Grb10 Mediated Akt Activation Is Required for Induction of CML Like Myeloproliferative Disease in Mice by BCR-ABL.

Abstract Chronic myelogenous leukaemia (CML) results from the neoplastic transformation of hematopoietic stem cells (HSC) and is characterized by a chromosomal translocation t(9;22)(q34;q11). This aberration leads to the expression of the oncogenic tyrosine kinase BCR-ABL, which mediates signals for proliferation, transformation and anti-apoptosis via various signalling pathways. Grb10, a member of the growth factor bound proteins, is known to bind activated tyrosine kinases like BCR-ABL and might be involved in the activation of the Akt signalling pathway. Here we report the impact of Grb10 for BCR-ABL mediated transformation. We exerted a siRNA based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both a Grb10 microRNA and the BCR-ABL oncogene on a single construct. This approach ensured knockdowns of more than 90% in every BCR-ABL transformed cell. Methylcellulose assays demonstrated that bone marrow coexpressing Grb10 microRNA and BCR-ABL had a 4-fold decreased colony forming ability compared to control cells. We then transduced bone marrow (BM) with retrovirus coexpressing Grb10 microRNA and p185 BCR-ABL and transplanted lethally irradiated recipient Balb/C mice. The onset and progression of leukaemia was significantly delayed in mice transplanted with Grb10 microRNA and BCR-ABL compared with the BCR-ABL transduced control microRNA group. However, we were not able to completely avoid the development of leukaemia by Grb10 knockdown. Mice transplanted with the Grb10 knockdown construct showed a delayed lymphoblastic disease, positive for B220, whereas the control group developed a rapid myeloproliferative disease, characterized by CD11b and Gr-1. In vitro analysis of BaF/3 and 32D cells showed that Grb10 knockdown in combination with BCR-ABL expression leads to a reduced phosphorylation of Akt. Taken together our data demonstrate that Grb10 is required for the development of a myeloproliferative disease by BCR-ABL in mice. Hereby, Grb10 seems to be critical for the BCR-ABL induced activation of the Akt pathway. In addition, this study describes a novel approach to express an oncogene and a microRNA using a single retroviral construct. This tool can be used to systematically screen for drugable signalling targets involved in oncogenesis.

Download Full-text

Raf1 Is Required for Induction of a Bcr-Abl Positive CML Like Myeloproliferative Disease in Mice

Blood ◽

10.1182/blood.v112.11.3207.3207 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3207-3207

Author(s):

Corinna Albers ◽

Anna Lena Illert ◽

Cornelius Miething ◽

Christian Peschel ◽

Justus Duyster

Keyword(s):

Bone Marrow ◽

Chromosomal Translocation ◽

Myelogenous Leukemia ◽

Myeloproliferative Disease ◽

Hematopoietic Stem ◽

Additional Function ◽

Murine Bone Marrow ◽

Interesting Approach ◽

Single Construct ◽

The Impact

Abstract Introduction: Chronic myelogenous leukemia (CML) results from neoplastic transformation of hematopoietic stem cells (HSC), characterized by a chromosomal translocation t(9;22)(q34;q11). This aberration leads to the expression of the oncogenic tyrosine kinase Bcr-Abl, which mediates signals for proliferation, transformation and anti-apoptosis via various different pathways including the Raf/MEK/ERK cascade. The cytoplasmic protein Raf1 is a key molecule within this cascade. Recent studies have revealed an additional function of the Raf-1 kinase that is independent of the activation of the MAPK cascade and whose effect is to increase resistance to apoptosis. Therefore Raf1 is an interesting target for molecular therapies and more effective Raf1 inhibitors have recently been developed by the pharmaceutical industry. Here we report the impact of Raf1 signalling for Bcr-Abl mediated transformation. Methods: We exerted a siRNA based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both the Raf1 microRNA and the Bcr-Abl oncogene on a single construct. This approach ensured knockdowns of more than 90% of Raf1 in every Bcr-Abl transformed cell. Results: Methylcellulose assays demonstrated that bone marrow coexpressing Raf1 microRNA and Bcr-Abl had a 2 fold decreased colony forming ability compared to control cells. We then transduced bone marrow (BM) with retrovirus coexpressing Raf1 microRNA and p185 Bcr-Abl and transplanted lethally irradiated recipient Balb/C mice. The onset and progression of leukemia was significantly delayed in mice transplanted with Raf1 microRNA and Bcr-Abl compared with the Bcr- Abl transduced control microRNA group. Raf1 knockdown mice showed only a moderate rise of white blood cell (WBC) counts and prolonged overall survival (median survival 39 ± 7.1 days) in comparison to control mice (23.3 ± 2.4 days). However, we were not able to completely avoid the development of leukemia by Raf1 knockdown. Conclusion: Taken together our data demonstrate that Raf1 is important for the development of a myeloproliferative disease by Bcr-Abl in mice. Therefore Raf1 inhibition in combination with Bcr-Abl kinase inhibition depicts an interesting approach towards eradication of Bcr- Abl positive leukemia. In addition, this study describes a novel and versatile approach to express an oncogene and a microRNA using a single retroviral construct. Thus this powerful tool can be used to systematically screen drugable signalling targets involved in oncogenesis.

