Mouse B-cell Lymphoma Research Articles

Abstract B16: Tumor suppressive functions by ATF2/7 transcription factors in a cMyc-driven B-cell lymphoma model

Abstract ATF2 and the highly homologous ATF7 are members of the AP-1 transcription factor family which regulate expression of genes involved in development and stress response. In cancer, ATF2 has been reported to function both as a tumor suppressor and tumor promoter. The aim of the project was to investigate the possible roles of ATF2/7 in cMyc-driven B-cell lymphoma development and to characterize their interaction with the cMyc oncogene. Previously, cMyc, which is frequently deregulated in Burkitt's lymphoma (Rabbitts et al, 1984), was shown to prolong the half-life and induce phosphorylation of ATF2 (Miethe et al, 2001). In addition, the MAP kinase JNK and its substrate ATF2 are frequently found upregulated in B-cell lymphomas (Kersten et al, 2004; Gururajan et al, 2005). We established a mouse B-cell lymphoma model by crossing B-cell specific knockouts of Atf2/7 (Atf2ΔBC, Atf7−/−) with Eμ-Myc transgenic animals. Eμ-Myc mice express the cMyc proto-oncogene driven by the immunoglobulin heavy chain enhancer (Eμ) and develop B-cell lymphomas with high frequency. We compared timing of tumor onset between Eμ-myc, Atf2ΔBC, Atf7−/− Eμ-myc, Atf2ΔBC/+, Atf7−/−, and Eμ-myc, Atf7−/−. In addition, we derived cell lines fom Eμ-Myc-driven lymphomas and characterized growth, apoptosis, and drug sensitivity in the presence or absence of functional Atf2/7. Atf2 and its phosphorylated (activated) form were found to be upregulated in cells obtained from tumor samples from Eμ-Myc mice in comparison to normal B lymphocytes. Analysis of tumor-free survival showed that Eμ-myc, AtfΔBC, Atf7−/− develop B-cell lymphomas at significantly faster rates than controls. In vitro deletion of Atf2 in cell lines derived from cMyc-driven lymphomas resulted in a marked reduction in the basal levels of apoptosis. Furthermore, Atf2/7-deficient Eμ-myc tumor cells were significantly more resistant to genotoxic stress (e.g., doxorubicin) induced apoptosis. Gene expression analysis identified proapoptotic members of the Bcl-2 family as possible transcriptional targets of Atf2. In this study, we show a novel tumor suppressive role of ATF2 in cMyc-induced lymphomagenesis and suggest the potential functions of ATF2 in regulating programmed cell death. The results also imply that the evaluation of levels of activated ATF2 in clinical samples of B-cell lymphoma may give an indication of their sensitivity to chemotherapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B16.

Separase Loss of Function Cooperates with the Loss of p53 in the Initiation and Progression of T- and B-Cell Lymphoma, Leukemia and Aneuploidy in Mice

BackgroundCohesin protease Separase plays a key role in faithful segregation of sister chromatids by cleaving the cohesin complex at the metaphase to anaphase transition. Homozygous deletion of ESPL1 gene that encodes Separase protein results in embryonic lethality in mice and Separase overexpression lead to aneuploidy and tumorigenesis. However, the effect of Separase haploinsufficiency has not been thoroughly investigated.Methodology/Principal FindingsHere we examined the effect of ESPL1 heterozygosity using a hypomorphic mouse model that has reduced germline Separase activity. We report that while ESPL1 mutant (ESPL1 +/hyp) mice have a normal phenotype, in the absence of p53, these mice develop spontaneous T- and B-cell lymphomas, and leukemia with a significantly shortened latency as compared to p53 null mice. The ESPL1 hypomorphic, p53 heterozygous transgenic mice (ESPL1 +/hyp, p53+/−) also show a significantly reduced life span with an altered tumor spectrum of carcinomas and sarcomas compared to p53+/− mice alone. Furthermore, ESPL1+/hyp, p53−/− mice display significantly higher levels of genetic instability and aneuploidy in normal cells, as indicated by the abnormal metaphase counts and SKY analysis of primary splenocytes.Conclusions/SignificanceOur results indicate that reduced levels of Separase act synergistically with loss of p53 in the initiation and progression of B- and T- cell lymphomas, which is aided by increased chromosomal missegregation and accumulation of genomic instability. ESPL1 +/hyp, p53−/− mice provide a new animal model for mechanistic study of aggressive lymphoma and also for preclinical evaluation of new agents for its therapy.

