Marilena Pontoriero

Human immunodeficiency virus-1 Tat activates NF-κB via physical interaction with IκB-α and p65

Nuclear factor (NF)-κB is a master regulator of pro-inflammatory genes and is upregulated in human immunodeficiency virus 1 (HIV-1) infection. Mechanisms underlying the NF-κB deregulation by HIV-1 are relevant for immune dysfunction in AIDS. We report that in single round HIV-1 infection, or single-pulse PMA stimulation, the HIV-1 Tat transactivator activated NF-κB by hijacking the inhibitor IκB-α and by preventing the repressor binding to the NF-κB complex. Moreover, Tat associated with the p65 subunit of NF-κB and increased the p65 DNA-binding affinity and transcriptional activity. The arginine- and cysteine-rich domains of Tat were required for IκB-α and p65 association, respectively, and for sustaining the NF-κB activity. Among an array of NF-κB-responsive genes, Tat mostly activated the MIP-1α expression in a p65-dependent manner, and bound to the MIP-1α NF-κB enhancer thus promoting the recruitment of p65 with displacement of IκB-α; similar findings were obtained for the NF-κB-responsive genes CSF3, LTA, NFKBIA and TLR2. Our results support a novel mechanism of NF-κB activation via physical interaction of Tat with IκB-α and p65, and may contribute to further insights into the deregulation of the inflammatory response by HIV-1.

In vivo targeting and growth inhibition of the A20 murine B-cell lymphoma by an idiotype-specific peptide binder.

B-cell lymphoma is a clonal expansion of neoplastic cells that may result in fatal outcomes. Here, we report the in vivo targeting and growth inhibition of aggressive A20 murine B-cell lymphoma by idiotype-specific peptide pA20-36. pA20-36 was selected from random peptide libraries and bound specifically to the B-cell receptor (BCR) of A20 cells in mice engrafted with A20 lymphoma, as shown by histology and positron emission tomographic analysis. BCR cross-linking of A20 cells with pA20-36 resulted in massive apoptosis of targeted tumor cells and in an increased survival of the diseased animals without any detectable evidence of toxicity. The pA20-36 treatment reverted the immune suppression of the tumor microenvironment as shown by reduced expression of vascular endothelial growth factor, interleukin-10, and transforming growth factor-beta cytokines together with a lower number of CD11b+Gr-1+ inhibitor myeloid-derived suppressor cells and Foxp3+CD4+ Treg cells. Furthermore, pA20-36 treatment was associated with an increased number of tumor-infiltrating, activated CD8+ T cells that exerted a tumor-specific cytolytic activity. These findings show that a short peptide that binds specifically to the complementarity-determining regions of the A20 BCR allows in vivo detection of neoplastic cells together with significant inhibition of tumor growth in vivo.

Btk regulation in human and mouse B cells via protein kinase C phosphorylation of IBtkγ.

The inhibitor of Bruton tyrosine kinase γ (IBtkγ) is a negative regulator of the Bruton tyrosine kinase (Btk), which plays a major role in B-cell differentiation; however, the mechanisms of IBtkγ-mediated regulation of Btk are unknown. Here we report that B-cell receptor (BCR) triggering caused serine-phosphorylation of IBtkγ at protein kinase C consensus sites and dissociation from Btk. By liquid chromatography and mass-mass spectrometry and functional analysis, we identified IBtkγ-S87 and -S90 as the critical amino acid residues that regulate the IBtkγ binding affinity to Btk. Consistently, the mutants IBtkγ carrying S87A and S90A mutations bound constitutively to Btk and down-regulated Ca(2+) fluxes and NF-κB activation on BCR triggering. Accordingly, spleen B cells from Ibtkγ(-/-) mice showed an increased activation of Btk, as evaluated by Y551-phosphorylation and sustained Ca(2+) mobilization on BCR engagement. These findings identify a novel pathway of Btk regulation via protein kinase C phosphorylation of IBtkγ.

Eukaryotic Initiation Factor 4H Is under Transcriptional Control of p65/NF-κB.

Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and spatial control of gene expression. Initiation of protein synthesis requires at least 13 translation initiation factors to assemble the 80S ribosomal initiation complex. Loss of translation control may result in cell malignant transformation. Here, we asked whether translational initiation factors could be regulated by NF-κB transcription factor, a major regulator of genes involved in cell proliferation, survival, and inflammatory response. We show that the p65 subunit of NF-κB activates the transcription of eIF4H gene, which is the regulatory subunit of eIF4A, the most relevant RNA helicase in translation initiation. The p65-dependent transcriptional activation of eIF4H increased the eIF4H protein content augmenting the rate of global protein synthesis. In this context, our results provide novel insights into protein synthesis regulation in response to NF-κB activation signalling, suggesting a transcription-translation coupled mechanism of control.

Design and characterization of a peptide mimotope of the HIV-1 gp120 bridging sheet.

The Bridging Sheet domain of HIV-1 gp120 is highly conserved among the HIV-1 strains and allows HIV-1 binding to host cells via the HIV-1 coreceptors. Further, the bridging sheet domain is a major target to neutralize HIV-1 infection. We rationally designed four linear peptide epitopes that mimic the three-dimensional structure of bridging sheet by using molecular modeling. Chemically synthesized peptides BS3 and BS4 showed a fair degree of antigenicity when tested in ELISA with IgG purified from HIV+ broadly neutralizing sera while the production of synthetic peptides BS1 and BS2 failed due to their high degree of hydrophobicity. To overcome this limitation, we linked all four BS peptides to the COOH-terminus of GST protein to test both their antigenicity and immunogenicity. Only the BS1 peptide showed good antigenicity; however, no envelope specific antibodies were elicited upon mice immunization. Therefore we performed further analyses by linking BS1 peptide to the NH2-terminus of the E2 scaffold from the Geobacillus Stearothermophylus PDH complex. The E2-BS1 fusion peptide showed good antigenic results, however only one immunized rabbit elicited good antibody titers towards both the monomeric and oligomeric viral envelope glycoprotein (Env). In addition, moderate neutralizing antibodies response was elicited against two HIV-1 clade B and one clade C primary isolates. These preliminary data validate the peptide mimotope approach as a promising tool to obtain an effective HIV-1 vaccine.

Impairment of T cell development and acute inflammatory response in HIV-1 Tat transgenic mice

Immune activation and chronic inflammation are hallmark features of HIV infection causing T-cell depletion and cellular immune dysfunction in AIDS. Here, we addressed the issue whether HIV-1 Tat could affect T cell development and acute inflammatory response by generating a transgenic mouse expressing Tat in lymphoid tissue. Tat-Tg mice showed thymus atrophy and the maturation block from DN4 to DP thymic subpopulations, resulting in CD4+ and CD8+ T cells depletion in peripheral blood. In Tat-positive thymus, we observed the increased p65/NF-κB activity and deregulated expression of cytokines/chemokines and microRNA-181a-1, which are involved in T-lymphopoiesis. Upon LPS intraperitoneal injection, Tat-Tg mice developed an abnormal acute inflammatory response, which was characterized by enhanced lethality and production of inflammatory cytokines. Based on these findings, Tat-Tg mouse could represent an animal model for testing adjunctive therapies of HIV-1-associated inflammation and immune deregulation.

CRL3IBTK Regulates the Tumor Suppressor Pdcd4 through Ubiquitylation Coupled to Proteasomal Degradation

Background: IBtkα is an uncharacterized protein belonging to the family of BTB proteins. Results: IBtkα is the substrate receptor for a Cullin3-dependent ubiquitin ligase promoting ubiquitylation and proteasomal degradation of Pdcd4. Conclusion: By regulating Pdcd4 stability, IBtkα can modulate the translation of specific mRNAs under different cellular conditions. Significance: The identification of new players in the ubiquitin/proteasome pathways contributes to a better understanding of protein homeostasis. The human inhibitor of Brutons tyrosine kinase isoform α (IBtkα) is a BTB protein encoded by the IBTK gene, which maps to chromosomal locus 6q14.1, a mutational hot spot in lymphoproliferative disorders. Here, we demonstrate that IBtkα forms a CRL3IBTK complex promoting its self-ubiquitylation. We identified the tumor suppressor Pdcd4 as IBtkα interactor and ubiquitylation substrate of CRL3IBTK for proteasomal degradation. Serum-induced degradation of Pdcd4 required both IBtkα and Cul3, indicating that CRL3IBTK regulated the Pdcd4 stability in serum signaling. By promoting Pdcd4 degradation, IBtkα counteracted the suppressive effect of Pdcd4 on translation of reporter luciferase mRNAs with stem-loop structured or unstructured 5′-UTR. IBtkα depletion by RNAi caused Pdcd4 accumulation and decreased the translation of Bcl-xL mRNA, a well known target of Pdcd4 repression. By characterizing CRL3IBTK as a novel ubiquitin ligase, this study provides new insights into regulatory mechanisms of cellular pathways, such as the Pdcd4-dependent translation of mRNAs.

