Jason M. God

A possible cross-talk between autophagy and apoptosis in generating an immune response in melanoma

Melanoma is the most aggressive form of skin cancer, responsible for the majority of skin cancer related deaths. Thus, the search for natural molecules which can effectively destroy tumors while promoting immune activation is essential for designing novel therapies against metastatic melanoma. Here, we report for the first time that a natural triterpenoid, Ganoderic acid DM (GA-DM), induces an orchestrated autophagic and apoptotic cell death, as well as enhanced immunological responses via increased HLA class II presentation in melanoma cells. Annexin V staining and flow cytometry showed that GA-DM treatment induced apoptosis of melanoma cells, which was supported by a detection of increased Bax proteins, co-localization and elevation of Apaf-1 and cytochrome c, and a subsequent cleavage of caspases 9 and 3. Furthermore, GA-DM treatment initiated a possible cross-talk between autophagy and apoptosis as evidenced by increased levels of Beclin-1 and LC3 proteins, and their timely interplay with apoptotic and/or anti-apoptotic molecules in melanoma cells. Despite GA-DM’s moderate cytotoxicity, viable cells expressed high levels of HLA class II proteins with improved antigen presentation and CD4+ T cell recognition. The antitumor efficacy of GA-DM was also investigated in vivo in murine B16 melanoma model, where GA-DM treatment slowed tumor formation with a significant reduction in tumor volume. Taken together, these findings demonstrate the potential of GA-DM as a natural chemo-immunotherapeutic capable of inducing a possible cross-talk between autophagy and apoptosis, as well as improved immune recognition for sustained melanoma tumor clearance.

Burkitt lymphoma: pathogenesis and immune evasion.

B-cell lymphomas arise at distinct stages of cellular development and maturation, potentially influencing antigen (Ag) presentation and T-cell recognition. Burkitt lymphoma (BL) is a highly malignant B-cell tumor associated with Epstein-Barr Virus (EBV) infection. Although BL can be effectively treated in adults and children, leading to high survival rates, its ability to mask itself from the immune system makes BL an intriguing disease to study. In this paper, we will provide an overview of BL and its association with EBV and the c-myc oncogene. The contributions of EBV and c-myc to B-cell transformation, proliferation, or attenuation of cellular network and immune recognition or evasion will be summarized. We will also discuss the various pathways by which BL escapes immune detection by inhibiting both HLA class I- and II-mediated Ag presentation to T cells. Finally, we will provide an overview of recent developments suggesting the existence of BL-associated inhibitory molecules that may block HLA class II-mediated Ag presentation to CD4+ T cells, facilitating immune escape of BL.

Elevation of c-MYC Disrupts HLA Class II–Mediated Immune Recognition of Human B Cell Tumors

Elevated levels of the transcription factor c-myc are strongly associated with various cancers, and in particular B cell lymphomas. Although many of c-MYC’s functions have been elucidated, its effect on the presentation of Ag through the HLA class II pathway has not been reported previously. This is an issue of considerable importance, given the low immunogenicity of many c-MYC–positive tumors. We report in this paper that increased c-MYC expression has a negative effect on the ability of B cell lymphomas to functionally present Ags/peptides to CD4+ T cells. This defect was associated with alterations in the expression of distinct cofactors as well as interactions of antigenic peptides with class II molecules required for the presentation of class II–peptide complexes and T cell engagement. Using early passage Burkitt’s lymphoma (BL) tumors and transformed cells, we show that compared with B lymphoblasts, BL cells express decreased levels of the class II editor HLA-DM, lysosomal thiol-reductase GILT, and a 47-kDa enolase-like protein. Functional Ag presentation was partially restored in BL cells treated with a c-MYC inhibitor, demonstrating the impact of this oncogene on Ag recognition. This restoration of HLA class II–mediated Ag presentation in early passage BL tumors/cells was linked to enhanced HLA-DM expression and a concurrent decrease in HLA-DO in BL cells. Taken together, these results reveal c-MYC exerts suppressive effects at several critical checkpoints in Ag presentation, which contribute to the immunoevasive properties of BL tumors.

Reduction of myeloid-derived suppressor cells and lymphoma growth by a natural triterpenoid.

