Proliferation Of Megakaryocyte Progenitors Research Articles

Survival Outcomes of Acute Megakaryoblastic Leukemia in the United States: A 10-Year SEER-Based Study

Background: Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML), commonly observed in children with Down Syndrome (DS) but also seen in adults. The cytogenetic abnormality t(1;22)(p13.3;q13.1), has been reported in children with non-DS AMKL, which involves the fusion of RNA-binding motif protein-15 and megakaryoblastic leukemia-1 resulting in abnormal proliferation of megakaryocyte progenitors. Population-level data on outcomes of AMKL is scarce. Methods: We conducted a retrospective analysis using the Surveillance Epidemiology and End Results (SEER) database, a U.S. population-based registry maintained by the National Cancer Institute. We included all cases with histologically confirmed diagnoses of AMKL and AML with t(1;22)(p13.3;q13.1) between the years 2010 and 2019. Survival analysis was performed using the Kaplan-Meier method, and differences in overall survival (OS) were assessed using the log-rank test. Results: The analysis included a total of 217 cases, consisting of 54 patients with AML with t(1;22)(p13.3;q13.1) and 163 patients with AMKL. Among the AML cases with the cytogenetic abnormality, the median age was 58 years (ranging from 0 to 89 years), and 61% were female. 74% (n=40) of these patients received chemotherapy. The 1-year survival rate was 54.0% for the pediatric group (age < 18 years, n=10) and 44.6% for the adult group (age > 18 years, n=44) (Median OS 20 vs 8 months, p=0.69). Patients who received chemotherapy had a significantly higher 1-year survival rate of 57.9% compared to 14.3% in those who did not receive chemotherapy (p=0.0005). No significant difference in survival was observed when patients were grouped based on the year of diagnosis, with 1-year survival rates of 50.8% for the period 2010-2014 and 41.3% for 2015-2019 (p=0.66). Patients with AMKL were divided into two age groups: pediatric AMKL (age < 18 years, n=96) and adult AMKL (age > 18 years, n=67). The median age in this group was 3 years (ranging from 0 to 86 years), and 55% were male. 81% (n=132) of these patients were treated with chemotherapy. The 1-year survival rate for the pediatric group was 79.4%, whereas it was 17.6% for the adult group (p<0.0001). Chemotherapy significantly improved overall survival (OS) for both pediatric and adult groups, with 1-year survival rates of 82.5% vs. 35.7% in the pediatric group and 27.6% vs. 0.00% in the adult group in patients who did and did not receive chemotherapy, respectively (p=0.041 for pediatric group and p<0.0001 for adults). No significant differences in survival were observed among the groups based on the year of diagnosis, both in adults and the pediatric group. Conclusion: AMKL is commonly seen in infants and children, but it can affect individuals of all age groups. The prognosis is more favorable in pediatric patients. In contrast, outcomes for adults remain poor with current chemotherapeutic agents. Interestingly, AML with t(1;22)(p13.3;q13.1), which has been predominantly reported in children in the literature, was found to be more common in adults in this study. Regardless of age group, current treatments show poor outcomes. This study emphasizes on the urgent need for investigational agents.

Recombinant Human Thrombopoietin Combined with Thrombopoietin Receptor Agonists Can Facilitate the Recovery of Platelet after Allogeneic HSCT

Introduction:Thrombocytopenia is a common complication after hematopoietic stem cell transplantation (HSCT) and is strongly associated with transplant-related morbidity and mortality. According to the statistics, the incidence of post-HSCT thrombocytopenia is about 5%-37%. So far, there is no standard treatment regimen for thrombocytopenia after HSCT. Recombinant human thrombopoietin (rhTPO) can act on megakaryocytes alone and has been wildly used in clinical practice. Thrombopoietin receptor agonists (TPO-RAs), another type of drugs that can increase platelet count, has been available since 2008. It can bind to the TPO receptor and cause the conformational change of the receptor, and then activate the JAK2/STATE5 pathway to increase the proliferation of megakaryocyte progenitors and platelet production. There have been many prospective or retrospective studies discussing the efficacy and safety of rhTPO and TPO-RAs monotherapy in patients with thrombocytopenia. In clinical practice, it is very meaningful to clarify whether the combination of two types of drugs has a synergistic effect when one type of drug has poor efficacy. M ethods This study is a retrospective study in total 12 patients from 2022 to 2023. We selected a total of 6 patients who underwent HSCT in our center during the past year, and used rhTPO combined with avatrombopag to help platelet recovery as observation group. Then, 6 patients treated with rhTPO alone with no statistical difference at baseline were selected as the control group. The basic characteristics of these patients are shown in the table1. All the patients were informed and consented to use rhTPO+TPO-RA (avatrombopag) or rhTPO alone. If platelet count <20×10 9/L during treatment，which will be treated with platelet infusion. All the patients were treated with a modified BUCY regimen as the conditioning regimen and ATG+MMF+cyclosporine+short-range MTX were used to prevent graft vs host disease (GVHD). In observation group, the patients were treated with rhTPO 15000U qd i.m and avatrombopag 40mg qd po, while in control group the patients were treated with rhTPO 15000U qd i.m alone. We recorded the platelet counts of the two groups at day0,7,14,21. Based on this data, the percentage increase and growth rate of platelets were calculated. (the percentage increase: difference in platelet count before and after medication/platelet count before medication ×100%; the growth rate: difference in platelet count before and after medication/duration of medication.) The above two values were used to evaluated the efficacy of two treatment protocols. Adverse events occurred during treatment including fever, weakness, liver damage, joint pain, bleeding and thrombosis would be recorded to evaluate the safety. Results In observation group, patients' platelet counts increased by an average was 54×10 9/L (range 40 to 69). In control group, the average and median increase in their platelet counts was 22 (range -2 to 49). P=0.025 (P<0.05). In terms of the percentage increase in platelet count, the average increase was 452% in observation group VS. 110% in control group. p=0.022(P<0.05). Refer to Figure1a. The average growth rates of platelet counts in observation group were 2.03×10 9/L per day. The averagerate of increase in control group were 1.07×109/L per day. P=0.041(P<0.05). The mean platelet count of the two groups at various time points were shown in Figure1b. None of the patients had any adverse events occurred. No patient developed clinical meaningful abnormality of liver function and discontinued avatrombopag or TPO treatment due to intolerability and adverse effects. Conclusion Our data shows that the treatment protocol of avatrombopag combined with rhTPO may have a greater effect on platelet growth and boost a faster platelet growth rate. Importantly, no additional adverse effects occurred when the addition of avatrombopag.

