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B-cell receptor (BCR) signaling is essential for the development of B cells and has a critical role in B-cell neoplasia. Increasing evidence indicates an association between chronic hepatitis C virus (HCV) infection and B-cell lymphoma, however, the mechanisms by which HCV causes B-cell lymphoproliferative disorder are still unclear. Herein, we demonstrate the expression of HCV viral proteins in B cells of HCV-infected patients and show that HCV upregulates BCR signaling in human primary B cells. HCV nonstructural protein NS3/4A interacts with CHK2 and downregulates its activity, modulating HuR posttranscriptional regulation of a network of target mRNAs associated with B-cell lymphoproliferative disorders. Interestingly, the BCR signaling pathway was found to have the largest number of transcripts with increased association with HuR and was upregulated by NS3/4A. Our study reveals a previously unidentified role of NS3/4A in regulation of host BCR signaling during HCV infection, contributing to a better understanding of the molecular mechanisms underlying HCV-associated B-cell lymphoproliferative disorders.

Canonical nuclear factor kappaB (NF-κB) activation signals stimulate nuclear translocation of p50:p65, replacing inhibitory p50:p50 with activating complexes on chromatin. C/EBP interaction with p50 homodimers provides an alternative pathway for NF-κB target gene activation, and interaction with p50:p65 may enhance gene activation. We previously found that C/EBPα cooperates with p50, but not p65, to induce Bcl-2 transcription and that C/EBPα induces Nfkb1/p50, but not RelA/p65, transcription. Using p50 and p65 variants containing the FLAG epitope at their N- or C-termini, we now show that C/EBPα, C/EBPα myeloid oncoproteins, or the LAP1, LAP2, or LIP isoforms of C/EBPβ have markedly higher affinity for p50 than for p65. Deletion of the p65 transactivation domain did not increase p65 affinity for C/EBPs, suggesting that unique residues in p50 account for specificity, and clustered mutation of HSDL in the “p50 insert” lacking in p65 weakens interaction. Also, in contrast to Nfkb1 gene deletion, absence of the RelA gene does not reduce Bcl-2 or Cebpa RNA in unstimulated cells or prevent interaction of C/EBPα with the Bcl-2 promoter. Saturating mutagenesis of the C/EBPα basic region identifies R300 and nearby residues, identical in C/EBPβ, as critical for interaction with p50. These findings support the conclusion that C/EBPs activate NF-κB target genes via contact with p50 even in the absence of canonical NF-κB activation and indicate that targeting C/EBP:p50 rather than C/EBP:p65 interaction in the nucleus will prove effective for inflammatory or malignant conditions, alone or synergistically with agents acting in the cytoplasm to reduce canonical NF-κB activation. Mol Cancer Res; 9(10); 1395–405. ©2011 AACR.

Mutated CEBPA defines a subgroup of acute myeloid leukemia (AML). We have previously shown that C/EBPα or its AML mutants synergize with NF-κB p50 to activate antiapoptotic genes, including BCL2 and FLIP. Furthermore, p50 binds and activates the CEBPA gene in myeloid cells. We now report that C/EBPα or C/EBPα leucine zipper AML mutants bind in vivo to the nfkb1 (p50) promoter and induce its expression even in the presence of cycloheximide. Induction of p50 by C/EBPα depends on 2 conserved κB sites in the nfkb1 promoter. C/EBPα did not induce p65 expression. Thus, C/EBPα and p50 reciprocally regulate each other's expression, establishing a positive feedback relationship. Although p50 homodimers inhibit transcription, C/EBPα and p50 synergistically activate antiapoptotic genes. ChIP analysis showed that C/EBPα diminishes the occupation of histone deacetylase 1 (HDAC1) or HDAC3 on the endogenous FLIP promoter but not in mice lacking p50. Coimmunoprecipitation confirmed that C/EBPα, its AML variants, or C/EBPβ disrupt interaction between p50 and HDACs dependent on the C/EBP basic region. These findings suggest that C/EBPs displace HDACs from p50 homodimers bound to antiapoptotic genes, contributing to NF-κB dysregulation in leukemia, and that the C/EBPα:p50 complex is a potential therapeutic target.

