Retinoic acid/alpha-interferon combination inhibits growth and promotes apoptosis in mantle cell lymphoma through Akt-dependent modulation of critical targets

Abstract Mantle cell lymphoma (MCL) is characterized by a profound deregulation of the mechanisms controlling cell-cycle progression and survival. We herein show that the combination of 9-cis-retinoic acid (RA) and IFN-α induces marked antiproliferative and proapoptotic effects in MCL cells through the modulation of critical targets. Particularly, IFN-α enhances RA-mediated G0–G1 cell accumulation by downregulating cyclin D1 and increasing p27Kip1 and p21WAF1/Cip1 protein levels. Furthermore, RA/IFN-α combination also induces apoptosis by triggering both caspases-8 and -9 resulting in Bax and Bak activation. In particular, RA/IFN-α treatment downregulates the antiapoptotic Bcl-xL and Bfl-1 proteins and upregulates the proapoptotic BH3-only Noxa protein. Sequestration of Mcl-1 and Bfl-1 by upregulated Noxa results in the activation of Bid, and the consequent induction of apoptosis is inhibited by Noxa silencing. Noxa upregulation is associated with nuclear translocation of the FOXO3a transcription factor as consequence of RA/IFN-α–induced Akt inhibition. Pharmacologic suppression of Akt, but not of TORC1, increases Noxa protein levels and downregulates Bfl-1 protein supporting the conclusion that the inhibition of the Akt pathway, the resulting FOXO3a activation and Noxa upregulation are critical molecular mechanisms underlying RA/IFN-α—dependent MCL cell apoptosis. These results support the potential therapeutic value of RA/IFN-α combination in MCL management. Cancer Res; 72(7); 1825–35. ©2012 AACR.</jats:p

Broadening specificity and enhancing cytotoxicity of adoptive T cells for nasopharyngeal carcinoma immunotherapy

Although promising, clinical responses to adoptive immunotherapy for nasopharyngeal carcinoma (NPC) are still limited by the restricted number of Epstein-Barr virus (EBV) antigens that can be targeted and their poor immunogenicity. Our previous work indicated that the immunogenic features of the NPC-associated viral antigen BARF1 may be exploited for immunotherapeutic purposes. Nevertheless, T-cell lines obtained with current protocols include only negligible numbers of BARF1-specific cytotoxic T lymphocytes, pointing to the need to enrich these effectors in BARF1 specificities. Considering that in B lymphocytes BARF1 is mainly a lytic EBV antigen, we tested different EBV lytic-cycle inducers (TPA/butyric acid, doxorubicin, and cisplatin) used at suboptimal concentrations for their ability to upregulate BARF1 expression in lymphoblastoid B-cell lines (LCL), the commonly used antigen-presenting cells, without compromising their survival. The LCLs treated with doxorubicin (DX-LCL) can reproducibly and efficiently generate EBV-specific effectors enriched in BARF1 specificities from both healthy donors and NPC patients. These DX-LCLs also had more pronounced immunogenic properties, including HLA class I upregulation and expression of immunogenic cell death markers, such as enhanced calreticulin exposure and HMGB1 release. In particular, doxorubicin triggers an HMGB1 autocrine/paracrine loop with its receptor, TLR4, which is also upregulated in DX-LCLs and is responsible for NF-\u3baB activation and a delayed apoptosis that allows a prolonged stimulation of EBV-specific T-cell precursors. This protocol may thus constitute a valid alternative to the use of engineered LCLs to generate EBV-specific T-cell lines for adoptive immunotherapy, being relatively simple, easily upgradable to Good Manufacturing Practice standards, and therefore more broadly applicable

Broadening specificity and enhancing cytotoxicity of adoptive T Cells for nasopharyngeal carcinoma immunotherapy

Although promising, clinical responses to adoptive immunotherapy for nasopharyngeal carcinoma (NPC) are still limited by the restricted number of Epstein-Barr virus (EBV) antigens that can be targeted and their poor immunogenicity. Our previous work indicated that the immunogenic features of the NPC-associated viral antigen BARF1 may be exploited for immunotherapeutic purposes. Nevertheless, T-cell lines obtained with current protocols include only negligible numbers of BARF1-specific cytotoxic T lymphocytes, pointing to the need to enrich these effectors in BARF1 specificities. Considering that in B lymphocytes BARF1 is mainly a lytic EBV antigen, we tested different EBV lytic-cycle inducers (TPA/butyric acid, doxorubicin, and cisplatin) used at suboptimal concentrations for their ability to upregulate BARF1 expression in lymphoblastoid B-cell lines (LCL), the commonly used antigen-presenting cells, without compromising their survival. The LCLs treated with doxo-rubicin (DX-LCL) can reproducibly and efficiently generate EBV-specific effectors enriched in BARF1 specificities from both healthy donors and NPC patients. These DX-LCLs also had more pronounced immunogenic properties, including HLA class I upregulation and expression of immunogenic cell death markers, such as enhanced calreticulin exposure and HMGB1 release. In particular, doxorubicin triggers an HMGB1 autocrine/paracrine loop with its receptor, TLR4, which is also upregulated in DX-LCLs and is responsible for NF-kappa B activation and a delayed apoptosis that allows a prolonged stimulation of EBV-specific T-cell precursors. This protocol may thus constitute a valid alternative to the use of engineered LCLs to generate EBV-specific T-cell lines for adoptive immunotherapy, being relatively simple, easily upgradable to Good Manufacturing Practice standards, and therefore more broadly applicable

