Reduced Hspa9 Expression Alters IL-7 Signaling In B-Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1569-1569
Author(s):  
Kilannin Krysiak ◽  
Justin Tibbitts ◽  
Matthew J. Walter
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Bone Marrow Cells ◽  
Cell Development ◽  
B Cell Development ◽  
Driving Mechanism ◽  
Wild Type ◽  
Fold Reduction ◽  
Marrow Cells

Abstract Myeloid and erythroid differentiation defects and cytopenias are most commonly described in myelodysplastic syndromes (MDS), however, a reduction in B-cell progenitors exists. The genetic events contributing to this reduction are poorly understood. Interstitial deletion or loss of one copy of the long arm of chromosome 5 (del5q) is the most common cytogenetic abnormality associated with MDS. Two commonly deleted regions on del(5q) have been described and no biallelic mutations have been identified implicating haploinsufficiency of genes on this interval as a driving mechanism. We, and others, have identified several del(5q) candidate genes, including RPS14, EGR1, CTNNA1, APC, NPM1, DIAPH1, miR145, miR146a, and HSPA9. Consistent with haploinsufficiency, HSPA9 mRNA levels are 50% reduced in del(5q) patients. We previously showed that knockdown of Hspa9by shRNA in a murine bone marrow transplant model resulted in a significant reduction in murine B-cells in the bone marrow, spleen and peripheral blood. To further characterize the role of Hspa9 in hematopoiesis, we created Hspa9 heterozygous mice (Hspa9+/-). Heterozygotes express 50% less Hspa9 protein and are born at normal Mendelian frequencies (N>100). No significant differences in mature lineage markers, complete blood counts, and hematopoietic organ cellularity, have been identified up to 12 months of age. However, as early as 2 months of age, Hspa9+/- mice show a significant reduction in CFU-PreB colonies compared to their wild-type littermates, indicating B-cell progenitor defects (14 vs. 48 colonies/100,000 bone marrow cells plated, respectively, N=10 mice/genotype, p<0.001). Following long-term engraftment of transplanted bone marrow cells from Hspa9+/-or littermate controls into lethally irradiated recipients, we also observed a 5.8-fold reduction in bone marrow CFU-PreB colonies (N=7-9 mice/genotype, p=0.002), confirming the B-cell progenitor defect is hematopoietic cell-intrinsic. Despite the reduction in CFU-PreB colony numbers, frequencies of freshly isolated early B-cell progenitor and precursor populations in the bone marrow and spleen of Hspa9+/- mice are not different than wild-type littermate controls when assessed by flow cytometry (common lymphoid progenitor, Hardy fractions A-F). We hypothesized that these mice were able to compensate for B-cell alterations caused by loss of Hspa9 in vivo. Consistent with our hypothesis, the reduction in CFU-PreB colony numbers was partially rescued by increasing the concentration of IL-7 in the media. Hspa9+/- colony numbers increased 1.8 fold when the IL-7 concentration was increased from 10ng/mL to 50ng/mL compared to 0.80 fold for wild-type littermates (p=0.03, N=6 mice/genotype). This effect was unique to IL-7. Adding increasing concentrations of Flt-3 ligand, another cytokine that contributes to early B-cell development, did not alter CFU-PreB colony formation. We isolated B220+ cells from Day 7 CFU-PreB cultures for gene expression array analysis and observe reduced expression of genes promoting B-cell proliferation and activation in Hspa9+/- compared to Hspa9+/+ cells. Since IL-7 is the only supportive cytokine in the methylcellulose media, can partially rescue the reduced CFU-PreB phenotype, and is required for early B-cell development and survival, we hypothesized that Hspa9 haploinsufficiency inhibits transduction of IL-7 signaling. We tested this hypothesis using an IL-7 dependent mouse B-cell line (B7 cells; Ba/F3 cells that stably express the IL-7 receptor). Knockdown of Hspa9 by siRNAs resulted in a 8-fold reduction in cell number after 4 days in culture (p=0.004, confirmed with two independent siRNAs) and was associated with an increase in apoptosis and reduction in cells in S-phase of the cell cycle. Knockdown of Hspa9 in B7 cells resulted in reduced levels of phosphorylated Stat5, an immediate downstream target of IL-7 receptor stimulation, compared to cells treated with a non-targeting siRNA (measured at 5, 10, 15 and 30 minutes following 10ng/mL IL-7 stimulation, p≤0.03). Ongoing studies will further interrogate the effects of Hsap9 knockdown on Jak-Stat signaling. Collectively, these data implicate that loss of HSPA9 alters IL-7 signaling, potentially contributing to the reduction of B-cell progenitors observed in patients with del(5q)-associated MDS. Disclosures: No relevant conflicts of interest to declare.