Download Full-text

Expression of Jak2V617F causes a polycythemia vera–like disease with associated myelofibrosis in a murine bone marrow transplant model

Blood ◽

10.1182/blood-2005-12-4824 ◽

2006 ◽

Vol 107 (11) ◽

pp. 4274-4281 ◽

Cited By ~ 357

Author(s):

Gerlinde Wernig ◽

Thomas Mercher ◽

Rachel Okabe ◽

Ross L. Levine ◽

Benjamin H. Lee ◽

...

Keyword(s):

Bone Marrow ◽

Polycythemia Vera ◽

Extramedullary Hematopoiesis ◽

Marrow Transplant ◽

Myeloproliferative Disease ◽

Murine Bone Marrow ◽

Retroviral Integration ◽

Vitro Analysis

AbstractAn acquired somatic mutation, Jak2V617F, was recently discovered in most patients with polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), and essential thrombocythemia (ET). To investigate the role of this mutation in vivo, we transplanted bone marrow (BM) transduced with a retrovirus expressing either Jak2 wild-type (wt) or Jak2V617F into lethally irradiated syngeneic recipient mice. Expression of Jak2V617F, but not Jak2wt, resulted in clinicopathologic features that closely resembled PV in humans. These included striking elevation in hemoglobin level/hematocrit, leukocytosis, megakaryocyte hyperplasia, extramedullary hematopoiesis resulting in splenomegaly, and reticulin fibrosis in the bone marrow. Histopathologic and flow cytometric analyses showed an increase in maturing myeloid lineage progenitors, although megakaryocytes showed decreased polyploidization and staining for acetylcholinesterase. In vitro analysis of primary cells showed constitutive activation of Stat5 and cytokine-independent growth of erythroid colony-forming unit (CFU-E) and erythropoietin hypersensitivity, and Southern blot analysis for retroviral integration indicated that the disease was oligoclonal. Furthermore, we observed strain-specific differences in phenotype, with Balb/c mice demonstrating markedly elevated leukocyte counts, splenomegaly, and reticulin fibrosis compared with C57Bl/6 mice. We conclude that Jak2V617F expression in bone marrow progenitors results in a PV-like syndrome with myelofibrosis and that there are strain-specific modifiers that may in part explain phenotypic pleiotropy of Jak2V617F-associated myeloproliferative disease in humans.

Download Full-text

Cdx4 Differs from Cdx2 in Its Oncogenic Potential and Its Regulation of Hox Gene Expression in the Murine Bone Marrow Transplantation Model.

Blood ◽

10.1182/blood.v112.11.1187.1187 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1187-1187

Author(s):

Silvia Thoene ◽

Vijay P.S. Rawat ◽

Vegi M. Naidu ◽

Wolfgang Hiddemann ◽

Michaela Feuring-Buske ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Expression Profile ◽

Ectopic Expression ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Integration Sites ◽

