scholarly journals Infectious Origins of Childhood Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 751-751
Author(s):  
Lars Klemm ◽  
Srividya Swaminathan ◽  
Anthony M Ford ◽  
Klaus Schwarz ◽  
David G. Schatz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
Childhood All ◽  
Leukemic Clone ◽  
Germinal Center B Cells

Abstract Abstract 751 Background: In most cases, childhood acute lymphoblastic leukemia can be retraced to a recurrent genetic lesion in utero, which establishes a pre-leukemic clone. The TEL-AML1 fusion gene, for instance, arises prenatally and defines the most frequent subtype of childhood ALL. Strikingly, ∼1 of 100 healthy newborns carry a TEL-AML1 pre-leukemic clone, but only <1% of these children will eventually develop leukemia. Encounter of infectious antigen in B cell typically leads to activation of the mutator enzyme AID. While AID is required for class switch recombination and somatic hypermutation of immunoglobulin genes during affinity maturation of germinal center B cells, its premature activation may be deleterious. The underlying questions for this project were (1) how are B cells during their early development safeguarded from pre-mature AID expression and (2) whether pre-mature expression of AID in early B cell development is deleterious in the sense that it pre-disposes to the clonal evolution of a pre-leukemic B cell clone in the bone marrow. Results: We performed a comprehensive analysis of human B cell development in bone marrow samples from two children carrying deleterious mutations of the IL7RA gene encoding one chain of the human IL7 receptor. As opposed to normal human pre-B cells, pre-B cells from IL7RA-mutant patients carried somatically mutated immunoglobulin genes consistent with aberrant expression of AID in these cells. This led to the hypothesis that signaling via IL7Ra suppresses premature activation of AID-dependent hypermutation. To test this hypothesis, we stimulated mouse pre-B cells with LPS in the presence or absence of IL7, which is normally abundantly present in the bone marrow. While pre-B cells did not respond to LPS in the presence of IL7, IL7 withdrawal dramatically sensitized pre-B cells to LPS exposure: in the absence of IL7, LPS-stimulation of pre-B cells resulted in similar AID protein levels as in splenic germinal center B cells, where AID is normally active. We confirmed these observations studying pre-B cells from an AID-GFP reporter transgenic mouse strain. While LPS resulted in ∼2% AID-GFP+ cells in the presence of IL7, the fraction of AID-GFP+ cells increased to ∼45% when IL7 was removed. Since IL7Ra signaling involves Stat5 phosphorylation, we studied inducible Cre-mediated deletion of Stat5, which had the same effect as IL7 withdrawal and led to transcriptional de-repression of AID. IL7Ra/Stat5 signaling likely involves negative regulation of FoxO3A via AKT since expression of a constitutively active FoxO3A mutant potentiated AID expression in pre-B cells. We next searched for a normal pre-B cell subset, in which loss of IL7Ra/Stat5 signaling occurs naturally. Since inducible activation of pre-B cell receptor signaling results in downregulation of IL7Ra surface expression, we tested pre-B cell receptor-positive stages of B cell development. Interestingly, AID mRNA levels were increased by >10-fold at the transition from IL7Ra-positive Fraction C' pre-B cells to IL7Ra-negative Fraction D pre-B cells. Conclusion: AID is a tightly controlled mutator enzyme in mature germinal center B cells. The factors that prevent premature expression of AID during early B cell development were not known. Here, we here we report a novel, IL7Ra/Stat5-dependent mechanism by which pre-B cells are rendered non-responsive to antigen-dependent upregulation of AID. Attenuation of the IL7Ra/Stat5 signal occurs naturally in Fraction D pre-B cells. As a consequence, Fraction D pre-B cells express significant levels of AID for a short time. We propose that Fraction D pre-B cells represent a subset of increased genetic vulnerability in the natural history of childhood ALL. Disclosures: No relevant conflicts of interest to declare.