Stathmin 1 deficiency induces erythro-megakaryocytic defects leading to macrocytic anemia and thrombocythemia in Stathmin 1 knock out mice

Stathmin 1 (STMN1) is a cytosolic phosphoprotein that was discovered as a result of its high level of expression in leukemic cells. It plays an important role in the regulation of mitosis by promoting depolymerization of the microtubules that make up the mitotic spindle and, aging has been shown to impair STMN1 levels and change microtubule stability. We have previously demonstrated that a high level of STMN1 expression during early megakaryopoiesis is necessary for proliferation of megakaryocyte progenitors and that down-regulation of STMN1 expression during late megakaryopoiesis is important for megakaryocyte maturation and platelet production. In this report, we examined the effects of STMN1 deficiency on erythroid and megakaryocytic lineages in the mouse. Our studies show that STMN1 deficiency results in mild thrombocytopenia in young animals which converts into profound thrombocythemia as the mice age. STMN1 deficiency also lead to macrocytic changes in both erythrocytes and megakaryocytes that persisted throughout the life of STMN1 knock-out mice. Furthermore, STMN1 knock-out mice displayed a lower number of erythroid and megakaryocytic progenitor cells and had delayed recovery of their blood counts after chemotherapy. These studies show an important role for STMN1 in normal erythro-megakaryopoietic development and suggests potential implications for disorders affecting these hematopoietic lineages.

Ex vivo Engineering of Platelets

Platelet transfusion is necessary for patients in thrombocytopenic states. Due to short shelf life of platelet product, the shortage of blood donors in the younger population as the consequence of aging societies in developed countries and platelet transfusion refractoriness (PTR) caused by alloimmune response, the risk of platelet product shortage has been a concern. Therefore, many attempts to substitute donor-dependent platelet product have been proposed. Induced pluripotent stem cell (iPSC) derived-platelet like particle product (which we refer as iPS-platelets) is aimed to provide measures against alloimmune PTR as well as to complement the current blood donor-dependent system.To generate the huge number of platelets required for transfusion into thrombocytopenia patients (200-300 billion per transfusion) ex vivo, megakaryopoiesis and subsequent platelet biogenesis process from megakaryocytes needed to be both substantially elucidated in mechanical systems. For the former, our key measure is to expand the immortalized megakaryocyte cell lines (imMKCLs) derived from iPSCs, whereby c-MYC, BMI-1, and Bcl-XL are overexpressed under doxycycline-inducible system to regulate proliferation of megakaryocyte progenitors (Cell Stem Cell, 2014). For the latter, in vivovisualization of mouse bone marrow revealed the presence of turbulence adjacent to megakaryocytes actively releasing platelets, which prompted us to utilize a unique turbulent flow-incorporated bioreactor to produce iPS-platelets. Furthermore, we succeeded in identifying turbulent energy and shear stress as essential physical parameters and further determined the optimal values, thereby to enable efficient and intact ex vivoplatelet manufacture, as exemplified by lowered Annexin V binding values in iPS-platelets, comparable with donor-human platelets. Simultaneously, we developed a culture medium cocktail including a thrombopoietin (TPO) mimetic small compound, an ADAM10/17 inhibitor for the maintenance of GPIb-alpha expression on platelets, an arylhydrocarbon receptor inhibitor, and a ROCK inhibitor for feeder cell-independent megakaryocyte maturation (Stem Cells Transl Med, 2017; Blood Adv, 2017; Blood Adv, 2018). By scaling up of the bioreactor, 100 billion iPS-platelets were produced in 8 L scale. In vitro and in vivo evaluation of iPS-platelets showed the functionality comparable with blood donor-derived platelets (Cell, 2018), including the newly developed thrombocytopenia rabbit model (Transfusion, 2017). Meanwhile, an iPS cell-derived human leukocyte antigen (HLA) class-I-null imMKCL as a universal HLA platelet source is potentially the best solution for alloimmune PTR. However, the immunogenic properties are still unclear, especially regarding natural killer (NK) cells that attack HLA-downregulated cells. Therefore, we evaluated the immunological reaction between HLA-null platelets and NK cells by using our newly developed alloimmune PTR in vivo model in which human NK cells are highly reconstituted in MISTRG mice (Suzuki and Sugimoto et al.,manuscript in revision). This pre-clinical study model should provide a proof-of concept for the clinical application of HLA-null iPS-platelets as a universal platelet product for future transfusion. References Nakamura S, et al. Expandable Megakaryocyte Cell Lines Enable Clinically Applicable Generation of Platelets from Human Induced Pluripotent Stem Cells. Cell Stem Cell. 2014, 14, 535-548.Hirata S, et al. Selective Inhibition of ADAM17 Efficiently Mediates Glycoprotein IbαRetention During Ex Vivo Generation of Human Induced Pluripotent Stem Cell-Derived Platelets. Stem Cells Transl Med. 2017, 6, 720-730.Aihara A, et al. Novel TPO receptor agonist TA-316 contributes to platelet biogenesis from human iPS cells. Blood Adv. 2017, 1, 468-476.Seo H, Chen SJ, et al. A β1-tubulin-based megakaryocyte maturation reporter system identifies novel drugs that promote platelet production. Blood Adv. 2018, 2, 2262-2272.Ito Y, Nakamura S, et al. Turbulence Activates Platelet Biogenesis to Enable Clinical Scale Ex Vivo Production. Cell. 2018, 174, 636-648.Watanabe N, et al. Refined methods to evaluate the in vivo hemostatic function and viability of transfused human platelets in rabbit models. Transfusion. 2017, 57, 2035-2044. Disclosures Eto: Megakaryon Co. Ltd.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Our published results that include some drugs, i.e., TA-316 (Blood Advances, 1(7):468-476, 2017), KP457 (Stem Cells Transl Med, 6(3):720-730, 2017), and SR1 (Cell, 174(3):636-648, 2018)

