0.011), were predictive of mortality. Conclusion. The results of a bone marrow biopsy can be used to assess the pathology, and, in addition, myeloid/erythroid ratio and abnormal megakaryocyte distribution can predict mortality in chronic hemodialysis patients.

Presence of Calreticulin Mutations in JAK2-Negative Polycythemia Vera

Calreticulin (CALR) mutations have recently been reported in JAK2- and MPL-negative Myeloproliferative Neoplasms (MPN), particularly essential thrombocythemia (ET) and primary myelofibrosis (PMF).The clinical course of sporadic CALR-mutated patients seems to be more indolent than that of JAK2-mutated patients. In contrast, no CALR mutation has been found in the 647 published cases of Polycythemia Vera (PV) patients tested. Consequently, CALR mutations were considered exclusive to JAK2 and MPL mutations. Since 98% of PV patients harbor a JAK2 mutation (mostly the V617F mutation in exon 14 and more rarely, in exon 12), the absence of CALR mutations in PV seemed logical. Here, we describe two JAK2V617F-negative PV patients who presented with a CALR mutation at the time of diagnosis.Patient # 1 had hemoglobin at 168 g/L, hematocrit at 51.3%, and increased red cell mass (RCM) at 128% associated with a normal erythropoietin level. The bone marrow biopsy showed hypercellularity for age, panmyelosis associated with normal megakaryocytes and rare isolated abnormal enlarged forms. Using reticulin stain, no myelofibrosis was noted. Patient # 2 had hemoglobin at 194 g/L, hematocrit at 53% and low erythropoietin level without any dehydration. Both had moderately elevated platelet counts (658 and 575 x109/L respectively) with normal leukocyte counts. They were negative for BCR-ABL. No mutation was found in JAK2 exons 12, 13 and 14 by HRM and allele-specific real-time PCR or in MPL exon 10. Using HRM analysis, CALR mutations were suspected in both patients and confirmed using Sanger sequencing and product sizing analyses: CALR mutations were in both patients type 1 deletions (52-bp deletion; c.1092_1143del). To complete genomic tests made on peripheral blood granulocytes, we performed colony assays in methylcellulose and in collagen, picked single BFU-E colonies grown after 14 days in the presence of erythropoietin, and genotyped each colony individually for CALR. Of the 27 colonies genotyped, 6 had no PCR amplification and 21 harboured the same CALR mutation observed in peripheral blood granulocytes, i.e 52-bp deletion; c.1092_1143del. BFU-E were found heterozygous for CALR, with a mean allele burden of 49%. To our knowledge, these patients are the first cases of CALR-mutated PV to be reported. However, since a biclonalJAK2V617F and CALR MPN case recently reported, we cannot rule out the possibility of a biclonal disease involving a yet unknown mutation associated with a CALR mutation. On the other hand, the presence of a CALR mutation both in peripheral granulocytes and in BFU-E suggests that the CALR mutation plays a role in the polycythemia phenotype. Our observations highlight the fact that in the absence of JAK2 mutation, CALR mutations can also be associated with PV.In conclusion, our data indicate that testing JAK2-negative PV patients for CALR mutations may be useful. DisclosuresNo relevant conflicts of interest to declare.

In Newborn Mice, Tpo Signaling Is Required for Normal Megakaryocyte Proliferation, Maturation, Platelet Formation, and Platelet Survival

Congenital amegakaryocytic thrombocytopenia (CAMT) is a disorder caused by c-mpl mutations that block or severely reduce thrombopoietin (Tpo) signaling, leading to amegakaryocytic thrombocytopenia and ultimately aplastic anemia. In neonates and infants, CAMT can have different clinical manifestations than in adults, and cases have been described of newborn infants who presented with severe thrombocytopenia but normal numbers of immature-appearing megakaryocytes (MKs) in the bone marrow. To elucidate the potential mechanisms underlying the developmental differences in the manifestations of Tpo deficiency, we evaluated MKs and platelets in newborn c-mpl-/- mice on postnatal day 1 (P1). Since the liver is the main site of megakaryopoiesis at that developmental stage (Liu et al., Blood 2014), liver MKs in P1 mice were immunohistochemically stained for VWF, quantified, measured, and ultrastructurally evaluated using transmission electron microscopy (TEM), as previously described. Histological analysis showed that MKs in newborn c-mpl-/- mice were present at a concentration of 30% and were significantly smaller compared to WT pups, a finding previously reported in adult mice. Unlike in adult c-mpl-/- mice, however, which have ultrastructurally normal MKs, TEM analysis of neonatal c-mpl-/- liver MKs revealed a significant shift toward immature cells and maturational abnormalities in 68% of MKs, including a disorganized demarcation system, reduced granules, and an abnormally wide peripheral zone. Ultrastructural analysis of platelets from c-mpl-/- P5 mice also revealed a larger open canalicular system and more glycosomes compared to age-matched WT platelets. These findings were not associated with increased levels of baseline P-selectin expression, suggesting that they were not the result of activation during processing. Next, we serially evaluated platelet counts in WT and c-mpl-/- mice during the transition from fetal/neonatal to adult megakaryopoiesis. At P2, c-mpl-/- platelet counts were 170±54x103/µl, representing 26% of those in P2 WT mice (648±137x103/µl). Over the first two weeks of life, WT platelet counts doubled and reached nearly adult levels, as previously described. In contrast, c-mpl-/- platelet counts failed to increase, and remained stably low at P14. Since we previously showed that the rise in platelet counts during this period was associated with a one-day prolongation of the neonatal platelet lifespan (Liu et al., Blood 2014), we evaluated platelet survival in c-mpl-/- compared to WT mice, using in vivo biotinylation. These studies suggested that the platelet lifespan was significantly shorter in c-mpl-/- compared to WT newborn (P2) mice. In contrast, we found no differences in platelet lifespan between c-mpl-/- and WT adult mice. Taken together, our findings suggest that, in fetal and neonatal life, Tpo signaling is required for normal MK proliferation, maturation, platelet formation, and platelet survival. This is in contrast to adult life, when Tpo deficiency leads only to reduced MK and platelet counts. We hypothesize that these differences reflect the lack of Tpo-independent pathways of MK maturation in the environment of the fetal/neonatal liver, and have implications in the developmental stage-specific clinical manifestations of CAMT. DisclosuresNo relevant conflicts of interest to declare.

