Caspase Inhibition Modulates Monocyte-Derived Macrophage Polarization in Damaged Tissues.

Circulating monocytes are recruited in damaged tissues to generate macrophages that modulate disease progression. Colony-stimulating factor-1 (CSF-1) promotes the generation of monocyte-derived macrophages, which involves caspase activation. Here, we demonstrate that activated caspase-3 and caspase-7 are located to the vicinity of the mitochondria in CSF1-treated human monocytes. Active caspase-7 cleaves p47PHOX at aspartate 34, which promotes the formation of the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase complex NOX2 and the production of cytosolic superoxide anions. Monocyte response to CSF-1 is altered in patients with a chronic granulomatous disease, which are constitutively defective in NOX2. Both caspase-7 down-regulation and radical oxygen species scavenging decrease the migration of CSF-1-induced macrophages. Inhibition or deletion of caspases prevents the development of lung fibrosis in mice exposed to bleomycin. Altogether, a non-conventional pathway that involves caspases and activates NOX2 is involved in CSF1-driven monocyte differentiation and could be therapeutically targeted to modulate macrophage polarization in damaged tissues.

Hemostatic disorders in a JAK2V617F-driven mouse model of myeloproliferative neoplasm.

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.

JAK2 and MPL protein levels determine TPO-induced megakaryocyte proliferation vs differentiation.

Megakaryopoiesis is a 2-step differentiation process, regulated by thrombopoietin (TPO), on binding to its cognate receptor myeloproliferative leukemia (MPL). This receptor associates with intracytoplasmic tyrosine kinases, essentially janus kinase 2 (JAK2), which regulates MPL stability and cell-surface expression, and mediates TPO-induced signal transduction. We demonstrate that JAK2 and MPL mediate TPO-induced proliferation arrest and megakaryocytic differentiation of the human megakaryoblastic leukemia cell line UT7-MPL. A decrease in JAK2 or MPL protein expression, and JAK2 chemical inhibition, suppress this antiproliferative action of TPO. The expression of JAK2 and MPL, which progressively increases along normal human megakaryopoiesis, is decreased in platelets of patients diagnosed with JAK2- or MPL-mutated essential thrombocytemia and primary myelofibrosis, 2 myeloproliferative neoplasms in which megakaryocytes (MKs) proliferate excessively. Finally, low doses of JAK2 chemical inhibitors are shown to induce a paradoxical increase in MK production, both in vitro and in vivo. We propose that JAK2 and MPL expression levels regulate megakaryocytic proliferation vs differentiation in both normal and pathological conditions, and that JAK2 chemical inhibitors could promote a paradoxical thrombocytosis when used at suboptimal doses.

JAK2 inhibition has different therapeutic effects according to myeloproliferative neoplasm development in mice.

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.

JAK2V617F expression in mice amplifies early hematopoietic cells and gives them a competitive advantage that is hampered by IFNα.

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.

Kit-activating mutations cooperate with Spi-1/PU.1 overexpression to promote tumorigenic progression during erythroleukemia in mice.

The erythroleukemia developed by spi-1/PU.1 transgenic mice is a multistage process characterized by an early arrest of the proerythroblast differentiation followed later on by malignant transformation. Herein, we report the presence of acquired mutations in the SCF receptor gene (Kit) in 86% of tumors isolated during the late stage of the disease. Kit mutations affect codon 814 or 818. Ectopic expression of Kit mutants in nonmalignant proerythroblasts confers erythropoietin independence and tumorigenicity to cells. Using PP1, PP2, and imatinib mesylate, we show that Kit mutants are responsible for the autonomous expansion of malignant cells via Erk1/2 and PI3K/Akt activations. These findings represent a proof of principle for oncogenic cooperativity between one proliferative and one differentiation blocking event for the development of an overt leukemia.

SRSF2-P95H decreases JAK/STAT signaling in hematopoietic cells and delays myelofibrosis development in mice.

