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Human Leukocyte Antigen (HLA) Peptides Derived from Tumor Antigens Induced by Inhibition of DNA Methylation for Development of Drug-facilitated Immunotherapy

2016; Elsevier BV; Volume: 15; Issue: 9 Linguagem: Inglês

10.1074/mcp.m116.060350

ISSN

1535-9484

Autores

Bracha Shraibman, Dganit Melamed Kadosh, Eilon Barnea, Arie Admon,

Tópico(s)

Peptidase Inhibition and Analysis

Resumo

Treatment of cancer cells with anticancer drugs often fails to achieve complete remission. Yet, such drug treatments may induce alteration in the tumor's gene expression patterns, including those of Cancer/Testis Antigens (CTA). The degradation products of such antigens can be presented as HLA peptides on the surface of the tumor cells and be developed into anticancer immunotherapeutics. For example, the DNA methyl transferase inhibitor, 5-aza-2′-deoxycytidine (Decitabine) has limited antitumor efficacy, yet it induces the expression of many genes, including CTAs that are normally silenced in the healthy adult tissues. In this study, the presentation of many new HLA peptides derived from CTAs and induced by Decitabine was demonstrated in three human Glioblastoma cell lines. Such presentation of CTA-derived HLA peptides can be exploited for development of new treatment modalities, combining drug treatment with anti-CTA targeted immunotherapy. The Decitabine-induced HLA peptidomes include many CTAs that are not normally detected in healthy tissues or in cancer cells, unless treated with the drug. In addition, the study included large-scale analyses of the simultaneous effects of Decitabine on the transcriptomes, proteomes and HLA peptidomes of the human Glioblastoma cells. It demonstrates the poor correlations between these three levels of gene expression, both in their total levels and in their response to the drug. The proteomics and HLA peptidomics data are available via ProteomeXchange with identifier PXD003790 and the transcriptomics data are available via GEO with identifier GSE80137. Treatment of cancer cells with anticancer drugs often fails to achieve complete remission. Yet, such drug treatments may induce alteration in the tumor's gene expression patterns, including those of Cancer/Testis Antigens (CTA). The degradation products of such antigens can be presented as HLA peptides on the surface of the tumor cells and be developed into anticancer immunotherapeutics. For example, the DNA methyl transferase inhibitor, 5-aza-2′-deoxycytidine (Decitabine) has limited antitumor efficacy, yet it induces the expression of many genes, including CTAs that are normally silenced in the healthy adult tissues. In this study, the presentation of many new HLA peptides derived from CTAs and induced by Decitabine was demonstrated in three human Glioblastoma cell lines. Such presentation of CTA-derived HLA peptides can be exploited for development of new treatment modalities, combining drug treatment with anti-CTA targeted immunotherapy. The Decitabine-induced HLA peptidomes include many CTAs that are not normally detected in healthy tissues or in cancer cells, unless treated with the drug. In addition, the study included large-scale analyses of the simultaneous effects of Decitabine on the transcriptomes, proteomes and HLA peptidomes of the human Glioblastoma cells. It demonstrates the poor correlations between these three levels of gene expression, both in their total levels and in their response to the drug. The proteomics and HLA peptidomics data are available via ProteomeXchange with identifier PXD003790 and the transcriptomics data are available via GEO with identifier GSE80137. Grade IV astrocytoma, also called glioblastoma multiforme (GBM) 1The abbreviations used are:GBMGlioblastoma multiformeHLAhuman leukocyte antigenMHCmajor histocompatibility complexCTACancer/Testis AntigensTAtumor antigensTSAtumor-specific antigensDecitabine5-aza-2′-deoxycytidineTMZTemozolamide., is the most common and aggressive primary brain tumor, with a