|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 1 July 2007, Vol. 110, No. 1, pp. 345-353. Prepublished online as a Blood First Edition Paper on March 20, 2007; DOI 10.1182/blood-2006-10-054502.
NEOPLASIA Proteasome inhibitor bortezomib impairs both myelofibrosis and osteosclerosis induced by high thrombopoietin levels in mice1 Institut National de la Santé et de la Recherche Médicale (INSERM), U790, Université Paris XI, Villejuif, France; 2 Institut Gustave Roussy, Villejuif, France; 3 Assistance Publique des Hôpitaux de Paris (AP-HP), Laboratoire d'Hématologie, Hôpital Henri Mondor, Université Paris XII, Créteil, France; 4 AP-HP, Laboratoire d'Anatomo-pathologie, Hôpital Cochin, Paris, France; 5 Université Paris XI, IFR54, Service Commun d'Expérimentation Animale, Institut Gustave Roussy, Villejuif, France
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-  B 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 TPOhigh 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-B activation in vitro in murine megakaryocytes. It also inhibited NF-B activation in vivo in TPOhigh mice leading to decreased IL-1 plasma levels. After 4 weeks of treatment, bortezomib decreased TGF-ß1 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 TPOhigh mouse survival (89% vs 8% at week 52). We conclude that bortezomib appears as a promising therapy for future treatment of PMF patients.
Primary myelofibrosis (PMF) is a myeloproliferative disorder1 known as a clonal stem-cell disorder, whereas the associated stromal reaction in the bone marrow environment, leading to fibrosis (excessive deposits of extracellular matrix proteins) and osteosclerosis (new bone formation), is considered to be reactive and cytokine mediated.2,3 Because the description of mice overexpressing thrombopoietin (TPO), known as TPOhigh mice,4 featuring numerous aspects of the human disease including dysmegakaryopoiesis, the implication of the TPO/MPL pathway in PMF has been demonstrated. Recently, two activating mutations of the TPO receptor MPL, MPLW515L and MPLW515K, have been detected in 5% of PMF patients and have been shown to induce fibrosis in mice.5,6 Activated MPL is known to stimulate the members of the Janus family of protein tyrosine kinases, JAKs. Indeed, the activating JAK2V617F mutation, directly linked to deregulated MPL signaling, is present in 50% of PMF patients7–10 and also induces fibrosis in mice.11,12 However, how these unique JAK2 or MPL mutations may lead to bone marrow fibrosis development is not yet understood. Notably, both mutations can be found in essential thrombocythemia,6–10 a myeloproliferative disorder without fibrosis. Thus, PMF is related to TPO/MPL pathway alterations and the TPOhigh model, mimicking deregulated MPL signaling, appears to be the most relevant to test drugs in vivo.
Several lines of evidence obtained from both studies of patients with PMF and of murine models ending with myelofibrosis are in favor of a crucial role (i) of the pleiotropic cytokine transforming growth factor ß1 (TGF-ß1),13 released by clonal proliferation of megakaryocytes or monocytes or both, in reticulin fiber deposition14–17; (ii) and of stroma-derived osteoprotegerin (OPG) in osteosclerosis development.17–19 The NF-
We therefore hypothesized that the NF-
Reagents and antibodies Bortezomib was obtained from Millennium Pharmaceuticals (Cambridge, MA). Stock bortezomib solution (0.5 mg/mL) was prepared in sodium chloride (NaCl) and stored at +4°C for up to a week prior to use. The stocks solutions were diluted in NaCl immediately before use.
