Mouse B-cell Research Articles

Parity History Determines a Systemic Inflammatory Response to Spread of Ovarian Cancer in Naturally Aged Mice.

Aging intersects with reproductive senescence in women by promoting a systemic low-grade chronic inflammation that predisposes women to several diseases including ovarian cancer (OC). OC risk at menopause is significantly modified by parity records during prior fertile life. To date, the combined effects of age and parity on the systemic inflammation markers that are particularly relevant to OC initiation and progression at menopause remain largely unknown. Herein, we profiled a panel of circulating cytokines in multiparous versus virgin C57BL/6 female mice at peri-estropausal age and investigated how cytokine levels were modulated by intraperitoneal tumor induction in a syngeneic immunocompetent OC mouse model. Serum FSH, LH and TSH levels increased with age in both groups while prolactin (PRL) was lower in multiparous respect to virgin mice, a finding previously observed in parous women. Serum CCL2, IL-10, IL-5, IL-4, TNF-α, IL1-β and IL-12p70 levels increased with age irrespective of parity status, but were specifically reduced following OC tumor induction only in multiparous mice. Animals developed hemorrhagic ascites and tumor implants in the omental fat band and other intraperitoneal organs by 12 weeks after induction, with multiparous mice showing a significantly extended survival. We conclude that previous parity history counteracts aging-associated systemic inflammation possibly by reducing the immunosuppression that typically allows tumor spread. Results suggest a partial impairment of the M2 shift in tumor-associated macrophages as well as decreased stimulation of regulatory B-cells in aged mice. This long term, tumor-concurrent effect of parity on inflammation markers at menopause would be a contributing factor leading to decreased OC risk.

CTLA-4 Limits Anti-CD20–Mediated Tumor Regression

The inhibition of tumor growth by anti-CD20 antibody (Ab) treatment is mediated by Ab- and complement-dependent cytotoxicity in xenograft tumor models. In addition, anti-CD20 therapy for B-cell lymphoma can result in intrinsic and extrinsic tumor resistance to further Ab treatment. However, adaptive immune response-related resistance has not been well studied in anti-CD20-mediated tumor control, and adaptive immunity has long been underestimated. The purpose of this study was to explore whether T cells are involved in mediating the effects of anti-CD20 therapy and what factors contribute to adaptive immune response-related resistance. Using a syngeneic mouse B-cell lymphoma model, we investigated the role of CD8+ T cells in anti-CD20-mediated tumor regression. Furthermore, we revealed how the tumor-specific T-cell response was initiated by anti-CD20. Finally, we studied adaptive immune response-related resistance in advanced B-cell lymphoma. CD8+ T cells played an essential role in anti-CD20-mediated tumor regression. Mechanistically, anti-CD20 therapy promoted dendritic cell (DC)-mediated cross-presentation. Importantly, macrophages were also necessary for the increase in the tumor-specific CTL response after anti-CD20 treatment, via the production of type I IFN to activate DC function. Furthermore, adaptive resistance is gradually developed through the CTLA-4 pathway in Treg cells in larger lymphomas. Further blockade of CTLA-4 can synergize with anti-CD20 treatment in antitumor activities. The therapeutic function of anti-CD20 depends on tumor-specific CD8+ T-cell responses initiated by anti-CD20 through macrophages and DCs. CTLA-4 blockade can synergize with anti-CD20 to overcome adaptive immune response-related resistance in advanced B-cell lymphoma. Clin Cancer Res; 23(1); 193-203. ©2016 AACR.

Crebbp Mutations Disrupt Dynamic Enhancer Acetylation in B-Cells, Enabling HDAC3 to Drive Lymphomagenesis

