RESUMEN
Classical BCR-ABL-negative myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoietic stem cells (HSCs) caused mainly by recurrent mutations in genes encoding JAK2 (JAK2), calreticulin (CALR), or the thrombopoietin receptor (MPL). Interferon α (IFNα) has demonstrated some efficacy in inducing molecular remission in MPNs. To determine factors that influence molecular response rate, we evaluated the long-term molecular efficacy of IFNα in patients with MPN by monitoring the fate of cells carrying driver mutations in a prospective observational and longitudinal study of 48 patients over more than 5 years. We measured the clonal architecture of early and late hematopoietic progenitors (84 845 measurements) and the global variant allele frequency in mature cells (409 measurements) several times per year. Using mathematical modeling and hierarchical Bayesian inference, we further inferred the dynamics of IFNα-targeted mutated HSCs. Our data support the hypothesis that IFNα targets JAK2V617F HSCs by inducing their exit from quiescence and differentiation into progenitors. Our observations indicate that treatment efficacy is higher in homozygous than heterozygous JAK2V617F HSCs and increases with high IFNα dose in heterozygous JAK2V617F HSCs. We also found that the molecular responses of CALRm HSCs to IFNα were heterogeneous, varying between type 1 and type 2 CALRm, and a high dose of IFNα correlates with worse outcomes. Our work indicates that the long-term molecular efficacy of IFNα implies an HSC exhaustion mechanism and depends on both the driver mutation type and IFNα dose.
Asunto(s)
Células Madre Hematopoyéticas/efectos de los fármacos , Factores Inmunológicos/uso terapéutico , Interferón-alfa/uso terapéutico , Mutación/efectos de los fármacos , Trastornos Mieloproliferativos/tratamiento farmacológico , Calreticulina/genética , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Factores Inmunológicos/farmacología , Interferón-alfa/farmacología , Janus Quinasa 2/genética , Estudios Longitudinales , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Estudios Prospectivos , Receptores de Trombopoyetina/genética , Células Tumorales CultivadasRESUMEN
Thrombosis is the main cause of morbidity and mortality in patients with JAK2V617F myeloproliferative neoplasms. Recent studies have reported the presence of JAK2V617F in endothelial cells of some patients with myeloproliferative neoplasms. We investigated the role of endothelial cells that express JAK2V617F in thrombus formation using an in vitro model of human endothelial cells overexpressing JAK2V617F and an in vivo model of mice with endothelial-specific JAK2V617F expression. Interestingly, these mice displayed a higher propensity for thrombus. When deciphering the mechanisms by which JAK2V617F-expressing endothelial cells promote thrombosis, we observed that they have a pro-adhesive phenotype associated with increased endothelial P-selectin exposure, secondary to degranulation of Weibel-Palade bodies. We demonstrated that P-selectin blockade was sufficient to reduce the increased propensity of thrombosis. Moreover, treatment with hydroxyurea also reduced thrombosis and decreased the pathological interaction between leukocytes and JAK2V617F-expressing endothelial cells through direct reduction of endothelial P-selectin expression. Taken together, our data provide evidence that JAK2V617F-expressing endothelial cells promote thrombosis through induction of endothelial P-selectin expression, which can be reversed by hydroxyurea. Our findings increase our understanding of thrombosis in patients with myeloproliferative neoplasms, at least those with JAK2V617F-positive endothelial cells, and highlight a new role for hydroxyurea. This novel finding provides the proof of concept that an acquired genetic mutation can affect the pro-thrombotic nature of endothelial cells, suggesting that other mutations in endothelial cells could be causal in thrombotic disorders of unknown cause, which account for 50% of recurrent venous thromboses.