Murine IL-3, murine IL-6, and murine stem cell factor (SCF) were purchased from R&D Systems (Oxon, United Kingdom). Recombinant human TPO (rhTPO) was kindly provided by Kirin Brewery (Tokyo, Japan), rhGM-CSF was a gift from Novartis (Basel, Switzerland), and TNF-
Polyclonal rabbit antibodies (Abs) against NF- The following rat monoclonal Abs were purchased from Pharmingen (San Diego, CA) and used for lineage-positive depletion: anti–Ly-6/GR-1 (RB6–8C5), anti-CD11b/MAC-1 (M1/70), anti-B220 (RA3–6B2), anti-CD4 (GK1.5), anti-CD8 (Lyt-1), and antierythroid TER-119. Mice and bone marrow transduction All procedures were approved by the local Institut Gustave Roussy (IGR) ethics committee. C57BL/6J mice (Janvier, Le Genest, France) were maintained at the IGR animal facility under specific pathogen-free conditions. Six- to 8-week-old male mice were used as bone marrow donors. Seven- to 10-week-old female mice were recipients. The infection was performed as previously described.4,19,23 Briefly, 4 days after 5-fluorouracil treatment (1 injection of 150 mg/kg administered intraperitoneally), total bone marrow cells were collected and cocultured with 106 MPZenTPO virus–producing GP+E-86 cells in Dulbecco modified Eagle medium (DMEM) supplemented with antibiotics, 20% fetal calf serum (FCS), murine IL-3 (100 U/mL), murine IL-6 (20 ng/mL), and murine SCF (20 ng/mL). After 4 days, nonadherent cells were harvested. An aliquot was immediately used in clonogenic progenitor assays to determine the percentage of infected colony-forming cells (CFCs) as previously described.4,19 CFCs were picked from methylcellulose and analyzed by polymerase chain reaction (PCR) with specific primers for the viral TPO gene and actin primers to ascertain the presence of material. The remaining cells were inoculated intravenously via the retro-orbital sinus into irradiated hosts (9.5 Gy, X-ray apparatus, single dose) in a ratio of one donor per one recipient. Three independent infection experiments were performed with a total of 80 engrafted hosts. The percentage of CFCs demonstrating the integrated TPO cDNA was comparable in the 3 experiments (95% ± 2%). Bortezomib administration One month after engraftment with TPO-overexpressing hematopoietic cells, 4 groups of 20 immunocompetent C57BL/6J mice displaying similar myeloproliferation (evaluated by similar platelet number) were constituted. Engrafted mice were treated intravenously twice a week, with either bortezomib in NaCl or vehicle (NaCl), for 4 to 12 weeks. Mice were weighed before each injection. Hematologic evaluation Blood from the orbital plexus was collected in citrated tubes at indicated times. Nucleated blood cells, hematocrit level, and platelet counts were determined using an automated blood counter calibrated for mouse blood (MS9; Melet Schloessing, Cergy-Pontoise, France). Differential cell counts were performed after May-Grünwald-Giemsa staining.
Platelet-poor plasma was used for determination of TPO, IL-1 Fresh spleen and blood cells (105) were grown in semisolid medium CFCs analyzed as previously described.4,19 Extracellular fluids of bone marrow were prepared by flushing 1 femur and 1 tibia freshly excised with 700 µL DMEM supplemented with 10% heat-inactivated FCS and antibiotics. Histopathology For histologic analysis, bones were excised and cleaned of soft tissue. One femur and 1 tibia were fixed in formaldehyde, decalcified, and paraffin embedded. Spleen, liver, and pulmonary samples were fixed and embedded in the same manner. Sections (4.5 µm) were stained with hematoxylin eosin, periodic acid Schiff, and Giemsa for overall cytology analysis. Reticulin fibers were revealed by silver staining according to Gordon Sweet method. Images were obtained using a Leica DMRB microscope (Leica, Solms, Germany) with 25x/0.85 NA (magnification x250), with 10x/0.3 NA (magnification x100) and with 2.5x/0.075 NA (magnification x25) objectives, and acquired with a Video 3 charge-coupled device (CCD) Sony Leica Power hole accumulated diode (HAD) camera (Sony, Tokyo, Japan). Determination of chimerism Fluorescent in situ hybridization (FISH) analysis of Y chromosome was performed on bone marrow cells from mice killed 8 weeks after engraftment as previously described.23 Enzyme-linked immunosorbent assay (ELISA)
