Hassan Jumaa

Bruton's tyrosine kinase cooperates with the B cell linker protein SLP-65 as a tumor suppressor in Pre-B cells.

Expression of the pre-B cell receptor (pre-BCR) leads to activation of the adaptor molecule SLP-65 and the cytoplasmic kinase Btk. Mice deficient for one of these signaling proteins have an incomplete block in B cell development at the stage of large cycling pre-BCR+CD43+ pre-B cells. Our recent findings of defective SLP-65 expression in ∼50% of childhood pre-B acute lymphoblastic leukemias and spontaneous pre-B cell lymphoma development in SLP-65−/− mice demonstrate that SLP-65 acts as a tumor suppressor. To investigate cooperation between Btk and SLP-65, we characterized the pre-B cell compartment in single and double mutant mice, and found that the two proteins have a synergistic role in the developmental progression of large cycling into small resting pre-B cells. We show that Btk/SLP-65 double mutant mice have a dramatically increased pre-B cell tumor incidence (∼75% at 16 wk of age), as compared with SLP-65 single deficient mice (<10%). These findings demonstrate that Btk cooperates with SLP-65 as a tumor suppressor in pre-B cells. Furthermore, transgenic low-level expression of a constitutive active form of Btk, the E41K-Y223F mutant, prevented tumor formation in Btk/SLP-65 double mutant mice, indicating that constitutive active Btk can substitute for SLP-65 as a tumor suppressor.

LAT links the pre-BCR to calcium signaling.

SLP-65(-/-) mice display a partial block at the pre-B cell stage of development. Here, we show that LAT is required for the differentiation of SLP-65(-/-) pre-B cells. We show that LAT and SLP-76 are recruited to the pre-BCR and associated with Ig-alpha upon pre-BCR engagement, whereas LAT interaction with SLP-76 is already detected in untreated pre-B cells. Reconstitution of LAT or SLP-65 expression in SLP-65/LAT(-/-) pre-B cells restored their calcium (Ca2+) mobilization capacity, led to downregulation of surface pre-BCR, and induced differentiation to BCR+ cells. Together, our results suggest that the adaptor proteins LAT and SLP-76 are involved in pre-BCR signaling, thereby rescuing arrested murine SLP-65(-/-) pre-B cells.

BCL6 is critical for the development of a diverse primary B cell repertoire

BCL6 protects germinal center (GC) B cells against DNA damage–induced apoptosis during somatic hypermutation and class-switch recombination. Although expression of BCL6 was not found in early IL-7–dependent B cell precursors, we report that IL-7Rα–Stat5 signaling negatively regulates BCL6. Upon productive VH-DJH gene rearrangement and expression of a μ heavy chain, however, activation of pre–B cell receptor signaling strongly induces BCL6 expression, whereas IL-7Rα–Stat5 signaling is attenuated. At the transition from IL-7–dependent to –independent stages of B cell development, BCL6 is activated, reaches expression levels resembling those in GC B cells, and protects pre–B cells from DNA damage–induced apoptosis during immunoglobulin (Ig) light chain gene recombination. In the absence of BCL6, DNA breaks during Ig light chain gene rearrangement lead to excessive up-regulation of Arf and p53. As a consequence, the pool of new bone marrow immature B cells is markedly reduced in size and clonal diversity. We conclude that negative regulation of Arf by BCL6 is required for pre–B cell self-renewal and the formation of a diverse polyclonal B cell repertoire.

Role of PI3K in the generation and survival of B cells

Summary:u2002 Engagement of the B‐cell antigen receptor (BCR) or its precursor, the pre‐BCR, induces a cascade of biochemical reactions that regulate the differentiation, selection, survival, and activation of B cells. This cascade is initiated by receptor‐associated tyrosine kinases that activate multiple downstream signaling pathways. Since it is required for metabolism, cell growth, development, and survival, the activation of phosphoinositide 3‐kinase (PI3K)‐dependent pathways represents a crucial event of BCR/pre‐BCR signaling. The phosphorylated substrates of the PI3K promote specific recruitment of selected signaling proteins to the plasma membrane, where important signaling complexes are formed to mediate the above‐mentioned biological processes. Here, we review the principles of PI3K signaling and highlight the role of an important PI3K‐driven module in VDJ recombination of immunoglobulin (Ig) genes during early B‐cell development as compared with class switch recombination of Ig genes in mature B cells after activation by specific antigens. Furthermore, we discuss the role of PI3K in the survival of mature B cells, which is strictly dependent on BCR expression and basal BCR signaling.

