Mechanisms of Pre-B Cell Receptor-Inactivation In Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 147-147
Author(s):  
Cihangir Duy ◽  
Daniel Nowak ◽  
Lars Klemm ◽  
Rahul Nahar ◽  
Carina Ng ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Signaling Molecules ◽  
B Cell Receptor ◽  
Dominant Negative ◽  
Leukemia Cells ◽  
Immunoglobulin Gene ◽  
Receptor Inactivation

Abstract Abstract 147 Background: We recently established that the pre-B cell receptor functions as a tumor suppressor in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). The pre-B cell receptor promotes differentiation of normal pre-B cells and couples the immunoglobulin μ -chain to activating tyrosine kinases (e.g. SYK) via linker molecules (e.g. BLNK). In virtually all cases of Ph+ ALL, pre-B cell receptor function is compromised and its reconstitution induces rapid cell cycle arrest. However, genomic deletions in pre-B cell receptor pathway are rare and the mechanisms of inactivation are not known. Here we report that pre-B cell receptor inactivation occurs at multiple levels and involves at least four different mechanisms, namely (1) deleterious immunoglobulin gene rearrangement, (2) defective splicing of pre-B cell receptor signaling molecules, (3) expression of dominant-negative PAX5 fusion genes and (4) overexpression of inhibitory signaling molecules. Result: (1) Studying progressive transformation of pre-B cells in BCR-ABL1-transgenic mice, we observed that surface expression of the immunoglobulin μ -chain was downregulated after 60 days of age, which was a prerequisite for the onset of full-blown leukemia. While the repertoire of immunoglobulin gene rearrangements was polyclonal in wildtype pre-B cells, BCR-ABL1-transgenic pre-B cells show clonal expansions, which are derived from one ancestral productive immunoglobulin gene rearrangement in the transformed pre-B cell. However, the ancestral immunoglobulin gene rearrangements were rendered non-functional through deleterious secondary rearrangements. Likewise, in 47 of 57 cases of primary human Ph+ ALL, we detected traces of pre-B cell receptor-inactivation through secondary deleterious recombination events at the immunoglobulin μ -chain locus. (2) We studied pre-B cell receptor signaling molecules in primary human pre-B cells and 10 patient-derived Ph+ ALL samples by Western blotting and RT-PCR. As opposed to normal bone marrow pre-B cells, in all 10 cases of Ph+ ALL defective splice variants of the SYK tyrosine kinase and its linker molecule BLNK were found. Sequence analysis revealed a frequent 4 bp slippage during SYK pre-mRNA splicing which resulted in a truncated protein lacking the kinase domain, as confirmed by Western blot. To study the functional significance of defective Syk expression in Ph+ ALL cells, we transformed pre-B cells from Syk-fl/fl mice with BCR-ABL1 and deleted the Syk kinase using tamoxifen-inducible Cre. As opposed to Syk-fl/fl leukemia cells, inducible ablation of Syk rendered the leukemia cells insensitive to forced expression of the pre-B cell receptor. Multiple defective transcript variants of BLNK were found that all lacked exon 16 encoding the central part of the BLNK SH2 domain. In the absence of exon 16, BLNK splice variants were detached from the pre-B cell receptor and function in a dominant-negative way as they reduce Ca2+-mobilization in response to pre-B cell receptor stimulation. In a titration experiment, BLNK−/− leukemia cells were reconstituted with full-length and exon 16-deficient BLNK. Dominant-negative BLNK interfered with pre-B cell receptor-mediated tumor suppression at a ratio of 0.1 relative to full-length BLNK. Of note, we found somatic mutations within the splice site of exon 16 in 2 of 6 primary Ph+ ALL cases. (3) Ph+ ALL cells often carry chromosomal translocations leading to the expression of dominant-negative PAX5-fusion molecules. In a systematic gene expression analysis, we observed that ectopic expression of the dominant-negative PAX5-C20orf112 fusion led to downregulation of immunoglobulin μ -chain and the signaling molecules including SYK and BLNK. As a consequence, Ca2+-mobilization in response to pre-B cell receptor stimulation was significantly diminished. (4) Correction of defective immunoglobulin-μ chain and BLNK expression results in compensatory overexpression of a broad array of inhibitory signaling molecules. These molecules share an ITIM signaling motif, which attenuates pre-B cell receptor signal transduction through recruitment of inhibitory phosphatases. Conclusion: Even though loss of pre-B cell receptor function represents the uniform outcome of a diverse spectrum of lesions, individual Ph+ ALL subclones exhibit a complex pattern of shared and distinct defects involving one or more of these 4 mechanisms. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The BCR-ABL1 Kinase Bypasses Selection for the Expression of a Pre–B Cell Receptor in Pre–B Acute Lymphoblastic Leukemia Cells

