73 Animal Models of Myelofibrosis - Springer

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that, either incidentally discovered or not (Table 73–1), have .... Early and progressive osteopetrosis, splenomegaly, and extramedullary hematopoiesis73.




(BM) structural changes that include fibrosis, neoangiogenesis, and osteosclerosis, and by constitutive circulation of stem/progenitor cells with dacryocytes, nucleated erythroid cells, and immature myeloid cells in the peripheral blood (PB); in some cases, MMM represents the disease evolution of a preexisting PV or ET (so called, post-polycythemic/thrombocythemic MMM), and has an intrinsic tendency to evolve into acute leukemia.1 Accumulated evidence indicates that the changes in the structure of the BM that are characteristic of MMM represent a typical reactive process (the cells of the stroma do not belong to the neoplastic clone), likely mediated by cytokines abnormally released by clonal hematopoietic cells. On the other hand, the primary lesion occurring in the hematopoietic stem/progenitor cell of MMM is still not well understood, although recently an acquired point mutation in the JAK2 gene, consisting of a valineto-phenylalanine substitution (JAK2V617F) in the autoinhibitory JH2 (JAK homology) domain of the molecule, has been found to occur in most PV patients and in about half of those with ET or MMM.5–8 An even more infrequent point mutation of the TPO receptor, MPLW515L, has been detected in less than 10% of MMM patients.9,10 Although both these mutations when expressed in murine models recapitulate several aspects of MMM (see below), their exact positioning in the sequence of a multistep series of events forming the basis of the pathogenesis of the disease remains to be defined. A frequent observation in MMM patients has been the occurrence of the discrete involvement of the Mk lineage in BM biopsies11; actually, the morphological appearance of the BM Mk population, beyond its quantitative expansion, is one of the key points in the WHO classification, allowing differential diagnosis of MMM from other MPDs, as well as the identification of a “prefibrotic” form of IM.12 This critical role of Mks has been corroborated by the study of animal models of the disease that, either incidentally discovered or not (Table 73–1), have been consistently characterized by the accumulation of Mks eventually associated with thrombocytosis. This chapter will review the production and characterization of two extensively studied models of MMM, represented by TPOhigh and GATA-1low mice, together with the newest findings concerning the development of IM in mice carrying hematopoietic cells mutated at the JAK2 or MPL locus.

The pathogenesis of idiopathic myelofibrosis has not yet been completely elucidated, but the availability of animal models of the disease has made it possible, in the past few years, to focus on the cells (megakaryocytes) and cytokines (transforming growth factor-β, among others) potentially involved. This review will deal with the two best characterized animal models (the TPO-overexpressing and the GATA-1low mouse) as well as with the newest ones that have been developed following the identification of the JAK2V617F and MPLW515K/L mutations in the human form of the disease. Each of these unique animal models conceivably represents an invaluable tool for further characterizing the disease mechanisms as well as for testing novel therapeutic agents. Key Words: Myelofibrosis, Mpl, GATA-1, Animal models, Transforming growth factor-β, Megakaryocyte, Osteosclerosis.

INTRODUCTION Idiopathic myelofibrosis (IM), also called myelofibrosis with myeloid metaplasia (MMM),1 is a malignant hematological disorders that together with polycythemia vera (PV), essential thrombocythemia (ET), and other less frequent entities belongs to the Philadelphia-negative chronic myeloproliferative disorders (MPD), according to the World Health Organization (WHO) classification of myeloid neoplasms.2 All these are clonal disorders that originate at the level of a pluripotent hematopoietic stem/progenitor cell that, while retaining a relatively normal differentiation program unlike that in acute leukemia, has lost some of the regulatory mechanisms that control proliferation; indeed, one of the best characterized abnormalities of hematopoietic cells in these disorders is their substantial independence from regulatory cytokines, in particular erythropoietin (EPO)3 or thrombopoietin (TPO),4 the two main cytokines for the erythroid and megakaryocytic (Mk) lineage, respectively. MMM is characterized by anemia, extramedullary hematopoiesis, bone marrow

From: Sourcebook of Models for Biomedical Research (P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.




Table 73–1 Additional murine models presenting myelofibrosisa Model

Main features

MPSV-induced mice

MPSV; splenic and medullary fibrosis; splenic myeloid hyperplasia; myelemia; anemia; thrombocytopenia; possibly due to high levels of IL-1a, IL-6, GM-CSF, G-CSF, CSF-1, and TNF-α62–64 Overproduction of mature neutrophils and dysplastic megakaryocytes in the bone marrow; splenomegaly with extramedullary hematopoiesis; high levels of IL-3 and GM-CSF65–67 Extramedullary hematopoiesis, intensive myeloproliferation in the bone marrow and the spleen with splenomegaly, granulocytic leukocytosis; high levels of IL-3 and GM-CSF66,68 Transgenic mice bearing human BACH1 cDNA under the control of the GATA1 locus; significant thrombocytopenia associated with impaired maturation of megakaryocytes and development of myelofibrosis69 Extensive osteoclast deficiency, resulting in bone marrow fibrosis and extramedullary hematopoiesis70,71 Developed in DBA/2 mice engrafted with FDC-P1 cells overexpressing LIF; myelosclerosis, splenomegaly, extramedullary hematopoiesis in the liver, neutrophil leukocytosis72 Early and progressive osteopetrosis, splenomegaly, and extramedullary hematopoiesis73 Splenomegaly, extramedullary megakaryocytopoiesis; increased megakaryocytic, and decreased erythroid, progenitor content in the bone marrow74 Myelofibrosis, splenomegaly, extramedullary hematopoiesis, leukocytosis with a few immature myeloid cells; myeloproliferative disease has a latency of 40–60 days and does not evolve into AML75 Splenomegaly with fibrosis and extramedullary hematopoiesis, hyperplasia of the megakaryocytic lineage, increased number of erythroid cells in the spleen but decreased in bone marrow; defects of B lymphopoiesis; leukocytosis and thrombocytosis; increased proliferative responses of hematopoietic progenitors to cytokines such as SCF, IL-3, and IL-776

