The DNA damage-dependent expression of ligands for cytotoxic NK-cell receptors: Impact of the DNA damage response on inside out signaling in CLL

细胞毒性 NK 细胞受体配体的 DNA 损伤依赖性表达：DNA 损伤反应对 CLL 中由内而外信号传导的影响

• 批准号: 234151796
• 负责人: Professorin Dr. Elke Pogge von Strandmann
• 金额: --
• 依托单位: Zentrum für Tumor- und Immunbiologie (ZTI)
• 依托单位国家: 德国
• 项目类别: Clinical Research Units
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/234151796?language=en
• 关键词: DNA; damage; dependent; expression; ligands

Aim of this project is to elucidate the role of genetic lesions and the cellular DNA damage response for the immune escape mechanisms in CLL. Normal cells respond to DNA damage in two ways. One response includes cell cycle arrest followed by DNA repair or, after severe damage, by apoptosis. The other response aims at signaling the comprised state of the dangerous cells to the innate immune system. This communication occurs through the induced expression of cellular ligands for activating immune receptors such as NKG2D, NKp30 and NKp46.The expression of ligands or "danger molecules" on released vesicles (exosomes) and on the surface of affected cells allows recognition and killing of the damaged cell by NK cells. Recently, we and others observed that the activity of NK cells from CLL patients is impaired and serum levels for soluble ligands engaging NKG2D and NKp30 are significantly elevated in comparison to healthy donors. Soluble ligands cause a general NK cell inhibition and high serum levels correlate with a poor prognosis indicating that NK cells are critically involved in immunosurveillance of CLL. The molecular mechanisms directing the inducible expression of ligands for NKG2D are not fully defined but known to be ATM/ATR-dependent. To uncover the impact of the DDR for ligand upregulation in CLL we will analyze the proposed ATM-dependent induction of the ligands using RNAi technology and established knockout mice. Specifically, we will cross the TCL1 transgenic mouse with ATM, TP53 and CBP/P300 knockouts. These compound mutant mice will be compared to TCL1 transgenic counterparts to test the hypothesis that components of the DDR contribute to the inducible ligand expression and to the generation of an anti-tumor immune response. Based on previous data we expect that loss of ATM, p53 and CBP/p300 in the CLL mouse model is essential to sensitize CLL cells to the NK cell attack and promotes tumor rejection by the innate immune system.Finally we plan to restore NK cell function by a rescue of ligand expression on malignant CLL cells to prove that NK cell inhibition results from the impaired expression of NKR-ligands. We will design bispecific fusion proteins (immunoligands) of ligands for NKG2D fused to an antibody fragment detecting the B-CLL-specific antigen ROR1. The construct is used to decorate ROR1-expressing CLL cells with NKG2D ligands rendering the cells more susceptible to NK cell-mediated killing. The effect of the restored ligand expression/decoration will be evaluated in the CLL mouse models and also ex vivo using NK cells derived from healthy donors or CLL patients. We aim to derive novel, genetically-informed combination therapies consisting of frontline DNA-damaging agents with innovative approaches that harness NK cells for cancer therapy.

本项目的目的是阐明遗传损伤和细胞DNA损伤反应在慢性淋巴细胞性白血病免疫逃逸机制中的作用。正常细胞对DNA损伤有两种反应方式。一种反应包括细胞周期停滞，然后是DNA修复，或者在严重损伤后，通过细胞凋亡。另一种反应旨在向先天免疫系统发出危险细胞组成状态的信号。这种交流是通过诱导细胞配体的表达来激活免疫受体，如NKG2D、NKp30和NKp46。在释放的囊泡(外体)和受影响细胞表面的配体或危险分子的表达允许NK细胞识别和杀死受损细胞。最近，我们和其他人观察到，与健康献血者相比，CLL患者的NK细胞活性受损，与NKG2D和NKp30结合的可溶性配体的血清水平显著升高。可溶性配体可引起全身NK细胞抑制，且血清水平高与预后不良相关，提示NK细胞在CLL的免疫监测中起重要作用。指导NKG2D配体诱导表达的分子机制尚未完全确定，但已知是依赖于ATM/ATR的。为了揭示DDR对CLL中配体上调的影响，我们将使用RNAi技术分析拟议的ATM依赖的配体诱导，并建立基因敲除小鼠。具体地说，我们将TCL1转基因小鼠与ATM、TP53和CBP/P300基因敲除进行杂交。这些复合突变小鼠将与TCL1转基因小鼠进行比较，以检验DDR组件有助于诱导配体表达和产生抗肿瘤免疫反应的假设。根据以前的数据，我们认为在CLL小鼠模型中ATM、P53和CBP/p300的丢失是使CLL细胞对NK细胞攻击敏感并通过天然免疫系统促进肿瘤排斥反应的关键。最后，我们计划通过挽救恶性CLL细胞上的配体表达来恢复NK细胞的功能，以证明NK细胞抑制是由于NKR-配体表达受损所致。我们将设计NKG2D配体的双特异性融合蛋白(免疫配体)，将其与检测B-CLL特异性抗原ROR1的抗体片段融合。该构建物用于用NKG2D配体修饰表达ROR1的CLL细胞，使其更容易受到NK细胞介导的杀伤。恢复的配体表达/修饰的效果将在CLL小鼠模型中进行评估，也将在体外使用来自健康捐赠者或CLL患者的NK细胞进行评估。我们的目标是获得新颖的、遗传信息的组合疗法，由一线DNA损伤剂组成，并采用创新的方法利用NK细胞进行癌症治疗。