Tuning MPL signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia progenitors.

Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable for both basic research and clinical utility. We developed a series of surrogate protein ligands for TPO-R, in the form of diabodies (DBs), that homodimerize TPO-R on the cell surface in geometries that are dictated by the DB receptor binding epitope, in effect "tuning" downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism, thus decoupling the dual functions of TPO/TPO-R. Using single-cell RNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in essential thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R.

Thrombopoietin contributes to the formation and the maintenance of hematopoietic progenitor-containing cell clusters in the aorta-gonad-mesonephros region.

In the midgestation mouse embryo, hematopoietic cell clusters containing hematopoietic stem/progenitor cells arise in the aorta-gonad-mesonephros (AGM) region. We have previously reported that forced expression of the Sox17 transcription factor in CD45lowc-Kithigh AGM cells, which are the hematopoietic cellular component of the cell clusters, and subsequent coculture with OP9 stromal cells in the presence of three cytokines, stem cell factor (SCF), interleukin-3 (IL-3), and thrombopoietin (TPO), led to the formation and the maintenance of cell clusters with cells at an undifferentiated state in vitro. In this study, we investigated the role of each cytokine in the formation of hematopoietic cell clusters. We cultured Sox17-transduced AGM cells with each of the 7 possible combinations of the three cytokines. The size and the number of Sox17-transduced cell clusters in the presence of TPO, either alone or in combination, were comparable to that observed with the complete set of the three cytokines. Expression of TPO receptor, c-Mpl was almost ubiquitously expressed and maintained in Sox17-transduced hematopoietic cell clusters. In addition, the expression level of c-Mpl was highest in the CD45lowc-Kithigh cells among the Sox17-transduced cell clusters. Moreover, c-Mpl protein was highly expressed in the intra-aortic hematopoietic cell clusters in comparison with endothelial cells of dorsal aorta. Finally, stimulation of the endothelial cells prepared from the AGM region by TPO induced the production of hematopoietic cells. These results suggest that TPO contributes to the formation and the maintenance of hematopoietic cell clusters in the AGM region.

Normal and pathological dynamics of platelets in humans.

We develop a mathematical model of platelet, megakaryocyte, and thrombopoietin dynamics in humans. We show that there is a single stationary solution that can undergo a Hopf bifurcation, and use this information to investigate both normal and pathological platelet production, specifically cyclic thrombocytopenia. Carefully estimating model parameters from laboratory and clinical data, we then argue that a subset of parameters are involved in the genesis of cyclic thrombocytopenia based on clinical information. We provide model fits to the existing data for both platelet counts and thrombopoietin levels by changing four parameters that have physiological correlates. Our results indicate that the primary change in cyclic thrombocytopenia is an interference with, or destruction of, the thrombopoietin receptor with secondary changes in other processes, including immune-mediated destruction of platelets and megakaryocyte deficiency and failure in platelet production. This study contributes to the understanding of the origin of cyclic thrombocytopenia as well as extending the modeling of thrombopoiesis.

Bone marrow niche in immune thrombocytopenia: a focus on megakaryopoiesis.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by increased bleeding tendency and thrombocytopenia. In fact, the precise pathogenesis of this disease is still not clear. Megakaryopoiesis involves complete differentiation of megakaryocyte (MK) progenitors to functional platelets. This complex process occurs in specific bone marrow (BM) niches composed of several hematopoietic and non-hematopoietic cell types, soluble factors, and extracellular matrix proteins. These specialized microenvironments sustain MK maturation and localization to sinusoids as well as platelet release into circulation. However, MKs in ITP patients show impaired maturation and signs of degradation. Intrinsic defects in MKs and their extrinsic environment have been implicated in altered megakaryopoiesis in this disease. In particular, aberrant expression of miRNAs directing MK proliferation, differentiation, and platelet production; defective MK apoptosis; and reduced proliferation and differentiation rate of the MSC compartment observed in these patients may account for BM defects in ITP. Furthermore, insufficient production of thrombopoietin is another likely reason for ITP development. Therefore, identifying the signaling pathways and transcription factors influencing the interaction between MKs and BM niche in ITP patients will contribute to increased platelet production in order to prevent incomplete MK maturation and destruction as well as BM fibrosis and apoptosis in ITP. In this review, we will examine the interaction and role of BM niches in orchestrating megakaryopoiesis in ITP patients and discuss how these factors can be exploited to improve the quality of patient treatment and prognosis.

Update on the inherited platelet disorders.

