Overexpression of BCL9, a Wnt/β-Catenin Transcriptional Co-Activator, in Transgenic Mice Promotes Spontaneous Development of B-Cell Malignancies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 441-441
Author(s):  
Tomasz Sewastianik ◽  
Jianjun Zhao ◽  
Meng Jiang ◽  
Peter S. Dennis ◽  
Myles Brown ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
Signaling Pathway ◽  
Transcriptional Activity ◽  
Hematological Malignancies ◽  
B Cell Malignancies ◽  
Human Cancers ◽  
Stop Cassette

Abstract Dysregulation of the Wnt signaling pathway underlies the pathogenesis of a wide range of human cancers, including hematological malignancies such as multiple myeloma (MM). The terminal effector of this signaling pathway is a transcriptional complex formed by β-catenin and BCL9. This complex is of particular interest because the BCL9 locus resides on the frequently recurring 1q21 chromosomal amplification in MM, which has been linked to poor clinical prognosis and outcome. Our previous studies indicate that BCL9-mediated enhancement of β-catenin activity increases cells proliferation, migration, invasion, and the metastatic potential of MM cells. Therefore, in order to: (I) unequivocally determine the oncogenic role of BCL9, (II) better understand its mechanism of action, and (III) develop mouse preclinical model of cancer with dysregulated Wnt/β-catenin/BCL9 activity, we generated transgenic mouse models. To overcome problems inherently related to embryonic lethality, we generated BCL9fl/- conditional transgenic mice using site-specific transgene integration into the mouse ColA1 gene in embryonic stem cells. To remove the stop cassette and activate BCL9 expression in vivo, we generated AID-Cre+/-; BCL9fl/- and ERT2-Cre+/-; BCL9fl/- compound mice. Recombinase activity driven by AID (activation-induced cytidine deaminase) gene promoter or ER receptor after tamoxifen administration, caused removal of the stop cassette and expression of BCL9 in germinal center (GC) B cells or several tissues, respectively, as confirmed by immunoblot, immunohistochemical (IHC) and PCR analysis. Since BCL9 is a β-catenin co-activator, next we generated cohorts of AID-Cre+/-; BCL9fl/-; TCF/Lef1-lacZ+/- and ERT2-Cre+/-; BCL9fl/-; TCF/Lef1-lacZ+/- triple compound transgenic mice carrying the Wnt reporter system that expresses β-galactosidase (β-gal), to determine whether Wnt/β-catenin transcriptional activity is increased as a consequence of BCL9 overexpression in vivo. β-gal stain was increased in frequency and intensity in cells within GCs but not outside them in AID-Cre+/-; BCL9fl/-; TCF/Lef1-lacZ+/- compared to control mice. In ERT2-Cre+/-; BCL9fl/-; TCF/Lef1-lacZ+/- mice β-gal staining was primarily detected in cells outside the GCs, not within them. Overall, these results indicate that Wnt transcriptional activity is increased in B-cells as a consequence of Cre-induced expression of BCL9 and that AID-Cre+/- and ERT2-Cre+/- target expression of BCL9 to GC and non-GC B cells, respectively. Because BCL9 is involved in the pathogenesis of human cancers, we evaluated whether our transgenic mice develop hematological malignancies. Except for mild splenic enlargement, BCL9-transgenic mice were indistinguishable from control mice between 8 and 30 weeks of age as assessed by weight and posture. However, after 40 weeks of age and at variable times thereafter, 80% (32/40) of AID-Cre+/-; BCL9fl/- and 70% (28/40) of ERT2-Cre+/-; BCL9fl/- mice but none from control cohorts showed signs of disease. Gross pathologic examination of euthanized animals with BCL9 overexpression revealed enlargement of the spleen and LNs. Two distinct patterns of clonal hematological malignancies were identified after detailed histological, IHC and molecular examination. In AID-Cre+/-; BCL9fl/- mice tumors resembled human plasmacytomas (PCs), whereas in ERT2-Cre+/-; BCL9fl/- mice B-cell acute lymphoblastic leukemia (B-ALL). This later result is of particular interest, since BCL9 was first identified by cloning the t(1;14)(q21;q32) translocation from a patient with B-ALL. These findings indicate that BCL9 overexpression at different stages of B-cell development leads to distinct subtypes of B-cell malignancies. Finally, we investigated the BCL9 expression in human extramedullary plasmocytomas (EMP) and B-ALL. 32% of EMP cases analyzed by IHC expressed BCL9 at significant levels. Utilizing gene expression data available in the public domain we also showed that BCL9 is significantly overexpressed in ETV6-RUNX1 and TCF3-PBX1 subtypes of human B-ALL when compared to normal bone marrow counterparts, suggesting that BCL9 may play important roles in the pathogenesis of EMP as well as B-ALL in humans. Since BCL9 is highly expressed in tumors but not in the cells of origin and its interaction with β-catenin is specific, these results imply BCL9 as a promising candidate for targeted therapy. Disclosures No relevant conflicts of interest to declare.

