C-Kit Signaling Blocks B Cell Differentiation of Common Lymphoid Progenitor Cells by Negatively Regulating Stat5-Dependent Activation of Ebf Expressionc-Kit Signaling Blocks B Cell Differentiation of Common Lymphoid Progenitor Cells by Negatively Regulating Stat5-Dependent Activation of Ebf1 Expression

Abstract Abstract 1209 B-lymphocyte commitment from common lymphoid progenitor cells (CLP) is orchestrated by a number of transcription factors and external cues such as IL-7, stem cell factor (SCF) and Flt3 ligand. IL-7 signaling plays a critical role in promoting B-cell differentiation by inducing the expression of transcription factor Ebf1 via the Jak-Stat5 signaling pathway (Kikuchi et al., 2007; Tsapogas et al., 2011). Previously, it has been shown that Flt3 ligand acts synergistically with IL-7 during the earliest stages of B-cell commitment (CLP and pre-pro-B fraction)(Åhsberg et al., 2010), but is downregulated by the pre-B stage to ensure normal development of B lymphocytes (Holmes et al., 2006). The role of SCF signaling in this early cell fate decision, however, remains unclear. Here, we demonstrate that SCF dominantly inhibits IL-7-induced B-cell differentiation from CLP by negatively regulating the Jak-Stat5 signaling pathway. Co-culture of bone marrow Lin-Kit+Sca-1+(LSK) cells with OP9 feeder cells in the presence of IL-7 (10 ng/ml) and SCF (10 ng/ml) resulted in 1.5–2-fold reduction in the frequency and absolute numbers of B220+CD19+ cells compared to cultures stimulated with IL-7 alone. To test whether c-Kit signaling inhibits B cell commitment in vivo, BM progenitor cells overexpressing a constitutively active form of KIT (MSCV-Kit-V814D-IRES-VEX) were transplanted into lethally irradiated mice. VEX+ cells in reconstituted mice had a very limited potential for B-lymphoid lineage differentiation, as judged by severe reduction in the frequency of B220+ cells in BM at 7 weeks post-transplant [B220+CD19-IgM-: 1.5%±0.3 (control) vs 0.6%±0.2 (V814D), p<0.05, n=6); B220+CD19+: 19.4%± 2.7 vs 2.1%±0.6, p<0.001; CD19+IgM+: 6.7%±1.3 vs 0.9%±0.3, p<0.01)]. Analysis of CLP cells isolated from IL-7−/− mice revealed that co-culture with IL-7 and SCF inhibited IL-7-induced upregulation of Ebf1 as well as other Stat5 target genes (Socs3, Cish, Osm). Furthermore, short-term treatment of BM cells isolated from IL-7−/− mice with a combination of SCF and IL-7 resulted in a significant reduction of phospho-Stat5 in CLP cells compared to cells treated with IL-7 alone. Class III receptor tyrosine kinases such as Flt3 and c-Kit are able to activate multiple signaling pathways via a juxtamembrane SH2-docking site. Mutation of the SH2 domain in the context of the Kit-V814D mutation (Kit-V814D-Y567/569F) did not rescue B-cell development when this mutant protein was expressed in primary murine bone marrow cells in vivo. In contrast, expression of a mutant form of Kit-V814D that inhibited binding of the SH2B adapter protein 2 (APS) (Kit-V814D-I570A/L937A) rescued B-lymphopoiesis, indicating that a signaling pathway coupled to APS dominantly suppresses B cell commitment when CLP are co-stimulated with SCF and IL-7. Based on the observations that APS is a known adapter for the E2-ubiquitin ligase, c-Cbl, which has been shown to negatively regulate Stat5 in other systems, we speculate that c-Kit signaling inhibits IL-7-induced Stat5 activation via an APS-Cbl pathway during early B-cell development. Further characterization of this inhibitory developmental pathway will be discussed. Disclosures: No relevant conflicts of interest to declare.