Somatic mutations in CREBBP occur frequently in germinal center derived lymphomas such as DLBCL and FL. However whether or how these mutations might contribute to lymphomagenesis is still largely unknown. Most CREBBP mutations are predicted to result in loss of function since they target the histone acetyltransferase (HAT) domain or give rise to premature stop codon prior to the HAT domain. Here, we show that Crebbp shRNA knockdown (KD) accelerated lymphomagenesis in VavP-Bcl2 transgenic mice, a model that recapitulates human GC-derived lymphomas. The median time to lymphoma onset in VavP-Bcl2/CrebbpKD mice was 114 days, significantly shorter than control VavP-Bcl2/GFP mice (193 days, p=0.04). Histopathology revealed that VavP-Bcl2/CrebbpKD lymphomas were more aggressive and widely disseminated than VavP-Bcl2/GFP lymphomas.CREBBP can regulate gene enhancer function through H3K27 acetylation. ChIP-seq in VavP-Bcl2/CrebbpKD lymphoma cells revealed significant reduction of H3K27ac peaks compared to control lymphoma cells (N = 1717, Kolmogorov-Smirnov test, p<2.2E-16). Loss of H3K27ac was markedly skewed towards enhancers. We observed similar loss of enhancer H3K27ac in human DLBCL cells after CREBBP shRNA KD. Enhancer H3K27ac loss was significantly associated with repression of nearby genes in both murine (FDR q=0.044) and human lymphoma cells (FDR q=0). RNA-seq performed in three independent FL or DLBCL patient cohorts revealed a characteristic CREBBP mutant gene expression signature featuring prominent transcriptional repression (p=1.32E-14, p=0.001, and p=0.0002 respectively). Notably, the human patient CREBBP mutant signature was highly enriched in murine and human cell line CREBBP shRNA profiles (FDR=0, GSEA), indicating that CREBBP KD signature was highly similar to CREBBP mutant signature in humans. Functional analysis of the CREBBP mutant/KD signature showed significant enrichment of GC exit pathways including genes induced by CD40, IRF4 and plasma cell differentiation; as well as immune response processes including antigen processing and presentation, such as MHC class II genes (BH-adjusted p<0.05).To better understand mechanism we performed an integrative analysis of CREBBP signatures against databases of B-cell transcription factor and epigenome profiles. This analysis yielded significant enrichment (BH-adjusted p<0.05) for i) enhancers bound by the BCL6 transcriptional repressor and its SMRT/HDAC3 corepressor complex, ii) enhancers that are normally deacetylated in GC B-cells, and iii) genes induced by BCL6 siRNA. This is notable because in normal GCs BCL6 represses enhancers by recruiting SMRT/HDAC3 complexes to deacetylate H3K27. Hence our data suggest that CREBBP is a counteracting HAT to BCL6/SMRT/HDAC3. Indeed, conditional knockout of Hdac3 in GC B-cells in mice resulted in impaired GC formation and a transcriptional signature featuring upregulation of the same genes that are repressed by CREBBP KD (GSEA FDR=0). Moreover, CREBBP KD in DLBCL cells resulted in H3K27ac loss at BCL6/SMRT/HDAC3 regulated enhancers, including those nearby CDKN1A, NFATC1, FOXP1, and MHC II genes, such as HLA-DQA1 and HLA-DRB5. CREBBP KD also resulted in silencing of these genes. Since we show HDAC3 is the opposing HDAC to CREBBP then we reasoned that CREBBP mutant DLBCLs might be especially dependent on HDAC3. Indeed we observed that HDAC3 shRNA resulted in profound suppression of CREBBP mutant DLBCL cells in vitro and in vivo (DLBCL xenografts in mice, p=0.005), whereas CREBBP WT cell lines were barely affected by HDAC3 KD.The opposing effects of BCL6/SMRT/HDAC3 and CREBBP on MHC class II could have implications for immune surveillance. Accordingly CREBBP KD induced significant loss of cell surface HLA-DR molecules (p<0.05), and these cells exhibited up to 90% less capability to stimulate T-cell response in allogeneic mixed lymphocyte reaction experiments. The loss of MHC class II molecules and T-cell response was rescued when CREBBP loss of function cells were exposed to a specific HDAC3 inhibitor. In summary, CREBBP mutations drive lymphomagenesis by enabling unopposed suppression of enhancers by BCL6/SMRT/HDAC3 complexes, resulting in a repressive transcriptional programming that disrupts GC exit and evades immune surveillance. HDAC3 targeted therapy may rescue these effects and serve as a precision approach for CREBBP mutant lymphomas. DisclosuresScott:Celgene: Consultancy; Roche: Honoraria; Janssen: Consultancy; BC Cancer Agency: Patents & Royalties: Inventor on a patent licensed to NanoString Technologies. Tam:Millennium Pharmaceuticals, Inc.: Consultancy. Melnick:Janssen: Research Funding.

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

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. DisclosuresNo relevant conflicts of interest to declare.

