Characterization of Splenic CD34+ Cells From Patients with Primary Myelofibrosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2810-2810 ◽  
Author(s):  
Xiaoli Wang ◽  
Sonam Prakash ◽  
Min Lu ◽  
Yan Li ◽  
Attilio Orazi ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Human Lymphocytes ◽  
Myeloproliferative Neoplasm ◽  
Myeloid Cells ◽  
Primary Myelofibrosis ◽  
Human Cells ◽  
Glycophorin A ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Jak2 Inhibitors

Abstract Abstract 2810 Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by abnormal trafficking of hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC), resulting in their constitutive mobilization and the establishment of extramedullary hematopoiesis. The mechanism by which HSC/HPC preferentially migrate, reside, proliferate and differentiate in the spleen remains poorly understood. We phenotypically and functionally characterized PMF splenic CD34+ cells (PMFSC) (n=8). Greater numbers of CD34+ (5.9±1.9%), CD34+CD38- (3.2±1.6%), CD34+CD90+ (2.0±1.5%), CD34+Lin- (4.4±1.8%) cells and assayable HPCs (4513±240/106 mononuclear cells (MNC)) were detected in PMF spleens as compared with PMF peripheral blood (PB) (CD34+: 1.4±0.6%; CD34+CD38-: 0.8±0.6%; CD34+CD90+: 0.1±0%; CD34+Lin-: 0.8±0.4%; HPC: 2496±677/106 MNCs). In addition, the expression of CXCR4 by CD34+ cells present in the spleen (20.8±3.7%) was shown to be greater than that previously observed in PMF PB CD34+ cells (PMFPBC) (7.4±2.2%, P<0.05). We next examined PMF spleens for the presence of SCID repopulating cells by analyzing human cells in the marrow and spleen of NOD/SCID γcnull mice transplanted with PMFSC using flow cytometry. Five months following transplantation, 12.1% of the marrow cells were hCD45+ in mice receiving 2×105 PMFSC as compared to 3.4% in mice receiving same number of PMFPBC from the same patient. The percentage of hCD45+ cells in the marrow significantly increased to 40.6% in mice receiving 2×106 PMFSC, while 2.7% hCD45+ cells were detected in the marrow of mice receiving similar number of PMFPBC. Surprisingly, 0.6% of the cells were hCD45+ in the spleens of mice receiving 2×104 PMFSC, while 33.9% of the cells were hCD45+ in the spleens of mice receiving 2×105 PMFSC. By contrast, as few as 0.1% hCD45+ cells were detected in the spleens of mice transplanted with equal numbers of PMFPBC. Moreover, the spleens of mice transplanted with PMFSC contained greater number of hCD34+ cells, while no hCD34+ cells were observed in the spleens of mice transplanted with PMFPBC. The human cells in the marrow of mice receiving PMFSCs were further found to be capable of differentiating into not only myeloid cells (CD33+, 8.7%; glycophorin A+, 1.1% and CD34+, 1.3%) but also B (2.3%) and T cells (2.9%). While, the human cells in the marrow of mice receiving similar numbers of PMFPBC were composed of myeloid cells (CD33+, 0.5%; glycophorin A+, 0.1% and CD34+, 0.1%) but very few CD19+ (0.1%) and no CD3+ cells. Moreover, both myeloid cells (CD33+, 14.5%; glycophorin A+, 3.9% and CD34+, 6.8%) and CD19+ (6.7%) and CD3+ cells (23.1%) were detected in the spleens of mice receiving PMFSC. The engraftment of human cells in the spleen and marrow of mice receiving PMFSC and the distinct differentiative potential of PMFSC in mice were further confirmed by immunohistochemical analysis. Greater numbers of hCD45+ cells were observed in the marrow and spleen of mice receiving PMFSC and the degree of human cell engraftment was consistent with the number of PMFSC transplanted. Moreover, the white pulps of the spleens of mice receiving PMFSC were composed primarily of human lymphocytes. Both human granulocytic cells and human lymphocytes were observed in red pulps of the spleens of mice transplanted with PMFSC. In addition, following the infusion of 5×105 PMFSC and PMFPBC, reduced numbers of PMFSC (90.3±16.8/106 BMCs) were detected in the marrows of these mice as compared with G-CSF mobilized PB (mPB) CD34+ cells (196±19/106 BMCs; P<0.05), and the number of PMFSC that homed to the marrow of the mice was lower than PMFPBC (101.7±8.8/106 BMCs; P=0.09). By contrast, similar numbers of PMFSC and PMFPBC and mPB CD34+ cells were detected in the spleens of these mice. Recently the use of JAK2 inhibitors has been shown to dramatically reduce the size of the spleen in patients with PMF. PMFSC and PMFPBC were treated with a JAK2 inhibitor, AZD1480. Similar reductions in the number of total cells, CD34+ cells and assayable HPCs were observed as compared with cells exposed to cytokines alone. These findings suggest that multipotent HSCs are present in the spleen but not the PB of PMF patients and that PMFSC have a distinct differentiative program and migratory behavior that distinguishes them from PMF and normal PB CD34+ cells. In addition treatment with JAK2 inhibitors does not appear to lead to a reduction of splenomegly by preferentially inhibiting splenic HPCs. Disclosures: No relevant conflicts of interest to declare.

