NME-2 蛋白在 HLA-A2+ 细胞中作为肿瘤相关抗原发挥作用，并通过 Bcr/Abl 依赖性转录后机制在 CML 中过度表达。,Blood

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NME-2 蛋白在 HLA-A2+ 细胞中作为肿瘤相关抗原发挥作用，并通过 Bcr/Abl 依赖性转录后机制在 CML 中过度表达。
Blood ( IF 21.0 ) Pub Date : 2009-11-20 , DOI: 10.1182/blood.v114.22.3266.3266
Sabine Tschiedel ₁ , Melanie Adler ₁ , Karoline Schubert ₁ , Annette Jilo ₁ , Enrica Mueller ₁ , Song-Y Wang ₁ , Michael Cross ₁ , Thoralf Lange ₁ , Dietger Niederwieser ₁

Affiliation

摘要 3266 海报板 III-1 介绍：NmE2（Nm23-H2，NDP 激酶 B）是催化γ-磷酸在三磷酸核苷和二磷酸核苷之间转移的蛋白质家族之一。两个主要家族成员 NmE1 和 NmE2 通过相互作用控制分化、增殖、迁移和凋亡，这些相互作用通常与其激酶活性无关，NmE2 是 c-myc 基因的转录激活剂。我们最近在一名 CML 患者中将 NmE2 鉴定为与肿瘤相关的 HLA-A32+ 限制性抗原，并发现该蛋白质（但不是 mRNA）在 CML 中普遍过度表达，但在其他血液系统恶性肿瘤中没有。我们还在移植后 5 年的患者的外周血细胞中检测到特定的 T 细胞反应。这表明 NmE2 是 CML 分子治疗和免疫治疗的潜在靶点。然而，免疫治疗方法的发展将取决于 NmE2 在常见 HLA 背景中作为肿瘤抗原发挥作用的能力。本研究的目的首先是研究 NmE2 在 HLA-A2 背景（占白种人人口的 50% 以上）中的抗原性，其次是使用细胞系表征 Bcr/Abl 和 NmE2 之间的调节关系CML 模型。材料和方法：将 5 种具有预测的 HLA-A2 锚定氨基酸的 NmE2 肽以 10μM 的浓度分别加载到表达 HLA-A2 的抗原呈递细胞上。在 HCT 后的不同时间点，使用从 CML 患者中直接分离的 CD8+ MLLC（用于鉴定抗原肽）或 CD8+ 细胞进行 Elispot 检测。用伊马替尼和尼罗替尼 (0 – 10 μM) 处理稳定表达野生型和突变型 Bcr/Abl 的 Ba/F3 细胞 48 小时。Bcr/Abl 活性通过 FACS 使用对 CrkL 和 Stat5 的磷酸化形式具有特异性的抗体进行评估。NmE2 和 c-Myc 蛋白通过免疫细胞化学和蛋白质印迹法检测到特异性抗体 [Santa Cruz，分别为 L-16 和 9E10 克隆]。nme2 和 c-myc mRNA 的水平通过实时定量 PCR 确定。结果：全长 NmE2 蛋白和 5 个测试的 HLA-A2 锚定肽中的 2 个（NmE2132-140 和 NmE2112-120）被 HLA-A2+ CD8+ MLLC 特异性识别，证明了 NmE2 在体外 HLA-A2 背景中的抗原性。此外，虽然来自移植的 HLA-A2+ CML 患者的 CD8+ T 细胞在 HCT 后的前 10 个月表现出很少或没有特异性反应性，但在后期阶段明显的反应性（高达 0.6% NmE2 反应性 CD8+ T 细胞）变得明显，一致随着体内针对 NmE2 表达细胞的免疫反应的发展。在此期间，患者的 bcr/abl 转录本保持阴性。与亲本 BA/F3 细胞系相比，BA/F3 Bcr/Abl 细胞表达的 NmE2 蛋白（但不是 mRNA）水平增加。有趣的是，用伊马替尼或尼罗替尼治疗降低了 BA/F3 Bcr/Abl 中 NmE2 蛋白的表达，但在表达对相应抑制剂具有抗性的 Bcr/Abl 突变体的细胞中没有。用 PI3K 抑制剂 Ly294002 处理 BA/F3 Bcr/Abl 细胞导致 Bcr/Abl 活性降低，c-Myc 和 NmE2 蛋白水平相应降低，但不影响 mRNA 水平。结论：NmE2 的过度表达与 Bcr/Abl 激酶活性密切相关，主要的调节水平是转录后的并依赖于 PI-3K 活性。NmE2 蛋白受 HLA-A2 和 HLA-A32 限制。干细胞移植后 CML 患者中 NmE2 特异性 T 细胞反应的发展表明，NmE2 在体内作为 HLA-A2+ 患者的肿瘤抗原，可能与 CML 的长期免疫控制有关。因此，NmE2 是开发用于治疗 CML 的新免疫治疗策略的有希望的候选者。披露：兰格：BMS：Honoraria；诺华：酬金，实体董事会或咨询委员会的成员，研究资金。Niederwieser：BMS：Honoraria，实体董事会或咨询委员会的成员；诺华：酬金，研究经费。

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NME-2 Protein Functions as a Tumour Associated Antigen in HLA-A2+ Cells and Is Over Expressed in CML Via a Bcr/Abl Dependent Post Transcriptional Mechanism.

