Involvement of a B1 Progenitor In a Murine Model of BCR-FGFR1 Induced Leukemogenesis.

Abstract Abstract 1221 Constitutive activation of the FGFR1 kinase domain through rearrangement with dimerization domains from various proteins leads to an atypical myeloproliferative disease (MPD). Although the majority of these rearrangements result in development of T-cell leukemia/lymphoma, in the case of the BCR-FGFR1 rearrangement the phenotype is predominantly a myeloid and B cell MPD. To investigate the etiology of this disease, a murine model of BCR-FGFR1 was created using a bone marrow transduction/transplantation approach. Consistent with the human disease, recipient mice developed clear myeloproliferations marked by CML-like basophilia as well as extramedullary leukemic transformation containing myelo- or lymphoblasts, with neoplasms of myeloid and B cell lineages. Flow analysis demonstrated a CD43+ phenotype in leukemic B cells suggesting a block in the differentiation of pro-B progenitor cells. A cell line (BBC1) has now been established with this immature B cell immunophenotype albeit B220-, consistent with a recently described B1 progenitor. These cells can differentiate in vitro with characteristics of both macrophage and dendritic lineages. When BBC1 cells are injected into normal mice, leukemogenesis marked by a hypercellular bone marrow and splenomegaly is induced. Although BBC cells show the same progenitor B cell immunophenotype seen in the parent cells throughout the hematopoietic system, tumor cells in the peritoneum lose CD43 expression, down-regulate CD19 expression and upregulate CD11b and F4/80 demonstrating a capacity to differentiate in this environment. In this model, therefore, the etiology of BCR-FGFR1 disease closely mimics that seen in humans and we have identified a likely B cell subtype involved in leukemic transformation. Disclosures: No relevant conflicts of interest to declare.

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SRC Is a Critical Signaling Mediator in FLT3-ITD Positive AML.

Blood ◽

10.1182/blood.v114.22.477.477 ◽

2009 ◽

Vol 114 (22) ◽

pp. 477-477

Author(s):

Hannes Leischner ◽

Rebekka Grundler ◽

Corinna Albers ◽

Anna Lena Illert ◽

Karsten Spiekermann ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Point Mutations ◽

Kinase Domain ◽

Sh2 Domain ◽

Control Group ◽

Tyrosine Kinase Domain ◽

Myeloproliferative Disease ◽

Activating Mutations ◽

Tandem Duplications

Abstract Abstract 477 Activating mutations of FLT3 are frequent in patients with AML. Two types of mutations are most common: Internal tandem duplications (ITD) of the juxtamembrane domain in approximately 30% of patients and point mutations within the second tyrosine kinase domain (TKD) in about 7% of AML patients. Patients carrying the FLT3-ITD mutation have a significantly worse prognosis whereas FLT3-TKD mutations do not appear to influence the clinical outcome. Studies have shown that mice receiving a transplant of bone marrow expressing FLT3 ITD develop a myeloproliferative disease. In contrast, mice which were transplanted with FLT3 TKD infected bone marrow, suffer from a lymphoid disease. Thus, both FLT3 mutations seem to exert different biological functions. Interestingly, FLT3-ITD but not FLT3-TKD or FLT3-WT leads to a strong activation of the STAT5 signaling pathway. Therefore, STAT5 activation may be responsible for the observed differences in biology. Here we investigated the signalling pathways leading to STAT5 activation downstream of FLT3-ITD. FLT3-ITD does not bind STAT5 directly nor does it activate the classical JAK2 pathway. Instead FLT3-ITD utilizes c-Src to activate STAT5. Co-immunoprecipitations and GST pull downs revealed a strong and exclusive interaction between Src and FLT3 ITD, which is mediated by the Src-SH2 domain. This interaction is absent in FLT3-TKD or FLT3-WT after ligand stimulation. The sequence duplication in FLT3-ITD leads to additional potential Src-SH2 binding sites. We identified tyrosines 589 and 591 of FLT3-ITD to be essential for Src binding and subsequent STAT5 activation. Specific Src inhibitors or Src-siRNA blocked STAT5 activation and growth induced by FLT3-ITD but not FLT3-TKD. FLT3-ITD positive cells with a stable Src knockdown injected into syngenic mice led to a leukemic disease with a significant delayed onset and prolonged survival in comparison to the control group. Finally, a combination of FLT3 and Src inhibitors was tested. This combination was highly efficient in FLT3-ITD positive cells but not in FLT-TKD positive cells. Together these findings show that Src plays an important role in the signalling of FLT3-ITD but not FLT3-TKD. Thus, Src might be an interesting therapeutic target for FLT3-ITD positive AML. Disclosures: No relevant conflicts of interest to declare.