Mmu-Mir-181a Regulates Terminal Erythroid Nuclear Maturation By Regulating Levels of Xpo7

Mammalian terminal erythroid differentiation involves cell cycle arrest, termination of transcription, and finally progressive chromatin condensation culminating in enucleation. The mechanisms underlying these final events are not completely understood. We have previously published that an erythroid-specific variant of the protein Exportin 7 (Xpo7) plays an important role in terminal erythroid nuclear maturation. Xpo7 is highly induced and highly abundant in late erythroblasts. Xpo7 knockdown in mouse erythroblasts inhibits the final stages of normal erythroid development such as chromatin condensation and enucleation without affecting such important processes as cell surface Ter119 acquisition or hemoglobin accumulation. Migration of nuclear proteins such as core histones into the cytoplasm during normal erythropoiesis is also inhibited by Xpo7 knockdown (1).In order to interrogate the function of Xpo7 in erythropoiesis, we next investigated Xpo7’s genomic structure and how it is regulated during mouse red blood cell development. Xpo7 exerts its effect on the erythroid nucleus after erythroblasts have left the cell cycle and transcription has ended so the timing of its induction must be tightly regulated.We have previously published that Xpo7 has an erythroid–specific start site and first exon (1). Using 5’ RACE, we determined that the erythroid variant of Xpo7 has a different 5’ UTR than the non-erythroid transcript. In contrast, the 3’ UTR is identical in both erythroid and non-erythroid transcripts, and contains a miR-181a binding site. MiR-181a has been previously shown to be abundant in hematopoietic stem cells and to regulate normal B-cell (2) and megakaryocyte development (3). However, in erythropoiesis, miR-181a decreases in expression just as Xpo7 increases, with lowest levels of miR-181a when Xpo7 is most abundant and presumably exerting its effect on erythroid nuclear maturation. Through luciferase reporter assay experiments, we have found that miR-181a directly binds the 3-UTR binding site of Xpo7. Moreover, miR-181a overexpression results in decreased Xpo7 transcript levels via qPCR and decreased enucleation via flow cytometry. Based on these results, we conclude that miR-181a regulates enucleation by repressing Xpo7 expression while the nucleus is active during terminal erythroid development. Our next step is to examine how miR-181a itself is downregulated during erythroid development, such as perhaps through the expression of a complementary long-noncoding RNA.1. Hattangadi, SM, et. al. Histones to the cytosol: Exportin 7 plays an important role in terminal erythroid nuclear maturation. Blood 2014, in press.2. Chen,CZ; Li, L; Lodish, HF; Bartel,DP. MicroRNAs modulate hematopoietic lineage differentiation. Science 2004; 303: 83–86 3. Li, X; Zhang J; Gao, L; McClellan, S; Finan, MA; Butler TW; Owen, BL; Piazza, GA; Xi, Y. MiR-181 mediates cell differentiation by interrupting the Lin28 and let-7 feedback circuit. Cell Death and Differentiation (2012) 19, 378–386. DisclosuresNo relevant conflicts of interest to declare.

Selinexor-Induced Thrombocytopenia Results from the Inhibition of Megakaryocyte Progenitor Cells in the Early Stage of Megakaryopoiesis

BackgroundSelinexor (KPT-330), a selective inhibitor of nuclear export (SINE) Exportin 1 (XPO1, CRM1) antagonist, has shown potent activity against solid and hematological malignancies in phase 1 clinical trials. Inactivation of XPO1 by selinexor results in accumulation of tumor suppressor proteins in the nucleus and activation of cell cycle checkpoints. This leads to transient cell cycle arrest in normal cells and irreversible arrest and cell death in cancer cells. Thrombocytopenia is a common side effect of selinexor and we aim to define the mechanism of selinexor-induced thrombocytopenia to potentially help in its clinical management. In this study, we have examined the effects of selinexor on platelet count in humans and mice. Additionally, we investigated the effects of selinexor on differentiation and maturation of megakaryocytes (MKs) in culture as a model system for understanding the effects on platelet levels in humans.MethodsPlatelet counts were measured in solid tumor patients in a Phase 1 trial of selinexor QoDx2 weekly. For in vivo studies, CD1 mice were treated with high-dose selinexor (20 mg/kg QoDx3) for 3 weeks and platelet number was determined in peripheral blood. In addition, femur bone marrow samples were analyzed histologically. In vitro, MK progenitors were isolated from fetal mouse livers, and MK maturation and platelet production was analyzed by microscopy, flow cytometry and immunofluorescence. In addition, whole protein lysates from selinexor-treated MKs were analyzed to detect changes in XPO1 and its cargo proteins.ResultsA majority of solid tumor patients treated with selinexor QoDx2 weekly in a Phase 1 trial for at least 1 cycle (4 wks) had >40% reduction in platelet count within the first cycle (72%, N=50), with an average maximal reduction of 50% after 20 days. These reductions were independent of pre-dose platelet count, did not typically decrease further after the first cycle, and recovered following dose reduction or cessation of treatment. Also, administration of the thrombopoietin (TPO) receptor agonists romiplostim and eltrombopag were found to reduce selinexor mediated thrombocytopenia. As in the clinical studies, selinexor reduced the platelets in mice by 40-50% within 2-3 weeks. In studies of MKs in cell culture, selinexor inhibited XPO1 mediated nuclear export as shown by nuclear localization of IkBa and FOXO3a proteins, but was not cytotoxic to MKs or platelets in vitro and did not affect platelet activation. However, MK progenitor cell development was significantly blocked in a dose dependent fashion, with 200 nM selinexor inducing a reduction of 81% in the number of progenitor cells that differentiated into MKs. The same dose of selinexor also affected endomitosis, a marker of MK maturation, shifting ploidy from predominantly 16N to 2N. Importantly, these effects were substantially reversible. When selinexor was washed out after 6 hours of treatment, the number of progenitor cells that differentiated into MKs was 65%, accompanied by reversion to 16N ploidy. Interestingly, MK inhibition was decreased when cells were treated later in the differentiation process and treatment of mature MKs did not decrease pro-platelet formation or release, suggesting selinexor affected an earlier stage in MKs development.ConclusionsSelinexor induces reductions in platelets in humans and mice. Our results suggest that this adverse effect is likely due to inhibition of the early commitment and differentiation phase of MK maturation from progenitor cells, and not a cytotoxic effect on normal stem cells or MKs. The reversibility of the effect in cell culture is congruent with the observations that selinexor-induced thrombocytopenia is reversible in humans upon dose interruption or reduction and/or the use of TPO receptor agonists by relieving the progenitor cell to MK differentiation block. Based on these results, the recommended Phase 2 dosing regimen of selinexor is twice weekly (days 1 and 3) for 3 weeks, followed by a 10-day dosing holiday and treatment with a TPO agonist if platelet counts are very low. DisclosuresMachlus:Karyopharm Therapeutics: Research Funding. Wu:Karyopharm Therapeutics: Research Funding. Carlson:Karyopharm Therapeutics: Employment. Friedlander:Karyopharm Therapeutics: Employment. Kashyap:Karyopharm Therapeutics: Employment. Kalid:Karyopharm Therapeutics: Employment. Shacham:Karyopharm Therapeutics: Employment. Rashal:Karyopharm Therapeutics: Employment. Shacham:Karyopharm Therapeutics: Employment. Italiano:Karyopharm Therapeutics: Research Funding. Landesman:Karyopharm Therapeutics: Employment.

