IL-1 Signaling Promotes Clonal Expansion and Progression of Bone Marrow Fibrosis in JAK2V617F-Induced Myeloproliferative Neoplasm

Abstract Myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell derived myeloid malignancies characterized by aberrant production of myeloid, erythroid or megakaryocytic lineage cells. JAK2V617F is the most common somatic driver mutation associated with MPN. Interestingly, JAK2V617F mutation can also be detected in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) who do not exhibit overt changes in blood counts. This suggests that other factors might be involved in association with JAK2 mutation in clonal expansion and initiation/progression of MPN. Chronic inflammation is frequently associated with MPN. Interleukin 1 (IL-1) is a major regulator of inflammation. IL-1 consists of two related cytokines IL-1α and IL-1β. Both IL-1α and IL-1β bind to the IL-1 receptor 1 (IL-1R1) to initiate downstream signaling. Although elevated expression of IL-1α and IL-1β has been observed in MPN, their role in the pathogenesis of MPN has remained elusive. In this study, we investigated the role of IL-1 signaling in JAK2V617F-induced MPN using a Jak2V617F knock-in mouse model. We observed elevated levels of IL-1α and IL-1β in mice expressing heterozygous (Jak2 VF/+) and homozygous Jak2V617F (Jak2 VF/VF) compared with WT control animals. Notably, IL-1α and IL-1β expression was significantly higher in Jak2 VF/VF mice exhibiting extensive bone marrow (BM) fibrosis compared with Jak2 VF/+ mice exhibiting polycythemia vera (PV), consistent with elevated levels of IL-1 in patients with myelofibrosis (MF). Since both IL-1α and IL-1β levels were elevated in Jak2 VF/VF mice exhibiting MF, we utilized conditional IL-1R1 knockout (IL-1R1cKO) and Jak2 VF/VF mice to assess the role of IL-1 signaling in the initiation/progression of MF. As expected, Jak2 VF/VF mice exhibited a significant increase in WBC, neutrophil and platelet counts compared to WT control mice. Deletion of IL-1R1in Jak2 VF/VF mice (IL-1R1cKO; Jak2 VF/VF) significantly reduced the WBC, neutrophil and platelet counts to almost control levels. Flow cytometric analysis also showed a significant reduction of myeloid (Gr-1 +) and megakaryocytic (CD41 +) precursors in the BM and spleens of IL-1R1cKO; Jak2 VF/VF mice compared to Jak2 VF/VF mice. Moreover, deletion of IL-1R1 significantly reduced hematopoietic stem and progenitor cells (HSPC) in the BM of IL-1R1cKO; Jak2 VF/VF mice compared to Jak2 VF/VF mice. Spleen weight was significantly reduced in IL-1R1cKO; Jak2 VF/VF mice compared with Jak2 VF/VF mice and they were comparable to control WT mice. More importantly, deletion of IL-1R1 markedly reduced BM fibrosis in Jak2 VF/VF mice. These data suggest an important role of IL-1 signaling in the progression of BM fibrosis in Jak2V617F-induced MPN. To test whether IL-1 signaling contributes to clonal expansion of JAK2 mutant HSPC, we performed competitive transplantation assays by mixing Mx1Cre; Jak2 VF/+ and Mx1Cre; IL-1R1 F/F; Jak2 VF/+ mice BM cells with CD45.1 + WT mice BM cells at a ratio of 1:1 and transplanted into lethally irradiated CD45.1 + recipient animals. At 4 weeks after BMT, the recipient animals were injected with pI-pC to induce Jak2V617F expression and IL-1R1 deletion. We observed significantly higher percentages of total CD45.2 + cells as well as CD45.2 + myeloid (Gr-1 +), B- and T-cells in the peripheral blood of chimeric mice receiving Jak2 VF/+ BM compared with chimeric mice receiving IL-1R1cKO; Jak2 VF/+ BM. We also observed significantly reduced percentages of CD45.2 + LSK, LK, Gr-1 + and CD41 + cells in the BM of chimeric recipient animals receiving IL-1R1cKO; Jak2 VF/+ BM compared with Jak2 VF/+ BM. These results suggest a role of IL-1 signaling in clonal expansion of Jak2V617F mutant HSPC. Additionally, we tested the effects of blocking IL-1R1 using an anti-IL-1R1 antibody in the homozygous Jak2V617F knock-in mouse model of MF. We observed that anti-IL-1R1 antibody treatment significantly reduced peripheral blood WBC and neutrophil counts and decreased HSPC and myeloid precursors in the BM of Jak2 VF/VF mice. Furthermore, anti-IL-1R1 antibody treatment significantly reduced splenomegaly and markedly reduced BM fibrosis in Jak2 VF/VF mice, suggesting that therapies targeting IL-1R1 could be useful for the treatment of myelofibrosis. Overall, our results suggest that IL-1 signaling contributes to clonal expansion of Jak2V617F mutant HSPC and progression of bone marrow fibrosis in MPN. Disclosures No relevant conflicts of interest to declare.