IL7Rα Signaling Prevents Premature Expression of AID In Human Pre-B Cells: Implications for Clonal Evolution of Childhood Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 26-26
Author(s):  
Srividya Swaminathan ◽  
Lars Klemm ◽  
Soo-mi Kweon ◽  
Anthony Ford ◽  
Klaus Schwarz ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Cell Clone ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Cell Receptor ◽  
B Cell Development ◽  
B Cell Receptor ◽  
Childhood All

Abstract Abstract 26 Background: Childhood acute lymphoblastic leukemia (ALL) typically arises from a pre-leukemic pre-B cell clone, which was established in utero (Greaves and Wiemels, 2003). This led to a scenario, in which the initial prenatal lesion is followed by a series of additional transforming events, which ultimately cause malignant transformation of a pre-B cell clone. For instance, the TEL-AML1 gene rearrangement defines the most frequent type of childhood ALL and is detected in ∼1% cord blood samples compared to the cumulative risk for TEL-AML1 ALL at 1:14,000. These findings support the notion that covert pre-leukemic clones are frequent but only a small minority of them develop into frank pre-B leukemia after critical secondary genetic lesions were acquired. The postnatal mechanism(s) that drive the evolution of the fetal pre-leukemic clone towards childhood ALL are not known. Hypothesis: We have recently demonstrated that aberrant somatic hypermutation activity of AID propagates progression of CML into lymphoid blast crisis (Klemm et al., 2009) and clonal evolution of acute lymphoblastic leukemia (Gruber et al., 2010). Here we test the hypothesis that premature expression of AID in human pre-B cells promotes the acquisition of secondary genetic lesions and propagates the clonal evolution of a pre-leukemic pre-B cell towards childhood ALL. Results: We performed a comprehensive analysis of human B cell development in bone marrow samples from two children carrying deleterious mutations of the IL7RA gene encoding one chain of the human IL7 receptor. As opposed to normal human pre-B cells, pre-B cells from IL7RA-mutant patients carried somatically mutated immunoglobulin genes. Premature hypermutation in IL7Rα-deficient pre-B cells was consistent with aberrant expression of AID in these cells. This led to the hypothesis that signaling via IL7Rα suppresses premature activation of AID-dependent hypermutation. To test this hypothesis, we stimulated mouse pre-B cells with LPS in the presence or absence of IL7, which is normally abundantly present in the bone marrow. While pre-B cells did not respond to LPS in the presence of IL7, IL7 withdrawal dramatically sensitized pre-B cells to LPS exposure: in the absence of IL7, LPS-stimulation of pre-B cells resulted in similar AID protein levels as in splenic germinal center B cells, where AID is normally active. We confirmed these observations studying pre-B cells from an AID-GFP reporter transgenic mouse strain. While LPS resulted in ∼2% AID-GFP+ cells in the presence of IL7, the fraction of AID-GFP+ cells increased to ∼45% when IL7 was removed. Since IL7Rα signaling involves Stat5 phosphorylation, we studied inducible deletion of both Stat5a and Stat5b in Stat5-fl/fl pre-B cells. Inducible deletion of Stat5a and Stat5b in pre-B cells had the same effect as IL7 withdrawal and led to transcriptional de-repression of AID. IL7Rα/Stat5 signaling likely involves negative regulation of FoxO3A via AKT since expression of a constitutively active FoxO3A mutant potentiated AID expression in pre-B cells. We next searched for a normal pre-B cell subset, in which loss of IL7Rα/Stat5 signaling occurs naturally. Since inducible activation of pre-B cell receptor signaling results in downregulation of IL7Rα surface expression, we tested pre-B cell receptor-positive stages of B cell development. Interestingly, AID mRNA levels were increased by >10-fold at the transition from IL7Rα-positive Fraction C’ pre-B cells to IL7Rα-negative Fraction D pre-B cells. Conclusion: AID is a tightly controlled mutator enzyme, which diversifies immunoglobulin genes upon antigen-encounter of germinal center B cells. The factors that prevent premature expression of AID in pre-germinal center stage B cells were not known. Here, we here we report a novel, IL7Rα/Stat5-dependent mechanism by which pre-B cells are rendered non-responsive to antigen-dependent upregulation of AID. Attenuation of the IL7Rα/Stat5 signal occurs naturally in Fraction D pre-B cells. As a consequence, Fraction D pre-B cells express significant levels of AID for a short time. We propose that Fraction D pre-B cells represent a subset of increased genetic vulnerability in the natural history of childhood ALL. Enlargement of the Fraction D pool or extension of the time window during which pre-B cells are at the Fraction D stage, may increase the risk to acquire secondary genetic lesions towards the development of childhood ALL. Disclosures: No relevant conflicts of interest to declare.