Asunto(s)
Células Endoteliales/metabolismo , Janus Quinasa 2/biosíntesis , Selectina-P/biosíntesis , Trombosis/metabolismo , Animales , Modelos Animales de Enfermedad , Células Endoteliales/patología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Hidroxiurea/farmacología , Janus Quinasa 2/genética , Ratones , Ratones Transgénicos , Selectina-P/genética , Trombosis/tratamiento farmacológico , Trombosis/genética , Trombosis/patologíaRESUMEN
Primary myelofibrosis is characterized by the development of fibrosis in the bone marrow that contributes to ineffective hematopoiesis. Bone marrow fibrosis is the result of a complex and not yet fully understood interaction among megakaryocytes, myeloid cells, fibroblasts, and endothelial cells. Here, we report that >30% of the endothelial cells in the small vessels of the bone marrow and spleen of patients with primary myelofibrosis have a mesenchymal phenotype, which is suggestive of the process known as endothelial-to-mesenchymal transition (EndMT). EndMT can be reproduced in vitro by incubation of cultured endothelial progenitor cells or spleen-derived endothelial cells with inflammatory cytokines. Megakaryocytes appear to be implicated in this process, because EndMT mainly occurs in the microvessels close to these cells, and because megakaryocyte-derived supernatant fluid can reproduce the EndMT switch in vitro. Furthermore, EndMT is an early event in a JAK2-V617F knock-in mouse model of primary myelofibrosis. Overall, these data show for the first time that microvascular endothelial cells in the bone marrow and spleen of patients with primary myelofibrosis show functional and morphologic changes that are associated to the mesenchymal phenotype.
Asunto(s)
Médula Ósea/patología , Mielofibrosis Primaria/patología , Bazo/patología , Animales , Modelos Animales de Enfermedad , Humanos , Megacariocitos/patología , RatonesRESUMEN
Frameshift mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and myelofibrosis patients. To address the contribution of the CALR mutants to the pathogenesis of myeloproliferative neoplasms, we engrafted lethally irradiated recipient mice with bone marrow cells transduced with retroviruses expressing these mutants. In contrast to wild-type CALR, CALRdel52 (type I) and, to a lesser extent, CALRins5 (type II) induced thrombocytosis due to a megakaryocyte (MK) hyperplasia. Disease was transplantable into secondary recipients. After 6 months, CALRdel52-, in contrast to rare CALRins5-, transduced mice developed a myelofibrosis associated with a splenomegaly and a marked osteosclerosis. Monitoring of virus-transduced populations indicated that CALRdel52 leads to expansion at earlier stages of hematopoiesis than CALRins5. However, both mutants still specifically amplified the MK lineage and platelet production. Moreover, a mutant deleted of the entire exon 9 (CALRdelex9) did not induce a disease, suggesting that the oncogenic property of CALR mutants was related to the new C-terminus peptide. To understand how the CALR mutants target the MK lineage, we used a cell-line model and demonstrated that the CALR mutants, but not CALRdelex9, specifically activate the thrombopoietin (TPO) receptor (MPL) to induce constitutive activation of Janus kinase 2 and signal transducer and activator of transcription 5/3/1. We confirmed in c-mpl- and tpo-deficient mice that expression of Mpl, but not of Tpo, was essential for the CALR mutants to induce thrombocytosis in vivo, although Tpo contributes to disease penetrance. Thus, CALR mutants are sufficient to induce thrombocytosis through MPL activation.
Asunto(s)
Calreticulina/metabolismo , Mutación INDEL , Megacariocitos/metabolismo , Mielofibrosis Primaria/metabolismo , Receptores de Trombopoyetina/metabolismo , Trombocitosis/metabolismo , Animales , Calreticulina/genética , Mutación del Sistema de Lectura , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Megacariocitos/patología , Ratones , Ratones Mutantes , Mielofibrosis Primaria/etiología , Mielofibrosis Primaria/genética , Mielofibrosis Primaria/patología , Receptores de Trombopoyetina/genética , Trombocitosis/complicaciones , Trombocitosis/genética , Trombocitosis/patologíaRESUMEN
Thrombosis is common in patients suffering from myeloproliferative neoplasm (MPN), whereas bleeding is less frequent. JAK2(V617F), the main mutation involved in MPN, is considered as a risk factor for thrombosis, although the direct link between the mutation and hemostatic disorders is not strictly established. We investigated this question using conditional JAK2(V617F) knock-in mice with constitutive and inducible expression of JAK2(V617F) in hematopoietic cells, which develop a polycythemia vera (PV)-like disorder evolving into myelofibrosis. In vitro, thrombosis was markedly impaired with an 80% decrease in platelet-covered surface, when JAK2(V617F) blood was perfused at arterial shear over collagen. JAK2(V617F) platelets presented only a moderate glycoprotein (GP) VI deficiency not responsible for the defective platelet accumulation. In contrast, a decreased proportion of high-molecular-weight von Willebrand factor multimers could reduce platelet adhesion. Accordingly, the tail bleeding time was prolonged. In the FeCl3-induced thrombosis model, platelet aggregates formed rapidly but were highly unstable. Interestingly, vessels were considerably dilated. Thus, mice developing PV secondary to constitutive JAK2(V617F) expression exhibit a bleeding tendency combined with the accelerated formation of unstable clots, reminiscent of observations made in patients. Hemostatic defects were not concomitant with the induction of JAK2(V617F) expression, suggesting they were not directly caused by the mutation but were rather the consequence of perturbations in blood and vessel homeostasis.
