Bruton’s Tyrosine Kinase and SLP-65 Regulate Pre-B Cell Differentiation and the Induction of Ig Light Chain Gene Rearrangement

Bruton's tyrosine kinase (Btk) and the adapter protein SLP-65 (Src homology 2 domain-containing leukocyte-specific phosphoprotein of 65 kDa) transmit precursor BCR (pre-BCR) signals that are essential for efficient developmental progression of large cycling into small resting pre-B cells. We show that Btk- and SLP-65-deficient pre-B cells have a specific defect in Ig λ L chain germline transcription. In Btk/SLP-65 double-deficient pre-B cells, both κ and λ germline transcripts are severely reduced. Although these observations point to an important role for Btk and SLP-65 in the initiation of L chain gene rearrangement, the possibility remained that these signaling molecules are only required for termination of pre-B cell proliferation or for pre-B cell survival, whereby differentiation and L chain rearrangement is subsequently initiated in a Btk/SLP-65-independent fashion. Because transgenic expression of the antiapoptotic protein Bcl-2 did not rescue the developmental arrest of Btk/SLP-65 double-deficient pre-B cells, we conclude that defective L chain opening in Btk/SLP-65-deficient small resting pre-B cells is not due to their reduced survival. Next, we analyzed transgenic mice expressing the constitutively active Btk mutant E41K. The expression of E41K-Btk in Ig H chain-negative pro-B cells induced 1) surface marker changes that signify cellular differentiation, including down-regulation of surrogate L chain and up-regulation of CD2, CD25, and MHC class II; and 2) premature rearrangement and expression of κ and λ light chains. These findings demonstrate that Btk and SLP-65 transmit signals that induce cellular maturation and Ig L chain rearrangement independently of their role in termination of pre-B cell expansion

Transcription Factor Sp3 Knockout Mice Display Serious Cardiac Malformations▿

Mice lacking the zinc finger transcription factor specificity protein 3 (Sp3) die prenatally in the C57BL/6 background. To elucidate the cause of mortality we analyzed the potential role of Sp3 in embryonic heart development. Sp3 null hearts display defective looping at embryonic day 10.5 (E10.5), and at E14.5 the Sp3 null mutants have developed a range of severe cardiac malformations. In an attempt to position Sp3 in the cardiac developmental hierarchy, we analyzed the expression patterns of >15 marker genes in Sp3 null hearts. Expression of cardiac ankyrin repeat protein (Carp) was downregulated prematurely after E12.5, while expression of the other marker genes was not affected. Chromatin immunoprecipitation analysis revealed that Sp3 is bound to the Carp promoter region in vivo. Microarray analysis indicates that small-molecule metabolism and cell-cell interactions are the most significantly affected biological processes in E12.5 Sp3 null myocardium. Since the epicardium showed distension from the myocardium, we studied expression of Wt1, a marker for epicardial cells. Wt1 expression was diminished in epicardium-derived cells in the myocardium of Sp3 null hearts. We conclude that Sp3 is required for normal cardiac development and suggest that it has a crucial role in myocardial differentiation

Malignant transformation of Slp65-deficient pre-B cells involves disruption of the Arf-Mdm2-p53 tumor suppressor pathway

The adapter protein Slp65 is a key component of the precursor-B (pre-B) cell receptor. Slp65-deficient mice spontaneously develop pre-B cell leukemia, but the mechanism by which Slp65(-/-) pre-B cells become malignant is unknown. Loss of Btk, a Tec-family kinase that cooperates with Slp65 as a tumor suppressor, synergizes with deregulation of the c-Myc oncogene during lymphoma formation. Here, we report that the presence of the immunoglobulin heavy chain transgene V(H)81X prevented tumor development in Btk(-/-)Slp65(-/-) mice. This finding paralleled the reported effect of a human immunoglobulin heavy chain transgene on lymphoma development in E mu-myc mice, expressing transgenic c-Myc. Because activation of c-Myc strongly selects for spontaneous inactivation of the p19(Arf)-Mdm2-p53 tumor suppressor pathway, we investigated whether disruption of this pathway is a common alteration in Slp65(-/-) pre-B cell tumors. We found that combined loss of Slp65 and p53 in mice transformed pre-B cells very efficiently. Aberrations in p19(Arf), Mdm2, or p53 expression were found in all Slp65(-/-)(n = 17) and Btk(-/-)Slp65(-/-)(n = 32) pre-B cell leukemias analyzed. In addition, 9 of 10 p53(-/-)Slp65(-/-) pre-B cell leukemias manifested significant Mdm2 protein expression. These data indicate that malignant transformation of Slp65(-/-) pre-B cells involves disruption of the p19(Arf)-Mdm2-p53 tumor suppressor pathway. (Blood. 2010; 115: 1385-1393