Permissive roles of cytokines interleukin-7 and Flt3 ligand in mouse B-cell lineage commitment

Hematopoietic cells are continuously generated throughout life from hematopoietic stem cells, thus making hematopoiesis a favorable system to study developmental cell lineage commitment. The main factors incorporating environmental signals to developing hematopoietic cells are cytokines, which regulate commitment of hematopoietic progenitors to the different blood lineages by acting either in an instructive or a permissive manner. Fms-like tyrosine kinase-3 (Flt3) ligand (FL) and Interleukin-7 (IL-7) are cytokines pivotal for B-cell development, as manifested by the severely compromised B-cell development in their absence. However, their precise role in regulating B-cell commitment has been the subject of debate. In the present study we assessed the rescue of B-cell commitment in mice lacking IL-7 but simultaneously overexpressing FL. Results obtained demonstrate that FL overexpression in IL-7-deficient mice rescues B-cell commitment, resulting in significant Ebf1 and Pax5 expression in Ly6D+CD135+CD127+CD19- precursors and subsequent generation of normal numbers of CD19+ B-cell progenitors, therefore indicating that IL-7 can be dispensable for commitment to the B-cell lineage. Further analysis of Ly6D+CD135+CD127+CD19- progenitors in IL-7- or FL-deficient mice overexpressing Bcl2, as well as in IL-7 transgenic mice suggests that both FL and IL-7 regulate B-cell commitment in a permissive manner: FL by inducing proliferation of Ly6D+CD135+CD127+CD19- progenitors and IL-7 by providing survival signals to these progenitors.

The mitochondrial translation machinery as a therapeutic target in Myc-driven lymphomas.

The oncogenic transcription factor Myc is required for the progression and maintenance of diverse tumors. This has led to the concept that Myc itself, Myc-activated gene products, or associated biological processes might constitute prime targets for cancer therapy. Here, we present an in vivo reverse-genetic screen targeting a set of 241 Myc-activated mRNAs in mouse B-cell lymphomas, unraveling a critical role for the mitochondrial ribosomal protein (MRP) Ptcd3 in tumor maintenance. Other MRP-coding genes were also up regulated in Myc-induced lymphoma, pointing to a coordinate activation of the mitochondrial translation machinery. Inhibition of mitochondrial translation with the antibiotic Tigecycline was synthetic-lethal with Myc activation, impaired respiratory activity and tumor cell survival in vitro, and significantly extended lifespan in lymphoma-bearing mice. We have thus identified a novel Myc-induced metabolic dependency that can be targeted by common antibiotics, opening new therapeutic perspectives in Myc-overexpressing tumors.

MEF2C protects bone marrow B-lymphoid progenitors during stress haematopoiesis.

DNA double strand break (DSB) repair is critical for generation of B-cell receptors, which are pre-requisite for B-cell progenitor survival. However, the transcription factors that promote DSB repair in B cells are not known. Here we show that MEF2C enhances the expression of DNA repair and recombination factors in B-cell progenitors, promoting DSB repair, V(D)J recombination and cell survival. Although Mef2c-deficient mice maintain relatively intact peripheral B-lymphoid cellularity during homeostasis, they exhibit poor B-lymphoid recovery after sub-lethal irradiation and 5-fluorouracil injection. MEF2C binds active regulatory regions with high-chromatin accessibility in DNA repair and V(D)J genes in both mouse B-cell progenitors and human B lymphoblasts. Loss of Mef2c in pre-B cells reduces chromatin accessibility in multiple regulatory regions of the MEF2C-activated genes. MEF2C therefore protects B lymphopoiesis during stress by ensuring proper expression of genes that encode DNA repair and B-cell factors.

AMP-Activated Protein Kinase Suppresses Autoimmune Central Nervous System Disease by Regulating M1-Type Macrophage-Th17 Axis.