2004 ◽  
Vol 199 (5) ◽  
pp. 673-685 ◽  
Author(s):  
Florian Klein ◽  
Niklas Feldhahn ◽  
Lana Harder ◽  
Hui Wang ◽  
Maria Wartenberg ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Leukemia Cells ◽  
Region Gene ◽  
B Cell Receptor Signaling ◽  
Selection For ◽  
Cell Receptor Signaling

The BCR-ABL1 kinase expressed in acute lymphoblastic leukemia (ALL) drives malignant transformation of human pre–B cells. Comparing genome-wide gene expression profiles of BCR-ABL1+ pre–B ALL and normal bone marrow pre–B cells by serial analysis of gene expression, many genes involved in pre–B cell receptor signaling are silenced in the leukemia cells. Although normal pre–B cells are selected for the expression of a functional pre–B cell receptor, BCR-ABL1+ ALL cells mostly do not harbor a productively rearranged IGH allele. In these cases, we identified traces of secondary VH gene rearrangements, which may have rendered an initially productive VH region gene nonfunctional. Even BCR-ABL1+ ALL cells harboring a functional VH region gene are unresponsive to pre–B cell receptor engagement and exhibit autonomous oscillatory Ca2+ signaling activity. Conversely, leukemia subclones surviving inhibition of BCR-ABL1 by STI571 restore responsiveness to antigen receptor engagement and differentiate into immature B cells expressing immunoglobulin light chains. BCR-ABL1 kinase activity is linked to defective pre–B cell receptor signaling and the expression of a truncated isoform of the pre–B cell receptor–associated linker molecule SLP65. Also in primary leukemia cells, truncated SLP65 is expressed before but not after treatment of the patients with STI571. We conclude that inhibition of BCR-ABL1 reconstitutes selection for leukemia cells expressing a functional (pre–) B cell receptor.

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.

Ikaros Mutation Confers Integrin-Dependent Survival Of Pre-B Cells and Progression To Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1259-1259
Author(s):  
Nilamani Jena ◽  
Ila Joshi ◽  
Toshimi Yoshida ◽  
Xiaoqing Qi ◽  
Jiangwen Zhang ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
Dna Binding ◽  
B Cell ◽  
Focal Adhesion ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Mapk Signaling ◽  
Dominant Negative ◽  
B Cell Differentiation