Treatment in Vitro with a Combination of Bcl-Xl Inhibitor-ABT-737 and a JAK2 Inhibitor Selectively Eliminates JAK2V617F MPN Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 752-752 ◽  
Author(s):  
Min Lu ◽  
Wei Zhang ◽  
Jiapeng Wang ◽  
Yan Li ◽  
Xiaoli Wang ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Lower Percentage ◽  
Normal Cells ◽  
Jak2 Inhibitor ◽  
Cd34 Cells ◽  
Hel Cells ◽  
Jak2 Inhibitors

Abstract Abstract 752 The Ph- myeloproliferative neoplasm (MPN) are associated with excessive production of red cells, platelets and granulocytes which largely determines their clinical manifestations. A mutation in the JAK2 tyrosine kinase (JAK2V617F) was identified in the majority of patients with MPNs. The JAK2V617F mutation has been shown to play a pivotal role in the pathogenesis of MPNs. We have reported that erlotinib (Tarceva), a kinase inhibitor which inhibits the epidermal growth factor induced kinase activity, is also a potent inhibitor of JAK2V617F activity. It has been shown that erythroblasts from patients with polycythemia vera (PV) express elevated levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL. In addition, we have recently documented that megakaryocytes derived from patients with primary myelofibrosis (PMF) undergo a delayed pattern of apoptosis in vitro which might be attributed to the over-expression of Bcl-xL. We hypothesize that a combination of a JAK2V617F inhibitor and a Bcl-xL inhibitor might be capable of selectively eliminating MPN cells while sparing normal cells, and therefore, providing an optimal treatment strategy for Ph− MPNs. We then evaluated the combinations of a JAK2V617 inhibitor (erlotinib or INCB018424) and a Bcl-xL inhibitor (ABT-737) for their ability to selectively eliminate MPN (JAK2V617F positive) cells while sparing normal cells in vitro using a variety of screening systems. We first tested the ability of each of the JAK2 inhibitors alone or in combination with ABT-737 to induce death of HEL cells, which harbors JAK2V617F. Among these treatments, ABT-737 (0.25 uM) plus either erlotinib (1.0 uM), or INCB018424 (1 nM) were shown to have similar capability of inducing HEL cell apoptosis (50-70%) which was significantly greater than that by each of the single agents (<5%). However, the combination of ABT-737 (0.25 uM) plus erlotinib (1.0 uM) was the least potent of inducing normal CD34+ cells to undergo apoptosis (∼5%) as compared to ABT-737 plus INCB018424 (∼20%). We next examined the effects of each of the JAK2 inhibitors alone or in combination with ABT-737 on CD34+ cells isolated from patients with PV or PMF. PV or PMF CD34+ cells were incubated in the absence or presence of agent(s) for 4 days; the percentage of apoptotic cells was then determined using Annexin V/PI staining by flow cytometry. A fraction of both untreated and treated CD34+ cells were assayed for hematopoietic colonies in the presence of SCF, IL-6, IL-3, G-CSF and EPO; individual colonies were then randomly plucked for JAK2V617F genotyping using nested allele-specific PCR. Similar to HEL cells, the percentage of MPD CD34+ cells undergoing apoptosis were similar when cells were treated with ABT-737 (0.25 uM) plus either erlotinib (1.0 uM), or INCB018424 (1.0 nM) (∼40%), but were significantly higher than that of MPN CD34+ cells treated with each of the agents alone (∼5%). Interestingly, when the percentages of JAK2V617F positive colonies within each of the treated and untreated cell populations were analyzed, MPN CD34+ cells treated with ABT-737 plus each of the JAK2 inhibitors contained a significantly lower percentage of JAK2V617F positive colonies than those untreated or treated with each of the single agents. These data indicate that the combination of ABT-737 plus a JAK2 inhibitor specifically targets on the JAK2V617F positive progenitor cells. Collectively, we identified an agent combination of ABT-737 (0.25 uM) plus a JAK2 inhibitor that is capable of selectively eliminating JAK2V617F positive MPN progenitor cells while sparing normal progenitor cells in vitro. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Plasma EDA Fibronectin in Primary Myelofibrosis Correlates with Bone Marrow Fibrosis and Predicts Splenomegaly

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1688-1688
Author(s):  
Alessandro Malara ◽  
Cristian Gruppi ◽  
Margherita Massa ◽  
Vittorio Rosti ◽  
Giovanni Barosi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Philadelphia Chromosome ◽  
Myeloproliferative Neoplasm ◽  
Primary Myelofibrosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Costal Margin ◽  
Cell Transplant ◽  
Healthy Controls ◽  
Hematopoietic Stem