The phosphorylation of eIF4E1 at serine 209 by MNK1 or MNK2 has been shown to initiate oncogenic mRNA translation, a process that favours cancer development and maintenance. Here, we interrogate the MNK-eIF4E axis in diffuse large B-cell lymphoma (DLBCL) and show a distinct distribution of MNK1 and MNK2 in germinal centre B-cell (GCB) and activated B-cell (ABC) DLBCL. Despite displaying a differential distribution in GCB and ABC, both MNKs functionally complement each other to sustain cell survival. MNK inhibition ablates eIF4E1 phosphorylation and concurrently enhances eIF4E3 expression. Loss of MNK protein itself downregulates total eIF4E1 protein level by reducing eIF4E1 mRNA polysomal loading without affecting total mRNA level or stability. Enhanced eIF4E3 expression marginally suppresses eIF4E1-driven translation but exhibits a unique translatome that unveils a novel role for eIF4E3 in translation initiation. We propose that MNKs can modulate oncogenic translation by regulating eIF4E1-eIF4E3 levels and activity in DLBCL., Diffuse large B-cell lymphoma (DLBCL) is a highly aggressive and heterogeneous type of non-Hodgkin’s lymphoma. Here the authors demonstrate that the differential regulation of eIF4E1 and eIF4E3 by the MAPK-interacting kinases is involved in DLBCL aetiology through modification of the cellular translatome.

Human diffuse large B-cell lymphomas (DLBCLs) often aberrantly express oncogenes that generally contain complex secondary structures in their 5' untranslated region (UTR). Oncogenes with complex 5'UTRs require enhanced eIF4A RNA helicase activity for translation. PDCD4 inhibits eIF4A, and PDCD4 knockout mice have a high penetrance for B-cell lymphomas. Here, we show that on B-cell receptor (BCR)-mediated p70s6K activation, PDCD4 is degraded, and eIF4A activity is greatly enhanced. We identified a subset of genes involved in BCR signaling, including CARD11, BCL10, and MALT1, that have complex 5'UTRs and encode proteins with short half-lives. Expression of these known oncogenic proteins is enhanced on BCR activation and is attenuated by the eIF4A inhibitor Silvestrol. Antigen-experienced immunoglobulin (Ig)G(+) splenic B cells, from which most DLBCLs are derived, have higher levels of eIF4A cap-binding activity and protein translation than IgM(+) B cells. Our results suggest that eIF4A-mediated enhancement of oncogene translation may be a critical component for lymphoma progression, and specific targeting of eIF4A may be an attractive therapeutic approach in the management of human B-cell lymphomas.

Insulin-loaded alginate-dextran nanospheres were prepared by nanoemulsion dispersion followed by triggered in situ gelation. Nanospheres were characterized for mean size and distribution by laser diffraction spectroscopy and for shape by transmission electron microscopy. Insulin encapsulation efficiency and in vitro release were determined by Bradford protein assay and bioactivity determined in vitro using a newly developed Western blot immunoassay and in vivo using Wistar diabetic rats. Nanospheres ranged from 267 nm to 2.76 [mu]m in diameter and demonstrated a unimodal size distribution. Insulin encapsulation efficiency was 82.5%. Alginate-dextran particles suppressed insulin release in acidic media and promoted a sustained release at near neutral conditions. Nanoencapsulated insulin was bioactive, demonstrated through both in vivo and in vitro bioassays http://www.sciencedirect.com/science/article/B6T25-4MTC6GD-1/1/c26097e450ad242243e8df9feae8643f

Dysregulation of mRNA translation can alter the cellular phenotype leading to cancer initiation, maintenance, progression, invasion or metastasis. Cap-dependent translation is the primary mechanism of mRNA translation in eukaryotic cells. The conventional cap-binding complex, eIF4F consists of three components including eIF4A, eIF4G and eIF4E1. eIF4E1 binds the 7-methyl-guanosine-cap (cap) structure in the mRNA and functions to bridge mRNA to the ribosome. eIF4E3 is another member of the eIF4E family that has been recently shown to bind cap using biophysical approach. We explored the biological function of eIF4E3 in a diffuse large B-cell lymphoma model. We found that in the absence or lack of eIF4E1 phosphorylation, eIF4E3 expression is enhanced, and this increases the cap-binding ability of eIF4E3. We find that eIF4E3 also physically associates with the components of the cap-binding complex eIF4A and eIF4G, essentially forming a novel cap-binding complex. We interrogated the functional capacity of the eIF4E3-mediated translation by analyzing the translatome and transcriptome of eIF4E3 expressing cells. eIF4E3 and eIF4E1 mutually regulate a high fraction of total mRNA transcripts, while exclusively modulating translation of select messages. Interestingly, we identified selective motif enrichments in the 5'UTR that may facilitate transcript selection by eIF4E1 or eIF4E3. Together, we have identified a novel cap-binding complex consisting of eIF4A, eIF4G and eIF4E3, which can initiate mRNA translation of select messages. Citation Format: Ari L. Landon, Parameswary A Muniandy, Simone Houng, Amol Shetty, Kevin Becker, Katherine Borden, Ronald B. Gartenhaus. eIF4E3 forms a novel cap-binding complex for mRNA translation initiation. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B08.