Asunto(s)
Modelos Animales de Enfermedad , Trastornos Hemostáticos/genética , Janus Quinasa 2/genética , Mutación Missense , Trastornos Mieloproliferativos/genética , Animales , Aorta/metabolismo , Aorta/patología , Aorta/fisiopatología , Tiempo de Sangría , Plaquetas/metabolismo , Citometría de Flujo , Técnicas de Sustitución del Gen , Humanos , Immunoblotting , Ratones Transgénicos , Trastornos Mieloproliferativos/sangre , Activación Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/metabolismo , Policitemia Vera/sangre , Policitemia Vera/genética , Mielofibrosis Primaria/sangre , Mielofibrosis Primaria/genética , Trombosis/sangre , Trombosis/genética , Vasodilatación/genética , Factor de von Willebrand/metabolismoRESUMEN
JAK2 inhibition therapy is used to treat patients suffering from myeloproliferative neoplasms (MPN). Conflicting data on this therapy are reported possibly linked to the types of inhibitors or disease type. Therefore, we decided to compare in mice the effect of a JAK2 inhibitor, Fedratinib, in MPN models of increasing severity: polycythemia vera (PV), post-PV myelofibrosis (PPMF) and rapid post-essential thrombocythemia MF (PTMF). The models were generated through JAK2 activation by the JAK2(V617F) mutation or MPL constant stimulation. JAK2 inhibition induced a correction of splenomegaly, leucocytosis and microcytosis in all three MPN models. However, the effects on fibrosis, osteosclerosis, granulocytosis, erythropoiesis or platelet counts varied according to the disease severity stage. Strikingly, complete blockade of fibrosis and osteosclerosis was observed in the PPMF model, linked to correction of MK hyper/dysplasia, but not in the PTMF model, suggesting that MF development may also become JAK2-independent. Interestingly, we originally found a decreased in the JAK2(V617F) allele burden in progenitor cells from the spleen but not in other cell types. Overall, this study shows that JAK2 inhibition has different effects according to disease phenotypes and can (i) normalize platelet counts, (ii) prevent the development of marrow fibrosis/osteosclerosis at an early stage and (iii) reduce splenomegaly through blockage of stem cell mobilization in the spleen.
Asunto(s)
Janus Quinasa 2/antagonistas & inhibidores , Policitemia Vera/tratamiento farmacológico , Mielofibrosis Primaria/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirrolidinas/administración & dosificación , Sulfonamidas/administración & dosificación , Trombocitemia Esencial/tratamiento farmacológico , Animales , Progresión de la Enfermedad , Ratones , Recuento de Plaquetas , Policitemia Vera/sangre , Policitemia Vera/fisiopatología , Mielofibrosis Primaria/sangre , Mielofibrosis Primaria/fisiopatología , Esplenomegalia/tratamiento farmacológico , Trombocitemia Esencial/sangre , Trombocitemia Esencial/fisiopatologíaRESUMEN
The acquired gain-of-function V617F mutation in the Janus Kinase 2 (JAK2(V617F)) is the main mutation involved in BCR/ABL-negative myeloproliferative neoplasms (MPNs), but its effect on hematopoietic stem cells as a driver of disease emergence has been questioned. Therefore, we reinvestigated the role of endogenous expression of JAK2(V617F) on early steps of hematopoiesis as well as the effect of interferon-α (IFNα), which may target the JAK2(V617F) clone in humans by using knock-in mice with conditional expression of JAK2(V617F) in hematopoietic cells. These mice develop a MPN mimicking polycythemia vera with large amplification of myeloid mature and precursor cells, displaying erythroid endogenous growth and progressing to myelofibrosis. Interestingly, early hematopoietic compartments [Lin-, LSK, and SLAM (LSK/CD48-/CD150+)] increased with the age. Competitive repopulation assays demonstrated disease appearance and progressive overgrowth of myeloid, Lin-, LSK, and SLAM cells, but not lymphocytes, from a low number of engrafted JAK2(V617F) SLAM cells. Finally, IFNα treatment prevented disease development by specifically inhibiting JAK2(V617F) cells at an early stage of differentiation and eradicating disease-initiating cells. This study shows that JAK2(V617F) in mice amplifies not only late but also early hematopoietic cells, giving them a proliferative advantage through high cell cycling and low apoptosis that may sustain MPN emergence but is lost upon IFNα treatment.