Introduction: Primary myelofibrosis (PMF) is a Philadelphia chromosome negative myeloproliferative neoplasm with adverse prognosis characterized by bone marrow (BM) fibrosis and extramedullary hematopoiesis. Fibronectin (FN) is an extracellular matrix glycoprotein that plays vital roles during tissue repair and regeneration. It exists in different forms. Plasma FN is synthesized by hepatocytes and secreted into the blood plasma, where circulates at a concentration of 300-600 μg/ml in a soluble, compact form. Differently, cellular FN is synthesized by several cell types, such as fibroblasts, endothelial cells, chondrocytes and myocytes. The alternative splicing of EDA and EDB and more complex splicing of the V domain, during transcription of FN1 gene, allows different isoforms of FN to be expressed in a tissue-dependent and temporally regulated manner. Very low levels (1.3-3 μg/ml) of FN containing EDA and/or EDB are present in plasma. Although its function is not well understood, EDA containing FN (EDA-FN) is known to agonize Toll like receptor 4 (TLR4), resulting in NF-κβ-dependent cytokine release; to induce myofibroblast differentiation during wound healing; and to increase agonist-induced platelet aggregation and thrombus formation in vivo. We previously showed that EDA-FN levels are increased in plasma and BM biopsies of PMF patients. Mechanistically, BM EDA-FN sustains megakaryocyte proliferation through TLR4 binding and confer a pro-inflammatory phenotype to cell niches promoting fibrosis progression in Romiplostim-treated mice. In this work we measured the plasma levels of EDA-FN in 104 well characterized patients with PMF to determine whether elevated levels of EDA-FN predict the occurrence of disease-related events. Methods: Plasma circulating EDA FN was measured with an enzyme linked immunosorbent assay developed at the University of Pavia, by our group. We obtained plasma EDA-FN concentration values and health care data of persons with PMF from the data-base of the Centre for the Study of Myelofibrosis at the IRCCS Policlinico S. Matteo Foundation in Pavia. We sequentially excluded persons treated with disease-modifying drugs at any time before or on the date of base-cohort entry, and those who had been splenectomized or had received a stem cell transplant. We also excluded persons with acute inflammatory diseases, autoimmune diseases, other neoplasms, and severe liver or renal dysfunction. For this study we selected everyone giving written informed consent and the study was approved by the local Ethic Committee. Immunofluorescence was performed on spleen sections from PMF patients who underwent splenectomy either because of anemia or symptomatic splenomegaly, or both; and healthy controls that were splenectomized following traumatic lesion of the spleen. Data were analyzed using STATISTICA software. Results: A homozygous JAK2V617F genotype was the major determinant of elevated plasma EDA-FN. Elevated EDA-FN levels were associated with anemia, increased levels of high-sensitivity C-reactive protein, BM fibrosis and splanchnic vein thrombosis at diagnosis. We interpreted these associations as reflecting the role EDA-FN plays in tissue remodeling, inflammation and vascular injury. Interestingly, EDA-FN levels resulted also associated with spleen size, and elevated levels of EDA-FN at diagnosis predicted large splenomegaly (more than 10 cm from the left costal margin) outcome. The evidence that plasma EDA-FN levels were not associated with the CD34+ hematopoietic stem cells mobilization, drove us to hypothesize that EDA-FN could reflect spleen endothelial cell activation and/or neoangiogenesis. Immunofluorescence analysis of spleen specimens from PMF patients and healthy controls revealed that high levels of EDA-FN were present in pathological spleens in strong association with endothelial neoangiogenesis. Conclusions: Quantification of EDA-FN level in PMF strongly correlates with BM fibrosis and may be the first marker of an altered spleen microvasculature that contributes to splenomegaly. Understanding the role of this FN isoform in PMF would be useful for testing new mechanisms of disease progression and new hypotheses about the treatment of splenomegaly in PMF. Disclosures No relevant conflicts of interest to declare.

JAK2 Inhibitors Do Not Affect Stem Cells Present in the Spleens of Patients with Myelofibrosis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1752-1752
Author(s):  
Xiaoli Wang ◽  
Joseph Tripodi ◽  
Jesse Novak ◽  
Min Lu ◽  
Yan Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chromosomal Abnormality ◽  
Conflicts Of Interest ◽  
Clinical Investigation ◽  
Lymphoid Cells ◽  
Hematopoietic Stem ◽  
Cytogenetic Abnormalities ◽  
Jak2 Inhibitor ◽  
Cd34 Cells ◽  
Jak2 Inhibitors