The AMP-activated protein kinase, AMPK, is an energy-sensing, metabolic switch implicated in various metabolic disorders; however, its role in inflammation is not well defined. We have previously shown that loss of AMPK exacerbates experimental autoimmune encephalomyelitis (EAE) disease severity. In this study, we investigated the mechanism through which AMPK modulates inflammatory disease like EAE. AMPKα1 knockout (α1KO) mice with EAE showed severe demyelination and inflammation in the brain and spinal cord compared with wild-type due to higher expression of proinflammatory Th17 cytokines, including IL-17, IL-23, and IL-1β, impaired blood-brain barrier integrity, and increased infiltration of inflammatory cells in the CNS. Infiltrated CD4 cells in the brains and spinal cords of α1KO with EAE were significantly higher compared with wild-type EAE and were characterized as IL-17 (IL-17 and GM-CSF double-positive) CD4 cells. Increased inflammatory response in α1KO mice was due to polarization of macrophages (Mϕ) to proinflammatory M1 type phenotype (IL-10(low)IL-23/IL-1β/IL-6(high)), and these M1 Mϕ showed stronger capacity to induce allogenic as well as Ag-specific (myelin oligodendrocyte glycoprotein [MOG]35-55) T cell response. Mϕ from α1KO mice also enhanced the encephalitogenic property of MOG35-55-primed CD4 T cells in B6 mice. The increased encephalitogenic MOG-restricted CD4(+) T cells were due to an autocrine effect of IL-1β/IL-23-mediated induction of IL-6 production in α1KO Mϕ, which in turn induce IL-17 and GM-CSF production in CD4 cells. Collectively, our data indicate that AMPK controls the inflammatory disease by regulating the M1 phenotype-Th17 axis in an animal model of multiple sclerosis.

TSLP or IL-7 provide an IL-7Rα signal that is critical for human B lymphopoiesis.

Thymic stromal lymphopoietin (TSLP) and IL-7 are cytokines that signal via the IL-7 receptor alpha (IL-7Rα) to exert both overlapping and unique functions during early stages of mouse B-cell development. In human B lymphopoiesis, the requirement for IL-7Rα signaling is controversial and the roles of IL-7 and TSLP are less clear. Here, we evaluated human B-cell production using novel in vitro and xenograft models of human B-cell development that provide selective IL-7 and human TSLP (hTSLP) stimulation. We show that in vitro human B-cell production is almost completely blocked in the absence of IL-7Rα stimulation, and that either TSLP or IL-7 can provide a signal critical for the production and proliferation of human CD19(+) PAX5(+) pro-B cells. Analysis of primary human bone marrow stromal cells shows that they express both IL-7 and TSLP, providing an in vivo source of these cytokines. We further show that the in vivo production of human pro-B cells under the influence of mouse IL-7 in a xenograft scenario is reduced by anti-IL-7 neutralizing antibodies, and that this loss can be restored by hTSLP at physiological levels. These data establish the importance of IL-7Rα mediated signals for normal human B-cell production.

Conditional disruption of Raptor reveals an essential role for mTORC1 in B cell development, survival, and metabolism.

Abstract Mechanistic target of Rapamycin (mTOR) senses diverse environmental conditions including nutrient, growth factor and stress conditions, to regulate cellular growth, division, and differentiation. Studies examining the role of mTOR signaling in immune function have revealed critical roles for mTOR in regulating T-cell differentiation and function. However, there have been few studies investigating the role of mTOR in early B cell development and function. We found that mTORC1 activity is highly activated during the pro- and pre-B stages of mouse B cell development. Disruption of mTORC1 signaling in developing mouse B-cells resulted in a developmental block at the pre-B cell stage, with a corresponding lack of peripheral B-cells and loss of antigen-specific antibody production. Pre-B cell survival and proliferation were also significantly reduced in B-cells from mTORC1-signaling deficient mice. mTORC1-signaling deficiency resulted in a significant decrease in both oxidative phosphorylation and glycolysis indicating that loss of mTORC1 signaling in B cells results in altered cellular metabolism. Forced expression of a transgenic B-cell receptor on mTORC1-signaling deficient B-cells failed to rescue the developmental block. Collectively, our data reveal a previously uncharacterized role of mTORC1 signaling in early B-cell development and metabolism.

A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice.

Interleukin-12 family cytokines have emerged as critical regulators of immunity with some members (IL-12, IL-23) associated with disease pathogenesis while others (IL-27, IL-35) mitigate autoimmune diseases. Each IL-12 family member is comprised of an α and a β chain, and chain-sharing is a key feature. Although four bona fide members have thus far been described, promiscuous chain-pairing between alpha (IL-23p19, IL-27p28, IL-12/IL-35p35) and beta (IL-12/IL-23p40, IL-27/IL-35Ebi3) subunits, predicts six possible heterodimeric IL-12 family cytokines. Here, we describe a new IL-12 member composed of IL-23p19 and Ebi3 heterodimer (IL-39) that is secreted by LPS-stimulated Bcells and GL7(+) activated Bcells of lupus-like mice. We further show that IL-39 mediates inflammatory responses through activation of STAT1/STAT3 in lupus-like mice. Taken together, our results show that IL-39 might contribute to immunopathogenic mechanisms of systemic lupus erythematosus, and could be used as a possible target for its treatment.