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Idiopathic myelofibrosis mimicking hemolytic anemia

Journal of Pathology of Nepal ◽

10.3126/jpn.v2i4.6888 ◽

2012 ◽

Vol 2 (4) ◽

pp. 324-327

Author(s):

R Baral ◽

G Aryal ◽

KC Shiva Raj

Keyword(s):

Bone Marrow ◽

Clinical Course ◽

Myeloproliferative Disorder ◽

Severe Anemia ◽

Idiopathic Myelofibrosis ◽

Blood Picture ◽

Bone Marrow Fibrosis ◽

Hematopoietic Stem ◽

Chronic Myeloproliferative Disorder ◽

Marrow Fibrosis

Idiopathic Myelofibrosis is an infrequent chronic myeloproliferative disorder characterized by varying degrees of bone marrow fibrosis and extra medullary hematopoiesis, with the fibrosis being a reactive phenomenon to a neoplastic proliferation of a pluripotent hematopoietic stem cell. Idiopathic Myelofibrosis is heterogeneous in presentation and clinical course, with anemia being one of the most important problems. We present a case of a 59 year old male who presented with severe anemia, the peripheral blood picture mimicking hemolysis with numerous schistocytes and teardrop cells.Journal of Pathology of Nepal (2012) Vol. 2, 323-327DOI: http://dx.doi.org/10.3126/jpn.v2i4.6888

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Differential Role for VLA-5 in Mobilization of Heamotopoieic Stem Cells Toward Peripheral Blood and Homing to Bone Marrow and Spleen.

Blood ◽

10.1182/blood.v106.11.2190.2190 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2190-2190 ◽

Cited By ~ 1

Author(s):