Abstract Abstract 1752 Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by abnormal trafficking of hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC), resulting in their constitutive mobilization and the establishment of extra-medullary hematopoiesis. At present, there is no known therapeutic approach capable of altering the natural history of MF, except for allogeneic stem cell transplantation. Treatment with JAK2 inhibitors has been shown to lead to a rapid and dramatic reduction of splenomegaly although having only a modest effect on the JAK2V617F allele burden and not resulting in the elimination of cytogenetic abnormalities or correction of histopathological abnormalities (Verstovsek S, et al. N Engl J Med. 2010; 363:1117-27). To date, the mechanism underlying the reduction of splenomegaly observed following the treatment with a JAK2 inhibitor remains the subject of speculation. Recently, we observed the presence of MF-stem cells (MF-SC) in the spleens of MF patients and demonstrated that these splenic MF-SCs have a distinct differentiation program that distinguishes them from MF peripheral blood (PB) counterparts (Wang X, et al. Journal of Clinical Investigation, 2012. In Press). We therefore explored the effect of a JAK2 Inhibitor, AZD1480, on splenic MF-SCs in order to provide an explanation for the dramatic effects on MF spleen size. Treatment of splenic or PB MF CD34+ cells with cytokines+AZD1480 (CAZD, 150nM) for 3 days resulted in a significant reduction in the number of total cells, CD34+ cells and assayable HPC (CFU-GM, BFU-E and CFU-Mix) as compared with splenic or PB MF CD34+ cells treated with cytokines alone (CA), respectively (P all <0.05, n=6). Moreover, the numbers of CD34+CD90+ cells and CD34+CXCR4+ cells generated in cultures of splenic MF CD34+ cells with CAZD were each half of that achieved in cultures containing CA. The numbers of CD34+CD90+ cells and CD34+CXCR4+ cells present in cultures of PB MF CD34+ cells with CAZD were each 1/3 of that observed in cultures with CA. However, exposure of splenic or PB MF CD34+ cells to CAZD did not result in an alteration of the proportion of JAK2V617F positive HPCs. Furthermore, the treatment of splenic MF CD34+cells with CAZD did not affect the number of colonies with a marker chromosomal abnormality. We have reported that the transplantation of PB MF CD34+ cells into NOD/SCID/IL2R γnull mice resulted in a limited degree of donor cell chimerism and a differentiation program skewed toward myeloid lineages (Wang X, et al. Blood. 2010; 116: 5972–5982), while the transplantation of splenic MF CD34+ cells achieved a higher level of chimerism and generated both myeloid and lymphoid cells which contained molecular or cytogenetic abnormalities indicating their malignant origin. Only splenic MF CD34+ cells were able to sustain hematopoiesis for prolonged time periods (9 months) and were able to engraft secondary recipients. These data document the existence of MF-SCs that reside in the spleens of MF patients (Wang X, et al. Journal of Clinical Investigation 2012. In Press). We, therefore, examined the effect of AZD1480 on splenic MF-SCs by analyzing the behavior of splenic MF SCID repopulating cells following the transplantation of splenic MF CD34+ cells treated with CA or CAZD (n=3) into NOD/SCID/IL2R γnull mice. Six months after transplantation of splenic MF CD34+ cells treated with CA or CAZD, similar numbers of human CD45+ cells were detected in both the BM and spleens of recipient mice, respectively. Furthermore, human CD45+ cells were harvested and isolated from the BM and spleen of the mice receiving splenic CD34+ cells treated with CA or CAZD from a MF patient who had a deletion of the long arms of chromosome 20 [del (20q)] and the presence of del(20q) in human CD45+ cells were determined. All the human BM CD45+ cells isolated from the mice transplanted with splenic CD34+ cells treated with either CA or CAZD had this specific chromosomal abnormality, del (20q). These findings suggest that JAK2 inhibitor treatment does not affect splenic MF-SCs, indicating that the rapid reduction in splenomegaly following such therapy or it enlargement following the cessation of such therapy is either due to the effects of JAK2 inhibitors on more differentiated hematopoietic cells or on the splenic microenvironment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Microenvironment Induced Myelophthisis Caused By CYP26 Deficiency

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1297-1297
Author(s):  
Laura Palau ◽  
Jessica M. Synder ◽  
Traci Beth Topping ◽  
Cathryn Hogarth ◽  
Nina Isoherranen ◽  
...  
Keyword(s):  
Histological Analysis ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Myeloproliferative Neoplasm ◽  
Primary Myelofibrosis ◽  
Differentiation Potential ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) reside in a complex microenvironment that enforces the balance between self-renewal and differentiation. The exact physiologic mechanisms by which the niche controls HSC fate remain elusive. Retinoic acid (RA) is a powerful morphogen that controls stem cell behavior across a variety of systems. In bone marrow (BM), mesenchymal stroma cells (MSCs) express cytochrome P450 (CYP)26 enzymes and can inactivate endogenous and pharmacological retinoids (Ghiaur G et al PNAS 2013, Su M et al. PlosOne 2015, Alonso S et al. 2016). Stromal CYP26 activity is required to maintain human HSCs ex vivo (Ghiaur G et al PNAS 2013). Here we set to study the role of CYP26 in HSC homeostasis in vivo. For this, we induced CYP26 knockout via injection of Tamoxifen in ROSA26CreERT CYP26A1loxP/loxPCYP26B1loxP/loxPmice (CYP26KO) and ROSA26CreERT wildtype mice (CTR). After 5 daily tamoxifen injections, the knockout was confirmed at DNA and RNA level in multiple tissues of CYP26KO mice. Within 4 weeks, CYP26KO mice showed profound leukocytosis (5.97 ±0.37 vs. 21.12 ±3.81 k/mm3, n=4, p<0.01 CTR vs. CYP26KO) with neutrophilia and monocytosis compared to CTR mice. By 6 weeks, they experience profound weight loss, became moribund and had to be sacrificed. At that time, they had massive splenomegaly and lymphadenopathy as well as brittle/pale bones compared to CTR (Figure A). Histological analysis revealed presence of extramedullary hematopoiesis with a predominantly myeloid infiltrate in the spleen (confirmed by flow cytometry analysis) and lymph nodes but also multiple clusters megakaryocytes (Figure B). The mice also displayed decreased BM cellularity (11.25 ±1.01 vs. 5.62 ±0.36 x106/femur, n=5, p<0.01, CTR vs. CYP26KO), increased frequency of CFU-C (44.33 ±3.72 vs. 69.83 ±8.10 CFU/25000 BM mononuclear cells (MNCs), n=10, p=0.02, CTR vs. CYP26KO) with a myeloid bias (26.76±3.89 vs. 44.57 ±3.21 %CFU-GM/G/M, n=10, p<0.01, CTR vs. CYP26KO). When cellularity was taken into account, total CFU-C per femur was comparable between the two groups. The mice also had increased frequency of LSK cells in the BM (0.26 ±0.12 vs 0.73 ±0.18 % LSK of MNCs, n=2, CTR vs. CYP26KO) and an increased frequency of circulating CFU-C in the peripheral blood (37 ±4.04 vs. 179.5 ±43.06 per 200 ml of blood, n=4, p=0.01, CTR vs. CYP26KO). On histological analysis, the BM is dominated by a myeloid infiltrate and shows a striking decrease in radial bone diameter (Figure C,D). MSCs derived from CYP26KO mice have lower levels of CXCL12 and SCF and have impaired osteoblast differentiation potential compared to MSCs derived from control mice (Figure E). These findings were confirmed with ex vivo generated CYP26KO MSCs via retroviral mediated Cre recombination of CYP26A1loxP/loxPCYP26B1loxP/loxPstroma cells. More so, preliminary studies suggest that the hematopoietic phenotype observed depends on cell extrinsic presence of CYP26 activity as transplantation of CYP26A1loxP/loxPCYP26B1loxP/loxP BM cells into wildtype BoyJ recipients did not reproduce the phenotype upon injection with Tamoxifen of the recipient mice in spite of 100% donor derived hematopoiesis. Alternatively, transplant of wildtype cells into CYP26A1loxP/loxPCYP26B1loxP/loxPrecipients, did reproduce the phenotype after injection of Tamoxifen. In conclusion, we show here that dysregulated RA homeostasis in the BM impairs MSCs function and result in egress of hematopoiesis to extramedullary sites. These results come to complement data from RARγKO (Walkley CR et al Cell 2007) and SMRTmRIDmice models (Hong S-H et al PNAS 2013) and suggest a pivotal role of RA signaling in pathogenesis of myeloproliferative neoplasm and myelofibrosis. To what extent these findings correlate with human primary myelofibrosis is currently under investigation. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Prediction of Overall Survival in 520 Patients with Primary Myelofibrosis: Outcome Update of the Dynamic International Prognostic Scoring System (DIPSS) Patient Cohort