Sprouty2 regulates proliferation and survival of multiple myeloma byinhibiting activation of the ERK1/2 pathway invitro and invivo.

Multiple myeloma (MM) is an incurable disease, and its pathogenesis remains unclear. MicroRNA (miR)-21 was detected at a high level in MM and plays a key role in the pathogenesis of MM. However, Sprouty2 (spry2), a downstream target of miR-21, has low expression, and its mechanism in MM is unknown. We investigated whether spry2 could exert an antimyeloma effect and further studied the potential pathogenesis and progression of MM. To address the functional consequences of spry2, we assessed the expression levels of spry2 in several myeloma cell lines and detected low expression levels in MMcells. Overexpression of spry2 suppressed growth and colony formation ability and decreased the phosphorylation of extracellular signal-regulated kinases 1 and 2. Spry2 also decreased secretion of vascular endothelial growth factor and partially enhanced the sensitivity of MMcells to an inhibitor of mitogen-activated protein kinases 1 and 2. Additionally, spry2 inhibited the tumorigenesis and angiogenesis of MMcells invivo. In summary, we report for the first time that spry2 can inhibit MMcell growth and survival with a concomitant reduction in phosphorylation of extracellular signal-regulated kinases 1 and 2 invitro and invivo.

CXCR2 MediatesBrucella-Induced Arthritis in Interferon γ–Deficient Mice

Brucella species are facultative intracellular gram-negative bacteria that cause brucellosis, a common global zoonosis. Infection of the joints is the most common focal complication of brucellosis in humans. The purpose of this study was to identify mediators of focal inflammation during brucellosis. Wild-type (WT) mice are naturally resistant to Brucella infection; therefore, we infected anti-interferon γ (IFN-γ)-treated, or IFN-γ(-/-) mice with Brucella to induce osteoarticular and musculoskeletal inflammation, as we previously described. Mice were infected intraperitoneally with Brucella melitensis, and the clinical course of disease, histopathologic changes, and cytokine levels were compared among groups. Rag1(-/-) mice (B- and T-cell deficient) and µMT(-/-) mice (B-cell deficient) developed paw inflammation at a similar rate and severity as WT mice following infection with B. melitensis and treatment with anti-IFN-γ. Joints from B. melitensis-infected IFN-γ(-/-) mice had markedly increased levels of CCR2 and CXCR2 ligands. While anti-IFN-γ-treated CCR2(-/-) and WT mice behaved similarly, anti-IFN-γ-treated CXCR2(-/-) or IFN-γ(-/-)/CXCR2(-/-) mice had strikingly reduced focal swelling relative to anti-IFN-γ-treated WT or IFN-γ(-/-) mice, respectively. Additionally, neutrophil recruitment was dependent on CXCR2. Adaptive immune cells and CCR2 are dispensable, while CXCR2 is necessary for Brucella-induced focal neutrophil recruitment and inflammation.

Abstract IA21: Exploiting metabolic alterations in therapy-induced senescence and drug resistance in a transgenic mouse lymphoma model by reverse and forward omics

