Characterization of ARF-BP1/HUWE1 interactions with CTCF, MYC, ARF and p53 in MYC-driven B cell neoplasms.

Transcriptional activation of MYC is a hallmark of many B cell lineage neoplasms. MYC provides a constitutive proliferative signal but can also initiate ARF-dependent activation of p53 and apoptosis. The E3 ubiquitin ligase, ARF-BP1, encoded by HUWE1, modulates the activity of both the MYC and the ARF-p53 signaling pathways, prompting us to determine if it is involved in the pathogenesis of MYC-driven B cell lymphomas. ARF-BP1 was expressed at high levels in cell lines from lymphomas with either wild type or mutated p53 but not in ARF-deficient cells. Downregulation of ARF-BP1 resulted in elevated steady state levels of p53, growth arrest and apoptosis. Co-immunoprecipitation studies identified a multiprotein complex comprised of ARF-BP1, ARF, p53, MYC and the multifunctional DNA-binding factor, CTCF, which is involved in the transcriptional regulation of MYC, p53 and ARF. ARF-BP1 bound and ubiquitylated CTCF leading to its proteasomal degradation. ARF-BP1 and CTCF thus appear to be key cofactors linking the MYC proliferative and p53-ARF apoptotic pathways. In addition, ARF-BP1 could be a therapeutic target for MYC-driven B lineage neoplasms, even if p53 is inactive, with inhibition reducing the transcriptional activity of MYC for its target genes and stabilizing the apoptosis-promoting activities of p53.

Expression of the cyclin-dependent kinase inhibitor p27 and its deregulation in mouse B cell lymphomas.

CDKN1B (p27) regulates cell-cycle progression at the G1-S transition by suppressing the cyclin E/CDK2 kinase complex. In normal lymphocytes and most human B cell non-Hodgkin lymphomas (NHL), there is an inverse correlation between proliferative activity and expression of p27; however, a subset of NHL with high mitotic indices expresses p27, which is inactive due to sequestration in nuclear protein complexes or due to cytoplasmic retention. Our studies of mouse B cell NHL also identified cases with high proliferative activity and high levels of p27 at a surprisingly high frequency. Here, p27 was complexed with D-type cyclins 1 and 3 and with the COPS9 protein, JAB1. In addition, we found cytoplasmic sequestration following phosphorylation by activated AKT.

Regulation of the germinal center gene program by interferon (IFN) regulatory factor 8/IFN consensus sequence-binding protein.

Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphomas showed that IRF8 transcripts were expressed at highest levels in centroblasts, either from secondary lymphoid tissue or transformed cells. In addition, staining for IRF8 was most intense in tonsillar germinal center (GC) dark-zone centroblasts. To discover B cell genes regulated by IRF8, we transfected purified primary tonsillar B cells with enhanced green fluorescent protein-tagged IRF8, generated small interfering RNA knockdowns of IRF8 expression in a mouse B cell lymphoma cell line, and examined the effects of a null mutation of IRF8 on B cells. Each approach identified activation-induced cytidine deaminase (AICDA) and BCL6 as targets of transcriptional activation. Chromatin immunoprecipitation studies demonstrated in vivo occupancy of 5' sequences of both genes by IRF8 protein. These results suggest previously unappreciated roles for IRF8 in the transcriptional regulation of B cell GC reactions that include direct regulation of AICDA and BCL6.

Insertion of c-Myc into Igh induces B-cell and plasma-cell neoplasms in mice.