prognosis of about three months if untreated. Despite advances in treatment, and even after applying a combination of maximal surgical resection, followed by radiation and Temozolamide (TMZ) chemotherapy, the survival rate for GBM has not changed much over the past 50 years, remaining largely an incurable disease with a median survival of about 15 months (1.Alifieris C. Trafalis D.T. Glioblastoma multiforme: Pathogenesis and treatment.Pharmacol. Ther. 2015; 152: 63-82Crossref PubMed Scopus (456) Google Scholar). Hence, there is a pressing need to develop better therapeutic modalities, such as immunotherapies, which are emerging as promising treatment options for otherwise incurable cancers, such as GBM (2.Mohme M. Neidert M.C. Regli L. Weller M. Martin R. Immunological challenges for peptide-based immunotherapy in glioblastoma.Cancer Treat. Rev. 2014; 40: 248-258Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 3.Weiss T. Weller M. Roth P. Immunotherapy for glioblastoma: concepts and challenges.Curr. Opin. 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Currently, 87 clinical trials of epigenetic cancer therapy are registered at Clinical Trials (http://www.clinicaltrials.gov) (30.Valdespino V. Valdespino P.M. (Potential of epigenetic therapies in the management of solid tumors.Cancer Manag. Res. 2015; 7: 241-251Crossref PubMed Scopus (44) Google Scholar). Identification of HLA peptides as tumor antigens and candidates for immunotherapy is complicated by the difficulty of detection of their presentation on tumor cells. HLA peptidome analysis is limited by the yield of immunoaffinity purified HLA molecules and by the technical limitations of the chromatography and mass spectrometry analysis of the recovered peptides (7.Comber J.D. Philip R. MHC class I antigen presentation and implications for developing a new generation of therapeutic vaccines.Therap. Adv. Vaccines. 2014; 2: 77-89Crossref PubMed Scopus (81) Google Scholar, 8.de Verteuil D. Granados D.P. Thibault P. Perreault C. Origin and plasticity of MHC I-associated self peptides.Autoimmunity Rev. 2012; 11: 627-635Crossref PubMed Scopus (40) Google Scholar, 9.Granados D.P. Laumont C.M. Thibault P. Perreault C. The nature of self for T cells-a systems-level perspective.Curr. Opin. Immunol. 2014; 34C: 1-8Google Scholar, 41.Caron E. Kowalewski D.J. Chiek Koh C. Sturm T. Schuster H. Aebersold R. Analysis of major histocompatibility complex (MHC) immunopeptidomes using mass spectrometry.Mol. Cell. Proteomics. 2015; 14: 3105-3117Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). To this end, analysis of the tumor cells' exomes and transcriptomes may provide useful data from which the presentation of HLA peptides can be inferred (9.Granados D.P. Laumont C.M. Thibault P. Perreault C. The nature of self for T cells-a systems-level perspective.Curr. Opin. Immunol. 2014; 34C: 1-8Google Scholar, 13.Schumacher T.N. Schreiber R.D. 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The obtained data are potentially useful for advancing the development of new approaches for immunotherapy, based on the induction by drugs of tumor antigens that are not normally expressed by the cancer cells and can be used for combined drug and immunotherapy cancer treatment. Three human Glioblastoma cell lines, U-87, T98G, and LNT-229 (obtained from the ATCC, Manassas, VA) were maintained in DMEM supplemented with 10% FBS, 1% l-glutamine, 1% Na-pyruvate, 0.1 mg/ml streptomycin, and 100 μ/ml penicillin and 0.1% HEPES. The cells were kept in 5% CO2 humidified incubator at 37 °C. The monoclonal antibodies W6/32 (anti-HLA class I) were affinity purified from growth medium of the HB95 hybridoma (obtained from the ATCC). Cells were counted and plated in new 150 mm Petri dishes 1 day prior to the treatment. The following day, the media was removed and replaced with fresh medium with or without 1 μm of 5′-Aza-2′-deoxycytidine (Decitabine, AdooQ Bioscience, Irvine, CA). After incubation with Decitabine for 72 h, the cells were