Azacytidine Inhibits Megakaryopoiesis Via the Induction of Immunogenic RNA Species and Activation of Type-I Interferon Signaling

Management of hematologic disease- and therapy-related thrombocytopenia remains a serious clinical issue, especially in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The ribonucleoside and DNA-demethylating agent azacytidine (AZA), has proven useful for the treatment of patients with MDS and AML not eligible for stem cell transplantation. While low-dose AZA therapy induces clinical remissions in up to 50% of treated patients, it comes at the cost of aggravating pre-existing thrombocytopenia which is observed in a subset of patients; this can lead to increased bleeding and bleeding-associated mortality, and importantly, often requires dose modifications and delays of therapy. Thus, identification of strategies alleviating ineffective megakaryopoiesis will likely lead to increased therapeutic efficacy for patients with MDS/AML. Eltrombopag (EP), a second-generation small molecule thrombopoietin receptor (TPO-R) agonist was effective in raising platelet counts in patients with MDS as a single agent, as well as in combination with certain standard of care therapies. However, it failed to stimulate platelet production during the first four cycles of AZA treatment as uncovered by a recent phase III placebo-controlled clinical study (SUPPORT; NCT02158936). The goals of this study were to identify the cellular and molecular underpinnings of AZA-associated inhibition of megakaryopoiesis and to assess the ineffectiveness of EP in mitigating AZA treatment-associated thrombocytopenia. Our results demonstrate that at a clinically-equivalent and non-cytotoxic dose, AZA rapidly induces transient activation of interferon type I (IFN-I) signaling in various hematopoietic cell types, including stem and lineage-committed progenitor cells (HSPCs). We detected IFNα and IFNβ production and release using ELISA and intracellular flow cytometry on primary total mononuclear cell- and purified CD34-positive HSPC populations derived from cord blood, bone marrow from healthy volunteers or patients with MDS/AML. AZA-mediated activation of Type I IFNs in healthy control- and MDS/AML cells was preceded by an accumulation of double-stranded RNA (dsRNA) species and decreased total RNA cytosine methylation measured by immunocytochemistry and intracellular FACS analysis; this suggested that AZA triggered the accumulation of immunogenic RNA species which elicit an IFN-I response. In support, we found Toll like receptor 3 (TLR3) activation and phosphorylation of STAT1 in CD34+ HPSC, along with premature activation of Suppressor of Cytokine Signaling 1 (SOCS1), a well-known JAK/STAT-dependent signaling attenuator. This rapid AZA-induced viral mimicry response led to abrogation of thrombopoietin (TPO) or EP-stimulated TPO-R signaling and inhibition of ex vivo megakaryocyte progenitor proliferation quantified by colony formation in semi-solid medium. Importantly, inhibition of IFN-I signal activation using the JAK3 inhibitor decernotinib, the IFNα/β-blocking peptide, B18R, or RNA interference-mediated knock-down of SOCS1 counteracted the inhibitory effects of AZA on TPO-R stimulation and restored megakaryopoiesis. Given these observations, we pre-clinically tested a revised treatment protocol, in which primary cells were first exposed to AZA for four days followed by TPO-R stimulation using TPO or EP. This new treatment strategy alleviated AZA's inhibitory effects at the molecular and cellular levels, demonstrating that upon resolution of the AZA-mediated vial mimicry response, EP and TPO can effectively stimulate TPO-R signaling and megakaryopoiesis. Together, our data reveal a mechanistic basis of AZA-mediated inhibition of megakaryopoiesis in patients with MDS/AML. Additionally, we show that EP cannot overcome the megakaryopoiesis-inhibitory effects of acute IFN-I signaling activation upon AZA exposure. Findings of our study are consistent with and provide a molecular explanation for the observations made in the context of the SUPPORT study. In the future, it will be critical to better understand and potentially counteract the megakaryopoiesis-inhibitory effects by IFN-I pathway activation upon AZA therapy in patients with MDS/AML. Disclosures Okoye-Okafor: Novartis Pharmaceuticals: Research Funding. Pallaud:Novartis Pharmaceuticals: Employment. Marques Ramos:Novartis Pharmaceuticals: Employment. Verma:Janssen: Research Funding; BMS: Research Funding; Celgene: Honoraria; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding. Will:Novartis Pharmaceuticals: Research Funding.

Knock-in of murine Calr del52 induces essential thrombocythemia with slow-rising dominance in mice and reveals key role of Calr exon 9 in cardiac development.

Frameshifting mutations (-1/+2) of the calreticulin (CALR) gene are responsible for the development of essential thrombocythemia (ET) and primary myelofibrosis (PMF). The mutant CALR proteins activate the thrombopoietin receptor (TpoR) inducing cytokine-independent megakaryocyte progenitor proliferation. Here, we generated via CRISPR/Cas9 technology two knock-in mouse models that are heterozygous for a type-I murine Calr mutation. These mice exhibit an ET phenotype with elevated circulating platelets compared with wild-type controls, consistent with our previous results showing that murine CALR mutants activate TpoR. We also show that the mutant CALR proteins can be detected in plasma. The phenotype of Calr del52 is transplantable, and the Calr mutated hematopoietic cells have a slow-rising advantage over wild-type hematopoiesis. Importantly, a homozygous state of a type-1 Calr mutation is lethal at a late embryonic development stage, showing narrowed ventricular myocardium walls, similar to the murine Calr knockout phenotype, pointing to the C terminus of CALR as crucial for heart development.