The Impact

Abstract Cdx4 is known to be of importance for specification of cell fate in embryonic hematopoiesis with defects leading to severe perturbation of blood formation. When overexpressed in a murine hematopoietic stem cell line, Cdx4 is capable to enhance progenitor formation in vitro and promote lymphoid reconstitution of lethally irradiated, transplanted mice in vivo. In line with this important function of Cdx4 in early hematopoiesis, we analyzed expression of Cdx4 in highly purified subpopulations isolated from murine bone marrow (BM) cells by TaqMan qPCR. Cdx4 showed an expression profile known from other stem cell regulatory genes with high expression in early hematopoietic progenitors followed by decreasing expression towards the more differentiated stages of hematopoiesis, with a more than 1200-fold lower expression in total BM cells compared to progenitor enriched 5-FU BM cells (n=3). To test the impact of Cdx4 on murine progenitors, we retrovirally transduced 5-FU BM cells with Cdx4. Overexpression of Cdx4 induced growth of BM cells in liquid expansion assay (Cdx4 5.7×108±2.2×108 SEM, EGFP 2.6×106±9×105 SEM, p=0.020; cell numbers after 14 days in cytokine supplemented medium, n=5). In addition, expression of Cdx4 conferred serial replating capacity to murine BM progenitors compared to empty vector control (CFU total after 3rd replating: 4.5×109±1.3×109 SEM/500 input cells in 1st CFC, n=5). This effect was significantly stronger compared to hematopoietic progenitors overexpressing the leukemogenic Cdx2 (p=0.008). Immunophenotyping of cells after 3rd replating showed expression of mainly myeloid antigens and cytospin preparation revealed a mature myeloid morphology. Interestingly, these colonies were able to engraft lethally irradiated mice and showed multilineage engraftment (lymphoid:myloid ratio week 16 after transplantation: 0.5:1, n=2), indicating the ability of Cdx4 expressing colonies to maintain stem cell properties in vitro. In contrast to Cdx2-transplanted mice which showed a severe myeloid bias, regular peripheral blood analysis of mice transplanted with Cdx4 overexpressing BM cells showed multilineage engraftment confirmed by immunophenotyping and normal hematological parameters (RBC 6.7×109±4.2×108, WBC 5.8×106±5.19×105; lymphoid:myeloid ratio 1.4:1; week 8–28). Of note, with a median latency of 309 days after transplantation, nine out of ten mice transplanted with Cdx4-transduced BM cells died of transplantable leukemia. In six out of seven cases we found single retroviral integration sites, indicating a monoclonal origin of the disease. We could determine three different integration sites located between 200 and 700 bp upstream of coding sequences (n=4; Opa3, Akap1, Sema4d). The integration sites of two other mice were located intragenic (Zfyve2, Zfp407), indicating that insertional mutagenesis might be a necessary factor for Cdx4 induced leukemogenesis. Moreover, qRT-PCR revealed that Cdx4 in contrast to Cdx2 did not induce ectopic expression of the leukemogenic Hoxb8 and was associated with a significant lower (7.8-fold) expression of the leukemogenic Hoxb6 in transduced murine BM cells. Taken together, these data indicate that Cdx4 plays a major role in the regulation of early hematopoiesis. Its expression profile and its hematopoietic activity in different hematopoietic assays clearly differs from Cdx2, which was shown to be highly leukemogenic in mice and to be ectopically expressed in human AML. Murine models analyzing the impact of Cdx4 and Cdx2 expression on hematopoietic development will help to delineate critical differences between the two related genes.

Download Full-text

Limited Regeneration but Functional Preservation of Hematopoietic Stem Cells in the Mice Developing Leukemia via Constitutive Expression of Notch1.

Blood ◽

10.1182/blood.v110.11.204.204 ◽

2007 ◽

Vol 110 (11) ◽

pp. 204-204 ◽

Cited By ~ 1

Author(s):

Xiaoxia Hu ◽

Hongmei Shen ◽

Hui Yu ◽

Feng Xu ◽

Jianmin Wang ◽

...

Keyword(s):