Pieter K. Wierenga ◽

Ellen Weersing ◽

Bert Dontje ◽

Gerald de Haan ◽

Ronald P. van Os

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Hematopoietic Stem ◽

Late Antigen ◽

Cell Mobilization

Abstract Adhesion molecules have been implicated in the interactions of hematopoietic stem and progenitor cells with the bone marrow extracellular matrix and stromal cells. In this study we examined the role of very late antigen-5 (VLA-5) in the process of stem cell mobilization and homing after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3 % of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 36±4%. The lineage negative fraction of MPB cells migrating in vitro towards SDF-1α (M-MPB) demonstrated a further decrease to 3±1% of VLA-5 expressing cells. These data are suggestive for a downregulation of VLA-5 on hematopoietic cells during mobilization. Next, MPB cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. Three hours after transplantation an increase in VLA-5 expressing cells was observed which remained stable until 24 hours post-transplant. When MPB cells were used the percentage PKH-67GL+ Lin− VLA-5+ cells increased from 36% to 88±4%. In the case of M-MPB cells the number increased from 3% to 33±5%. Although the increase might implicate an upregulation of VLA-5, we could not exclude selective homing of VLA-5+ cells as a possible explanation. Moreover, we determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first three hours post-tranpslant. Finally, we separated the MPB cells in VLA-5+ and VLA-5− cells and plated these cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be demonstared that 98.8±0.5% of the progenitor cells and 99.4±0.7% of the stem cells were present in the VLA-5+ fraction. Hence, VLA-5 is not downregulated during the process of mobilization and the observed increase in VLA-5 expressing cells after transplantation is indeed caused by selective homing of VLA-5+ cells. To shed more light on the role of VLA-5 in the process of homing, BM and MPB cells were treated with an antibody to VLA-5. After VLA-5 blocking of MPB cells an inhibition of 59±7% in the homing of progenitor cells in bone marrow could be found, whereas homing of these subsets in the spleen of the recipients was only inhibited by 11±4%. For BM cells an inhibition of 60±12% in the bone marrow was observed. Homing of BM cells in the spleen was not affected at all after VLA-5 blocking. Based on these data we conclude that mobilization of hematopoietic progenitor/stem cells does not coincide with a downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor/stem cells to the bone marrow after transplantation apparantly requires adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen seems to be independent of VLA-5 expression. These data are indicative for different adhesive pathways in the process of homing to bone marrow or spleen.

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The Prognostic Role of Jagged-1 Protein Expression In Acute Myeloid Leukemia Patients

Blood ◽

10.1182/blood.v116.21.2726.2726 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2726-2726 ◽

Cited By ~ 1

Author(s):

Agnieszka Wierzbowska ◽

Agnieszka Pluta ◽

Konrad Stepka ◽

Magdalena Czemerska ◽

Barbara Cebula-Obrzut ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Protein Expression ◽

Hodgkin Lymphoma ◽

Peripheral Blood ◽

Univariate Analysis ◽

Response To Treatment ◽

Notch 1 ◽

Hematopoietic Stem

Abstract Abstract 2726 Objectives: Jagged-1 is a member of the Delta/Serrate/Lag-2 (DSL) family of proteins that are ligands for Notch receptors. Aberrant Jagged-1/Notch-1 signaling is posited to promote the development of AML by inducing excessive self-renewal with a concomitant block in cell differentiation. Moreover, Notch-1 signaling has been identified as a critical factor involved in the maintenance of a pool of self-renewing hematopoietic stem cells (HSC) as well as AML stem cells. So far there were no reports on the clinical role of Notch-1 and Jagged-1 expression in AML. In this study we evaluated the expression of Jagged-1 and Notch-1 proteins in AML blasts and CD34+ peripheral blood stem cells (PBSC) collected during mobilization procedures before autologous stem cell transplantation. In addition, in AML patients we correlated the expression of both proteins with known prognostic factors and response to treatment. Methods: The expression of Notch-1 and Jagged-1 proteins was examined in leukemic blasts isolated from bone marrow or peripheral blood of 53 de novo AML patients with median age 57 years (range 21–82). CD34+ collected from 13 lymphoma patients (11 multiple myeloma, 1 Hodgkin lymphoma, 1 non-Hodgkin lymphoma) with median age 57 (range 21–69 years) served as a control. All measurements were carried out using multi-colour flow cytometry. In parallel, the isotype controls were performed for all measurements. Protein expression was assessed as a percentage of Notch-1 and Jagged-1 positive cells. The cut-off 20% was used to subdivide patients into “low-expressers” and “high-expressers” group. Results: We found that the median expression of Jagged-1 was significantly higher in AML blasts (18,2%; range 0,9–62,4%) as compared to CD34+ PBSC (3,0%; range 0,9–21%); p<0.0001. In contrast, the expression of Notch-1 in AML patients (median 1,4%; range 0,1–24,8%) was lower than in the control CD34+ cells (median 3,85%; range 0,7–16%); p<0.004. There was no correlation between Jagged-1 and Notch-1 protein expression in both AML blasts and PBSC. Jagged-1 expression was significantly higher in AML patients with WBC ≤20G/L (median 21,2%) compared to group with WBC >20 G/L (median 9,85%); p<0.004. Consequently, we found the significant negative correlation between Jagged-1 expression and WBC count (p<0.02). Patients with good-risk karyotype according to SWOG classification showed significantly higher expression of Jagged-1 protein as compared to intermediate and poor risk group (medians 21,8% vs. 11,5% respectively; p< 0.02). Thirty two out of 53 AML patients received standard induction chemotherapy with daunorubicine and cytarabine (“3+7”), 21/53 received non-intensive therapy. Nineteen (61%) of intensively treated patients achieved complete remission (CR). We observed that the CR rate in “high-expressers” of Jagged-1 was significantly higher than in the “low-expressers” group (80% vs. 43% respectively; p=0.04). Additionally, a good karyotype and a high expression of Jagged-1 protein were the only factors associated with higher probability of CR (p=0.05, p<0.01, respectively) in univariate analysis. There was no statistical association between the Notch-1 expression and response to treatment, karyotype or tumour size associated risk factors as: WBC, percentage of leukemic blasts in bone marrow and LDH. Moreover, no correlations between Notch-1 and CD34 expression as well as Notch-1 and differentiation markers (CD13, CD14, CD15, CD33) expressions in AML blasts were found. Conclusions: Jagged-1 protein is highly expressed in AML blasts and correlates with better response to standard chemotherapy, favorable karyotype and lower WBC in AML patients. These data clearly demonstrate an important role of Jagged-1 in AML biology. A better understanding of autonomous Jadded-1 signaling in AML may create new options for therapeutic interventions in AML. Disclosures: Robak: Johnson & Johnson: Research Funding.