Evidence of shared epitopic reactivity among independent B-cell clones in chronic lymphocytic leukemia patients

Evidence of shared epitopic reactivity among independent B-cell clones in chronic lymphocytic leukemia patients

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

Abstract 1814: Harnessing the potential of idiotypic peptide therapy for the treatment of multiple myeloma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BACKGROUND: The idiotype determinant of surface Immunoglobulin B-Cell Receptor (IgBCR) is the most reliable clonal marker of tumor cell population and can be exploited for novel targeting approaches. We previously developed a novel experimental approach to identify peptide ligands of the IgBCR idiotype determinant of neoplastic B cells, named “Id-peptides” (Palmieri C., Blood 2010). These Id-peptides were validated as a tool for specific targeting of B-cell malignancies with a potential role as modulators of tumor microenvironment via sIgBCR signalling. In Multiple Myeloma (MM), both mature and clonogenic tumor stem cells (clonotype B-lymphocytes, CBLs) share the same antigenic specificity, making these cells a suitable target for Id-peptides. In this regard, we have recently developed Id-peptides for tumor targeting in primary neoplastic plasma cells as well as in the 5T33MM mouse model of MM. RESULTS: Tumor-specific Id-peptides of MM (n=8), or MGUS (n=2) patients were selected by screening phage-displayed random peptide libraries using the cognate serum IgG paraprotein as bait. The selected Id-peptides specifically bound to the cognate primary neoplastic plasma cells, as shown by FACS analysis. By confocal microscopy, we observed that each patient-specific Id-peptide co-localized with the sIgG-BCR at the cell surface of the cognate neoplastic plasma cell. Further, the Id-peptides were able to target CD138pos differentiated plasma cells as well as a number of less differentiated CD138neg, IgGpos and CD19pos bone marrow B-cells, suggesting that they tracked the clonogenic precursors of MM cells. Using the same screening approach in the 5T33MM mouse model of MM, Id-peptides were able to specifically detect a significant proportion of target cancer cells in the peripheral blood of diseased animal. Interestingly, the in vivo exposure of 5T33 murine MM cells to Id-peptides resulted in a reduced expression of immunosuppressive cytokines genes (Tgfb1 and Il10) and pro-angiogenic genes (Vegf, Tbxas, SDF, Il8, CXCr4). CONCLUSION: This study demonstrates that Id-peptides can be used to identify and purify the clonotypic precursors of MM cells, thus providing a unique tool for assessing both the phenotypic and functional features of the still poorly characterized MM “cancer stem cell” compartment. Moreover, the Id-peptides can inhibit the expression of genes that are crucial to promote a functional MM microenvironment and angiogenesis, thus representing a potential tool for MM therapy. Note: This abstract was not presented at the meeting. Citation Format: Enrico Iaccino, Selena Mimmi, Cristina Falcone, Eleonora Vecchio, Roberta Crescenzo, Giuseppina Maggisano, Cesare Carvelli, Samuela Russo, Annamaria de Laurentiis, Marilena Pontoriero, Antonio Pisano, Simona Ceglia, Francesca Fasanella Masci, Annarita Scialdone, Annalisa Rossi, Giuseppe Fiume, Camillo Palmieri, Ileana Quinto, Giuseppe Scala. Harnessing the potential of idiotypic peptide therapy for the treatment of multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1814. doi:10.1158/1538-7445.AM2014-1814