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Trisomy of the Down Syndrome Critical Region Suppresses Precursor B-Cell Differentiation and Promotes B-Cell Transformation Associated with Altered Expression of Polycomb Repressor Complex 2 Targets

Blood ◽

10.1182/blood.v120.21.115.115 ◽

2012 ◽

Vol 120 (21) ◽

pp. 115-115

Author(s):

Andrew A. Lane ◽

Diederik van Bodegom ◽

Bjoern Chapuy ◽

Gabriela Alexe ◽

Timothy J Sullivan ◽

...

Keyword(s):

Bone Marrow ◽

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Cell Transformation ◽

B Cell Differentiation ◽

Wild Type ◽

Repressor Complex

Abstract Abstract 115 Extra copies of chromosome 21 (polysomy 21) is the most common somatic aneuploidy in B-cell acute lymphoblastic leukemia (B-ALL), including >90% of cases with high hyperdiploidy. In addition, children with Down syndrome (DS) have a 20-fold increased risk of developing B-ALL, of which ∼60% harbor CRLF2 rearrangements. To examine these associations within genetically defined models, we investigated B-lineage phenotypes in Ts1Rhr mice, which harbor triplication of 31 genes syntenic with the DS critical region (DSCR) on human chr.21. Murine pro-B cell (B220+CD43+) development proceeds sequentially through “Hardy fractions” defined by cell surface phenotype: A (CD24−BP-1−), B (CD24+BP-1−) and then C (CD24+BP-1+). Compared with otherwise isogenic wild-type littermates, Ts1Rhr bone marrow harbored decreased percentages of Hardy fraction B and C cells, indicating that DSCR triplication is sufficient to disrupt the Hardy A-to-B transition. Of note, the same phenotype was reported in human DS fetal liver B-cells, which have a block between the pre-pro- and pro-B cell stages (analogous to Hardy A-to-B). To determine whether DSCR triplication affects B-cell proliferation in vitro, we analyzed colony formation and serial replating in methylcellulose cultures. Ts1Rhr bone marrow (B6/FVB background) formed 2–3-fold more B-cell colonies in early passages compared to bone marrow from wild-type littermates. While wild-type B-cells could not serially replate beyond 4 passages, Ts1Rhr B-cells displayed indefinite serial replating (>10 passages). Ts1Rhr mice do not spontaneously develop leukemia, so we utilized two mouse models to determine whether DSCR triplication cooperates with leukemogenic oncogenes in vivo. First, we generated Eμ-CRLF2 F232C mice, which express the constitutively active CRLF2 mutant solely within B-cells. Like Ts1Rhr B-cells, (but not CRLF2 F232C B-cells) Ts1Rhr/CRLF2 F232C cells had indefinite serial replating potential. In contrast with Ts1Rhr B-cells, Ts1Rhr/CRLF2 F232C B-cells also engrafted into NOD.Scid.IL2Rγ−/− mice and caused fatal and serially transplantable B-ALL. Second, we retrovirally transduced BCR-ABL1 into unselected bone marrow from wild-type and Ts1Rhr mice and transplanted into irradiated wild-type recipients. Transplantation of transduced Ts1Rhr cells (106, 105, or 104) caused fatal B-ALL in recipient mice with shorter latency and increased penetrance compared to recipients of the same number of transduced wild-type cells. By Poisson calculation, the number of B-ALL initiating cells in transduced Ts1Rhr bone marrow was ∼4-fold higher than in wild-type animals (1:60 vs 1:244, P=0.0107). Strikingly, transplantation of individual Hardy A, B, and C fractions after sorting and BCR-ABL1 transduction demonstrated that the increased leukemia-initiating capacity almost completely resides in the Ts1Rhr Hardy B fraction; i.e., the same subset suppressed during Ts1Rhr B-cell differentiation. To define transcriptional determinants of these phenotypes, we performed RNAseq of Ts1Rhr and wild-type B cells in methylcellulose culture (n=3 biologic replicates per genotype). As expected, Ts1Rhr colonies had ∼1.5-fold higher RNA abundance of expressed DSCR genes. We defined a Ts1Rhr signature of the top 200 genes (false discovery rate (FDR) <0.25) differentially expressed compared with wild-type cells. Importantly, this Ts1Rhr signature was significantly enriched (P=0.02) in a published gene expression dataset of DS-ALL compared with non-DS-ALL (Hertzberg et al., Blood 2009). Query of >2,300 signatures in the Molecular Signatures Database (MSigDB) C2 Chemical and Genetic Perturbations with the Ts1Rhr signature identified enrichment in multiple gene sets of polycomb repressor complex (PRC2) targets and H3K27 trimethylation. Most notably, SUZ12 targets within human embryonic stem cells were more highly expressed in Ts1Rhr cells (P=1.2×10−6, FDR=0.003) and the same SUZ12 signature was enriched in patients with DS-ALL compared to non-DS-ALL (P=0.007). In summary, DSCR triplication directly suppresses precursor B-cell differentiation and promotes B-cell transformation both in vitro and by cooperating with proliferative alterations such as CRLF2 activation and BCR-ABL1 in vivo. Pharmacologic modulation of H3K27me3 effectors may overcome the pro-leukemogenic effects of polysomy 21. Disclosures: No relevant conflicts of interest to declare.