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Combination of c-SRC and FLT3 Inhibitors Has An Additive Inhibitory Effect on FLT3 ITD but Not on FLT3 TKD Positive Cells

Blood ◽

10.1182/blood.v116.21.2892.2892 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2892-2892 ◽

Cited By ~ 1

Author(s):

Hannes Leischner ◽

Rebekka Grundler ◽

Corinna Albers ◽

Anna Lena Illert ◽

Katharina Götze ◽

...

Keyword(s):

Bone Marrow ◽

Growth Inhibition ◽

Conflicts Of Interest ◽

Point Mutations ◽

Inhibitory Effect ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Myeloproliferative Disease ◽

Activating Mutations ◽

Flt3 Inhibitors

Abstract Abstract 2892 Activating mutations of FLT3 are frequent in patients with AML. Two types of mutations are most common: Internal tandem duplications (ITD) of the juxtamembrane domain in approximately 30% of patients and point mutations within the second tyrosine kinase domain (TKD) in about 7% of AML patients. Patients carrying the FLT3-ITD mutation have a significantly worse prognosis whereas FLT3-TKD mutations do not appear to influence the clinical outcome. Studies have shown that mice receiving a transplant of bone marrow expressing FLT3 ITD develop a myeloproliferative disease. In contrast, mice which were transplanted with FLT3 TKD infected bone marrow, suffer from a lymphoid disease. Thus, both FLT3 mutations seem to exert different biological functions. Interestingly, FLT3-ITD but not FLT3-TKD or FLT3-WT leads to a strong activation of the STAT5 signaling pathway. Recently we have shown that c-SRC is the crucial signaling mediator of FLT3 ITD to activate STAT5. Based on these findings we investigated the effect of FLT3 inhibitors (Midostaurin, Sorafenib and Sunitinib) in combination with c-SRC inhibitors (Dasatinib and PD166-326) on FLT3 ITD and FLT3 TKD murine and human cell lines as well as on primary patient material. In FLT3 ITD expressing murine myeloid 32D cells c-SRC inhibitors in combination with FLT3 inhibitors showed clear additive effects on growth inhibition, apoptosis and activation of STAT5. In contrast, c-SRC inhibitors had no additional effects in FLT3 TKD expressing cells. Accordingly, a strong additive effect of c-SRC and FLT3 inhibitors could also be demonstrated in the FLT3 ITD positive human AML cell line MV4-11. Finally FLT3 ITD and FLT3 TKD positive primary human AML cells were investigated. We were able to detect a significant additional growth inhibition of FLT3 ITD positive human cells by combining c-SRC and FLT3 inhibitors. In contrast, no further growth inhibition was observed by c-SRC inhibition in primary AML cells expressing the FLT3 TKD mutation. Together our results confirm c-SRC as a crucial signaling mediator in FLT3-ITD but not FLT3-TKD positive AML. The combination of FLT3 and c-SRC inhibitors warrants further investigation in FLT3 ITD positive AML. Disclosures: No relevant conflicts of interest to declare.