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.

Myeloproliferative neoplasm induced by constitutive expression of JAK2V617F in knock-in mice.

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.

A major role of TGF-beta1 in the homing capacities of murine hematopoietic stem cell/progenitors.

Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic cytokine with major in vitro effects on hematopoietic stem cells (HSCs) and lymphocyte development. Little is known about hematopoiesis from mice with constitutive TGF-beta1 inactivation largely because of important embryonic lethality and development of a lethal inflammatory disorder in TGF-beta1(-/-) pups, making these studies difficult. Here, we show that no sign of the inflammatory disorder was detectable in 8- to 10-day-old TGF-beta1(-/-) neonates as judged by both the number of T-activated and T-regulator cells in secondary lymphoid organs and the level of inflammatory cytokines in sera. After T-cell depletion, the inflammatory disease was not transplantable in recipient mice. Bone marrow cells from 8- to 10-day-old TGF-beta1(-/-) neonates showed strikingly impaired short- and long-term reconstitutive activity associated with a parallel decreased in vivo homing capacity of lineage negative (Lin(-)) cells. In addition an in vitro-reduced survival of immature progenitors (Lin(-) Kit(+) Sca(+)) was observed. Similar defects were found in liver cells from TGF-beta1(-/-) embryos on day 14 after vaginal plug. These data indicate that TGF-beta1 is a critical regulator for in vivo homeostasis of the HSCs, especially for their homing potential.

A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera.

Myeloproliferative disorders are clonal haematopoietic stem cell malignancies characterized by independency or hypersensitivity of haematopoietic progenitors to numerous cytokines. The molecular basis of most myeloproliferative disorders is unknown. On the basis of the model of chronic myeloid leukaemia, it is expected that a constitutive tyrosine kinase activity could be at the origin of these diseases. Polycythaemia vera is an acquired myeloproliferative disorder, characterized by the presence of polycythaemia diversely associated with thrombocytosis, leukocytosis and splenomegaly. Polycythaemia vera progenitors are hypersensitive to erythropoietin and other cytokines. Here, we describe a clonal and recurrent mutation in the JH2 pseudo-kinase domain of the Janus kinase 2 (JAK2) gene in most (> 80%) polycythaemia vera patients. The mutation, a valine-to-phenylalanine substitution at amino acid position 617, leads to constitutive tyrosine phosphorylation activity that promotes cytokine hypersensitivity and induces erythrocytosis in a mouse model. As this mutation is also found in other myeloproliferative disorders, this unique mutation will permit a new molecular classification of these disorders and novel therapeutical approaches.

Multistage hematopoietic stem cell regulation in the mouse: A combined biological and mathematical approach.

We have reconciled steady-state and stress hematopoiesis in a single mathematical model based on murine in vivo experiments and with a focus on hematopoietic stem and progenitor cells. A phenylhydrazine stress was first applied to mice. A reduced cell number in each progenitor compartment was evidenced during the next 7 days through a drastic level of differentiation without proliferation, followed by a huge proliferative response in all compartments including long-term hematopoietic stem cells, before a return to normal levels. Data analysis led to the addition to the 6-compartment model, of time-dependent regulation that depended indirectly on the compartment sizes. The resulting model was finely calibrated using a stochastic optimization algorithm and could reproduce biological data in silico when applied to different stress conditions (bleeding, chemotherapy, HSC depletion). In conclusion, our multi-step and time-dependent model of immature hematopoiesis provides new avenues to a better understanding of both normal and pathological hematopoiesis.

Adenoviral-mediated TGF-beta1 inhibition in a mouse model of myelofibrosis inhibit bone marrow fibrosis development.