Lymphomagenic properties of a HIV p17 variant derived from a splenic marginal zone lymphoma occurred in a HIV-infected patient

Despite antiretroviral therapy, HIV+ individuals still have increased risk to develop lymphomas, including marginal zone lymphomas, suggesting that factors other than HIV-related immunosuppression are probably acting as lymphomagenic factors in the HIV setting. The possible pathogenic involvement of HIV p17 protein variants was investigated in a particularly informative case of HIV-related splenic marginal zone lymphoma, which was negative for oncogenic virus infections, thus allowing us to assess the possible direct contribution of these HIV-encoded proteins to lymphomagenesis. The presence of p17 protein was analyzed by immunohistochemistry in lymphoma tissue. Recombinant p17 protein derived from the dominant sequence detected in plasma and lymphoma biopsy was characterized for B-cell proliferation, clonogenicity in soft agar, in vitro tube formation and wound healing. Intracellular signaling was investigated by immunoblotting. HIV p17 protein was detected in reactive lymphoid follicles but not within lymphoma cells. An identical dominant variant p17 sequence, p17-Lyrm, carrying a 117 to 118 Ala-Ala insertion was detected in both plasma and lymphoma tissue. Recombinant p17-Lyrm enhanced B-cell proliferation and clonogenicity promoted the formation of capillary-like structures and enhanced endothelial cell migration. Unlike reference p17, the p17-Lyrm variant enhanced the activation of Akt and ERK, critical kinases in lymphomagenesis. p17-Lyrm clonogenic activity was dependent on the activation of Akt but not of ERK1/2. These results indicated that HIV p17 variants with distinct molecular signatures and functional properties may accumulate in lymphoid tissues of HIV-infected individuals where they may act as a local stimulus promoting the development of lymphomas

Detection of HIV-1 matrix protein P17 quasispecies variants in plasma of chronic HIV-1-infected patients by ultra-deep pyrosequencing

Background:The HIV-1 matrix protein p17 (p17MA) is a pleiotropic protein that plays a key role in the HIV-1 life cycle. It has been long believed to have a highly conserved primary amino acid sequence and a well-preserved structural integrity to avoid severe fitness consequences. However, recent data revealed that the carboxy (COOH)-terminus of p17MA possesses high levels of predicted intrinsic disorder, which would subtend to at least partially unfolded status of this region. This finding pointed to the need of investigating p17MA heterogeneity.Methods:The degree of intrapatient variations in the p17MA primary sequence was assessed on plasma viral RNA by using ultra-deep pyrosequencing.Results:Data obtained support a complex nature of p17MA quasispecies, with variants present at variable frequency virtually in all patients. Clusters of mutations were scattered along the entire sequence of the viral protein, but they were more frequently detected within the COOH-terminal region of p17MA. Moreover, deletions and insertions also occurred in a restricted area of the COOH-terminal region.Conclusions:On the whole, our data show that the intrapatient level of sequence diversity in the p17MA is much higher than predicted before. Our results pave the way for further studies aimed at unraveling possible correlations between the presence of distinct p17MA variants and peculiar clinical evolutions of HIV-1 disease. The presence of p17MA quasispecies diversity may offer new tools to improve our understanding of the viral adaptation during the natural history of HIV-1 infection

A natural HIV p17 protein variant up-regulates the LMP-1 EBV oncoprotein and promotes the growth of EBV-infected B-lymphocytes: implications for EBV-driven lymphomagenesis in the HIV setting

Human immunodeficiency virus p17 matrix protein is released by infected cells and may accumulate within lymphoid tissues where it may deregulate the biological activities of different cell populations by binding to CXCR1 and CXCR2 cellular receptors. S75X, a natural p17 variant, was recently shown to enhance the malignant properties of lymphoma cells. We investigated a reference p17 protein and the S75X variant for their ability to bind to Epstein-Barr virus (EBV)-infected primary and fully transformed B-lymphocytes and trigger downstream effects of potential pathogenic relevance. We demonstrate that EBV infection of primary B-lymphocytes or the ectopic expression of the latent membrane protein-1 viral oncoprotein in EBV-negative B-cells up-regulates CXCR2, but not CXCR1. Multispectral imaging flow cytometry showed that EBV-infected primary B-cells more efficiently bind and internalize p17 proteins as compared with activated B-lymphocytes. The S75X variant bound more efficiently to EBV-infected primary and fully transformed B-lymphocytes compared with reference p17, because of a higher affinity to CXCR2, and enhanced the proliferation of these cells, an effect associated with cyclin D2 and D3 up-regulation and increased interleukin-6 production. Notably, the S75X variant markedly up-regulated latent membrane protein-1 expression at both mRNA and protein levels and enhanced the activation of Akt, ERK1/2 and STAT3 signaling, thereby contributing to EBV+ B-cell growth promotion. These results indicate that EBV infection sensitizes B-lymphocytes to CXCR2-mediated effects of p17 proteins and provide evidence supporting a possible contribution of natural p17 variants to EBV-driven lymphomagenesis in the human immunodeficiency virus setting.