Identification of Novel HLA-A*24:02-Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies

PLOS ONE, Dec 2019

The homeobox protein, PEPP2 (RHOXF2), has been suggested as a cancer/testis (CT) antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs) is unknown. In this study, we revealed that PEPP2 gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2271-279). The CTLs induced by PEPP2271-279 recognized PEPP2-positive target cells in an HLA-A*24:02-restricted manner. We also found that a demethylating agent, 5-aza-2’-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2’-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients.

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Identification of Novel HLA-A*24:02-Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies

January Identification of Novel HLA-A 24:02- Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies Maiko Matsushita 0 1 Yohei Otsuka 0 1 Naoya Tsutsumida 0 1 Chiaki Tanaka 0 1 Akane Uchiumi 0 1 Koji Ozawa 0 1 Takuma Suzuki 0 1 Daiju Ichikawa 0 1 Hiroyuki Aburatani 0 1 Shinichiro Okamoto 0 1 Yutaka Kawakami 0 1 Yutaka Hattori 0 1 0 1 Division of Clinical Physiology and Therapeutics, Keio University, Faculty of Pharmacy, Tokyo, Japan, 2 Division of Pharmacy, National Cancer Center Hospital , Tokyo , Japan , 3 Genome Science Laboratory Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan, 4 Division of Hematology, Keio University, School of Medicine, Tokyo, Japan, 5 Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University, School of Medicine , Tokyo , Japan 1 Editor: Natalia Lapteva, Baylor College of Medicine, UNITED STATES The homeobox protein, PEPP2 (RHOXF2), has been suggested as a cancer/testis (CT) antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs) is unknown. In this study, we revealed that PEPP2 gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2271-279). The CTLs induced by PEPP2271-279 recognized PEPP2-positive target cells in an HLA-A*24:02-restricted manner. We also found that a demethylating agent, 5-aza-2'-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2'-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by a Grant-in-Aid for Scientific Research and a grant from the Private University Strategic Research Base Development Program of MEXT (the Ministry of Education, Culture, Sports, Science and Technology) of Japan, Friends of Leukemia Research Fund, and Keio Gijuku Academic Development Funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction The development of chemotherapy regimens and targeted therapies has improved the survival of patients with leukemia. However, this disease continues to recur following conventional therapies in some patients, leading to poor prognosis [ 1,2 ]. Therefore, eradication of residual disease by additional treatment is necessary for these therapy-resistant patients. By this rationale, immunotherapy using antigen specifically expressed by leukemic cells might be an attractive strategy to cure leukemia patients [ 3,4,5 ]. Competing Interests: The authors have declared that no competing interests exist. Several leukemia-associated antigens (LAAs) have been reported, which include Wilms’ tumor 1 (WT1), proteinase-3, bcr-abl or PML-RARα[ 3,6 ]. Some of these are now in clinical trials [ 7,8,9 ]. However, their clinical benefits remain to be proven in some cases, although these LAAs can elicit antigen-specific immune responses in patients. One of the reasons for this discrepancy might be that recognition of only one antigen is not sufficient to eradicate leukemic cells in patients, because cancer cells could escape the immune response in various ways including down-regulating antigens. Recent studies have shown that using multiple epitopes in a vaccine setting is more effective than using a single peptide, therefore, identification of novel LAAs is necessary to enhance anti-tumor effects [ 10,11 ]. Another key to improving the efficacy of LAA-targeted immunotherapy is to choose LAAs expressed by leukemic stem cells (LSCs). Evidence suggests that LSCs are resistant to chemotherapy or targeted therapy such as tyrosine kinase inhibitors by several mechanisms, including maintenance of a resting state or the expression of a high number of drug-efflux pumps [ 12,13,14 ]. Immunotherapy targeting antigens expressed by LSCs would not be affected by these characteristics of LSCs and, therefore, could eliminate these cells. PEPP2, which is also called RHOXF2, was first identified by Wayne et al [ 15 ] as a homologue of the mouse Pem1 gene, which belongs to the homeobox genes regulating sperm differentiation. This gene has been suggested to be a cancer/testis antigen (CTA) based on its expression pattern [ 16,17 ]. Cancer/testis antigens are known to be good therapeutic targets, since their expression in normal ti (...truncated)


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Maiko Matsushita, Yohei Otsuka, Naoya Tsutsumida, Chiaki Tanaka, Akane Uchiumi, Koji Ozawa, Takuma Suzuki, Daiju Ichikawa, Hiroyuki Aburatani, Shinichiro Okamoto, Yutaka Kawakami, Yutaka Hattori. Identification of Novel HLA-A*24:02-Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies, PLOS ONE, 2016, Volume 11, Issue 1, DOI: 10.1371/journal.pone.0146371