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A Putative Role for VEGFR-1 (FLT-1) in B Cell Commitment and Differentiation.

Blood ◽

10.1182/blood.v108.11.1152.1152 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1152-1152

Author(s):

Rita Fragoso ◽

Catia Igreja ◽

Claudia Appleton ◽

Alexandra Henriques ◽

Nuno Clode ◽

...

Keyword(s):

Bone Marrow ◽

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Peripheral Blood ◽

Myeloid Differentiation ◽

B Cell Differentiation ◽

Cd34 Cells ◽

Cell Commitment

Abstract VEGF and its receptors are expressed in the hematopoietic system. A role for FLT-1 in particular was described in monocyte-macrophage migration and lineage differentiation (Sawano A et al, 2001), megakaryocytes maturation (Casella I et al, 2003) and dendritic cell differentiation (Dikov M et al, 2005). Given that the expression of this receptor in the lymphoid lineage is not known, we to studied FLT-1 expression and a putative function in normal lymphoid progenitors. To address this question we induced in vitro CD34+ cells differentiation into the B cell lineage using a well established assay (on S17 stromal cells). With this approach, we observed that FLT-1 is expressed throughout B cell differentiation increasing along the differentiation process, and reaching its highest at the “immature B cell” stage. We also neutralized FLT-1 during B cell differentiation in vitro. Surprisingly, in the presence of the FLT-1 neutralizing antibody (6.12 monoclonal Ab, from ImClone systems), at the end of the assays (4 different experiments) a significantly higher number of CD19+ cells (mainly immature B cells) were detected. Analyzing some of the transcription factors known to be involved in the commitment and differentiation of lymphoid B cells, we observed that the expression of PU.1, Pax5 and E47 was up-regulated by FLT-1 neutralization. Next, given that FLT-1 function was mainly associated with cell migration, and since it is expressed in B cells that are ready to exit the bone marrow into secondary lymphoid organs, we reasoned that FLT-1 might have a role in B cells exit from the bone marrow. For this purpose, we treated mice with the FLT-1 neutralizing Ab for 3 days and analyzed B cells levels in bone marrow and peripheral blood. FLT-1 neutralization led to a significant decrease (p<0.05) in B cells in the bone marrow and peripheral blood. Taken together, our data supports a clear role for FLT-1 in B cell commitment. To understand if VEGF/PlGF signalling through FLT-1 promotes myeloid differentiation, suppresses B cell differentiation or simply regulates the quiescent state of hematopoietic stem cells, we differentiated in vitro CD34+/FLT-1− cells and CD34+/FLT-1+ cells (10% of CD34+ cells) using the assay described above. Interestingly, CD34+/FLT-1− differentiation in vitro largely promoted B cell differentiation, while CD34+/FLT-1+ cells originated mostly myeloid cell differentiation. We are currently exploiting the molecular basis whereby FLT-1 signalling may impair B cells commitment and possibly promotes myeloid differentiation.