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IL-6 Deficiency Reverses Leukemic Transformation in an MDS Mouse Model

Blood ◽

10.1182/blood-2020-137026 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 36-36

Author(s):

Yang Mei ◽

Yijie Liu ◽

Xu Han ◽

Jing Yang ◽

Peng Ji

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Inflammatory Cytokines ◽

Conflicts Of Interest ◽

Double Knockout ◽

Leukemic Transformation ◽

Hematopoietic Stem ◽

Cell Counts ◽

Age Related

Myelodysplastic syndromes (MDS) are a group of age-related myeloid malignancies that are characterized by ineffective hematopoiesis and increased incidence of developing acute myeloid leukemia (AML). The mechanisms of MDS to AML transformation are poorly understood, which is partially due to the scarcity of leukemia transformation mouse models. Recently, we established a mDia1/miR146a double knockout (DKO) mouse model mimicking human del(5q) MDS. DKO mice present with pancytopenia with aging due to myeloid suppressive cell (MDSC) expansion and over-secretion of pro-inflammatory cytokines including TNF-a and interlukine-6 (IL-6). In the current study, we found that most of the DKO mice underwent leukemic transformation at 12-14 months of age. The bone marrow of these mice was largely replaced by c-Kit+ blasts in a background of fibrosis. Flow cytometry analysis and in vitro colony formation assay demonstrated that hematopoietic stem progenitor cells (HSPCs) in DKO bone marrow were dramatically declined. The leukemic DKO mice had elevated white blood cell counts and circulating blasts, which contributes to the myeloid cell infiltration in non-hematopoietic organs including liver and lung. Moreover, the splenocytes from DKO old mice efficiently reconstitute the hematopoiesis, but led to a 100% disease occurrence with rapid lethality in gramma irradiated recipient mice, suggesting the leukemic stem cells enriched in DKO spleen were transplantable. Given the significant roles of the inflammatory cytokines in the pathogenesis of the DKO mice, we crossed DKO mice with IL-6 knockout mice and generated mDia1/miR-146a/IL-6 triple knockout (TKO) mice. Strikingly, the TKO mice showed dramatic rescue of the leukemic transformation of the DKO mice in that all the aforementioned leukemic phenotypes were abolished. In addition, IL-6 deficiency normalized the cell comparts and prevented leukemic transplantation ability in TKO spleen. Single cell RNA sequencing analyses indicated that DKO leukemic mice had increased monocytic blast population with upregulation of Fn1, Csf1r, and Lgals1, that was completely diminished with IL-6 knockout. Through a multiplex ELISA, we found IL-6 deficiency attenuated the levels of multiple inflammatory cytokines in TKO serum. In summary, we report a mouse model with MDS leukemic transformation during aging, which could be reverted with the depletion of IL-6. Our data indicate that IL-6 could be a potential target in high risk MDS. Disclosures No relevant conflicts of interest to declare.

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Developmental Stage Specific B-Progenitor Expansion in Normal Fetal Bone Marrow Is Absent in Down Syndrome: Implications for Infant ALL

Blood ◽

10.1182/blood.v124.21.4331.4331 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4331-4331

Author(s):

Anindita Roy ◽

Georg Bohn ◽

Katerina Goudevenou ◽

Gillian Cowan ◽

Neha Bhatnagar ◽

...

Keyword(s):