Abstract Treatment failure is the key determinant of poor outcome in lymphoma therapy. Unveiling the underlying molecular mechanisms is critical to overcome drug insensitivity, may identify novel targets and direct the development of conceptual treatment alternatives. We utilize transgenic mouse lymphoma models as valuable tools for the molecular dissection of treatment responsiveness. Notably, we previously demonstrated the predictive cross-species power of our murine lymphoma model for patients diagnosed with diffuse large B-cell lymphoma (DLBCL) (Reimann-M et al., Cancer Cell, 2010; Jing-H et al., Genes Dev., 2011). Here, we employ two different approaches: reverse genomics, allowing us to test the dependency of certain effector mechanisms such as apoptosis or senescence (Dörr-JR et al., Nature, 2013) on distinct genetics, and forward omics, a multitude of omics-based investigations, namely (epi-)genomics, transcriptomics, proteomics, and metabolomics to decipher mechanisms of patient-reminiscent treatment resistance in the well-established Eµ-myc-driven lymphoma mouse model. Technically, we transplanted primary Eµ-myc transgenic mouse B-cell lymphomas with or without defined genetic lesions into immunocompetent mice, and expose the recipients to cyclophosphamide (CTX) chemotherapy upon tumor manifestation. Whole-exome sequencing, copy number alteration analysis, array-based transcriptomics and kinomics, mass spectrometry-based proteomics and metabolomics as well as functional assays (e.g. apoptosis, cellular senescence) were applied, and the data subjected to bioinformatics processing to unveil molecular mechanisms of treatment resistance. After treatment of lymphoma-bearing mice, lasting remissions (reflecting cure) were observed in about half of the cohort (comparable to DLBCL patients after induction therapy). Repetitive treatments of relapsing mice resulted in progressively shortened remission times and finally full-blown resistance, thereby recapitulating clinical courses of patients with drug-insensitive aggressive lymphomas. Multipe omics technologies were applied to the large sample panel to compare curable vs. relapse-prone and resistant lymphomas, all with or without an additional short-term exposure to CTX to acutely challenge drug-specific response programs. Candidate findings will be discussed at the conference. In the reverse genomics approaches, findings related to distinct mutations in key lymphoma drivers, their metabolic implications for treatment outcome, and their specific role in therapy-induced senescence and senescence-related vulnerabilities will be presented at the meeting. Dörr, J.R., Y. Yu, M. Milanovic, G. Beuster, C. Zasada, S. Lee, and C.A. Schmitt. 2013. Synthetic lethal metabolic targeting of cellular senescence in cancer therapy. Nature 501: 421-425. Jing, H., J. Kase, J.R. Dörr, M. Milanovic, D. Lenze, C.A. Schmitt, and S. Lee. 2011. Opposing roles of NF-κB in anti-cancer treatment outcome unveiled by cross-species investigations. Genes Dev. 25: 2137-2146. Reimann, M., S. Lee, C. Loddenkemper, J.R. Dörr, V. Tabor, T. Jenuwein, and C.A. Schmitt. 2010. Tumor stroma-derived TGF-β limits Myc-driven lymphomagenesis via Suv39h1-dependent senescence. Cancer Cell 17: 262-272. Citation Format: Clemens A. Schmitt. Exploiting metabolic alterations in therapy-induced senescence and drug resistance in a transgenic mouse lymphoma model by reverse and forward omics. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr IA21.

The structure-dependent toxicity, pharmacokinetics and anti-tumour activity of HPMA copolymer conjugates in the treatment of solid tumours and leukaemia

Polymer drug carriers that are based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been widely used in the development and synthesis of high-molecular-weight (HMW) drug delivery systems for cancer therapy. In this study, we compared linear (Mw ~27kDa, Rh ~4nm) and non-degradable star (Mw ~250kDa, Rh ~13nm) HPMA copolymer conjugates bearing anthracycline antibiotic doxorubicin (DOX) bound via pH-sensitive hydrazone bond. We determined the in vitro and in vivo toxicity of both conjugates and their maximum tolerated dose (MTD). We also compared their anti-tumour activity in mouse B-cell leukaemia (BCL1) and a mouse T-cell lymphoma (EL4) model. We found that MTD was higher for the linear conjugate (85mgDOX/kg) and lower for the star conjugate (22.5mgDOX/kg). An evaluation of the intestinal barrier integrity using FITC-dextran as a gut permeability tracer proved that no pathology was caused by the MTD of either conjugate. However, free DOX showed some damage to the gut barrier. The therapy of BCL1 leukaemia by both of the polymeric conjugates using the MTD or its fraction (i.e., equitoxic dosage) showed better results in the case of the star conjugate. On the other hand, treatment of EL4 lymphoma seemed to be more efficient when the linear conjugate was used. We suppose that the anti-cancer treatment of solid tumours and leukaemias requires different types of drug conjugates. We hypothesise that the most suitable HPMA copolymer-DOX conjugate for the treatment of solid tumours should have an HMW structure with increased Rh that would be stable for three to four days after the conjugate administration and then rapidly disintegrate in the short polymer chains, which are excretable from the body by glomerular filtration. On the other hand, the treatment of leukaemia requires a drug conjugate with a long circulation half-life. This would provide an active drug, whilst slowly degrading to excretable fragments.

