Infantile (fetal and neonatal) megakaryocytes (Mks) have a distinct phenotype consisting of hyperproliferation, limited morphogenesis, and low platelet production capacity. These properties ...contribute to clinical problems that include thrombocytopenia in neonates, delayed platelet engraftment in recipients of cord blood stem cell transplants, and inefficient ex vivo platelet production from pluripotent stem cell-derived Mks. The infantile phenotype results from deficiency of the actin-regulated coactivator, MKL1, which programs cytoskeletal changes driving morphogenesis. As a strategy to complement this molecular defect, we screened pathways with the potential to affect MKL1 function and found that DYRK1A inhibition dramatically enhanced Mk morphogenesis in vitro and in vivo. Dyrk1 inhibitors rescued enlargement, polyploidization, and thrombopoiesis in human neonatal Mks. Mks derived from induced pluripotent stem cells responded in a similar manner. Progenitors undergoing Dyrk1 inhibition demonstrated filamentous actin assembly, MKL1 nuclear translocation, and modulation of MKL1 target genes. Loss-of-function studies confirmed MKL1 involvement in this morphogenetic pathway. Expression of Ablim2, a stabilizer of filamentous actin, increased with Dyrk1 inhibition, and Ablim2 knockdown abrogated the actin, MKL1, and morphogenetic responses to Dyrk1 inhibition. These results delineate a pharmacologically tractable morphogenetic pathway whose manipulation may alleviate clinical problems associated with the limited thrombopoietic capacity of infantile Mks.
Drug-induced thrombocytopenia often results from dysregulation of normal megakaryocytopoiesis. In this study, we investigated the mechanisms responsible for thrombocytopenia associated with the use ...of Panobinostat (LBH589), a histone deacetylase inhibitor with promising anti-cancer activities. The effects of LBH589 were tested on the cellular and molecular aspects of megakaryocytopoiesis by utilizing an ex vivo system in which mature megakaryocytes (MK) and platelets were generated from human primary CD34+ cells. We demonstrated that LBH589 did not affect MK proliferation or lineage commitment but inhibited MK maturation and platelet formation. Although LBH589 treatment of primary MK resulted in hyperacetylation of histones, it did not interfere with the expression of genes that play important roles during megakaryocytopoiesis. Instead, we found that LBH589 induced post-translational modifications of tubulin, a nonhistone protein that is the major component of the microtubule cytoskeleton. We then demonstrated that LBH589 treatment induced hyperacetylation of tubulin and alteration of microtubule dynamics and organization required for proper MK maturation and platelet formation. This study provides new insights into the mechanisms underlying LBH589-induced thrombocytopenia and provides a rationale for using tubulin as a target for selective histone deacetylase inhibitor therapies to treat thrombocytosis in patients with myeloproliferative neoplasms.
Recently, interactions between thrombopoietin (TPO) and its receptor, the myeloproliferative leukemia (MPL) virus oncogene, have been shown to play a role in the development and progression of ...myeloproliferative neoplasms including myelofibrosis (MF). These observations have led to the development of strategies to disrupt the association of TPO with its receptor as a means of targeting MF hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). In this report, we show that although both splenic and peripheral blood MF CD34+ cells expressed lower levels of MPL than normal CD34+ cells, TPO promoted the proliferation of MF CD34+ cells and HPCs in a dose-dependent fashion. Furthermore, the treatment of MF but not normal CD34+ cells with a synthesized MPL antagonist, LCP4, decreased the number of CD34+Lin− cells and all classes of assayable HPCs (colony-forming unit–megakaryocyte CFU-MK, CFU-granulocyte/macrophage, burst-forming unit–erythroid/CFU-erythroid, and CFU-granulocyte/erythroid/macrophage/MK) irrespective of their mutational status. In addition, LCP4 treatment resulted in the depletion of the number of MF HPCs that were JAK2V617F+. Moreover, the degree of human cell chimerism and the proportion of malignant donor cells were significantly reduced in immunodeficient mice transplanted with MF CD34+ cell grafts treated with LCP4. These effects of LCP4 on MF HSCs/HPCs were associated with inhibition of JAK-STAT activity, leading to the induction of apoptosis. These findings demonstrate that such specific anti-cytokine receptor antagonists represent a new class of drugs that are capable of targeting MF HSCs.
•Treatment of MF CD34+ cells with a TPO receptor antagonist selectively depletes MF HSCs and HPCs.•Agents that target the TPO receptor represent potentially new approaches for the treatment of MF patients.