PURPOSE OF REVIEW: The inherited platelet disorders have witnessed a surge in our understanding of molecular mechanisms of disease in the past few years due in large to part to the introduction of next-generation sequencing for discovery of novel genes. The purpose of this review is to update the reader on the novel discoveries with regard to the inherited platelet disorders, with a particular focus on describing the novel disorders described most recently. RECENT FINDINGS: The description of novel mechanisms of disease including mutations in PRKACG, in a family with severe macrothrombocytopenia, RUNX1 and FLI1 mutations in patients with inherited mild platelet function disorders and CalDAG-GEFI resulting in a severe platelet bleeding phenotype show that there is still much to be learned from studying families and molecular sequencing of patients with well phenotyped platelet disorders. SUMMARY: The implications for clinical practice of the continually growing list of genes described in small numbers of families makes whole exome/genome tempting as an option for evaluation of patients, but use outside of the research setting still needs to be done with extreme caution as interpretation of variants is likely to require additional studies.

Critical role of Jak2 in the maintenance and function of adult hematopoietic stem cells.

Jak2, a member of the Janus kinase family of nonreceptor protein tyrosine kinases, is activated in response to a variety of cytokines, and functions in survival and proliferation of cells. An activating JAK2V617F mutation has been found in most patients with myeloproliferative neoplasms, and patients treated with Jak2 inhibitors show significant hematopoietic toxicities. However, the role of Jak2 in adult hematopoietic stem cells (HSCs) has not been clearly elucidated. Using a conditional Jak2 knockout allele, we have found that Jak2 deletion results in rapid loss of HSCs/progenitors leading to bone marrow failure and early lethality in adult mice. Jak2 deficiency causes marked impairment in HSC function, and the mutant HSCs are severely defective in reconstituting hematopoiesis in recipient animals. Jak2 deficiency also causes significant apoptosis and loss of quiescence in HSC-enriched LSK (Lin(-)Sca-1(+)c-Kit(+)) cells. Jak2-deficient LSK cells exhibit elevated reactive oxygen species levels and enhanced p38 MAPK activation. Mutant LSK cells also show defective Stat5, Erk, and Akt activation in response to thrombopoietin and stem cell factor. Gene expression analysis reveals significant downregulation of genes related to HSC quiescence and self-renewal in Jak2-deficient LSK cells. These data suggest that Jak2 plays a critical role in the maintenance and function of adult HSCs.

Characterization of thrombopoietin (TPO)-responsive progenitor cells in adult mouse bone marrow with in vivo megakaryocyte and erythroid potential.

Hematopoietic progenitor cells are the progeny of hematopoietic stem cells that coordinate the production of precise numbers of mature blood cells of diverse functional lineages. Identification of cell-surface antigen expression associated with hematopoietic lineage restriction has allowed prospective isolation of progenitor cells with defined hematopoietic potential. To clarify further the cellular origins of megakaryocyte commitment, we assessed the in vitro and in vivo megakaryocyte and platelet potential of defined progenitor populations in the adult mouse bone marrow. We show that megakaryocytes arise from CD150(+) bipotential progenitors that display both platelet- and erythrocyte-producing potential in vivo and that can develop from the Flt3(-) fraction of the pregranulocyte-macrophage population. We define a bipotential erythroid-megakaryocyte progenitor population, the CD150(+)CD9(lo)endoglin(lo) fraction of Lin(-)cKit(+)IL7 receptor alpha(-)FcγRII/III(lo)Sca1(-) cells, which contains the bulk of the megakaryocyte colony-forming capacity of the bone marrow, including bipotential megakaryocyte-erythroid colony-forming capacity, and can generate both erythrocytes and platelets efficiently in vivo. This fraction is distinct from the CD150(+)CD9(hi)endoglin(lo) fraction, which contains bipotential precursors with characteristics of increased megakaryocytic maturation, and the CD150(+)CD9(lo)endoglin(hi) fraction, which contains erythroid lineage-committed cells. Finally, we demonstrate that bipotential erythroid-megakaryocyte progenitor and CD150(+)CD9(hi)endoglin(lo) cells are TPO-responsive and that the latter population specifically expands in the recovery from thrombocytopenia induced by anti-platelet serum.

Thrombopoietin from beginning to end.