B-cell receptor (BCR) signaling is critical for the development of normal B-cells and B-cell lymphoma. Increasing epidemiological evidence indicates an association between chronic hepatitis C virus (HCV) infection and B-cell lymphoma, however, the mechanisms by which HCV causes B-cell lymphoproliferative disorder are still unclear. Herein, we demonstrate the expression of HCV viral proteins in B-cells of HCV-infected patients and show that HCV up-regulates BCR signaling in human primary B-cells. HCV nonstructural protein NS3/4A interacts with CHK2 and down-regulates its activity, modulating HuR posttranscriptional regulation of a network of target mRNAs associated with B-cell lymphoproliferative disorders. Strikingly, the BCR signaling pathway was found to have the largest number of transcripts with increased association with HuR and up-regulated by NS3/4A. Our study reveals a previously unidentified role of NS3/4A in regulation of host BCR signaling during HCV infection, lending further insight into the molecular mechanisms underlying HCV-associated B-cell lymphoproliferative disorders. Citation Format: Bojie Dai, Ari Landon, Simone Houng, Ronald B. Gartenhaus. Hepatitis C virus NS3/4A hijacks the host B-cell receptor signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-035. doi:10.1158/1538-7445.AM2015-LB-035

Abstract 708 Nuclear Factor-κB (NF-κB) is a key mediator of the inflammatory response and also inhibits apoptosis. Activation of NF-κB is prevalent in AML, Hodgkin's lymphoma and subsets of non-Hodgkin's lymphoma, as well as in multiple solid tumors. NF-κB p50 (NF-κB1) and p65 (Rel A) are the most prevalent NF-κB subunits. NF-κB p65 has 30–50 fold higher affinity than p50 for the cytoplasmic protein IκB, and so under basal conditions p50:p65 heterodimers are held in the cytoplasm whereas p50:p50 homodimers are found in the nucleus bound to chromatin where they repress NF-κB target genes via association with HDACs. Canonical NF-κB activation signals lead to IκB proteosome-mediated degradation and translocation of p50:p65 heterodimers into the nucleus to replace inhibitory p50:p50 dimers with activating complexes on chromatin. We postulate an alternative means to activate NF-κB target genes wherein induction or activation of C/EBP proteins allows them to bind chromatin-bound p50 and displace HDACs in cells lacking canonical NF-κB activation. We found that C/EBPα induces bcl-2 and FLIP RNA expression via promoter interaction dependent upon the presence of NF-κB p50, that C/EBPα present in two myeloid leukemia cell lines preferentially binds p50 compared with p65, and that mutation of the C/EBPα basic region prevents bcl-2 induction and weakens interaction with p50 but not p65 (Paz-Priel et al 2005; 2009; Wang et al 2009). Using p50 and p65 variants containing the FLAG epitope at either their N- or C-termini we now demonstrate using reciprocal co-immunoprecipitation that p50 has much higher affinity than p65 for C/EBPα, for N-terminal or leucine zipper mutants of C/EBPα expressed in a subset of AML cases, and for the longer LAP or shorter LIP isoforms of C/EBPβ. The FLAG-tagged p65 proteins retained the ability to bind DNA in a gel shift assay and to activate a reporter containing two κB sites as effectively as untagged p65. Deletion of the p65 trans-activation domain (TAD) did not increase p65 affinity for C/EBPα, indicating that an inhibitory effect of the TAD does not account for specificity of C/EBPα:p50 interaction. Moreover, the increased affinity of the p50 rel homolog domain (RHD) for C/EBPα compared with the p65 RHD suggests that residues that differ between these RHDs rather than those in common make key contacts wih the C/EBP basic region. Germline deletion of the gene encoding p65, by injecting p65(flox/flox); Mx1-CRE mice with pIpC, did not reduce C/EBPα expression, prevent interaction of C/EBPα with the bcl-2 promoter in a ChIP assay, or reduce bcl-2 RNA expression, in contrast to our prior findings with p50 gene deletion. A C/EBPα variant harboring a single point mutation in the basic region, C/EBPα(R300A), has minimal affinity for p50, and further saturating mutagenesis of the C/EBPα basic region identifies an additional block of six amino acids, RERNNI, identical in C/EBPβ and critical for interaction with NF-κB p50. In conclusion, increased affinity of C/EBP proteins for p50 compared with p65 lends support to the hypothesis that C/EBP interaction with p50:p50 complexes provides an alternative means for normal cells or cancer cells to activate NF-κB target genes, for example in response to C/EBP induction or activation. C/EBP interaction with p50:p65 in activated cells may also occur, via p50, to further activate genes regulated by NF-κB. In addition, these findings suggest that targeting the C/EBP:p50 interface rather than C/EBP:p65 interaction will prove more effective in the therapy of inflammatory or malignant conditions, and our mapping of relevant basic region residues and the finding that the p50 RHD retains increased affinity for C/EBPα compared wih the p65 RHD provides a first step towards rational design of such inhibitors. Disclosures: No relevant conflicts of interest to declare.