Abstract Deletion of the IKAROS DNA-binding domain generates dominant-negative isoforms that interfere with the transcriptional activity of the IKAROS family and correlate with poor prognosis in human precursor B cell acute lymphoblastic leukemias (B-ALL). In this study, we defined the role of the Ikaros family during pre-B cell differentiation, the stage from which human B-ALLs arise, by conditionally inactivating IKAROS DNA binding in the immediate precursors of pre-B cells in mice. We demonstrate a novel niche-dependent phase in early pre-B cell differentiation that supports self-renewal and proliferative expansion. Expression of dominant-negative IKAROS arrests cells in this state by augmenting integrin and MAPK signaling and attenuating pre-B cell receptor signaling and differentiation. Up-regulated genes in Ikaros mutant pre-B cells were highly enriched in pathways involved in focal adhesion and remodeling of the actin cytoskeleton. The mutant pre-B cells had increased β1 integrin-mediated adhesion and elevated levels of activated focal adhesion kinase (FAK), whereas treatment with a small molecule FAK inhibitor greatly reduced pre-B cell stromal adhesion and selectively induced apoptosis in Ikaros mutant but not WT pre-B cells. Transplantation of polyclonal Ikaros mutant pre-B cells into recipient mice resulted in long-latency oligoclonal pre-B-ALL, demonstrating that loss of IKAROS contributes to multistep B-leukemogenesis. The highly proliferative and aberrantly self-renewing phenotype of Ikaros-deficient pre-B cells illuminates mechanisms underlying human IKAROS mutant B-ALL and suggests new therapeutic strategies for treatment of this aggressive leukemia. Disclosures: Van Etten: Bristol Myers Squibb: Consultancy; Deciphera Pharmaceuticals: Consultancy; TEVA Pharmaceuticals: Consultancy, Research Funding.

scholarly journals Engagement of the B-cell receptor of chronic lymphocytic leukemia cells drives global and MYC-specific mRNA translation

Blood ◽  
2016 ◽  
Vol 127 (4) ◽  
pp. 449-457 ◽  
Author(s):  
Alison Yeomans ◽  
Stephen M. Thirdborough ◽  
Beatriz Valle-Argos ◽  
Adam Linley ◽  
Sergey Krysov ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mrna Translation ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Key Points ◽  
Specific Mrna

Key Points BCR stimulation promotes mRNA translation in CLL cells, including of the oncoprotein, MYC, and is inhibited by ibrutinib or tamatinib. Differences in mechanisms of regulation of mRNA translation in CLL and normal blood B cells may highlight potential targets for therapy.

scholarly journals Mimicry of a constitutively active pre–B cell receptor in acute lymphoblastic leukemia cells

2005 ◽  
Vol 201 (11) ◽  
pp. 1837-1852 ◽  
Author(s):  
Niklas Feldhahn ◽  
Florian Klein ◽  
Jana L. Mooster ◽  
Paul Hadweh ◽  
Mieke Sprangers ◽  
...  
Keyword(s):  
B Cell ◽  
Kinase Activity ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Full Length ◽  
Leukemia Cells ◽  
Domain 3 ◽  
Survival Signals ◽  
Constitutively Active

Pre–B cells undergo apoptosis unless they are rescued by pre–B cell receptor–dependent survival signals. We previously showed that the BCR-ABL1 kinase that is expressed in pre–B lymphoblastic leukemia bypasses selection for pre–B cell receptor–dependent survival signals. Investigating possible interference of BCR-ABL1 with pre–B cell receptor signaling, we found that neither SYK nor SLP65 can be phosphorylated in response to pre–B cell receptor engagement. Instead, Bruton's tyrosine kinase (BTK) is constitutively phosphorylated by BCR-ABL1. Activated BTK is essential for survival signals that otherwise would arise from the pre–B cell receptor, including activation of PLCγ1, autonomous Ca2+ signaling, STAT5-phosphorylation, and up-regulation of BCLXL. Inhibition of BTK activity specifically induces apoptosis in BCR-ABL1+ leukemia cells to a similar extent as inhibition of BCR-ABL1 kinase activity itself. However, BCR-ABL1 cannot directly bind to full-length BTK. Instead, BCR-ABL1 induces the expression of a truncated splice variant of BTK that acts as a linker between the two kinases. As opposed to full-length BTK, truncated BTK lacks kinase activity yet can bind to BCR-ABL1 through its SRC-homology domain 3. Acting as a linker, truncated BTK enables BCR-ABL1–dependent activation of full-length BTK, which initiates downstream survival signals and mimics a constitutively active pre–B cell receptor.

scholarly journals RasGRP1 Sensitizes an Immature B Cell Line to Antigen Receptor-induced Apoptosis