Bone Marrow ◽

Molecular Mechanisms ◽

Lymphoblastic Leukemia ◽

Hematopoietic Cells ◽

Constitutive Expression ◽

Hematopoietic Progenitor Cell ◽

Control Group ◽

Extrinsic Factors ◽

Hematopoietic Stem ◽

The Impact

Abstract Leukemia development is a complex process involving both intrinsic and extrinsic factors. While many environmental factors have been studied, the impact of leukemic environment on normal hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) has not been definitively investigated. In this study, we have formally addressed this important issue by examining the potential functional alterations of HSC and HPC in the mice bearing Notch1-induced T acute lymphoblastic leukemia (T-ALL). The MSCV retrovirus vector containing cDNA encoding oncogenic intracellular domain of Notch1 (ICN1) pseudotyped with VSV-G was used to infect Lin−Sca-1+ cells in order to induce leukemic development. Normal hematopoietic cells from the B6.SJL strain (CD45.1+) were co-transplanted with Notch1 transduced Lin−Sca-1+ cells (CD45.2+) into lethally irradiated recipients. In this robust leukemia model with 100% penetrance, the normal hematopoietic cell compartment marked by CD45.1 in the leukemic marrow was sorted for phenotypic analyses and functional assays at different time points. Same numbers of the normal hematopoietic cells without Notch1-transduced cells were transplanted into the irradiated recipients as controls. As expected, progressive hematopoietic suppression was observed at both HSC and HPC levels in the leukemic mice. The frequency of HSC enriched population (Lin−c-Kit+Sca-1+, LKS) in the leukemic group was 7 times lower than that in the control at the 4th week of leukemogensis. When normalized to the bone marrow cellularity, the absolute yield of each population was 246 times lower in the leukemic group than that in the control group. These data were highly consistent with significantly lower yields of colony forming unit (CFU) and cobblestone area forming cell (CAFC). To measure the long-term engraftment of HSCs from leukemic environment, we performed the competitive bone marrow transplantation (cBMT), in which equal numbers of CD45.1+ cells isolated from leukemic or control mice and competitor cells (CD45.1/.2) at the 2nd week of leukemogenesis were co-transplanted into lethally irradiated C57BL/6J recipients. Unexpectedly, the multilineage engraftment of the hematopoietic cells isolated from the leukemic mice was 3 times more than that of the control group. Moreover, HSCs from the leukemic environment remained functional in serial transplant recipients. Finally, to explore the underlying molecular mechanisms for the enhanced function of normal HSC in the cBMT model, we examined a number of cell cycle and self-renewal regulators in HSC and HPC from leukemic marrow and control group at the time of harvest prior to transplantation by qRT-PCR. There was a significant decrease in p18 expression when compared with the control, whereas p21 expression was significantly increased. Notch1, Gfi1 and c-myc signalings were also elevated in the HSCs from leukemic environment. In summary, our current work provides the first definitive evidence for the reversible inhibition of normal HSC growth by the leukemic environment, thereby having important implications for HSC transplantation as well as leukemogenesis.

Download Full-text

Requirement for p85α Regulatory Subunit of Class IA PI3Kinase and Rac2 GTPase in Myeloproliferative Disease Driven by Activation Loop Mutant of KIT.

Blood ◽

10.1182/blood.v110.11.89.89 ◽

2007 ◽

Vol 110 (11) ◽

pp. 89-89

Author(s):

Veerendra Munugalavadla ◽

Emily C. Sims ◽

Stephen D. Lenz ◽

Reuben Kapur

Keyword(s):

Bone Marrow ◽

Mast Cells ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Regulatory Subunit ◽

Activation Loop ◽

Myeloproliferative Disease ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract Oncogenic activation-loop mutants of KIT, the receptor for stem cell factor (SCF), are commonly observed in acute myeloid leukemia (AML) and systemic mastocytosis (SM); however, unlike the KIT juxtamembrane mutants (found in patients with gastrointestinal stromal tumors [GISTs]), the activation-loop mutants are commonly insensitive to inhibition by tyrosine kinase inhibitors. Furthermore, little is known about the signaling pathways that contribute to oncogenic KIT-induced transformation in SM or AML. We demonstrate that expression of KITD814V (KIT activation-loop mutant) in primary hematopoietic stem and progenitor cells induces constitutive KIT autophosphorylation, promotes ligand-independent hyperproliferation, skews myeloid differentiation towards the granulocytic lineage, and promotes promiscuous cooperation with multiple cytokines, including G-CSF, M-CSF and IL-3. KITD814V expressing primary mast cells also demonstrated hyperproliferation in response to SCF, IL-3, IL-4 and IL-10. Biochemical analyses of KITD814V expressing cells revealed constitutively elevated levels of phosphatidylinositol-3-kinase (PI3K) and its downstream substrate, the Rho family GTPase Rac. Genetic disruption of p85a, the regulatory subunit of class IA PI-3Kinase, but not of p85β, or genetic disruption of the hematopoietic cell-specific Rho GTPase, Rac2, normalized KITD814V-induced ligand independent hyperproliferation in vitro. Additionally, deficiency of p85α or Rac2 corrected the promiscuous hyperproliferation observed in response to multiple cytokines in both KITD814V expressing stem/progenitor cells as well as mast cells in vitro. Although p85α is hyperphosphorylated and constitutively bound to KITD814V in bone marrow cells in vitro; its physiologic role in transformation in vivo is not known. To address this, we generated a new mouse model to study KITD814V induced transformation in myeloid cells as opposed to previously described models that primarily result in the generation of phenotypes resembling acute lymphocytic leukemia via this mutation. Our results show that transplantation of KITD814V expressing bone marrow cells from C57/BL6 strain of mice into syngeneic recipients results in a fatal myeloproliferative disease (MPD) characterized by leukocytosis, splenomegaly, disruption of the splenic architecture as well as myeloid cell infiltration in the lung and liver. Importantly, in this model, transplantation of KITD814V expressing p85α deficient bone marrow cells rescued the MPD phenotype, including splenomegaly, peripheral blood leukocytosis and the reduced life span associated with the transplantation of KITD814V expressing wildtype bone marrow cells. Treatment of KITD814V-expressing hematopoietic progenitors with either a Rac inhibitor (NC23766) or rapamycin showed a dose-dependent suppression in KITD814V induced growth. Taken together, our results describe the generation of a new murine transplant model to study KITD814V induced transformation and identify p85a and Rac2 as potential novel therapeutic target for the treatment of KITD814V-bearing diseases including SM and AML.