Therapies Targeting the MAPK Pathway Improve Bone Marrow (BM) Fibrosis Induced By JAK2V617F

In primary myelofibrosis patients, somatic mutations such as JAK2V617F(JAKVF) and MPLW515 that activate JAK-STAT signaling are often seen. Small-molecule JAK2 inhibitors are effective for organomegaly and constitutional symptoms, but the drugs have little effect on BM fibrosis. To clarify the mechanism by which MPN cells with JAK2 mutations cause BM fibrosis, we compared the gene expression patterns of Lin−Sca1+ BM cells in JAK2VF transgenic mice (JAK2VF-TG), which develop myelofibrosis (MF), with that in WT mice. We found that TGFb1 and HOXB4, the target genes of transcription factor USF1 were highly expressed. TGFβ1, which is secreted by hematopoietic cells, is essential for fibrotic development in a murine model of MF (Chagraoui et al. Blood 2002), and increased expression of HOXB4 enhances human megakaryocytic development (Zhong et al. BBRC 2010).To investigate the mechanism of the high expression of these genes downstream of JAK2 signaling, USF1 and a cytokine receptor gene (MPL, EPOR or CSF3R) were co-transfected into 293T cells along with either a TGF-β1/HOXB4 promoter-driven or a STAT5 response element-driven luciferase reporter. Stimulation of MPL with TPO enhanced USF1 transcriptional activity about 3 fold, but stimulation of EPOR with EPO or of CSF3R with G-CSF did not change this activity. However, stimulation with any of the 3 types of cytokines enhanced STAT5 transcriptional activity. JAK2VF upregulated USF1 and STAT5 much more highly than JAK2WT without TPO stimulation. This USF1 upregulation specifically to TPO/MPL signaling was suppressed by a dominant negative mutant of USF1, JAK2 inhibitors (AG490, NS-018) or MEK inhibitors (U0126, PD325901). Inhibition of PI3K or p38MAPK did not affect the USF1 activation. Co-treatment with JAK2 and MEK inhibitors showed a synergistic effect in blocking both USF1 upregulation and STAT5 activation induced by JAK2VF.Next, we tested the MEK inhibitor, PD325901, in combination with the JAK2 inhibitor, NS-018, in the JAK2VF-TG mice. After disease was established 12 weeks after birth, JAK2VF-TG mice were divided into the following 4 groups: vehicle control; PD325901 monotherapy; NS-018 monotherapy; and combined therapy. PD325901 (5 mg/kg) and NS-018 (50 mg/kg) were orally administered once and twice daily, respectively. After 12 weeks of treatment, we evaluated the effect on BM fibrosis. The grading of MF in each group (n = 5-6) was as follows: vehicle control (MF-0: 0/6, MF-1 or 2: 6/6); PD325901 monotherapy (MF-0: 4/5, MF-1 or 2: 1/5); NS-018 monotherapy (MF-0: 0/6, MF-1 or 2: 6/6); and combined therapy (MF-0: 3/6, MF-1 or 2: 3/6). In the 2 groups treated with PD325901, 50~80% of mice showed MF-0. In contrast, in vehicle-treated or NS-018 monotherapy groups, all mice showed MF-1 or 2. Consistent with the MF grading, BM cellularity was significantly increased in the PD325901 monotherapy or combined therapy groups compared with the vehicle-treated group. A significant reduction was seen in the plasma TGFβ1 concentration in the PD325901 monotherapy and combined therapy groups compared with the vehicle-treated group (9.7 ng/ml, 8.1 ng/ml vs. 18.2 ng/ml, respectively). The TGFβ1 concentration in the extracellular fluid of BM (Wagner et al blood 2007) was also significantly reduced (5.6 ng/ml, 6.8 ng/ml vs. 9.1 ng/ml, respectively). BM cellularity and the TGFβ1 concentration in the NS-018 monotherapy group were comparable to those in the vehicle-treated group. Interestingly, megakaryocytes in the PD325901 monotherapy and combined therapy groups were decreased in number and were smaller than those in the vehicle-treated or NS-018 monotherapy groups. Regarding the effect on splenomegaly, spleen weight was significantly reduced in the NS-018 monotherapy and combined therapy groups compared with the vehicle-treated group (0.83 g, 0.69 g vs. 1.18 g, respectively). PD325901 monotherapy had little effect on splenomegaly.It is known that MEK-ERK1/2 pathway is critical in normal megakaryocyte development. In vitro data suggest that JAK2VF activates this pathway downstream of MPL and may contribute to TGFβ1 overproduction and dysmegakaryopoiesis, causing BM fibrosis via transcriptional enhancement of USF1. In vivo data suggest that MEK inhibition has the potential to improve dysmegakaryopoiesis and BM fibrosis. The combined therapy of JAK2 inhibitors with MEK inhibitors might be a promising therapy for improving both splenomegaly and BM fibrosis. DisclosuresNo relevant conflicts of interest to declare.

