Myeloma stem cell and cancer testis antigens

描述（由申请人提供）：多发性骨髓瘤（MM）是一种常见的血液癌症，采用标准治疗很少能治愈。虽然接受免疫调节剂和/或化疗治疗的骨髓瘤患者可能会出现完全临床缓解，但大多数患者最终会复发。更积极的治疗，包括异基因干细胞移植可能是治愈性的，但高发病率和死亡率的清髓性预处理排除了在许多患者中使用。该项目的目标是开发一种免疫治疗靶向骨髓瘤干细胞的临床前模型，从长远来看，该模型可以发展成为一种新型的临床治疗策略，从而改善骨髓瘤患者的预后。我们和其他人先前已经描述了一群骨髓瘤干细胞，它们具有克隆形成潜力，对化疗药物具有高度抗性，并且可以启动肿瘤发生。干细胞用来限制其暴露于化疗的机制之一是表达可以泵出药物的转运蛋白。干细胞的这种特性可以用于它们的纯化，因为相同的转运蛋白流出Hoechst染料，在流式细胞术分析中形成不同的侧群。这些细胞可能在疾病复发中发挥关键作用。我们现在建议用针对癌症睾丸抗原（CTA）的免疫疗法来靶向这些骨髓瘤干细胞。CTA在多发性骨髓瘤中异常表达，并且由于其免疫原性和在正常组织中的限制性表达使得T细胞耐受性不发展，使得CTA成为骨髓瘤中有希望的抗原靶标。此外，最近报道了一些CTA，包括NAPs、NY-ESO、法师-1和SSX在人间充质干细胞（MSC）中表达。这表明CTA表达不仅是配子发生的标志，也是干细胞标志。基于这些观察结果，我们假设骨髓瘤干细胞优先表达CTA，因此容易被CTA特异性T细胞杀死。此外，CTA转录部分受甲基化调节，因此脱甲基剂可用于增强低表达肿瘤中的CTA表达，并增加这些肿瘤细胞对CTA特异性T细胞杀伤的易感性。我们的假设得到了我们的初步数据的支持，即来自人骨髓瘤细胞系RPMI 8226的骨髓瘤干细胞比成熟骨髓瘤细胞具有更高的几种CTA表达，包括PRAME、CT 7和NY-ESO 1（第C.1节）。此外，我们已经产生了可以杀死PRAME阳性骨髓瘤细胞的PRAME特异性细胞毒性T细胞（CTL）。在目标1中，我们将分析大量骨髓瘤肿瘤样本，以确定CTA表达是否在原代骨髓瘤干细胞中保留和/或富集。在目标2中，我们将使用鼠骨髓瘤模型确定针对CTA的CTL是否可以直接杀死骨髓瘤干细胞。 公共卫生相关性：该项目的长期目标是通过使用细胞毒性T细胞靶向在肿瘤发生和疾病复发中起关键作用的癌症干细胞，改善多发性骨髓瘤（MM）的结局，MM是目前无法治愈的浆细胞来源的常见血液癌症。该提案的主要目标是生成使用细胞毒性T细胞靶向骨髓瘤干细胞的临床前数据。建立的基本模型和从该建议中获得的知识将代表未来设计长期治愈骨髓瘤的新方法的关键。

DESCRIPTION (provided by applicant): Multiple myeloma (MM) is a common hematological cancer that with standard treatment is rarely curable. Although myeloma patients treated with immunomodulatory agents and or chemotherapy may experience a complete clinical response, the majority will eventually relapse. More aggressive treatments including allogeneic stem cell transplantation may be curative, but the high morbidity and mortality of myeloablative conditioning precludes use in many patients. The objective of this project is to develop a preclinical model of targeting myeloma stem cells with immunotherapy that in the long-term can be developed into a novel clinical therapeutic strategy that will improve outcomes for patients with myeloma. We and others have previously described a population of myeloma stem cells that possess clonogenic potential, are highly resistant to chemotheraputics agents, and can initiate tumorigenesis. One of the mechanisms that stem cells use to limit their exposure to chemotherapy is to express transport proteins that can pump out drug. This property of stem cells can be exploited for their purification, since the same transporters efflux Hoechst dye forming a distinct side population on flow cytometric analysis. These cells are likely to play a key role in disease relapse. We now propose to target these myeloma stem cells with immunotherapy directed against cancer testis antigens (CTAs). CTAs are expressed aberrantly in multiple myeloma, and by virtue of their immunogenecity and restricted expression in normal tissue such that T-cell tolerance does not develop, makes CTAs a promising antigen target in myeloma. Additionally, it was recently reported that some CTAs including NRAGE, NY-ESO, MAGE-1, and SSX are expressed in human mesenchymal stem cells (MSCs). This suggests that CTA expression may not only be a hallmark of gametogenesis but also a stem cell marker. Based on these observations, we hypothesize that myeloma stem cells preferentially express CTA and are therefore susceptible to killing by CTA specific T-cells. In addition, CTA transcription is partially regulated by methylation, and therefore demethylating agents maybe used to enhance CTA expression in low expressing tumors and increase susceptibility of these tumor cells to CTA-specific T-cell killings. Our hypothesis is supported by our preliminary data that myeloma stem cells from human myeloma cell line, RPMI 8226, have higher expression of several CTAs, including PRAME, CT7 and NY-ESO1, than the mature myeloma cells (Section C.1). Furthermore, we have already generated PRAME specific cytotoxic T- cells (CTLs) that can kill PRAME positive myeloma cells. In aim 1 we will analyze a large panel of myeloma tumor samples to determine if CTA expression is preserved and/or enriched in primary myeloma stem cells. In aim 2 we will determine if CTLs directed to CTA can directly kill myeloma stem cells using a murine myeloma model. PUBLIC HEALTH RELEVANCE: The long-term objective of this project is to improve the outcome of multiple myeloma (MM), a currently incurable common hematological cancer of plasma cell origin, by targeting the cancer stem cell which play a key role in tumorigenesis and disease relapse using cytotoxic T-cells. The primary goal of this proposal is to generate pre- clinical data for using cytotoxic T-cells to target myeloma stem cells. The fundamental models established and knowledge obtained from this proposal will represent the key for the future design of novel methods toward the long-term cure of myeloma.