TEL/PDGFRβR transgenic mice TEL/JAK2 and TEL/TRKC transgenic mice Bach1 transgenic mice

VEGFR-1 transgenic mice (op/opFlt1TK−/−) LIF overexpressing mice OPG/OCIF transgenic mice BUBR1 mutant mice (BUBR+/−) FLT3-ITD mutant mice Lnk mutant mice (Lnk−/−)

a MPSV, myeloproliferative sarcoma virus; IL, interleukin; GM-CSF, granulocyte-macrophage colony-stimulating factor; TNF, tumor necrosis factor; LIF, leukemia inhibitory factor; AML, acute myeloid leukemia; SCF, stem cell factor.

THE TPOhigh MODELS TPO, also known as MPL ligand or megakaryocyte growth and development factor (MGDF), is the major physiological humoral regulator of megakaryopoiesis and platelet production.13,14 The TPOhigh model of myelofibrosis was discovered during investigations aimed to determine pathologies and side effects associated with long-term exposure to high doses of the recently discovered TPO, an obvious drug to increase platelet levels in patients. Several models are described, including the simplest approach by systemic repeated administration of a truncated version of recombinant TPO associated with a polyethylene glycol tail (PEG-rhMDGF)15 and more sophisticated approaches by which TPO overexpression was achieved by adenovirus vectors,16–19 retrovirus vectors,20,21 or by transgenesis.22,23 Except for one model of transgenic mice,22 all the other models described the occurrence of BM fibrosis in mice. We will mainly focus on the retroviral models and adenoviral models that have been studied extensively. TPOhigh MODEL BY SYSTEMIC REPEATED ADMINISTRATION OF PEG-RH-MDGF Immunocompetent BALB/c mice were injected daily for 14 days with increasing doses of PEG-rhMDGF from 10 to 500 µg/kg.15 All animals developed dosedependent thrombocytosis, moderate leukocytosis, anemia, splenomegaly, and extramedullary hematopoiesis. A BM fibrosis was observed at the highest dose injected, which reversed rapidly when the injections were stopped. Transient myelofibrosis was

also observed in rats receiving PEG-rh-MDGF.24 The effects of systemic administration of TPO in the GATA-1low model of myelofibrosis will be described below.25 TPOhigh MODELS BY ADENOVIRAL VECTORS An alternative approach was developed using replication-deficient adenovirus vectors. All the studies were performed using injections of adenovirus carrying a human TPO cDNA. However, the promoter driving the expression of TPO was either a cytomegalovirus (CMV) or a Rous sarcoma virus (RSV) promoter. Furthermore, several routes of injection were used (subcutaneous,16 intratracheal,17 intraperitoneal,19 and intravenous18). Finally, different stains of mice were treated: immunocompetent BALB/c mice and immunodeficient nonobese diabetic/severe combined immunodeficient (NOD/SCID) and SCID mice. Immunocompetent mice did not develop fibrosis because an immune thrombocytopenia occurred after a brief thrombocytosis that peaked at day 7, accompanied by Mk hyperplasia in the spleen. This immune reaction was due to the generation of neutralizing antibodies directed against the transgene human TPO that cross-reacted with murine TPO.26 To avoid this reaction, models were developed in immunodeficient mice.16,17,19,26 SCID (severe combined immunodeficient; T and B cell deficient) mice developed thrombocytosis, leukocytosis, and marked Mk hyperplasia in the spleen and BM with a progressive myelofibrosis and osteosclerosis as early as 6 weeks after injection. Surprisingly, no fibrotic or sclerotic changes were seen in NOD/SCID [T and B cell deficient as well as presenting multiple other defects in innate immunity including natural


Figure 73–1. A representation of the procedure used for the adoptive transfer of bone marrow cells infected by a retrovirus expressing the gene of interest (TPO, JAK2V617F). BMT, bone marrow transplantation; 5FU, 5-fluorouracil.

5FU treatment

715 Lethal irradiation

Infection 4-5 days

Lethal irradiation





(3-5 days) BM exposed to virus supernatant Primary recipients Seconday recipients or virus producing cells Disease phenotype in the presence of cytokines

Generation of the retrovirus producing cells Gene of interest

Packaging constructs (containing gag, pol, env)

MESV LTR (promoter)

Packaging cell line (GP+E86, 293T, ...)