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1729-1729 ◽  
Author(s):  
Margherita Maffioli ◽  
Elisa Rumi ◽  
Francisco Cervantes ◽  
Alessandro M. Vannucchi ◽  
Enrica Morra ◽  
...  
Keyword(s):  
Median Time ◽  
Scoring System ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Primary Myelofibrosis ◽  
Outcome Data ◽  
International Prognostic Scoring System ◽  
Risk Category ◽  
Hematopoietic Stem

Abstract Abstract 1729 Background: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm whose survival at diagnosis is predicted by the International Prognostic Scoring System (IPSS), which is based on the presence of the following five risk factors: age greater than 65 years, presence of constitutional symptoms, hemoglobin level below 10 g/dL, leukocyte count greater than 25 ×109/L, and circulating blast cells 1% or greater (Cervantes et al, Blood 2009). To allow dynamic prognostication at any time during follow up, we further developed the Dynamic International Prognostic Scoring System (DIPSS), based on the same IPSS-factors, but with different score values (one point for each risk factor, two points for acquisition of anemia) and with a distinct score model (low risk, LR, 0 points; intermediate-1 risk, Int-1R, 1–2 points; intermediate-2 risk, Int-2R, 3–4 points; high risk, HR, 5–6 points) (Passamonti et al, Blood 2010). The DIPSS model was also efficient in the prediction of acute myeloid leukemia (AML) evolution (Passamonti et al, Blood 2010) and in the assessment of survival and non-relapse mortality after allogeneic hematopoietic stem cell transplantation (Scott et al, Blood 2012). Aim: The aim of the present study is to update outcome data of PMF patients included in the original series used to generate the DIPSS model and to assess the DIPSS prediction of survival in PMF patients with a longer follow up. The Institutional Review Board approved the study, and the procedures followed were in accordance with the Declaration of Helsinki. Patients and methods: This study was performed on 520 of 525 regularly followed DIPSS-PMF patients, as five patients have been lost to follow up after the original publication. Results: Updated median follow up was of 4.1 years (range, 0.1–30.1). At the time of analysis 326 (63%) patients died, of whom 194 due to known causes: 69 AML, 16 non-AML disease progression, 21 bleeding, 17 thrombosis, 33 infections, 38 other. Median survival was 6 years (95% CI: 5.1–6.7). DIPSS stratification allowed different survivals in PMF patients even with a longer follow-up (Figure 1). Hence, to assess the time to DIPSS-category progression, we evaluated the median time spent within each risk group. This estimate revealed that the median time spent in each risk category was: 4.9 years in LR (range, 0–26.7), 2.1 years in Int-1R (range, 0–18.7), 1.7 years in Int-2R (range, 0–13.4), and 0.74 years in HR (range, 0–13.7). To investigate the prognostic role of the DIPSS score on survival, we analyzed the score as a categorical time-dependent covariate in a Cox survival regression model: the hazard ratio of shifting category from LR to Int-1R was 5.0 (95% CI: 2.4–10.6; P <0.001), it was 3.6 when shifting from Int-1R to Int-2R (95% CI: 2.6–4.9; P <0.001), and 2.7 (95% CI: 2.0–3.6; P <0.001) from Int-2R to HR. Conclusion: The updated analysis shows that the DIPSS model continues to predict survival in patients with PMF. Disclosures: No relevant conflicts of interest to declare.