Helicobacter pylori cytotoxin-associated gene A (CagA) subverts the apoptosis-stimulating protein of p53 (ASPP2) tumor suppressor pathway of the host

Type I strains of Helicobacter pylori (Hp) possess a pathogenicity island, cag, that encodes the effector protein cytotoxin-associated gene A (CagA) and a type four secretion system. After translocation into the host cell, CagA affects cell shape, increases cell motility, abrogates junctional activity, and promotes an epithelial to mesenchymal transition-like phenotype. Transgenic expression of CagA enhances gastrointestinal and intestinal carcinomas as well as myeloid and B-cell lymphomas in mice, but the mechanism of the induced cancer formation is not fully understood. Here, we show that CagA subverts the tumor suppressor function of apoptosis-stimulating protein of p53 (ASPP2). Delivery of CagA inside the host results in its association with ASPP2. After this interaction, ASPP2 recruits its natural target p53 and inhibits its apoptotic function. CagA leads to enhanced degradation of p53 and thereby, down-regulates its activity in an ASPP2-dependent manner. Finally, Hp-infected cells treated with the p53-activating drug Doxorubicin are more resistant to apoptosis than uninfected cells, an effect that requires ASPP2. The interaction between CagA and ASPP2 and the consequent degradation of p53 are examples of a bacterial protein that subverts the p53 tumor suppressor pathway in a manner similar to DNA tumor viruses. This finding may contribute to the understanding of the increased risk of gastric cancer in patients infected with Hp CagA+ strains.

Synergistic effect of HPMA copolymer-bound doxorubicin and dexamethasone in vivo on mouse lymphomas

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer—drug conjugates bearing the antiinflammatory and anti-proliferative drug dexamethasone (DEX) and the anti-cancer drug doxorubicin (DOX) bound to either the polymer carrier separately or in combination, were synthesized. Both the in vitro and in vivo anti-cancer activity on mouse B-cell (38C13) and T-cell (EL4) lymphoma was studied. The conjugates were fairly stable in model buffers at pH 7.4, simulating blood pH, and the drug was released by chemical hydrolysis at pH values based on the endosomal and lysosomal environments in the target cells (pH 5—6). As a control, polymer conjugates containing only a single-drug (DOX or DEX) attached to the HPMA copolymer carrier were synthesized and tested. Contrary to the treatment with either single drug conjugate, where no significant long-term survival was observed, a synergistic effect of the two drugs was manifested in vivo. Treatment of mice bearing B-cell lymphoma 38C13 formulated as a mixture of both single drug-containing conjugates or a as a conjugate with both drugs in combination gave long-term survivors.

Inflammation driven by tumour-specific Th1 cells protects against B-cell cancer

The immune system can both promote and suppress cancer. Chronic inflammation and proinflammatory cytokines such as interleukin (IL)-1 and IL-6 are considered to be tumour promoting. In contrast, the exact nature of protective antitumour immunity remains obscure. Here, we quantify locally secreted cytokines during primary immune responses against myeloma and B-cell lymphoma in mice. Strikingly, successful cancer immunosurveillance mediated by tumour-specific CD4+ T cells is consistently associated with elevated local levels of both proinflammatory (IL-1α, IL-1β and IL-6) and T helper 1 (Th1)-associated cytokines (interferon-γ (IFN-γ), IL-2 and IL-12). Cancer eradication is achieved by a collaboration between tumour-specific Th1 cells and tumour-infiltrating, antigen-presenting macrophages. Th1 cells induce secretion of IL-1β and IL-6 by macrophages. Th1-derived IFN-γ is shown to render macrophages directly cytotoxic to cancer cells, and to induce macrophages to secrete the angiostatic chemokines CXCL9/MIG and CXCL10/IP-10. Thus, inflammation, when driven by tumour-specific Th1 cells, may prevent rather than promote cancer.