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Transcriptional Repressor Gfi-1 Integrates Cytokine-Receptor Signals Controlling B Cell Differentiation.

Blood ◽

10.1182/blood.v106.11.204.204 ◽

2005 ◽

Vol 106 (11) ◽

pp. 204-204 ◽

Cited By ~ 3

Author(s):

Chozhavendan Rathinam ◽

Christoph Klein

Keyword(s):

Bone Marrow ◽

Transcription Factors ◽

Cell Differentiation ◽

B Cells ◽

B Cell ◽

B Cell Development ◽

B Cell Differentiation ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Deficient Mice

Abstract Hematopoietic stem cell differentiation is specified by cytokines and transcription factors but the mechanisms controlling instructive and permissive signalling networks are poorly understood. We provide evidence that common lymphoid progenitor (CLP)-1-dependent IL7-receptor mediated B cell differentiation is critically controlled by the transcriptional repressor Gfi1. Gfi1 is expressed selectively in CLP1 but not in CLP2 cells. Furthermore, in Gfi1-deficient hematopoiesis, CLP1 cells are significantly reduced, while CLP2 cells are present in normal numbers. Hardy fractions B/C, proposed to represent IL7-sensitive stages of early B cell development, are also reduced in Gfi1-deficient bone marrow. In contrast to bone marrow B cells, the phenotype of Gfi1-deficient thymic B cells was comparable to wildtype mice, suggesting that IL7-mediated signalling is perturbed. This hypothesis was further substantiated by a side-by-side comparison of Gfi1- and IL7-deficient mice; both knockout mice revealed a similar B cell phenotype in bone marrow and thymus. In vitro, Gfi1-deficient Sca1+ckit+lin- hematopoietic stem cells did not differentiate into B220+IgM+ B cells in the presence of SCF and IL7, suggesting that instructive IL7-mediated signals are not operative in the absence of Gfi1. Upon retroviral Gfi1 gene transfer, B differentiation was re-established Furthermore, whereas a combination of SCF and IL7 maintained the viability and induced proliferation of Gfi1-deficient HSC, their viability and proliferative capacity was significantly reduced in the presence of IL7 only, suggesting that both trophic and proliferative responses to IL7 depend on Gfi1. In addition, RT-PCR analysis revealed that in early B cell development the coordinated upregulation of the B cell specific transcription factors E2A, EBF, and Pax5 was significantly reduced in sorted Hardy fractions obtained from Gfi1-deficient mice. We reasoned that defective IL7-mediated responses might be due to defective Jak/Stat signalling. Indeed, phosphorylation of STAT5 was almost undectable in Gfi1-deficient B-progenitor cells. This may be due to enhanced expression levels of SOCS3 as determined by RT-PCR and Western Blot analysis. Thus, Gfi1 selectively specifies IL7-dependent development of B cells from CLP1 progenitors, providing clues to the transcriptional networks integrating cytokine signals and lymphoid differentiation.

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Individual CD34+CD38lowCD19−CD10− Progenitor Cells From Human Cord Blood Generate B Lymphocytes and Granulocytes

Blood ◽

10.1182/blood.v89.10.3554 ◽

1997 ◽

Vol 89 (10) ◽

pp. 3554-3564 ◽

Cited By ~ 88

Author(s):

Anna C. Berardi ◽

Eric Meffre ◽

Françoise Pflumio ◽

Andre Katz ◽

William Vainchenker ◽

...