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Adenoviral Mediated TGF-β1inhibition in a Murine Model of Myelofibrosis Can Prevent and Cure Bone Marrow Fibrosis Development.

Blood ◽

10.1182/blood.v104.11.794.794 ◽

2004 ◽

Vol 104 (11) ◽

pp. 794-794

Author(s):

Thomas Gastinne ◽

Orianne Wagner-Ballon ◽

Micheline Tulliez ◽

Hedia Chagraoui ◽

William Vainchenker ◽

...

Keyword(s):

Bone Marrow ◽

Murine Model ◽

Bone Marrow Cells ◽

Fold Increase ◽

Clinical Feature ◽

Transduction Efficiency ◽

Soluble Receptor ◽

Bone Marrow Fibrosis ◽

Marrow Fibrosis

Abstract Myelofibrosis is a clinical feature of several hematopoietic disorders and is most prominent in idiopathic myelofibrosis. It is characterized by excessive deposits of extracellular matrix proteins which occur as a marrow microenvironment reactive response to cytokines released from the clonal malignant myeloproliferation. The observation that mice exposed to high systemic levels of thrombopoietin (TPO) invariably develop a myelofibrosis within a period of 4 to 8 weeks has allowed the demonstration of the crucial role of TGF-b1 released by hematopoietic cells in the promotion of myelofibrosis (Chagraoui et al Blood, 2002 ; 100 :3495). The aim of this study was to investigate whether TGF-b1 inhibition could directly inhibit the fibrosis development in a curative approach of this murine model. An adenovirus encoding for TGF-b1 soluble receptor was constructed. SCID mice were infected with this adenovirus to measure the TGF-b1 inhibitory effect. We measure TGF-b1-Rs (TGF-b1 soluble receptor) concentration and TGF-b1 levels in 5 groups of mice injected with 0- 6.5 106- 6.5 107- 6.5 108-and 6.5 109 pfu/mice. We demonstrated that 6.5 108 pfu/mice was able to completely inhibit 10 fold increase of circulating TGF-b1 and that 1 microgram of soluble receptor was able to inhibit 1 ng of TGF-b1. All subsequent experiments were performed by injecting 1 109 pfu/mice either shortly after transplantation (preventive) or after 30 days (curative). Briefly, 46 mice (2 individuals experiments n= 16 and n= 30) were transplanted with 5-FU treated syngeneic bone marrow cells transduced with a retrovirus encoding for murine TPO. Retroviral transduction efficiency varied from 67% to 87%. All mice developed a myeloproliferative syndrome characterized by an increase in platelet count, a leukocytosis, an increase in progenitor cells in the blood circulation and a 1000-fold increase in TPO level. TGF-b Rs (13 to 32 micrograms/ml) was detected in the blood of all mice injected with the TGF-b1-Rs Adeno. A dramatic decrease in TGF-b1 level was noticed in all these treated mice in comparison to the control groups. Histological analysis demonstrated that the two approaches (curative or preventive) were identical to abolish fibrosis development in bone marrow as well as in the spleen. However, all mice which received the TGF-b1-RS adenovirus developed a hepatitis leading to death 6 weeks to 3 months after infections. In conclusion our approach support the major role of TGF-b1 in myelofibrosis development as previously demonstrated by our group using TGF-b1 KO mice. Moreover, the present results suggest that TGF-b1 inhibition could be a relevant approach to treat patients presenting a TGF-b1 mediated bone marrow fibrosis.