2004 ◽  
Vol 279 (19) ◽  
pp. 19523-19530 ◽  
Author(s):  
Benoit Guilbault ◽  
Robert J. Kay
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wehi 231 ◽  
Induced Apoptosis

RasGRP1 is a guanine nucleotide exchange factor that activates Ras GTPases and is activated downstream of antigen receptors on both T and B lymphocytes. Ras-GRP1 provides signals to immature T cells that confer survival and proliferation, but RasGRP1 also promotes T cell receptor-mediated deletion of mature T cells. We used the WEHI-231 cell line as an experimental system to determine whether RasGRP1 can serve as a quantitative modifier of B cell receptor-induced deletion of immature B cells. A 2-fold elevation in RasGRP1 expression markedly increased apoptosis of WEHI-231 cells following B cell receptor ligation, whereas a dominant negative mutant of RasGRP1 suppressed B cell receptor-induced apoptosis. Activation of ERK1 or ERK2 kinases was not required for RasGRP1-mediated apoptosis. Instead, elevated RasGRP1 expression caused down-regulation of NF-κB and Bcl-xL, which provide survival signals counter-acting apoptosis induction by B cell receptor. Inhibition of NF-κB was sufficient to enhance B cell receptor-induced apoptosis of WEHI-231 cells, and ligation of co-stimulatory receptors that activate NF-κB suppressed the ability of RasGRP1 to promote B cell receptor-induced apoptosis. These experiments define a novel apoptosis-promoting pathway leading from B cell receptor to the inhibition of NF-κB and demonstrate that differential expression of RasGRP1 has the potential to modulate the sensitivities of B cells to negative selection following antigen encounter.

The BCR-ABL1 Kinase Interferes with Pre-B Cell Receptor Signal Transduction in B Cell Precursor Leukemia Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1894-1894
Author(s):  
Florian Klein ◽  
Niklas Feldhahn ◽  
Peter Wernet ◽  
Markus Müschen
Keyword(s):  
Signal Transduction ◽  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Leukemia Cells ◽  
Cell Precursor ◽  
Receptor Signal ◽  
B Cell Receptor Signaling ◽  
Vh Gene

Abstract The BCR-ABL1 kinase expressed in B cell precursor leukemia drives malignant transformation of human pre-B cells. To identify novel target genes of BCR-ABL1-mediated transformation and to elucidate how the oncogenic BCR-ABL1 kinase may interfere with normal pre-B cell receptor signaling, we compared genome-wide gene expression of BCR-ABL1+ B cell precursor leukemia cells with their normal counterpart. While pre-B cell receptor signals are critical for differentiation and survival of normal pre-B cells, nearly all components of pre-B cell receptor-dependent signal transduction were downregulated in the leukemia cells. Surprisingly, BCR-ABL1+ leukemia cells mostly even do not harbor a productively rearranged IGH allele. In these cases, we identified traces of secondary VH gene rearrangements, which may have rendered an initially productive VH gene rearrangement nonfunctional. However, even BCR-ABL1+ leukemia cells harboring a functional VH gene rearrangement are unresponsive to pre-B cell receptor engagement. Independently from engagement, BCR-ABL1 kinase activity induces activation of PLCg1, resulting in an autonomous oscillatory Ca2+ signaling. BCR-ABL1 kinase activity is also linked to the expression of a truncated isoform of the pre-B cell receptor-associated linker molecule SLP65, which acts as a tumor suppressor in mice. Conversely, leukemia subclones surviving inhibition of BCR-ABL1 show responsiveness to antigen receptor engagement and express only full-length SLP65. These cells differentiate into immature B cells expressing Ig light chains. We conclude that BCR-ABL1 interferes with pre-B cell receptor signal transduction in B cell precursor leukemia cells, while its inhibition reconstitutes the selection for functional (pre-)B cell receptor signaling. Hence, a functional pre-B cell receptor signal-transduction cascade may provide these cells with a potential mechanism to escape apoptotic cell death induced by inhibition of BCR-ABL1. Figure Legend: BCR-ABL1 + leukemia cells were incubated for 48 hours in the presence or absence of 10 μmol/l STI571. Differentiating subclones among the STI571-treated cells replace their pre-B cell receptor by a B cell receptor and were enriched for surface IgM-expression by MACS. Figure Figure

scholarly journals IGLV3-21R110 identifies an aggressive biological subtype of chronic lymphocytic leukemia with intermediate epigenetics