Imetelstat (GRN163L), a Telomerase Inhibitor Selectively Affects Malignant Megakaryopoiesis in Myeloproliferative Neoplasms (MPN)

Imetelstat is a telomerase inhibitor which has been shown to have therapeutic activity in patients with myelofibrosis (MF) (Tefferi A. et al., 2013 ASH Annual Meeting, Abstract 662) and essential thrombocythemia (ET) (Baerlocher G. et al., 2012 ASH Annual Meeting, Abstract 179). In clinical trials involving patients with a variety of other malignancies, the primary dose-limiting toxicity of GRN163L has been thrombocytopenia. We utilized GRN163L in order to explore the possible role of telomerase on human megakaryocytes (MK) and the mechanisms underlying the drug’s inhibitory effects on platelet production. MK were generated from normal primary CD34+ cells using an ex vivo culture system previously described by our laboratory (Iancu-Rubin C. et al, Blood 2011, 117:17; 4580-4589). We first showed that both telomerase activity and the expression of its catalytic unit hTERT were elevated in proliferating normal CD34+ cells, declined transiently during the initial stages of MK differentiation but were then partially restored during the final stages of terminal maturation. Treatment of normal CD34+ cells with GRN163L did not affect the numbers of CFU-MK assayed. Furthermore, exposure to GRN163L during the first 7 days in a liquid culture system did not interfere with the ability of CD34+ cells to commit to MK. When the same cultures were allowed to mature for 7 additional days, the proportion of CD34+/CD41+ MK precursors in drug-treated cultures was twice that observed in control and the drug-treated cultures contained 70% fewer mature CD41+/CD42b+MK than control cultures. The inhibitory effects of GRN163L on MK maturation were supported by observations showing that the cultures treated with the drug contained 60% fewer polyploid MK than control cultures. These results suggest that that GRN163L-mediated inhibition of telomerase does not affect normal MK progenitors but blocks the maturation of MK precursor cells.Previous studies have shown that GRN163L inhibits neoplastic CFU-MK colony formation by CD34+ cells from patients with ET (Brunold C. et al., 2011 ASH Annual Meeting, Abstract 3843). We, therefore, examined the effects of GRN163L on malignant MK from patients with MF and ET. Unlike normal CD34+ cells, treatment of MPN PB-MNCs with GRN163L decreased the numbers of assayable CFU-MK from 6 out of 11 patients. CFU-MK colony formation by PB-MNCs from these 6 patients was reduced by 33% (ranging from 13% to 50% reduction in CFU-MK formation) as compared to PB-MNCs treated with an inactive form of the drug (p value= 0.00473). Of note, in two out of 5 patients in which the total number of CFU-MK was not affected by GRN163L treatment, the drug decreased the size of the CFU-MK colonies formed. The ability of MPN PB-MNCs to differentiate into MK was next assessed. Although the total number of cells in PB-MNC liquid cultures exposed to GRN163L was decreased as compared to those treated with the inactive drug (n=6; p value=0.03204), the proportion of CD34+/CD41+MK precursors generated was not significantly affected. We then evaluated the effects of GRN163L on the genotype of MPN MK generated in the presence and absence of GRN163L by assessing the JAK2V617F allele burden. Treatment with GRN163L but not the inactive form of the drug reduced the JAK2V617F allele burden in MK derived from two patients: in one patient, JAK2V617F allele burden in MK generated in the presence of the inactive drug was 91.86% while MK generated in the presence of GRN163L was 19.75%; MK generated from a second patient had a JAK2V617F allele burden of 10.84% in the presence of the inactive drug which was reduced to 2.14% in the presence of GRN163L.We conclude that GRN163L-mediated inhibition of telomerase affects normal megakaryopoiesis by blocking the terminal maturation of CD34+/CD41+ MK precursors, providing a possible explanation for GRN163L’s propensity to induce thrombocytopenia in patients with normal bone marrow. By contrast, GRN163L treatment inhibited the ability of MPN CD34+ cells but not normal CD34+ cells to form CFU-MK colonies and drug treatment reduced the numbers of malignant MK generated. We propose that the amelioration of fibrosis observed in a clinical trial of MF patients treated with GRN163L might be due to, at least in part, two potential modes of action: 1) inhibiting malignant MK progenitors cells and 2) preventing terminal MK maturation thus depleting the pool of mature MKs which are the major source of fibrogenic cytokines in MF. DisclosuresIancu-Rubin:Geron Corporation: Research Funding. Parker:Geron Corporation: Employment. Eng:Geron Corporation: Employment. Hoffman:Geron Corporation: Consultancy.

Impaired proplatelet formation in immune thrombocytopenia: a novel mechanism contributing to decreased platelet count

The pathophysiological mechanisms contributing to the decreased platelet count in immune thrombocytopenia (ITP) are not entirely understood. Here, we investigated the key step of proplatelet formation (PPF) by studying the effect of ITP plasma in thrombopoiesis. Normal cord blood-derived mature megakaryocytes were cultured in the presence of recalcified plasma from ITP patients, and PPF was evaluated by microscopic analysis. Patient samples induced a dose-dependent inhibition in PPF, as well as decreased complexity of proplatelet architecture. Although slightly increased, plasma-induced megakaryocyte apoptosis was not related to PPF impairment. Purified IgG reproduced the inhibitory effect, while platelet-adsorbed plasma induced its reversion, suggesting the involvement of auto-antibodies in the inhibition of thrombopoiesis. Impaired PPF, induced by ITP plasmas bearing anti-GPIIb-IIIa antibodies, was related to their ability to interfere with the normal function of this integrin, as assessed by megakaryocyte PAC-1 binding and β3 integrin phosphorylation while the presence of anti-glycoprotein Ia-IIa auto-antibodies was associated with loss of normal inhibition of PPF induced by type I collagen. In conclusion, abnormal thrombopoiesis comprising decreased PPF and morphological changes in proplatelet structure are induced by patient samples, unveiling new mechanisms contributing to decreased platelet count in ITP.

Novel function of the chromosome 7 open reading frame 41 gene to promote leukemic megakaryocyte differentiation by modulating TPA-induced signaling

12-O-tetradecanoylphorbol-13-acetate (TPA) activates multiple signaling pathways, alters gene expression and causes leukemic cell differentiation. How TPA-induced genes contribute to leukemic cell differentiation remains elusive. We noticed that chromosome 7 open reading frame 41 (C7ORF41) was a TPA-responsive gene and its upregulation concurred with human megakaryocyte differentiation. In K562 cells, ectopic expression of C7ORF41 significantly increased CD61 expression, enhanced ERK and JNK signaling, and upregulated RUNX1 and FLI1, whereas C7ORF41 knockdown caused an opposite phenotype. These observations suggest that C7ORF41 may promote megakaryocyte differentiation partially through modulating ERK and JNK signaling that leads to upregulation of RUNX1 and FLI1. In supporting this, C7ORF41 overexpression rescued megakaryocyte differentiation blocked by ERK inhibition while JNK inhibition abrogated the upregulation of FLI1 by C7ORF41. Furthermore, we found that Y34F mutant C7ORF41 inhibited megakaryocyte differentiation. nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was the major activator of C7ORF41 that in turn repressed NF-κB activity by inhibiting its phosphorylation at serine 536, while MAPK/ERK was the potent repressor of C7ORF41. Finally, we showed that C7ORF41 knockdown in mouse fetal liver cells impaired megakaryocyte differentiation. Taken together, we have identified the function of a novel gene C7ORF41 that forms interplaying regulatory network in TPA-induced signaling and promotes leukemic and normal megakaryocyte differentiation.