Myelofibrosis is characterized by excessive deposits of extracellular matrix proteins, which occur as a marrow microenvironment reactive response to cytokines released from the clonal malignant myeloproliferation. The observation that mice exposed to high systemic levels of thrombopoietin (TPO) invariably developing myelofibrosis has allowed demonstration of the crucial role of transforming growth factor (TGF)-beta1 released by hematopoietic cells in the onset of myelofibrosis. The purpose of this study was to investigate whether TGF-beta1 inhibition could directly inhibit fibrosis development in a curative approach of this mice model. An adenovirus encoding for TGF-beta1 soluble receptor (TGF-beta-RII-Fc) was injected either shortly after transplantation (preventive) or 30 days post-transplantation (curative). Mice were transplanted with syngenic bone marrow cells transduced with a retrovirus encoding for murine TPO. All mice developed a myeloproliferative syndrome. TGF-beta-RII-Fc was detected in the blood of all treated mice, leading to a dramatic decrease in TGF-beta1 level. Histological analysis show that the two approaches (curative or preventive) were successful enough to inhibit bone marrow and spleen fibrosis development in this model. However, lethality of TPO overexpression was not decreased after treatment, indicating that in this mice model, myeloproliferation rather than fibrosis was probably responsible for the lethality induced by the disorder.

A miR-150/TET3 pathway regulates the generation of mouse and human non-classical monocyte subset.

Non-classical monocyte subsets may derive from classical monocyte differentiation and the proportion of each subset is tightly controlled. Deregulation of this repartition is observed in diverse human diseases, including chronic myelomonocytic leukemia (CMML) in which non-classical monocyte numbers are significantly decreased relative to healthy controls. Here, we identify a down-regulation of hsa-miR-150 through methylation of a lineage-specific promoter in CMML monocytes. Mir150 knock-out mice demonstrate a cell-autonomous defect in non-classical monocytes. Our pulldown experiments point to Ten-Eleven-Translocation-3 (TET3) mRNA as a hsa-miR-150 target in classical human monocytes. We show that Tet3 knockout mice generate an increased number of non-classical monocytes. Our results identify the miR-150/TET3 axis as being involved in the generation of non-classical monocytes.

HSP27 is a partner of JAK2-STAT5 and a potential therapeutic target in myelofibrosis.

Heat shock protein 27 (HSP27/HSPB1) is a stress-inducible chaperone that facilitates cancer development by its proliferative and anti-apoptotic functions. The OGX-427 antisense oligonucleotide against HSP27 has been reported to be beneficial against idiopathic pulmonary fibrosis. Here we show that OGX-427 is effective in two murine models of thrombopoietin- and JAKV617F-induced myelofibrosis. OGX-427 limits disease progression and is associated with a reduction in spleen weight, in megakaryocyte expansion and, for the JAKV617F model, in fibrosis. HSP27 regulates the proliferation of JAK2V617F-positive cells and interacts directly with JAK2/STAT5. We also show that its expression is increased in both CD34+ circulating progenitors and in the serum of patients with JAK2-dependent myeloproliferative neoplasms with fibrosis. Our data suggest that HSP27 plays a key role in the pathophysiology of myelofibrosis and represents a new potential therapeutic target for patients with myeloproliferative neoplasms.

Role for the nuclear factor kappaB pathway in transforming growth factor-beta1 production in idiopathic myelofibrosis: possible relationship with FK506 binding protein 51 overexpression.

The release of transforming growth factor-beta1 (TGF-beta1) in the bone marrow microenvironment is one of the main mechanisms leading to myelofibrosis in murine models and probably in the human idiopathic myelofibrosis (IMF). The regulation of TGF-beta1 synthesis is poorly known but seems regulated by nuclear factor kappaB (NF-kappaB). We previously described the overexpression of an immunophilin, FK506 binding protein 51 (FKBP51), in IMF megakaryocytes. Gel shift and gene assays show that FKBP51's overexpression in a factor-dependent hematopoietic cell line, induces a sustained NF-kappaB activation after cytokine deprivation. This activation correlates with a low level of IkappaBalpha. A spontaneous activation of NF-kappaB was also detected in proliferating megakaryocytes and in circulating CD34(+) patient cells. In normal cells, NF-kappaB activation was only detected after cytokine treatment. The expression of an NF-kappaB superrepressor in FKBP51 overexpressing cells and in derived megakaryocytes from CD34(+) of IMF patients revealed that NF-kappaB activation was not involved in the resistance to apoptosis after cytokine deprivation of these cells but in TGF-beta1 secretion. These results highlight the importance of NF-kappaB's activation in the fibrosis development of this disease. They also suggest that FKBP51's overexpression in IMF cells could play an important role in the pathogenesis of this myeloproliferative disorder.