The Ex-Vivo Expansion of Megakaryocytic Progenitors from Hematopoietic Stem Cells for Thrombocytopenia in NOD/SCID Mice

Objective: Thrombocytopenia is a common clinical problem in patients with cancer or bone marrow transplantation. Currently it is mainly managed by platelet transfusion. Repeated platelet transfusions are associated with the risks of transfusion reactions/alloimmunisation and may lead to platelet refractoriness. Infusion of ex vivo expanded megakaryocytic (MK) progenitor cells is other strategy for the treatment of thrombocytopenia. This study aimed to establish efficient conditions for the expansion of the MK progenitors from enriched CD34(+) cells of umbilical cord blood.Methods: This study investigated the effect of flt-3 ligang (FL), stem cell factor (SCF) and platelet-derived growth factor (PDGF) in combination with other megakaryocyte-promoting cytokines such as thrombopoietin (TPO) on the differentiation and proliferation of megakaryocytic progenitors. As an early acting growth factor, FL may promote the ex vivo expansion of hematopoietic stem and progenitor cells. We compared the effects of FL and SCF in combination with other megakaryocyte-promoting cytokines in megakaryocytic progenitors.Results: In liquid cultures of enriched CD34+ cells from human umbilical cord blood for 14 days, FL plus TPO, interleukin-3 (IL-3), and IL-6 promoted the expansion of nucleated cells, CD34+ cells, CD34+ CD38- cells, and megakaryocyte colony-forming units (CFU-MK) by 300 +/- 115-, 23.8 +/- 11.3-, 33.9 +/- 28.6-, and 584 +/- 220-fold, respectively. Replacing FL with SCF significantly decreased the yield of all cell types. While one human acute lymphoblastic leukemia sample expressed high levels of flt-3 receptor, the four megakaryocytic cell lines (Meg-01, CHRF-288-11, M-07e, and Dami) did not show any positive expression. Our data suggest that the effect of FL in augmenting the expansion of MK progenitors might be due to the early action of FL at the pluripotent stem cell stage. Our results also demonstrated that TPO alone produced a high proportion of CD61(+)CD41(+) cells but a low total cell count and high cell death, resulting in an inferior expansion. The addition of in IL-1 beta, FL and to a lesser extent IL-3 improved the expansion outcome. The treatment groups with three to five cytokines produced efficient expansions of CFU-MK up to 400-fold with the highest yield observed in the presence of TPO, IL-1 beta, IL-3, IL-6 and FL. CD34(+) cells were expanded by five to 22-fold. PDGF improved the expansion of all cell types with CD61(+)CD41(+) cells, CFU-MK and CD34(+) cells increased by 101%, 134% and 70%, respectively. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in NOD/SCID mice.Conclusions: This study showed that the present cytokine combination and expansion conditions provide an effective and potentially useful system for the clinical expansion of cord blood for bone marrow transplantation (BMT). PDGF might be a suitable growth factor to improve the ex vivo expansion of MK progenitors for clinical applications. DisclosuresYang:National Natural Science Foundation of China: Other: National Natural Science Foundation of China(81270580).

Regulation of Hematopoiesis by Cholesterol

Leukocytosis is a risk factor for athero-thrombotic disease in humans, and develops in animal models of atherosclerosis in response to feeding high-fat, high-cholesterol diets. The ATP binding cassette transporters ABCA1 and ABCG1 promote cholesterol efflux to apoA-1 and high density lipoprotein (HDL), respectively and are targets of liver X receptor (LXR) transcription factors. Mice lacking ABCA1/G1 develop a dramatic myeloproliferative phenotype with monocytosis and neutrophilia, associated with expansion and proliferation of hematopoietic stem and myeloid progenitor populations (HSPCs). The transporters are highly expressed in HSPCs where they act to control proliferative responses to growth factors (IL-3, GM-CSF) by regulating plasma membrane lipid rafts and cell surface expression of the common β subunit of the IL-3/GM-CSF receptor. ABCG4 is closely related to ABCG1 but is expressed primarily in the megakaryocyte progenitor (MkP) population of the bone marrow. ABCG4-deficient mice have MkP proliferation and expansion, thrombocytosis, increased platelet/leukocyte aggregates and accelerated atherosclerosis. ABCG4 promotes cholesterol efflux onto HDL, and thereby reduces the cell surface expression of the thrombopoietin (TPO) receptor. This appears to involve membrane cholesterol enrichment and interruption of a negative feedback loop involving the TPO receptor and mediated by Lyn Kinase and c-CBL which mediate ubiquitination and internalization/degradation of the receptor. Overall results suggest that ATP binding cassette transporters promote cholesterol efflux, decrease membrane lipid raft formation and enhance the feedback downregulation of growth factor receptors in response to growth factor binding, with anti-proliferative responses that may be beneficial in atherosclerosis and myeloproliferative neoplasms. DisclosuresNo relevant conflicts of interest to declare.

Deficiency of ATP binding cassette transporter b6 in megakaryocyte progenitors accelerates atherosclerosis in mice

Objective— The ATP-binding cassette (ABC) transporter B6 (ABCB6) is highly expressed in megakaryocyte progenitors, but its role in platelet production and disease has not been elucidated. Approach and Results— Among various ABC transporters, ABCB6 was highly expressed in megakaryocyte progenitors, exhibiting the same pattern of expression of genes involved in heme synthesis pathway. Transplantation of Abcb6 −/− bone marrow into Ldlr −/− recipient mice resulted in expansion and proliferation of megakaryocyte progenitors, attributable to increased reactive oxygen species production in response to porphyrin loading. The enhanced megakaryopoiesis in Abcb6 −/− bone marrow–transplanted mice was further illustrated by increased platelet counts, mean platelet volume, and platelet activity. Platelets from Abcb6 −/− bone marrow–transplanted mice had higher levels of chemokine (C-C motif) ligand 5, which was associated with increased plasma chemokine (C-C motif) ligand 5 levels. There were also increased platelet–leukocyte aggregates, which resulted in leukocyte activation. Abcb6 −/− bone marrow–transplanted mice had accelerated atherosclerosis which was associated with deposition of the chemotactic agent, chemokine (C-C motif) ligand 5 in atherosclerotic plaques, resulting in increased macrophage accumulation. Conclusions— Our findings identify a new role of ABCB6 in preventing atherosclerosis development by dampening platelet production, reactivity, and chemokine (C-C motif) ligand 5 deposition in atherosclerotic lesions.