In the two decades since its cloning, thrombopoietin (TPO) has emerged not only as a critical haematopoietic cytokine, but also serves as a great example of bench-to-bedside research. Thrombopoietin, produced by the liver, is the primary regulator of megakaryocyte progenitor expansion and differentiation. Additionally, as TPO is vital for the maintenance of haematopoietic stem cells, it can truly be described as a pan-haematopoietic cytokine. Since recombinant TPO became available, the molecular mechanisms of TPO function have been the subject of extensive research. Via its receptor, c-Mpl (also termed MPL), TPO activates a wide array of downstream signalling pathways, promoting cellular survival and proliferation. Due to its central, non-redundant role in haematopoiesis, alterations of both the hormone and its receptor contribute to human disease; congenital and acquired states of thrombocytosis and thrombocytopenia and aplastic anaemia as a result from dysregulated TPO expression or functional alterations of c-Mpl. With TPO mimetics now in clinical use, the story of this haematopoietic cytokine represents a great success for biomedical research.

Hereditary thrombocythemia caused by a thrombopoietin (THPO) gain-of-function mutation associated with multiple myeloma and congenital limb defects.

Hereditary thrombocythemia (HT) has been described as a rare benign disorder caused by mutations in the thrombopoietin (THPO) or the c-Mpl receptor genes. Here we report two families with HT resulting from a THPO c.13+1 G>C mutation in the splice donor of intron 3. In one family there were coexisting distal limb defects, whereas in the other one member developed early-onset multiple myeloma. These observations, together with previously reported patients, suggest that THPO gain of function may dysregulate the hemangioblast and disturb vasculogenesis and hematopoietic development. Overstimulation of the THPO pathway might therefore predispose to clonal hematopoietic disease and to congenital abnormalities.

Thrombopoietin and thrombopoietin mimetics in the treatment of thrombocytopenia.

Although the thrombopoietin receptor was discovered in 1991 and thrombopoietin (TPO) was purified in 1994, the development of a clinically useful TPO was hampered by the appearance of neutralizing antibodies to some forms of recombinant TPO. However, in 2008 two new drugs that mimic the effect of TPO became available to treat thrombocytopenia. Romiplostim is a TPO peptide mimetic given by subcutaneous injection that activates the TPO receptor by binding to the distal hematopoietic receptor domain just like TPO. Eltrombopag is a TPO nonpeptide mimetic administered orally that activates the TPO receptor by binding to the transmembrane domain. Both increase the platelet count in healthy humans as well as in >80% of patients with immune thrombocytopenic purpura (ITP). Although initially restricted to the second-line treatment of ITP, both agents could help treat many thrombocytopenic disorders. Both agents are well tolerated, with mild headache being the most common complaint. Potential long-term complications include thrombosis, increased bone marrow reticulin, rebound worsening of thrombocytopenia upon discontinuation, and increased blast formation. Ongoing studies should establish the incidence of these complications and determine the efficacy of these new agents in a variety of other thrombocytopenic conditions.

Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis.

The molecular pathways involved in the differentiation of hematopoietic progenitors are unknown. Here we report that chemokine-mediated interactions of megakaryocyte progenitors with sinusoidal bone marrow endothelial cells (BMECs) promote thrombopoietin (TPO)-independent platelet production. Megakaryocyte-active cytokines, including interleukin-6 (IL-6) and IL-11, did not induce platelet production in thrombocytopenic, TPO-deficient (Thpo(-/-)) or TPO receptor-deficient (Mpl(-/-)) mice. In contrast, megakaryocyte-active chemokines, including stromal-derived factor-1 (SDF-1) and fibroblast growth factor-4 (FGF-4), restored thrombopoiesis in Thpo(-/-) and Mpl(-/-) mice. FGF-4 and SDF-1 enhanced vascular cell adhesion molecule-1 (VCAM-1)- and very late antigen-4 (VLA-4)-mediated localization of CXCR4(+) megakaryocyte progenitors to the vascular niche, promoting survival, maturation and platelet release. Disruption of the vascular niche or interference with megakaryocyte motility inhibited thrombopoiesis under physiological conditions and after myelosuppression. SDF-1 and FGF-4 diminished thrombocytopenia after myelosuppression. These data suggest that TPO supports progenitor cell expansion, whereas chemokine-mediated interaction of progenitors with the bone marrow vascular niche allows the progenitors to relocate to a microenvironment that is permissive and instructive for megakaryocyte maturation and thrombopoiesis. Progenitor-active chemokines offer a new strategy to restore hematopoiesis in a clinical setting.

[Signal transduction pathway mediated by thrombopoietin in the inflammation model of microglia].