Blood ◽  
2020 ◽  
Author(s):  
Ferran Nadeu ◽  
Romina Royo ◽  
Guillem Clot ◽  
Martí Duran-Ferrer ◽  
Alba Navarro ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Immunoglobulin Gene ◽  
Biological Features ◽  
Mutational Status ◽  
Bcr Signaling

B-cell receptor (BCR) signaling is crucial for chronic lymphocytic leukemia (CLL) biology. IGLV3-21-expressing B-cells may acquire a single point mutation (R110) that triggers autonomous BCR signaling conferring aggressive behavior. Epigenetic studies have defined three CLL subtypes based on methylation signatures reminiscent of naïve-like (n-CLL), intermediate (i-CLL) and memory-like B-cells (m-CLL) with different biological features. i-CLL carry a borderline IGHV mutational load and a significant higher usage of IGHV3-21/IGLV3-21. To determine the clinical and biological features of IGLV3-21R110 CLL and its relationship to these epigenetic subtypes we have characterized the immunoglobulin gene of 584 CLL cases using whole-genome/exome and RNA sequencing. IGLV3-21R110 was detected in 6.5% of cases, being 30/79 (38%) i-CLL, 5/291 (1.7%) m-CLL and 1/189 (0.5%) n-CLL. All stereotype subset #2 cases carried IGLV3-21R110 while 62% of IGLV3-21R110 i-CLL had non-stereotyped B-cell receptor immunoglobulins. IGLV3-21R110 i-CLL had significantly higher number of SF3B1 and ATM mutations, and total number of driver alterations. Nonetheless, the R110 mutation was the sole alteration in one i-CLL and accompanied only by del(13q) in three. Although composite regarding IGHV mutational status, IGLV3-21R110 i-CLL transcriptomically resembled naïve-like/unmutated IGHV CLL with a specific signature including WNT5A/B overexpression. Contrarily, i-CLL lacking the IGLV3-21R110 mirrored memory-like/mutated IGHV cases. IGLV3-21R110 i-CLL had a short time to first treatment and overall survival similar to n-CLL/unmutated IGHV cases whereas non-IGLV3-21R110 i-CLL had a good prognosis similar to memory-like/mutated IGHV. Altogether, IGLV3-21R110 defines a CLL subgroup with specific biological features and an unfavorable prognosis independent of the IGHV mutational status and epigenetic subtypes.

Aberrant Splicing of the SLP65 SH2 Domain Enables Pre-B Cell Transformation and Compromises the Leukemia-Suppressive Function of the Pre-B Cell Receptor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 294-294
Author(s):  
Cihangir Duy ◽  
Lars Klemm ◽  
Rahul Nahar ◽  
Peter van Essen ◽  
Mieke Sprangers ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
B Cell Receptor ◽  
Dominant Negative ◽  
Full Length ◽  
Aberrant Splicing ◽  
Cell Stage ◽  
Suppressive Function