Successful treatment of myelodysplastic syndrome and chronic hepatitis C using combined peginterferon‐α‐2b and ribavirin therapy

We report a patient with myelodysplastic syndrome (MDS) and hepatitis C virus (HCV) infection who was successfully treated with a combination of peginterferon and ribavirin therapy. A 65-year-old man was referred to our hospital for treatment of chronic hepatitis C and close examination of pancytopenia. MDS of "refractory cytopenia with multilineage dysplasia" was diagnosed on the basis of bone marrow findings. Although the patient was not a good candidate for interferon (IFN) therapy because of his pancytopenia, we decided to proceed with IFN therapy for the following reasons: his elevated transaminases could not be controlled; he had a high possibility of recovery from chronic hepatitis C in consideration of his HCV genotype 2a and relatively low RNA titer; and his pancytopenia was expected to worsen in the future. After combination peginterferon/ribavirin therapy, the patient achieved sustained viral response, and the bone marrow findings showed neutrophils with normal granulation and megakaryocytes with normal morphological features. Additionally, the normal 46, XY karyotype converted from 45, X0 which was found before IFN therapy. This suggested that the patient's MDS was completely resolved.

Ulcerative Colitis and Acute Thrombocytopenia in a Pediatric Patient: A Case Report and Review of the Literature

We report the case of a 14-year-old boy affected by ulcerative colitis (UC) and acute thrombocytopenic purpura (ITP) with simultaneous onset. UC diagnosis was based on symptoms, endoscopy and histology findings. ITP diagnosis was based on the normal bone marrow megakaryocyte count, the presence of platelet associated IgG and the absence of splenomegaly. Medical treatments including high doses of steroids, intravenous immunoglobulins were ineffective on ITP course, while UC course was mild for several months after the onset. When colonic inflammation became untractable not responsive at steroids and immunosuppressive agents, colectomy resolved both pathologies.

Activation of p53 by the MDM2 inhibitor RG7112 impairs thrombopoiesis

The tumor suppressor p53 is thought to play a role in megakaryocyte (MK) development. To assess the influence of the p53 regulatory pathway further, we studied the effect of RG7112, a small molecule MDM2 antagonist that activates p53 by preventing its interaction with MDM2, on normal megakaryocytopoiesis and platelet production. This drug has been previously been evaluated in clinical trials of cancer patients where thrombocytopenia was one of the major dose-limiting toxicities. In this study, we demonstrated that administration of RG7112 in vivo in rats and monkeys results in thrombocytopenia. In addition, we identified two distinct mechanisms by which RG7112-mediated activation of p53 affected human megakaryocytopoiesis and platelet production in vitro. RG7112 promoted apoptosis of MK progenitor cells, resulting in a reduction of their numbers and RG7112 affected mature MK by blocking DNA synthesis during endomitosis and impairing platelet production. Together, the disruption of these events provides an explanation for RG7112-induced thrombocytopenia and insight into the role of the p53-MDM2 auto-regulatory loop in normal megakaryocytopoiesis.

Megakaryocyte morphology and its impact in predicting response to steroid in immune thrombocytopenia

Pathogenetic mechanisms of primary immune thrombocytopenia (ITP) include antibody-mediated destruction of platelets; cell-mediated destruction and suppressed thrombopoiesis. Various morphological changes in megakaryocytes are reported in ITP patients, but never these were correlated with the response to various forms of therapy. In the present study, we intended to find out the impact of megakaryocytic abnormalities on the response to steroid, the first line of treatment. The mean age of patients (n = 33) was 28.9 ± 7.6 years. Male/female ratio was 1/1.8. The mean platelet count at presentation was 5.6 ± 4.4 × 109/L. 63.6% (21/33) patients showed response to steroid. The non-responders were given 50 mg/d TPO-RA (eltrombopag) in addition to steroid, to which, 83.3% (10/12) responded. Two-third patients (66.7%, n = 22) had normal megakaryocyte morphology. Those with abnormal morphology commonly had hypolobated forms and micromegakaryocytes. Ninety-five percent of steroid responders had normal megakaryocytic morphology. Among steroid non-responders, most patients (n = 10, 83.3%) had abnormal megakaryocytic morphology. 80% steroid non-responders with abnormal morphology responded to the addition of eltrombopag. The findings suggest that the abnormal megakaryocyte morphology can be an evidence of dominant pathogenetic mechanism and thereby can help us in individualizing the treatment of ITP.

Aurora A Kinase Is Required For Hematopoiesis and Couples Polyploidization With Terminal Differentiation In Megakaryocytes Through Phosphorylation Of NF-E2

