Van S. Tompkins

Nucleophosmin (B23) Targets ARF to Nucleoli and Inhibits Its Function

ABSTRACT The ARF tumor suppressor is a nucleolar protein that activates p53-dependent checkpoints by binding Mdm2, a p53 antagonist. Despite persuasive evidence that ARF can bind and inactivate Mdm2 in the nucleoplasm, the prevailing view is that ARF exerts its growth-inhibitory activities from within the nucleolus. We suggest ARF primarily functions outside the nucleolus and provide evidence that it is sequestered and held inactive in that compartment by a nucleolar phosphoprotein, nucleophosmin (NPM). Most cellular ARF is bound to NPM regardless of whether cells are proliferating or growth arrested, indicating that ARF-NPM association does not correlate with growth suppression. Notably, ARF binds NPM through the same domains that mediate nucleolar localization and Mdm2 binding, suggesting that NPM could control ARF localization and compete with Mdm2 for ARF association. Indeed, NPM knockdown markedly enhanced ARF-Mdm2 association and diminished ARF nucleolar localization. Those events correlated with greater ARF-mediated growth suppression and p53 activation. Conversely, NPM overexpression antagonized ARF function while increasing its nucleolar localization. These data suggest that NPM inhibits ARFs p53-dependent activity by targeting it to nucleoli and impairing ARF-Mdm2 association.

NF-κB/STAT3/PI3K signaling crosstalk in iMycEμ B lymphoma

BackgroundMyc is a well known driver of lymphomagenesis, and Myc-activating chromosomal translocation is the recognized hallmark of Burkitt lymphoma, an aggressive form of non-Hodgkins lymphoma. We developed a model that mimics this translocation event by inserting a mouse Myc cDNA gene into the immunoglobulin heavy chain locus, just upstream of the intronic Eμ enhancer. These mice, designated iMycEμ, readily develop B-cell lymphoma. To study the mechanism of Myc-induced lymphoma, we analyzed signaling pathways in lymphoblastic B-cell lymphomas (LBLs) from iMycEμ mice, and an LBL-derived cell line, iMycEμ-1.ResultsNuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) were constitutively activated in iMycEμ mice, not only in LBLs but also in the splenic B-lymphocytes of young animals months before tumors developed. Moreover, inhibition of either transcription factor in iMycEμ-1 cells suppressed growth and caused apoptosis, and the abrogation of NF-κB activity reduced DNA binding by both STAT3 and Myc, as well as Myc expression. Inhibition of STAT3 signaling eliminated the activity of both NF-κB and Myc, and resulted in a corresponding decrease in the level of Myc. Thus, in iMycEμ-1 cells NF-κB and STAT3 are co-dependent and can both regulate Myc. Consistent with this, NF-κB and phosphorylated STAT3 were physically associated with one another. In addition, LBLs and iMycEμ-1 cells also showed constitutive AKT phosphorylation. Blocking AKT activation by inhibiting PI3K reduced iMycEμ-1 cell proliferation and caused apoptosis, via downregulation of NF-κB and STAT3 activity and a reduction of Myc levels. Co-treatment with NF-κB, STAT3 or/and PI3K inhibitors led to additive inhibition of iMycEμ-1 cell proliferation, suggesting that these signaling pathways converge.ConclusionsOur findings support the notion that constitutive activation of NF-κB and STAT3 depends on upstream signaling through PI3K, and that this activation is important for cell survival and proliferation, as well as for maintaining the level of Myc. Together, these data implicate crosstalk among NF-κB, STAT3 and PI3K in the development of iMycEμ B-cell lymphomas.

HHV-8–encoded viral IL-6 collaborates with mouse IL-6 in the development of multicentric Castleman disease in mice

Human herpes virus 8 (HHV-8) or Kaposi sarcoma-associated herpes virus is the etiologic agent of Kaposi sarcoma, primary effusion lymphoma, and plasma cell-type multicentric Castleman disease (MCD). HHV-8 encodes a viral homolog of human IL-6, called viral IL-6 (vIL-6), which does not require the cellular IL-6 receptor for binding to the ubiquitously expressed gp130 receptor subunit and subsequent JAK-STAT signaling. Thus, in contrast to IL-6, vIL-6 can stimulate virtually all cells in the body. To elucidate the mechanism by which vIL-6 drives human diseases, we generated transgenic mice that constitutively express vIL-6 under control of the MHC class I promoter. The mice were found to exhibit vIL-6 serum levels comparable with those observed in HHV-8-infected patients, to contain elevated amounts of phosphorylated STAT3 in spleen and lymph nodes, where vIL-6 was produced, and to spontaneously develop key features of human plasma cell-type MCD, including splenomegaly, multifocal lymphadenopathy, hypergammaglobulinemia, and plasmacytosis. Transfer of the vIL-6 transgene onto an IL-6-deficient genetic background abrogated MCD-like phenotypes, indicating that endogenous mouse IL-6 is a crucial cofactor in the natural history of the disease. Our results in mice suggest that human IL-6 plays an important role in the pathogenesis of HHV-8-associated MCD.