Bone Marrow ◽

B Cell ◽

Cell Development ◽

B Cell Development ◽

Fetal Life ◽

Second Trimester ◽

Leukemic Transformation ◽

Developmentally Regulated ◽

Cd34 Cells

Abstract Although distinct fetal and adult B-cell lineage development has been demonstrated in murine studies, human fetal B-lymphopoiesis remains poorly understood. Previous work from our lab identified a population of PreProB progenitors (CD34+CD19+CD10-) in fetal liver (FL), co-existing with adult-type CD34+CD19+CD10+ ProB progenitors (Roy, A., Cowan G et al. PNAS: 109, 17579-17584; 2012). This supports other work demonstrating a PreProB progenitor population in normal cord blood (Sanz, E., et al. PNAS: 107, 5925-5930; 2010) and suggests that fetal pathways of B-cell development are also likely to be markedly different from adult bone marrow (AdBM) in humans. FL PreProB progenitors give rise solely to B-cells in vitro and display a gene expression pattern of successive activation of B-cell transcription factors as determined by Fluidigm RQ-PCR. In addition, increasing evidence indicates that infant ALL and many cases of childhood ALL arise in fetal life, suggesting that ontogeny-related changes in B-cell development may provide the cellular and microenvironmental context for in utero leukemia initiation. We therefore investigated B-cell development in normal human fetal BM from the onset of BM hematopoiesis in late first trimester. The composition and function of the early lymphoid and committed B-progenitor compartments of fetal BM(12-22 weeks; n=20) were compared with FL (n=25) at the same gestation, paediatric (Paed) BM (n=6) and AdBM (n=7), by multiparameter flow cytometry, differentiation in stromal co-culture assays and clonogenic assays. All stages of B-cell development were demonstrable in human fetal BM. However, there was a significantly higher frequency of B-progenitors in fetal BM (45.8±2.7% of CD34+ cells) compared to FL (10.3±0.97%; p<0.0001), PaedBM (28.2±4.2%; p=0.001) and AdBM (25.8±2.8%; p<0.0001). As in FL, both CD34+CD19+CD10- (PreProB) and CD34+CD19+10+ (ProB) progenitors were identified in fetal BM. PreProB progenitors were significantly higher in fetal BM (21.9±2.3% of CD34+ cells) compared to FL (3.8±0.4%), PaedBM (4.2±0.9%) and AdBM (3.4±0.9%) (p<0.0001 for all). Fetal BM PreProB progenitors gave rise solely to B-cells when co-cultured on MS5 stromal cells with FLT3L, SCF and IL7 in contrast to multilineage output of fetal BM HSC and LMPP cultured under identical conditions. Furthermore, fetal BM Lin-CD10-CD34+ cells cultured in vitro acquire CD19 expression before CD10 expression (n=6) consistent with a unique, fetal-specific B-cell differentiation pathway. The progressive decline in the proportion of PreProB progenitors from 47.8% to 36.8%, 14.8% and 13% of total B-progenitors in fetal BM, FL, PaedBM and AdBM respectively points towards a developmental stage-specific emergence of these progenitors. Finally, the fetal BM B-progenitor: B-cell ratio falls rapidly from ~4:1 at 12 weeks gestation to 1:1 at 18 weeks gestation as mature B-cell production in fetal BM gradually increases. This is consistent with a developmentally-regulated drive to B-progenitor proliferation, at the expense of differentiation, early in the second trimester which might represent a target population vulnerable to leukemic transformation in fetal life. Since children with Down syndrome (DS) do not develop infant ALL, we examined DS fetal BM as they may lack susceptible target cells for leukemic transformation. Consistent with this, PreProB progenitors in DS fetal BM (n=7) were >6-fold lower than normal fetal BM (3.3±0.8% vs. 21.9±2.3% of CD34+ cells; p<0.0001). Furthermore, CD19+ cells with an infant ALL-associated immunophenotype (CD19+CD10-CD20-) are detectable in normal fetal BM mononuclear cells, but are rarely found in DS fetal BM. Conclusion: There is a marked expansion of PreProB progenitors in normal second trimester human fetal BM which is virtually absent in DS fetal BM, in normal PaedBM and in normal AdBM. We suggest that developmentally-regulated, functional and molecular characteristics of these fetal-specific B-progenitors may provide the 'oncogenic' cellular context necessary to co-operate with genetic events, such as MLL rearrangements, to induce ALL in infants without DS. Disclosures No relevant conflicts of interest to declare.

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EXEL-8232, a Small Molecule JAK2 Inhibitor, Effectively Treats Thrombocytosis and Extramedullary Hematopoiesis in a Murine Model of Myeloproliferative Disease Induced by MPLW515L

Blood ◽

10.1182/blood.v112.11.3741.3741 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3741-3741

Author(s):

Gerlinde Wernig ◽

Michael G. Kharas ◽

Dena S. Leeman ◽

Rachel Okabe ◽

Maricel Gozo ◽

...

