ALBERT

Description: HLA-DR is one of the markers associated with hematopoietic cell differentiation, since expression of this molecule is modulated throughout hematopoiesis. We have previously described and cloned the gene encoding factor IK, which inhibits both interferon gamma (IFN-γ)-induced and constitutive HLA-DR expression. The current study demonstrates that IK gene transcripts are present in CD34+ cells purified from human umbilical cord blood. IK expression increased and was therefore inversely correlated with the gradual loss of HLA-DR during growth factor–induced CD34+ cell proliferation and differentiation. To study the possible role of IK in hematopoiesis, antisense probes were used. IK expression was specifically inhibited by an antisense oligodeoxynucleotide containing two phosphorothioate internucleotide linkages at each of the 3′ and 5′ ends and corresponding to the initiation site of IK mRNA. A control oligonucleotide was also tested in parallel. A specific decrease of IK transcripts was correlated with an increase of HLA-DR antigen expression level. In colony-forming assays, IK antisense oligonucleotide inhibited colony formation by multilineage early erythroid and granulomonocytic CD34+ progenitors. The mean colony size was decreased 70% by IK antisense oligonucleotide in comparison to controls. These results provide evidence that the IK molecule participates in the regulation of HLA-DR expression on hematopoietic cells and plays a role in growth factor–dependent CD34+ cell proliferation and differentiation by modulating HLA-DR expression.

Description: Background: Dysregulation and overexpression of the tumorigenic gene HMGA2 has been demonstrated in the mononuclear cells of 12 consecutive MMM patients (pts) and thus appears a hallmark of the disease [Andrieux & al. Genes, Chromosomes and Cancer 2004]. Apart from pathogenetic considerations, the consistency of this finding raises the question of its specificity and usefulness as a differential diagnosis tool able to distinguish proliferative forms of MMM from other chronic Phi negative myeloproliferative diseases and on the other hand cytopenic forms of MMM from myelodysplasia. Material and methods: We assessed the level of HMGA2 expression in 30 MMM pts (the former series and 18 additional pts), 15 healthy volunteers as controls, 20 pts with Polycythemia vera (PV), 5 having evolved to post-polycythemic myelofibrosis (PPMF), 12 pts with Essential Thrombocytemia (ET), 3 of whom having developed fibrosis (PTEMF), 1 pt with a MMM-like picture due to systemic mastocytosis and 15 patients presenting with various types of MDS, 3 of whom having marked medullary fibrosis. RNA was extracted from peripheral blood leukocytes separated by standard Ficoll centrifugation and QT-PCR was performed on cDNA with TaqMan technology. Results: While controls were all negative, a substantial expression of HMGA2 was confirmed in 28/30 MMM pts, significantly higher than in any other situation; PV and ET patients were negative except for those having developed myelofibrosis; the pt with myelofibrosis related to mastocytosis was also negative. In the short and heterogeneous series of MDS pts, a weak expression was occasionnaly found with no obvious correlation with myelofibrosis or the subtype of MDS. Conclusions: dysregulation and overexpression on HMGA2 characterize MMM and allow a definite discrimination from other Phi-negative MPD; in PV or ET, the gene is not expressed during the chronic proliferative phase but becomes activated in the case of an evolution towards myelofibrosis. These findings, together with the presence of transcripts in MDS, suggest that the re-activation of HMGA2 is not directly linked to the myeloproliferation nor to the myelofibrosis itself but rather associated with dysplastic hematopoïeisis either primitive or subsequent to myelofibrosis.

Description: Background: Myelofibrosis with myeloïd metaplasia (MMM) is a rare myeloproliferative disorder which associates clonal proliferation of multipotent hematopoietic progenitors and reactive fibrosis. Deregulation and overexpression of HMGA2 has recently been demonstrated in MMM. Using quantitative RT-PCR on blood mononuclear cells from 27 patients, we found various levels of expression. It could be hypothesized that HMGA2 expression reflected the variable increase in circulating CD34+ progenitors in MMM though we failed to establish significant correlation between the level of expression and the blood enumeration of CD34+ progenitors. Material and Methods: We studied 24 patients diagnosed as primary MMM according to the set of criteria recently updated through an Italian Consensus Conference. Six individuals free from hematologic malignancy were used as controls. We also studied-purified CD34+ cells: 8 samples were isolated from the PB of MMM patients; given the very low concentrations of progenitors in normal PB, 2 samples pooling 8 healthy donors each were also prepared. In addition, 7 CD34+ cell samples were purified from the BM of consenting subjects collected during surgery for hip replacement. Results: We assayed HMGA2 expression in purified CD34+ populations from MMM patients and controls: both expressed HMGA2 but markedly more the CD34+ cells from the patients. We also evidenced transcripts in their CD15+ granulocytic cells suggesting that the enhanced expression of the gene affects the whole pathological clone. HMGA2, not expressed in normal blood cells, is involved in benign solid tumors of mesenchymal origin. Conclusion: Our data support the hypothesis that its reactivation in the clonal progenitors contribute to the pathogenesis of MMM.