T and B cell development in pituitary deficient insulin-like growth factor-II transgenic dwarf mice

1997 ◽  
Vol 155 (1) ◽  
pp. 165-170 ◽  
Author(s):  
R Kooijman ◽  
SC van Buul-Offers ◽  
LE Scholtens ◽  
RG Reijnen-Gresnigt ◽  
BJ Zegers
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Bone Marrow Cells ◽  
T Cell Development ◽  
Cell Development ◽  
B Cell Development ◽  
Igf I ◽  
Dwarf Mice

Treatment of mice with IGF-I stimulates T and B cell development. We showed that overexpression of IGF-II in transgenic FVB/N mice only stimulated T cell development. In the present study, we further addressed the in vivo effects of IGF-II in the absence of IGF-I to get more insight into the potential abilities of IGF-II to influence T and B cell development. To this end, we studied lymphocyte development in IGF-II transgenic Snell dwarf mice that are prolactin, GH and thyroid-stimulating hormone deficient and as a consequence show low serum IGF-I levels. We showed that T cell development was stimulated to the same extent as in IGF-II transgenic FVB/N mice. Furthermore, IGF-II increased the number of nucleated bone marrow cells and the number of immature B cells without having an effect on the number of mature B cells in spleen and bone marrow. Our data show that IGF-II has preferential effects on T cell development compared with B development, and that these preferential effects also occur in the absence of measurable IGF-I levels.

scholarly journals The Complex Role of KDM6A in B-Cell Development and Function

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 513-513
Author(s):  
Ling Tian ◽  
Monique Chavez ◽  
Lukas D Wartman
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Bone Marrow Cells ◽  
Cell Development ◽  
Young Female ◽  
B Cell Development ◽  
And Function

Abstract Loss-of-function mutations in KDM6A, an X-linked H3K27 demethylase, occur recurrently in B-cell lymphoid malignancies, including B-cell acute lymphoblastic leukemia and non-Hodgkin lymphoma. Germline inactivating mutations in KDM6A cause a neurodevelopmental disorder called Kabuki syndrome that is associated with recurrent infections and hypogammaglobulinemia.1 The role of KDM6A in normal B-cell development and function, as well as how the somatic loss of KDM6A contributes to B-cell malignancies, has not been completely defined. To address this issue, we generated a conditional knockout mouse of the KDM6A gene (with LoxP sites flanking the 3rd exon) and crossed these mice with Vav1-Cre transgenic mice to selectively inactivate KDM6A in hematopoietic stem/progenitor cells. We characterized normal hematopoiesis from young (6 to 8 week old) and aged (50 to 55 week old) male and female KDM6A conditional KO mice. We found a significant shift from lymphoid to myeloid differentiation in the bone marrow and peripheral blood of these mice. Young, female KDM6A-null mice had mild splenomegaly. Their spleens had an increased number of neutrophils (Gr-1+CD11b+ cells) and erythrocyte progenitors (CD71+Ter119+ cells) and a decreased number of B-cells (B220+ cells). These changes became more pronounced with age and were specific to the female, homozygous KDM6A knockout mice. Furthermore, analysis of B-cell maturation showed that the loss of KDM6A was associated with decreased immature (B220+IgM+ cells) and mature, resting B-cells (B220+IgD+ cells) in the spleen. Similar changes were present in the bone marrow (decreased B220+IgM+ cells and B220+CD19+ cells) and peripheral blood (decreased B220+IgM+, B220+IgD+ and B220+CD19+ cells). Early B-cell development is also altered in KDM6A-null mice. Flow cytometry showed a decrease in multipotent progenitor cells (MPPs) with a decrease in both common lymphoid progenitors (CLPs) and B cell-biased lymphoid progenitors (BLPs) in young, female KDM6A-null mice bone marrow. Next, we performed flow cytometry to catergorize the Hardy fractions of early B-cell development on bone marrow isolated from young, female KDM6A-null mice. B-cell progenitor analysis (Hardy profiles) showed an increase in Fraction A with a concomitant decrease in Fraction B/C and Fraction D, which was likely indicative of an incomplete block in B-cell differentiation after the Fraction A stage. When bulk bone marrow cells isolated from young, female KDM6A-null mice were plated in methylcellulose supplemented with interleukin-7, we observed a significantly decreased colony formation compared with bone marrow cells isolated from wildtype littermates. This pre-B lymphoid progenitor cell plating phenotype was expected given the flow cytometry results of decreased B-cell progenitors outlined above. We examined the effect of the loss of KDM6A expression on germinal center (GC) formation in the spleen following immunization with NP-CGG (4-Hydroxy-3-nitrophenylacetyl-Chicken Gamma Globulin, Ratio 16). Two weeks after NP-CGG immunization, we observed a significant decrease in follicular B-cells (FO) and a significant increase in GC B-cells as compared to wildtype littermates (Figure 1). The result is significant as GC B-cells are thought to be the cell-of-origin of follicular and DLBCL. To determine if inactivation of KDM6A affected antibody production, we measured IgM, IgG, IgE and IgA levels by ELISA from serum isolated from young, female KDM6A-null mice. Results revealed higher levels of IgM and lower levels of IgG in serum from KDM6A-null mice, which is suggestive of a class switch recombination (CSR) defect. Concordant with this result, we observed that the loss of KDM6A impaired CSR to IgG1 in splenic B cells after in vitro stimulation for three days with lipopolysaccharide (LPS), an anti-CD180 antibody and interleukin-4. Moreover, we observed a striking defect in the production of plasma cells from KDM6A-null B-cells after LPS stimulation. Taken together, our data shows that KDM6A plays an important, but complex, role in B-cell development and that loss of KDM6A impedes the B-cell immune response in a specific manner that may contribute to infection and B-cell malignancies.Stagi S, et al. Epigenetic control of the immune system: a lesson from Kabuki syndrome. Immunol Res. 2016; 64(2):345-359. Disclosures No relevant conflicts of interest to declare.