Keyword(s):

Bone Marrow ◽

Small Molecule ◽

Murine Model ◽

Extramedullary Hematopoiesis ◽

Response To Treatment ◽

Myeloproliferative Disease ◽

Wild Type ◽

Murine Bone Marrow

Abstract Approximately 50% of patients with essential thrombocythemia (ET) or myelofibrosis (MF) lack activating mutations in JAK2. Among these patients, ~10% harbor an activating mutation in the thrombopoietin receptor, MPLW515L. We have reported that expression of MPLW515L in a murine bone marrow transplant model recapitulates many features of ET and MF, including severe fibrosis and thrombocytosis, that are not observed in the JAK2V617F model. These observations provide an opportunity to assess the efficacy of small molecule JAK2 inhibitors on a myeloproliferative disease (MPD) induced by MPLW515L in vivo, and to determine whether such inhibitors attenuate thrombocytosis. We have tested EXEL-8232 for efficacy in suppression of thrombocytosis in vivo, and for its ability to attenuate JAK2V617F-negative MPD mediated by MPLW515L. EXEL-8232 is a potent small molecule inhibitor of JAK2 and is structurally similar to XL019, a compound currently in clinical trials for MF and polycythemia vera. EXEL-8232 is selective for JAK2 with a biochemical IC50 of 2 nM, and abolished constitutive phosphorylation of JAK2 and STAT5, as well as cytokine-independent growth, of Ba/F3 cells in vitro. After disease was established 12 days post-bone marrow transplantation, EXEL-8232 was administered for 28 days q12h by oral gavage at doses of 30mg/kg or 100mg/kg respectively. Animals treated with 100mg/kg normalized high platelet counts in excess of2 million/ml and normalized leukocytosis from a median of 134,000/ml in vehicle treated controls. Furthermore, drug treatment eliminated extramedullary hematopoiesis in the spleen, as well as bone marrow fibrosis. Of note, EXEL-8232 had no impact on erythrocytosis in diseased animals or in wild type controls, and wild type animals treated with either dosage of 30mg/kg or 100mg/kg did not develop thrombocytopenia. Consistent with these clinical responses, the surrogate endpoints for response to treatment included a reduction of genomic disease burden in the 100mg/kg treated arm (p<0.05) as assessed by quantitative PCR, a reduction of endogenous colony growth, as well as a inhibition of activation of P-STAT5, P-STAT3 and P-S6K1 kinase as assessed by flow cytometry in immature erythroid and myeloid primary cells both in vitro and upon treatment in vivo. We conclude that EXEL-8232 has efficacy in treatment of thrombocytosis in vivo in a murine model of ET and MF, and may be of therapeutic benefit for patients with JAK2V617F-negative MPD.

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The MiR-23a MicroRNA Cluster Inhibits B Cell Development.

Blood ◽

10.1182/blood.v114.22.1465.1465 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1465-1465

Author(s):