Breast cancer cell lines are widely used tools to investigate breast cancer biology and to develop new therapies. Breast cancer tissue contains molecularly heterogeneous cell populations. Thus, it is ...important to understand which cell lines best represent the primary tumor and have similarly diverse phenotype. Here, we describe the development of five breast cancer cell lines from a single patient's breast cancer tissue. We characterize the molecular profiles, tumorigenicity and metastatic ability in vivo of all five cell lines and compare their responsiveness to 4-hydroxytamoxifen (4-OHT) treatment.
Five breast cancer cell lines were derived from a single patient's primary breast cancer tissue. Expression of different antigens including HER2, estrogen receptor (ER), CK8/18, CD44 and CD24 was determined by flow cytometry, western blotting and immunohistochemistry (IHC). In addition, a Fluorescent In Situ Hybridization (FISH) assay for HER2 gene amplification and p53 genotyping was performed on all cell lines. A xenograft model in nude mice was utilized to assess the tumorigenic and metastatic abilities of the breast cancer cells.
We have isolated, cloned and established five new breast cancer cell lines with different tumorigenicity and metastatic abilities from a single primary breast cancer. Although all the cell lines expressed low levels of ER, their growth was estrogen-independent and all had high-levels of expression of mutated non-functional p53. The HER2 gene was rearranged in all cell lines. Low doses of 4-OHT induced proliferation of these breast cancer cell lines.
All five breast cancer cell lines have different antigenic expression profiles, tumorigenicity and organ specific metastatic abilities although they derive from a single tumor. None of the studied markers correlated with tumorigenic potential. These new cell lines could serve as a model for detailed genomic and proteomic analyses to identify mechanisms of organ-specific metastasis of breast cancer.
Red cell production is primarily determined by the action of erythropoietin. Additional erythropoiesis-regulatory factors include molecules and cellular interactions occurring within the bone marrow ...(BM) microenvironment. Sotatercept (ACE-011) is an activin receptor ligand trap that binds several members of the TGF-β superfamily. Treatment with ACE-011 reverses bone loss and reduces the degree of osteoporosis, but it is accompanied by elevated hemoglobin and hematocrit levels. The mechanisms underlying the beneficial effects of ACE-011 on red cell production remain unknown. This study explores the means by which ACE-011 promotes erythropoiesis. We showed that ACE-011 does not directly affect erythroid differentiation of human CD34+ cells in vitro. We next tested whether ACE-011 acts indirectly by affecting BM accessory cells. Conditioned media produced by BM stromal cells (SCs) inhibited erythroid differentiation of CD34+ cells while maintained their ability to proliferate. However, conditioned media from SCs treated with ACE-011 partially restored erythropoiesis, coinciding with changes in the molecular and secretory profile of SCs, including the expression and secretion of erythropoiesis-modulatory factors. We conclude that inhibitory factors produced by BM SCs in vitro might control erythropoiesis in vivo and that agents that reverse these microenvironmental signals could provide an approach to attenuate anemia in clinical conditions.
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.
Infantile (fetal and neonatal) megakaryocytes (Mks) have a distinct phenotype consisting of hyperproliferation, limited morphogenesis, and low platelet production capacity. These properties ...contribute to clinical problems that include thrombocytopenia in neonates, delayed platelet engraftment in recipients of cord blood stem cell transplants, and inefficient ex vivo platelet production from pluripotent stem cell-derived Mks. The infantile phenotype results from deficiency of the actin-regulated coactivator, MKL1, which programs cytoskeletal changes driving morphogenesis. As a strategy to complement this molecular defect, we screened pathways with the potential to affect MKL1 function and found that DYRK1A inhibition dramatically enhanced Mk morphogenesis in vitro and in vivo. Dyrk1 inhibitors rescued enlargement, polyploidization, and thrombopoiesis in human neonatal Mks. Mks derived from induced pluripotent stem cells responded in a similar manner. Progenitors undergoing Dyrk1 inhibition demonstrated filamentous actin assembly, MKL1 nuclear translocation, and modulation of MKL1 target genes. Loss-of-function studies confirmed MKL1 involvement in this morphogenetic pathway. Expression of Ablim2, a stabilizer of filamentous actin, increased with Dyrk1 inhibition, and Ablim2 knockdown abrogated the actin, MKL1, and morphogenetic responses to Dyrk1 inhibition. These results delineate a pharmacologically tractable morphogenetic pathway whose manipulation may alleviate clinical problems associated with the limited thrombopoietic capacity of infantile Mks.