Abstract 3266 Poster Board III-1 Introduction: NmE2 (Nm23-H2, NDP kinase B) is one of a family of proteins that catalyze the transfer of gamma-phosphate between nucleoside-triphosphates and diphosphates. The two major family members, NmE1 and NmE2 are strongly implicated in the control of differentiation, proliferation, migration and apoptosis via interactions which are often independent of their kinase activity, NmE2 being a transcriptional activator of the c-myc gene. We recently identified NmE2 as a tumour associated, HLA-A32+ restricted, antigen in a patient with CML and found the protein (but not the mRNA) to be generally over expressed in CML but not in other haematological malignancies. We also detected a specific T-cell response in peripheral blood cells of a patient 5 years after transplantation. This identifies NmE2 as a potential target for both molecular and immunotherapy of CML. However, the development of immunotherapeutic approaches will depend on the ability of NmE2 to function as a tumour antigen in common HLA backgrounds. The aims of this study were firstly to investigate the antigenicity of NmE2 in the HLA-A2 background (which accounts for more than 50% of the Caucasian population), and secondly to characterise the regulatory relationship between Bcr/Abl and NmE2 using a cell line model of CML. Materials and Methods: 5 nonameric NmE2 peptides with predicted anchor amino acids for HLA-A2 were loaded at concentrations of 10μM separately onto HLA-A2 expressing antigen presenting cells. Elispot Assays were carried out with CD8+ MLLCs (for the identification of antigenic peptides) or CD8+ cells isolated directly from a CML patient at different time points after HCT. Ba/F3 cells stably expressing wild type and mutant forms of Bcr/Abl were treated with imatinib and nilotinib (0 – 10 μM) for 48h. Bcr/Abl activity was assessed by FACS using antibodies specific for the phosphorylated forms of CrkL and Stat5. NmE2 and c-Myc protein were detected by immunocytochemistry and Western blotting with specific antibodies [Santa Cruz, clones L-16 and 9E10 respectively]. Levels of nme2 and c-myc mRNA were determined by quantitative real time PCR. Results: Full length NmE2 protein and 2 of 5 HLA-A2 anchor-containing peptides tested (NmE2132–140 and NmE2112–120) were specifically recognized by the HLA-A2+ CD8+ MLLC, demonstrating the antigenicity of NmE2 in the HLA-A2 background in vitro. Furthermore, while CD8+ T-cells from a transplanted HLA-A2+ CML patient showed little or no specific reactivity in the first 10 months after HCT, a distinct reactivity (up to 0.6 % NmE2 reactive CD8+ T cells) became apparent at later stages, consistent with the development of an immune response against NmE2-expressing cells in vivo. The patient remained negative for bcr/abl transcripts throughout this period. BA/F3 Bcr/Abl cells expressed increased levels of NmE2 protein (but not mRNA) compared to the parent BA/F3 line. Interestingly, treatment with imatinib or nilotinib reduced NmE2 protein expression in BA/F3 Bcr/Abl, but not in cells expressing Bcr/Abl mutants resistant to the respective inhibitors. Treatment of BA/F3 Bcr/Abl cells with the PI3K inhibitor Ly294002 resulted in reduced Bcr/Abl activity and a corresponding reduction in both c-Myc and NmE2 protein levels, without affecting mRNA levels. Conclusion: The over expression of NmE2 is closely linked to Bcr/Abl kinase activity, the predominant level of regulation being post-transcriptional and dependent on PI-3K activity. The NmE2 protein is restricted by HLA-A2 as well as by HLA-A32. The development of an NmE2-specific T-cell response in a CML patient after stem cell transplantation suggests that NmE2 functions as a tumour antigen in HLA-A2+ patients in vivo and may be relevant to the long term immune control of CML. NmE2 is therefore a promising candidate for the development of new immunotherapeutic strategies for the treatment of CML. Disclosures: Lange:BMS: Honoraria; Novartis: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding. Niederwieser:BMS: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Novartis: Honoraria, Research Funding.

更新日期：2009-11-20

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