LYL-1 deficiency induces a stress erythropoiesis.

OBJECTIVE: LYL-1 is a transcription factor containing a basic helix-loop-helix motif closely related to SCL/TAL-1, a regulator of erythroid differentiation. Because LYL-1 is expressed in erythroid cell populations, we addressed its role in erythropoiesis using knockin mice. MATERIALS AND METHODS: Erythropoiesis of LYL-1(-/-) mice was studied by progenitor assays, flow cytometry, reconstitution assays, and functional tests. Expression of LYL-1, SCL, and GATA-1 was assessed at messenger RNA level by quantitative reverse transcription polymerase chain reaction. RESULTS: LYL-1(-/-) mice displayed decreased erythropoiesis with a partial arrest in differentiation, and enhanced apoptosis associated with decreased Bcl-x(L) expression in the bone marrow (BM). In addition, LYL-1(-/-) BM cells were severely impaired in their abilities to reconstitute the erythroid lineage in competitive assays, suggesting a cell autonomous abnormality of erythropoiesis. In parallel, erythroid progenitor and precursor cells were significantly increased in the spleen of LYL-1(-/-) mice. Expression of LYL-1 was differentially regulated during maturation of erythroblasts and strikingly different between spleen- and BM-derived erythroblasts. Expression of LYL-1 decreased during erythroid differentiation in the spleen whereas it increased in the BM to reach the same level in mature erythroblasts as in the soleen. Loss of Lyl-1 expression was accompanied with an increase of SCL/TAL-1 and GATA-1 transcripts in spleen but not in BM-derived erythroblasts. Furthermore, phenylhydrazine-induced stress erythropoiesis was elevated in LYL-1(-/-) mice and mutant BM and spleen erythroid progenitors were hypersensitive to erythropoietin. CONCLUSIONS: Taken together, these results suggest that LYL-1 plays a definite role in erythropoiesis, albeit with different effects in BM specifically regulating basal erythropoiesis, and spleen, controlling stress-induced erythropoiesis.

Putative innate immunity of antiatherogenic paraoxanase-2 via STAT5 signal transduction in HIV-1 infection of hematopoietic TF-1 cells and in SCID-hu mice.

Paraoxanase-2 (PON2) activity was increased upon HIV-1 infection of the CD34+CD4+ hematopoietic cell line TF-1. Thymocytes derived from the human fetal conjoint thymus/liver hematopoietic organ of SCID-hu mice also exhibited an increase in PON2 activity. Additionally, a remarkable increase of PON2 mRNA expression was also observed in both TF-1 and thymocytes following HIV-1 infection. The phosphorylation of STAT5 was decreased in TF-1 cells upon HIV-1 infection. Interestingly, phosphorylation of STAT5 does not occur in GM-CSF "starved" TF-1 cells; however, PON2 protein, activity and mRNA expression are increased under these conditions, similar to HIV-1 infection. We conclude that PON2 is induced in HIV-1 infection through a mechanism that may involve STAT5 inactivation.

Ligand-independent thrombopoietin mutant receptor requires cell surface localization for endogenous activity.