We have recently shown that small molecule inhibitors of Aurora A kinase (AURKA) including dimethyfasudil and MLN827 induce polyploidization and differentiation of normal and malignant megakaryocytes, as evidenced by increased DNA content and upregulation of the cell surface markers CD41 and CD42. Furthermore, our pre-clinical studies demonstrate that MLN8237 shows potent anti-leukemia activity and anti-myelofibrotic activity in MPNs by promoting polyploidization and terminal differentiation of abnormal megakaryocytes. To determine the mechanism by which AURKA inhibitors ameliorate the leukemic and myelofibrotic phenotypes, we have examined the functional requirement for Aurka in adult hematopoiesis. To circumvent the embryonic lethality of the germline knockout and study the requirement for AURKA in adult hematopoiesis, we induced deletion in Aurka floxed mice with Mx1-Cre. Complete loss of AURKA caused a rapid and profound defect in hematopoiesis, with the mice developing pancytopenia and marked hypocellularity of the bone marrow. Notably, we observe an increase in the proportion of CD41 and CD42-positive megakaryocytes in contrast to the deficiency of myeloid and lymphoid cells in the bone marrow. To determine whether the observed defects are cell autonomous, we transplanted Aurkafl/fl, Mx1-Cre bone marrow cells to lethally irradiated recipients, confirmed engraftment, and induced deletion with pIpC. Upon AURKA deletion, the transplanted mice develop an identical phenotype to the one observed in the conditional knockout mice, demonstrating a cell autonomous requirement for AURKA during hematopoiesis. Moreover, in competitive transplantation experiments, Aurka-/- cells are selectively lost and fail to contribute to hematopoiesis. Mechanistically, loss of AURKA led to a significant degree of apoptosis in hematopoietic cells, likely due to mitotic catastrophe resulting from impaired chromosome segregation. To bypass the requirement for AURKA in progenitor cells and examine AURKA function in the megakaryocyte lineage, we deleted AURKA in megakaryocytes ex vivo by infecting Aurka floxed bone marrow with MSCV-Cre. We found that deletion of AURKA resulted in increased CD41 and CD42 expression as well as increased ploidy of the megakaryocyte fraction. Together these results are consistent with a selective differentiation effect of AURKA deficiency on megakaryocytes.To investigate whether AURKA modulates differentiation of megakaryocytes through interactions with lineage specific transcription factors, we performed co-immunoprecipitation experiments between AURKA and megakaryocyte transcription factors containing consensus AURKA phosphorylation sites in primary megakaryocytes. Results confirmed a robust interaction between AURKA and p45 NF-E2. Additionally, we found by an in vitro kinase assay that AURKA phosphorylates p45 NF-E2 on the S170 residue, as the S170A mutant of NF-E2 cannot be phosphorylated by AURKA. To explore the functional importance of NF-E2 phosphorylation at S170, we created a phosphomimetic mutant, S170D, and assayed its ability to induce megakaryocyte differentiation in primary cultures. While overexpression of wild-type NF-E2 significantly increased the expression of the megakaryocyte differentiation markers CD41 and CD42, overexpression of the S170D mutant was markedly less effective in promoting megakaryocyte differentiation, indicating that the loss of phosphorylation promotes NF-E2 activity and that NF-E2 activity may be suppressed by AURKA phosphorylation. Finally, to determine if AURKA inhibitors promote megakaryocyte differentiation by allowing activation of NF-E2, we knocked-down down NF-E2 in megakaryocytic cell lines and subsequently treated with diMF or MLN8237. Strikingly, cells with NF-E2 knocked-down displayed significantly less CD41 and CD42 expression in response to AURKA inhibitor treatment compared to control knockdowns. This experiment demonstrates that NF-E2 is required for the differentiation effect caused by AURKA inhibition. Taken together, our data show that Aurora A kinase is required for adult hematopoiesis and that Aurora A regulates NF-E2 function during megakaryocyte differentiation. Our results also reveal that inhibition of AURKA kinase activity couples polyploidization and terminal differentiation of megakaryocytes. Disclosures:Crispino: Sanofi: Research Funding.

The Zinc Finger Transcription Factor (Growth Factor Independence 1b) Gfi1b Regulates Megakaryocyte Proliferation and Their Ability To Produce and Release Platelets

Under normal conditions, humans maintain a blood content of 150-400 x 109 platelets per liter, whereas mice can reach 1000 x 109 platelets per liter. Thrombocytopenia occurs when the level of platelets becomes too low, a situation that increases the risk of spontaneous bleeding and hemorrhage. Although the number of platelets is continuously very high, they are produced by a rare cell population the megakaryocytes (MKs), which in turn are produced by megakaryocyte-erythrocyte precursors (MEPs) in the bone marrow. The first report of a potential role for the zinc finger transcription factor Gfi1b in megakarypoiesis and thrombopoiesis showed that the full knock-out of this gene in mice leads to a severe impairment of both erythropoiesis and megakaryopoiesis, translating into a severe thrombocytopenia and a lethality by day e15.5 (Saleque et al Genes Dev 2002). Because of this developmental arrest at mid-gestation, the function of Gfi1b in adult differentiated hematopoietic cells could not be analyzed. We have thus generated conditionally deficient mice carrying floxed Gfi1b alleles, to study the role of Gfi1b in adult hematopoiesis. To ablate Gfi1b expression, we crossed Gfi1b flox/flox mice with animals carrying two different Cre transgenes: a ROSA-Cre-ERT2 transgene that quickly deactivates Gfi1b in all cells upon treatment with tamoxifen, allowing to almost instantly measure effects on already differentiated MKs and ii) a PF4-Cre transgene that constitutively expresses the Cre specifically in MKs, excluding any adverse effects due to other cell types.With these mice, we could show that Gfi1b ablation leads to a strong proliferation and expansion of both MEPs, MK precursors and MKs, which was surprisingly associated with an almost complete loss of platelets (∼99.9% reduction compared to controls in PF4-Cre, Gfi1bflox/flox mice). Most striking was the strong increase in the number of MKs, even from an early stage of differentiation, when Gfi1b was deleted. It was not clear, however, if this expansion of MKs was caused directly by the loss of Gfi1b in early progenitors, or if it was a consequence of the severe thrombocytopenia that could stimulate megakaryopoiesis through a feedback loop. An in vivo kinetic study of platelet loss and MK proliferation in the ROSA-Cre-ERT2, Gfi1bflox/flox mice revealed that the expansion of MKs started as early as 3 days after administration of tamoxifen (over 4-fold increase compared to age matched controls). On the other hand, platelet counts started only to decrease noticeably 4 days after tamoxifen administration (about 400 x 109/L vs 1200 x109/L), although reticulated platelets started to decrease as early as day 2 after tamoxifen injection and almost completely disappeared by day 4 after tamoxifen treatment. This suggests an arrest in platelet release in the absence of Gfi1b. After 4 days of tamoxifen treatment, platelet counts decreased quickly to reach a minimum around day 7-8 (60 x 109/L vs 1350 x 109/L in controls). By this time, the number of MKs literally exploded to reach levels up to 15 fold higher than in wild type controls. These results indicate that the number of MKs increases prior to the decrease in circulating platelet when Gfi1b is deleted. However, the results may also suggest that a feedback loop could contribute to this phenomenon by boosting MK expansion upon platelet loss. A platelet lifespan analysis on the rare remaining platelets in PF4-Cre, Gfi1bflox/flox mice revealed that the low platelet level was not due to accelerated platelet clearance, confirming that the platelet loss was the result of an arrest in platelet release. Gfi1b deficient MKs are still polyploidy, but are significantly smaller and have a different nuclear cytoplasmic ratio than their wt counterparts. In addition, in contrast to normal wt MKs, Gfi1b deficient MKs were unable to properly spread or migrate on fibronectin or fibrinogen surfaces, showed lower F-actin content and an increased expression of the platelet glycoprotein IIb of IIb/IIIa complex (CD41/CD61) on their surface. These data suggest that Gfi1b controls the signaling of the platelet specific integrins to restrict MK proliferation, control MK size and their ability to produce platelets. Disclosures:No relevant conflicts of interest to declare.