Asunto(s)
Apoptosis , Células Madre Hematopoyéticas/citología , Interferón-alfa/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Mutación , Animales , Células de la Médula Ósea/citología , Trasplante de Médula Ósea , Ciclo Celular , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Humanos , Linfocitos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Trastornos Mieloproliferativos/genética , Fenotipo , Policitemia Vera/genética , Mielofibrosis Primaria/metabolismo , Células Madre/citologíaRESUMEN
The JAK2(V617F) mutation is present in the majority of patients with polycythemia vera and one-half of those with essential thrombocythemia and primary myelofibrosis. JAK2(V617F) is a gain-of-function mutation resulting in constitutive JAK2 signaling involved in the pathogenesis of these diseases. JAK2(V617F) has been shown to promote S-phase entry. Here, we demonstrate that the CDC25A phosphatase, a key regulator of the G1/S cell-cycle transition, is constitutively overexpressed in JAK2(V617F)-positive cell lines, JAK2-mutated patient CD36(+) progenitors, and in vitro-differentiated proerythroblasts. Accordingly, CDC25A is overexpressed in BM and spleen of Jak2(V617F) knock-in mice compared with wild-type littermates. By using murine FDC-P1-EPOR and human HEL and SET-2 cell lines, we found that JAK2(V617F)-induced CDC25A up-regulation was caused neither by increased CDC25A transcription or stability nor by the involvement of its upstream regulators Akt and MAPK. Instead, our results suggest that CDC25A is regulated at the translational level through STAT5 and the translational initiation factor eIF2α. CDC25A inhibition reduces the clonogenic and proliferative potential of JAK2(V617F)-expressing cell lines and erythroid progenitors while moderately affecting normal erythroid differentiation. These results suggest that CDC25A deregulation may be involved in hematopoietic cells expansion in JAK2(V617F) patients, making this protein an attracting potential therapeutic target.
Asunto(s)
Janus Quinasa 2/genética , Fosfatasas cdc25/genética , Sustitución de Aminoácidos/fisiología , Animales , Ciclo Celular/genética , Células Cultivadas , Activación Enzimática/genética , Regulación Leucémica de la Expresión Génica/genética , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Células Madre Hematopoyéticas/metabolismo , Humanos , Janus Quinasa 2/metabolismo , Ratones , Proteínas Mutantes/genética , Oncogenes/genética , Fenilalanina/genética , Regulación hacia Arriba , Valina/genética , Fosfatasas cdc25/metabolismoRESUMEN
The constitutively active JAK2 V617F mutant is the major determinant of human myeloproliferative neoplasms (MPNs). We show that coexpression of murine JAK2 V617F and the murine thrombopoietin (Tpo) receptor (TpoR, c-MPL) in hematopoietic cell lines or heterozygous knock-in of JAK2 V617F in mice leads to down-modulation of TpoR levels. Enhanced TpoR ubiquitinylation, proteasomal degradation, reduced recycling, and maturation are induced by the constitutive JAK2 V617F activity. These effects can be prevented in cell lines by JAK2 and proteasome inhibitors. Restoration of TpoR levels by inhibitors could be detected in platelets from JAK2 inhibitor-treated myelofibrosis patients that express the JAK2 V617F mutant, and in platelets from JAK2 V617F knock-in mice that were treated in vivo with JAK2 or proteasome inhibitors. In addition, we show that Tpo can induce both proliferative and antiproliferative effects via TpoR at low and high JAK2 activation levels, respectively, or on expression of JAK2 V617F. The antiproliferative signaling and receptor down-modulation by JAK2 V617F were dependent on signaling via TpoR cytosolic tyrosine 626. We propose that selection against TpoR antiproliferative signaling occurs by TpoR down-modulation and that restoration of down-modulated TpoR levels could become a biomarker for the treatment of MPNs.