Cooperation Between Aid and the Rag1/Rag2 V(D)J Recombinase Drives Clonal Evolution of Childhood Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 519-519
Author(s):  
Srividya Swaminathan ◽  
Lars Klemm ◽  
Anthony M. Ford ◽  
Klaus Schwarz ◽  
Rafael Casellas ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Cell Development ◽  
B Cell Development ◽  
Childhood All ◽  
Leukemic Clone ◽  
Late Stages

Abstract Abstract 519 Background and Rationale: In many cases, childhood acute lymphoblastic leukemia (ALL) can be retraced to a recurrent genetic lesion in utero which establishes a pre-leukemic clone. The TEL-AML1 fusion gene for instance, arises prenatally and defines the most frequent subtype of childhood ALL. Strikingly, ∼1 of 100 healthy newborns carry a TEL-AML1 pre-leukemic clone, but only less than 1% of these children eventually develop leukemia. Encounter of infectious antigen leads to activation of the mutator enzyme AID in mature B cells. While AID is required for somatic hypermutation and class switching during late stages of B cell development, its pre-mature activation may be deleterious. The underlying questions for this project were: (1) how are B cells safeguarded from pre-mature AID expression during their early development and (2) whether pre-mature AID expression during early B cell development is deleterious in the sense that it promotes the clonal evolution of a pre-leukemic B cell clone in the bone marrow. Results: Studying gene expression in a clinical trial for children with high risk pre-B ALL (COG P9906; n=207), we found that high expression levels of AID at the time of diagnosis is predictive of poor overall survival and a higher frequency of leukemia relapse. These findings suggest that AID may be a contributing factor to the clonal evolution of childhood pre-B ALL. Previous work by Michael Lieber's group proposed cooperation of AID and the V(D)J recombinase Rag1/Rag2 as a key mechanism leading to the acquisition of chromosomal translocations in human B cell malignancies (Tsai et al., 2008). Activity of Rag1/Rag2 V(D)J recombinase and AID is segregated to early and late stages of B cell development, respectively. However, we found that experimental withdrawal of IL7 receptor (IL7R) signaling in pre-B cells not only activates Rag1/Rag2 expression and V(D)J recombinase but also rendered pre-B cells responsive to antigen (LPS) encounter with strong upregulation of AID. We found that upon withdrawal of IL7, transcription of AID and Rag1/Rag2 is activated by the same elements through a Pten/FoxO-dependent pathway. To test whether IL7R signaling also negatively regulates AID activation in human pre-B cells, we performed a comprehensive analysis of human B cell development in bone marrow samples from two children carrying deleterious mutations of the IL7RA and IL2RG genes encoding the two chains of the human IL7R. As opposed to normal human pre-B cells, pre-B cells from IL7RA and IL2RG-mutant patients carried somatically mutated immunoglobulin genes consistent with aberrant expression of AID in these cells. Based on these observations, we propose that Fraction D pre-B cells represent a subset of increased genetic vulnerability owing to concomitant expression of both AID and Rag1/Rag2. To test whether the vulnerability of Fraction D pre-B cells is relevant in the clonal evolution of childhood ALL, we challenged pre-B cells carrying the TEL-AML1 fusion gene with 5 consecutive cycles of IL7 withdrawal (−IL7) and LPS stimulation (+LPS). To distinguish between the potential contribution of AID and Rag1/Rag2 to secondary genetic lesions, -IL7/+LPS-challenges were performed with wildtype pre-B cells, AID−/−, Rag1−/− and AID−/− Rag1−/− double knockout pre-B cells. TEL-AML1-bearing pre-B cells were labeled with firefly luciferase and then 25 million cells were injected into 7 recipient animals per group. While wildtype TEL-AML1 pre-B cells that went through 5 rounds of -IL7/+LPS-challenge caused leukemia in all recipient mice, TEL-AML1 pre-B cells lacking either AID or Rag1 failed to give rise to full-blown leukemia in transplant recipients. Conclusion: While one in 100 newborns carry the TEL-AML1 fusion molecule, the mechanisms that lead to the acquisition of critical secondary genetic lesions are not known. Here, we report a novel, IL7R/Stat5-dependent mechanism by which pre-B cells are rendered non-responsive to LPS-dependent upregulation of AID. We propose that Fraction D pre-B cells represent a subset of increased natural genetic vulnerability in the context of concomitant activativity of AID and Rag1/Rag2. Frequent exposure to infectious antigens (e.g. LPS) in this constellation may propagate clonal evolution towards full-blown leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A generalized quantitative antibody homeostasis model: regulation of B-cell development by BCR saturation and novel insights into bone marrow function