Description: Abnormal mobilization of CD34+ hematopoietic stem/progenitor cells in peripheral blood (PB) characterizes patients (pts) with Primary Myelofibrosis (PMF), and is attributed to both disruption of CXCR4/SDF-1 axis by a bone marrow (BM) proteolytic environment (Xu M, Blood 2005) and reduced CXCR4 expression on CD34+ cells (Rosti V, BCMD 2007) due to a transcriptional defect (Guglielmelli P, Stem Cells 2007). CXCR4 expression on CD34+ cells was inversely related to their number in PB and associated with advanced disease. We aimed at addressing potential mechanisms of reduced CXCR4 transcription in PMF CD34+ cells. To rule out cytokine-induced transcriptional regulation, CD34+ cells from controls and PMF pts were exposed to a range of SDF-1, G-CSF, TGF-beta and Interferon-gamma concentrations, and CXCR4 protein and mRNA level were concurrently measured by flow cytometry and quantitative PCR (RTQ-PCR), respectively. SDF-1 at 500 ng/mL for 24 hrs reduced CXCR4+/CD34+ cells to less than 10% basal value while CXCR4 mRNA level was unchanged, suggesting ligand-induced receptor internalization but no effect on gene transcription. Other cytokines tested were ineffective. Since abnormal methylation of CXCR4 has been implicated in reduced CXCR4 expression in pancreatic cancer cells (Sato N, Canc Biol Ther 2005), we evaluated methylation pattern of five CpG islands in CXCR4 promoter in PMF CD34+ cells and in HEL, K562 and HL-60 cell lines as control, using both methylation-specific PCR and bisulphite sequencing. A correlation was found between CXCR4 expression and extent of CpG island methylation in cell lines: K562 had 8±6% CXCR4+ cells and all five CpG islands were methylated while in HL-60, that showed bright CXCR4 expression in 90% of the cells, the promoter was in a completely un-methylated state; in HEL cell line, that expressed CXCR4 in 37±10% of cells, partial methylation of CpG island 1 and 5 was observed. On the other hand, CXCR4 promoter CpG island 1 hypermethylation was found in CD34+ cells of 15 PMF pts, and in a minority of them also of CpG island 4 or 5. The effect of hypomethylating agent 5-azacytydine (5-AZA; 1 uM up to 72 hrs) was evaluated in CD34+ cells from 10 PMF pts; starting from 12 hr incubation, significantly increased proportion (from 3- to greater than 10-fold basal level) of CD34+ cells expressed CXCR4, and a three- to five-fold induction of CXCR4 RNA by RTQ-PCR was detectable at 8–12hr. Similar effects were observed after 5-AZA treatment of HEL, while in K562 cells changes in CXCR4 expression were minimal. Concurrent incubation with 5-AZA and HDAC inhibitor SAHA induced effects similar to hypomethylating agent alone. In both PMF CD34+ and HEL cells treated with 5-AZA, hypermethylation of CpG 1 was almost completely reverted at 24–48 hr. Finally, 5-AZA treated CD34+ cells showed enhanced migration in response to SDF-1. These data point to abnormal methylation of CXCR4 promoter as a mechanims for reduced CXCR4 expression in PMF CD34+ cells, possibly contributing to their constitutive migration in PB. Additionally, owing demonstration of an effect of 5-AZA combined with HDAC inhibitor TSA on the proportion of JAK2617V〉F PMF mutant clones in culture (Shi J, Canc Res 2007), they represent a proof-of-principle for exploring use of hypomethylating agents in PMF.