CD19 as Foreign Antigen Elicits a Specific T-Cell Response and Mediates Rejection of High Grade B-Cell Lymphoma In Mice

AbstractAbstract 4907Targeting selfantigens by specific T-cells for anti-tumor therapy bears the risk of fatal autoimmunity. Ideal self antigens are characterized by restricted expression in a specific cell compartment and are ideally highly expressed in malignant cells. Potential autoimmunity against those antigens must be tolerable for the host and loss of the expressing cell compartment has to be acceptable. In addition such self antigens should deliver survival signals for the tumor making escape variants highly unlikely or even impossible. The CD19 molecule as a member of the Ig superfamily is a transmembrane protein whose expression is restricted to B-cells in mouse and man. Aside from other proteins (CD81, CD21 etc.) it associates with the B-cell receptor (BCR) to augment its signal and appears to be of critical importance in the development and differentiation of B-cells. CD19 is expressed on virtually all B-cell malignancies and may provide a survival signal for at least some B cell malignancies. Targeting CD19 by antibodies appears to be effective in disease control making it an interesting target for T-cell therapy.We have developed a murine model for high grade B-cell lymphoma using lambda-human-cmyc transgenic mice. These animals spontaneously develop high grade B-cell lymphomas with similarities to human Burkittxs lymphoma. From these mice we established various cell lines which can be cultured long term and transferred onto recipients to form new locally or systemically growing lymphomas. Using homozygous CD19cre mice as a model for CD19 deficiency we show here that transfer of lethal doses of lymphoma cells (0.1-1.0 Mio 291cells) which form lymphomas in more than 90% of wild type recipients can be rejected long term (100 days) in up to 60% (9/14) of CD19 deficient mice (p=0.001, left figure). After s.c. injection 9/14 of CD19-/- animals transiently form tumors at the site of injection (right figure CD19-/-B6 rejection) which eventually disappear as a sign of rejection. STAT1-/- deficient animals served as a positive control since they harbour a NK- and T-cell defect. In case of outgrowing tumors (5/14), we still observe a strong infiltration by T-cells which is completely absent in wild type recipients. Heterotypic prime-boost-vaccination of CD19 deficient mice using a CD19 expression vector and long peptides results in a strong T-cell response as shown by IFNgamma ELISPOT and ELISA assay which is absent in wt mice or control vaccinated animals (chicken ovalbumine, OVA). Using peptide prediction several epitopes within the murine CD19 protein could be identified giving rise to mainly CD4 responses in the context of MHC H2b. Vaccination of CD19 deficient animals is being done to further improve survival of tumor-challenged animals. Collectively, our data indicate that CD19 serves as a rejection antigen in our model system and that protective T-cell responses can be generated to provide long survival after tumor challenge. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

UCH-L1, a Potent Oncogene That Drives the Development of B-Cell Lymphoma, Promotes Akt Signaling by Regulating Mtor Complex Assembly

AbstractAbstract 4182De-ubiquitinating enzymes (DUBs) are important regulators of many cellular processes through their ability to reverse the modifications installed by ubiquitin ligases. Several members of the DUB family are implicated in human cancers, where they may act as oncogenes or tumor suppressors. There is little direct in vivo evidence, however, for their roles in cancer. We recently reported that the DUB UCH-L1 is a potent oncogene that drives the development of spontaneous B-cell lymphoma in transgenic mice, and is required for the survival of malignant B-cells in vitro. The mechanism of these effects involves the strong induction of Akt signaling that is observed in culture and in lymphoid tissues of Uchl1 transgenic mice. Akt is phosphorylated on two distinct sites by the kinases PDK1 and mTOR, with both phosphorylation events required for maximal signaling. The activity of mTOR towards its substrates is dependent on the binding partners to which it is bound. When mTOR binds to raptor, it forms mTORC1, an important regulator of nutrient sensitive protein translation, whereas when complexed with rictor and SIN1, it forms mTORC2, a critical regulator of Akt signaling. It is unclear how the cell regulates the formation of mTORC1/2. Here we show a novel mechanism by which UCH-L1 regulates mTOR-dependent Akt signaling. By co-immunoprecipitating mTOR and its binding partners, we find that UCH-L1 expression promotes the binding of mTOR with rictor at the expense of raptor, without changing the level of either protein. These data suggest a novel ubiquitin-dependent regulatory step in mTOR complex assembly. In addition, UCH-L1 expression leads to a reduction in the levels of the Akt antagonist phosphatase PHLPP1 in vitro and in vivo. This decline is completely dependent on UCH-L1 DUB activity as expression of a catalytic mutant does not affect levels of PHLPP1. We find that the mechanism of this decline is not due to proteasome degradation, nor transcriptional control. We therefore hypothesize that the UCH-L1 associated shift in cellular mTOR activities leads to reduced mTORC1-dependent translation of PHLPP1. To test this, we inhibited mTORC1 activity by depleting raptor using shRNA and found a dramatic decline in PHLPP1 levels, whereas depletion of rictor had no effect on PHLPP1. We conclude that UCH-L1 boosts Akt signaling through a novel two-step mechanism by which it promotes the generation of mTORC2, reduces the levels of mTORC1, and thereby impairs the translation of PHLPP1. These data have important implications for understanding the roles of ubiquitin, mTOR, and Akt, in lymphoma biology and may lead to more intelligent use of next generation mTOR inhibitors in the treatment of lymphoma. Disclosures:No relevant conflicts of interest to declare.

