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

本项目旨在阐明遗传病变和细胞DNA损伤反应在CLL免疫逃逸机制中的作用。正常细胞对DNA损伤有两种反应。一种反应包括细胞周期阻滞，随后进行DNA修复，或者在严重损伤后发生细胞凋亡。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。这种通讯通过诱导表达细胞配体来激活免疫受体，如NKG2D、NKp30和NKp46。在释放的囊泡（外泌体）和受影响细胞表面上的配体或“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CLL患者NK细胞的活性受损，与健康供者相比，血清中参与NKG2D和NKp30的可溶性配体水平显著升高。可溶性配体引起NK细胞普遍抑制，高血清水平与不良预后相关，表明NK细胞在CLL的免疫监视中起关键作用。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技术和已建立的敲除小鼠分析提出的atm依赖的配体诱导。具体来说，我们将在TCL1转基因小鼠中进行ATM、TP53和CBP/P300基因敲除。将这些化合物突变小鼠与TCL1转基因小鼠进行比较，以验证DDR成分有助于诱导配体表达和产生抗肿瘤免疫反应的假设。基于先前的数据，我们预计CLL小鼠模型中ATM、p53和CBP/p300的缺失对于CLL细胞对NK细胞攻击的敏感性和先天免疫系统促进肿瘤排斥是必不可少的。最后，我们计划通过挽救配体在恶性CLL细胞上的表达来恢复NK细胞的功能，以证明NK细胞的抑制是由NK配体的表达受损引起的。我们将设计NKG2D配体的双特异性融合蛋白（免疫配体）与检测b - cll特异性抗原ROR1的抗体片段融合。该结构体用于用NKG2D配体修饰表达ror1的CLL细胞，使细胞更容易受到NK细胞介导的杀伤。修复的配体表达/修饰的效果将在CLL小鼠模型和体外使用来自健康供体或CLL患者的NK细胞进行评估。我们的目标是获得新颖的，遗传信息的联合疗法，包括一线dna损伤剂与利用NK细胞进行癌症治疗的创新方法。

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.