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R-Ras Regulates Hematopoietic Stem/Progenitor Cell Adhesion, Migration, and Mobilization through Rac GTPase-Mediated Signals.

Blood ◽

10.1182/blood.v110.11.221.221 ◽

2007 ◽

Vol 110 (11) ◽

pp. 221-221

Author(s):

Xun Shang ◽

Lina Li ◽

Jose Concelas ◽

Fukun Guo ◽

Deidre Daria ◽

...

Keyword(s):

Bone Marrow ◽

Cell Adhesion ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Wild Type ◽

Rac Gtpase ◽

Hematopoietic Stem ◽

And Migration ◽

Marrow Cells

Abstract Hematopoietic stem/progenitor cells (HSPCs) are maintained by strictly regulated signals in the bone marrow microenvironment. One challenge in understanding the complex mode of HSPC regulation is to link intracellular signal components with extracellular stimuli. R-Ras is a member of the Ras family small GTPases. Previous mouse genetic studies suggest that R-Ras mRNA is primarily expressed in endothelial cells and R-Ras is involved in vascular angiogenesis. In clonal cell lines, although dominant mutant overexpression studies suggest a possible role of R-Ras in regulating cell adhesion and spreading, proliferation and/or differentiation in a cell-type dependent manner, it remains controversial whether R-Ras activity may promote or inhibit cell adhesion and migration. Here, in a mouse knockout model, we have examined the role of R-Ras in HSPC regulation by a combined in vivo and in vitro approach. Firstly, we found that R-Ras is expressed in the Lin− low density bone marrow cells of wild-type mice, and R-Ras activity in the cells is downregulated by cytokines and chemokines such as SCF and SDF-1a (∼ 20% and 40% of unstimulated control, respectively). Secondly, R-Ras deficiency did not significantly affect peripheral blood CBC, nor alter the frequency or distribution of long-term and short-term hematopoietic stem cells (defined by IL7Ra−Lin−Sca-1+c-Kit+CD34− and IL7Ra−Lin−Sca-1+c-Kit+CD34+ genotypes, respectively) in the bone marrow, peripheral blood and spleen. Competitive repopulation experiments using the wild-type and R-Ras−/− bone marrow cells at 1:1 ratio in lethally irradiated recipient mice showed no significant difference of blood cells of the two genotypes in the recipients up to 6 months post-transplantation. R-Ras−/− bone marrow cells did not show a detectable difference in colony forming unit activities assayed in the presence of various combinations of SCF, TPO, EPO, IL3, G-CSF and serum, compared with the matching wild-type cells. Thirdly, upon challenge with G-CSF, a HSPC mobilizing agent, R-Ras−/− mice demonstrated a markedly enhanced ability to mobilize HSPCs from bone marrow to peripheral blood as revealed by genotypic and colony-forming unit analyses (WT: 150 vs. KO: 320 per 200uL blood, p=0.018), and R-Ras−/− HSPCs exhibit significantly decreased homing activity (WT: 4.3% vs. KO: 2.8%, p<0.001). Fourthly, isolated R-Ras−/− HSPCs displayed a constitutively assembled cortical actin cytoskeleton structure in the absence of cytokine or chemokine stimulation, similar to that of activated wild-type HSPCs. The R-Ras−/− HSPCs were defective in adhesion of cobblestone area-forming cells to a bone marrow-derived stroma cell line (FBMD-1) and in adhesion to fibronectin CH296 fragment, and showed a drastically increased ability to migrate toward a SDF-1a gradient (WT: 16% vs. KO: 38%, p<0.001). These data point to a HSPC-intrinsic role of R-Ras in adhesion and migration. Finally, the functional changes of R-Ras−/− cells were associated with a ∼3 fold increase in Rac-GTP species and constitutively elevated Rac downstream signals of phsopho-PAK1 and phospho-myosin light chain. Partial inhibition of Rac activity by NSC23766, a Rac GTPase-specific inhibitor, readily reversed the migration phenotype under SDF-1a stimulation. Taken together, these studies demonstrate that R-Ras is a critical signal regulator for HSPC adhesion, homing, migration, and mobilization through a mechanism involving Rac GTPase-regulated cytoskeleton and adhesion machinery.