Lymphoma is a potentially life threatening disease. The goal of this study was to investigate the therapeutic potential of a natural triterpenoid, Ganoderic acid A (GA‐A) in controlling lymphoma growth both in vitro and in vivo. Here, we show that GA‐A treatment induces caspase‐dependent apoptotic cell death characterized by a dose‐dependent increase in active caspases 9 and 3, up‐regulation of pro‐apoptotic BIM and BAX proteins, and a subsequent loss of mitochondrial membrane potential with release of cytochrome c. In addition to GA‐As anti‐growth activity, we show that lower doses of GA‐A enhance HLA class II‐mediated antigen (Ag) presentation and CD4+ T cell recognition of lymphoma cells in vitro. The therapeutic relevance of GA‐A treatment was also tested in vivo using the EL4 syngeneic mouse model of metastatic lymphoma. GA‐A‐treatment significantly prolonged survival of EL4 challenged mice and decreased tumor metastasis to the liver, an outcome accompanied by a marked down‐regulation of STAT3 phosphorylation, reduction myeloid‐derived suppressor cells (MDSCs), and enhancement of cytotoxic CD8+ T cells in the host. Thus, GA‐A not only selectively induces apoptosis in lymphoma cells, but also enhances cell‐mediated immune responses by attenuating MDSCs, and elevating Ag presentation and T cell recognition. The demonstrated therapeutic benefit indicates that GA‐A is a candidate for future drug design for the treatment of lymphoma. J. Cell. Biochem. 116: 102–114, 2015.

Mechanisms regulating enhanced human leukocyte antigen class II-mediated CD4 + T cell recognition of human B-cell lymphoma by resveratrol

Abstract Malignant B-cells express measurable levels of human leukocyte antigen (HLA) class II proteins, but often escape immune recognition by CD4 + T cells. Resveratrol (Resv) has been the focus of numerous investigations due to its potential chemopreventive and anti-cancer effects, but it has never been tested in the regulation of immune components in B-cell tumors. Here, we show for the first time that Resv treatment enhances HLA class II-mediated immune detection of B-cell lymphomas by altering immune components and class II presentation in tumor cells. Resv treatment induced an up-regulation of both classical and non-classical HLA class II proteins (DR and DM) in B-lymphoma cells. Resv also altered endolysosomal cathepsins (Cat S, B and D) and a thiol reductase (GILT), increasing HLA class II-mediated antigen (Ag) processing in B-cell lymphomas and their subsequent recognition by CD4 + T cells. Mechanistic study demonstrated that Resv treatment activated the recycling class II pathway of Ag presentation through up-regulation of Rab 4B protein expression in B-lymphoma cells. These findings suggest that HLA class II-mediated immune recognition of malignant B-cells can be improved by Resv treatment, thus encouraging its potential use in chemoimmunotherapy of B-cell lymphoma.

Disruption of HLA class II antigen presentation in Burkitt lymphoma: implication of a 47 000 MW acid labile protein in CD4+ T-cell recognition

While Burkitt lymphoma (BL) has a well‐known defect in HLA class I‐mediated antigen presentation, the exact role of BL‐associated HLA class II in generating a poor CD4+ T‐cell response remains unresolved. Here, we found that BL cells are deficient in their ability to optimally stimulate CD4+ T cells via the HLA class II pathway. This defect in CD4+ T‐cell recognition was not associated with low levels of co‐stimulatory molecules on BL cells, as addition of external co‐stimulation failed to elicit CD4+ T‐cell activation by BL. Further, the defect was not caused by faulty antigen/class II interaction, because antigenic peptides bound with measurable affinity to BL‐associated class II molecules. Interestingly, functional class II–peptide complexes were formed at acidic pH 5·5, which restored immune recognition. Acidic buffer (pH 5·5) eluate from BL cells contained molecules that impaired class II‐mediated antigen presentation and CD4+ T‐cell recognition. Biochemical analysis showed that these molecules were greater than 30 000 molecular weight in size, and proteinaceous in nature. In addition, BL was found to have decreased expression of a 47 000 molecular weight enolase‐like molecule that enhances class II‐mediated antigen presentation in B cells, macrophages and dendritic cells, but not in BL cells. These findings demonstrate that BL likely has multiple defects in HLA class II‐mediated antigen presentation and immune recognition, which may be exploited for future immunotherapies.

HLA class II defects in Burkitt lymphoma: bryostatin-1-induced 17 kDa protein restores CD4+ T-cell recognition.