killer (NK) and monocyte–macrophage function] mice.26 Because NOD/SCID mice differ from SCID mice by impaired mononuclear phagocyte functions, it was postulated that monocytes and macrophages are required for promotion of myelofibrosis in TPOhigh mice. However, this theory is not supported by another recent study demonstrating that NOD/SCID mice exposed to high and sustained TPO levels develop BM fibrosis.27 TPOhigh MODELS BY RETROVIRAL VECTORS Two models were described, one by Yan et al.20 and one by our group.21 In both investigations, the murine TPO cDNA was cloned into a retroviral vector and infectious nonreplicative recombinant retroviral particles were produced from the transfected GP + E86 packaging cell line. A high virus titer-producing clone was selected. In our study, this clone, called GP122, also produced very high TPO level (6000 U/ml).21 The principle consists of transducing hematopoietic stem cells (HSC) of a mouse with the virus in order to sustain high levels of TPO production by the hematopoietic system throughout the life of a mouse. The procedure is based on BM transplantation (BMT). BM cells, collected from donor mice, are infected with the retrovirus and injected into recipient mice depleted of their own hematopoietic system as a result of lethal irradiation (9.5 Gy for the C57BL/6 strain). To transduce HSC efficiently, which will ensure the lifetime maintenance of a transduced progeny, donor mice are treated with 5fluorouracil (5FU, a single intraperitoneal or intravenous injection at 150 mg/kg for 4 days). The duration of treatment allows the maximal number of HSC to be infected by the retrovirus, corresponding to the maximal number of cycling HSC. The infection is carried out by a coculture of the 5FU-treated cells (two femurs) with the virus-producing cells (105 cells in a 10-cm tissue culture Petri dish) for 4–5 days. The number of adherent virus-producing cells is calculated in order to obtain a confluent culture at the day of BM cell collection. The infection is carried out in the presence of cytokines able to induce HSC proliferation and survival. We used a spleen cell-conditioned medium, but this can be replaced with a mixture of early-acting cytokines such as interleukin 3 (IL-3), stem cell factor (SCF), and IL-6 (TPO being secreted by the virus-producing cells).20 The infected BM cells are collected as the nonadherent cells, and care should be taken not to detach too many virus-producing cells, which will be detrimental to the mice during the injection (these cells, derived from Swiss mice,

GFP Reporter gene (eGFP, NeoR, ...)

Construction of the retroviral vector

will be rejected by the immunocompetent C57BL/6J recipient mice). In the study by Yan et al., the infection was not carried by coculture but by serial exposures of the 5FU-treated cells to a virus-producing cell supernatant.20 Lethally irradiated syngeneic mice (2 months old) are finally injected with 106 BM cells (Figure 73–1). The mice are not studied before full blood reconstitution (5 weeks for myeloid and B cells and 7 weeks for T cells). The result of this procedure is the genomic integration of the virus in HSC and their progeny that could be analyzed by Southern blot to evaluate the numbers of both provirus copies, compared to the endogenous gene, and of individual transduced HSC marked by the sites of provirus integration. Provirus, as efficient vectors of expression, produced high level of TPO under the control of the viral long terminal repeat (LTR) promoter. Using this strategy, we obtained a two-step disease that mimicked the evolution of human MMM and resulted in the death of 100% of the animals within 10 months after transplantation.21 The first phase of the disease (2 months after BMT) is characterized by thrombocytosis (a 4-fold increase), leukocytosis (up to a 10fold increase), and decrease of the hematocrit. Giant platelets were observed in PB smears. A striking elevation in the PB, BM and spleen progenitor cell numbers, except the colony-forming unit (CFU)-E, was noticed. Massive hyperplasia of Mks, often organized in clusters, and of granulocytes was observed in BM and spleen with a hypoplasia of erythroblasts in the BM. From the third month post-BMT, platelets, leukocytes, and red cell numbers dropped dramatically and mice developed a severe pancytopenia. Progenitor cell numbers dropped in BM and spleen but increased in the PB and peritoneal cavity. Extramedullary hematopoiesis was observed in liver, kidney, lymph nodes, and lung. Histological examination revealed severe fibrosis in the BM and spleen and osteosclerosis in BM. Notably, 2 mice out of 32 primary recipients and 1 mouse out of 30 secondary recipients died prematurely from undifferentiated acute leukemia. Thus, this fatal myeloproliferative disorder mimicked most of the features observed in the human disease. Yan et al. reported a less dramatic phenotype.20 The platelet counts increased 5-fold and remained at a steady level for up to 73 weeks. A moderate anemia was observed and a slight increase of the number of PB nucleated cells was described. Mice developed extramedullary hematopoiesis and splenomegaly with



hyperplasia of Mks and granulocytes. BM was also obturated with a dense network of reticulin fibers and new bone trabeculae. However, mice did not display the second phase of our disease model characterized by severe thrombocytopenia, anemia, and leukopenia. They remained alive and active over a year and no leukemic transformation was described. The difference may be related to the plasma level of TPO; in the study by Yan et al.,20 plasma TPO reached 30 ng/ml compared to up to 1000 ng/ml in our study.21 TPOhigh MODELS BY TRANSGENESIS Two models of transgenic mice with contradictory results were developed. In the first one,22 transgenic mice overexpressing human TPO under the control of a liver-specific apolipoprotein E enhancer/promoter did not display fibrosis despite elevated plasma TPO levels (3 ng/ml), and a 4-fold increase in platelet counts and in tissue Mk content for more than 16 months. In contrast, in another recent transgenic model,23 expressing full-length murine TPO under the control of the mouse H chain enhancer/promoter, similar mild leukocytosis (2-fold), anemia (a 10% decrease in hematocrit), thrombocytosis (3-fold), and increase of plasma TPO level (3 ng/ml) were associated with BM fibrosis starting at 9 months with further progression at 12 months of age. Local production of TPO in hematopoietic tissues in the second model, in contrast to the first one, might have been the reason for the occurrence of fibrosis.23 All these TPOhigh models showed that TPO overexpression in mice leads to a myeloproliferative disorder featuring most aspects of MMM: leukocytosis, anemia, thrombocytosis, and in some cases pancytopenia, splenomegaly, extramedullary hematopoiesis, fibrosis, and osteosclerosis. Depending on the approach used, the severity of the disease (pancytopenia, leukemia, massive splenomegaly, fibrosis, and osteosclerosis) seems to be linked to the levels of TPO, the duration of exposure, as well as to the site of TPO production.28