Download Full-text

JAK2 Rearrangements, Including the Novel SEC31A-JAK2 Fusion, Are Recurrent in Classical Hodgkin Lymphoma.

Blood ◽

10.1182/blood.v114.22.140.140 ◽

2009 ◽

Vol 114 (22) ◽

pp. 140-140 ◽

Cited By ~ 3

Author(s):

Katrien Van Roosbroeck ◽

Luk Cox ◽

Idoya Lahortiga ◽

Olga Gielen ◽

Thomas Tousseyn ◽

...

Keyword(s):

Bone Marrow ◽

Chromosomal Translocation ◽

Marrow Transplant ◽

Chromosomal Translocations ◽

The Novel ◽

Oncogenic Potential ◽

Murine Bone Marrow ◽

Transplant Model

Abstract Abstract 140 Molecular mechanisms underlying the pathogenesis of classical Hodgkin lymphoma (cHL) are poorly understood. Although no characteristic chromosomal translocation has been identified in cHL, gain and amplification of the 9p24 region harbouring JAK2 has been observed in up to 50% of cHLs. JAK2 encodes a protein tyrosine kinase (PTK) that plays a key role in the JAK/STAT signalling pathway. Chromosomal translocations and gain-of-function mutations involving JAK2 occur in several haematological malignancies. The aim of this study was to characterize a novel t(4;9)(q21;p24) found in a case of nodular sclerosis HL (NSHL), and to determine the in vitro and in vivo consequences of the fusion associated with this translocation. FISH with BAC clones flanking JAK2/9p24 was used to identify the 9p breakpoint and demonstrated involvement of JAK2. A BAC- and fosmid-walking interphase FISH strategy was further applied to identify the 4q21 breakpoint which was eventually mapped in the region of SEC31A. SEC31A is ubiquitously expressed in human cells and is known to play a role in ER-to-Golgi vesicular transport. Further molecular studies led to the identification of a SEC31A-JAK2 in-frame fusion transcript in which exon 24 of SEC31A is fused to exon 17 of JAK2. Of note, our recent studies showed involvement of SEC31A as a partner of ALK in ALK+ LBCL (Van Roosbroeck et al., Haematologica 2009, in press). To determine the in vitro oncogenic potential of SEC31A-JAK2, a chimeric expression construct was designed and introduced into mouse haematopoietic IL3-dependent Ba/F3 cells. SEC31A-JAK2 was found to transform Ba/F3 cells to IL3-independent growth, demonstrating its implication in oncogenic transformation. The fusion protein is likely to function as a constitutively activated tyrosine kinase, due to SEC31A-mediated oligomerization of JAK2. Attempts to identify the SEC31A domain responsible for the constitutive JAK2 activation are ongoing. Initial experiments with deletion mutants containing or lacking the WD40-like repeats of SEC31A exclude these repeats to be the driving force of JAK2 activation. An in vivo role of the fusion was assessed with a murine bone marrow transplant model. All six recipients of SEC31A-JAK2 transduced bone marrow cells developed a fatal disease after 107 – 174 days, showing involvement of the blood, bone marrow and spleen, and in a subset of mice also of lymph nodes and thymus. FACS and histopathological examination of the involved tissues in 3 mice revealed the development of a T-lymphoblastic lymphoma. Analysis of the remaining mice is still ongoing. In addition, we showed that the T-lymphoblastic disease is transplantable to secondary recipients. Downstream of the SEC31A-JAK2 fusion we could demonstrate constitutive activation of the ERK pathway in Ba/F3 cells bearing the SEC31A-JAK2 construct as well as in the reconstituted mouse tissues. To determine the incidence of JAK2 rearrangements in cHL, we screened 60 unselected cHL cases, including 25 with NSHL, by FISH and cDNA-based nested PCR. Using this approach, we identified one additional case with a SEC31A-JAK2 fusion showing 4q21 and 9p24 breakpoints identical to these in the index case. Moreover, we found a third case with a JAK2 rearrangement and two extra copies of the 3'JAK2. As SEC31A is not involved in the latter aberration, further studies aiming at the identification of the JAK2 partner in this case of cHL are ongoing. The vast majority (80%) of the remaining cHL cases analyzed by FISH revealed recurrent gains/amplifications of JAK2. In summary, we proved that JAK2 is recurrently targeted by chromosomal translocations in cHL. We identified and molecularly characterized the novel t(4;9)(q21;p24) resulting in a SEC31A-JAK2 fusion found in two NSHL cases and identified another not yet characterized JAK2 rearrangement in the third cHL case. We demonstrated the oncogenic potential of the SEC31A-JAK2 fusion both in vitro in the mouse haematopoietic IL3-dependent Ba/F3 cell line and in vivo in a murine bone marrow transplant model. Of note, this is the first report of a recurrent translocation associated with cHL. Although aberrant expression of various PTKs including JAK2 has already been documented in cHL, our results indicate that at least in some cHL cases, this aberration can be driven by a chromosomal translocation. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Prolonged Thrombocytopenia After Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) and Its Association with an Increase in CD8+ CX3CR1+ Cells in Bone Marrow.