Description: MMM is a myeloproliferative disorder characterized by extramedullary hematopoiesis and reactive myelofibrosis. Recently, HMGA2 dysregulation has been demonstrated in 2 MMM patients showing 12q15 rearrangement and confirmed in 25 consecutive MMM patients without cytogenetic abnormalities (Andrieux, 2004). HMGA2 proteins belong to the high mobility group A (HMGA) family of architectural transcription factors regulating the expression of several genes. As MMM is a clonal disorder of CD34+ hematopoietic progenitors, we analyzed HMGA2 expression in peripheral blood sub-populations of 5 MMM patients and 7 healthy donors to determine in which sub-population HMGA2 was dysregulated. RNA was extracted from peripheral blood mononuclear cells (PBMC) and CD15+ granulocytic cells (PBCD15+) separated through Ficoll centrifugation or from immunomagnetically selected circulating CD34+ cells (PBCD34+). Real-time quantitative PCR (RQ-PCR) using Taqman technology was performed on cDNA. As different isoforms were described in malignancies, we used two primer sets : the first one allowing the amplification of all HMGA2 isoforms (exon 1 to 3) (HMGA2 1–3), the second one allowing the amplification of the full length HMGA2 isoform (exon 1 to 5)(HMGA2 1–5). In healthy donors and in MMM, PBMC HMGA2 expression levels were heterogeneous, depending of the cellular sub-population purity. HMGA2 1–3 or HMGA2 1–5 were both expressed in MMM and normal PBCD34+ cells, but with a higher expression level for HMGA2 1–3 as compared to HMGA2 1–5. Furthermore, both HMGA2 1–3 and HMGA2 1–5 expression levels were significantly increased in PBCD34+ MMM patients (p

Description: Introduction Primary myelofibrosis (PMF) is accompanied by an increase in the bloodstream circulation of some adult progenitor cells. Extramedullary hematopoiesis observed in this setting might remind some features related to foetal hematopoiesis. Material and methods We looked for evidence in favour of this hypothesis in blood samples of a small cohort of untreated patients with PMF (4 pre-fibrotic (PF) and 4 fibrotic (F), defined according to the WHO and Thiele's histopathology score (Blood, 2011)). Patient baseline characteristics are shown below. We performed a) flow-cytometric analysis for cell subsets related to VSEL, PEC, MPC, HPC; b) RT-PCR for embryonic transcriptional factors NANOG, OCT4, SOX2, LIN28 from MNC fraction (positive control hES, negative control CPRE2 c) in-vitro development of embryonic stem like cells (ESlC) under specific culture conditions. In addition we looked for SRSF2 mutations in order to better characterize PMF stages. Results As expected we detected high numbers of circulating CD34+ cells (HPC) (mean 233083±307148/ml (range 4600-783000), with similar numbers in PF- (231125±289553/ml) and F-PMF (235040 ±369156/ml). We were able to detect small numbers of the following cell subsets related to VSEL (size 2-4m) (Fig 1) Lin-/CD45-/CD34+ (mean 124±239/ml), Lin-/CD45-/CD133+ (mean 1178±971/ml), Lin-/CD45-/CXCR4+ (mean 1572±1622/ml). Lin-/CD45-CD34+AC133+CXCR4+ cells were detected in 6 of 8 patients (mean 186±375/ml) with F-PMF patients showing higher numbers (279±416/ml) than PF-PMF (63±71/ml). NANOG and OCT4 expression was detected by RT-PCR in all the patients tested. Mean OCT4 expression was about 50% the level of hES, but F-PMF showed higher levels. NANOG expression was similar to that of hES, whereas Sox2 and Lin28 were not expressed in most patients. We failed to observe the in-vitro development of ESlC in the 2 tested patients. PEC (Lin-/CD45-CD34+AC133+KDR+) were detected in all the PF-PMF (185±332/ml) and in 1 of 4 F-PMF (mean 9±18/ml). MPC (Lin-/CD45-CD90+CD105+) were detected in higher numbers in PF-PMF (mean 413±528/ml) than in F-PMF (mean 157±216/ml). We were not able to detect mutations in the hot spot of SRSF2 (codons 93,94,95). Conclusions Small numbers of cell subsets displaying morphologic and immunophenotypic features of VSELs were detected in PMF patients. However, we are not able to define these as fully specific VSELs according to previous works that defined them (Kucia, Leukemia 2006). Interestingly Lin-/CD45-CD34+AC133+CXCR4+ cells were observed in higher numbers in F-PMF, supporting in part our hypothesis that PMF evolution can be associated to the recruitment and circulation of some primitive progenitors (dormant in the adult life) as it can be observed during the foetal period. This recruitment also involves HPC. Moreover although all patients expressed OCT4 and NANOG, OCT4 expression was higher in F-PMF. As expected PEC circulate in higher numbers in PF-PMF compared to F-PMF. Interestingly both F-PMF and PF-PMF were associated to the circulation of significant numbers of MPC but higher numbers observed in PF-MFP might be interpreted either as a necessary recruitment to establish extra-medullary stroma or due to the exit from bone marrow of highly proliferative MPC. Whether all these different circulating progenitor cells, are clonally or not clonally related to the PMF pathogenesis or to unspecific mobilisation secondary to bone marrow microenvironment injury cannot be determined from these preliminary results. Disclosures: No relevant conflicts of interest to declare.