RETRACTED: Expression of a truncated Hmga1b gene induces gigantism, lipomatosis and B-cell lymphomas in mice

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).This article has been retracted at the request of the Editor-in-Chief.A reader contacted the Editors of the European Journal of Cancer regarding possible duplications in Figures 1D (‘RT-PCR analysis of the expression of the CMV-Hmgaib/T transgene in a panel of adult tissues from transgenic mice’) and in Figure 4D (‘RT-PCR analysis showing the expression of V-preB and Rag2 genes in TG and WT spleens’). The authors are unable to provide the Editors with the original source files that were used to generate the Figures. Therefore, the paper has been retracted from the Journal.The corresponding author disagrees with the retraction decision and claims that the original files were lost in the transfer of the laboratory in 2003. Further, the author stands behind the results noted in the paper and asserts that further experiments have validated these results.

Aurora kinases A and B are up-regulated by Myc and are essential for maintenance of the malignant state

Myc oncoproteins promote continuous cell growth, in part by controlling the transcription of key cell cycle regulators. Here, we report that c-Myc regulates the expression of Aurora A and B kinases (Aurka and Aurkb), and that Aurka and Aurkb transcripts and protein levels are highly elevated in Myc-driven B-cell lymphomas in both mice and humans. The induction of Aurka by Myc is transcriptional and is directly mediated via E-boxes, whereas Aurkb is regulated indirectly. Blocking Aurka/b kinase activity with a selective Aurora kinase inhibitor triggers transient mitotic arrest, polyploidization, and apoptosis of Myc-induced lymphomas. These phenotypes are selectively bypassed by a kinase inhibitor-resistant Aurkb mutant, demonstrating that Aurkb is the primary therapeutic target in the context of Myc. Importantly, apoptosis provoked by Aurk inhibition was p53 independent, suggesting that Aurka/Aurkb inhibitors will show efficacy in treating primary or relapsed malignancies having Myc involvement and/or loss of p53 function.

Changes in H2A.Z occupancy and DNA methylation during B-cell lymphomagenesis

The histone variant H2A.Z has been implicated in the regulation of gene expression, and in plants antagonizes DNA methylation. Here, we ask whether a similar relationship exists in mammals, using a mouse B-cell lymphoma model, where chromatin states can be monitored during tumorigenesis. Using native chromatin immunoprecipitation with microarray hybridization (ChIP-chip), we found a progressive depletion of H2A.Z around transcriptional start sites (TSSs) during MYC-induced transformation of pre-B cells and, subsequently, during lymphomagenesis. In addition, we found that H2A.Z and DNA methylation are generally anticorrelated around TSSs in both wild-type and MYC-transformed cells, as expected for the opposite effects of these chromatin features on promoter competence. Depletion of H2A.Z over TSSs both in cells that are induced to proliferate and in cells that are developing into a tumor suggests that progressive loss of H2A.Z during tumorigenesis results from the advancing disease state. These changes were accompanied by increases in chromatin salt solubility. Surprisingly, ∼30% of all genes showed a redistribution of H2A.Z from around TSSs to bodies of active genes during the transition from MYC-transformed to tumor cells, with DNA methylation lost from gene bodies where H2A.Z levels increased. No such redistributions were observed during MYC-induced transformation of wild-type pre-B cells. The documented role of H2A.Z in regulating transcription suggests that 30% of genes have the potential to be aberrantly expressed during tumorigenesis. Our results imply that antagonism between H2A.Z deposition and DNA methylation is a conserved feature of eukaryotic genes, and that transcription-coupled H2A.Z changes may play a role in cancer initiation and progression.