Impairment of Myeloid Dendritic Differentiation and Role in the Support of Splenic Dysmegakaryopoiesis in Patients with Primary Myelofibrosis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1762-1762
Author(s):  
Christophe Desterke ◽  
Bernadette Guerton ◽  
Sophie Amsellem ◽  
Agnes Charpentier ◽  
Brigitte Dupriez ◽  
...  
Keyword(s):  
Cell Culture ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Calf Serum ◽  
Myeloproliferative Neoplasm ◽  
Primary Myelofibrosis ◽  
Adherent Cell ◽  
Boyden Chamber ◽  
Hla Dr ◽  
Cd34 Cells

Abstract 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.

Role of MicroRNA-486-5p Overexpression In CML CD34+ Cells In Modulating BCR-ABL Mediated Hematopoietic Stem/Progenitor Cell Transformation and Imatinib Sensitivity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1667-1667
Author(s):  
Li-Sheng Wang ◽  
Ling Li ◽  
Liang Li ◽  
Keh-Dong Shiang ◽  
Min Li ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Ectopic Expression ◽  
Myeloid Cells ◽  
Chromosome 8 ◽  
Mirna Sequence ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Abl Kinase ◽  
Cd34 Cells

Abstract Abstract 1667 MicroRNAs are key regulators of gene expression that regulate normal differentiation and contribute to malignant transformation of hematopoietic cells. Using microRNA microarrays we identified increased expression of miR-486 in chronic myeloid leukemia (CML) compared to normal CD34+ cells. In both normal and CML cells, miR-486 expression level was significantly higher in MEP compared to HSC, GMP and CMP populations. Treatment with Imatinib resulted in reduced expression of miR-486-5p in CML CD34+ cells, suggesting that upregulation of miR-486-5p expression was at least in part BCR-ABL kinase dependent. Consistent with this ectopic expression of BCR-ABL in cord blood CD34+ cells using retroviral vectors resulted in 4.2 fold increase in miR-486-5p expression. miR-486-5p is located within the last intron of the Ankyrin-1 gene on chromosome 8 and is enriched in muscle cells. However, the role of miR-486-5p in normal and leukemic hematopoiesis has not been evaluated. To explore the role of miR-486-5p in growth and differentiation of hematopoietic progenitor cells (HSPC), we first overexpressed hsa-miR-486-5p pre-microRNA in normal CD34+ cells using lentiviral vectors. CB CD34+ cells overexpressing miRNA-486-5p generated modestly increased numbers of cells (1.22 fold) in culture with SCF, IL-3, GM-CSF, G-CSF and EPO for 6 days compared to cells expressing control vectors, with increased numbers of erythroid cells and reduced numbers of myeloid cells. We further investigated the role of miR-486-5p on growth and differentiation of normal and leukemic HSPC by inhibiting miR-486-5p expression using a modified pmiRZip lentivirus vector expressing an anti-miR-486-5p sequence and comparing to cells expressing a control scrambled anti-miRNA sequence. Expression of anti-miR-486-5p resulted in reduced proliferation of normal CD34+ cells (32±10% inhibition) and BCR-ABL transformed CD34+ cells (38±7 % inhibition) with significantly greater inhibition of erythroid compared to myeloid cells. Anti-miR486-5p expression resulted in significantly increased apoptosis of BCR-ABL-transformed CD34+ cells but not normal CD34+ cells (CML CD34+ cells: scramble 11.1±2.4%, anti-miR-486-5p 14.7±1.7 % p=0.02; Normal CD34+ cells: scramble 9.7±5.4%, anti-486-5p 13.4±7.9% p=0.15). Importantly, anti-miR-486-5p significantly enhanced the sensitivity of BCR-ABL transformed CD34+ cells to imatinib-mediated apoptosis [combination of scramble with IM: 17.7±8.1%; anti-miR-486-5p with IM: 26.4±13%]. A search for conserved miR-486-5p target genes in the TargetScan database identified the important hematopoietic negative regulatory factors Foxo1 and Pten amongst the highest ranking targets. Using pMIR-REPORT constructs containing miR-486-5p seed sites within the Foxo1 and Pten 3'-UTR we showed that Foxo1 and Pten are direct targets of miR-486-5p. Expression of anti-miR-486-5p increased Foxo1 and Pten protein expression and decreased active Akt in normal and CML CD34+ cells. Knockdown of Foxo1 using shRNA partly blocked the suppressive effects of anti-miR486-5p on the growth of CD34+ cells. In summary, we have shown that miR-486-5p expression is modulated during hematopoietic differentiation and plays an important role in regulating hematopoietic progenitor growth and differentiation towards the erythroid lineage. We further show that miR-486-5p expression is enhanced in CML CD34+ cells, related at least in part to BCR-ABL kinase activity, and contributes to enhanced progenitor growth and survival. Inhibition of miR-486-5p results in enhanced sensitivity of CML CD34+ cells to IM-induced apoptosis. miR-486-5p effects are mediated at least in part through inhibition of Foxo1 and Pten expression. We conclude that miR-486-5p represents a novel regulatory mechanism that promotes erythroid differentiation in normal hematopoiesis and modulates Bcr-Abl-mediated transformation and tyrosine kinase inhibitor sensitivity in CML progenitors. Disclosures: No relevant conflicts of interest to declare.