THE GATA-1low MODEL GATA-1 is a transcription factor, encoded by a gene located on the X chromosome, that plays essential roles in the development of the erythroid,29 megakaryocytic,30 eosinophilic,31 and mast cell32 lineages, and that has been clarified by the phenotype of mice harboring experimentally induced alterations of its expression. Animal knockouts for the gene die in utero at day E10.5






because of severe anemia,33 while those expressing ectopic levels of Gata1 showed an increased rate of erythroid differentiation.34 GATA-1 is member of the family of GATA transcription factors that controls the expression of target genes by binding to the consensus WGATAR sequence; since they share a highly conserved structure, the cellular functions of GATA-1 are strictly dependent on the appropriate lineage and on the differentiation stage of activation.35 As a result of studies performed in the laboratory of Dr. Stuart Horkin in Boston, concerning the role of the region upstream to Gata1 in regulating gene expression in erythroid and Mk differentiation, McDevitt et al. generated a strain of mice (Gata1neoδHS) characterized by the targeted deletion of an ≅8-kb upstream region that included a DNase hypersensitive (HS) site (HSI) replaced by a neomycin-resistance cassette by homologous recombination.36 A targeting vector, containing a loxP siteflanked neomycin resistance gene driven by the mouse phosphoglycerate kinase promoter and a herpes simplex virus thymidine kinase gene, was used to disrupt the region of interest. The construct was electroporated into 129S4/SvJae-derived J1 embryonic stem cells and correctly targeted embryonic stem cells were injected into C57BL/6 blastocysts36 (Figure 73–2). The resulting Gata1neoδHS mice were anemic and demonstrated marked impairment in erythroid cell maturation36 because of an increased apoptotic rate due to a 4- to5-fold decrease in GATA-1 expression. However, the most striking finding was that these mutants were severely thrombocytopenic because of the almost complete lack of Gata1 expression in Mks that remain immature and release a few abnormally large platelets.37,38 The proliferative rate of GATA1low Mks is increased, while the expression of lineage-specific genes, including the mpl, glycoproteins Ibα and Ibβ, and platelet factor 4, is reduced.37,38 At the ultrastructural level, the Mks are blocked between stage I and II of maturation, and show poor organization of the α-granules due to a reduced expression of some α-granule-specific proteins, such as von Willebrand factor, or an atypical localization of other proteins, such as P-selectin, that, although being expressed at normal levels, are found on the demarcation membrane systems (DMS) rather than in the granules themselves39 (see also below). In the original C57BL/6 mutant strain, the mortality was >90% of the mutants around birth; however, when the mutation was introduced in the CD1 background in our laboratory by backcross-

Coding exons

8 kb

NeoδHS Neo

Erythroid cells



Megakaryocytes Wild-type NeoδHS


b2-mg b-glob




Figure 73–2. Generation and characterization of the GATA-1low mice. (A) The procedure employed to generate GATA-1low mice in the original C57BL/6 strain is described.34 HS, DNase hypersensitive site I. IT and IE, “testis” and “erythroid” alternative first GATA1 exons, respectively. (B) The expression levels (by semiquantitative RT-PCR) of GATA-1 in erythroid and megakaryocytic cells, to demonstrate the selective abrogation of GATA-1 in the latter, are presented.



Figure 73–3. The age-dependent development of myelofibrosis in GATA-1low mice.

ing to CD1 mice, starting from one breeder pair of B6 mice provided by Dr. S. Orkin, most of the mutants survived to reach adulthood, and, although severely anemic at birth, recovered from their anemia within the first month. On the other hand, marked thrombocytopenia (with counts less than 10% of the normal value) is maintained throughout their life. Thrombocytopenia is due to impaired Mk maturation, since the number of Mks in the BM and spleen is markedly increased; these cells are of varying size, with a population of large, almost mature Mk together with smaller forms, which are often found in clusters, especially in the spleen.40 These morphological features, which resembled those observed in IM biopsies and in hematopoietic tissues from the TPOhigh mice, prompted us to investigate whether these mice would develop myelofibrosis. By analyzing a large cohort of GATA-1low mice, we have been able to describe the natural history of myelofibrosis in these mice (Figure 73–3).40,41 During their first year, the only overt hematological abnormality of these mice is thrombocytopenia and splenic enlargement due to the expansion of the Mks lineage; after the first month, they are no longer anemic, although a greater than normal degree of erythroid progenitor cell apoptosis persists, and they are able to mount a supraadequate response to anemia induction or EPO treatment.42 From the first year of life, the first signs of MMM appear, with a progressive reduction (up to one-fifth the normal value) of the nucleated cell content in the BM cavity, the appearance of BM reticulin fibrosis that proceeds to collagen fiber deposition, and eventually osteosclerosis. The size of the spleen increases further (more than 5- to 10-fold that of the wild-type animals), and extramedullary hematopoiesis in the liver and less frequently in other organs can be detected. A moderate leukocytosis, with a few immature myeloid cells but no blasts, and dacryocytes with nucleated erythroblasts can be found in the PB. Thus, at 15–18 months of age, a full myelofibrotic phenotype is presented by the mice. However, unlike the mice overexpressing TPO, there is no leukemic terminal phase in the GATA-1low animals, and the survival of the mice is not significantly reduced, since most die of old age at 20–24 months.40 It is of interest that

these genetically modified animal models have been made possible by their development in a CD1 background, since the high mortality rate in the original C57BL/6 strain would have likely precluded their recognition. Furthermore, when the mutation was introduced in the DBA/2 background, these mice showed severe anemia but did not express a full myelofibrotic phenotype, including extramedullary hematopoiesis and osteosclerosis. These observations point to the possible role of gene modifiers in the different strains, and might also configure a model to study the variability of the clinical phenotype of the disease in humans.43 The GATA-1low mice in the CD1 background are available from the Jackson Laboratories (at http://jaxmice.jax.org/, Strain Name: STOCK Gata1tm2Sho, Stock Number 004655).