CDDO and CDDO-Im Reduce Tumor Burden in a Transgenic Mouse Model of CLL.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3477-3477
Author(s):  
Juan M. Zapata ◽  
Christina L. Kress ◽  
Marina Konopleva ◽  
Maryla Krajewska ◽  
Mark Hyer ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Chemotherapeutic Agents ◽  
Tumor Burden ◽  
Leukemic Cells ◽  
Medium Size ◽  
Control Group

Abstract Transgenic mice over-expressing in B lymphocytes both Bcl-2 and a TRAF2 mutant lacking the N-terminal RING and zinc finger domains (TRAF2DN), which mimics TRAF1, develop small B cell lymphoma and leukemia that have remarkably similar characteristics to human chronic lymphocytic leukemia (CLL). TRAF2DN/Bcl-2 mice develop over time leukemia, severe splenomegaly, and lymphadenopathy, which are associated with monoclonal and oligoclonal B cell neoplasms. The lifespan of TRAF2DN/Bcl-2 mice is markedly reduced compared to Bcl-2 and TRAF2DN single transgenics or wild-type littermates. The expanded B cell population in the blood of leukemic TRAF2DN/Bcl-2 double transgenic mice is primarily comprised of small-medium size, non-cycling B220M/IgMH/IgDL/CD21L/CD23−/CD11b+/CD5+ cells that were Bcl-6 negative, consistent with a B-1 phenotype, closely resembling their human CLL counterparts. Indeed, these B cells showed comparable proliferation rates to normal B-cells, but exhibited markedly increased survival and were resistant to apoptosis induced by chemotherapeutic agents and glucocorticoids. We studied the effects of synthetic triterpenoid 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic Acid (CDDO) and its imidazolide derivative (CDDO-Im) on cultured B-cells from the TRAF2DN/Bcl-2 transgenic mice. Both CDDO and CDDO-Im efficiently induced apoptosis of these cells in vitro, although CDDO-Im was approximately 10-times more potent than CDDO (LD50: 0.35μM CDDO-Im vs 3.8 μM CDDO). To study the effect of CDDO and CDDO-Im in vivo, groups of TRAF2DN/Bcl-2 mice that had developed leukemia were injected i.v. with liposomes alone or liposomes containing either CDDO or CDDO-Im, at a dose of 20 mg/kg/day. Each mouse received a total of nine injections administered over a period of 22 days. The concentration of B cells in the blood of these mice was monitored daily after each injection, using a mini-FACS (Guava Technologies, Inc.). CDDO-treated mice showed a steady reduction in the number of leukemic cells in blood during the treatment and this tendency was maintained 10 days after the last treatment. In contrast, CDDO-Im treated mice showed a striking increase in the concentration of B cells in blood (B220+ events) immediately after the first inoculation. One mouse of this group died after the first injection, and 2 more mice died after 5 injections. Only 2 mice treated with CDDO-Im survived the full treatment, showing a striking reduction of leukemic cells in blood by the end of the treatment. Administration of empty liposomes had no inhibitory effect on the leukemia, and mice in this control group had massive splenomegaly (1431±323 mg; n=3) and severe disseminated lymphadenopathy. In contrast, CDDO-treated mice had less severe splenomegaly (938±234; n=4) but still had severe lymphadenopathy. CDDO-Im treated mice showed a dramatic reduction in the spleen size that was evident also in those mice that died after 5 injections (474±185 mg; n=4) and had no signs of lymphadenopathy. Although preliminary, these results indicate that in vivo administration of CDDO and CDDO-Im reduced the tumor burden in a transgenic model of CLL, and illustrate the potential of triterpenoids as single agents for the treatment of CLL.