Jason Mullenix ◽

Kimi Y Kong ◽

Kristin Severns Owens ◽

Jason Rogers ◽

Shannon FitzPatrick ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Fate ◽

Conflicts Of Interest ◽

Myeloid Cells ◽

Mature Mirnas ◽

Cell Development ◽

B Cell Development

Abstract Abstract 1465 Poster Board I-488 The miR-23a microRNA (miRNAs) cluster inhibits both [ITALIC]in vitro[/ITALIC] and [ITALIC]in vivo[/ITALIC] B cell development. When murine hematopoietic progenitor cells expressing the 23a cluster miRNAs were cultured in B cell promoting conditions we observed over a five-fold decrease in the generation of CD19+ B cells compared to control cultures. Conversely, we observed over a five-fold increase in CD11b+ myeloid cells. When irradiated mice were transplanted with bone marrow expressing the miR-23a cluster we observed a two-fold decrease in bone marrow and splenic B cells, 8 weeks post-transplant compared to control mice. The miR-23a cluster codes for a single pri-transcript, which when processed yields three mature miRNAs: miR-23a, miR-27a, and miR-24-2. All three mature miRNAs are more abundant in myeloid cells compared to other hematopoietic cells. In vitro miR-24 alone is necessary and sufficient to inhibit B cell development. The promoter for the cluster contains conserved binding sites for the essential myeloid transcription factors PU.1 and C/EBP alpha. Chromatin immunoprecipitations demonstrated that PU.1 and C/EBP alpha are associated with the promoter in myeloid cells. In addition, C/EBP alpha is bound to several highly conserved regions upstream of the promoter. Both PU.1 and C/EBP alpha promote myeloid development at the expense of lymphopoiesis. Our work suggests that the miR-23a cluster may be a critical downstream target of PU.1 and C/EBP alpha in the specification of myeloid cell fate. Although miRNAs have been identified downstream of PU.1 and C/EBP alpha in mediating the development of monocytes and granulocytes, the 23a cluster is the first downstream miRNA target implicated in the regulating lymphoid cell fate acquisition. We are currently identifying targets of miR-24 that may mediate the inhibitory effect on B lymphopoiesis. Disclosures No relevant conflicts of interest to declare.

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Loss Of BTG1 Function Promotes ETV6-RUNX1-Mediated Leukemic Transformation

Blood ◽

10.1182/blood.v122.21.2545.2545 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2545-2545

Author(s):

Esther J.H. Tijchon ◽

Liesbeth van Emst ◽

Dorette van Ingen Schenau ◽

Laurens T. van der Meer ◽

Owen Williams ◽

...

Keyword(s):

B Cell ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Lymphoblastic Leukemia ◽

Point Mutations ◽

Wild Type ◽

Leukemic Transformation ◽

Genetic Event ◽

Gene Deletions

Abstract Background Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy and is characterized by the acquisition of recurrent genetic aberrations, which include chromosomal translocations, submicroscopic deletions and point mutations. The t(12;21) ETV6-RUNX1 translocation is present in about 25% of all pediatric B-cell precursor ALL (BCP-ALL) cases and represents an early genetic event, which can already be detected in utero. However, ETV6-RUNX1 expression is insufficient to induce leukemia and requires other cooperating genetic lesions for BCP-ALL to develop. Results Using extensive genomic profiling of pediatric BCP-ALL we and others have demonstrated that several recurrent gene deletions can be found in ETV6-RUNX1-positive leukemia. These include focal deletions affecting the B-cell translocation gene 1 (BTG1), which is a member of the BTG/Tob family of anti-proliferation genes. BTG1 deletions are present in 9% of all BCP-ALL cases, but appear to be specifically enriched (20%) in ETV6-RUNX1-positive leukemia. BTG1 protein displays no intrinsic enzymatic activity but may act by recruiting effector molecules like protein arginine methyltransferase 1 (PRMT1) to specific transcription factors, including RUNX1 and ATF4. To investigate whether loss of BTG1 function cooperates with ETV6-RUNX1 in leukemic transformation we developed an in vitro transformation assay. To this end, primitive fetal liver progenitors (FLPs) were purified as c-Kit+ Ter119- cells from fetal livers of embryonic day 13.5 (E13.5) of C57Bl6/J wild-type and Btg1-/- mice. After transduction with control pMSCV-IRES-GFP (pMIG) and pMIG-ETV6-RUNX1 retrovirus, cells were serially replated in methylcellulose or liquid culture in the presence of cytokines SCF, FLT3L and IL-7. We observed a proliferative growth advantage of ETV6-RUNX1 over control virus, and in BTG1-deficient FLPs as compared to wild-type FLPs. Notably, the proliferative advantage of BTG1-deficient FLPs was even further enhanced by co-expression of ETV6-RUNX1. By immunoprecipitation from FLPs, we could demonstrate that endogenous PRMT1 binds to ETV6-RUNX1, and this interaction is lost in BTG1-deficient FLPs, arguing that BTG1 is required for the interaction between PRMT1 and ETV6-RUNX1. In summary, our data indicate that loss of BTG1 function promotes leukemic transformation induced by oncogenic fusion protein ETV6-RUNX1, which implies that BTG1 gene deletions can act as a cooperating event in ETV6-RUNX1-driven leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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Gene Therapy of Human Blnk Deficiency in NOD/SCID/Gc Ko Murine Model