Stat3 Negatively Regulates Myeloproliferative Neoplasm Induced By Jak2V617F

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 111-111
Author(s):  
Dongqing Yan ◽  
Golam Mohi
Keyword(s):  
Blood Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
White Blood Cells ◽  
Flow Cytometric Analysis ◽  
Myeloproliferative Neoplasm ◽  
Primary Myelofibrosis ◽  
Signaling Molecules ◽  
Hematopoietic Stem ◽  
Knock Out

Abstract The JAK2V617F mutation has been found in most patients with Ph-negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Expression of JAK2V617F results in constitutive activation of several signaling molecules/pathways, such as Stat5, Stat3, Akt and Erk. Unraveling the contribution of these signaling pathways in MPNs will improve our understanding of the pathogenesis of MPNs and allow us to develop more effective targeted therapies. We have previously reported the generation of a conditional Jak2V617F knock-in mouse, which exhibits all the clinical features of human PV. Using this mouse model, we have demonstrated that Stat5 is absolutely required for the pathogenesis of PV induced by Jak2V617F. However, the contribution of other signaling molecules activated by Jak2V617F in the development and progression of MPNs still remains elusive. Stat3, a member of the family of signal transducer and activator of transcription (Stat), is often found activated in solid tumors and hematologic malignancies including MPNs. Although Stat3 is known to play a tumor-promoting function in various human malignancies, recent studies also have found a tumor suppressive function of Stat3 in certain malignancies. For instance, Stat3 negatively regulates BRAFV600E-induced thyroid tumorigenesis (Couto et al., Pro Natl Acad Sci USA 2012) or suppresses PTEN loss-induced malignant transformation of astrocytes (Iglesia et al., Genes Dev 2008). Thus, Stat3 can positively or negatively regulate cell growth and tumor progression. Here, we sought to determine the role of Stat3 in Jak2V617F-evoked MPN using conditional Stat3 knock-out (Stat3 floxed) and Jak2V617F knock-in mice. Whereas expression of Jak2V617F resulted an increase in red blood cells (RBC), hemoglobin, hematocrit, white blood cells (WBC), neutrophils and platelets in the peripheral blood of the Jak2V617F knock-in mice, deletion of Stat3 did not cause any significant change in RBC, hemoglobin, hematocrit and platelet numbers in Jak2V617F knock-in mice. Strikingly, Stat3 deficiency significantly increased nertrophil counts in mice expressing Jak2V617F. Flow cytometric analysis showed that deletion of Stat3 increased the hematopoietic stem cell (HSC) compartments (LSK, LT-HSC, ST-HSC) and GMP populations in the bone marrow (BM) and spleens of mice expressing Jak2V617F. However, MEP population was unaffected by Stat3 deletion. Cell cycle analysis using Hoechst/Pyronin Y staining revealed that Jak2V617F expression alone resulted in increased cycling of HSC-enriched LSK cells, and Stat3-deficiency further enhanced the cycling of Jak2V617F-expressing LSK cells. Stat3-deficiency also caused a marked expansion of Gr-1+/Mac-1+ population in the BM and spleens of mice expressing Jak2V617F. As a consequence, CD71+/Ter119+ population was proportionally reduced in Stat3-deficient Jak2V617F-expressing mice BM. Histopathologic analysis showed marked increase in granulocytes in the BM and spleens of Stat3-deficient Jak2V617F-expressing mice compared with mice expressing Jak2V617F. Stat3-deficient Jak2V617F-expressing mice also exhibited marked infiltration of neutrophils in their livers. Furthermore, deletion of Stat3 significantly reduced the survival of Jak2V617F knock-in mice. Together, these results suggest a negative role for Stat3 in Jak2V617F-induced MPN. Thus, Stat3 may not be a suitable therapeutic target for treatment of PV and other JAK2V617F-positive MPNs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Focus on Osteosclerotic Progression in Primary Myelofibrosis

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 122
Author(s):  
Mariarita Spampinato ◽  
Cesarina Giallongo ◽  
Alessandra Romano ◽  
Lucia Longhitano ◽  
Enrico La Spina ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Marrow Cell ◽  
Myeloproliferative Neoplasm ◽  
Primary Myelofibrosis ◽  
Hematopoietic Stem ◽  
Clonal Proliferation ◽  
Cytokines And Chemokines ◽  
Bone Damage ◽  
Underlying Mechanisms ◽  
Modulating Functions

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hematopoietic stem-cell-derived clonal proliferation, leading to bone marrow (BM) fibrosis. Hematopoiesis alterations are closely associated with modifications of the BM microenvironment, characterized by defective interactions between vascular and endosteal niches. As such, neoangiogenesis, megakaryocytes hyperplasia and extensive bone marrow fibrosis, followed by osteosclerosis and bone damage, are the most relevant consequences of PMF. Moreover, bone tissue deposition, together with progressive fibrosis, represents crucial mechanisms of disabilities in patients. Although the underlying mechanisms of bone damage observed in PMF are still unclear, the involvement of cytokines, growth factors and bone marrow microenvironment resident cells have been linked to disease progression. Herein, we focused on the role of megakaryocytes and their alterations, associated with cytokines and chemokines release, in modulating functions of most of the bone marrow cell populations and in creating a complex network where impaired signaling strongly contributes to progression and disabilities.