Abstract 3637 The NF-κB transcription factors are involved in inflammation, proliferation, and apoptosis. The p50:p65 heterodimer, the most common transcription activating form of NF-κB, is retained in the cytosol and translocates into the nucleus only upon stimulation. In contrast, the p50 homodimer is the primary nuclear NF-κB species in unstimulated cells, and recruit HDACs to repress NF-κB-target gene expression. Dysregulation of NF-κB frequently occurs in AML and lymphoma. C/EBPα is mutated in approximately 10% of patients with AML, resulting in expression of C/EBPαp30 or C/EBPαLZ oncoproteins. We showed that C/EBPα or its oncoproteins, including leucine zipper variants (C/EBPαLZ) that cannot bind DNA directly, protects hematopoietic cells from apoptosis. Through interaction with NF-κB p50, C/EBPα or its AML mutants activate several anti-apoptotic genes including BCL2 and FLIP. C/EBPβ or its truncated LIP isoform are over-expressed in Hodgkin lymphoma and breast cancer. Using Ba/F3 cell lines carrying an estrogen receptor fusion of C/EBPα or C/EBPαLZ, we now show that activation of C/EBPα isoforms results in a significant 90% reduction in the association of endogenous HDAC1 or HDAC3 with the FLIP promoter, within 5 hours. In contrast, activation of the basic region mutant C/EBPαBR that cannot bind p50 did not have an effect. Accordingly, ChIP analysis demonstrated that on the FLIP promoter, the expression of C/EBPα or C/EBPαLZ resulted in a 2.5 or 4-fold increase in histone H3 or H4 acetylation, respectively. To study the specific displacement of HDACs from p50 we used Eμ-C/EBPα transgenic mice which express C/EBPα in splenic lymphoid cells, in contrast to control littermates. The ectopic presence of C/EBPα resulted in a significant reduction of endogenous HDAC1 or HDAC3 on the FLIP promoter to 17 or 4% of the expression in wild type mice. In contrast, in p50-/- mice, the C/EBPα transgene had a minimal effect on HDAC1 or a modest reduction of HDAC3 occupancy on the FLIP promoter, to 90 or 50% of levels seen in p50-/- controls. These findings suggest that on the FLIP promoter C/EBPα displaces primarily p50-bound HDACs. HDAC1 or HDAC3 co-ip with p50 in transiently transfected 293T cells. The co-expression of C/EBPα, its C/EBPαLZ or C/EBPαp30 AML mutants, or C/EBPβ interrupted interaction of p50 with HDAC1 and to a lesser extent with HDAC3. In striking correlation to our previous findings, the basic region mutant C/EBPαBR does not interrupt this interaction. As a control, we confirmed that C/EBPα or C/EBPβ do not disrupt the NF-κB p50:p65 co-ip. We have shown that interaction with p50 is required for induction of bcl-2 and FLIP by C/EBPα and that p50 binds and induces the CEBPA gene. Using ChIP analysis, we now show that in U937 AML cells endogenous C/EBPα, C/EBPβ and p50 specifically bind the proximal segment of the nfkb1 (p50) promoter. In addition, C/EBPαLZ expressed from the metallothionein promoter in Ba/F3 cells binds a proximal site in the nfkb1 promoter. C/EBPα or C/EBPαLZ induced p50 RNA expression 3.5-fold, while C/EBPαBR was not effective. Treating the cells with the translation inhibitor cycloheximide did not change p50 induction, suggesting direct activation. C/EBPα, C/EBPαLZ but not C/EBPαBR activated to a similar degree luciferase reporters containing 1800 or 250 bp fragments of the nfkb1 promoter. In addition, C/EBPβ or its truncated LIP isoform activated these reporters, although LIP cannot activate a (CEBP)2TK-Luc reporter. The murine nfkb1 promoter contains 2 proximal κB sites that are 100% conserved between mouse and human. p50 but not C/EBP avidly binds either of these sites in a gel shift assay. Disruption of either κB site abrogated 65 to 80% of reporter activation, and mutating both resulted in activation of less than 10% by either C/EBPα or C/EBPαLZ. In summary, we show that C/EBPs displace HDAC from NF-κB p50, providing a mechanism whereby C/EBPα or its AML mutants can derepress NF-κB target genes and contribute to their dysregulated expression. In addition, C/EBPα, a C/EBPαLZ AML variant that cannot bind DNA directly, or C/EBPβ bind and activate the nfkb1 promoter through interaction with NF-κB bound to two proximal, highly conserved κB sites. Together these findings lend support to the idea that disrupting the C/EBP:NF-κB p50 complex may offer a complementary approach for targeting NF-κB activity in the nucleus to favor apoptosis in AML or other malignancies in which C/EBP family members are expressed. Disclosures: No relevant conflicts of interest to declare.