Depletion of Normal and Malignant B cells with a CD37-specific SMIP molecule

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 3063-3063
Author(s):  
C. Cerveny ◽  
L. Grosmaire ◽  
E. Espling ◽  
R. Bader ◽  
C. Nilsson ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Target Cell ◽  
Growth Arrest ◽  
Xenograft Model ◽  
B Cell Malignancies ◽  
B Lymphoma ◽  
Anti Tumor Activity

3063 Background: CD37 is a member of the tetraspanin family expressed at high levels by normal mature B cells and by most B cell malignancies. Previously, an antibody to CD37 has been labeled with 131I and tested in clinical trials for therapy of NHL. Treatment with 131I-MB-1, resulted in durable tumor remissions in patients lasting from 4 to 11 months (Press OW, Eary JF, Badger CC, et al. Treatment of refractory non-Hodgkin’s lymphoma with radiolabeled MB-1 (anti-CD37) antibody. J Clin Oncol. 1989;7:1027–1038). Here we assess the functional properties and therapeutic potential of a small modular immunopharmaceutical (SMIP) targeting CD37. Methods: Growth arrest and apoptosis of B lymphoma cell lines was assessed. ADCC activity was evaluated using BJAB targets and human peripheral blood mononuclear cells (PBMC) effectors. Drug-drug interactions were assessed by the Combination Index method. In vivo studies were performed utilizing established human B cell tumor xenografts in nude mice. Results: A CD37-directed SMIP drug candidate mediated growth arrest, apoptosis and ADCC, but not CDC, towards B lymphoma cell lines. The protein showed significant anti-tumor activity in a mouse xenograft model, and selectively depleted normal human B cells in short term cultures of PBMC. When combined with rituximab, the molecule increased apoptosis, C1q binding, and C’ dependent target cell death in vitro, and increased anti-tumor activity in vivo in a xenograft model. Conclusions: In vitro and in vivo characterization of the CD37-targeted SMIP drug suggest a potent capacity to eliminate target cells through combined effects of direct target cell signaling and effector cell recruitment. CD37-mediated growth was synergistic with standard chemotherapies in vitro and showed additive in vivo activity with CD20-targeted therapy. On the basis of these data CD37-directed SMIP therapy is being developed for clinical evaluation against B cell malignancies. No significant financial relationships to disclose.

scholarly journals KLF4 suppresses transformation of pre-B cells by ABL oncogenes

Blood ◽  
2006 ◽  
Vol 109 (2) ◽  
pp. 747-755 ◽  
Author(s):  
Michael G. Kharas ◽  
Isharat Yusuf ◽  
Vanessa M. Scarfone ◽  
Vincent W. Yang ◽  
Julia A. Segre ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
Tumor Suppressor ◽  
B Cell ◽  
Cell Activation ◽  
Cell Transformation ◽  
Cell Lineage ◽  
Cell Types ◽  
B Cell Malignancies