scholarly journals Interactions Between c-kit and Stem Cell Factor Are Not Required for B-Cell Development In Vivo

Blood ◽  
1997 ◽  
Vol 89 (2) ◽  
pp. 518-525 ◽  
Author(s):  
Shunichi Takeda ◽  
Takeyuki Shimizu ◽  
Hans-Reimer Rodewald
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Fetal Liver ◽  
Critical Role ◽  
Cell Development ◽  
B Cell Development ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract The receptor-type tyrosine kinase, c-kit is expressed in hematopoietic stem cells (HSC), myeloid, and lymphoid precursors. In c-kit ligand-deficient mice, absolute numbers of HSC are mildly reduced suggesting that c-kit is not essential for HSC development. However, c-kit− HSC cannot form spleen colonies or reconstitute hematopoietic functions in lethally irradiated recipient mice. Based on in in vitro experiments, a critical role of c-kit in B-cell development was suggested. Here we have investigated the B-cell development of c-kitnull mutant (W/W ) mice in vivo. Furthermore, day 13 fetal liver cells from wild type or W/W mice were transferred into immunodeficient RAG-2−/− mice. Surprisingly, transferred c-kit− cells gave rise to all stages of immature B cells in the bone marrow and subsequently to mature conventional B2, as well as B1, type B cells in the recipients to the same extent as transferred wild type cells. Hence, in contrast to important roles of c-kit in the expansion of HSC and the generation of erythroid and myeloid lineages and T-cell precursors, c-kit− HSC can colonize the recipient bone marrow and differentiate into B cells in the absence of c-kit.

G-CSF Induces Alterations in the Bone Marrow Microenvironment That Suppress B Lymphopoiesis,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3243-3243
Author(s):  
Ryan B Day ◽  
Adam Greenbaum ◽  
Mahil Rao ◽  
Daniel Link
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Bone Marrow Microenvironment ◽  
B Lymphopoiesis ◽  
Wild Type ◽  
Treatment Results ◽  
A Cell