Asunto(s)
Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/fisiología , Inhibidores de Proteasoma , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Trombopoyetina/genética , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/fisiología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense/fisiología , Fenilalanina/genética , Receptor Cross-Talk/efectos de los fármacos , Receptor Cross-Talk/fisiología , Receptores de Trombopoyetina/metabolismo , Transducción de Señal/efectos de los fármacos , Valina/genéticaRESUMEN
ABSTRACT: Cerebral venous sinus thrombosis (CVST) is an uncommon venous thromboembolic event accounting for <1% of strokes resulting in brain parenchymal injuries. JAK2V617F mutation, the most frequent driving mutation of myeloproliferative neoplasms, has been reported to be associated with worse clinical outcomes in patients with CVST. We investigated whether hematopoietic JAK2V617F expression predisposes to specific pathophysiological processes and/or worse prognosis after CVST. Using an in vivo mouse model of CVST, we analyzed clinical, biological, and imaging outcomes in mice with hematopoietic-restricted Jak2V617F expression, compared with wild-type Jak2 mice. In parallel, we studied a human cohort of JAK2V617F-positive or -negative CVST. Early after CVST, mice with hematopoietic Jak2V617F expression had increased adhesion of platelets and neutrophils in cerebral veins located in the vicinity of CVST. On day 1, Jak2V617F mice had a worse outcome characterized by significantly more frequent and severe intracranial hemorrhages (ICHs) and higher mortality rates. Peripheral neutrophil activation was enhanced, as indicated by higher circulating platelet-neutrophil aggregates, upregulated CD11b expression, and higher myeloperoxydase plasma level. Concurrently, immunohistological and brain homogenate analysis showed higher neutrophil infiltration and increased blood-brain barrier disruption. Similarly, patients with JAK2V617F-positive CVST tended to present higher thrombotic burden and had significantly higher systemic immune-inflammation index, a systemic thromboinflammatory marker, than patients who were JAK2V617F-negative. In mice with CVST, our study corroborates that Jak2V617F mutation leads to a specific pattern including increased thrombotic burden, ICH, and mortality. The exacerbated thromboinflammatory response, observed both in mice and patients positive for JAK2V617F, could contribute to hemorrhagic complications.
Asunto(s)
Inflamación , Janus Quinasa 2 , Mutación , Trombosis de los Senos Intracraneales , Animales , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Ratones , Trombosis de los Senos Intracraneales/genética , Humanos , Pronóstico , Inflamación/genética , Modelos Animales de Enfermedad , Masculino , Femenino , Neutrófilos/metabolismoRESUMEN
Aberrant JAK2 signalling plays a central role in myeloproliferative neoplasms (MPN). JAK2 inhibitors have proven to be clinically efficacious, however, they are not mutation-specific and competent enough to suppress neoplastic clonal haematopoiesis. We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated. To this end we investigated the efficacy of BEZ235, a dual PI3K/mTOR inhibitor, alone and in combination with the JAK1/JAK2 inhibitor ruxolitinib, in different preclinical models of MPN. Single-agent BEZ235 inhibited the proliferation and induced cell cycle arrest and apoptosis of mouse and human JAK2V617F mutated cell lines at concentrations significantly lower than those required to inhibit the wild-type counterpart, and preferentially prevented colony formation from JAK2V617F knock-in mice and patients' progenitor cells compared with normal ones. Co-treatment of BEZ235 and ruxolitinib produced significant synergism in all these in-vitro models. Co-treatment was also more effective than single drugs in reducing the extent of disease and prolonging survival of immunodeficient mice injected with JAK2V617F-mutated Ba/F3-EPOR cells and in reducing spleen size, decreasing reticulocyte count and improving spleen histopathology in conditional JAK2V617F knock-in mice. In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Janus Quinasa 2/genética , Trastornos