Abstract Genes that are strongly repressed after B-cell activation are candidates for being inactivated, mutated, or repressed in B-cell malignancies. Krüppel-like factor 4 (Klf4), a gene down-regulated in activated murine B cells, is expressed at low levels in several types of human B-cell lineage lymphomas and leukemias. The human KLF4 gene has been identified as a tumor suppressor gene in colon and gastric cancer; in concordance with this, overexpression of KLF4 can suppress proliferation in several epithelial cell types. Here we investigate the effects of KLF4 on pro/pre–B-cell transformation by v-Abl and BCR-ABL, oncogenes that cause leukemia in mice and humans. We show that overexpression of KLF4 induces arrest and apoptosis in the G1 phase of the cell cycle. KLF4-mediated death, but not cell-cycle arrest, can be rescued by Bcl-XL overexpression. Transformed pro/pre-B cells expressing KLF4 display increased expression of p21CIP and decreased expression of c-Myc and cyclin D2. Tetracycline-inducible expression of KLF4 in B-cell progenitors of transgenic mice blocks transformation by BCR-ABL and depletes leukemic pre-B cells in vivo. Collectively, our work identifies KLF4 as a putative tumor suppressor in B-cell malignancies.

scholarly journals The LMP2A ITAM Is Essential for Providing B Cells with Development and Survival Signals In Vivo

2000 ◽  
Vol 74 (19) ◽  
pp. 9115-9124 ◽  
Author(s):  
Mark Merchant ◽  
Robert G. Caldwell ◽  
Richard Longnecker
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
Tyrosine Kinases ◽  
Cell Receptor ◽  
Lytic Replication ◽  
Barr Virus ◽  
Bcr Signaling ◽  
Survival Signals

ABSTRACT In Epstein-Barr virus-transformed B cells, known as lymphoblastoid cell lines (LCLs), LMP2A binds the tyrosine kinases Syk and Lyn, blocking B-cell receptor (BCR) signaling and viral lytic replication. SH2 domains in Syk mediate binding to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) in LMP2A. Mutation of the LMP2A ITAM in LCLs eliminates Syk binding and allows for full BCR signaling, thereby delineating the significance of the LMP2A-Syk interaction. In transgenic mice, LMP2A causes a developmental alteration characterized by a block in surface immunoglobulin rearrangement resulting in BCR-negative B cells. Normally B cells lacking cognate BCR are rapidly apoptosed; however, LMP2A transgenic B cells develop and survive without a BCR. When bred into the recombinase activating gene 1 null (RAG−/−) background, all LMP2A transgenic lines produce BCR-negative B cells that develop and survive in the periphery. These data indicate that LMP2A imparts developmental and survival signals to B cells in vivo. In this study, LMP2A ITAM mutant transgenic mice were generated to investigate whether the LMP2A ITAM is essential for the survival phenotype in vivo. LMP2A ITAM mutant B cells develop normally, although transgene expression is comparable to that in previously described nonmutated LMP2A transgenic B cells. Additionally, LMP2A ITAM mutant mice are unable to promote B-cell development or survival when bred into the RAG−/− background or when grown in methylcellulose containing interleukin-7. These data demonstrate that the LMP2A ITAM is required for LMP2A-mediated developmental and survival signals in vivo.

Enforced BCL6 Expression Inhibits B Cell Development in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1535-1535
Author(s):  
Davide F. Robbiani ◽  
Kaity Colon ◽  
Kruti Naik ◽  
Helen Nickerson ◽  
Maurizio Affer ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
Light Chain ◽  
Germinal Center ◽  
Regulatory Elements ◽  
Cell Development ◽  
B Cell Development ◽  
Kappa Light Chain