Description: BACKGROUND The dynamic relationship between HSC, their niches and the trabecular bone is disrupted in MPN as primary myelofibrosis (PMF). PMF is associated to extramedullary hematopoiesis (EMH) usually with splenomegaly. Several tracers are used in nuclear medicine to assess EMH and bone marrow (BM) activity. Colloids labelled with Technetium 99m (99mTc) show the reticulo-endothelial system (RES) and Indium111 chloride (111In-Cl3) transferrin scintigraphy reflects the hematopoietic activity. Cell imaging using single photon emission tomography (SPECT) utilizes gamma emitting radiopharmaceuticals and 3D acquisition allowing for imaging planes reconstruction. It is often coupled to a computed tomography (CT) (SPECT/CT). Few data exist about hematopoiesis assessment in PMF using hybrid imaging (HIm). We have shown its interest in EMH diagnosis. In PMF, we looked at patterns of active/inactive hematopoiesis in order to study later the impact of targeted therapies. MATERIAL & METHODS We performed HIm with 99mTc nanocolloids and 111In-Cl3 (coupled to SPECT/CT) in 5 untreated PMF patients and in 1 with essential thrombocytemia (ET) who was developing a secondary MF (Table1). RESULTS AND DISCUSSION We observed a lower fixation of both tracers in the axial skeleton (AxS) and conversely a hyperfixation of 111In-Cl3 in the distal skeleton (DiS) and the spleen, this latter considered as pathognomonic of PMF or secondary MF (Figs1-2. Table1). The more advanced the process of marrow fibrosis and HSC externalization the more 111In-Cl3 splenic and DiS fixation. When associated to a low 111In-Cl3 fixation in the AxS it is suggestive of PMF. No other EMH localizations besides spleen were observed. Liver fixation interpretation was uneasy because both tracers show a significant fixation. Some questions remain unsolved as the mechanisms involved in the DiS recruitment in circumstances that this hematopoietic area was progressively inactive since birth and replaced with adipose tissue. Is DiS reactivation a consequence of HSCs that cannot seed in the physiological homing hematopoietic areas, which take part essentially in the AxS? Or is the fruit of HSC reactivation (and their microenvironment) already present in the DiS but simply dormant? In this latter case we do not know if HSCs carry the same molecular abnormalities of the malignant PMF clone. Interestingly in the post ET MF patient, there was no 111In-Cl3 hyperfixation at the DiS. Conversely we observed a significant splenic fixation with both tracers and a low fixation with 99mTc in the AxS (Figs1-2). This evocates a sequence of hematopoietic reactivation starting in the spleen, followed by DiS recruitment and associated to a progressive hematopoietic regression in the AxS. HiM using these 2 radionuclides and SPECT/CT is a non-invasive procedure allowing larger and more precise visualization of the active/inactive hematopoietic areas in PMF. Moreover, regarding deep regions or high-weight patients, attenuation correction can show more uptake focus, potentially hidden because of soft-tissue interposition between emitting source and detector. This can represent an alternative to BM biopsies that also have the inconvenient to be limited in their investigation to the crista iliac. They may reflect the AxS but do not give any information neither on DiS nor on liver and spleen hematopoiesis. CONCLUSIONS The use of HIm allowed a good assessment of the extent and intensity of PMF hematopoiesis. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1762 Introduction The primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by myeloproliferation, splenomegaly with hematopoietic metaplasia and dysmegakaryopoieisis. We have previously described an increase in Flt3 ligand (FL) in plasma and spleen of patients with PMF and its role in the dysmegakaryopoiesis (Desterke and al, Cancer Res, 2011). Account the importance of FL in development of splenic myeloid dendritic cells (mDC), we studied the differentiation of mDC in patients and its potential impact on dysmegakaryopoiesis. Patients and Methods The mDCs were obtained from cell culture of blood