OBJECTIVE: To explore the signal transduction pathway mediated by thrombopoietin (TPO) in the inflammation model of microglia induced by lipopolysaccharide (LPS). METHODS: The inflammation model of microglia BV2 cells was prepared by LPS of 0.5 and 1.0 µg/mL stimulation. The expression of TPO and ERK mRNA in BV2 cells was detected by real time quantitative PCR. Western blot was used to evaluate the expression of TPO and ERK protein in BV2 cells. TPO and IL-6 contents in the culture supernatant fluid were measured using ELISA. RESULTS: LPS stimulation increased significantly the mRNA and protein expression of TPO and ERK in BV2 cells, especially at the concentration of 1.0 µg/mL for 12 hrs stimulation. There was a significant positive correlation between the mRNA and protein expression of TPO and ERK. CONCLUSIONS: Signal transduction pathway of ERK1/2 participates in the activation of TPO in inflammatory injury of BV2 cells.

Development of romiplostim for the treatment of patients with chronic immune thrombocytopenia: from bench to bedside.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterised by abnormally low platelet counts (<100 x 10(9)/l), purpura, and bleeding episodes, and can be categorised in three phases: newly-diagnosed, persistent, and chronic. As many patients become refractory to standard treatments (corticosteroids, danazol, azathioprine, splenectomy), there is an urgent need for alternative treatments. The successful isolation and cloning of thrombopoietin (TPO) in the mid-1990s and identification of its key role in platelet production was a major breakthrough, rapidly followed by the development of the recombinant thrombopoietins, recombinant human TPO and a pegylated truncated product, PEG-rHuMGDF. Both agents increased platelet counts but development was halted because of the development of antibodies that cross-reacted with native TPO, resulting in prolonged treatment-refractory thrombocytopenia. Experimentation with novel platforms for extending the circulating half-life of therapeutic peptides by combining them with antibody fragment crystallisable (Fc) constructs led to the development of a new family of molecules termed 'peptibodies'. The 60Da recombinant peptibody romiplostim was finally produced by linking several copies of an active TPO-binding peptide sequence to a carrier Fc fragment. In clinical trials, romiplostim was effective in ameliorating thrombocytopenia in patients with chronic ITP, was well tolerated and did not elicit cross-reacting antibodies. Romiplostim has recently been approved for the treatment of adults with chronic ITP.

Eltrombopag: an update on the novel, non-peptide thrombopoietin receptor agonist for the treatment of immune thrombocytopenia.

Immune thrombocytopenia (ITP) is characterised by a transient or persistent decrease in platelets accompanied by an increased risk of bleeding, which can have a significant negative impact on patients' health-related quality of life. The condition has long been associated with an increased rate of immune-mediated platelet destruction, and traditional treatments have targeted the reduction in platelet destruction; however, some interventional drugs are limited by transient efficacy and side effects. Recent advances in our understanding of ITP pathogenesis have highlighted the role of impaired platelet production, which has led to the advent of a new generation of thrombopoietin (TPO)-receptor agonist therapies, including eltrombopag and romiplostim. The oral, non-peptide TPO-receptor agonist eltrombopag has shown considerable promise in both preclinical and clinical trials. Eltrombopag has a unique mechanism of action and binds to a transmembrane region of the TPO receptor that is distant from the TPO binding site. As such, eltrombopag may confer synergistic effects with endogenous TPO rather than competing for binding. Eltrombopag also induces activation of the TPO receptor and downstream signalling in a distinct manner to TPO and does not have a significant impact on platelet function. Clinical evidence demonstrates that eltrombopag produces a rapid and sustainable increase in platelet counts that significantly reduces bleeding and is well tolerated in patients with ITP. Eltrombopag therefore represents an important addition to the therapeutic armamentarium for ITP.

[Megakaryocyte development and platelet production in normal and disease states].

Megakaryocytopoiesis involves the commitment of hematopoietic stem cell, the proliferation and terminal differentiation of the megakaryocytic progenitors and maturation to platelet producing megakaryocytes (MK). MKs align adjacent to bone marrow vascular endothelial cells, and form proplatelets. Platelets are released, or sheared from the MK directly into the circulation from the tips of proplatelets which protrude into the vascular lumen. The regulation of megakaryocytopoiesis is mediated through multiple hematopoietic growth factors, chemokines and cellular interactions via signal transduction pathways and integrated transcription factors. The primary physiological growth factor that regulates megakaryocytopoiesis and platelet production is thrombopoietin. Circulating Levels of thrombopoietin (TPO) induce concentration-dependent proliferation and maturation of MK progenitors by binding to the c-Mpl receptor and signaling induction. Increased concentration of free TPO resulting from decreased platelet turnover rates enables the compensatory response of marrow MKs to drive amplified platelet production. C-MpL signaling is orchestrated by a complex cascade of signaling molecules inducing the action of specific transcription factors to drive MK proliferation and maturation. Newly developed thrombopoietic agents operating via c-Mpl receptor have now been proven useful in supporting platelet production in thrombocytopenic states. In this article, the authors review the regulation of megakaryocytopoiesis and platelet production in normal and disease states, and new approaches of thrombopoietic therapy.