Abstract The B-Cell Lymphoma 6 (BCL6) gene encodes for a zinc finger motifs containing transcriptional repressor that is frequently dysregulated by chromosomal translocations in germinal center lymphomas. A putative protooncogene, its transforming ability in vivo was reported in I-mu-HA-BCL6 knock-in mice by Cattoretti et al last year. We also tested this assumption in transgenic mice expressing BCL6 in B cells under the control of kappa light chain regulatory elements. We replaced the murine C-kappa locus with the 16kb human BCL6 genomic locus in a construct containing the murine kappa light chain regulatory elements (Vk, EiK, 3′RR). While control transgenics were readily obtained (5/32 founders), only 3/68 founders were positive for the BCL6 transgene, of which only one (bearing a single copy of the transgene) was able to transmit the transgene to its progeny, thus suggesting embryonal toxicity of exogenous BCL6. In the bone marrow, flow cytometry revealed a nearly complete block of B cell development at the pro-B to pre-B transition. This was also the stage at which we first detected expression of EGFP in control reporter mice that were generated in parallel. Spleens of transgenic mice weighed about 50% of control spleens and less than 5% of splenocytes were CD19+ B cells. These were IgM high, IgD intermediate, corresponding to an immature B cell phenotype. Lymph nodes were smaller and B cells barely detected. Peyers’ patches were not visible. Combined, our analysis of 6–8 weeks old VkHABCL6 transgenic mice reveals that enforced expression of BCL6 early in development results in a profound block of B lymphocyte differentiation. How transgenic BCL6 modulates this effect at the transcriptional level remains to be investigated. To test the oncogenic potential of BCL6 in B cells, it will be interesting to precisely turn on this gene in the germinal center.

In Vivo Depletion of Cynomolgus Monkey B Cells Targeted by a Novel Monoclonal Antibody (Xi-20H5) Specific for a Shared Portion of the Human Immunoglobulin Variable Light Chain Lambda 1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2354-2354
Author(s):  
Thierry Sornasse ◽  
Keri Tate ◽  
Kimberly Milner ◽  
Thomas Theriault ◽  
Dan W. Denney ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Light Chain ◽  
Peripheral Blood ◽  
Cynomolgus Monkey ◽  
Patient Specific ◽  
B Cell Malignancies ◽  
Variable Regions ◽  
Variable Light

Abstract Genitope is developing a novel, personalized treatment for surface immunoglobulin (Ig) positive B cell malignancies. This treatment is based on a panel of monoclonal antibodies (Mabs) directed against shared epitopes expressed on different subsets of the variable regions of human Ig. The concept of targeting surface tumor Ig was explored in a series of clinical trials performed by Dr. Ronald Levy and his colleagues at Stanford. In these studies, Mabs directed against patient specific (idiotypic) determinants of the surface tumor Ig produced significant clinical benefit in relapsed follicular non-Hodgkin’s lymphoma patients. A number of these patients have had long term remissions. In Genitope’s planned clinical use, each patient will receive a single Mab from the panel selected based on its reactivity with the patient’s tumor. The selected Mab will react with the patient’s tumor and a minority of normal B cells, leaving the majority of the normal B cell repertoire intact. The ability of the panel members to provide therapeutic effect requires binding to tumor surface Ig in the presence of serum containing soluble Ig molecules. Mab Xi-20H5, a member of this panel of antibodies, is specific for a shared determinant on the human Ig variable light chain lambda 1. It binds to 25 to 35% of normal human B cells from peripheral blood and to 20 to 30% of normal cynomolgus monkey B cells from peripheral blood. In this study, we sought to demonstrate that, despite the presence of serum Ig, Mab Xi-20H5 would bind to surface Ig expressed on monkey B cells in vivo, resulting in specific depletion of target B cells. Six naïve cynomolgus monkeys received 8 intravenous infusions of the Mab Xi-20H5 at a dose of 40 mg/kg on days 1, 2, 3, 4, 7, 10, 14 & 17. Two naïve control animals received 8 infusions of vehicle only following the same schedule. The frequencies of lymphocyte sub-populations and of target B cells were monitored by flow cytometry on plasma-depleted whole blood samples. Samples were collected 23 hours after each infusion. In addition, two baseline samples were collected prior to treatment. The frequencies of lymphocyte sub-populations and of target B cells were compared to the average of the two baseline measurements. Frequencies of target B cells bound by Mab Xi-20H5 decreased in all treated animals while no significant change was detectable in the control animals. The bulk of the reduction in target B cell frequencies was observed 23 hours after the first infusion (range: 22% – 62%, average 41%). Frequencies of target B cells continued to decrease moderately with additional daily infusions (days 2 – 4), resulting in maximum reduction in target B cell frequency at 23 h post infusion 4 (range: 39% – 78%, average 54%). The frequencies of total B and T lymphocytes did not significantly change during the treatment. In vivo administration of Mab Xi-20H5 results in depletion of target B cells in a manner consistent with the expectation of an immunotherapeutic Mab aimed at treating surface Ig expressing B cell malignancies.