Arginine Methylation Of RBM15 By PRMT1 Controls Alternative Splicing Of Genes Involved In Megakaryocyte Differentiation

More than 90% of under one year old infants with acute megakaryoblastic leukemia (AMKL) have chromosome translocation t(1;22)(p13;q13) with RBM15 fused to MKL1. RBM15 encodes an RNA binding protein important for hematopoietic stem cell self-renewal and differentiation. In agreement with its roles in AMKL, RBM15 is required for normal megakaryocyte differentiation. We found that higher expression of PRMT1 (Protein Arginine Methyltransferase) is commonly seen in M7 leukemia patient samples than other types of myeloid leukemia and that RBM15 is a bona fide methylation target for PRMT1. Using mass spectrometry analysis, we mapped the PRMT1 mediated mono-methylated site. The enzymatic activity of the PRMT1 V2 isoform is required for RBM15 degradation, as both shRNA molecules knocking down PRMT1 and small chemical PRMT1 inhibitors stabilize the RBM15 protein. Mutation of the methylation site to lysine blocks the ubiquitylation mediated degradation. Thus the degradation is a methylation dependent process. We identified the E3 ligase responsible for the degradation. Down-regulation of the RBM15 protein changes the isoform ratio of genes including GATA1 critical megakaryocyte differentiation. We found that RBM15 regulates its interaction with SF3B1A in methylation dependent manner during alternative splicing of GATA1 pre-mRNA. Thus, via methylation triggered RBM15 degradation, the megakaryocyte progenitor cells maintain a delicate balance between differentiation and proliferation by keeping the proper ratio of GATA1s and GATA1-full length mRNA. SF3B1A has been shown to be mutated in myeloid dysplasia syndrome and in several different types of leukemia. Methylation by PRMT1 links the two types of leukemic genes into a single pathway. Our results imply that targeting PRMT1/RBM15 pathway might be a potential therapy for AMKL and other blood malignancies. Disclosures:No relevant conflicts of interest to declare.

Imetelstat, a Telomerase Inhibitor, Induces Morphologic and Molecular Remissions In Myelofibrosis and Reversal Of Bone Marrow Fibrosis

BackgroundJAK inhibitors, including ruxolitinib, are to date incapable of inducing complete (CR) or partial (PR) remissions, reversal of bone marrow (BM) fibrosis or molecular responses in myelofibrosis (MF). This is consistent with the fact that JAK2 mutations are neither specific nor pathogenetically essential for the disease. Other currently available drugs in MF are equally ineffective in terms of disease-modifying activity. MethodsIn an investigator-sponsored single-center study (ClinicalTrials.gov Identifier: NCT01731951), imetelstat, a lipid-conjugated oligonucleotide inhibitor of human telomerase, was administered to patients with high or intermediate-2 risk MF (JCO 2011). Adverse events were monitored by common terminology criteria (Version 4.03) and responses by the International Working Group criteria (Blood 2013). Eligibility criteria included platelets ≥50 x 10(9)/L. Study drug and funding were provided by Geron Corporation (Menlo Park, CA, USA).Imetelstat was administered by a 2-hour intravenous infusion (9.4 mg/kg) every three weeks (cohort A) or weekly x 3 followed by every three weeks (cohort B). Mutations with prognostic (ASXL1 and SRSF2) or phenotypic (SF3B1 and U2AF1) relevance were screened by DNA sequencing. Quantitative PCR was used to measure JAK2V617F burden (assay sensitivity 0.01%). Laboratory correlative studies included analyses of granulocyte telomere length, mononuclear cell telomerase activity and human telomerase reverse transcriptase (hTERT) isoforms. ResultsThirty-three patients were accrued; the first 18 patients enrolled and followed for a minimum of 3 months or discontinued are presented in this abstract: 11 cohort A and 7 cohort B; 44% PMF, 33% post-PV MF and 22% post-ET MF. Median age was 68 years and baseline risk was high in 56% and intermediate-2 in 44%. Seven patients were transfusion-dependent. Median spleen size was 13 cm and 11 patients had constitutional symptoms. Karyotype was abnormal in 7 patients and 89% were JAK2-mutated. Fifteen (83%) patients were previously treated including 7 with a JAK inhibitor and 3 with pomalidomide.i) ToxicityAt a median f/u of 3.2 months, 16 (89%) patients remain on treatment; the two discontinuations were from unrelated death and disease progression. In cohort A, there were no grade-4 treatment-related adverse events; grade-3 events were limited to thrombocytopenia in 27% and anemia in 9%. In cohort B, two (29%) patients experienced grade-4 thrombocytopenia; grade-3 events were limited to thrombocytopenia, neutropenia and anemia in one patient each. Dose reduction was necessary in only two (11%) patients because of grade 3 or 4 myelosuppression.ii) EfficacyOverall response rate was 44%. This included five (28%) patients who met the BM and peripheral blood morphologic criteria for CR (n=4) or PR (n=1) and 3 patients with clinical improvement, pending validation of response duration and resolution of drug-induced grade-1 thrombocytopenia. The four (22%) CR patients experienced reversal of BM fibrosis and recovery of normal megakaryocyte morphology. Two CR patients were transfusion-dependent at baseline and became transfusion-independent. Complete molecular responses were documented in 2 CR patients: one had U2AF1Q157P and 10% JAK2V617F and the other SF3B1K666E and 50% JAK2V617F. A third CR patient had a >50% reduction in U2AF1 469_insAGTATG mutation. Among 13 patients with leukocytosis, 10 (77%) normalized their count or had >50% reduction. Eleven (61%) patients had complete or partial resolution of leukoerythroblastosis.iii) Laboratory correlative studiesThree (50%) of 6 spliceosome-mutated vs. 1 (8%) of 12 unmutated (p=0.045) achieved CR. Spliceosome-mutated patients were also more likely to experience grade-3/4 myelosuppression (67% vs. 25% ; p=0.09). Treatment was associated with suppression of telomerase activity, shortening of telomere length and alteration of hTERT isoform pattern. ConclusionsThe current study signifies the potential value of telomerase-based treatment strategies in MF and identifies imetelstat as an active drug in that regard. The observed morphologic and molecular remissions confirm selective anti-clonal activity, which has thus far eluded other drugs in MF, including JAK inhibitors. The association between response and spliceosome mutations suggests a broader application for the drug in myeloid malignancies.</abstract> Disclosures:Stuart:Geron: Consultancy.