2016 ◽  
Author(s):  
József Prechl
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Quantitative Model ◽  
Cell Development ◽  
B Cell Development ◽  
Marginal Zone B Cells ◽  
Humoral Immune ◽  
Narrow Region

In a pair of articles we present a generalized quantitative model for the homeostatic function of clonal humoral immune system. In this first paper we describe the cycles of B-cell expansion and differentiation driven by B-cell receptor engagement.The fate of a B cell is determined by the signals it receives via its antigen receptor at any point of its lifetime. We express BCR engagement as a function of apparent affinity and free antigen concentration, using the range of 10−14 to 10−3 M for both factors. We assume that for keeping their BCR responsive B cells must maintain partial BCR saturation, which is a narrow region defined by [Ag]≈KD. To remain in this region, B cells respond to changes in [Ag] by proliferation or apoptosis and modulate KD by changing BCR structure. We apply this framework to various niches of B-cell development, such as the bone marrow, blood, lymphoid follicles and germinal centers. We propose that clustered B cells in the bone marrow and in follicles present antigen to surrounding B cells by exposing antigen captured on complement and Fc receptors. The model suggests that antigen-dependent selection in the bone marrow results in 1) effector BI cells, which develop in blood as a consequence of the inexhaustible nature of soluble antigens, 2) memory cells that survive in antigen rich niches, identified as marginal zone B cells. Finally, the model implies that memory B cells could derive survival signals from abundant non-cognate antigens.

scholarly journals Transcription factors of the alternative NF-κB pathway are required for germinal center B-cell development

2016 ◽  
Vol 113 (32) ◽  
pp. 9063-9068 ◽  
Author(s):  
Nilushi S. De Silva ◽  
Michael M. Anderson ◽  
Amanda Carette ◽  
Kathryn Silva ◽  
Nicole Heise ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Alternative Pathway ◽  
Cell Development ◽  
B Cell Development ◽  
Cell Surface Receptor ◽  
Signaling Cascade

The NF-κB signaling cascade relays external signals essential for B-cell growth and survival. This cascade is frequently hijacked by cancers that arise from the malignant transformation of germinal center (GC) B cells, underscoring the importance of deciphering the function of NF-κB in these cells. The NF-κB signaling cascade is comprised of two branches, the canonical and alternative NF-κB pathways, mediated by distinct transcription factors. The expression and function of the transcription factors of the alternative pathway, RELB and NF-κB2, in late B-cell development is incompletely understood. Using conditional deletion of relb and nfkb2 in GC B cells, we here report that ablation of both RELB and NF-κB2, but not of the single transcription factors, resulted in the collapse of established GCs. RELB/NF-κB2 deficiency in GC B cells was associated with impaired cell-cycle entry and reduced expression of the cell-surface receptor inducible T-cell costimulator ligand that promotes optimal interactions between B and T cells. Analysis of human tonsillar tissue revealed that plasma cells and their precursors in the GC expressed high levels of NF-κB2 relative to surrounding lymphocytes. Accordingly, deletion of nfkb2 in murine GC B cells resulted in a dramatic reduction of antigen-specific antibody-secreting cells, whereas deletion of relb had no effect. These results demonstrate that the transcription factors of the alternative NF-κB pathway control distinct stages of late B-cell development, which may have implications for B-cell malignancies that aberrantly activate this pathway.

scholarly journals Essential Immunoregulatory Role for BCAP in B Cell Development and Function

2002 ◽  
Vol 195 (5) ◽  
pp. 535-545 ◽  
Author(s):  
Tetsuo Yamazaki ◽  
Kiyoshi Takeda ◽  
Kumiko Gotoh ◽  
Hiroshi Takeshima ◽  
Shizuo Akira ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Phosphoinositide 3 Kinase ◽  
And Function ◽  
B Cell Deficiency

BCAP was recently cloned as a binding molecule to phosphoinositide 3-kinase (PI3K). To investigate the role of BCAP, mutant mice deficient in BCAP were generated. While BCAP-deficient mice are viable, they have decreased numbers of mature B cells and B1 B cell deficiency. The mice produce lower titers of serum immunoglobulin (Ig)M and IgG3, and mount attenuated responses to T cell–independent type II antigen. Upon B cell receptor cross-linking, BCAP-deficient B cells exhibit reduced Ca2+ mobilization and poor proliferative responses. These findings demonstrate that BCAP plays a pivotal immunoregulatory role in B cell development and humoral immune responses.