Efficient Inhibition of Human B-cell Lymphoma in SCID Mice by Synergistic Antitumor Effect of Human 4-1BB Ligand/Anti-CD20 Fusion Proteins and Anti-CD3/Anti-CD20 Diabodies

Here we constructed and produced a recombinant human 4-1BB ligand (4-1BBL)/anti-CD20 fusion protein and examined its antitumor activity, alone and in combination with an anti-CD3/anti-CD20 bispecific diabody. The 4-1BBL/anti-CD20 fusion protein retained both the costimulatory activity of 4-1BBL on T cells and the tumor targeting ability of CD20 antibody on B cells. The fusion protein bound as efficiently to 4-1BB- and CD20-positive cells as its respective parental antibodies, and was capable of cross-linking human T lymphocytes and CD20-positive tumor cells. Combination treatment with 4-1BBL/anti-CD20 fusion protein and anti-CD3/anti-CD20 diabody led to significantly increased T-cell cytotoxicity to human B-lymphoma cells in vitro and drastically more potent tumor inhibitory activity in vivo in xenografted B-cell lymphoma in severe combined immunodeficiency disease mice. Mechanistic studies revealed that the combination treatment remarkably inhibited apoptosis of human peripheral blood lymphocytes, accompanied by upregulation of Bcl-XL and Bf1-1, perforin and granzyme B mRNA, and increased interleukin-2 production. Taken together, these results suggest that targeted delivery of 4-1BBL to the tumor site, when combined with anti-CD3/anti-CD20 diabody, could strongly potentiate the antitumor activity of the diabody, thus may have significant clinical application in the treatment of human CD20-positive B-cell malignancies.

Transactivation of the dopamine receptor 3 gene by a single provirus integration results in development of B-cell lymphoma in transgenic mice generated from retrovirally transduced embryonic stem cells

AbstractGene transfer vectors based on retroviruses are commonly used in gene therapy applications because of their unique ability to integrate efficiently into host genomes. This ability also forms the basis of a transformation event that can be induced in transduced cells by transactivation of proto-oncogenes near the vector integration sites. Here, we report on the development of lymphoma in mice generated from embryonic stem cells transduced with an enhanced green fluorescent protein. The cells expressed B220, CD5, Mac1, and IgM on their surfaces and expressed transcription factors characteristic of B-cell lymphoma. Importantly, each mouse had a single copy of the provirus in its genome; the copy was integrated into the second intron of the dopamine receptor 3 (D3) gene, and high-level expression of D3 was detected only in the lymphoma cells. Ectopic expression of D3 in murine marrow cells resulted in preferential proliferation of cells at the pre–B-cell stage in response to a D3-specific agonist, but this proliferation was not observed in vivo. Cells cotransduced with D3 and Bcl-xL genes had a phenotype similar to that of lymphoma in vivo, suggesting that the leukemogenesis induced by retroviral integration required “second hit” mutations of additional genes.

Construction of retroviral vectors to functionally dissect the mir‐17‐92 oncogenic cluster

MicroRNAs (miRNAs) are small non‐coding ribonucleic acids involved in gene regulation by inhibiting translation through binding with perfect or almost perfect complementarity with the 3’untranslated region of target mRNAs. Investigations by He et.al.describe a miRNA cluster, the mir‐17‐92 polycistron, overexpressed in B‐cell lymphomas and comprised of the six miRNAs: miR‐17, 18, 19a, 20, 19b, and 92. Enforced expression of this cluster acted with c‐myc expression to accelerate tumor development in a mouse B‐cell lymphoma model and prevented the apoptosis of tumors derived from hematopoietic stem cells. However, the individual potential of the seven miRNAs in the cluster to accumulate tumor formation is largely unknown. To access this problem PCR amplified primary forms of individual miRNAs were used to produce retroviral constructs with the vector CMSV Sfi A, B T7 ires thCD4‐1.ab 1(hCD4). Molecular clones were obtained of miR‐17,18, 19a, 20,1792 and mutated miRNA to unexpress 19a and 92(miR‐Δ19 and 1792Δ92). The constructs where produced to isolate cells expressing the miRNAs by magnetic labeling using the MACS Select System. However, to understand the individual role of these miRNAs further screening and expression assays need to be preformed. Due to the novelty and complexity of miRNAs involvement in cancer progression the quest to solve the miRNA puzzle has just begun.