Interferon (IFN-α) is effective therapy for polycythemia vera (PV) patients, but it is frequently interrupted because of adverse events. To permit the long-term use of IFN, we propose combining low ...doses of IFN with Nutlin-3, an antagonist of MDM2, which is also capable of promoting PV CD34+ cell apoptosis. Combination treatment with subtherapeutic doses of Peg IFN-α 2a and Nutlin-3 inhibited PV CD34+ cell proliferation by 50% while inhibiting normal CD34+ cells by 30%. Combination treatment with Nutlin-3 and Peg IFN-α 2a inhibited PV colony formation by 55%-90% while inhibiting normal colony formation by 22%-30%. The combination of these agents also decreased the proportion of JAK2V617F-positive hematopoietic progenitor cells in 6 PV patients studied. Treatment with low doses of Peg IFN-α 2a combined with Nutlin-3 increased phospho-p53 and p21 protein levels in PV CD34+ cells and increased the degree of apoptosis. These 2 reagents affect the tumor suppressor p53 through different pathways with Peg IFN-α 2a activating p38 MAP kinase and STAT1, leading to increased p53 transcription, whereas Nutlin-3 prevents the degradation of p53. These data suggest that treatment with low doses of both Nutlin-3 combined with Peg IFN-α 2a can target PV hematopoietic progenitor cells, eliminating the numbers of malignant hematopoietic progenitor cells.
Platelet (PTL) transfusions are currently the most effective treatment for patients with thrombocytopenia. Demand for PTL transfusions has steadily increased in recent years, straining a PTL supply ...that is already limited due to dependency on volunteer donors, short shelf life, risk of infections, and alloimmunization. This dilemma has stimulated the search for alternative approaches for generating PTLs ex vivo from different sources of hematopoietic stem cells (HSCs). Although PTLs have been successfully generated in cultures initiated with primary human CD34+ cells and pluripotent stem cells, the generation of a clinically relevant PTL product ex vivo faces significant obstacles due to scalability, reproducibility and shelf life. We propose an alternative approach to overcome such obstacles by developing a cryopreservable cell product consisting of megakaryocytes (MK) that can produce PTL in vivo after transfusion into patients.
Umbilical cord blood units (CBU) are FDA-approved, readily available sources for allogeneic HSC for transplantation in patients with various blood disorders. Our method utilizes a previously developed two-step culture system of megakaryopoiesis from CB CD34+ cells to generate an MK culture composed of defined MK populations: CD34+/CD41+/CD42b- MK precursors (MKP), immature CD34-/CD41+/CD42b- MK (iMK) and mature CD34-/CD41+/CD42b+ MK (mMK). While robust, the yield of MKs obtained in these cultures is restricted due to limited numbers of HSCs in CB. Our group has recently demonstrated that the numbers of CB CD34+ can be significantly expanded by epigenetic reprogramming following treatment with valproic acid (VPA). Here, we report the integration and optimization of HSC expansion with MK differentiation in order to generate a clinically relevant MK cell product. We tested 20 different culture conditions in which CD34+ cells were cultured for 5 to 8 days in the absence or presence of VPA in serum-free media with various cytokines to allow for HSC expansion. The resulting HSC pool is cultured for additional 4 to 7 days in MK differentiation/maturation media. The overall yield of CD41+ MKs obtained ranged from 8 to 33 MK per input CD34+ cell expanded in the presence of cytokines alone (n=10; mean 19.8 MK) and from 9 to 34 MK per input CD34+ cell expanded in the presence of cytokines plus VPA (n=10; mean 20.7 MK). Given that up to 2x106 CD34+ cells can be isolated from one CBU, it is anticipated that a culture yielding 28 or more MK per one CD34+ cell would generate over 56x106 MK or the equivalent of 7x105 CD41+ MK/kg/body weight for infusion into an 80 kg recipient. The culture conditions resulting in a yield of 28 or more MK per one CD34+ cell input are currently optimized to further maximize the fraction of MK generated which currently varies between 15-57% of culture. The predominant sub-population of MK resulted in these conditions consists of mMKs, regardless of VPA treatment. However, in the presence of VPA, the cultures contain a greater number of assayable CFU-MKs as compared to cytokines alone. Furthermore, preliminary studies suggest that transplantation of ex vivo generated MK leads to detectable human CD41+ cells into the BM and human PTL into the PB of NSG recipient mice. These results indicate that a MK cell product derived from CB HSCs expanded by VPA comprises not only mMK and iMK capable of immediate PTL release but also MKP and HPCs which are capable of sustained MK and PTL production. Another major advantage of a transfusion product composed of nucleated MKs is the possibility of storage by cryopreservation. Due to the fragility of mMK, we tested the cryopreservation of heterogeneous and purified MK cultures. Viability of cryopreserved MK cultures post-thaw was between 68.4-70% and no changes in the MK phenotype. Studies are ongoing to test the ex vivo and in vivo functionality of the cryopreserved MKs. In summary, starting with expanded CB HSC we created a cryopreservable cell product composed of different MK sub-populations within the MK hierarchy which is being developed into a clinically relevant therapy for treatment of thrombocytopenia.