TPO, IL-1 Cell culture Human factor-dependent cell line UT7/c-MPL (clone 11OC1) was maintained in DMEM supplemented with 10% heat-inactivated FCS, antibiotics, and 5 ng/mL of rhGM-CSF. Murine megakaryocytes were derived from fetal livers of 14-day-old embryos. Single-cell suspensions were enriched for progenitor cells (Lin– fraction) by immunomagnetic selection (Dynabeads M-450; Dynal AS, Oslo, Norway) using lineage-specific Abs. The Lin– fraction was grown for 3 to 5 days as previously described.26
Adherent murine stromal cells were cultured as initially described by Dexter et al.27 Briefly, total bone marrow cells from one femur and one tibia were cultured in Immunofluorescence and Western-blot analysis
For in vitro analysis, UT7/c-mpl cells and murine megakaryocytes (4-day-old) were cytokine deprived for 16 hours in DMEM supplemented with 1% FCS or IMDM supplemented with 3% FCS, respectively. For stimulation, TNF- For in vivo analysis, fresh spleen cells from mice were dissociated and nuclear extracts were prepared and analyzed by Western blotting with the adequate Abs as previously described.28 Statistical analysis Differences between data groups were evaluated for significance using the Wilcoxon test. A P value of less than .05 was considered significant. The data are presented as the mean plus or minus the standard error of the mean (±SEM). Dose effect was evaluated using an analysis of variance of the linear model. A P(DE) value of less than .05 was considered significant. Survival was analyzed using the log-rank test.
Bortezomib inhibits TPO-induced NF- B activation in vitro in UT7/c-MPL cells and in murine megakaryocytes
Bortezomib has been demonstrated, both in vitro and in vivo, to block degradation of I
We first confirmed that TPO was able to activate the NF-
Bortezomib inhibits NF- B activation in vivo in TPOhigh mice
We assessed whether NF- Four weeks after engraftment with the transduced hematopoietic cells, 4 groups of 20 mice each displaying similar TPO-induced myeloproliferation (evaluated by similar platelet number) were constituted. Engrafted mice were treated intravenously twice a week, with either bortezomib in NaCl (1 mg/kg, 0.5 mg/kg, and 0.25 mg/kg) or vehicle (NaCl), for 4 to 12 weeks. Plasma levels of TPO were monitored over time using an ELISA. Four weeks after engraftment, the TPO concentration in plasma was more than 1000-fold higher in engrafted mice than in the controls. No difference between treated and untreated TPOhigh mice was observed at week 8 (Figure 1E) and at week 16 (data not shown). Chimerism was analyzed by FISH on Y chromosome (bone marrow donors were male, recipients were female) on whole nucleated bone marrow cells. Chimerism levels were more than 90% and were similar in both treated and untreated mice (data not shown).
We studied what bortezomib would effect on NF- The most common side effect of bortezomib treatment in mice is weight loss; therefore, treatment was adapted to each mouse weight, and their weights were closely monitored throughout the follow-up. A bortezomib dose of 1 mg/kg led to the death of 50% of the animals within 4 weeks accompanied with statistically significant weight loss. Surviving mice were nevertheless analyzed. This toxicity of bortezomib at 1 mg/kg was higher than previously reported and may be related to the total body irradiation regimen that preceded the bortezomib administration. We then chose to repeat experiments with lower doses of bortezomib. Mice treated with 0.5 mg/kg and 0.25 mg/kg doses lost weight rapidly after the first bortezomib injections but progressively recovered their initial weights over time. Bortezomib reduces myeloproliferation induced by high TPO levels We then assessed the effects of bortezomib treatment on the TPOhigh myeloproliferative syndrome. We studied blood and spleen parameters (Figure 2). Leukocytosis (Figure 2A) and thrombocytosis (Figure 2B) displayed by TPOhigh mice were decreased in treated mice in a statistically significant dose-dependent fashion. Conversely, bortezomib did not significantly improve anemia of TPOhigh mice (Figure 2C).