harvested, counted and prepared for FACS, HLA peptidomics, proteomics analysis, or for RNA sequencing. Three Glioblastoma cell lines (U-87, T98G, and LNT-229), treated and untreated with Decitabine, were used for the HLA peptidomics analysis, proteomics analysis, and for the RNA sequencing and quantification. Three HLA peptidomics and proteomics analyses biological replicates were performed with each of the treated and untreated Glioblastoma cell lines. In addition, two out of these three replicates of each of the cells lines were also used for RNA-seq analyses. About 2 × 105 Decitabine-treated and untreated cells were used for each flow cytometric analysis. The cells were tagged with the W6/32 monoclonal antibody (anti-HLA-A, B and C) produced from the HB95 hybridoma and used at a final concentration of 0.5 μg/μl. Secondary antibodies were anti-mouse IgG conjugated to FITC (Sigma, St. Louis, MO). FACS analysis was performed using LSRII instrument (BD Biosciences, San Jose, CA) and the data were analyzed using FCS Express 5 Plus (DeNovo Software, Glendale, CA). HLA class I molecules were purified from three biological replicates for each Glioblastoma cell line. Each replica with about 5 × 108 cells was lysed with 0.25% sodium deoxycholate, 0.2 mm iodoacetamide, 1 mm EDTA, 1:200 Protease Inhibitors Mixture (Sigma), 1 mm PMSF, and 1% octyl-β-D glucopyranoside (Sigma) in PBS at 4 °C for 1 h. The cell extracts were cleared by centrifugation for 45 min at 18,000 rpm, 4 °C. The recovered HLA class I molecules were immunoaffinity purified using the W6/32 mAb bound to Amino-Link beads (Thermo Scientific, Waltham, MA) as in (49.Milner E. Gutter-Kapon L. Bassani-Strenberg M. Barnea E. Beer I. Admon A. The effect of proteasome inhibition on the generation of the human leukocyte antigen (HLA) peptidome.Mol. Cell. Proteomics. 2013; 12: 1853-1864Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The HLA molecules with their bound peptides were eluted from the affinity column with five column volumes of 0.1 N acetic acid. The eluted HLA class I proteins and the released peptides were loaded on disposable C18 columns (Harvard Apparatus, Holliston, MA) and the peptides fraction was recovered with 30% acetonitrile in 0.1% TFA. The peptide fractions were dried using vacuum centrifugation, reconstituted in 100 μl of 0.1% TFA, reloaded on Stage-Tips (50.Ishihama Y. Rappsilber J. Mann M. Modular stop and go extraction tips with stacked disks for parallel and multidimensional Peptide fractionation in proteomics.J. Proteome Res. 2006; 5: 988-994Crossref PubMed Scopus (224) Google Scholar), eluted with 80% ACN, dried, and reconstituted with 0.1% formic acid for μLC-MS-MS analysis. Two approaches for proteome analysis were employed in this study. The first was in-solution tryptic digest followed by resolution of the peptides by long (3 h) one-dimensional reversed phase capillary chromatography and tandem mass spectrometry and the other was based on in-gel proteolysis of 5 gel slices from each lane, followed by two hours μLC-MS-MS of the tryptic peptides from each gel slice, as in (51.Bourdetsky D. Schmelzer C.E. Admon A. The nature and extent of contributions by defective ribosome products to the HLA peptidome.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: E1591-E1599Crossref PubMed Scopus (74) Google Scholar). Thirty μg of total protein were used for the in-gel digest and μLC-MS-MS and 2.5 μg of proteins were used for the in-solution digest and μLC-MS-MS analysis. The HLA and tryptic peptides were resolved by capillary chromatography and electrospray tandem mass spectrometry with Q-Exactive-plus mass spectrometers (Thermo Scientific). The reversed phase capillary chromatographies were performed with home-packed a 30 cm long, 75 μm inner diameter column with 3.5 μm silica ReproSil-Pur C18-AQ resin (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). HLA and tryptic peptides were eluted with a linear gradient of 5–28% of buf

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