Blood ◽

10.1182/blood.v118.21.4717.4717 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4717-4717

Author(s):

Chantal Lagresle-Peyrou ◽

Michèle Milili ◽

Julien Rouiller ◽

Capucine Picard ◽

Marina Cavazzana-Calvo ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Murine Model ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

B Cell Differentiation ◽

Cell Stage ◽

Immune Disorder ◽

Marrow Cells

Abstract Abstract 4717 Blnk deficiency is an autosomal recessive immune disorder characterized by the absence of B cells in periphery and the absence of any seric immunoglobulins due to an early blocage at the pro-B cell stage in the bone marrow. The very rare patients affected by blnk deficiency develop severe infections. In the murine model of the disease, a similar blocage in B cell development is described as well as susceptibility to infections and to pre-B lymphomas. A homozygote stop mutation in blnk gene was identified in a 8-yr old boy. Bone marrow cells analysis revealed that CD34+CD10+CD24-CD19- lymphoid progenitors were present as well as CD34+CD10+CD24+CD19- early B cells and CD34+CD19+ pro B cells. However, no surface IgM or seric Ig were detected in this patient. To demonstrate the implication of Blnk in the B-cell differentiation process, we transduced CD34+ sorted bone marrow cells from this patient with a lentiviral construct containing human wild type Blnk cDNA. The transduced cells were intravenously injected into irradiated NOD/SCID/IL2rg knock-out mice. Twelve weeks after transplantation, recipients were analysed. Human engraftment was detected in bone marrow and spleen. Among bone marrow human CD45+ cells, more than 80% were CD19+ and 6 to 8% express surface IgM. In the spleen, between 14 and 30% of CD19+ cells were detected. Eight to 42% of CD19+ cells expressed surface IgM. This is the first demonstration that Blnk is absolutely required for the differentiation of pro-B cells toward mature B cells. Disclosures: No relevant conflicts of interest to declare.

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Identification of immature and mature myeloma cells in the bone marrow of human myelomas

Blood ◽

10.1182/blood.v82.2.564.564 ◽

1993 ◽

Vol 82 (2) ◽

pp. 564-570 ◽

Cited By ~ 96

Author(s):

MM Kawano ◽

N Huang ◽

H Harada ◽

Y Harada ◽

A Sakai ◽

...

Keyword(s):

Bone Marrow ◽

Adhesion Molecules ◽

Proliferative Activity ◽

M Protein ◽

Color Analysis ◽

Myeloma Cells ◽

A Cell ◽

Immature Cells ◽

Human Myeloma Cells

Abstract With regard to the expression of adhesion molecules, human myeloma cells freshly isolated from bone marrow were heterogeneous. By two- color analysis with anti-VLA-5 antibody (PE staining) and FITC-labeled anti-CD38 antibody, we found all myeloma cells located at CD38-strong positive (CD38++) fraction and identified two subpopulations among these myeloma cells: CD38++ VLA-5-(VLA-5-) myeloma cells and CD38++ VLA- 5+ (VLA-5+) myeloma cells. To clarify the biologic character of these two subpopulations, the morphology, in vitro proliferative activity and in vitro M-protein secretion were examined in each fraction isolated by the purification procedure or a cell sorter. Morphologic examination showed that VLA-5- myeloma cells were mostly immature or plasmablastic and VLA-5+ cells were mature myeloma cells. Furthermore, VLA-5- myeloma cells proliferated markedly in vitro and responded to interleukin 6 (IL- 6), a growth factor for myeloma cells, while VLA-5+ myeloma cells showed very low uptakes of 3H-thymidine and no responses to IL-6 but secreted higher amounts of M-protein (immunoglobulin) in vitro significantly. Therefore, we could clarify here heterogeneity of human myeloma cells in the bone marrow with regard to the expression of VLA- 5, one of integrin adhesion molecules; VLA-5- myeloma cells were proliferative immature cells and VLA-5+ cells were mature myeloma cells.