Blood ◽

10.1182/blood.v120.21.3077.3077 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3077-3077

Author(s):

Xiao-hui Zhang ◽

Guo-xiang Wang ◽

Yan-rong Liu ◽

Lan-Ping Xu ◽

Kai-Yan Liu ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Surface Expression ◽

T Cell Subsets ◽

Control Group ◽

Hematopoietic Stem ◽

Cell Counts

Abstract Abstract 3077 Background: Since prolonged thrombocytopenia (PT) is an independent risk factor for poor clinical outcome after allogeneic hematopoietic stem cell transplantation (allo-HSCT), the underlying mechanisms need to be understood in order to develop selective treatments. Previous studies1–4 have suggested that abnormalities in B cells may play a role in the pathogenesis of PT. However, abnormalities in B cells alone do not fully explain the complete pathogenic mechanisms of PT. Our previous studies5 showed that the frequency of megakaryocytes with a ploidy value ≤ 8N was significantly increased in patients who developed PT after allo-HSCT compared to the control group. Mechanisms concerning the megakaryocyte hypoplasia in PT after allo-HSCT are not well understood. Design and Methods: PT was defined as a platelet count ≤80 × 109/L for more than 3 months after HSCT, recovery of all other cell counts, and no apparent cause for thrombocytopenia, such as aGVHD, disease recurrence, CMV infection, or antiviral drug treatment at three months post-HSCT when all other blood cell counts had return to normal.5 We analyzed T cell subsets in bone marrow (BM) and peripheral blood (PB) from allo-HSCT recipients with and without PT (n = 23 and 17, respectively) and investigated the expression characteristics of homing receptors CX3CR1, CXCR4 and VLA-4 by flow cytometry. Futhermore, Mononuclear cells (MNCs) from PT patients and controls were cultured with and without autologous CD8+ T cells in vitro, and clarify the effect of activated CD8+ T cells on the ploidy and apoptosis of megakaryocytes in the bone marrow. Results: The results demonstrated that the percentage of CD3+ T cells in the BM was significantly higher in PT patients than the experimental controls (76.00 ± 13.04% and 57.49 ± 9.11%, respectively, P < 0.001), whereas this difference was not significant for the PB (71.01 ± 11.49% and 70.49 ± 12.89%, respectively, P = 0.911). While, some T cell subsets in the BM and PB from allo-HSCT recipients with PT were not significantly different from that of the experimental control group, such as CD8+ T cells, CD4+ T cells, CD4+ CD25bright T cells (regulatory T cells), CD44hi CD62Llo CD8+ T cells and naive T cells (CD11a+ CD45RA+). Furthermore, the surface expression of homing receptor CX3CR1 on BM T cells (64.16 ± 14.07% and 37.45 ± 19.66%, respectively, P < 0.001) and CD8+ T cells (56.25 ± 14.54% and 35.16 ± 20.81%, respectively, P = 0.036), but not in blood, were significantly increased in PT patients compared to controls. For these two groups of patients, the surface expression of CXCR4 and VLA-4 on T cells and CD8+ T cells from both BM and PB did not show significant differences. Through the study in vitro, we found that the activated CD8+ T cells in bone marrow of patients with PT might suppress apoptosis (MNC group and Co-culture group: 18.02 ± 3.60% and 13.39 ± 4.22%, P < 0.05, respectively) and Fas expression (MNC group and Co-culture group: 21.10 ± 3.93 and 15.10 ± 2.33, P <0.05, respectively) of megakaryocyte. In addition, megakaryocyte with a ploidy value ≤ 8N (MNC group: 40.03 ± 6.42% and 24.54 ± 4.31%, respectively, P < 0.05) was significantly increased in patients with PT compared to the control group. Conclusions: In conclusion, an increased surface expression of CX3CR1 on T cells may mediate the recruitment of CD8+ T cells into the bone marrow in patients with PT who received an allo-HSCT. Moreover, CD8+CX3CR1+ T cells, which can have significantly increased numbers in bone marrow of patients with PT, likely caused a reduction in the megakaryocyte ploidy, and suppressed megakaryocyte apoptosis via CD8+ T cell-mediated cytotoxic effect, possibly leading to impaired platelet production. Therefore, treatment targeting CX3CR1 should be considered as a reasonable therapeutic strategy for PT following allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Differential homing and engraftment properties of hematopoietic progenitor cells from murine bone marrow, mobilized peripheral blood, and fetal liver