THE JAK2V617F MODEL The JAK2V617F model of myelofibrosis was established during investigations trying to determine the exact role of the JAK2V617F mutation recently identified in MPDs.5–8 To analyze the consequences of this mutation for normal hematopoiesis, two groups studied the in vivo effects of JAK2V617F expression in hematopoietic cells using an adoptive transfer in recipient mice of marrow cells transduced by a retrovirus expressing JAK2V617F.44,45 Both studies demonstrated that mice transplanted with HSC expressing JAK2V617F develop a disease mimicking human PV, including its evolution toward the “spent” phase characterized by myelofibrosis. In our study, we used a procedure similar to the one previously described for the TPO overexpressing model except that cocultures were carried out in the presence of IL-3, IL-6, SCF, and TPO.44 Furthermore, virus-producing clones were generated from two infections of the GP + E86 cells with vesicular stomatitis virus (VSV)-G pseudotyped virus. Wernig et al. used transient transfections of 293T cells with the retroviral vector and the packaging plasmids to produce viral supernatants that were used to transduce 5FU-treated BM cells.45 The infection procedure was carried out in the presence of IL-3, IL-6, and SCF and consisted of 1 day of prestimulation followed by 2 successive days of brief



exposure to the virus supernatant (90 min for 1800 × g centrifugations). Recipients were treated with two serial sets of irradiation (5.5 Gy each), instead of one (9.5 Gy) in our study, before injection of the transduced BM cells.44 In both studies, bicistronic viruses carrying an IRESeGFP cassette were used with similar promoters [murine stem cell virus (MSCV) LTR derived]. During the 2–4 months of transplantation, using both strategies, C57BL/6 mice rapidly developed polycythemia (hematocrit: 60–90%) with reticulocytosis. Wernig et al. reported low EPO levels.45 A mild leukocytosis was observed with an excess of polynuclear elements and rare immature forms. The platelet size was increased but platelet counts remained normal except in one of our low JAK2V617F expresser groups, where transient mild thrombocytosis was observed. Myeloid hyperplasia of the marrow with Mk clusters was observed. Mild splenomegaly (2- to 3-fold) developed with expansion of the red pulp and hyperplasia of myeloid and erythroid cells (4- to 5-fold) and clusters of dysplastic and apoptotic Mks with neutrophil emperipolesis. Wernig et al. described decreased Mk ploidy and extramedulary hematopoiesis in the liver with myeloid, erythroid, and Mk cells.45 The total number of myeloid and erythroid progenitor cells increased due to the expansion of splenic hematopoiesis. Endogenous erythroid colonies (growing without addition of EPO) developed from BM and spleen cells (around 30% of the total CFU-E) and constitutive activation of JAK2 downstream effectors (Stat5, ERK1/2) was detected in spleen and BM cells. No fibrosis was observed at this stage. In our study, mice were followed for 4 months after BMT.44 During this late phase, the disease evolved to a very different phenotype. We observed abatement of polycythemia, abnormal red blood cell morphology, giant platelets as a sign of dysmegakaryopoiesis, and fibrosis in the BM and spleen. Ultimately, development of severe fibrosis (with reticulin and collagen deposition) in the BM and spleen was associated with anemia, thrombocytopenia, severe granulocytosis, marrow hypocellularity, and significant splenomegaly (6-fold). Mild osteosclerosis was observed in femurs. C57BL/6 mice usually survived over the 6month survey; however, 3 out of 18 mice in Wernig’s study45 and 3 out of 65 mice in our study44 prematurely died with very elevated white blood cell counts (58–102 × 109 cell/ml) with no sign of acute leukemia. The disease phenotype was not transplantable in sublethally irradiated mice but was transferable in lethally irradiated mice. Southern blots revealed oligoclonal retrovirally marked hematopoiesis early or late after transplantation. Wernig et al. described a more dramatic phenotype in BALB/c than in C57B6 mice.45 These mice develop marked leukocytosis (up to 17-fold), huge splenomegaly (10-fold), and fibrosis at early stages (1 month post-BMT), and had a short survival (from 50 to 95 days), suggesting that genetic modifiers are important in disease phenotype.45 In summary, these models demonstrated that high levels of JAK2V617F expression induce a disease with features similar to those observed in PV patients, including evolution into a myelofibrosis-like disease. However, how a unique mutation can be involved in numerous diverse human disease phenotypes, such as in the MPD, is not explained by these models, and further work will be needed to establish the mechanisms leading to JAK2V617Fpositive pathologies that are not associated with erythrocytosis, especially as concerns MMM. Finally, this model offers a unique opportunity to assess novel therapeutic approaches for JAK2V617Fpositive PV.