Mieloproliferativos/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/enzimología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/fisiología , Humanos , Imidazoles/administración & dosificación , Concentración 50 Inhibidora , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Células K562 , Ratones , Ratones SCID , Ratones Transgénicos , Terapia Molecular Dirigida , Mutación Missense , Trastornos Mieloproliferativos/enzimología , Trasplante de Neoplasias , Nitrilos , Fosfatidilinositol 3-Quinasas/metabolismo , Pirazoles/administración & dosificación , Pirimidinas , Quinolinas/administración & dosificación , Esplenomegalia/prevención & control , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Current JAK2 inhibitors used for myeloproliferative neoplasms (MPN) treatment are not specific enough to selectively suppress aberrant JAK2 signalling and preserve physiological JAK2 signalling. We tested whether combining a JAK2 inhibitor with a series of serine threonine kinase inhibitors, targeting nine signalling pathways and already used in clinical trials, synergized in inhibiting growth of haematopoietic cells expressing mutant and wild-type forms of JAK2 (V617F) or thrombopoietin receptor (W515L). Out of 15 kinase inhibitors, the ZSTK474 phosphatydylinositol-3'-kinase (PI3K) inhibitor molecule showed strong synergic inhibition by Chou and Talalay analysis with JAK2 and JAK2/JAK1 inhibitors. Other pan-class I, but not gamma or delta specific PI3K inhibitors, also synergized with JAK2 inhibitors. Synergy was not observed in Bcr-Abl transformed cells. The best JAK2/JAK1 and PI3K inhibitor combination pair (ruxolitinib and GDC0941) reduces spleen weight in nude mice inoculated with Ba/F3 cells expressing TpoR and JAK2 V617F. It also exerted strong inhibitory effects on erythropoietin-independent erythroid colonies from MPN patients and JAK2 V617F knock-in mice, where at certain doses, a preferential inhibition of JAK2 V617F mutated progenitors was detected. Our data support the use of a combination of JAK2 and pan-class I PI3K inhibitors in the treatment of MPNs.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Janus Quinasa 2/antagonistas & inhibidores , Trastornos Mieloproliferativos/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Técnicas de Sustitución del Gen , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/enzimología , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Ratones , Ratones Desnudos , Ratones Transgénicos , Mutación Missense , Trastornos Mieloproliferativos/enzimología , Trasplante de Neoplasias , Nitrilos , Fosfatidilinositol 3-Quinasas/metabolismo , Pirazoles/administración & dosificación , Pirimidinas , Pirrolidinas/administración & dosificación , Transducción de Señal/efectos de los fármacos , Sulfonamidas/administración & dosificación , Triazinas/administración & dosificación , Células Tumorales CultivadasAsunto(s)
Síndrome de Budd-Chiari/genética , Síndrome de Budd-Chiari/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Sustitución de Aminoácidos , Animales , Síndrome de Budd-Chiari/patología , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Femenino , Humanos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Transforming growth factor-ß1 (TGF-ß1) is the most important cytokine involved in the promotion of myelofibrosis. Mechanisms leading to its local activation in the bone marrow environment remain unclear. As a recent study has highlighted the role of thrombospondin-1 (TSP-1) in platelet-derived TGF-ß1 activation, we investigated the role of TSP-1 in the TPO(high) murine model of myelofibrosis. Two groups of engrafted mice, WT TPO(high) and Tsp-1-null TPO(high), were constituted. All mice developed a similar myeloproliferative syndrome and an increase in total TGF-ß1 levels in the plasma and in extracellular fluids of marrow and spleen. Surprisingly, we were able to detect the active form of TGF-ß1 in Tsp-1-null TPO(high) mice. Accordingly, these mice developed marrow and spleen fibrosis, with intriguingly a higher grade than in WT TPO(high) mice. Our results show that TSP-1 is not the major activator of TGF-ß1 in TPO-induced myelofibrosis, suggesting the contribution of another mechanism in the megakaryocyte/platelet compartment.