and spleen mononuclear cells in presence of fetal calf serum (FCS) and bacterial lipolysaccharides (LPS). The megakaryocytes were obtained by culturing CD34+ cells from blood or spleen in specific medium serum free in presence of IL-3, IL-6, IL-11 and Tpo. Gene expression was quantified by microarray and RT-QPCR, protein analysis by immunofluorescence and flow cytometry, and migration experiments were performed in Boyden chamber. Results Transcriptome of circulating CD34+ cells from PMF patients showed an increase in expression of genes encoding for integrin CD11c and also TLR4 and a decrease in the expression of gene encoding TLR9: suggesting the presence of progenitor mDCs in the blood. These results have been confirmed 1/ in cytometry by an increase in the number of CD34+ CD11c+ HLA-Dr+ cells in the blood; 2/ in cell culture by the presence of adherent cell colonies positives for TLR4+ CD11c+ HLA-Dr+ in the blood. The in vitro differentiation of mDC cells and the proportion of mature mDCs HLA-Dr+ CD11c+ cells are decreased in blood of PMF patients. Myeloid nature of circulating DCs was confirmed by the absence expression of the plasmacytoid membrane marker CD123 and by an increased of TLR4 and myeloid PU-1 (myeloid transcription factor) expression in opposite to a decreased of plasmacytoid markers: IL-23, HMGB1 and TLR9. Moreover in PMF patients, circulating adherent mDCs overexpressed CXCL12 chemokine and also FL which have abnormal chemottractant ability with respect to PMF megakaryocytic precursors still expressing flt3 receptor. Finally, in PMF patients, coculture of MK with mDC promotes their survival, differentiation and maturation (MK ploidy and transcriptional program). Primary results confirmed the presence of these mDCs precursors (CD34+ CD11c+ HLA-Dr+) in the spleen of PMF patients which harbored also an extramedullary megakaryopoiesis. These mDCs precursors are absent of the spleen from healthy subjects. Conclusion Our results show an increased presence of mDC progenitor population CD34+ HLA-Dr+ CD11c+ in the blood and the spleen of PMF patients. They also suggest that these cells are involved in migration, survival and differentiation/maturation of megakaryocytes, particulary in the spleen of patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2897 Poster Board II-873 Introduction: We have shown in a deficient mouse model that the adaptor protein Lnk had an important role as negative regulator of cytokine signaling during hematopoiesis (Velazquez et al., J. Exp Med 2002). Lnk-/- animals display abnormal megakaryopoiesis sharing many features with that found in MPN patients. This phenotype is due to loss of Lnk inhibition of thrombopoietin (TPO)-mediated JAK2 activation (Tong et al., J Exp Med 2004). Recent studies have shown that Lnk, when expressed in hematopoietic cell lines, could bind and regulate two mutant proteins found in MPNs, JAK2V617F and MPLW515L. However, the role of Lnk in MPN pathogenesis is still unclear. In the present study, we studied in detail both Lnk expression and function in MPNs. Patients and methods: The study included a total of 82 MPN patients (pts), including 41 essential thrombocytemia (ET), 29 polycythemia vera (PV) and 12 primary myelofibrosis (PMF). Lnk expression was assessed by quantitative RT-PCR. Biochemical and cellular analyses of Lnk and JAK2 interaction were carried out using co-immunoprecipitation, GST pull-down and proliferation assays on primary hematopoietic cells and cell lines expressing either wild-type (WT) or mutant forms of both Lnk and JAK2. Results: Lnk mRNA was clearly overexpressed in platelets and CD34+ cells of most MPN pts compared to controls (P=0.005 and P=0.03, respectively). Moreover, this increased Lnk expression strongly correlated with JAK2V617F allele burden (P=0.02). In contrast, Lnk mRNA levels were reduced in the 18 pts treated with interferon-α compared to the 34 pts treated with hydroxyurea (P=0.04). TPO specifically upregulated Lnk expression at both mRNA and protein levels in both primary and UT7/Mpl megakaryocytic (MK) cells. Analysis of TPO-stimulated platelets from ET patients revealed the