Single Cell Analysis Of Protein Phosphorylation In Chronic Myeloid Leukemia Treated With Dasatinib, Eltrombopag, and Pegfilgrastim

IntroductionTyrosine kinase inhibitor (TKI) induced neutropenia and thrombocytopenia may be relieved with G-CSF and trombopoietin analogs. To characterize the signaling response in a CML patient treated with hematopoietic growth factors and TKI, we employed single cell analysis of phosphoproteins in serial samples. MethodsWe characterized the single cell phosphoprotein responses to treatment with dasatinib, eltrombopag, and pegfilgrastim in CML-like cell lines K562 and KCL-22, normal peripheral blood leukocytes and leukocytes from a patient starting on nilotinib. These data formed a basis for comparison with serial samples (Figure 1) from a CML patients after extensive wash-out before restarted on third-choice TKI, combined with eltrombopag and pegfilgrastim. For characterization of in vivo transformational potential CML patient bone marrow cells were engrafted in duplicate in NOG mice by femoral injection. Basal phosphorylation was determined using 17 phosphorylation specific antibodies: All staining panels contain the same 4 surface antibodies and a live-dead discriminator; CD33(P67.6) PerCP-Cy5.5, CD38(HB7) PE-Cy7, CD34(581) PE, CD45(MEM-28) PE-Dynamics590 and phospho c-PARP(Asp214) AlexaFlour-700. Two phosphospecific antibodies were added to each panel with the respective direct conjugated dye Alexa Flour 488 and Alexa Flour 647. The lowest median signal for each phosphoprotein in CML cells and lymphocytes, respectively, were used as reference value for calculation of basal phosphorylation. [Display omitted] ResultsA 56 y old male was diagnosed with CML (Sokal RR 0.75, Hasford RR 929, karyotype 46,XY,t(9;22)(q34;q11)[20], FISH 9q34 ABL/22q11.2 BCR dual fusion 62% of cells, PCR positive BCR-ABL e13e2/e14a2), developed prolonged thrombocytopenia and neutropenia on various dose levels of imatinib and nilotinib (neutrophile <1x109/L, <30x109/L) during the first 7 months after diagnosis. After a 6 months pause of TKI therapy: quantitative PCR BCR-ABL1/ABL1 25%IS, bone marrow biopsy with reduced normal megakaryocytes. Platelet supportive therapy with eltrombopag 50 mg daily was initiated 14 days before start with dasatinib 50 mg daily keeping platelet level above 50. After two more weeks pegfilgastim 6 mg was started, increasing neutrophils above 1.0x109/L.The CML bone marrow engrafted without symptomatic disease in NOG mice over 6 months monitoring, in contrast to engraftment with disseminated disease in an accelerated phase CML patient sample and the KCL-22 cell line. Phosphorylation of patient myeloid cells indicated STAT3(pY705) and STAT5(pY694) phosphorylation 2 and 3 hours after intake of eltrombopag. Dasatinib modestly inhibited SRC(pY418) and STAT3(pY705), but only transiently blunted the effect of eltrombopag. Pegfilgrastim strongly induced STAT3(pY705), STAT3(pS727), and SRC(pY418). Further analyses of cellular subsets and phospho-Abl will be presented. ConclusionEltrombopag kept stable levels of platelets on dasatinib treatment, and cell phosphorylation reflected persisting signaling induced by eltrombopag after seven days on combination therapy. Pegfilgrastim induced strong phosphorylation of STAT3(pY705) during dasatinib co-therapy. Phosphoprotein signal analysis of patient leukemic cells reflected the hematological response of dasatinib, eltrombopag, and pegfilgrastim and may be used to monitor therapy responses of and growth factors and targeted therapy.Phosphoprotein modulated early after start of treatment. (A) Each day 1-23 is analysed separately with its pretreatment sample as control and signal baseline. (B) Various treatment days are compared, using pretreatment control at day 1 used as baseline. (A) and (B) were analysed by flow cytometry at the same day, using bar code labelling, sample processing and analysis as previously described (Skavland et al. Blood Cancer J 2012). On day 16 dasatinib and eltrombopag is administrated concomitantly. Signalling responses in lymphocytes were distinct different from myeloid cells. Further determination of cellular subsets and their signalling responses will be presented. Disclosures:Popa:KinN Therapeutics AS: Employment.

Differential regulation of the apoptotic machinery during megakaryocyte differentiation and platelet production by inhibitor of apoptosis protein Livin

Livin is a member of the inhibitor of apoptosis proteins (IAP) family of intracellular antiapoptotic proteins that act by binding and inhibiting caspases. Upon strong apoptotic stimuli, it is then specifically cleaved by caspases to produce a truncated protein (tLivin) with a paradoxical proapoptotic activity. Intriguingly, we have detected robust protein levels of Livin in normal mature bone marrow megakaryocyte (MK) and platelets. To evaluate the potential role of Livin in thrombopoiesis, we used the human BCR-ABL+ cell line, LAMA-84, and cord blood CD34+ cells to induce differentiation toward MKs. Upon differentiation, induced by phorbol myristate acetate and concurrent with increase in Livin protein expression, LAMA-84 cells formed functional platelet-like particles. Livin overexpression in CD34+ progenitor cells induced higher endoreplication in the MKs generated. Furthermore, overexpression of Livin increased the ability of both primary MKs and differentiated LAMA-84 cells to produce functional platelets. In the differentiated LAMA-84 cells, we observed accumulation of proapoptotic tLivin concomitant with increased caspase-3 activity. Downregulation of Livin with small interfering RNA in both leukemic and primary MK cells decreased their ability to produce functional platelets. We suggest that Livin has a role in thrombopoiesis by regulating the apoptotic and antiapoptotic balance in MK endoreplication and platelet production.