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

Blood ◽  
2012 ◽  
Vol 119 (16) ◽  
pp. 3744-3756 ◽  
Author(s):  
Laurens P. Kil ◽  
Marjolein J. W. de Bruijn ◽  
Menno van Nimwegen ◽  
Odilia B. J. Corneth ◽  
Jan Piet van Hamburg ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
Cell Activation ◽  
Systemic Autoimmune Disease ◽  
B Cell Activation ◽  
Autoantibody Production ◽  
Autoreactive B Cells ◽  
Autoimmune Pathology

Abstract 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 overexpressing 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.

scholarly journals SOX11 Cooperates with CCND1 in Mantle Cell Lymphoma Pathogenesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1253-1253 ◽  
Author(s):  
Pei-Yu Kuo ◽  
Zewei Jiang ◽  
Deepak Perumal ◽  
Violetta V Leshchenko ◽  
Alessandro Lagana' ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Mouse Model ◽  
Transgenic Mice ◽  
B Cell ◽  
Transgenic Mouse ◽  
Cell Lymphoma ◽  
Mantle Cell ◽  
Sox11 Expression

Abstract MCL (Mantle cell lymphoma) is an aggressive and incurable B cell malignancy with a median survival of 5-6 years. Cyclin D1 (CCND1) overexpression is a key diagnostic feature of this disease, observed in more than 90% of MCL tumors. However, murine models over-expressing CCND1 in B cells do not recapitulate the phenotype of MCL. The SOX11 transcription factor is aberrantly expressed in 80-90% of primary MCL. Our published data demonstrated that SOX11 binds and functionally regulates key components in multiple oncogenic pathways in MCL such as WNT and TGFβ pathways. Recent studies have also showed that SOX11 regulates PAX5 and PDGFA to block differentiation and facilitate lymphoma growth. We thus hypothesize that SOX11 expression may contribute directly and functionally cooperate with CCND1 in MCL pathogenesis. To study the role of SOX11 in MCL tumorigenesis in vivo, we have generated a novel SOX11 transgenic mouse model with B cell-specific tissue expression under the E-mu enhancer and an IRES-eGFP tag to monitor the expression of SOX11. The presence of SOX11 can be readily detected in pre-pro-B stage in the bone marrow coincided with the activation of E-mu enhancer and was persistent through all stages of B cells. SOX11 over-expression in our mouse model led to an aberrant oligo-clonal expansion of CD19+/CD5+ B cells. This phenotype was evident in all SOX11 transgenic mice studied (100% penetrance, n= 42 mice) with an average of 7-12 fold increase (p<0.03) of the CD5+ B cell populations as compared to littermate controls starting from 1.5 months. Using Mass Cytometry (CyTOF), we further characterized this B cell population to be CD23-, CD21/35 dim, CD138-, high surface IgM, and variable IgD expression, a naive B cell phenotype consistent with an early precursor stage of human MCL. This MCL phenotype is most prominent in peripheral blood and spleen and, to a much lesser extent, in peritoneal cavity and bone marrow. Transplanting bone marrow from SOX11 transgenic mouse to lethally-irradiated wild type mice successfully transferred the observed phenotypic CD19+/CD5+/CD23- B cell hyperplasia, suggesting that SOX11 overexpression in early B cells drives this MCL phenotype. We next studied the cooperation between CCND1 and SOX11 by crossing SOX11 transgenic mice with a CCND1 transgenic mouse model, which over-expresses CCND1 in a B-cell specific manner under a similar E-mu enhancer. Overexpression of both CCND1 and SOX11 in the double transgenic mice model dramatically enhanced (average 10x, range 6x-30x) the aberrant MCL phenotype (CD19+/CD5+/CD23-) in peripheral blood, spleen, bone marrow, peritoneal cavity and lymph nodes compared to age-matched SOX11 and CCND1 single-transgenic mice. We report here the first direct evidence in vivo that SOX11 expression drives an aberrant expansion of B cells consistent with early human MCL and functionally collaborates with CCND1 in "full blown" MCL pathogenesis, mimicking the commonly observed co-expression of SOX11 and CCND1 in most human MCL tumors. This model captures the underpinning molecular pathogenesis events occurred in the majority of human MCL and overcomes constraints of previous MCL models that develop a phenotype after long latency or with low penetrance, making it a valuable tool for testing anti-MCL therapeutics. We are currently developing small molecule SOX11 inhibitors using SOX11 DNA binding domain models and consensus SOX11 binding nucleotides to screen a large library of compounds to identify new therapeutics for this fatal disease and gain better understanding of the molecular mechanisms of MCL tumorigenesis. Disclosures No relevant conflicts of interest to declare.