Abstract Abstract 3243 During infectious stress, there is a marked shift in the bone marrow from lymphopoiesis to granulopoiesis. Granulocyte colony-stimulating factor (G-CSF) is the principal cytokine regulating granulopoiesis, and its expression is induced during infection. In this study, we show that G-CSF treatment in mice is associated with a marked suppression of lymphopoiesis in the bone marrow. Specifically, after 5 days of G-CSF treatment (250 μg/kg), the number of B cells in the bone marrow was reduced 8.6 ± 1.3-fold, the number of T cells reduced 14.8 ± 3.8-fold, and the number of NK cells reduced 7.5 ± 1.6-fold. Though modest increases in splenic and blood lymphocytes were observed following G-CSF treatment, this did not account for the loss in the bone marrow. To assess B cell development, modified Hardy fractions were analyzed. All stages of B cell development were significantly reduced by G-CSF, but to different degrees. Fraction A (pre-pro B cells) declined 2.1 ± 0.5-fold; fraction B/C (mostly pro-B cells): 9.4 ± 1.7-fold; fraction D cells (pre-B cells): 5.9 ±1.1-fold; fraction E (immature B cells): 8.1 ± 1.6-fold; and fraction F (mature B cells): 87 ±13-fold. In addition, mature plasma cells declined 1.3 ± 0.07-fold while immature plasmablasts decreased 7.7 ± 1.7-fold. Interestingly, preliminary analysis suggests that there is no significant change in the number of common lymphoid progenitors in the bone marrow. Since there are reports of G-CSF receptor (G-CSFR) expression on certain B cell subsets, we next asked whether G-CSFR signals act in a cell-intrinsic fashion to suppress B lymphopoiesis. Mixed bone marrow chimeras were generated that contain both wild type and G-CSFR−/− bone marrow cells. G-CSF treatment of these mixed chimeras demonstrated equal suppression of wild type and G-CSFR−/− B cells. Thus, G-CSF works in a cell-extrinsic fashion to suppress B lymphopoiesis. Certain bone marrow stromal cell populations are known to regulate B lymphopoiesis, including osteoblasts and CXCL12-abundant reticular (CAR) cells. We previously showed that G-CSF treatment results in a loss of mature osteoblasts. To examine CAR cells, we analyzed mice in which green fluorescent protein (GFP) has been knocked-in to the Cxcl12 locus, allowing for CAR cell identification (Tokoyoda et al. 2004). Whereas G-CSF treatment did not alter the number of CAR cells, a significant decrease in GFP expression per CAR cell was observed. Consistent with this observation, we observed a significant decrease in CXCL12 mRNA expression in the bone marrow following G-CSF treatment. Interestingly, we also noted significant decreases in RNA and/or protein expression of a number of B-supportive cytokines, including interleukin-6, interleukin-7, and B cell activating factor (BAFF) protein. In summary, G-CSF treatment results in marked changes in the bone marrow microenvironment that lead to a suppression of B lymphopoiesis. The ability of G-CSF to disrupt homeostatic signals required for B cell maintenance at multiple stages of development suggest that upfront G-CSF treatment may be a novel strategy to sensitize certain B cell malignancies to chemotherapy. Disclosures: No relevant conflicts of interest to declare.

The Role of H2AFY in U2AF1 Mutant Cells and Normal Hematopoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 963-963
Author(s):  
Sanghyun Kim ◽  
Cara Lunn Shirai ◽  
Matthew J. Walter
Keyword(s):  
Bone Marrow ◽  
Alternative Splicing ◽  
B Cells ◽  
Blood Cell ◽  
B Cell ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Wild Type ◽  
K Cells ◽  
Marrow Cells