Asunto(s)
Mielofibrosis Primaria/inducido químicamente , Mielofibrosis Primaria/patología , Trombopoyetina/efectos adversos , Trombospondina 1/fisiología , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Plaquetas/metabolismo , Plaquetas/patología , Médula Ósea/metabolismo , Médula Ósea/patología , Femenino , Masculino , Megacariocitos/metabolismo , Megacariocitos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mielofibrosis Primaria/metabolismo , Bazo/metabolismo , Bazo/patologíaRESUMEN
Heterozygous mutation targeting proline 95 in Serine/Arginine-rich Splicing Factor 2 (SRSF2) is associated with V617F mutation in Janus Activated Kinase 2 (JAK2) in some myeloproliferative neoplasms (MPNs), most commonly primary myelofibrosis. To explore the interaction of Srsf2P95H with Jak2V617F, we generated Cre-inducible knock-in mice expressing these mutants under control of the stem cell leukemia (Scl) gene promoter. In transplantation experiments, Srsf2P95H unexpectedly delayed myelofibrosis induced by Jak2V617F and decreased TGFß1 serum level. Srsf2P95H reduced the competitiveness of transplanted Jak2V617F hematopoietic stem cells while preventing their exhaustion. RNA sequencing of sorted megakaryocytes identified an increased number of splicing events when the two mutations were combined. Focusing on JAK/STAT pathway, Jak2 exon 14 skipping was promoted by Srsf2P95H, an event detected in patients with JAK2V617F and SRSF2P95 co-mutation. The skipping event generates a truncated inactive JAK2 protein. Accordingly, Srsf2P95H delays myelofibrosis induced by the thrombopoietin receptor agonist Romiplostim in Jak2 wild-type animals. These results unveil JAK2 exon 14 skipping promotion as a strategy to reduce JAK/STAT signaling in pathological conditions.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Trastornos Mieloproliferativos , Mielofibrosis Primaria , Animales , Ratones , Janus Quinasa 2/genética , Quinasas Janus/genética , Mutación , Trastornos Mieloproliferativos/genética , Mielofibrosis Primaria/genética , Proteínas de Unión al ARN/genética , Transducción de Señal , Factores de Transcripción STAT/genéticaRESUMEN
The effects of Notch signaling on human megakaryocytic and erythroid differentiation were investigated by exposing human CD34(+) progenitor cells to an immobilized chimeric form of the Notch ligand, Delta-like4 (Dll4Fc). Exposure of human cord blood CD34(+) cells to Dll4Fc induced a modest enhancement of erythroid cell production. Conversely, under megakaryocytic culture conditions, Dll4Fc strongly impaired platelet production by reducing the generation of mature CD41a(+)CD42b(+) megakaryocytes (MKs) and platelet-forming cells. The inhibitory activity of Dll4 on terminal MK differentiation was confirmed by culturing CD34(+) cells onto Dll-4-expressing stroma cells (engineered to express the membrane-anchored form of Dll4). The reduced production of mature CD41a(+)CD42(+) cells was rescued by inhibiting Notch signaling either with the N-N-(3,5-difluorophenacetyl-L-alanyl)-S-phenylglycine t-butyl ester γ-secretase inhibitor or the dominant-negative version of Mastermind. Dll4 impaired the generation of mature CD41a(+)CD42b(+) cells and proplatelet formation without affecting earlier steps of MK differentiation, such as production of megakaryocytic/erythroid progenitors and colony-forming units-MKs. This blockade was accompanied by a modulation of the transcriptional program of megakaryocytic differentiation. All these results indicate that Dll4/Notch signaling inhibits human terminal MK differentiation.