THE MPLW515L MODEL Searching for novel mutations involving other components of the JAK-STAT signal transduction pathway in JAK2V617F-negative MPD, a novel somatic mutation in the transmembrane region of MPL was identified, resulting in a tryptophan-to-leucine substitution at codon 515 (MPLW515L).9 This mutation is present in approximately 5–10% of JAK2V617F-negative IM patients,9,10 and results in constitutive activation of JAK-STAT signaling. Hematopoietic cells expressing MPLW515L showed a selective proliferative advantage and a cytokine-independent growth when compared to wildtype cells.9 With the aim to evaluate the relevance of this mutation in the pathogenesis of IM, a murine transplant assay was developed. Bone marrow cells were transduced with murine stem cell virus–internal ribosome entry site–enhanced green fluorescent protein (MSCV-IRES-EGFP) vectors containing MPLWT or MPLW515L and transplanted into lethally irradiated BALB/c mice. Only mice transplanted with MPLW515L-transduced HSC developed, with a median latency of 18 days, a fully penetrant lethal myeloproliferative disease characterized by hepatomegaly and splenomegaly, leukocytosis, marked thrombocytosis, extramedullary hematopoiesis, and myelofibrosis.9 Compared to MPLWT mice, the BM of MPLW515L animals showed a predominance of maturing myeloid cells and atypical and dysplastic Mks, sometime collected in clusters, that frequently exhibited the phenomenon of emperipolesis of neutrophils in their cytoplasm. Similar findings were reported in the spleen and the liver. When cultured in vitro, spleen cells derived from MPLW515L-transduced mice demonstrated a marked increase of MK-CFUs that originated larger colonies as compared to those derived from BM cells.9 In summary, this model offered a novel mechanism for activation of downstream signal transduction pathways in JAK2V617F-negative MPD patients that results in myelofibrosis, and may represent a useful model for the study of drugs targeted to MPLW515L-harboring cells.

INSIGHTS FROM MURINE MODELS INTO THE PATHOGENESIS OF THE STROMAL REACTIONS IN MYELOFIBROSIS The stromal reaction observed in the BM microenvironment in MMM is characterized by fibrosis (excessive deposits of extracellular matrix proteins), osteosclerosis (new bone formation), and neoangiogenesis. This aberrant stromal reaction is known to be secondary to the stem cell disorder and is supposedly mediated by cytokines released by cells belonging to the abnormal clone, and especially from the Mks. Both the TPOhigh and the GATA-1low mouse model have provided new insight into the pathogenesis of the stromal reaction. The role of transforming growth factor-β1 (TGF-β1) and osteoprotegerin (OPG) in the promotion of BM fibrosis and osteosclerosis, respectively, has been elucidated. Additionally, a possible mechanism for release of cytokines in the BM microenvironment by abnormal Mks has been identified in the process of emperipolesis. Finally, the role of the monocyte/ macrophage lineage in the promotion of the stromal reaction has been addressed in the TPOhigh model. Thus, both TPOhigh and GATA-1low mouse models have proved to be useful tools with which to investigate both the cytokines and the cells involved in the modification of the BM microenvironment that occurs in MMM.


CYTOKINES INVOLVED IN THE STROMAL REACTION Role of Transforming Growth Factor-b1 In vivo and in vitro studies have involved several cytokines in the development of myelofibrosis, such as TGF-β1, platelet-derived growth factor (PDGF), or basic fibroblast growth factor (bFGF). Among these, TGF-β1 has been the most likely candidate based on indirect observations. First, it potently stimulates the production of extracellular matrix components, by inducing fibroblasts to proliferate and secrete extracellular matrix and cell adhesion proteins, and prevents matrix degradation through the enhanced expression of protease that inhibits enzymes involved in the degradation of the extracellular matrix.46 Second, TGF-β1 promotes fibrosis of a variety of nonhematopoietic tissues.46 More recently, it has been shown to be directly involved in the pathogenesis of BM fibrosis in hairy cell leukemia.49 Third, increased plasma levels of TGF-β1 have been reported both in patients with MMM and in TPOhigh mice, and increased levels of TGF-β1 were found in extracellular fluids of BM and spleen in TPOhigh and GATA-1low mice.25 The prominent role of TGF-β1 in the promotion of BM fibrosis has been confirmed using the TPOhigh model.50 Lethally irradiated wild-type hosts (used because TGF-b1−/− mice die at weaning age from a multifocal inflammatory syndrome) were transplanted for long-term reconstitution with HSC from mice genetically deficient for TGF-b1 (TGF-b1−/−) or from wild-type (TGF-b1+/+) mice infected with a retrovirus encoding the mouse TPO. Over the 4 months of follow-up, TPO levels in plasma were markedly elevated in both groups of mice and all animals typically developed a myeloproliferative syndrome characterized by thrombocytosis, leukocytosis, splenomegaly, increased numbers of progenitors in the PB, and extramedullary hematopoiesis. However, whereas prominent fibrosis was observed in BM and spleen from all mice engrafted with TGF-b1+/+ cells, none of the mice repopulated with TGF-b1−/− cells showed deposition of reticulin or collagen fibers. In accordance with the development of fibrosis, latent TGF-β1 levels in plasma and extracellular fluids of the spleen from mice engrafted with TGF-b1+/+ cells were increased 4- to 8-fold over baseline levels, whereas no increase was detected in hosts engrafted with TGF-b1−/− hematopoietic cells. Taken together, these data strongly supported the prominent role of TGF-β1 produced by hematopoietic cells in the pathogenesis of myelofibrosis induced by high TPO levels. Significantly, increased levels of TGF-β1 are contained in the extracellular fluids of BM and spleen in GATA-1low mice, but they are 2- to 6-fold lower than those reported in the TPOhigh mice; it has been suggested that the different disease evolution and morbidity in these two animal models might be correlated with the levels of TGF-β1 in the BM microenvironment.25 However, other growth factors, such as PDGF or bFGF, might also contribute to the development of fibrosis, and would require investigation. Finally, another mechanism remains unknown; this concerns the fact that TGF-β1 has to be activated to exert its biological effect. In fact, even if increased plasma levels of latent TGF-β1 were detected in TPOhigh mice, no increase in the active form of TGF-β1 was measured in the circulation.50 Accordingly, no fibrotic lesions were identified in distant organs known to be targets of the fibrogenic effect of TGF-β1, such as lung or kidney. This indicates that critical regulatory mechanisms controlling TGF-β1 activation must take place within the hematopoietic environment at secretion sites. These mechanisms responsible for the local activation of TGF-β1 are not yet understood, but might involve potent in vivo activators of TGF-β1, such