While the defects in HLA class I-mediated Ag presentation by Burkitt lymphoma (BL) have been well documented, CD4+ T-cells are also poorly stimulated by HLA class II Ag presentation, and the reasons underlying this defect(s) have not yet been fully resolved. Here, we show that BL cells are deficient in their ability to optimally stimulate CD4+ T cells via the HLA class II pathway. The observed defect was not associated with low levels of BL-expressed costimulatory molecules, as addition of external co-stimulation failed to result in BL-mediated CD4+ T-cell activation. We further demonstrate that BL cells express the components of the class II pathway, and the defect was not caused by faulty Ag/class II interaction, because antigenic peptides bound with measurable affinity to BL-associated class II molecules. Treatment of BL with broystatin-1, a potent modulator of protein kinase C, led to significant improvement of functional class II Ag presentation in BL. The restoration of immune recognition appeared to be linked with an increased expression of a 17 kDa peptidylprolyl-like protein. These results demonstrate the presence of a specific defect in HLA class II-mediated Ag presentation in BL and reveal that treatment with bryostatin-1 could lead to enhanced immunogenicity.

A Missing Link between Neuron Specific Enolase Release and Poor Prognosis in Aging Patients with B-cell Lymphoma

Rachel Polcyn1, Jason God1, Mollie Capone1, Denise Matzelle2,3, Naren L Banik1,2,3 and Azizul Haque1* 1Department of Microbiology and Immunology, Hollings Cancer Center, Medical University of South Carolina, Charleston, USA 2Department of Neurosurgery, Medical University of South Carolina, Charleston, USA 3Ralph H. Johnson Veterans Administration Medical Center, Charleston, SC, USA *Corresponding author: Azizul Haque, Department of Microbiology and Immunology, Hollings Cancer Center, Medical University of South Carolina, USA, Tel: 843-792-9466; Fax: 843-792-2464; E-mail: haque@musc.edu

Multiple Defects Impair the HLA Class II Antigen Presentation Capacity of Burkitt Lymphoma

Burkitt lymphoma (BL) is a B-cell malignancy which occurs with varying clinical manifestations and frequencies. The most well-known presentation of BL is found in areas which are holoendemic or hyperendemic for malaria. This endemic BL occurs primarily in children and presents as tumors of the jaw [1,2]. Though a strong association exists between endemic BL and malaria, the nature of the relationship remains unclear. A sporadic form of BL also occurs elsewhere in the world and typically causes tumors in the gut and upper respiratory tract [3]. There are various other contributing factors which may lead to development of BL, including Epstein-Barr Virus (EBV) and Human Immunodeficiency Virus (HIV) [4,5]. EBV has long been associated with development of BL and is found in nearly all cases of endemic BL but, in spite of intense research, the relationship between EBV and BL remains unclear. It is generally believed that infection with EBV somehow drives transformation which then leads to development of BL [6]. While the exact cause of BL has remained elusive (and is likely multi-factorial) the hallmark of all BL is the constitutive activation of the MYC gene resulting in its overexpression and cell transformation [7,8]. Most commonly, this occurs through translocation of the MYC gene to an immunoglobulin locus, which in B cells would result in constitutive expression [8]. Though translocation of MYC was long considered a diagnostic criterion for BL, recent evidence suggests other mechanisms may be responsible for MYC deregulation in a small percentage of BL cases [9].

Abstract 5641: Dual roles for ganoderic acid A in inducing apoptosis and enhancing HLA class II presentation in leukemia and lymphoma cells

Ganoderic acid A (GA-A), a natural extract isolated from the medicinal mushroom Ganoderma lucidum, was found to induce a remarkable cytotoxicity in various leukemia and lymphoma cell lines and primary tumors, with a minor toxicity to nonmalignant B-cells. The GA-A cytotoxicity and growth inhibitory effects resulted from an induction of apoptosis, which was characterized by a dose-dependent increase in expression and activity of activated caspases 3, 8, and 9. Mechanistic studies showed that GA-A treatment upregulated pro-apoptotic protein Bax proteins while downregulating anti-apototic proteins Bcl-2 and survivin. Data also demonstrated that cytochrome c and apoptotic protease activating factor-1 (Apaf-1) were significantly elevated in the cytoplasmic fractions of GA-A-treated cells, which may have activated caspases for cell death. Despite GA-A9s notable cytotoxic effects on leukemia and lymphoma cells, GA-A treatment enhanced HLA class II antigen (Ag) presentation and CD4+ T cell recognition of both malignant and nonmalignant B-cells. These data suggest that GA-A administration may play dual roles in inducing apoptosis and enhancing HLA class II Ag presentation, and could be used as novel adjunctive therapy for the treatment of hematological malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5641. doi:1538-7445.AM2012-5641