Deficiency of ATP-Binding Cassette Transporter B6 in Megakaryocyte Progenitors Accelerates Atherosclerosis in Mice

The ATP-binding cassette (ABC) transporter B6 (ABCB6) is highly expressed in megakaryocyte progenitors, but its role in platelet production and disease has not been elucidated. Among various ABC transporters, ABCB6 was highly expressed in megakaryocyte progenitors, exhibiting the same pattern of expression of genes involved in heme synthesis pathway. Transplantation of Abcb6 deficient (Abcb6(-/-)) bone marrow into low density lipoprotein receptor deficient recipient mice resulted in expansion and proliferation of megakaryocyte progenitors, attributable to increased reactive oxygen species production in response to porphyrin loading. The enhanced megakaryopoiesis in Abcb6(-/-) bone marrow-transplanted mice was further illustrated by increased platelet counts, mean platelet volume, and platelet activity. Platelets from Abcb6(-/-) bone marrow-transplanted mice had higher levels of chemokine (C-C motif) ligand 5, which was associated with increased plasma chemokine (C-C motif) ligand 5 levels. There were also increased platelet-leukocyte aggregates, which resulted in leukocyte activation. Abcb6(-/-) bone marrow-transplanted mice had accelerated atherosclerosis which was associated with deposition of the chemotactic agent, chemokine (C-C motif) ligand 5 in atherosclerotic plaques, resulting in increased macrophage accumulation. Our findings identify a new role of ABCB6 in preventing atherosclerosis development by dampening platelet production, reactivity, and chemokine (C-C motif) ligand 5 deposition in atherosclerotic lesions.

C‐Myb and GATA‐1 alternate dominant roles during megakaryocyte differentiation

Transcription factors are essential for blood cell formation. Mice expressing low levels of c-Myb (c-Myb(low)) have an increased number of bone marrow megakaryocytes (MKs) and corresponding thrombocytosis. In contrast, mice engineered to express low levels of GATA-1 (GATA-1(low)) in the megakaryocytic lineage exhibit aberrant megakaryocytopoiesis with hyperproliferation of progenitors and defective terminal differentiation leading to thrombocytopenia. These seemingly opposite roles may affect platelet turnover and thus be of clinical relevance. To determine how these two transcription factors act together to control megakaryocytopoiesis and platelet formation. We used a combination of cellular and molecular in vitro assays to examine the ability of bone marrow cells from mice expressing low levels of both c-Myb and GATA-1 (referred to as double(low)) to produce MKs and platelets. Double(low) cells, or those with low GATA-1 levels in which c-Myb is conditionally deleted, lack the hyperproliferative capacity of GATA-1(low) cells, allowing the cells to proceed towards more committed MKs that are, however, impaired in their capacity to produce fully differentiated cells, as confirmed by the abundance of morphologically aberrant cells that lack the ability to form proplatelets. c-Myb and GATA-1 act in concert to achieve correct megakaryocytic differentiation. GATA-1 regulates both the proliferation of megakaryocytic progenitors and their terminal maturation. c-Myb also acts at the level of the progenitor by influencing its commitment to differentiation, but in contrast to GATA-1 it does not have any effect on the process of terminal differentiation.

Lentiviral Short Hairpin RNA Silencing of DNM3Decreases Megakaryoycte Progenitor Proliferation and Inhibits the Formation of the Demarcation Membrane System.

Abstract 4594Dynamin 3 (DNM3) is a mechanochemical enzyme, which participates in membrane dynamics by hydrolyzing nucleotides to link cell membranes to the actin cytoskeleton. Recently, we identified DNM3 to be differentially up-regulated during megakaryocyte (MK) development and reported that DNM3 participates in the proliferation of MK progenitors. We report here that dynamin 3 not only participates in MK progenitor amplification, but is also involved in the formation of the demarcation membrane system (DMS). To explore the role of dynamin 3 during MK development, we used three different lentiviral DNM3 shRNA constructs to infect human umbilical cord blood (UCB) CD34+ cells that were then cultured to differentiate along the MK lineage. Relative to controls, CD34+ cells transfected with DNM3 shRNAs significantly decreased the total number of cells produced in culture as well as the number of colony forming unit-megakaryocytes (CFU-MKs). As a result, the total number of CD41+ and CD61+ progeny cells was also reduced. To investigate the role dynamin 3 plays during the maturation of MKs, a Src family kinase inhibitor, SU6656, was used to induce high polyploidization and the development of a complex DMS in the UT-7/mpl cell line. The styryl membrane dye di-8-ANEPPS was used to monitor the formation of the DMS. In the absence of SU6656 treatment, UT-7/mpl cells showed a peripheral staining pattern that was indicative of cells that did not have extensive plasma membrane invaginations. In contrast, the staining pattern of UT-7/mpl cells treated with SU6656 clearly showed a well-developed plasma membrane system with invaginations. When DNM3 was silenced in SU6656 treated UT-7/mpl cells the results showed a significant reduction in di-8-ANEPPS staining relative to empty vector and non-specific negative controls. These findings are consistent with the conclusion that dynamin 3 participates in the morphogenesis of the DMS. Disclosures:No relevant conflicts of interest to declare.

A Novel in Vitro Model of Focal Fibrosis in Bone Marrow Stromal Co-Culture of CD34+ Cells From Patients with Idiopathic Myelofibrosis.