scholarly journals A Novel Humanized Animal Model Reveals Clonal Architecture and Therapeutic Vulnerabilities in Myelofibrosis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
Hamza Celik ◽  
Ethan Krug ◽  
Hassan Bjeije ◽  
Nancy Issa ◽  
Wentao Han ◽  
...  
Keyword(s):  
Mouse Models ◽  
Myeloproliferative Neoplasm ◽  
Digital Pcr ◽  
Major Barrier ◽  
Hematopoietic Stem ◽  
Chronic Stage ◽  
Post Transplant ◽  
Clonal Architecture ◽  
Primary Disease ◽  
Cd34 Cells

Myelofibrosis (MF) is the deadliest subtype of myeloproliferative neoplasm (MPN) with a median survival of approximately 5 years. Ruxolitinib, a front line therapy for JAK2V617F mutant MPN, can alleviate symptoms of the disease, but does not eliminate the malignant clone and has minimal impact on BM fibrosis and overall survival. Current mouse models do not recapitulate the clinical heterogeneity, clonal genetic composition, or morphological features of MF. Most notably, these models do not generate robust reticulin fibrosis in the bone marrow, the most significant MF pathology. This lack of clinically relevant MF models presents a major barrier to deciphering the complex genetic drivers of the disease and developing effective therapies against it. We evaluated the ability of CD34+ hematopoietic stem and progenitor cells (HSPCs) from MF patients (that contain the MPN-disease initiating population) to give rise to MF in xenotransplanted NSGS mice. &gt;5x104 FACS-sorted CD34+ HSPCs from the peripheral blood of MF patients with JAK2V617F (n=12), CALRindels (n=7) and MPLW515L (n=2) were transplanted into sublethally irradiated (200rads) NSGS mice via X-ray guided intra-tibial injection. We observed robust engraftment of patient-derived cells at 12 weeks post-transplant regardless of their genetic background or donor patient disease severity (Fig 1A). Post-transplant, BM analysis revealed robust expansion of phenotypically defined MF HSCs relative to cord blood CD34+ control recipients, suggesting a permissive niche for MF HSCs to undergo self-renewal. Remarkably, transplantation of CD34+ cells produced other hallmarks of MF in recipient animals such as splenomegaly, thrombocytosis and most importantly BM reticulin fibrosis in all recipients (Fig 1B). We assessed the clonal architecture of engrafted human cells compared to the primary disease in the donor patients through exome sequencing of CD34+ cells prior to transplantation and hCD45+ cells from MF xenografts. We found that the clonal and subclonal mutational landscape observed in CD34+ cells prior to transplantation was maintained in recipient mice (Fig 1C), suggesting that the PDX model accurately reflects the cellular composition of the primary disease. Intriguingly, in two of the xenografted patient samples, we identified additional mutations that were not detected in the primary patient samples using standard sequencing - namely TP53R248Q and EZH2Y663H respectively. Two years after we detected these mutations in PDXs, these MF patients transformed into sAML with acquisition of TP53R248Q and EZH2Y663H mutations. We performed droplet digital PCR and demonstrated that indeed rare pre-leukemic subclones containing these mutations were present at low levels (&lt; 0.01% VAF) in chronic stage MF patients at least two years prior to sAML progression. These data also suggested that these rare subclones responsible for leukemic transformation expand significantly (&gt;300 fold) under the selective pressure of transplantation in NSGS mice. Additional validation of these findings in a further six pre-sAML MF patient samples is currently ongoing. If successful, this model could be used to prospectively identify rising leukemic clones in chronic stage MF patients, which are below the level of detect of standard sequencing as a mechanism to stratify such patients for more aggressive treatments. While sequencing can identify ultra-rare variants, it cannot discern their functional potential for sAML transformation, which is the advantage of this approach. Finally, we harnessed this system for pre-clinical studies, initially focusing on inhibiting the JAK/STAT signaling pathway. Ruxolitinib treatment in MF PDXs produced remarkably similar phenotypes as observed in patients. We observed a small, but significant reduction in engraftment of MF cells in the BM and a sharp reduction in spleen size in Ruxolitinib-treated group compared to vehicle control. Ruxoltinib treatment however did not reduce the frequency of MF HSCs, the disease initiating population or lessen the degree of reticulin fibrosis. These data suggest that this system can be used as a reliable, clinically-relevant drug screening platform. Taken together, we offer the field a critical, previously missing biologically relevant screening system for validation of MPN drug targets identified in cell lines or genetic mouse models prior to moving forward into clinical trials. Disclosures Oh: Blueprint Medicines: Consultancy; Celgene/BMS: Consultancy; Constellation: Consultancy; CTI Biopharma: Consultancy; Disc Medicine: Consultancy; Gilead Sciences: Consultancy; Incyte Corporation: Consultancy; Kartos Therapeutics: Consultancy; Novartis: Consultancy; PharmaEssentia: Consultancy.

Sign in / Sign up

Export Citation Format

Share Document