Abstract Mutations in genes that are involved in pre-mRNA splicing (i.e., spliceosome genes; U2AF1, SF3B1, SRSF2 and ZRSR2) are particularly frequent in MDS, affecting ~50% of MDS patients. Our group showed that expression of U2AF1(S34F), the most common U2AF1 mutant, causes altered splicing in human and mouse cells and altered hematopoiesis in mice. How U2AF1(S34F) expression contributes to altered hematopoiesis is unclear. We hypothesized that U2AF1(S34F) alters splicing of target genes that affect hematopoiesis. Using three independent sets of RNA-seq data from human acute myeloid leukemia (AML) patients, human CD34+ hematopoietic cells, and mouse common myeloid progenitor cells that express either wild-type or mutant U2AF1, we identified consistent alternative splicing of H2AFY. H2AFY, also known as macroH2A1, is a histone H2A variant implicated in transcription, development, and tumorigenesis. Alternative splicing of H2AFY generates two isoforms, H2AFY1.1 and H2AFY1.2. In many solid cancers, alternative splicing of H2AFY is observed, resulting in reductions in H2AFY1.1 isoform expression. Similarly, in all three RNA-seq datasets and RT-PCR of primary MDS samples, mutant U2AF1 samples express H2AFY1.1 at 30% of the levels observed in samples without a spliceosome mutation (p<0.01). In order to determine whether expression of H2AFY1.1 could rescue a cellular phenotype in U2AF1 mutant cells, we exogenously expressed H2AFY1.1 in doxycycline-inducible U2AF1(S34F) mouse bone marrow cells ex vivo using a lentivirus co-expressing GFP. We observed an attenuation of cell death induced by U2AF1(S34F) expression (%GFP+ live cells relative to uninduced cells: U2AF1(S34F) cells-45% [control virus] vs. 89% [H2AFY1.1 virus], p<0.001; wild-type cells-94% [control virus] vs. 97% [H2AFY1.1 virus], ns). Next, we asked whether loss of H2afy1.1 expression phenocopies any of the hematopoietic alterations observed in U2AF1(S34F) mice (reductions in white blood cell counts [WBC], B-cells and monocytes, or expansion of progenitor cells). To address this question, we first investigated the role of H2afy in normal mouse hematopoiesis. H2afy-/- mice (lacking both H2afy1.1 and H2afy1.2 isoforms) are viable and born at the expected Mendelian frequency. H2afy-/- and H2afy+/-mice exhibited reduced WBC compared to H2afy+/+ littermate mice (7.7, 8.1, and 9.9 K cells/µL, respectively, p<0.01 compared to wild-type mice, N=15 each genotype, 8-12 weeks old), whereas the red blood cell and platelet counts were no different. Flow cytometric analysis of the peripheral blood cell lineages revealed reduced B-cell numbers in H2afy-/- and H2afy+/-mice compared to H2afy+/+ mice (mean 4.0, 4.5, and 5.6 K cells/µL, respectively, p<=0.003). Stem (KLS and KLS-SLAM) and progenitor cells (CMP, GMP, MEP) were not different between genotypes. There was a 2-fold reduction in CFU-pre-B colonies in the H2afy-/- bone marrow cells compared to wild-type controls (p= 0.01, N=10), but CFU-C colonies were no different. To determine whether the reduction in B-cells is hematopoietic cell-intrinsic, we transplanted mouse bone marrow cells collected from H2afy+/+ or H2afy-/- mice into lethally irradiated congenic recipients. At 6 weeks post-transplant, mice who received the H2afy-/- bone marrow cells compared to mice who received H2afy+/+ cells exhibited a marked reduction in WBC (5.0, 9.1 K cells/µL, respectively, p<0.001, N=10) and peripheral blood B-cells (1.9, 5.3 K cells/µL, respectively, p<0.001), but not in myeloid cells. This suggests that hematopoietic expression of H2afy contributes to B-cell development in mice. To assess stem/progenitor cell function in vivo, we performed a competitive repopulation assay. As early as 1 month post-transplant (and persistent to 4 months), there was a reduction in H2afy-/- donor-derived B-cells in the peripheral blood compared to mice receiving H2afy+/+ cells (14%, 58%, respectively, p<0.001, N=14-15). The reduction in peripheral white blood cell counts and B-cells in H2afy-/- mice mirrors the phenotypic changes observed in U2AF1(S34F) mice, suggesting that alterations in H2afy may contribute to altered hematopoiesis in U2AF1 mutant mice. Ongoing studies are designed to address whether the B-cell phenotype in H2afy-/- and U2AF1 mutant mice is due to a specific loss of H2afy1.1 or 1.2 isoform expression in hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals The B Lymphocyte-Specific Coactivator BOB.1/OBF.1 Is Required at Multiple Stages of B-Cell Development

2001 ◽  
Vol 21 (5) ◽  
pp. 1531-1539 ◽  
Author(s):  
Jochen Hess ◽  
Peter J. Nielsen ◽  
Klaus-Dieter Fischer ◽  
Hermann Bujard ◽  
Thomas Wirth
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
B Lymphocyte ◽  
Bone Marrow Cells ◽  
Expression System ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Differentiation ◽  
Conditional Expression