Abstract 2896Poster Board II-872Chronic idiopathic myelofibrosis (IMF), essential thrombocythemia, polycythemia vera (PV) and chronic myelogenous leukemia (CML) are chronic myeloproliferative disorders (MPDs) characterized by the clonal proliferation of one or more hematopoietic lineage. In this study we investigated the interaction of MPDs stem/progenitor cells with bone marrow stroma. We established serum-free co-cultures of purified CD34+ cells from 59 peripheral blood (PB) samples from 30 MPD patients using the murine bone marrow stromal cell line OP9 transduced with an adenoviral vector expressing the human thrombopoietin gene (Adeno-Tpo). We observed that patients with IMF invariable had elevated numbers of CD34+ cells in PB, comparable to numbers reported in G-CSF mobilized PB (MPB). In co-cultures of CD34+cells from IMF PB or normal MPB there was an increase in total cells (IMF, 639±99 folds, N=31; MPB, 180±30, N=3) by 13-18 days with a high percentage expressing the megakaryocyte-platelet lineage marker CD41a (IMF 24-63%, MPB 18-35%) and <5% expressing CD14 (a monocytic marker). In co-culture of CD34+ cells from PV specimens, there was cellular expansion of 45±30 folds (N=3) but with <5% CD41a+ and >24 % CD14+ cells. CML PB cellular expansion was 147±45 folds (N=4) with 6-32 % CD41a+, 5-22% CD14+ and >60% CD15+ cells. In IMF stromal co-cultures, we observed the development of focal areas of OP9 stromal cell fibrosis (FF) at 12-14 days, associated with clonal proliferation of megakaryocyte progenitors and their differentiation to megakaryocytes and pro-platelets. This was associated with suppression of the spontaneous differentiation of OP9 cells to adipocytes. We obtained similar results in serum-free co-culture using the murine bone marrow stromal line MS-5 transduced with adeno-Tpo. This focal fibrosis phenomenon was not uniquely associated with IMF since we observed FF in normal MBP CD34+ co-culture in association with foci of magakaryocyte/platelet production, however the number of areas of FF was significantly higher when IMF CD34+ were compared to CD34+ cells from normal MPB or other types of MPDs. In IMF there were 84±17 FF/1,000 CD34+ (n=10), in PV <3 FF/1,000 (n=3) and in CML <9 FF/1,000 (n=3). Interferon α-2b is known to inhibit thrombopoietin-induced megakaryocytopoiesis and in clinical trials we have observed that IFNα-2b may retard progression of early IMF (Silver RT, Vandris K Leukemia 2009;23:1366). Addition of IFNα-2b to IMF CD34+ co-cultures dramatically reduced (>70%) total nucleated cells, CD41a+ cells, and FF-forming progenitor cells compared to control. This serum-free bone marrow stromal co-culture system distinguish different types of MPDs and may be useful in monitoring disease progression and patient response to therapy in IMF. The in vitro system appears to recapitulate the process of marrow fibrosis in the patient and can thus provide a sensitive bioassay to evaluate potential drugs for IMF therapy. Disclosures:Moore:Amgen: Patents & Royalties.

Role of mTOR in Hematopoiesis and Hematopoietic Stem Cell Regulation.

Abstract 1490Poster Board I-513The mammalian target of rapamycin (mTOR) integrates nutrients, growth factors, and cellular energy status to control protein synthesis that determines cell growth and metabolism. It is also known that mTOR plays an essential role in cell survival by regulating Akt/PKB signaling. By using the inhibitor rapamycin, mTOR has previously been suggested to regulate proliferation of megakaryocyte progenitors and late stage of megakaryocyte differentiation without a general impact on normal hematopoiesis or hematopoietic stem cell (HSC) function. Due to limitations of rapamycin and the early lethality of conventional mTOR gene targeted mice, the physiological role of mTOR in blood development remains undefined. In this study, we have utilized an inducible conditional mTOR knockout mouse model by crossbreeding mTORflox/flox mice with Mx-Cre mice that allow interferon-induced mTOR deletion in the bone marrow following a transplantation and polyI:C induction protocol, in an effort to determine the genetic role of mTOR in hematopoiesis. Depletion of mTOR drastically affected hematopoiesis in a blood cell autonomous manner in Mx-Cre;mTORflox/flox bone marrow transplant recipients: the mice showed marked reduction in BM cellularity and in the numbers of myeloid and lymphoid lineage cells, erythrocytes, and platelets in peripheral blood, bone marrow, and thymus, leading to bone marrow failure, blood cell exhaustion and lethality. In vitro colony-forming activities by bone marrow or spleen progenitors were completely abolished in the absence of mTOR. Interestingly, the number and frequency of HSCs in bone marrow (Lin−Sca-1+c-Kit+) increased transiently while the number of early progenitors (CMP, GMP, MEP, CLP) detected by cell surface markers remained unchanged or only mildly affected in the mutant mice within 14 days after polyI:C treatment. Concomitantly, mTOR deletion led to a massive egress of HSCs from bone marrow to distal organs including spleen (∼60-fold increase). Transplantation of mTOR−/− bone marrow cells into NOD-SCID mice or competitive transplantation of mTOR−/− bone marrow cells into BoyJ mice further demonstrated that mTOR deficiency caused a complete failure in HSC engraftment and repopulation. Surprisingly, at the cellular level these phenotypes are associated with increased proliferation of HSCs in vivo and in vitro by 60% and 2.5-fold, respectively, as assessed by 5-bromodeoxyuridine incorporation assays whereas the cell survival index appears to be unaffected. Moreover, mTOR−/− HSCs and progenitor cells displayed impaired adhesion to fibronectin CH296 fragment (∼30% decrease) and migration toward SDF-1α gradients (∼30% decrease). At the molecular level, gene chip microarray analysis of mTOR−/− HSCs revealed that the cell cycle regulators myb, wee1, FANCD2, and FANCE were significantly downregulated while Rb and E2F5 were upregulated, the survival/apoptosis regulators MCL1 and BCL2L1 were upregulated, and the actin cytoskeleton and cell extracellular matrix adhesion regulators Arp2/3 complex subunit 5, paxillin, laminin α5, integrin β3, and myosin light chain 6B were upregulated. Further, immunoblotting analysis of isolated Lin− cells showed that SCF-stimulated activation of translational regulators S6K and 4E-BP and survival regulator Akt were abolished upon mTOR deletion. Taken together, these data suggest that mTOR is a critical regulator of HSC quiescence, self-renewal, and engraftment through the regulation of cell cycle, survival and actin cytoskeleton signals, and is essential in multiple stages of hematopoiesis. DisclosuresCancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

Survivin Is Required for Proliferation, but Not Polyploidization of Megakaryocytes.