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Homeostatic Role of the Krüppel-Like Factor 4 (KLF4) in Proliferation and Differentiation of Primitive Hematopoietic Progenitor Cells.

Blood ◽

10.1182/blood.v114.22.1497.1497 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1497-1497 ◽

Cited By ~ 2

Author(s):

Chun Shik Park ◽

Takeshi Yamada ◽

H. Daniel Lacorazza

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Progenitor Cells ◽

Peripheral Blood ◽

Blood Cells ◽

Proliferative Potential ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Deficient Mice

Abstract Abstract 1497 Poster Board I-520 KLF4 is a tumor suppressor in the gastrointestinal tract known to induce cell cycle arrest in a cell context dependent manner. We recently reported that KLF4 maintains quiescence of T lymphocytes downstream of T-cell receptor signaling (Yamada et al., Nature Immunology, 2009). The role of KLF4 in reprogramming adult somatic cells into pluripotent stem cells along with Oct3/4, c-Myc and Sox2 suggests that KLF4 restricts proliferation of undifferentiated cells. In spite of a redundant role of KLF4 in fetal liver hematopoietic stem cells (HSC), its role in the maintenance of adult bone marrow HSCs has not been studied yet. To study the role of KLF4 in the hematopoietic system we used gain- and loss-of-function mouse models. Retroviral transfer of KLF4 into wild type bone marrow (BM) cells led to significant reduction of colony forming units (CFU) in methylcellulose cultures due to increased apoptosis and lower proliferation. Then, Mx1-Cre was used to induce deletion of Klf4-floxed mice by polyI:C administration. Analysis of peripheral blood cells up to 6-9 months post polyI:C administration showed significant reduction of monocytes, as previously reported, and expansion of CD8+CD44+ T cells due to their increased proliferative potential. BM cells from Klf4-deficient mice exhibited increased number of myeloid progenitor cells measured by flow cytometry (Lin-Sca-1-c-kit+FcRII/III+CD34+ cells), CFU and CFU-S8. Cytoablation with 5-fluorouracil (5-FU) showed lower nadir of peripheral white blood cells in Klf4-deficient mice compared to control mice. In spite of normal multilineage reconstitution in BM transplants experiments, competitive reconstitution with Klf4-deficient and normal BM cells resulted in reduced contribution of Klf4-deficient cells to peripheral blood, likely due to homing and proliferative differences. Collectively, our data shows that KLF4 has an important role in function of hematopoietic stem and progenitor cells. Disclosures: No relevant conflicts of interest to declare.