ABSTRACT The transcriptional coactivator BOB.1/OBF.1 confers B-cell specificity on the transcription factors Oct1 and Oct2 at octamer site-containing promoters. A hallmark of the BOB.1/OBF.1 mutation in the mouse is the absence of germinal center development in secondary lymphoid organs, demonstrating the requirement for BOB.1/OBF.1 in antigen-dependent stages of B-cell differentiation. Here we analyzed earlier stages of B lymphopoiesis in BOB.1/OBF.1-deficient mice. Examination of B-cell development in the bone marrow revealed that the numbers of transitional immature (B220+ IgMhi) B cells were reduced and that B-cell apoptosis was increased. When in competition with wild-type cells, BOB.1/OBF.1−/− bone marrow cells exhibited defects in repopulating the bone marrow B-cell compartment and were unable to establish a presence in the periphery of host mice. The defective bone marrow populations in BOB.1/OBF.1−/− mice were rescued by conditional expression of a BOB.1/OBF.1 transgene controlled by the tetracycline gene expression system. However, the restored populations did not restore the numbers of IgDhi B cells in the periphery, where the BOB.1/OBF.1 transgene was not expressed. These results show that BOB.1/OBF.1−/− B cells exhibit multistage defects in B-cell development, including impaired production of transitional B cells and defective maturation of recirculating B cells.

Transformation of murine bone marrow cells with combined v-raf-v-myc oncogenes yields clonally related mature B cells and macrophages

1990 ◽  
Vol 10 (7) ◽  
pp. 3562-3568
Author(s):  
M Principato ◽  
J L Cleveland ◽  
U R Rapp ◽  
K L Holmes ◽  
J H Pierce ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Hematopoietic Differentiation ◽  
Developmental Relationships ◽  
Murine Bone Marrow ◽  
B Lineage ◽  
Marrow Cells

Murine bone marrow cells infected with replication-defective retroviruses containing v-raf alone or v-myc alone yielded transformed pre-B cell lines, while a retroviral construct containing both v-raf and v-myc oncogenes produced clonally related populations of mature B cells and mature macrophages. The genealogy of these transformants demonstrates that mature myeloid cells were derived from cells with apparent B-lineage commitment and functional immunoglobulin rearrangements. This system should facilitate studies of developmental relationships in hematopoietic differentiation and analysis of lineage determination.

Precursor B Cell Acute Lymphoblastic Leukemia Induces Pro-Leukemic Changes in the Bone Marrow Microenvironment That Contribute to Therapeutic Resistance

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1466-1466
Author(s):  
Christopher D Chien ◽  
Elizabeth D Hicks ◽  
Paul P Su ◽  
Haiying Qin ◽  
Terry J Fry
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Development ◽  
B Cell Development ◽  
Bone Marrow Microenvironment ◽  
Therapeutic Resistance