Survivin is a member of chromosome passenger complex, which plays an important role in chromosome alignment, separation and cytokinesis. We recently reported that survivin is necessary for the proliferation and survival of hematopoietic stem and progenitor cells. Furthermore, we previously showed that reduced levels of survivin expression facilitates megakaryocyte development, whereas elevated levels of survivin inhibit their maturation and polyploidization. However, the extent to which survivin is necessary for polyploidization and terminal differentiation of committed megakaryocytes remains unclear. To determine whether survivin is required for megakaryocyte and platelet biogenesis, we mated mice with floxed alleles of survivin (sur fl/fl) to mice that express Cre recombinase under the control of the PF4 promoter. Compound mutant animals appeared grossly normal and harbored normal platelet counts. Furthermore, survivin deleted and control littermates displayed similar expression of CD41 and CD42, as well as similar DNA content within the CD41+ population. The only significant difference detected was an increase in annexin V staining of CD41+ cells within the bone marrow of the mice with survivin deletion. Analysis of DNA extracted from these bone marrows showed no evidence of the survivin deletion, indicating that the surviving cells all escaped excision. These in vivo findings are consistent with a requirement for survivin in the survival or proliferation of megakaryocyte progenitors. Next, to induce megakaryocyte development ex vivo, we cultured bone marrow from surv fl/fl mice in vitro in the presence of TPO. Using this approach, we were able to induce survivin deletion in 75% of the cells as evidenced by PCR. Despite the deletion of survivin, polyploidization of the ex vivo generated megakaryocytes was unaffected. Finally, we induced deletion of survivin by retroviral infection of surv fl/fl progenitors with MSCV-Cre and found that megakaryocyte polyploidization was actually increased in the excised population. Taken together, our results suggest that survivin is not required for polyploidization, but is necessary for proliferation of megakaryocyte progenitors.

GATA-2 Controls Proliferation of Megakaryocyte Progenitors and Its Expression Contributes to Human DS-AMKL.

GATA2 is an essential transcription factor that regulates multiple aspects of hematopoiesis. Mutations in GATA2 are associated with blast crisis phase of chronic myelogenous leukemia (CML) while overexpression of GATA2 is a hallmark of Down syndrome acute megakaryoblastic leukemia (DS-AMKL), a malignancy that is defined by the combination of trisomic chromosome 21 and a GATA1 mutation. Here, we show thatGATA2 is essential for megakaryocyte development from both wild-type and GATA-1mutant hematopoietic progenitors. Furthermore, we reveal that GATA2 is indispensable for cell cycle progression and that its expression cooperates with activated JAK3 to promote TPO-independent expansion of megakaryocytes. Genome-wide microarray analysis revealed that GATA2 regulates a wide set of genes including cell cycle regulators and lineage-specific genes that are essential for terminal differentiation of megakaryocytes. Of note, a subset of genes within the GATA2 signature is contained within human DS-AMKL patient specimens. These observations implicate overexpression of GATA2 in the aberrant gene expression and proliferation of AMKL. Taken together, our data demonstrate thatGATA2 is a critical regulator of normal and malignant megakaryopoiesis.

SH2-Inositol Phosphatase 1 Negatively Influences Early Megakaryocyte Progenitors

BackgroundThe SH2-containing-5′inositol phosphatase-1 (SHIP) influences signals downstream of cytokine/chemokine receptors that play a role in megakaryocytopoiesis, including thrombopoietin, stromal-cell-derived-Factor-1/CXCL-12 and interleukin-3. We hypothesize that SHIP might control megakaryocytopoiesis through effects on proliferation of megakaryocyte progenitors (MKP) and megakaryocytes (MK).Methodology and Principal FindingsHerein, we report the megakaryocytic phenotype and MK functional assays of hematopoietic organs of two strains of SHIP deficient mice with deletion of the SHIP promoter/first exon or the inositol phosphatase domain. Both SHIP deficient strains exhibit a profound increase in MKP numbers in bone marrow (BM), spleen and blood as analyzed by flow cytometry (Lin−c-Kit+CD41+) and functional assays (CFU-MK). SHIP deficient MKP display increased phosphorylation of Signal Transducers and Activators of Transcription 3 (STAT-3), protein kinase B (PKB/AKT) and extracellular signal-regulated kinases (ERKs). Despite increased MKP content, total body number of mature MK (Lin−c-kit−CD41+) are not significantly changed as SHIP deficient BM contains reduced MK while spleen MK numbers are increased. Reduction of CXCR-4 expression in SHIP deficient MK may influence MK localization to the spleen instead of the BM. Endomitosis, process involved in MK maturation, was preserved in SHIP deficient MK. Circulating platelets and red blood cells are also reduced in SHIP deficient mice.Conclusions/SignificanceSHIP may play an important role in regulation of essential signaling pathways that control early megakaryocytopoiesis in vivo.

HoxA2 Regulates Proliferation of an Embryonic Megakaryocyte Progenitor, Which Can Effectively Produce Platelets In Vitro.

Megakaryocytes are rare cells in the bone marrow, and are solely responsible for thrombopoiesis throughout the life of the organism. Various transcription factors regulating megakaryopoiesis have been identified, including GATA1, Fli1 and NF-E2, which are important for the maturation and differentiation of megakaryocytes and SCL and FOG1, which play important roles in the specification and proliferation of the early megakaryocyte progenitor but no single factor capable of sustaining long-term proliferation of the megakaryocyte progenitor has been identified. The rarity of this progenitor, and the difficulty of proliferating megakaryocytes in vitro have seriously hampered the effort to produce large quantities platelets in vitro, and to date the only source of platelets for transplantation remains human donors. In this study, through a gain of function screen of Hox genes in which each paralog group was tested, we identified HoxA2 as an important transcription factor for the proliferation of early embryonic megakaryocytic progenitors. HoxA2 promoted the outgrowth of undifferentiated megakaryocyte progenitors when transduced into precirculation yolk sac stem and progenitor cells, however it had no effect on megakaryocytic progenitors derived from definitive HSCs of adult bone marrow. To study this unique embryonic regulatory circuit further, we generated an inducible mouse embryonic stem cell line, in which the expression of murine HoxA2 is regulated by doxycycline. Embryoid body differentiation of these cells, followed by sorting and reculture of the Kit+/CD41+ hematopoietic progenitor fraction at day 6 in the presence of continual HoxA2 expression allowed undifferentiated c-Kit+/CD41+/CD45+ megakaryocyte progenitors to proliferate for upwards of 2 months. When doxycycline is removed, numerous gene expression changes take plase, and these progenitors differentiate into mature c-Kitneg/CD41high/CD45neg megakaryocytes, which undergo proplatelet formation, releasing large quantities of platelets (∼107/ml) into the medium. As this rate of production can be sustained for weeks, a highly efficient platelet bioreactor is enabled. The platelets express the integrin αIIb-β3 (GPIIb-IIIa complex), and upon stimulation they bind fibrinogen, demonstrating functionality. We are currently investigating the transplantation potential of these cells.Our data suggest that adult and embryonic megakaryopoiesis have distinct regulatory circuits, and demonstrate a novel and uniquely embryonic role for HoxA2, which opens up the possibility of producing industrial quantities of platelets in vitro for therapeutic purposes.