The activating W515L mutation in the thrombopoietin receptor (MPL) has been identified in primary myelofibrosis and essential thrombocythemia. MPL belongs to a subset of the cytokine receptor superfamily that requires the JAK2 kinase for signaling. We examined whether the ligand-independent MPL(W515L) mutant could signal intracellularly. Addition of the endoplasmic reticulum (ER) retention KDEL sequence to the receptor C terminus efficiently locked MPL(W515L) within its natural ER/Golgi maturation pathway. In contrast to cells expressing the parental MPL(W515L), MPL(W515L)-KDEL-expressing FDC-P1 cells were unable to grow autonomously and to produce tumors in nude mice. When observed, tumor nodules resulted from in vivo selection of cells leaking the receptor at their surface. JAK2 co-immunoprecipitated with MPL(W515L)-KDEL but was not phosphorylated. We generated disulfide-bonded MPL(W515L) homodimers by the S402C substitution, both in the normal and KDEL context. Unlike MPL(W515L)-KDEL, MPL(W515L-S402C)-KDEL signaled constitutively and exhibited cell surface localization. These data establish that MPL(W515L) with appended JAK2 matures through the ER/Golgi system in an inactive conformation and suggest that the MPL(W515L)/JAK2 complex requires membrane localization for JAK2 phosphorylation, resulting in autonomous receptor signaling.

P19INK4D links endomitotic arrest and megakaryocyte maturation and is regulated by AML-1.

The molecular mechanisms that regulate megakaryocyte (MK) ploidization are poorly understood. Using MK differentiation from primary human CD34(+) cells, we observed that p19(INK4D) expression was increased both at the mRNA and protein levels during ploidization. p19(INK4D) knockdown led to a moderate increase (31.7% +/- 5%) in the mean ploidy of MKs suggesting a role of p19(INK4D) in the endomitotic arrest. This increase in ploidy was associated with a decrease in the more mature MK population (CD41(high)CD42(high)) at day 9 of culture, which was related to a delay in differentiation. Inversely, p19(INK4D) overexpression in CD34(+) cells resulted in a decrease in mean ploidy level associated with an increase in CD41 and CD42 expression in each ploidy class. Confirming these in vitro results, bone marrow MKs from p19(INK4D) KO mice exhibited an increase in mean ploidy level from 18.7N (+/- 0.58N) to 52.7N (+/- 12.3N). Chromatin immunoprecipitation assays performed in human MKs revealed that AML-1 binds in vivo the p19(INK4D) promoter. Moreover, AML-1 inhibition led to the p19(INK4D) down-regulation in human MKs. These results may explain the molecular link at the transcriptional level between the arrest of endomitosis and the acceleration of MK differentiation.

Proteasome inhibitor bortezomib impairs both myelofibrosis and osteosclerosis induced by high thrombopoietin levels in mice.

Primary myelofibrosis (PMF) is the most serious myeloproliferative disorder, characterized by clonal myeloproliferation associated with cytokine-mediated bone marrow stromal reaction including fibrosis and osteosclerosis. Current drug therapy remains mainly palliative. Because the NF-kappaB pathway is implicated in the abnormal release of cytokines in PMF, the proteasome inhibitor bortezomib might be a potential therapy. To test its effect, we used the lethal murine model of myelofibrosis induced by thrombopoietin (TPO) overexpression. In this TPO(high) model, the development of the disease is related to a deregulated MPL signaling, as recently described in PMF patients. We first demonstrated that bortezomib was able to inhibit TPO-induced NF-kappaB activation in vitro in murine megakaryocytes. It also inhibited NF-kappaB activation in vivo in TPO(high) mice leading to decreased IL-1alpha plasma levels. After 4 weeks of treatment, bortezomib decreased TGF-beta1 levels in marrow fluids and impaired marrow and spleen fibrosis development. After 12 weeks of treatment, bortezomib also impaired osteosclerosis development through osteoprotegerin inhibition. Moreover, this drug reduced myeloproliferation induced by high TPO level. Finally, bortezomib dramatically improved TPO(high) mouse survival (89% vs 8% at week 52). We conclude that bortezomib appears as a promising therapy for future treatment of PMF patients.