Background The mechanistic target of rapamycin, (mTOR) kinase plays a pivotal role in controlling critical cellular growth and survival pathways, and its aberrant induction is implicated in cancer pathogenesis. Therefore, suppression of active mTOR signaling has been of great interest to researchers; several mTOR inhibitors have been discovered to date. Ethanol (EtOH), similar to pharmacologic mTOR inhibitors, has been shown to suppress the mTOR signaling pathway, though in a non-catalytic manner. Despite population studies showing that the consumption of EtOH has a protective effect against hematological malignancies, the mechanisms behind EtOH’s modulation of mTOR activity in cells and its downstream consequences are largely unknown. Here we evaluated the effects of EtOH on the mTOR pathway, in comparison to the active-site mTOR inhibitor INK128, and compared translatome analysis of their downstream effects in diffuse large B-cell lymphoma (DLBCL). Results Treatment of DLBCL cells with EtOH suppressed mTORC1 complex formation while increasing AKT phosphorylation and mTORC2 complex assembly. INK128 completely abrogated AKT phosphorylation without affecting the structure of mTORC1/2 complexes. Accordingly, EtOH less profoundly suppressed cap-dependent translation and global protein synthesis, compared to a remarkable inhibitory effect of INK128 treatment. Importantly, EtOH treatment induced the formation of stress granules, while INK128 suppressed their formation. Microarray analysis of polysomal RNA revealed that although both agents primarily affected cell growth and survival, EtOH and INK128 regulated the synthesis of mostly distinct genes involved in these processes. Though both EtOH and INK128 inhibited cell cycle, proliferation and autophagy, EtOH, in contrast to INK128, did not induce cell apoptosis. Conclusion Given that EtOH, similar to pharmacologic mTOR inhibitors, inhibits mTOR signaling, we systematically explored the effect of EtOH and INK128 on mTOR signal transduction, components of the mTORC1/2 interaction and their downstream effectors in DLBCL malignancy. We found that EtOH partially inhibits mTOR signaling and protein translation, compared to INK128’s complete mTOR inhibition. Translatome analysis of mTOR downstream target genes established that differential inhibition of mTOR by EtOH and INK128 distinctly modulates translation of specific subsets of mRNAs involved in cell growth and survival, leading to differential cellular response and survival. Electronic supplementary material The online version of this article (doi:10.1186/s12964-015-0091-0) contains supplementary material, which is available to authorized users.