UCH-L1 Cooperates with BCL6 and Identifies Patients with Aggressive Germinal Center Diffuse Large B-Cell Lymphoma

Gene expression profiling has identified two major subclasses of diffuse large B-cell lymphoma. Cases resembling germinal center B-cells (GCB-DLBCL) generally occur in younger patients, have a distinct molecular pathophysiology, and have improved outcomes compared with those similar to activated post-germinal center cells (ABC-DLBCL). We previously found that the ubiquitin hydrolase UCH-L1 is frequently overexpressed in mature B-cell malignancies and is a potent oncogene in mice. The cause for its overexpression in lymphoma, and whether it impacts the outcome of patients with DLBCL is unknown. Here we show that UCH-L1 reflects germinal center lineage in lymphoma and is an oncogenic biomarker of aggressive GCB-DLBCL. We find that UCH-L1 is specifically induced in germinal center B-cells in mice and humans, and that its expression correlates highly with the GCB subtype in DLBCL. Despite the typically good outcomes of GCB-DLBCL, increased UCHL1 identifies a subgroup with early relapses independent of MYC expression, suggesting biologic diversity in this subset of disease. We also find that UCH-L1 synergizes with BCL6 in a mouse model of germinal center B-cell lymphoma, but not with the development of multiple myeloma derived from post-germinal center cells. Consistent with this, forced Uchl1 overexpression had a substantial impact on gene expression in germinal center B-cells including pathways of cell cycle progression, cell death and proliferation, and DNA replication. These data demonstrate a novel role for UCH-L1 outside of the nervous system and suggest its potential use as a biomarker and therapeutic target in DLBCL. DisclosuresGalardy:Mission Therapeutics: Research Funding.

ATM deficiency promotes development of murine B-cell lymphomas that resemble diffuse large B-cell lymphoma in humans

AbstractThe serine-threonine kinase ataxia-telangiectasia mutated (ATM) plays a central role in maintaining genomic integrity. In mice, ATM deficiency is exclusively associated with T-cell lymphoma development, whereas B-cell tumors predominate in human ataxia-telangiectasia patients. We demonstrate in this study that when T cells are removed as targets for lymphomagenesis and as mediators of immune surveillance, ATM-deficient mice exclusively develop early-onset immunoglobulin M+ B-cell lymphomas that do not transplant to immunocompetent mice and that histologically and genetically resemble the activated B cell–like (ABC) subset of human diffuse large B-cell lymphoma (DLBCL). These B-cell lymphomas show considerable chromosomal instability and a recurrent genomic amplification of a 4.48-Mb region on chromosome 18 that contains Malt1 and is orthologous to a region similarly amplified in human ABC DLBCL. Of importance, amplification of Malt1 in these lymphomas correlates with their dependence on nuclear factor (NF)-κB, MALT1, and B-cell receptor (BCR) signaling for survival, paralleling human ABC DLBCL. Further, like some human ABC DLBCLs, these mouse B-cell lymphomas also exhibit constitutive BCR-dependent NF-κB activation. This study reveals that ATM protects against development of B-cell lymphomas that model human ABC DLBCL and identifies a potential role for T cells in preventing the emergence of these tumors.

Molecular Cloning, Recombinant Expression, and Funtional Characterization of APRIL (TNFSF13) in Cat (Felis catus)

ABSTRACTA proliferation-inducing ligand (APRIL) is a critical member of the tumor necrosis factor (TNF) superfamily, which is involved in immune regulation. In the present study, the cDNA of cat APRIL (cAPRIL) was successfully amplified. Sequence analysis showed that the open reading frame (ORF) of cAPRIL contains a putative furin protease cleavage site (R-R-K-R), a conserved putative N-glycosylation site (Asn124), and two conservative cysteine residues (Cys196 and Cys211). Real-time quantitative PCR (qPCR) analysis revealed that cAPRIL could be detected in various tissues. The phylogenetic analysis and predicted three dimensional (3D) structure revealed that it is similar to its counterparts. The extracellular soluble domain of the cAPRIL (csAPRIL) fragment was cloned into the expression vector pET43.1a. SDS-PAGE and Western blotting analysis indicated a high-level expression of csAPRIL protein in Escherichia coli BL21 (DE3). MTT assays revealed that purified recombinant csAPRIL protein was able to stimulate proliferation of mouse B-cells. These findings indicate that cAPRIL plays an important role in proliferation of B-cells and provide the basis for investigation on the roles of APRIL in this important domestic species.