The Loss of Kdm6a in B-Cell Development Causes Germinal Center Hyperplasia and Impedes the B-Cell Immune Response in a Specific Manner

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 5-5
Author(s):  
Ling Tian ◽  
Monique Chavez ◽  
Lukas D Wartman
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Cell Development ◽  
Young Female ◽  
B Cell Development ◽  
Cell Immune Response ◽  
Germinal Center Hyperplasia

Putative loss-of-function mutations in KDM6A, an X-linked H3K27 demethylase, occur recurrently in B-cell malignancies, including B-cell non-Hodgkin lymphoma. How the KDM6A in normal B cell development and function, as well as the mechanism(s) by which its loss contributes lymphomagenesis has not been 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. Our previous data have shown young Kdm6a-null mice have a myeloid skewing in the bone marrow, spleen and peripheral blood. These changes became more pronounced with age and were specific to the female, homozygous Kdm6a knockout mice. Early B-cell development is also altered in female 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. B-cell progenitor analysis (Hardy profiles) showed an increase in Fraction A with a concomitant decrease in Fraction B/C and Fraction D. The GC B-cells are thought to be the cell-of-origin of diffuse large B-cell lymphoma (DLBCL). To determine if the loss of Kmd6a could impact the mature B cells undergo germinal center (GC) reaction, we immunized the young, female Kdm6a-null mcie and wildtype littermates with T cell-dependent antigen sheep red blood cell (SRBC). Mice were scrificed 14 days after immunization, spleen cells were examined by flow cytometry. As expected, we observed a significant increase in the percentage of GC B cells (B220+GL7+CD95+) from female Kdm6a-null mice compared to control mice. We also observed differences in the percentage of other B-cell subsets between these mice, including an increase in plasma cells (B220-CD138+) and memory B cells (B220+CD19+CD27+), concomitant with an increase trend towards the elevated marginal zone B cells (B220+CD23loCD21+) and transitional B cells (B220+CD23-CD21-). In contrast, there was a decrease in the follicular zone B cells (B220+CD23-CD21-) and plasmablast (B220+CD138+). To analyze the levels of SRBC-specific Abs from immunized mice, serum was collected from blood at day 14. A flow cytometry-based assay was performed to detect the fluorescent-labeled SRBC-specfic Abs for immunoglobulin. Results showed that the abundance of non-class-switched anti-SRBC IgM level was significantly increased in female Kdm6a-null mice serum compared with control mice. In contrast, these mice had significantly decreased anti-SRBC IgA, IgG, IgG1, IgG3 and IgE levels indicating a isotype class switch defect. The aberrant GC phenotype induced by SRBC indeicated that kdm6a loss results in expansion of GC B cells, which subsequently enhances the plasma cell generation. This finding prompted us to investigate if the Kdm6a impairs the immunoglobulin affinity maturation. Therefore, we analyzed the ability of female Kdm6a-null mice and wildtype littermates to generate specific Abs against another T cell-dependent antigen NP-Chicken Gamma Globulin (NP-CGG). Mice were immunized with NP-CGG (29) and serum were collected weekly up to 8 weeks total. ELISA analysis of serum revealed that NP-specfic total Ig level were similar for both groups of mice over time. However, consistent with the SRBC immunization results, we did observed a sinificant reduction in the titers of NP-specific IgA and IgG1 Abs in female Kdm6a-null mice compared with control mice at each time point, while these mice had a sinificant increase in NP-specific IgM Abs, which indicating the loss of Kdm6a disrupts the balance between non-class-switched and class-switched NP-specific Abs isotypes (Figure 1A-D). Likewise, we also observed an increase in the percentage of GC B cells and plasma cells 8 weeks after NP-CGG immunization by flow cytometry. Again, our findings indicate the loss of Kdm6a causes germinal center hyperplasia, enhances plasma cell differentiation, and likely impairs class switch recombination (CSR). Taken together, our data shows that Kdm6a plays an important, but complex, role in B-cell transiting in the GC reaction and that loss of Kdm6a causes germinal center hyperplasia and impedes the B-cell immune response in a specific manner that may contribute to infection and B-cell malignancies. Disclosures Wartman: Novartis: Consultancy; Incyte: Consultancy.

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.

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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