Insights into signaling and function of hematopoietic stem cells at the single-cell level.

PURPOSE OF REVIEW: Development of a technique prospectively to isolate hematopoietic stem cells (HSCs) to near homogeneity has enabled clonal analysis and thus converted our understanding of HSCs from conceptual and qualitative to realistic and quantitative. Recent studies have revealed that despite their high proliferation potential, most HSCs are in G0 and enter cell cycle only after a long interval. This dormancy of HSCs, which seems to be important for long-term maintenance of 'stemness', appears to be regulated by the exchange of signals between HSCs and the bone marrow niche. Analysis of intersignaling and intrasignaling events in HSCs in and out of the bone marrow niche has begun. RECENT FINDINGS: With the help of advances in confocal microscopy, laser scanning microscopy, and personal computer computational power over the last decade, it has become evident that thrombopoietin/c-Mpl signaling plays a role in HSC self-renewal and AKT-forkhead box O signaling in HSC dormancy. Furthermore, transforming growth factor-beta has been indicated as a candidate niche signal to induce hibernation in HSCs. SUMMARY: Understanding of the signaling events between HSCs and niche is critical not only for stem cell biology in general and for transplantation medicine but also for the development of novel cancer therapy.

Multifaceted roles of thrombopoietin in hematopoietic stem cell regulation.

Thrombopoietin (Thpo) and its receptor myeloid proliferative leukemia (Mpl) were initially identified as the cytokine signaling that stimulates megakaryopoiesis and platelet production. However, Thpo-Mpl signaling has also been widely characterized as one of the few cytokine systems that directly regulates hematopoietic stem and progenitor cells. The ability of Thpo signaling to stimulate hematopoietic stem cell (HSC) self-renewal has led to the development and utilization of Thpo mimetic drugs to treat hematopoietic diseases with restricted function of HSCs, such as aplastic anemia. This review will cover the mechanisms by which Thpo-Mpl signaling regulates HSCs.

Thrombopoietin and its splicing variants: structure and functions in thrombopoiesis and beyond.

Since its cloning in 1994, several studies have reported that thrombopoietin (THPO) presents several alternative splicing products that differ from the full-length protein in its 5' UTR, N- or C-terminal regions. Most of these splice variants are evolutionarily conserved and have been detected in different tissues as well as in cell lines. Although the possible functions of the THPO isoforms are still elusive, different clues link them to the peculiar mechanism that regulates THPO production. Moreover, novel fields to explore possible roles of the THPO variants are opened by observations that this hormone can influence the formation of hematopoietic progenitors and its expression occurs in some tumors as well as in tissues not directly related to the thrombopoiesis. In this review, we summarize the structure and functions of THPO through the published evidence on its splicing isoforms and discuss about their involvement with physiopathologic phenomena.

Thrombopoietin inhibits murine mast cell differentiation.

We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin or addition of this growth factor to bone marrow-derived mast cell cultures severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target antiapoptotic gene Bcl2.

Thrombopoietin inhibits nerve growth factor-induced neuronal differentiation and ERK signalling.

Thrombopoietin (TPO), a hematopoietic growth factor regulating platelet production, and its receptor (TPOR) were recently shown to be expressed in the brain where they exert proapoptotic activity. Here we used PC12 cells, an established model of neuronal differentiation, to investigate the effects of TPO on neuronal survival and differentiation. These cells expressed TPOR mRNA. TPO increased cell death in neuronally differentiated PC12 cells but had no effect in undifferentiated cells. Surprisingly, TPO inhibited nerve growth factor (NGF)-induced differentiation of PC12 cells in a dose- and time-dependent manner. This inhibition was dependent on the activity of Janus kinase-2 (JAK2). Using phospho-kinase arrays and Western blot we found downregulation of the NGF-stimulated phosphorylation of the extracellular signal-regulated kinase p42ERK by TPO with no effect on phosphorylation of Akt or stress kinases. NGF-induced phosphorylation of ERK-activating kinases, MEK1/2 and C-RAF was also reduced by TPO while NGF-induced RAS activation was not attenuated by TPO treatment. In contrast to its inhibitory effects on NGF signalling, TPO had no effect on epidermal growth factor (EGF)-stimulated ERK phosphorylation or proliferation of PC12 cells. Our data indicate that TPO via activation of its receptor-bound JAK2 delays the NGF-dependent acquisition of neuronal phenotype and decreases neuronal survival by suppressing NGF-induced ERK activity.