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Distinct Effects of SCF and Hes1 On Normal Hematopoietic Stem and Progenitor Cells During Leukemic Cell Expansion in a T-ALL Mouse Model

Blood ◽

10.1182/blood.v120.21.1216.1216 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1216-1216

Author(s):

Chen Tian ◽

Zhipan Cao ◽

Qiao Li ◽

Jinhong Wang ◽

Zhenyu Ju ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Progenitor Cells ◽

Real Time ◽

Hematopoietic Cells ◽

Leukemic Cell ◽

Leukemic Cells ◽

Rt Pcr ◽

Hematopoietic Stem

Abstract Abstract 1216 During leukemia development, emerging leukemic cells out-compete normal hematopoietic cells and become predominant in the body. How hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) respond to the growth of leukemic cell population is an important, yet less investigated area. Our previous study demonstrated differential effects of a leukemic environment on normal HSCs and HPCs in the Notch1-induced T-ALL mouse model (Hu X, et al. Blood 2009). We found that normal HSCs were better preserved in the leukemic bone marrow in part due to increased quiescence of the HSCs and in contrast, HPCs were exhausted during the expansion of leukemic cells. Our current work is aimed to further explore the molecular mechanisms concerning the distinct impacts of leukemic environment on normal HSCs and HPCs in the T-ALL mouse model. Given the previous report by others showing that increased secretion of stem cell factor (SCF) by myeloid leukemia cells played an important role in inducing normal HSCs/HPCs out of their niche and thus allowing leukemic cells to occupy the niche in the human-NOD/SCID xeno-graft model (Sipkins DA et al, Science 2008), we first examined the expression of SCF by ELISA, Western blot and real-time RT PCR in both normal hematopoietic and leukemic cell fractions in the Notch1-induced T-ALL mouse model as previously reported. We found that while expression of SCF in peripheral blood (PB) or bone marrow (BM) was increased in the leukemic mice, both mRNA and protein levels of SCF in normal hematopoietic cells were higher than that in leukemic cells, thereby suggesting that elevated SCF might be mainly secreted by non-leukemic cells in the leukemic hosts of our model. Further assessments on the role of SCF in leukemogenesis with the mice specifically deficient in SCF in different niche cell types are currently under investigation in our laboratory. In order to define potential mediators in HSCs in response to leukemic cell growth, a microarray study on normal HSCs isolated from T-ALL leukemic mice and the control mice was conducted. Gene expression profiling showed significantly differed expression of 169 genes (127 up and 42 down). Especially, real-time RT PCR confirmed an increase of Hes1, p21, Fbxw11, IL-18R1 and Itgb3, and a decrease of CXCR4 and Mmp2. Interestingly, the expression of Hes1 and its target gene, p21 were elevated in normal HSCs but not in HPCs, letting us to hypothesize that Hes1 might be in part mediate the different responses of HSCs and HPCs to the T-ALL leukemic environment. To test this hypothesis, we ectopically expressed Hes1 in normal hematopoietic cells and then examined their functions under the leukemic condition. BM cells from B6.SJL mice were transduced with either MSCV-Hes1-IRES-GFP or control MSCV-GFP vector. After transduction, Hes1-GFP+or control-GFP+cells were co-transplanted with the Notch1-induced T-ALL cells into lethally irradiated C57BL/6J recipients. The engrafted cells from the leukemic BM were analyzed and Hes1-GFP+or control-GFP+cells were sorted for functional assessments. Interestingly, although over-expression of Hes1 inhibited the growth of colony forming cell (CFC) in vitro, it could potentiate the long-term repopulating cells by maintaining more cells in the quiescent (G0) state in vivo. Taken together, our current study supports a role of Hes1 in mediating the distinct responses of normal HSCs and HPCs to the T-ALL leukemic environment. Disclosures: No relevant conflicts of interest to declare.