Abstract Abstract 1466 Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Although cure rates for this disease are approximately 90%, ALL remains one of the leading causes cancer-related deaths in children. Thus, new treatments are needed for those patients that do not respond to or recur following standard chemotherapy. Understanding the mechanisms underlying resistance of pediatric ALL to therapy offers one approach to improving outcomes. Recent studies have demonstrated the importance of communication between cancer cells and their microenvironment and how this contributes to the progression and therapeutic resistance but this has not been well studied in the context of ALL. Since the bone marrow is presumed to be the site of initiation of B precursor ALL we set out in our study to determine how ALL cells utilize the bone marrow milieu in a syngeneic transplantable model of preB cell ALL in immunocompetent mice. In this model, intravenously injected preB ALL develops first in the bone marrow, followed by infiltration into the spleen, lymph node, and liver. Using flow cytometry to detect the CD45.2 isoform following injection into B6CD45.1+ congenic recipients, leukemic cells can be identified in the bone marrow as early as 5 days after IV injection with a sensitivity of 0.01%-0.1%. The pre-B ALL line is B220+/CD19+/CD43+/BP1+/IL-7Ralpha (CD127)+/CD25-/Surface IgM-/cytoplasmic IgM+ consistent with a pre-pro B cell phenotype. We find that increasing amounts of leukemic infiltration in the bone marrow leads to an accumulation of non-malignant developing B cells at stages immediately prior to the pre-pro B cell (CD43+BP1-CD25-) and a reduction in non-malignant developing pre B cells at the developmental stage just after to the pre-pro B cell stage (CD43+BP1+CD25+). These data potentially suggest occupancy of normal B cell developmental niches by leukemia resulting in block in normal B cell development. Further supporting this hypothesis, we find significant reduction in early progression of ALL in aged (10–12 month old) mice known to have a deficiency in B cell developmental niches. We next explored whether specific factors that support normal B cell development can contribute to progression of precursor B cell leukemia. The normal B cell niche has only recently been characterized and the specific contribution of this niche to early ALL progression has not been extensively studied. Using a candidate approach, we examined the role of specific cytokines such as Interleukin-7 (IL-7) and thymic stromal lymphopoietin (TSLP) in early ALL progression. Our preB ALL line expresses high levels of IL-7Ralpha and low but detectable levels of TLSPR. In the presence of IL-7 (0.1 ng/ml) and TSLP (50 ng/ml) phosphSTAT5 is detectable indicating that these receptors are functional but that supraphysiologic levels of TSLP are required. Consistent with the importance of IL-7 in leukemia progression, preliminary data demonstrates reduced lethality of pr-B cell ALL in IL-7 deficient mice. Overexpression of TSLP receptor (TSLPR) has been associated with high rates of relapse and poor overall survival in precursor B cell ALL. We are currently generating a TSLPR overepressing preBALL line to determine the effect on early ALL progression and are using GFP-expressing preB ALL cells to identify the initial location of preB ALL occupancy in the bone marrow. In conclusion, or model of early ALL progression provides insight into the role of the bone marrow microenvironment in early ALL progression and provides an opportunity to examine how these microenvironmental factors contribute to therapeutic resistance. Given recent advances in immunotherapy for hematologic malignancies, the ability to study this in an immunocompetent host will be critical. Disclosures: No relevant conflicts of interest to declare.

Dlk-1 Regulates B Cell Development Through Altered Bone Marrow Stromal Microenvironment, Not by Affecting Hematopoitic Stem/Progenitor Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3465-3465
Author(s):  
Edyta Pawelczyk ◽  
Heba A Degheidy ◽  
Allison L Branchaw ◽  
Kenn Holmbeck ◽  
Steven R Bauer
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cell ◽  
Methyl Cellulose ◽  
Cell Development ◽  
B Cell Development ◽  
Bone Marrow Microenvironment ◽  
Wild Type ◽  
Mineral Density ◽  
Hematopoietic Stem

Abstract Abstract 3465 Introduction: DLK-1(delta-like 1) is a member of the EGF-like homeotic protein family whose expression is known to influence cell fate decisions through cell-cell interactions. It is also known to influence the differentiation of bone marrow stromal cells (BMSC) and hematopoietic stem cells (HSC) in bone marrow. Recently, we reported the essential role of DLK-1 in B cell development, which showed that the absence of DLK-1 led to accumulation of the earliest B cell progenitors (pre-pro B cells or Fraction A (Fr A)) in bone marrow, an altered pattern of B cell development in the spleen, and an altered humoral immune response. The objective of this study was to determine whether alterations in the HSC compartment or the BMSC microenvironment contributed to Fr A accumulation in mdlk1−/− mice. Methods: The mdlk1−/− and wild type bone marrow osteoblast and HSC compartments were analyzed by multicolor flow cytometry and in vitro methyl-cellulose colony forming cell assays. Bone marrow harvested from mdlk1−/− and wild type mice was assessed for BMSCs colony forming efficiency (CFU-F) and cultured. Supernatants from cultured BMSCs were analyzed by protein arrays. Since osteoblasts are an important component of the bone marrow microenvironment, OPN+CD45-TER119-ALP+ osteoblasts were identified in the bone marrow and quantified by flow cytometry. Finally, the femurs of mdlk1−/− and wild type mice were analyzed by micro-computed tomography (uCT) scanning. Results: Using flow cytometry, we observed no statistically significant changes in the HSC and progenitor populations in the absence of DLK-1 in mice at 4 and 16 weeks of age. The results of methyl-cellulose assay confirmed the findings of flow cytometry experiments and showed no statistically significant differences in the number of CFU-G, CFU-GM, and CFU-M of 4 and 16 week old mdlk1−/− mice as compared to wild-type control mice. However, significant alterations in the microenvironment of the mdlkl −/− were observed. CFU-F efficiency of mdlk1−/− bone marrow BMSC isolated from 4 week old mice was significantly decreased when compared to age-matched controls. Furthermore, the uCT scans showed the mineral density of the femoral bone significantly decreased in 4 week old mdlk1−/− mice and the number of osteoblast cells analyzed by flow cytometry was decreased by 10%. The analysis of BMSC supernatants revealed a striking down regulation of factors associated with osteoblast function and differentiation such as osteoactivin, PF-4, Follstatin-like 1, Frizzled-6, IGF-1, M-CSF, DKK-1 and others. Conclusions: Our results indicate that accumulation of the earliest B cell progenitors with DLK-1 ablation is the result of multiple defects in the bone marrow microenvironment including decreased CFU-F, decreased number of osteoblasts, decreased bone mineral density or alterations in factors important for osteoblast function but not from increase in numbers of hematopoietic stem or progenitors cells. Our laboratory is investigating this further. Disclosures: Pawelczyk: Baxter Inc.: currently employed by Baxter Inc. Other.