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

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, Brutons 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 BCLX L. 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.

A leucine zipper in the N terminus confers membrane association to SLP-65

Membrane recruitment of adaptor proteins is crucial for coupling antigen receptors to downstream signaling events. Despite the essential function of the B cell adaptor SLP-65, the mechanism of its recruitment to the plasma membrane is not yet understood. Here we show that a highly conserved leucine zipper in the SLP-65 N terminus is responsible for membrane association. Alterations in the N terminus abolished SLP-65 membrane localization and activity, both of which were restored by replacement of the N terminus with a myristoylation signal. The N terminus is an autonomous domain that confers specific localization and function when transferred to green fluorescent protein or the adaptor protein SLP-76. Our data elucidate the mechanism of SLP-65 membrane recruitment and suggest that leucine zipper motifs are essential interaction domains of signaling proteins.

FoxO1 induces Ikaros splicing to promote immunoglobulin gene recombination

During murine B cell development, PI3 kinase inhibits Ig gene rearrangement by suppressing FoxO1, which mediates Ikaros mRNA splicing; Ikaros is needed for Ig gene recombination.

Insulin-like growth factor-1 receptor (IGF1R) as a novel target in chronic lymphocytic leukemia

The receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF1R) is implicated in various tumor entities including chronic lymphocytic leukemia (CLL), but its functional significance in this disease remains poorly characterized. Here, we show that the IGF1R protein is overexpressed in various CLL subsets, suggesting a contribution to CLL pathology. Indeed, we show that IGF1R knockdown in primary human CLL cells compromised their viability. Likewise, IGF1R inhibition with 3 structurally distinct compounds induced apoptosis, even in the presence of protective stroma components. Furthermore, IGF1R inhibition effectively limited CLL development in Eμ-TCL1 transgenic mice and of primary human CLL xenografts. In agreement with its prosurvival function, IGF1R inhibition affected the phosphorylation and/or expression of multiple signaling proteins. The multikinase inhibitor sorafenib yielded similar effects on these signaling elements as IGF1R inhibitors. Indeed, IGF1R appears to be a direct sorafenib target because sorafenib decreased IGF1R expression and phosphorylation, counteracted insulin-like growth factor-1 (IGF-1) binding to CLL cells, and lowered the in vitro kinase activity of recombinant, purified IGF1R. Thus, we demonstrate that blockade of IGF1R-mediated signaling represents a novel mechanism of action for sorafenib in CLL. Importantly, IGF1R inhibitors compromise CLL viability in their microenvironment context, implicating this RTK as a promising therapeutic target.

SOX4 enables oncogenic survival signals in acute lymphoblastic leukemia

The Sox4 transcription factor mediates early B-cell differentiation. Compared with normal pre-B cells, SOX4 promoter regions in Ph(+) ALL cells are significantly hypomethylated. Loss and gain-of-function experiments identified Sox4 as a critical activator of PI3K/AKT and MAPK signaling in ALL cells. ChIP experiments confirmed that SOX4 binds to and transcriptionally activates promoters of multiple components within the PI3K/AKT and MAPK signaling pathways. Cre-mediated deletion of Sox4 had little effect on normal pre-B cells but compromised proliferation and viability of leukemia cells, which was rescued by BCL2L1 and constitutively active AKT and p110 PI3K. Consistent with these findings, high levels of SOX4 expression in ALL cells at the time of diagnosis predicted poor outcome in a pediatric clinical trial (COG P9906). Collectively, these studies identify SOX4 as a central mediator of oncogenic PI3K/AKT and MAPK signaling in ALL.

Efficient generation of B lymphocytes by recognition of self‐antigens

Antibody diversity is generated by a random gene recombination process with the inherent risk of the production of autoreactive specificities. The current view suggests that B cells expressing such specificities are negatively selected at an early developmental stage. Using the knock‐in model system of the 3‐83 autoreactive B‐cell antigen receptor (BCR) in combination with precursor‐BCR (pre‐BCR) deficiency, we show here that the 3‐83 BCR mediates efficient generation of B cells in the presence, but not the absence, of a strongly recognized auto‐antigen. Experiments with mixed bone marrow chimeras showed that combining the 3‐83 BCR with the corresponding auto‐antigen resulted in efficient reconstitution of B‐cell development in immune‐deficient mice. These results suggest that B cells are positively selected by recognition of self‐antigens during developmental stages that precede receptor editing. Moreover, the data indicate that the pre‐BCR functions as a specialized autoreactive BCR to initiate positive selection at a stage where the cells express immunoglobulin heavy but not light chains.