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Dual Role Of MERIT40 In Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v122.21.797.797 ◽

2013 ◽

Vol 122 (21) ◽

pp. 797-797

Author(s):

Krasimira Rozenova ◽

Jing Jiang ◽

Chao Wu ◽

Junmin Wu ◽

Bernadette Aressy ◽

...

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Stem Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Bone Marrow Failure ◽

Adaptor Protein ◽

Self Renewal ◽

Hematopoietic Stem

Abstract The balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is maintained by cell intrinsic and extrinsic mechanisms, including tight regulation of signaling pathways such as Tpo-Mpl and SCF-ckit. Posttranslational modifications, such as phosphorylation and ubiquitination, regulate these pathways. While the role of protein phosphorylation is well established, the importance of ubiquitination in HSC self-renewal has not been well addressed. It is known that of the seven different lysines on ubiquitin, Lys48 polyubiquitination is a marker for protein degradation, and Lys63 polyubiquitination is associated with regulation of kinase activity, protein trafficking, and localization. In this study, we provide evidence that the adaptor protein MERIT40 has multiple roles in hematopoietic stem/progenitor cells (HSPCs). MERIT40 is a scaffolding protein shared by two distinct complexes with Lys63 deubiquitinase (DUB) activities: the nuclear RAP80 complex with a known role in DNA damage repair in breast/ovarian cancer cells, whereas the functions of the cytoplasmic BRISC remains less characterized. MERIT40 is important for integrity of both complexes, and its deficiency leads to their destabilization and a >90% reduction in deubiquitinase activity. By using MERIT40 knockout (M40-/-) mice, we found that lack of MERIT40 leads to a two-fold increase in phenotypic and functional HSCs determined by FACS and limiting dilution bone marrow transplantation (BMT), respectively. More importantly, M40-/- HSCs have increased regenerative capability demonstrated by increased chimerism in the peripheral blood after BMT of purified HSCs. The higher self-renewal potential of these HSCs provides a survival advantage to M40-/- mice and HSCs after repetitive administration of the cytotoxic agent 5-flurouracil (5FU). MERIT40 deficiency also preserves HSC stemness in culture as judged by an increase in peripheral blood chimerism in recipient mice transplanted with M40-/- Lin-Sca1+Kit+ (LSK) cells cultured in cytokines for nine days compared to recipient mice receiving cultured wildtype (WT) LSK cells. In contrast to the increased HSC homeostasis and superior stem cell activity due to MERIT40 deficiency, M40-/- mice are hypersensitive to DNA damaging agents caused by inter-cross linking (ICL), such as Mitomycin C (MMC) and acetaldehydes that are generated as side products of intracellular metabolism. MMC injection caused increased mortality in M40-/- mice compared to WT controls attributable to DNA damage-induced bone marrow failure. MMC-treated M40-/- mice showed marked reduction in LSK progenitor numbers accompanied by increased DNA damage, in comparison to WT mice. Consistent with the in vivo studies, M40-/- progenitor cells are hypersensitive to MMC and acetaldehyde treatment in a cell-autonomous manner in colony forming assays. ICL repair is known to require Fanconi Anemia (FA) proteins, an ICL repair network of which mutations in at least 15 different genes in humans cause bone marrow failure and cancer predisposition. Thus, M40-/- mice represent a novel mouse model to study ICL repair in HSPCs with potential relevance to bone marrow failure syndromes. Taken together, our data establishes a complex role of MERIT40 in HSPCs, warranting future investigation to decipher functional events downstream of two distinct deubiquitinating complexes associated with MERIT40 that may regulate distinct aspects of HSPC function. Furthermore, our findings reveal novel regulatory pathways involving a previously unappreciated role of K63-DUB in stem cell biology, DNA repair regulation and possibly bone marrow failure. DUBs are specialized proteases and have emerged as potential “druggable” targets for a variety of diseases. Hence, our work may provide insights into novel therapies for the treatment of bone marrow failure and associated malignancies that occur in dysregulated HSCs. Disclosures: No relevant conflicts of interest to declare.

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