Abstract 2186: Quantitative cell surface proteome profiling of CD4+ T cells to identify potential therapeutic targets for adult T-cell leukemia (ATL).
Authors: Ishihara Makoto , Araya Natsumi , Sato Tomoo , Utsunomiya Atae , Yamano Yoshihisa , Nakamura Yusuke , Nakagawa Hidewaki , Ueda Koji 
Published: August 2014

Human T-cell leukemia virus type 1 (HTLV-1) is one of retroviruses known as the causative agent of adult T cell leukemia/lymphoma (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), both of which develop after a long latency period over 30 years. ATL develops in HTLV-1-infected individuals at the prevalence of 5%, who exhibit only 20% overall 5-year survival rate. Recently, ATL treatment using Mogamulizumab showed 58 % of response rate in the clinical studies. However, adverse reactions were reported for all cases during the treatment. Moreover no effective treatment has been developed for the Mogamulizumab-resistant ATL patients. The objective of the present study is to discover molecular treatment targets for antibody therapies against ATL. For effective isolation of the therapeutic targets, comprehensive cell surface proteome profiling was performed. Since most of cell surface proteins are glycosylated, a glycopeptide enrichment technology IGEL (Isotopic Glycosidase Elution and labeling on Lectin column chromatography) was employed to focus on the cell surface proteins. The IGEL was previously developed in our group, which based on 96-well modified lectin column chromatography (Mol Cell Proteomics. 2010,9(9),1819). 1 × 106 of peripheral blood mononuclear cells (PBMCs) were lysed, reduced and alkylated followed by tryptic digestion. In this study, we used ConA lectin beads to enrich glycopeptides, which specifically recognized α-mannosyl or α-glucosyl residues of N-glycans. The eluates were lyophilized and subjected to LC/MS/MS analysis. The LC/MS/MS analysis identified 594 peptides derived from 268 proteins including 510 glycosylated peptides. Gene Ontology analysis revealed that 73.6% of identified proteins were assigned as the membrane associated proteins. For the therapeutic target screening experiment, CD4+ T cells were isolated from PBMCs derived from 16 uninfected volunteers, 25 asymptomatic carriers, 26 HAM/TSP patients, and 15 ATL patients. To perform relative quantification and subsequent statistical analysis of over 100,000 peptides among 82 samples, we employed the Expressionist proteome server system (Genedata AG). Although recent high-end mass spectrometers allow in-depth and comprehensive analysis of any proteomes, improvement of data processing infrastructures, such as Expressionist, should be essential for future clinical proteomics studies analyzing hundreds of specimens. Here we introduce a systematic workflow to identify ATL-specific cell surface antigens, accompanying further FACS-based replication experiments. In particular, our strategy could be useful for exploring any proteomic changes on cell surface, which include malignant transformation, metastasis, or cell development.

Citation Format: Makoto Ishihara, Natsumi Araya, Tomoo Sato, Atae Utsunomiya, Yoshihisa Yamano, Yusuke Nakamura, Hidewaki Nakagawa, Koji Ueda. Quantitative cell surface proteome profiling of CD4+ T cells to identify potential therapeutic targets for adult T-cell leukemia (ATL). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2186. doi:10.1158/1538-7445.AM2013-2186