We used gene targeting in mice to insert a His(6)-tagged mouse c-Myc cDNA, Myc(His), head to head into the mouse immunoglobulin heavy-chain locus, Igh, just 5' of the intronic enhancer, Emu. The insertion of Myc(His) mimicked both the human t(8;14)(q24;q32) translocation that results in the activation of MYC in human endemic Burkitt lymphomas and the homologous mouse T(12;15) translocation that deregulates Myc in certain mouse plasmacytomas. Beginning at the age of 6 months, Myc(His) transgenic mice developed B-cell and plasma neoplasms, such as IgM(+) lymphoblastic B-cell lymphomas, Bcl-6(+) diffuse large B-cell lymphomas, and CD138(+) plasmacytomas, with an overall incidence of 68% by 21 months. Molecular studies of lymphoblastic B-cell lymphoma, the most prevalent neoplasm (50% of all tumors), showed that the lymphomas were clonal, overexpressed Myc(His), and exhibited the P2 to P1 promoter shift in Myc expression, a hallmark of MYC/Myc deregulation in human endemic Burkitt lymphoma and mouse plasmacytoma. Only 1 (6.3%) of 16 lymphoblastic B-cell lymphomas contained a BL-typical point mutation in the amino-terminal transactivation domain of Myc(His), suggesting that most of these tumors are derived from naive, pregerminal center B cells. Twelve (46%) of 26 lymphoblastic B-cell lymphomas exhibited changes in the p19(Arf)-Mdm2-p53 tumor suppressor axis, an important pathway for Myc-dependent apoptosis. We conclude that Myc(His) insertion into Igh predictably induces B-cell and plasma-cell tumors in mice, providing a valuable mouse model for understanding the transformation-inducing consequences of the MYC/Myc-activating endemic Burkitt lymphoma t(8;14)/plasmacytoma T(12;15) translocation.

Evidence for selective transformation of autoreactive immature plasma cells in mice deficient in Fasl.

Germline mutations in Fas and Fasl induce nonmalignant T cell hyperplasia and systemic autoimmunity and also greatly increase the risk of B cell neoplasms. B lymphomas occurring in Fasl mutant (gld) mice usually are immunoglobulin (Ig) isotype switched, secrete Ig, and are plasmacytoid in appearance but lack Myc translocations characteristic of other plasma cell (PC) neoplasms. Here, we explore the relationship between B cell autoreactivity and transformation and use gene expression profiling to further classify gld plasmacytoid lymphomas (PLs) and to identify genes of potential importance in transformation. We found that the majority of PLs derive from antigen-experienced autoreactive B cells producing antinuclear antibody or rheumatoid factor and exhibit the skewed Ig V gene repertoire and Ig gene rearrangement patterns associated with these specificities. Gene expression profiling revealed that both primary and transplanted PLs share a transcriptional profile that places them at an early stage in PC differentiation and distinguishes them from other B cell neoplasms. In addition, genes were identified whose altered expression might be relevant in lymphomagenesis. Our findings provide a strong case for targeted transformation of autoreactive B cells in gld mice and establish a valuable model for understanding the relationship between systemic autoimmunity and B cell neoplasia.

Endophilins interact with Moloney murine leukemia virus Gag and modulate virion production.

BACKGROUND: The retroviral Gag protein is the central player in the process of virion assembly at the plasma membrane, and is sufficient to induce the formation and release of virus-like particles. Recent evidence suggests that Gag may co-opt the host cell's endocytic machinery to facilitate retroviral assembly and release. RESULTS: A search for novel partners interacting with the Gag protein of the Moloney murine leukemia virus (Mo-MuLV) via the yeast two-hybrid protein-protein interaction assay resulted in the identification of endophilin 2, a component of the machinery involved in clathrin-mediated endocytosis. We demonstrate that endophilin interacts with the matrix or MA domain of the Gag protein of Mo-MuLV, but not of human immunodeficiency virus, HIV. Both exogenously expressed and endogenous endophilin are incorporated into Mo-MuLV viral particles. Titration experiments suggest that the binding sites for inclusion of endophilin into viral particles are limited and saturable. Knock-down of endophilin with small interfering RNA (siRNA) had no effect on virion production, but overexpression of endophilin and, to a lesser extent, of several fragments of the protein, result in inhibition of Mo-MuLV virion production, but not of HIV virion production. CONCLUSIONS: This study shows that endophilins interact with Mo-MuLV Gag and affect virion production. The findings imply that endophilin is another component of the large complex that is hijacked by retroviruses to promote virion production.