Abstract A47: Gene regulation by Myc during B cell activation

Abstract Myc is required for the response of cells to proliferative stimuli, as shown for example in fibroblasts or B-cells. Cellular activation entails the regulation of specific transcriptional programs concomitant with a general increase in RNA production (also termed “amplification”), which will need to be carefully disentangled for a full understanding of the mitogenic response. Upon activation of quiescent fibroblasts with serum, c-myc is rapidly induced, and is required for the subsequent activation of a set of ca. 300 genes (Myc-Dependent Serum Response, or MDSR genes) in the early-mid G1 phase of the cell cycle (1). This is followed by RNA amplification, concomitant with the G1-S transition (2). Deletion of the c-myc gene prior to serum stimulation led to a loss of all of these responses: these data indicated that Myc primarily acts to selectively regulate specific gene programs, with RNA amplification occurring as a secondary consequence (2). This is in apparent contrast with previous studies on B-cells and tumors, in which Myc was proposed to directly drive transcriptional amplification at all active loci (3, 4). Here, we addressed Myc-dependent responses in primary mouse B-cells, based on conditional deletion of c-myc followed by LPS stimulation. Our data indicate that, as in fibroblasts, a discrete set of Myc-dependent genes can be identified in B-cells: the response of these genes occurs prior to general RNA amplification, and is not entirely attributable to overt promoter/enhancer invasion by Myc. Together with our previous studies (see poster by A. Sabò et al.) (2), these data provide a unifying view of Myc' s action in different cell types, with amplification occurring as an indirect effect, following from the action of specific Myc-target genes on cellular physiology and metabolism. Systematic work is needed to unravel which of the numerous Myc-regulated genes are critical in mediating this chain of events.

Abstract 013: Early Life Stress Increases Susceptibility to Develop Obesity and Metabolic Syndrome in a Sex-specific Manner

Recent epidemiological studies demonstrate that women have a greater prevalence of metabolic syndrome with associated increases in fasting glucose not seen in men. Lifestyle factors including diet and physical activity contribute to the risk of developing metabolic disease; however, it has been reported that exposure to early life stress (ELS) has enduring emotional, immune, and metabolic disturbances resulting in increased risk for obesity and type II diabetes. To investigate the effects of ELS as an independent risk factor for metabolic disease, we expose C57Bl/6 mice to maternal separation (MSep), an established behavioral stress model during postnatal life. At weaning, mice were placed on a low-fat diet (LFD, n=6-10) or high-fat diet (HFD, 60% fat calories, n=10) for 16 weeks. Body weight (BW) gain was not different between MSep and control (C) mice when fed a LFD; however, only female MSep mice display higher fat mass compared to C (6.7 ± 0.5 vs. 5.2±0.5 g, p&lt;0.05). Magnetic resonance spectroscopy revealed elevated levels of visceral fat in female MSep mice compared to C, suggesting that MSep increases central adiposity. HFD increased BW in male MSep mice vs C (54.2± 0.7 vs 51.3 ± 0.5, p&lt;0.05); however, BW was dramatically exaggerated in female MSep mice vs. C (48.2± 1.3 vs 34.4 ±2.3 g, p&lt;0.05). Accordingly, fat mass was increased in female MSep mice vs. C (18.2 ± 1.4 vs. 7.6 ± 0 g, p&lt;0.05). Only female MSep exhibited significant impaired glucose tolerance (AUC: 18445±507 vs 22070± 696 AU, p&lt;0.05), hepatomegaly, hypercholesterolemia (156±20 vs. 82 ± 2 mg/dl, p&lt;0.05), hyperleptinemia and hyperinsulinemia (p&lt;0.05) compared to C. Because impaired metabolic function has been linked to inflammation, we characterized splenocytes in female HFD-fed mice. We found a lower population of T-cells but no difference in B-cells in MSep mice vs. C. Functional studies of T cell differentiation demonstrated a reduced capability of naïve T cells to differentiate into anti-inflammatory T regulatory cells in MSep mice vs. C (54± 4 vs. 66± 1 % FoxP3+/CD25+ cells, p&lt;0.05, n=3). The ability to polarize into the pro-inflammatory Th17 phenotype remained intact. These data suggest that the mechanisms by which MSep primes the metabolic responses are sex-specific. NIH R00 HL111354