Role of the translocation partner in protection against AID-dependent chromosomal translocations

Chromosome translocations between Ig (Ig) and non-Ig genes are frequently associated with B-cell lymphomas in humans and mice. The best characterized of these is c-myc/IgH translocation, which is associated with Burkitt's lymphoma. These translocations are caused by activation-induced cytidine deaminase (AID), which produces double-strand DNA breaks in both genes. c-myc/IgH translocations are rare events, in part because ATM, p53, and p19 actively suppress them. To further define the mechanism of protection against the accumulation of cells that bear c-myc/IgH translocation, we assayed B cells from mice that carry mutations in cell-cycle and apoptosis regulator proteins that act downstream of p53. We find that PUMA, Bim, and PKCdelta are required for protection against c-myc/IgH translocation, whereas Bcl-XL and BAFF enhance c-myc/IgH translocation. Whether these effects are general or specific to c-myc/IgH translocation and whether AID produces dsDNA breaks in genes other than c-myc and Ig is not known. To examine these questions, we developed an assay for translocation between IgH and Igbeta, both of which are somatically mutated by AID. Igbeta/IgH, like c-myc/IgH translocations, are AID-dependent, and AID is responsible for lesions on IgH and the non-IgH translocation partners. However, ATM, p53, and p19 do not protect against Igbeta/IgH translocations. Instead, B cells are protected against Igbeta/IgH translocations by a BAFF- and PKCdelta-dependent pathway. We conclude that AID-induced double-strand breaks in non-Ig genes other than c-myc lead to their translocation, and that at least two nonoverlapping pathways protect against translocations in primary B cells.

MiR-19 is a key oncogenic component of mir-17-92

Recent studies have revealed the importance of multiple microRNAs (miRNAs) in promoting tumorigenesis, among which mir-17-92/Oncomir-1 exhibits potent oncogenic activity. Genomic amplification and elevated expression of mir-17-92 occur in several human B-cell lymphomas, and enforced mir-17-92 expression in mice cooperates with c-myc to promote the formation of B-cell lymphomas. Unlike classic protein-coding oncogenes, mir-17-92 has an unconventional gene structure, where one primary transcript yields six individual miRNAs. Here, we functionally dissected the individual components of mir-17-92 by assaying their tumorigenic potential in vivo. Using the Emu-myc model of mouse B-cell lymphoma, we identified miR-19 as the key oncogenic component of mir-17-92, both necessary and sufficient for promoting c-myc-induced lymphomagenesis by repressing apoptosis. The oncogenic activity of miR-19 is at least in part due to its repression of the tumor suppressor Pten. Consistently, miR-19 activates the Akt-mTOR (mammalian target of rapamycin) pathway, thereby functionally antagonizing Pten to promote cell survival. Our findings reveal the essential role of miR-19 in mediating the oncogenic activity of mir-17-92, and implicate the functional diversity of mir-17-92 components as the molecular basis for its pleiotropic effects during tumorigenesis.

Immunisation against Human c-MYC Elicits a T-Cell Response and Influences Growth of High Grade B-Cell Lymphoma in Mice.

Abstract 5120Overexpression of the proto-oncogene c-myc due to chromosomal translocation is the hallmark of Burkitt-lymphoma. The evolving high grade lymphoma is dependent on the overexpression of c-myc, which provides the necessary signal to drive uncontrolled proliferation. Therefore loss of function or recognition of c-myc overexpressing cells by c-MYC specific T-cells should result in killing of the target and a halt to lymphoma progression. C-myc is also expressed in a variety of other human malignancies. Peptide prediction reveals several potential foreign epitopes in the context of murine H2b due to 87% homology between human and mouse c-MYC. In this study we explored whether the human c-myc gene product can be a target for T-cell therapy. Wildtype C57BL/6 mice were immunized with recombinant human c-MYC protein in combination with incomplete Freund′s adjuvans and CpG, and were boosted at various time points thereafter using either c-MYC protein or 40mer peptides encompassing the non homologous regions. Control animals were vaccinated with recombinant GFP or OVA protein. C-MYC vaccinated animals displayed a higher IFNg release upon re-stimulation with c-MYC pulsed dendritic cells compared to control vaccinated animals. In ELISPOT assays we observed a higher number of IFNg positive cells (299±17 vs. 122±8.5 (GFP vaccinated) vs. 66±8.5 (OVA vaccinated)). Vaccination using single peptides revealed that peptides spanning the region from amino acid 87-123, 216-255 and 334-376 produced similar results. In addition, using a human c-MYC specific ELISA we were able to detect c-MYC specific antibodies in serum from immunized mice in a concentration up to 40mg/l. Using established cell lines from l-hu-c-myc transgenic mice, where the human c-myc gene is overexpressed due to the juxtaposition of elements of the immunoglobuline lambda locus as found in t(8;22) of Burkitt's lymphoma, we investigated whether vaccination with human c-MYC protein would influence lymphoma growth in a lymphoma transfer model. Animals were s.c. challenged with 0.1 Mio 291cells overexpressing human c-MYC and were monitored for lymphoma growth. C-MYC vaccinated animals (n=15) displayed a delay in tumor onset and a significantly better disease free survival (28 vs. 22 days, p=0.012) compared to control (OVA) vaccinated animals (n=10). This delayed growth was associated with an increased number of infiltrating CD3+/Perforin+ cells. However, all mice eventually succumbed to lymphoma growth, indicating that the T-cell response was not sufficient to control lymphoma growth in the long term. From these data we conclude that the human c-MYC is a possible target antigen for T-cells, but responses are weak and presumably low in frequency. DisclosuresNo relevant conflicts of interest to declare.