CD19 signaling pathways play a major role for murine AIDS induction and progression.

Infection of genetically susceptible mice with the LP-BM5 mixture of murine leukemia viruses including an etiologic defective virus (BM5def) causes an immunodeficiency syndrome called murine AIDS (MAIDS). The disease is characterized by interactions between B cells and CD4(+) T cells resulting in polyclonal activation of both cell types. It is known that BM5def is expressed at highest levels in B cells and that B cells serve as viral APC. The CD19-CD21 complex and CD22 on the surface of B cells play critical roles as regulators of B cell responses to a variety of stimuli, influencing cell activation, differentiation, and survival. CD19 integrates positive signals induced by B cell receptor ligation by interacting with the protooncogene Vav, which leads to subsequent tyrosine phosphorylation of this molecule. In contrast, CD22 negatively regulates Vav phosphorylation. To analyze the role of CD19, CD21, Vav, and CD22 in MAIDS, we infected mice deficient in CD19, CD21 (CR2), Vav-1, or CD22 with LP-BM5 murine leukemia viruses. Infected CR2(-/-) mice developed MAIDS with a time course and severity indistinguishable from that of wild-type mice. In contrast, CD19 as well as Vav-1 deficiency restricted viral replication and suppressed the development of typical signs of MAIDS including splenomegaly, lymphadenopathy, and hypergammaglobulinemia. Finally, CD22 deficiency was found to accelerate MAIDS development. These results provide novel insights into the B cell signaling pathways required for normal induction and progression of MAIDS.

Genomic instability in mouse Burkitt lymphoma is dominated by illegitimate genetic recombinations, not point mutations.

lambda-MYC-induced mouse Burkitt lymphoma (BL) harboring the shuttle vector pUR288, which includes a lacZ reporter gene to study mutagenesis, was employed to assess genomic instability associated with MYC deregulation. The frequency of lacZ mutations in lymphomas was elevated only 1.75-fold above that in normal tissue, indicating that mouse BL does not exhibit a phenotype of hypermutability. However, the nature of lacZ mutations was strikingly different in normal tissues and lymphomas. While point mutations comprised approximately 75% of the mutations found in normal tissues, apparent translocations, deletions and inversions constituted the majority of mutations ( approximately 65%) in lymphomas. Genomic instability in mouse BL thus seems characterized by a preponderance of illegitimate genetic rearrangements in the context of near-background mutant frequencies. SKY analyses of cell lines from primary BL tumors revealed substantial changes in chromosomal structure, confirming the lacZ studies. Bi-allelic deletions of the tumor suppressor p16(Ink4a) were detected in six out of 16 cell lines, illustrating cellular selection of advantageous mutations. Together, these approaches indicate that MYC may contribute to lymphomagenesis through the dominant mutator effect of inducing chromosomal instability. The results further suggest that a phenotype of hypermutability (elevated mutant frequency) may not always be required for oncogenesis to occur.

The Bcl6 locus is not mutated in mouse B-cell lineage lymphomas.

In normal human germinal centre (GC) B-cells and post-GC B-cell lymphomas, a region in the first intron of the BCL6 gene, termed the major mutations cluster (MMC) exhibits somatic point mutations and deletions with patterns very similar to those seen in the variable regions of immunoglobulin (Ig) genes. In studies of mouse post-GC diffuse large cell lymphoma, Burkitt lymphomas, and plasmacytomas, direct sequencing or cold SSCP analyses revealed no mutations within a 686-bp region in Bcl6 intron 1 with 72% identity to the human MMC. The mouse Bcl6 locus must be inaccessible to the mutational machinery responsible for somatic mutations of Ig and BCL6 in humans.