Abstract The linker molecule SLP65 is critical for pre-B cell receptor-mediated differentiation signals during early B cell development. Germline mutations of the SLP65 gene in mice and humans result in a profound differentiation block at an early pre-B cell stage. Likewise, B cell lineage acute lymphoblast leukemia (ALL) cells are phenotypically arrested at an early pre-B cell stage in the vast majority of cases. In about 2–5% of cases with ALL, mutations and deletions of the SLP65 gene are found (Sprangers et al., 2006; Mullighan et al., 2007). In a comprehensive study of SLP65 function in human ALL cells, we identified numerous aberrant SLP65 splice variants, which all lack one or more exons that encode the C-terminal SLP65 SH2 domain. A splice variant lacking exon 16, which encodes the central part of the SH2 domain was particularly abundant in ALL but not in normal pre-B cells (EMBL/GenBank Accession number AM180337). The SH2 domain may have critical function because it represents the main ligand for the non-ITAM Y204 in the Igα (CD79A) signaling chain of the pre-B cell receptor (Patterson et al., 2006) and thereby connects the pre-B cell receptor to downstream signaling molecules. To examine a potential role of the SLP65 SH2 domain during the process of malignant transformation in ALL, we measured mRNA levels of full-length SLP65 vs SLP65 splice variants, in which the SH2 domain was deleted (SLP65ΔSH2) in normal pre-B cells and primary human ALL cells by quantitative RT-PCR. While normal pre-B cells express full-length SLP65 at high and SLP65ΔSH2 at very low levels, the SLP65/SLP65ΔSH2 ratio was reduced in E2A-PBX1-driven ALL and particularly low in BCR-ABL1-driven ALL. Given that SLP65ΔSH2 is overexpressed in ALL cells at the expense of full-length SLP65, we investigated biochemical properties of SLP65ΔSH2. To this end, we transduced Slp65−/− pre-B cells with either full-length Slp65, SLP65ΔSH2 or a GFP empty vector control. As opposed to full-length SLP65, SLP65ΔSH2 does not interact with Igα and does not confer tyrosine phosphorylation of BTK downstream of the pre-B cell receptor. Also Ca2+-release in response to pre-B cell receptor engagement and induction of differentiation was drastically reduced in Slp65−/− pre-B cells that were reconstituted with SLP65ΔSH2 instead of SLP65. Co-expression experiments with SLP65 showed that SLP65ΔSH2 has a dominant-negative function and impairs pre-B cell receptor signaling. We next transformed Slp65−/− pre-B cells with a retroviral BCR-ABL1 vector and tested whether SLP65 and SLP65ΔSH2 have a different impact on BCR-ABL1-mediated pre-B cell transformation and BCR-ABL1-driven leukemic growth. Reconstitution of SLP65 expression in Slp65−/− BCR-ABL1-transformed pre-B ALL cells resulted in cell death and nine days after transduction more than 90% of the SLP65-transduced cells had undergone apoptosis. Conversely, reconstitution of Slp65−/− BCR-ABL1-transformed pre-B ALL cells with SLP65ΔSH2 had no significant effect on viability of the leukemia cells. In a titration experiment, we reconstituted SLP65−/−BCR-ABL1-driven ALL cells with SLP65 and SLP65ΔSH2 at various ratios and thereby confirmed that SLP65ΔSH2 has a dominant-negative effect and compromises the leukemia-suppressive function of the pre-B cell receptor. We next tested whether aberrant splicing also interferes with the tumor suppressor function of SLP65 in vivo: To this end, we engrafted SLP65−/−BCR-ABL1- driven ALL cells that were transduced with either SLP65-GFP, SLP65ΔSH2 or GFP into sublethally irradiated NOD/SCID mice. After 24 days, mice that were engrafted with ALL cells transduced with SLP65ΔSH2 or GFP became terminally leukemic, whereas mice injected with SLP65-transduced ALL cells showed no signs of disease. At this time, all mice were sacrificed and analyzed. Whereas mice with SLP65ΔSH2- or GFP-transduced ALL cells had enlarged spleens and substantial leukemic infiltration of the bone marrow (79 and 81% leukemia cell content), leukemic infitration was reduced if SLP65-transduced ALL cells were injected (1.8% leukemia infiltration). Whereas mutations and deletions of the SLP65 gene are rare, we conclude that aberrant splicing of the SLP65 SH2 domain represents a common mechanism in ALL cells to compromise the leukemia-suppressive function of the pre-B cell receptor.

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