Superantigen‐presentation by rat major histocompatibility complex class II molecules RT1.Bl and RT1.Dl

Rat major histocompatibility complex (MHC) class II molecules RT1.B(l) (DQ-like) and RT1.D(l) (DR-like) were cloned from the LEW strain using reverse transcription-polymerase chain reaction and expressed in mouse L929 cells. The transduced lines bound MHC class II-specific monoclonal antibodies in an MHC-isotype-specific manner and presented peptide antigens and superantigens to T-cell hybridomas. The T-cell-hybridomas responded well to all superantigens presented by human MHC class II, whereas the response varied considerably with rat MHC class II-transduced lines as presenters. The T-cell hybridomas responded to the pyrogenic superantigens Staphylococcus enterotoxin B (SEB), SEC1, SEC2 and SEC3 only at high concentrations with RT1.B(l)-transduced and RT1.D(l)-transduced cells as presenters. The same was true for streptococcal pyrogenic exotoxin A (SPEA), but this was presented only by RT1.B(l) and not by RT1.D(l). SPEC was recognized only if presented by human MHC class II. Presentation of Yersinia pseudotuberculosis superantigen (YPM) showed no MHC isotype preference, while Mycoplasma arthritidis superantigen (MAS or MAM) was presented by RT1.D(l) but not by RT1.B(l). Interestingly, and in contrast to RT1.B(l), the RT1.D(l) completely failed to present SEA and toxic shock syndrome toxin 1 even after transduction of invariant chain (CD74) or expression in other cell types such as the surface MHC class II-negative mouse B-cell lymphoma (M12.4.1.C3). We discuss the idea that a lack of SEA presentation may not be a general feature of RT1.D molecules but could be a consequence of RT1.D(l)beta-chain allele-specific substitutions (arginine 80 to lysine, asparagine 82 to aspartic acid) in the extremely conserved region flanking the Zn(2+)-binding histidine 81, which is crucial for high-affinity SEA-binding.

Expression of sprouty2 inhibits B-cell proliferation and is epigenetically silenced in mouse and human B-cell lymphomas

B-cell lymphoma is the most common immune system malignancy. TCL1 transgenic mice (TCL1-tg), in which TCL1 is ectopically expressed in mature lymphocytes, develop multiple B- and T-cell leukemia and lymphoma subtypes, supporting an oncogenic role for TCL1 that probably involves AKT and MAPK-ERK signaling pathway augmentation. Additional, largely unknown genetic and epigenetic alterations cooperate with TCL1 during lymphoma progression. We examined DNA methylation patterns in TCL1-tg B-cell tumors to discover tumor-associated epigenetic changes, and identified hypermethylation of sprouty2 (Spry2). Sprouty proteins are context-dependent negative or positive regulators of MAPK-ERK pathway signaling, but their role(s) in B-cell physiology or pathology are unknown. Here we show that repression of Spry2 expression in TCL1-tg mouse and human B-cell lymphomas and cell lines is associated with dense DNA hypermethylation and was reversed by inhibition of DNA methylation. Spry2 expression was induced in normal splenic B cells by CD40/B-cell receptor costimulation and regulated a negative feedback loop that repressed MAPK-ERK signaling and decreased B-cell viability. Conversely, loss of Spry2 function hyperactivated MAPK-ERK signaling and caused increased B-cell proliferation. Combined, these results implicate epigenetic silencing of Spry2 expression in B lymphoma progression and suggest it as a companion lesion to ectopic TCL1 expression in enhancing MAPK-ERK pathway signaling.