Blood ◽

10.1182/blood.v98.7.2108 ◽

2001 ◽

Vol 98 (7) ◽

pp. 2108-2115 ◽

Cited By ~ 82

Author(s):

Stephen J. Szilvassy ◽

Todd E. Meyerrose ◽

Penny L. Ragland ◽

Barry Grimes

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Fetal Liver ◽

Proliferative Potential ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Hematopoietic Reconstitution ◽

Late Antigen ◽

Mobilized Peripheral Blood

The rate of reconstitution following hematopoietic stem cell (HSC) transplantation differs widely depending on the tissue source of the cells infused. To test the hypothesis that variability in engraftment kinetics is related to differences in the efficiency with which intravenously transplanted HSCs “home” to the bone marrow (BM), the homing properties of murine fetal liver (FL), adult BM, and mobilized peripheral blood (MPB) cells were compared. Lethally irradiated mice transplanted with 2 × 106 FL, BM, or MPB cells exhibited sequentially slower recovery of circulating leukocytes and platelets that correlates with the progressively lower frequency of colony-forming cells (CFCs) in these tissues. However, differences in the rate and degree of early and long-term reconstitution were maintained even after infusing equal numbers of CFCs derived from FL, BM, and MPB. To compare the homing of progenitors from these tissues, cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. Three hours later, PKH26+ cells were reisolated from the BM and spleen by fluorescence-activated cell sorting and assayed for in vitro CFCs. Despite the higher level of very late antigen (VLA)-2, VLA-4, and VLA-5 on Sca-1+c-kit+ cells from FL compared to BM, 10-fold fewer FL CFCs homed to hematopoietic organs than those from BM. MPB cells homed slightly better, but still less efficiently than BM cells. Therefore, clonogenic cells from different tissues exhibit striking variations in homing efficiency that does not necessarily correlate with engraftment kinetics. Homing is likely counterbalanced by intrinsic differences in proliferative potential that ultimately determine the rate of hematopoietic reconstitution.

Download Full-text

Phenotype and hematopoietic potential of side population cells throughout embryonic development

Blood ◽

10.1182/blood-2003-01-0118 ◽

2003 ◽

Vol 102 (7) ◽

pp. 2436-2443 ◽

Cited By ~ 71

Author(s):

Brian M. Nadin ◽

Margaret A. Goodell ◽

Karen K. Hirschi

Keyword(s):

Bone Marrow ◽

Side Population ◽

Vascular Endothelial ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Embryonic Tissues ◽

Vascular Endothelial Cadherin ◽

Endothelial Receptor

Abstract Adult murine bone marrow hematopoietic stem cells (HSCs) can be purified by sorting Hoechst 33342-extruding side population (SP) cells. Herein we investigated whether SP cells reside within embryonic tissues and exhibit hematopoietic progenitor activity. We isolated yolk sac (YS) and embryonic tissues 7.5 to 11.5 days after coitus (dpc), resolved an SP in each, and demonstrated that these SP cells exhibit distinct phenotypic and functional characteristics throughout development. YS and embryonic SP isolated 8.0 dpc expressed vascular endothelial-cadherin (VE-cadherin) and vascular endothelial receptor 2 (Flk-1), markers not expressed by bone marrow SP but expressed by endothelial cells and progenitors. SP at this stage did not express CD45 or produce hematopoietic colonies in vitro. In contrast, SP isolated 9.5 to 11.5 dpc contained a significantly higher proportion of cells expressing cKit and CD45, markers highly expressed by bone marrow SP. Furthermore, YS SP isolated 9.5 to 11.5 dpc demonstrated 40- to 90-fold enrichment for hematopoietic progenitor activity over unfractionated tissue. Our data indicate that YS and embryonic SP cells detected prior to the onset of circulation express the highest levels of endothelial markers and do not generate blood cells in vitro; however, as development progresses, they acquire hematopoietic potential and phenotypic characteristics similar to those of bone marrow SP. (Blood. 2003;102:2436-2443)