A Novel Nuclear Interactor of ARF and MDM2 (NIAM) That Maintains Chromosomal Stability

The ARF tumor suppressor signals through p53 and other poorly defined anti-proliferative pathways to block carcinogenesis. In a search for new regulators of ARF signaling, we discovered a novel nuclear protein that we named NIAM (nuclear interactor of ARF and MDM2) for its ability to bind both ARF and the p53 antagonist MDM2. NIAM protein is normally expressed at low to undetectable levels in cells because of, at least in part, MDM2-mediated ubiquitination and proteasomal degradation. When reintroduced into cells, NIAM activated p53, caused a G1 phase cell cycle arrest, and collaborated with ARF in an additive fashion to suppress proliferation. Notably, NIAM retains growth inhibitory activity in cells lacking ARF and/or p53, and knockdown experiments revealed that it is not essential for ARF-mediated growth inhibition. Thus, NIAM and ARF act in separate anti-proliferative pathways that intersect mechanistically and suppress growth more effectively when jointly activated. Intriguingly, silencing of NIAM accelerated chromosomal instability, and microarray analyses showed reduced NIAM mRNA expression in numerous primary human tumors. This study identifies a novel protein with tumor suppressor-like behaviors and functional links to ARF-MDM2-p53 signaling.

Identification of Novel ARF Binding Proteins by Two-Hybrid Screening

The ARF tumor suppressor protects us against cancer through protein-protein interactionsin partially defined p53-dependent and p53-independent pathways. We performed a two-hybridscreen using ARF as bait and present the identification of several new ARF partners that mayregulate its growth inhibitory signaling. The potential physiological roles of these novel ARFbinding proteins in regulating ARF signaling are discussed.

Identification of Candidate B-Lymphoma Genes by Cross- Species Gene Expression Profiling

Comparative genome-wide expression profiling of malignant tumor counterparts across the human-mouse species barrier has a successful track record as a gene discovery tool in liver, breast, lung, prostate and other cancers, but has been largely neglected in studies on neoplasms of mature B-lymphocytes such as diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma (BL). We used global gene expression profiles of DLBCL-like tumors that arose spontaneously in Myc-transgenic C57BL/6 mice as a phylogenetically conserved filter for analyzing the human DLBCL transcriptome. The human and mouse lymphomas were found to have 60 concordantly deregulated genes in common, including 8 genes that Cox hazard regression analysis associated with overall survival in a published landmark dataset of DLBCL. Genetic network analysis of the 60 genes followed by biological validation studies indicate FOXM1 as a candidate DLBCL and BL gene, supporting a number of studies contending that FOXM1 is a therapeutic target in mature B cell tumors. Our findings demonstrate the value of the “mouse filter” for genomic studies of human B-lineage neoplasms for which a vast knowledge base already exists.

18 F-FDG-PET/CT imaging in an IL-6- and MYC-driven mouse model of human multiple myeloma affords objective evaluation of plasma cell tumor progression and therapeutic response to the proteasome inhibitor ixazomib

18F-fluorodeoxyglucose positron emission tomography (FDG-PET) and computed tomography (CT) are useful imaging modalities for evaluating tumor progression and treatment responses in genetically engineered mouse models of solid human cancers, but the potential of integrated FDG-PET/CT for assessing tumor development and new interventions in transgenic mouse models of human blood cancers such as multiple myeloma (MM) has not been demonstrated. Here we use BALB/c mice that contain the newly developed iMycΔEμ gene insertion and the widely expressed H2-Ld-IL6 transgene to demonstrate that FDG-PET/CT affords an excellent research tool for assessing interleukin-6- and MYC-driven plasma cell tumor (PCT) development in a serial, reproducible and stage- and lesion-specific manner. We also show that FDG-PET/CT permits determination of objective drug responses in PCT-bearing mice treated with the investigational proteasome inhibitor ixazomib (MLN2238), the biologically active form of ixazomib citrate (MLN9708), that is currently in phase 3 clinical trials in MM. Overall survival of 5 of 6 ixazomib-treated mice doubled compared with mice left untreated. One outlier mouse presented with primary refractory disease. Our findings demonstrate the utility of FDG-PET/CT for preclinical MM research and suggest that this method will play an important role in the design and testing of new approaches to treat myeloma.