B-Cell Receptor Signaling in the Genesis and Maintenance of B-Cell Lymphoma

Lymphomas constitute a number of different diseases that have been subdivided into two broad categories, Hodgkin’s and non-Hodgkin’s lymphoma (NHL). Hodgkin’s disease comprises a uniform set of malignancies primarily defined by the presence of Reed–Sternberg giant cells, whereas NHL is a heterogeneous set of clinical entities. The number of cases of NHL is almost tenfold that of Hodgkin’s disease [1]. In addition, the number of newly diagnosed cases of NHL has increased by almost 80% in the last 25 years. This dramatic increase does not correlate with age, gender or infectious agents, and cannot be accounted for by the onset of HIV-associated B-cell lymphomas [2]. As a result, NHL are currently the fifth most common form of cancer in the USA, after breast, prostate, lung and colon cancer [101]. NHL is one of the few cancers whose incidence and mortality rates have risen in the past 35 years. Despite the increase in the incidence of NHL, the etiology of these lymphomas remains elusive, and current therapeutic approaches rely on traditional, nonspecific chemotherapeutic approaches. The diagnosis of NHL encompasses different clinical entities. Approximately 85–90% of all NHL in the USA consist of B-cell lymphomas [3]. Among the B-cell NHLs, aggressive lymphomas account for 45–50% of new diagnoses. The two most common forms of aggressive NHL are diffuse large B-cell lymphoma and Burkitt’s lymphoma. These two types of malignancies involve neoplastic B cells that have a surface phenotype that is consistent with that of mature, activated B cells. Specifically, they express B-cell antigen receptors (BCR) on their surface, which contain mutations consistent with the process of affinity maturation during a germinal center reaction [4]. These cells also express other molecules that are normally expressed by postgerminal B cells [5]. There are other B-cell NHLs that have a similar cellular composition. These include follicular lymphomas, mucosal-associated lymphoid tissue lymphomas and mantle-cell lymphomas [3]. All of these B-cell NHL share a B-cell surface phenotype, including BCR expression. The nature of additional mutations, possibly involving oncogenes and tumor suppressor genes, has been postulated to explain their biological differences [6]. The one characteristic they have in common, namely BCR expression and evidence of antigen-dependent activation, may suggest that the BCR and an antigen may have an important role in the genesis of these tumors. Dameshek and Schwartz originally proposed that antigenic stimulus could contribute to the development of lymphomas nearly 50 years ago [7]. Throughout the years, a large amount of circumstantial evidence has implicated chronic inflammation in lymphomagenesis. There are prior hints that antigenic stimulus can play a role in lymphomagenesis. First, retroviral infection of mice elicits T-cell lymphomas only in those strains of mice that can mount an immune response to the virus [7,8]. Second, infection with Helicobacter pylori is an apparent cause of human lymphomas in mucosal-associated lymphoid tissue and gut-associated lymphoid tissue [9]. Treatment with antibiotics to eradicate infection elicits remission of these tumors, as if they might have been sustained by antigenic stimulus from the microbe [10,11]. Third, mice with graft versus host disease consequent to bone marrow transplantation frequently develop T-cell lymphomas; immunosuppression of the mice prevents the tumors [12]. Fourth, chronic antigenic stimulation by infection may contribute to the genesis of Burkitt’s lymphoma [13,14]. Fifth, the gene-expression profiles of diffuse large B-cell lymphomas resemble those of B cells that have mounted a response to antigen [15], and the tumor cells display high-affinity antigen receptors on their surface, as if they had been subjected to the selective pressure of an antigen [4,16–18]. Sixth, our own studies demonstrate a causal relationship between BCR-derived signals and elevated levels of MYC in the genesis of B-cell lymphomas in mice [19]. These findings prompt the hypothesis that an antigenic stimulus may cooperate with other tumorigenic influences in the genesis of lymphoma.

Stroma-Initiated Hedgehog Signaling Takes Center Stage in B-Cell Lymphoma

Hedgehog-mediated signaling has been shown to promote growth and dissemination of solid cancers, most prominently basal cell carcinomas and medulloblastoma. Recent findings indicate that hedgehog signals are also important for tumor growth in hematologic malignancies. Hedgehog ligands secreted by stromal cells could elicit Patched/Smoothened-mediated antiapoptotic signaling in mouse B-cell lymphomas. Inhibition of hedgehog signaling induced apoptosis in lymphoma cells and prolonged survival of lymphoma-bearing mice. Depletion of tumor cells proceeded in the absence of p53 via the mitochondrial apoptotic pathway. These and other recently published data on hedgehog inhibition in cancer cells and their implications will be discussed.