Download Full-text

Integrative Analysis of Primary SF3B1 mt Ring Sideroblasts Provides Fundamental Insights into MDS-RS Pathogenesis and Dyserythropoiesis

Blood ◽

10.1182/blood-2021-148321 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 146-146

Author(s):

Pedro Luis Moura ◽

Teresa Mortera-Blanco ◽

Isabel Juliana F Hofman ◽

Gabriele Todisco ◽

Warren W Kretzschmar ◽

...

Keyword(s):

Bone Marrow ◽

Erythroid Differentiation ◽

Integrative Analysis ◽

Model Systems ◽

Stress Factors ◽

Blue Staining ◽

Hematopoietic Stem ◽

Ring Sideroblasts ◽

The Impact

Abstract Myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal hematopoietic stem cell (HSC) disorders characterized by aberrant HSC differentiation, cytopenia, and an increased risk of progression to leukemia. The low-risk subtype MDS with ring sideroblasts (MDS-RS) is specifically characterized by expanded and ineffective erythropoiesis, with more than 80% of patients displaying mutations in the core spliceosome component SF3B1 (SF3B1 mt). A hallmark of the MDS-RS bone marrow (BM) is the progressive accumulation of ring sideroblasts (RS), erythroblasts displaying perinuclear mitochondria loaded with aberrant ferritin-iron complexes. Whilst several in vitro and in vivo model systems exist for studying the impact of SF3B1 mt on erythropoiesis and RS development, primary SF3B1 mt erythroid biology remains largely unexplored due to the inability to purify live SF3B1 mt cells or fully replicate BM conditions in vitro. To address this issue, we developed an innovative two-step method to isolate live ring sideroblasts from SF3B1 mt MDS-RS BM aspiration material with extremely high purity (as determined through droplet digital PCR-based genotyping [Fig. 1A] and morphology-based detection through Prussian blue staining [Fig. 1B,C]). Unexpectedly, evaluation of matching peripheral blood samples showed that circulating ring sideroblasts are strikingly common in MDS-RS (Fig. 1D), with their abundance being significantly positively associated with clinically-determined BM RS frequencies and serum erythropoietin levels, as well as negatively associated with hemoglobin levels. Through high-throughput Chromium 3'-based single-cell RNA sequencing (scRNAseq) analysis of purified RS, we then showed that these cells comprise a heterogeneous population encompassing all stages of the erythroid differentiation continuum, from early progenitors to orthochromatic erythroblasts (Fig. 1E). The RS transcriptome was shown to be dynamically regulated towards the maintenance of cell survival during late terminal erythroid differentiation (exemplified through parkin 1 [PINK1] expression), with SF3B1 K700E erythroblasts employing multiple strategies to preserve homeostasis despite undergoing extreme oxidative stress. These observations were confirmed through a parallel whole-transcript RNAseq investigation comprising CD34 + and GPA +-enriched samples obtained from normal bone marrow (NBM) donors and SF3B1 K700E MDS-RS patients, as well as purified RS samples. This bulk RNAseq experiment validated the RS transcriptomic signature observed in scRNAseq (Fig. 1F) and allowed for a detailed investigation of RNA splicing. SF3B1 K700E-associated alternative splicing in CD34 + and RS was consistent with previous literature, but also highly context-dependent and with substantial changes in scope and magnitude throughout erythroid differentiation (Fig. 1G-I). Finally, we substantiated these RNAseq results through Tandem Mass Tag-based semi-quantitative proteomic analysis of purified RS and GPA-enriched cells from NBM donors and MDS-RS patients. We confirmed that ring sideroblast survival is heavily dependent on redox balance modulation and suppression of ER stress via an increased dependence on glutamine, mirroring the molecular mechanisms observed in malignancy. Additionally, our data strongly indicate that the RS population is a major modulator of the MDS-RS BM microenvironment due to expression of stress factors (with particular emphasis on GDF15, erythroferrone and IL-18). In conclusion, our integrative analysis of primary RS constitutes a unique platform for the study of MDS-RS, with special interest for the investigation of potential drivers of disease severity or treatment avenues. Figure 1 Figure 1. Disclosures Kretzschmar: Vanadis Diagnostics, a PerkinElmer company.: Current Employment.

Download Full-text