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Mesenchymal Stromal Cells Orchestrate Follicular Lymphoma Cell Niche Through the CCL2-Dependent Recruitment and Polarization of Monocytes

Blood ◽

10.1182/blood.v118.21.1566.1566 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1566-1566

Author(s):

Fabien Guilloton ◽

Gersende Caron ◽

Cédric Ménard ◽

Céline Pangault ◽

Patricia Amé-Thomas ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Cell Growth ◽

Follicular Lymphoma ◽

B Cell ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Gene Set Enrichment Analysis ◽

Cell Niche

Abstract Abstract 1566 Accumulating evidence indicates that infiltrating stromal cells contribute directly and indirectly to tumor growth in a wide range of solid cancers and hematological malignancies. In follicular lymphoma (FL), malignant B cells are found admixed with heterogeneous lymphoid-like stromal cells within invaded lymph nodes and bone marrow (BM). In addition, in vitro functional studies have underlined that mesenchymal cells recruit malignant FL B cells and protect them from spontaneous and drug-induced apoptosis. In particular, we have previously demonstrated that mesenchymal stromal cells (MSC) efficiently support in vitro FL B-cell survival, especially after their engagement towards lymphoid differentiation through treatment with TNF-α and Lymphotoxin-α1β2 (TNF/LT) or after coculture with malignant B cells. However, the mechanisms of this supportive activity remain largely unknown. In this study, we used Affymetrix U133 Plus 2.0 microarrays, to compare the gene expression profile (GEP) of bone marrow-derived MSC (BM-MSC) obtained from 10 FL patients at diagnosis versus 6 age-matched healthy donors (HD). In these conditions, neither the CFU-F concentration in the BM nor the cumulative population doubling of BM-MSC significantly differed between HD and FL patients. Unsupervised analysis was able to perfectly segregate FL-MSC from HD-MSC and we identified, using supervised analyzes, a list of 408 probesets defining FL-MSC signature, including 320 nonredundant genes upregulated in FL-MSC compared to HD-MSC. We then defined the GEP of human lymphoid-like stroma using HD-MSC treated in vitro by TNF/LT and demonstrated, by a Gene Set Enrichment Analysis (GSEA) approach, that the FL-MSC signature is significantly enriched for genes associated with a lymphoid-like commitment. Interestingly, CCL2 was strongly overexpressed by FL-MSC, was upregulated in HD-MSC by coculture with malignant B cells, and was detected at a higher level in FL BM plasma compared to normal BM plasma (504.4 pg/mL [23.8-4413] versus 33.9 pg/mL [5-126.1]; P <.01). In agreement, FL-MSC triggered a more potent CCL2-dependent monocyte migration than HD-MSC. Moreover, FL-MSC and macrophages cooperated to sustain malignant B-cell growth through both protection from apoptosis and enhancement of cell proliferation. Finally, FL-MSC promoted monocyte differentiation towards a proangiogenic LPS-unresponsive phenotype close to that of tumor-associated macrophages. We unraveled a key role for the Notch pathway in this process and identified an overexpression of JAGGED1 in FL-MSC compared to HD-MSC. Altogether, these results highlight the complex role of FL stromal cells that promote direct tumor B-cell growth and orchestrate FL cell niche. The identification and characterization of this intricate network of cell interactions may provide novel therapeutic targets in this disease. Disclosures: No relevant conflicts of interest to declare.

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