Keyword(s):

Cell Differentiation ◽

B Cells ◽

Cord Blood ◽

B Cell ◽

Progenitor Cells ◽

B Cell Differentiation ◽

Human Cord Blood ◽

B Lymphoid

Abstract Identification of human hematopoietic stem cells and analysis of molecular mechanisms regulating their function require biological assays that permit differentiation in all hematopoietic lineages simultaneously. In this study, we established conditions that permit the joint expression of the B-lymphoid and myeloid potential from cord blood-derived CD34+CD38lowCD19−/CD10− primitive progenitors that lack B-specific markers and transcripts. When cocultured during 6 weeks with the murine stromal cells MS-5 in the absence of exogenous human cytokines, CD34+CD38lowCD19−CD10− cells generated a high number of CD19+ B cells. Virtually all of these cells expressed a CD34−CD10+CD19+cIgM− phenotype of late pro-B cells and transcripts of Pax-5, λ-like, and μ chain were detected. We further show that 7% of CD34+CD38lowCD19− cells from cord blood, when grown individually with MS-5 cells, generated both CD19+ and CD11b+ cells after 6 weeks. Efficient B-cell differentiation was also observed in vivo after transplantation of human cord blood-derived unfractionated mononuclear cells or CD34+CD19+CD10− cells into immune-deficient mice. In contrast to the in vitro situation, all stages of B-cell differentiation were observed in vivo, including pro-B, pre-B, and sIgM+ B cells. Interestingly, human progenitors with the ability to differentiate along both B-lymphoid and granulocytic pathways were also detected among human CD34+CD38low cells in the marrow of chimeric mice 6 to 7 weeks after transplantation. Both in vitro and in vivo systems will offer an invaluable tool to further identify the lymphoid and myeloid potentialities of primitive progenitor cells isolated from fetal as well as adult human hematopoietic tissues and characterize stromal-derived signals that regulate their function.

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Individual CD34+CD38lowCD19−CD10− Progenitor Cells From Human Cord Blood Generate B Lymphocytes and Granulocytes

Blood ◽

10.1182/blood.v89.10.3554.3554_3554_3564 ◽

1997 ◽

Vol 89 (10) ◽

pp. 3554-3564 ◽

Cited By ~ 3

Author(s):

Anna C. Berardi ◽

Eric Meffre ◽

Françoise Pflumio ◽

Andre Katz ◽

William Vainchenker ◽

...

Keyword(s):

Cell Differentiation ◽

B Cells ◽

Cord Blood ◽

B Cell ◽

Progenitor Cells ◽

B Cell Differentiation ◽

Human Cord Blood ◽

B Lymphoid

Identification of human hematopoietic stem cells and analysis of molecular mechanisms regulating their function require biological assays that permit differentiation in all hematopoietic lineages simultaneously. In this study, we established conditions that permit the joint expression of the B-lymphoid and myeloid potential from cord blood-derived CD34+CD38lowCD19−/CD10− primitive progenitors that lack B-specific markers and transcripts. When cocultured during 6 weeks with the murine stromal cells MS-5 in the absence of exogenous human cytokines, CD34+CD38lowCD19−CD10− cells generated a high number of CD19+ B cells. Virtually all of these cells expressed a CD34−CD10+CD19+cIgM− phenotype of late pro-B cells and transcripts of Pax-5, λ-like, and μ chain were detected. We further show that 7% of CD34+CD38lowCD19− cells from cord blood, when grown individually with MS-5 cells, generated both CD19+ and CD11b+ cells after 6 weeks. Efficient B-cell differentiation was also observed in vivo after transplantation of human cord blood-derived unfractionated mononuclear cells or CD34+CD19+CD10− cells into immune-deficient mice. In contrast to the in vitro situation, all stages of B-cell differentiation were observed in vivo, including pro-B, pre-B, and sIgM+ B cells. Interestingly, human progenitors with the ability to differentiate along both B-lymphoid and granulocytic pathways were also detected among human CD34+CD38low cells in the marrow of chimeric mice 6 to 7 weeks after transplantation. Both in vitro and in vivo systems will offer an invaluable tool to further identify the lymphoid and myeloid potentialities of primitive progenitor cells isolated from fetal as well as adult human hematopoietic tissues and characterize stromal-derived signals that regulate their function.

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