MCLA-117, a CLEC12AxCD3 bispecific antibody targeting a leukaemic stem cell antigen, induces T cell-mediated AML blast lysis

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Malignant transformation of Slp65-deficient pre-B cells involves disruption of the Arf-Mdm2-p53 tumor suppressor pathway

The adapter protein Slp65 is a key component of the precursor-B (pre-B) cell receptor. Slp65-deficient mice spontaneously develop pre-B cell leukemia, but the mechanism by which Slp65-/- pre-B cells become malignant is unknown. Loss of Btk, a Tec-family kinase that cooperates with Slp65 as a tumor suppressor, synergizes with deregulation of the c-Myc oncogene during lymphoma formation. Here, we report that the presence of the immunoglobulin heavy chain transgene VH81X prevented tumor development in Btk -/-Slp65-/- mice. This finding paralleled the reported effect of a human immunoglobulin heavy chain transgene on lymphoma development in Eμ-myc mice, expressing transgenic c-Myc. Because activation of c-Myc strongly selects for spontaneous inactivation of the p19Arf-Mdm2-p53 tumor suppressor pathway, we investigated whether disruption of this pathway is a common alteration in Slp65-/- pre-B cell tumors. We found that combined loss of Slp65 and p53 in mice transformed pre-B cells very efficiently. Aberrations in p19Arf, Mdm2, or p53 expression were found in all Slp65-/-(n = 17)andBtk-/-Slp65-/-(n = 32) pre-B cell leukemias analyzed. In addition, 9 of 10 p53-/-Slp65 -/- pre-B cell leukemias manifested significant Mdm2 protein expression. These data indicate that malignant transformation of Slp65 -/- pre-B cells involves disruption of the p19Arf-Mdm2-p53 tumor suppressor pathway

Btk levels set the threshold for B-cell activation and negative selection of autoreactive B cells in mice

On antigen binding by the B-cell receptor (BCR), B cells up-regulate protein expression of the key downstream signaling molecule Bruton tyrosine kinase (Btk), but the effects of Btk up-regulation on B-cell function are unknown. Here, we show that transgenic mice overexpressing Btk specifically in B cells spontaneously formed germinal centers and manifested increased plasma cell numbers, leading to antinuclear autoantibody production and systemic lupus erythematosus (SLE)-like autoimmune pathology affecting kidneys, lungs, and salivary glands. Autoimmunity was fully dependent on Btk kinase activity, because Btk inhibitor treatment (PCI-32765) could normalize B-cell activation and differentiation, and because autoantibodies were absent in Btk transgenic mice overex-pressing a kinase inactive Btk mutant. B cells overexpressing wild-type Btk were selectively hyperresponsive to BCR stimulation and showed enhanced Ca2+ influx, nuclear factor (NF)-κB activation, resistance to Fas-mediated apoptosis, and defective elimination of selfreactive B cells in vivo. These findings unravel a crucial role for Btk in setting the threshold for B-cell activation and counterselection of autoreactive B cells, making Btk an attractive therapeutic target in systemic autoimmune disease such as SLE. The finding of in vivo pathology associated with Btk overexpression may have important implications for the development of gene therapy strategies for X-linked agammaglobulinemia, the immunodeficiency associated with mutations in BTK

MCLA-117, a CLEC12AxCD3 bispecific antibody targeting a leukaemic stem cell antigen, induces T cell-mediated AML blast lysis

Objective: We report the characterization of MCLA-117, a novel T cell-redirecting antibody for acute myeloid leukaemia (AML) treatment targeting CD3 on T cells and CLEC12A on leukaemic cells. In AML, CLEC12A is expressed on blasts and leukaemic stem cells. Methods: The functional capacity of MCLA-117 to redirect resting T cells to eradicate CLEC12APOS tumor cells was studied using human samples, including primary AML samples. Results: Within the normal hematopoietic compartment, MCLA-117 binds to cells expressing CD3 and CLEC12A but not to early myeloid progenitors or hematopoietic stem cells. MCLA-117 induces T cell activation (EC50 = 44 ng/mL), T cell proliferation, mild pro-inflammatory cytokine release, and redirects T cells to lyse CLEC12APOS target cells (EC50 = 68 ng/mL). MCLA-117-induced targeting of normal CD34POS cells co-cultured with T cells spares erythrocyte and megakaryocyte differentiation as well as preserves mono-myelocytic lineage development. In primary AML patient samples with autologous T cells, MCLA-117 robustly induced AML blast killing (23–98%) at low effector-to-target ratios (1:3–1:97). Conclusion: These findings demonstrate that MCLA-117 efficiently redirects T cells to kill tumour cells while sparing the potential of the bone marrow to develop the full hematological compartment and support further clinical evaluation as a potentially potent treatment option for AML