The number of progenitor cells in the spleen and the blood was studied 8 weeks after engraftment. The increase of blood-circulating CFCs displayed by the TPOhigh mice was dramatically reduced by bortezomib treatment in a statistically significant dose-dependent manner (Figure 2D). Moreover, the high number of spleen CFCs was also impaired by bortezomib treatment (Figure 2E), in parallel to a dramatic decrease in the splenomegaly displayed by the TPOhigh mice (Figure 2F). Surprisingly, our data provide evidence that bortezomib is able to reduce the TPOhigh myeloproliferative syndrome in mice. Bortezomib impairs marrow and spleen fibrosis development induced by high TPO levels through TGF-ß1 inhibition The development of fibrosis is characterized by the excessive deposits of extracellular matrix proteins. Bone marrow fibrosis has been reported to be a direct consequence of high TGF-ß1 levels in blood and bone marrow fluids in the TPOhigh model.16 Furthermore, to exert its biologic effects, TGF-ß1 has to be activated at secretion sites within the hematopoietic environment. The mechanisms responsible for TGF-ß1 activation remain unclear. Total and active forms of TGF-ß1 were measured in treated and untreated mice (Figure 3A-C). As previously reported,16 the level of TGF-ß1 in the plasma increased as early as 4 weeks after engraftment and reached a level 4 times higher than in control mice by week 8 (Figure 3A). Moreover, 8 weeks after engraftment, we observed an augmentation in total TGF-ß1 levels in extracellular fluids of marrow in TPOhigh compared with control mice (Figure 3B). As expected, the active form of TGF-ß1 was detected only in engrafted mice and was absent in control mice (Figure 3C). Bortezomib treatment significantly decreased total TGF-ß1 plasma levels (Figure 3A) and both the total (Figure 3B) and active (Figure 3C) forms of TGF-ß1 in extracellular fluids of marrow in a statistically significant dose-dependent manner.
Three mice of each group were killed after 4 weeks of treatment (8 weeks after engraftment). Macroscopic examination of the femora of control mice, excised and cleaned of soft tissue, appeared dark red, full of marrow cells (Figure 3D left). In contrast, fibrotic bones from untreated TPOhigh mice appeared white (Figure 3D middle). We observed that the femora from treated TPOhigh mice (bortezomib 0.5 mg/kg) seemed to be more pinkish (Figure 3D right), suggesting fewer deposits of extracellular matrix proteins. As expected, histologic sections of femora and spleens of TPOhigh mice revealed a massive hyperplasia of dysmorphic megakaryocytes and granulocytic cells in both treated and untreated mice (Figure 3E,G,I,K). Silver impregnation, used to examine the degree of fibrosis, showed densification of the reticulin network with deposition surrounding megakaryocytes in untreated mice (Figure 3F bone marrow; Figure 3J spleen). In contrast, mice treated with bortezomib (1 mg/kg) displayed impaired reticulin fibers in both the bone marrow (Figure 3H) and the spleen (Figure 3L), confirming the macroscopic observation of their femora. Lower doses of bortezomib (0.5 and 0.25 mg/kg) have much fewer effects (data not shown). Bortezomib impairs osteosclerosis development induced by high TPO levels through OPG inhibition
Osteosclerosis is another stromal change displayed by TPOhigh mice. It has been demonstrated that OPG secreted by the bone marrow microenvironment, more precisely by the stromal cells, is required for this abnormal bone growth.19 The mechanism leading to stromal OPG up-regulation remains unknown but it is not due to the increased TGF-ß1 levels in TPOhigh mice.19 However, these mice display high plasma levels of IL-1
Osteosclerosis development, which occurs later than bone marrow fibrosis, was assessed 16 weeks after engraftment. Therefore, 3 mice of each group were killed after 12 weeks of treatment. As expected, histologic sections of femora from untreated TPOhigh mice revealed a dense new bone formation, almost filling the medullar cavity (Figure 4E-F). Since OPG levels were decreased in bone marrow extracellular fluids of 0.5 mg/kg bortezomib–treated TPOhigh mice, a rare bone growth was observed in femora collected from these mice (Figure 4G-H). A minimal dose of 0.25 mg/kg also dramatically reduced osteosclerosis development (data not shown). Bortezomib dramatically improves survival in TPOhigh mice