existence of a new interaction site between Lnk and JAK2 located in the N-terminal region of Lnk, in addition to the previously known interaction mediated by Lnk SH2 domain. This interaction resulted in Lnk phosphorylation. In JAK2V617F expressing platelets or cell lines, we observed both increased phosphorylation of Lnk, and stronger binding of JAK2 to the N-terminal region of Lnk compared to WT-JAK2 cells. Overexpression of Lnk in JAK2V617F cells showed a dose dependent growth inhibition, as seen in JAK2 WT cells. In addition, overexpression of various mutant forms of Lnk showed that this inhibition required a fully functional SH2 domain. Finally, expression of either WT or mutant forms of Lnk also demonstrated the crucial role of Lnk SH2 domain in growth inhibition of myeloid and MK progenitors in Lnk-/- hematopoietic cells. Conclusion: This first study of a large cohort of 82 patients allowed us to investigate the role of Lnk in MPN: (1) Lnk mRNA was found to be significantly overexpressed in MPN derived platelets and CD34+ cells, and correlated with JAK2V617F allele burden. (2) Lnk expression is upregulated by TPO, an effect likely mediated by JAK2 activation. (3) The Lnk SH2 domain plays a major role in the down-regulation of both normal and MPN-derived hematopoiesis. (4) We describe here a novel interaction site between the N-terminal region of Lnk and JAK2. Stronger interaction of the JAK2V617F mutant form with this N-terminal binding site may account for the dysregulated hematopoiesis observed in MPN patients despite Lnk overexpression. Disclosures: No relevant conflicts of interest to declare.

Description: Primary myelofibrosis (PMF) is myeloproliferative neoplasm characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in bone marrow and spleen. Mesenchymal stromal cells (MSCs) are reported to play a pivotal role in fibrosis and stromal changes are considered as a reactive counterpart of the cytokine production by clonal hematopoietic cells. The present study shows that MSCs from patients demonstrate functional abnormalities that are unexpectedly maintained ex-vivo, in culture. Material and Methods: we studied MSCs and bone marrow sections from PMF patients (n=12) as compared to healthy donors (HDs) (n=6). We tested their proliferation, immunophenotype, hematopoiesis supporting capacities, differentiation abilities, in-vivo osteogenic assays, and performed secretome and transcriptome analysis. Results: We found that PMF-MSCs exhibit similar proliferative capacity and long-term hematopoiesis supporting abilities as compare to healthy donors. They overproduce interleukin 6, VEGF, RANTES, PDGF, BMP-2 and surprisingly TGF-beta1. MSCs from fibrotic PMF patients express high levels of glycosaminoglycans. Adipocytes and chondrocytes differentiation abilities were not different as compared to HDs but PMF-MSCs exhibit an increased in vitro potential. Implementation on scaffold in nude mice confirmed, in vivo, this increased osteogenic potential. We then looked into gene expression and discovered that PMF-MSCs show an original transcriptome signature related to osteogenic lineage and TGF-beta1. Indeed, osteogenic genes such as Runx2, Dlx5, Twist1, Noggin, Sclerostin, GDF5 and Serpine1 are deregulated and suggest a potential osteoprogenitor priming of PMF-MSCs. These molecular results also advocated for a TGF-beta1 impregnation that prompted us to study its impact on PMF-MSCs osteogenic differentiation. First, we then showed that Smad2 is intrinsically over-activated in PMF-MSC and that stimulation by TGF-beta1 is associated with an increase phospho-Smad2 level and an enhancement of bone master gene regulator Runx2 expression. Then, we inhibited TGF-beta1 pathway by by SB-431542 and evidenced a specific behavior of osteogenic MSCs differentiation in patients, suggesting involvement of TGF-beta1 in osteogenic impairment. Conclusion: Altogether, our results identify a signature of PMF-MSCs and suggest that they participate in PMF osteogenic dysregulation independently from in vivo local stimulation by clonal hematopoietic cells Disclosures No relevant conflicts of interest to declare.