RNAi Screening Identifies A Novel Role for A-Kinase Anchoring Protein 12 (AKAP12) in B Cell Development and Function

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 855-855 ◽  
Author(s):  
Mutlu Kartal-Kaess ◽  
Luisa Cimmino ◽  
Simona Infantino ◽  
Mehmet Yabas ◽  
Jian-Guo Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Leukemia Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Leukemia Cell Line ◽  
B Lymphopoiesis ◽  
Catalytic Subunits ◽  
B Cell Leukemia

Abstract Abstract 855 The cAMP signaling pathway has emerged as a key regulator of hematopoietic cell proliferation, differentiation, and apoptosis. Signal specificity is achieved through local activation of signaling enzymes that are anchored to subcellular organelles and membranes. In particular, A-kinase anchoring proteins (AKAPs) coordinate and control cAMP responsive events. AKAPs were originally classified based on their ability to bind cAMP-dependent protein kinase (protein kinase A; PKA). The activity of PKA is regulated by its two regulatory subunits, which from a dimer that binds to the two catalytic subunits. Binding of cAMP to the regulatory dimer dissociates the catalytic subunits and activates PKA. Anchoring of PKA by AKAPs constrains PKA activity to a relevant subset of potential substrates. Thus, AKAPs contribute to the precision of intracellular signaling events by directing anchored enzyme pools to a subset of their physiological substrates at specific subcellular localizations. Using an in vitro short hairpin RNA (shRNA) screen against potentially druggable targets, we have uncovered a requirement for AKAP12 in the proliferation of a cultured pre-B cell leukemia cell line. In the hematopoietic system of mice and humans, expression of AKAP12 is tightly restricted to the pro/pre/immature stages of B lymphopoiesis, suggesting a potential role in pre-B cell receptor (pre-BCR) or BCR signaling. We find that retroviral knockdown or germline knockout of AKAP12 in mice leads to an increase in pre B and immature B cells in the bone marrow. In contrast, B cell numbers in the spleen are significantly reduced, as are recirculating B cells in the bone marrow. Transplantation of AKAP12 null hematopoietic stem and progenitor cells from fetal liver into wildtype recipients demonstrates an autonomous defect in the development of AKAP12−/− B cells. Competitive bone marrow transplantations confirm that this defect is cell autonomous and not due to a defective bone marrow environment or secretion of a B cell inhibitory factor. To identify AKAP12 interaction partners, we overexpressed FLAG-epitope tagged AKAP12 in a pre-B cell leukemia cell line. Affinity purification of AKAP12 showed a repeated co-immunoprecipitation of poorly characterized RIO kinase 1 (RIOK1). Our current efforts are focused on investigating the interaction between RIOK1 and AKAP12 and their role in the control of B cell development and cell cycle progression. Further, we are focusing on a likely role for AKAP12 in the scaffolding of PKA, PKC and phosphodiesterases by analyzing the activation of signaling cascades in cultured primary wildtype and AKAP12−/− pre B cells. Additionally, we are investigating the role of the BCR in vivo by testing if enforced expression of BCR components rescue B cell development in a AKAP12−/− BCR transgenic mouse model (SWHEL mouse). In summary, we have confirmed a novel role for AKAP12 in B lymphopoiesis. Disclosures: No relevant conflicts of interest to declare.

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