The TPOhigh model is known to induce a severe myeloproliferative disorder that mimics the evolution of the MMM disease in humans, leading to the death of all animals within 10 months after TPO-infected bone marrow cell engraftment.4 We assessed the effect of bortezomib treatment on TPOhigh mice survival after 12 weeks of treatment (from 16 to 52 weeks after engraftment). As TPOhigh mice invariably die with severe anemia,4 we decided not to bleed them anymore to avoid artificial death. Therefore, we observed a delayed lethality (up to 12 months) in untreated (vehicle) TPOhigh mice (Figure 5) compared with the survival curve initially described.4 Bortezomib (0.5 mg/kg) significantly improved survival in TPOhigh mice (89% vs 8% at week 52; P
We show here that bortezomib reduces the myeloproliferative disorder, impairs both bone marrow fibrosis and osteosclerosis development, and dramatically improves survival in a murine model mimicking PMF. This TPOhigh model, induced by systemic TPO overexpression, is one of the two extensively studied experimental models of myelofibrosis in mice, together with the GATA-1low model.32 In the latter model, the knockout mice specifically express a low amount of the transcriptional factor GATA-1 in the megakaryocyte lineage. They display a megakaryocyte hyperplasia in the bone marrow and the spleen, like the TPOhigh mice. Myeloproliferation observed in both models does not originate from a clonal malignant event, as is the case in the human disease,2,3 but leads to similar stromal changes including bone marrow fibrosis and osteosclerosis. No alteration in either the structure or the expression of GATA-133 or its cofactor FOG-134 genes has yet been described in PMF. Conversely, three activating mutations implicating the TPO/MPL pathway (JAK2V617F, MPLW515L, and MPLW515K) have recently been identified.5–10 JAK2V617F and MPLW515L induce fibrosis in mice.5,11,12 However, JAK2V617F mice display a disease mimicking more polycythemia vera progressing to bone marrow fibrosis than PMF.11,12 MPLW515L mice develop a myeloproliferative disorder, fatal within 30 days after engraftment.5 In fact, this model is not really relevant, since it does not behave like a slowly evolving disorder such as PMF. Indeed, the activating MPL is transduced in total bone marrow cells, regardless of the hematopoietic lineage. On the contrary, in the TPOhigh model, only hematopoietic cells physiologically expressing MPL respond to TPO overexpression, mimicking deregulated MPL signaling in PMF cells. Thereby, the TPOhigh model appears more relevant to human PMF than either the MPLW515L or the JAK2V617F model. Moreover, TPOhigh mice develop a myeloproliferative disorder with associated myelofibrosis and osteosclerosis and all the engrafted animals display a delayed death, like the natural evolution of human PMF. Therefore the TPOhigh model is an interesting in vivo model for testing candidate drugs.
In this study, we assessed the effects of the proteasome inhibitor bortezomib in the stromal changes displayed by the TPOhigh mice. Because the minimal dose of 1 mg/kg recommended by Millennium Pharmaceuticals for in vivo studies led to the death of all our irradiated and engrafted mice, we successfully decreased the dose to 0.5 and 0.25 mg/kg. Bortezomib impaired bone marrow fibrosis development through inhibition of TGF-ß1 in a dose-dependent fashion (the minimal dose 0.25 mg/kg had few effects), as well as the myeloid proliferation. Conversely, bortezomib considerably reduced osteosclerosis development through OPG inhibition, regardless of dosage. It also dramatically improved TPOhigh mice survival but not at the minimal dose of 0.25 mg/kg. Thus, bortezomib seems to have dissociated effects on fibrosis and osteosclerosis development induced by TPO overexpression. Even a lower dose of0.25 mg/kg dramatically reduced OPG production and osteosclerosis without significantly improving mice survival. Hence, osteosclerosis does not seem to be a major determinant in TPOhigh mouse lethality. In addition, our results shed light on osteosclerosis pathogenesis. Indeed, we have demonstrated that IL-1
Furthermore, reduction of reticulin fiber deposition appears to be dose dependent and paralleled the decreased in TGF-ß1 secretion. TGF-ß1 production seems to be directly related to the megakaryocyte/platelet compartment. In TPOhigh mice, a 2- to 4-fold increase in the TGF-ß1 plasma level has been described with a similar augmentation in circulating platelet number. Therefore, only a slight increase in TGF-ß1 transcript was found in TPOhigh platelets, unlike the IL-1 Altogether, our results allow us to propose a physiopathologic model for the stromal reaction in TPOhigh mice, probably recapitulating bone marrow changes observed in the human disease (Figure 6).