Cross-Talk between c-Myb and GATA-1 Regulates the Entry into Megakaryocyte Differentiation.

Megakaryocytic differentiation and platelet formation are regulated by the combined function of multiple transcription factors. Previously, we have shown that knockdown (KD) mice expressing low levels of c-Myb have an increased number of bone marrow megakaryocytes and a corresponding thrombocytosis. In contrast, mice engineered to express low levels of GATA-1 in the megakaryocytic lineage exhibit aberrant megakaryocytopoiesis with hyperproliferation of progenitors and defective terminal differentiation leading to thrombocytopenia. Here, making use of mice expressing low levels of both c-Myb and GATA-1 (referred to as “double-KD”) and comparison with the single-KD counterparts and wild type animals, we describe how c-Myb and GATA-1 act together to control megakaryocytopoiesis. Few double-KD mice are born live, most likely as a result of hypocellularity in the hematopoietic organs, and platelet numbers are low, indicating that they are not capable of overcoming the thrombocytopenia characteristic of the GATA-1 KD mice. Proliferation assays performed using E14 fetal liver cells, growing in liquid cultures containing thrombopoietin to induce megakaryocytic differentiation, revealed that double-KD cells lacked the hyperproliferative capacity of GATA-1 KD cells. A similar conclusion was reached using colony assays in semi-solid media capable of supporting multilineage differentiation. The colony assays also revealed that double-KD hematopoietic progenitors could only give rise to megakaryocyte and erythroid lineage cells, unlike the single-KD fetal liver cells, which additionally yielded both macrophage and neutrophil-containing colonies. The reduced hyperproliferative capacity of megakaryocytic progenitors in the double-KD compared to the GATA-1 KD suggests that c-Myb and GATA-1 have opposing effects on megakaryocytic progenitors, the reduced level of c-Myb overcoming the block to differentiation imposed by low expression of GATA-1. However, the persistent thrombocytopenia observed in double-KD mice suggests that the low level of GATA-1 prevents terminal differentiation even when commitment has been enhanced by the reduction in c-Myb. To better define the consequences of this functional interaction between c-Myb and GATA-1, we compared the phenotype of the megakaryocytic cells deriving from single- and double-KD progenitors. We observed that the c-Kit+CD41+ megakaryocytic progenitor population present in GATA-1 KD fetal liver is replaced in the double-KD by a more differentiated c-Kit-CD41+ population, similar to that seen in wild type and c-Myb-KD fetal liver. However, the levels of CD41 on double-KD cells are low, suggesting that megakaryocytic terminal differentiation is affected. This was confirmed by staining for acetylcholinesterase, which revealed an abundance of morphologically aberrant megakaryocytes that are incapable of forming proplatelets, as has been described for the GATA-1 KD megakaryocytes. Based on these observations, we conclude that c-Myb and GATA-1 act in concert to achieve correct megakaryocytic differentiation. GATA-1 regulates both the proliferation of megakaryocytic progenitors and their terminal differentiation towards platelet-producing megakaryocytes. c-Myb also acts at the level of the progenitor by influencing its commitment to differentiation, but in contrast to GATA-1 it does not have any effect on the process of terminal differentiation.

Eltrombopag, a novel, oral platelet growth factor, increases platelet counts in thrombocytopenic patients and healthy subjects

8602 Background: Eltrombopag (SB-497115) is a novel, first in class, orally bioavailable, thrombopoietin receptor agonist that induces differentiation and proliferation of megakaryocyte progenitors and has been shown to increase platelet counts in preclinical and clinical studies. Methods: In two randomized, placebo-controlled trials, eltrombopag was administered as oral tablets, once daily for 10 days to 73 healthy male subjects at doses of 5–75mg in an ascending dose cohort study, and to 103 (64 female/39 male) adult chronic immune thrombocytopenic purpura (ITP) patients, with a platelet count of &lt;30×109/L, once daily for 6 weeks at doses of 30–75mg in a parallel dose cohort study. The primary efficacy endpoint in the Phase II ITP trial was the proportion of subjects with a platelet count &gt;50×109/L after 6 weeks of dosing. Results: In healthy subjects, eltrombopag induced a dose dependent increase in the platelet counts. Mean maximal platelet count increases were 24.1 % at 30mg, 42.9 % at 50mg, and 50.4 % at 75mg. In 95 eligible ITP patients, platelet counts increased from &lt;30 to &gt;50×109/L in 16% (4/25) of subjects on placebo, and in the eltrombopag groups in 28% (7/25, p=ns) on 30mg, 67% (16/24, p&lt;0.001) on 50mg and 86% (18/21, p&lt;0.001) on 75mg eltrombopag. The median platelet counts in each treatment arm after 6 weeks of dosing were 16×109/L on placebo, 29×109/L on 30mg, 132×109L on 50mg, and 202×109/L on 75mg. The dose dependent effect was not significantly affected by the splenectomy status, background immunosuppressant use, or baseline platelet count (greater than or less than 15×109/L). Conclusions: The platelet count data from these clinical studies suggests that eltrombopag could be an effective therapy for the treatment of thrombocytopenia. Eltrombopag is being tested in further studies involving patients with ITP and chronic liver disease, and cancer patients receiving thrombocytopenic chemotherapy. [Table: see text]