NK Cells Contribute Significantly to the Innate Immune Effector Role of CD37-Specific SMIP in CLL and NHL.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 135-135 ◽  
Author(s):  
Xiaobin B. Zhao ◽  
Joshi Trupti ◽  
Rosa Lapalombella ◽  
Carolyn Cheney ◽  
Aruna Gowda ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Nk Cells ◽  
Therapeutic Efficacy ◽  
T Regulatory Cells ◽  
Raji Cell ◽  
Regulatory Cells ◽  
B Cell Malignancies

Abstract CD37 is a lineage-specific B-cell antigen that represents an attractive target for immunotherapy in B-cell malignancies, especially in chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL). CD37-specific small modular immuno pharmaceutical (CD37-SMIP™) drug is an engineered protein therapeutic directed to the CD37 antigen using a single chain variable region (scFv) linked to a modified human IgG1 hinge, CH2, and CH3 domains. We have previously presented that CD37-SMIP™ drug induces both ADCC and apoptosis against primary CLL cells and B-cell lymphoma cells and therapeutic efficacy was observed in a Raji cell disseminated leukemia xenograft model. Herein, we sought to determine the effector cell type(s) mediating ADCC and explore if agents that deplete NK cell inhibitory T-regulatory cells influence CD37-SMIP™ efficacy. Immunostaining of human peripheral blood mononuclear cells (PBMCs) from CLL patients demonstrated no expression of CD37 on CD3+ T cells, CD16+ or CD56+ NK cells, or CD64+ monocytes whereas CD37 was highly expressed on CD19+ B cells. Using purified human PBMCs as effector cells and Cr-51 labelled CLL B cells as targets, we found the CD37-SMIP™ dependent ADCC was predominantly mediated by NK cells, but not naïve or activated monocytes. Consistent with these in-vitro results, the in-vivo therapeutic efficacy of CD37-SMIP™ drug was significantly compromised by depletion of NK cells in the Raji cell disseminated leukemia xenograft SCID mouse model. The median survival time of CD37-SMIP™ treated mice decreased from 51 days (95% CI: 38, 78) to 27 days (95% CI: 25, 37) (p=0.017) with the depletion of NK cells. Consistent with previous studies, ADCC is not diminished by fludarabine, an agent that may deplete T-regulatory cells, suggesting that the anti-CD37 protein and fludarabine might combine for increased efficacy in-vivo. We therefore examined the effect of fludarabine on CD37-SMIP™ in-vitro apoptotic activity. These data demonstrate that direct cell death mediated by CD37-SMIP™ drug (5 ug/mL) synergizes with fludarabine-induced caspase-dependent apoptosis, as measured by both the MTT assay and annexin V/PI staining of CLL cells (combination index, CI=0.44). Overall, these data suggest that the CD37-SMIP™ is a promising therapeutic agent against CD37+ B-cell malignancies as either monotherapy or in combination with fludarabine. Further clinical development is warranted to investigate the effect of CD37-SMIP™ drug in CLL and NHL. (SMIP trademark is owned by Trubion Pharmaceuticals).

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