as thrombospondin-1 (TSP-1), integrins αvβ6, αIIbβ3, and αvβ3, or proteases. MKs and platelets synthesize and store TSP-1 in the same organelles as TGF-β1, and, moreover, they express integrins αIIbβ3 and αvβ3. Thus, Mks may participate in the local activation of TGF-β1; additionally, monocytes/macrophages are sources of cytokines that can activate TGF-β1 in vivo as well. Role of Osteoprotegerin The rapid development in TPOhigh mice of osteosclerosis also offered a model to investigate the underlying causes of abnormal bone growth. Bone remodeling depends on the tightly integrated activity of two distinct cell types, the osteoblasts (the bone-forming cells) and the osteoclasts (the bone-resorbing cells). Osteoblasts derive from mesenchymal progenitors through the regulatory action of cell–cell and cell–matrix interactions and the actions of several growth factors such as TGF-β1, which could exert both stimulatory and inhibitory effects. Osteoclasts are monocyte-derived multinucleated cells. They require macrophage-colony-stimulating factor (M-CSF) for proliferation and receptor activation of nuclear factor-Kappa-ligand (RANK-L), via its binding to the receptor RANK expressed at the surface of osteoclast progenitors, for differentiation and maturation. OPG is a decoy soluble receptor that binds RANK-L and inhibits osteoclastogenesis. The role of OPG in the development of osteosclerosis in TPOhigh mice was demonstrated using OPG-deficient mice (OPG−/−).51 Because these animals are viable and develop normally, the four combinations of graft/host were performed, allowing the discrimination of the potential role of hematopoietic OPG (from donor cells) or stromal OPG (host cells). Regardless of the combination, all animals developed the expected myeloproliferative syndrome with BM fibrosis associated with increased TGF-β1 plasma levels. Osteosclerosis occurred only in OPG+/+ hosts regardless of donor cell genotype with a marked elevation of OPG plasma levels. In contrast, only rare bone growth was observed in OPG−/− hosts with no OPG detectable in the circulation. These findings strongly suggest the role of the stromal OPG secreted by the host microenvironment in the promotion of osteosclerosis. The mechanism leading to stromal OPG upregulation remains unknown, but it is not mediated by increased TGF-β1 in TPOhigh mice. Indeed, retrospective analysis of TPOhigh mice engrafted with TGF-b1−/− donor cells showed no correlation between TGF-β1 levels and OPG levels.51 All mice displayed similarly increased OPG plasma levels regardless of donor cell genotype (TGF-b1−/− or TGF-b1+/+). However, TPOhigh mice engrafted with TGF-b1−/− donor cells developed a delayed osteosclerosis compared to the control group, suggesting the involvement of TGF-β1 in this process, likely by stimulating osteoblasts proliferation. Furthermore, a recent study showed that TPOhigh mice also displayed also high plasma levels of IL-1, which was demonstrated to originate in part from platelets.27 This cytokine may be involved in stromal OPG upregulation as well. Osteosclerosis and increased bone mRNA levels for osteocalcin are also developed by GATA-1low mice, starting around the sixth month of age. By crossing experiments, it has been observed that the extent of osteosclerosis is dependent on the strain employed, since bone marrow formation is maximal in C57BL/6 mice, very limited in DB/2 mice, and intermediate in the standard CD1 strain;43 thus these models offer the opportunity to study novel factors implied in the process of osteosclerosis. Although GATA-1low Mks are able to promote osteoblast proliferation in vitro by direct cell contact,52 we failed to demonstrate an in vivo correlation between the



number of Mks in the BM of different mutated strains and the degree of osteosclerosis.43 CELLS INVOLVED IN THE STROMAL REACTION Prominent Role of Megakaryocytes and the Process of Emperipolesis Several lines of evidence, obtained both from studies of patients with MMM and from murine models as described above, favor a crucial role of Mks in the development of the stromal reaction (Figure 73–4). First, Mks and platelets are the main source of TGF-β1. Second, hyperplasia and disturbed differentiation of Mks are observed in MMM patients and in both TPOhigh and GATA-1low mice. In addition, on BM biopsies, Mks are often found in close proximity to or surrounded by reticulin fibers, both in humans and mice. Third, BM fibrosis is associated with other diseases involving Mks, such as megakaryoblastic leukemia or gray platelets syndrome, and is also presented by other genetically modified animals (Table 73–1). TPOhigh mice display Mk hyperplasia with dysmorphic Mks, suggesting that a long-lasting high production of TPO could affect Mk maturation. However, since GATA-1low mice display normal levels of plasma TPO, notwithstanding their severe lifelong thrombocytopenia (likely because of TPO sequestration by the enlarged Mk mass), it seems unlikely that myelofibrosis in these mice is linked to TPO overstimulation. However, interestingly enough, Mks from mice treated with high TPO doses expressed low GATA-1 levels, positioning TPO/MPL and GATA-1 in a downstream relationship in the pathogenesis of the development of abnormal megakaryocytopoiesis and myelofibrosis.25 How a high level of TPO can induce a dysmegakaryopoiesis remains unknown. One hypothesis is that a long-lasting TPO stimulation leads to MPL traffic defects and altered signaling (S. Constantinescu, unpublished data). One possible mechanism for inappropriate cytokine release in MMM could be a pathological form of emperipolesis. This rare