No relevant conflicts of interest to declare.
Abstract 1082
Megakaryocyte (MK) development is characterized by polyploidization, cytoplasmic maturation and proplatelet formation, which culminates in the release of platelets into the circulation. ...The tumor suppressor p53 plays a critical role in the regulation of both cell cycle and apoptosis; its function is tightly controlled by the murine double minute (MDM2) protein which facilitates p53 degradation and inhibits p53 transcriptional activity. MK ploidy results from a disruption of normal cell cycle progression termed endomitosis while platelet release is believed to depend on apoptotic processes. The role of p53-MDM2 in MK in these two processes has not been clearly defined. A small molecule RG7112, which disrupts MDM2-p53 interaction, has shown promising anti-tumor effects in phase I clinical trials. This beneficial outcome has, however, been associated with the development of thrombocytopenia. We, therefore, used RG7112 as pharmacological probe to examine the effects of disruption of the MDM2-p53 regulatory loop on MK. We determined the effects of RG7112 on primary human MK by utilizing an in vitro system in which MK were generated from BM-derived CD34+ cells. We first demonstrated that both p53 and MDM2 transcripts are up-regulated as MK differentiation progresses. The ability of CD34+ cells to proliferate in the absence or presence of various concentrations of RG7112 was then evaluated both in liquid cultures and in CFU-MK colony assays. CD34+ cells exposed to 10 μM RG7112 for 7 days generated 70% fewer viable cells as compared to control cells exposed to the inactive form of the drug (p value = 0.0038). Furthermore, CD34+ cells treated with RG7112 formed up to 40% less CFU-MK colonies as compared to untreated cells. An assessment of apoptosis of MK precursors generated in the presence of RG7112 revealed that 69.5+2.1% were Annexin V positive as compared to 31.5+3.5% present in control cultures. These findings are consistent with the previously reported role of RG7112 in inducing p53 activation and apoptosis. Interestingly, phenotypical characterization of the viable cells generated under identical culture conditions, showed that RG7112 treatment did not interfere with the ability of CD34+ cells to acquire markers of MK differentiation during the first 7 days of culture since similar degrees of CD41 and CD42 expression were observed in the absence and in the presence of the drug. Likewise, exposure of MK precursors to the drug for 7 additional days (i.e. later stages of maturation) did not influence CD41 and CD42 expression. By contrast, cells differentiated in the presence of 5 μM RG7112 generated 50% fewer polyploid MK with greater than 4N DNA content as compared to those treated with the inactive form of the drug. Moreover, the negative effects on ploidy were associated with p53 activation, as assessed by the increased levels of p21 protein, a direct target of p53 which is known to limit polyploidization of primary MK. Finally, platelets generated in vitro were analyzed phenotypically and quantitated by dual labeling with anti-CD41 antibodies and thiazole orange (TO). The number of CD41+/TO+platelets derived from MK generated in the presence of RG7112 was reduced by 22% as compared to control. Based on these findings, we conclude that RG7112 impacts megakaryopoiesis by two potential mechanisms: 1) Impairing the ability of CD34+ cells to generate MK precursors due to increased apoptosis; 2) Limiting polyploidization during the late stages of development due to phamacological activation of p53. A combination of these two effects may provide an explanation for thrombocytopenia observed in patients receiving this drug and suggests that p53 plays an important role in normal human thrombocytopoiesis.
Iancu-Rubin:Roche: Research Funding. Hoffman:Roche: Research Funding.
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