Asunto(s)
Diferenciación Celular , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Megacariocitos/citología , Receptores Notch/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Unión al Calcio , Células Cultivadas , Técnicas de Cocultivo , Células Precursoras Eritroides/metabolismo , Sangre Fetal/citología , Sangre Fetal/metabolismo , Citometría de Flujo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Megacariocitos/metabolismo , Ratones , ARN Mensajero/genética , Receptores Notch/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/metabolismoRESUMEN
The Jak2(V617F) mutation is found in most classical BCR/ABL-negative myeloproliferative neoplasms (MPNs). Usually, heterozygosity of the mutation is associated with essential thrombocythemia (ET) and homozygosity with polycythemia vera (PV). Retrovirally transduced or transgenic animal models have shown that the mutation is sufficient for MPN development but that the level of expression is crucial for MPN phenotypes. Therefore we investigated the effect of an endogenous heterozygous expression of Jak2(V617F) in knock-in (KI) mice. These animals displayed constitutive JAK2 activation and autonomous erythroid progenitor cell growth. Mice suffered from marked polycythemia, granulocytosis and thrombocytosis. Spleens and marrows displayed myeloid trilineage hyperplasia. Most animals survived to develop advanced fibrosis in these organs at around 9 months of age. In conclusion, constitutive heterozygous expression of JAK2(V617F) in mice is not embryo-lethal but results in severe PV-like disease with secondary myelofibrosis and not in ET-like disease as expected from patient study.
Asunto(s)
Janus Quinasa 2/genética , Trastornos Mieloproliferativos/etiología , Policitemia Vera/genética , Sustitución de Aminoácidos , Animales , Médula Ósea/patología , Linaje de la Célula , Cruzamientos Genéticos , Técnicas de Sustitución del Gen , Heterocigoto , Humanos , Hiperplasia , Janus Quinasa 2/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , Trastornos Mieloproliferativos/enzimología , Trastornos Mieloproliferativos/genética , Mutación Puntual , Policitemia Vera/enzimología , Mielofibrosis Primaria/enzimología , Mielofibrosis Primaria/etiología , Mielofibrosis Primaria/genética , Bazo/patología , Trombocitemia Esencial/enzimología , Trombocitemia Esencial/genéticaRESUMEN
Over the past decades, a variety of MPN mouse models have been developed to express in HSC the main mutations identified in patients: JAK2V617F, CALRdel52 or ins5 and MPLW515L. These models mimic quite faithfully human PV or ET with their natural evolutions into MF and their hemostasis complications, demonstrating the driver function of these mutations in MPN. Here, we review these models and show how they have improved our general understanding of MPN regarding (1) the mechanisms of fibrosis, thrombosis/hemorrhages and disease initiation, (2) the roles of additional mutations and signaling pathways in disease progression and (3) the preclinical development of novel therapies. We also address controversial results between these models and remind how these models may differ from human MPN onset and also how basically mice are not humans, encouraging caution when one draw lessons from mice to humans. Furthermore, the contribution of germline genetic predisposition, HSC and niche aging, metabolic, oxidative, replicative or genotoxic stress, inflammation, immune escape and additional mutations need to be considered in further investigations to encompass the full complexity of human MPN in mice.
Asunto(s)
Trombocitemia Esencial , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Predisposición Genética a la Enfermedad , Humanos , Ratones , Mutación , Receptores de Trombopoyetina/genética , Trombocitemia Esencial/genéticaRESUMEN
Current murine models of myeloproliferative neoplasms (MPNs) cannot examine how MPNs progress from a single bone marrow source to the entire hematopoietic system. Thus, using transplantation of knock-in JAK2V617F hematopoietic cells into a single irradiated leg, we show development of polycythemia vera (PV) from a single anatomic site in immunocompetent mice. Barcode experiments reveal that grafted JAK2V617F stem/progenitor cells migrate from the irradiated leg to nonirradiated organs such as the contralateral leg and spleen, which is strictly required for development of PV. Mutant cells colonizing the nonirradiated leg efficiently induce PV in nonconditioned recipient mice and contain JAK2V617F hematopoietic stem/progenitor cells that express high levels of carbonic anhydrase 1 (CA1), a peculiar feature also found in CD34+ cells from patients with PV. Finally, genetic and pharmacologic inhibition of CA1 efficiently suppresses PV development and progression in mice and decreases PV patients' erythroid progenitors, strengthening CA1 as a potent therapeutic target for PV. SIGNIFICANCE: Follow-up of hematopoietic malignancies from their initiating anatomic site is crucial for understanding their development and discovering new therapeutic avenues. We developed such an approach, used it to characterize PV progression, and identified CA1 as a promising therapeutic target of PV. This article is highlighted in the In This Issue feature, p. 265.