physiological process is defined by the random passage of different types of BM cells through the Mk cytoplasm without any important consequences for either the host or the invading cells. An increased frequency of neutrophil polymorphonuclear (PMN) cell emperipolesis within Mks is a common feature shared by the majority of patients suffering from extreme thrombocytosis (either reactive or myeloproliferative), but is not necessarily associated with BM fibrosis.53 On the other hand, both in patients with MMM and in TPOhigh and GATA-1low mice, increased neutrophil and eosinophil PMN cell emperipolesis related to abnormal P-selectin localization on the DMS has been described.54 The Mks from both these mice contain very few normal, mature α-granules; rather they have the appearance of large empty vacuoles derived from the Golgi apparatus and are almost devoid of normal constituents. In particular, P-selectin (GMP-140, CD62), a leukocyte receptor and a normal constituent of α-granules from which it is released during platelet activation, is found in small vacuolar structures scattered in the cytoplasm and on the membranes of DMS.39 A model has been proposed by which the increased PMN emperipolesis in Mks, favored by the abnormal P-selectin localization on the DMS, would result in paraapoptosis of Mks due to the release of neutrophil enzymes in the cytoplasm; in fact, the cytoplasm of engulfed PMN is almost devoid of myeloperoxidase-positive granules, which conversely can be found in the Mk cytoplasm. This pathological and specific interaction between Mks and PMN cells could lead progressively to PMN-mediated Mk destruction, especially by causing α-granule lysis and the discharge of growth factors, including TGF-β1 normally contained in the granules, outside the Mk itself in the BM microenvironment through the DMS. This would finally result in the stimulation of reticulin and collagen synthesis and deposition by fibroblasts.37 Eventually, this same sequence of events might play a role in the constitutive mobilization of HSC in the circulation, which is

STROMAL CELLS STIMULATION (Polyclonal fibroblasts)






IL-1 TGF-b1


Figure 73–4. A pathogenetic model for the cytokine-mediated stromal reaction observed in MMM. (See color insert.)


characteristic of both the murine models and patients with MMM.55–57 The PMN proteases released through the process of emperipolesis might result in the cleavage of adhesion receptors from the surface of the cells constituting the hematopoietic niche in the BM, as well as from the membrane of HSC, similar to what happens in the HSC mobilization induced by granulocyte-colonystimulating factor (G-CSF). Minor Role of Monocyte/Macrophage A central role for the changes occurring in the monocyte/macrophage lineage in the BM microenvironment in MMM has been supported by two different observations. First, Rameshwar et al reported that monocytes from patients with MMM were spontaneously activated and abnormally secreted TGF-β1.58 Second, Frey et al. reported that TPO overexpression in SCID mice, but not in NOD/SCID mice, led to BM fibrosis, indicating a prominent role for the monocyte/ macrophage lineage in the promotion of the stromal reaction.19 However, a recent study demonstrated that NOD/SCID mice exposed to high TPO levels developed marrow fibrosis, strongly suggesting that fully functional monocytes are not required for the stromal reaction to occur, and pointing again to Mk as the key cell responsible for the pathogenesis of the changes in the BM microenvironment.27

RELEVANCE OF MURINE MODELS FOR HUMAN MYELOFIBROSIS Since TPOhigh models were first described, the role played by the TPO/MPL pathway in MMM has been elucidated. Recently two activating mutations of MPL, MPLW515L and MPLW515K, have been described in 10% of MMM patients and have been shown to induce fibrosis in mice.9,10 Furthermore, the activating JAK2V617F mutation, found in 50% of MMM, also induces fibrosis in mice59 and is directly linked to deregulated MPL signaling. On the other hand, although mutations in GATA1 have not been reported to occur in MMM, the levels of GATA-1 in the MKs from MMM patients are significantly reduced, independent of JAK2 mutational status, which points to a defect in the GATA-1-dependent pathway(s).60 Thus, there is enough to suggest that the downstream relationship existing between the TPOhigh and the GATA1low animal models would mirror the situation in human disease. Furthermore, it is likely that the prominent role of TGF-β1 in fibrosis and of OPG in the promotion of osteosclerosis, shown with these models, will also be confirmed in the new animal models of MMM; of interest, abnormalities in OPG levels have already been described in human MMM.61 In conclusion, even though the newly discovered molecular lesions as a basis for MMM in humans appear to be different from those found in the animal models, these models have been shown to recapitulate the same cascade of events that culminate in the specific phenotypic abnormalities of the human disorder. Thus, these animal models are expected to further improve our understanding of the pathogenesis of MMM, with potential clinical implications, not to mention their usefulness for testing novel therapeutic agents to treat this incurable disease.62–76

ACKNOWLEDGMENTS Supported by a grant from Associazione Italiana per la Ricerca sul Cancro (Milano, I) to A.M.V., La Ligue Nationale contre le Cancer (équipe labellisée 2004) to J.L.V. and O.W.B., and NIH to A.R.M.


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