Here, we used bortezomib to target the NF- B signaling pathway.24 However, this molecule has much broader effects and can interfere with synthesis of numerous cytokines, especially at the level of bone marrow environment. Anderson's group (Roccaro et al44) has shown that bortezomib has antiangiogenic effects in multiple myeloma. In TPOhigh mice, there are some abnormalities of angiogenesis.45 PMF patients display a similar increase in bone marrow microvessel density, and neo-angiogenesis appears to be a component of the bone marrow stromal reaction in PMF.46 Thus we cannot exclude that part of bortezomib effects may also be related to an inhibition of neo-angiogenesis.
Moreover, the sole inhibition of the NF- In summary, on one hand, this study has improved our understanding of the pathogenesis of stromal reaction induced by TPO overexpression, which may be extended to the human disease; on the other hand, nowadays only allogeneic hematopoietic stem-cell transplantation might be curative but remains inappropriate for most of the PMF patients. Therefore, bortezomib might be a promising drug for the treatment of human PMF as presented in the TPOhigh model.
Contribution: O.W.B. performed cell-culture studies, animal studies, ELISA, FISH, and immunofluorescence; generated figures; helped design the study; and wrote the manuscript. D.F.P. performed Western blot analysis and helped generate figures and write the manuscript. T.G. performed animal studies and ELISA. M.T. performed histologic analysis. R.C. performed cell-culture and Western blot analysis. C.L. performed bortezomib injections. F.A. performed murine megakaryocytes cultures and helped generate figures. J.-L.V. provided MPZenTPO virus–producing GP+E86 cells. P.G. performed statistical analysis. W.V. and S.G. designed studies, analyzed data, and drafted and edited the manuscript. D.F.P. and T.G. contributed equally to this work. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Stéphane Giraudier, INSERM U790, Institut Gustave Roussy, PR1, 39 rue Camille Desmoulins, 94805 Villejuif, France; e-mail: sgiraudi{at}igr.fr or stephane.giraudier{at}hmn.aphp.fr.
This work was supported by grants from Institut National de la Santé et la Recherche Médicale (INSERM) and La Ligue Nationale contre le Cancer (équipe labellisée 2004 and 2007). O.W.B. was supported by a fellowship from INSERM. T.G. was supported by La Ligue Nationale contre le Cancer. D.F.P. was supported by Institut Gustave Roussy. We are grateful to Annie Rouchès and Patrice Ardouin for managing the animals and to Caroline Lefebvre and Caroline Marty for improving the English manuscript. We thank Anna-Lila Kaushik and Sébastien Giroux for providing embryos.
Submitted October 26, 2006; accepted March 16, 2007.
Prepublished online as Blood First Edition Paper, March 20, 2007
DOI: 10.1182/blood-2006-10-054502
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
.001). Results are presented as the mean plus or minus the SEM of 12 animals per experimental group (except for the "1 mg/kg" group that included only 6 surviving mice at week 8). Results of statistical analysis with the Wilcoxon test are as follows: treated (bortezomib 0.25 mg/kg, 0.5 mg/kg, and 1 mg/kg) versus untreated (Vehicle) mice; *P < .05. Bzb indicates bortezomib.