Residues in the alternative reading frame tumor suppressor that influence its stability and p53-independent activities.

The Alternative Reading Frame (ARF) protein suppresses tumorigenesis through p53-dependent and p53-independent pathways. Most of ARFs anti-proliferative activity is conferred by sequences in its first exon. Previous work showed specific amino acid changes occurred in that region during primate evolution, so we programmed those changes into human p14ARF to assay their functional impact. Two human p14ARF residues (Ala(14) and Thr(31)) were found to destabilize the protein while two others (Val(24) and Ala(41)) promoted more efficient p53 stabilization and activation. Despite those effects, all modified p14ARF forms displayed robust p53-dependent anti-proliferative activity demonstrating there are no significant biological differences in p53-mediated growth suppression associated with simian versus human p14ARF residues. In contrast, p53-independent p14ARF function was considerably altered by several residue changes. Val(24) was required for p53-independent growth suppression whereas multiple residues (Val(24), Thr(31), Ala(41) and His(60)) enabled p14ARF to block or reverse the inherent chromosomal instability of p53-null MEFs. Together, these data pinpoint specific residues outside of established p14ARF functional domains that influence its expression and signaling activities. Most intriguingly, this work reveals a novel and direct role for p14ARF in the p53-independent maintenance of genomic stability.

Nuclear interactor of ARF and Mdm2 regulates multiple pathways to activate p53

The p53 tumor suppressor is controlled by an interactive network of factors that stimulate or inhibit its transcriptional activity. Within that network, Mdm2 functions as the major antagonist of p53 by promoting its ubiquitylation and degradation. Conversely, Tip60 activates p53 through direct association on target promoters as well as acetylation of p53 at lysine 120 (K120). This study examines the functional relationship between Mdm2 and Tip60 with a novel p53 regulator, NIAM (nuclear interactor of ARF and Mdm2). Previous work showed NIAM can suppress proliferation and activate p53 independently of ARF, indicating that other factors mediate those activities. Here, we demonstrate that NIAM is a chromatin-associated protein that binds Tip60. NIAM can promote p53 K120 acetylation, although that modification is not required for NIAM to inhibit proliferation or induce p53 transactivation of the p21 promoter. Notably, Tip60 silencing showed it contributes to but is not sufficient for NIAM-mediated p53 activation, suggesting other mechanisms are involved. Indeed, growth-inhibitory forms of NIAM also bind to Mdm2, and increased NIAM expression levels disrupt p53–Mdm2 association, inhibit p53 polyubiquitylation, and prevent Mdm2-mediated inhibition of p53 transcriptional activity. Importantly, loss of NIAM significantly impairs p53 activation. Together, these results show that NIAM activates p53 through multiple mechanisms involving Tip60 association and Mdm2 inhibition. Thus, NIAM regulates 2 critical pathways that control p53 function and are altered in human cancers, implying an important role for NIAM in tumorigenesis.

Piperlongumine inhibits LMP1/MYC-dependent mouse B-lymphoma cells

Piperlongumine (PL), isolated from the fruit of Long pepper, Piper longum, is a cancer-inhibiting compound that selectively kills tumor cells while sparing their normal counterparts. Here we evaluated the efficacy with which PL suppresses malignant B cells derived from a newly developed, double-transgenic mouse model of human endemic Burkitt lymphoma (BL), designated mCD40-LMP1/iMyc(Eμ). PL inhibited tumor cell proliferation in a concentration-dependent manner and induced apoptosis of neoplastic but not normal B cells. Treatment with PL resulted in downregulation of EBV-encoded LMP1, cellular Myc, constitutive NF-κB activity, and a host of LMP1-Myc-NF-κB-regulated target genes including Aurka, Bcat1, Bub1b, Ccnb1, Chek1, Fancd2, Tfrc and Xrcc6. Of note, p21(Cip1)-encoding Cdkn1a was suppressed independent of changes in Trp53 mRNA levels and p53 DNA-binding activity. Considering the central role of the LMP1-NF-κB-Myc axis in B-lineage neoplasia, these findings further our understanding of the mechanisms by which PL inhibits B-lymphoma and provide a preclinical rationale for the inclusion of PL in new interventions in blood cancers.