Molecular determinants of PARP inhibitor sensitivity in 11q-deleted malignancy

 描述(由申请人提供)：11q缺失在人类恶性肿瘤中很常见，并与不良的临床结果相关。在此背景下，含有共济失调-毛细血管扩张突变(ATM)基因的11q22缺失在B细胞淋巴瘤(BCL)中常见，并与第二等位基因的突变相结合，使这些肿瘤ATM缺失。相反，11q23处包含组蛋白H_2AX基因的缺失导致了神经母细胞瘤(NB)亚群中H_2AX的单等位基因表达。鉴于ATM激酶及其底物组蛋白H_2AX在DNA双链断裂(DSB)的信号和修复中的重要作用，它们的拷贝数的变化可能导致癌细胞对DNA损伤剂的不同反应。在这种背景下，PARP抑制物(PARPI)是一类新型的DNA损伤化疗药物，它优先消除复制相关DSB中存在缺陷的细胞。基于这些先前的观察，我们在这里提出了一种假设，即11q缺失的恶性肿瘤中H_2AX的单等位基因表达使它们对PARPI敏感，这是通过赋予同源重组(HR)介导的人类癌细胞中PARPI诱导的DSB修复的缺陷来实现的。为了支持我们的假设，我们提供了强有力的遗传证据，证明H_2AX缺乏是具有PARP抑制的合成致死作用。首先，我们发现， H2AX和两个主要的PARPI靶点PARP1或PARP2中的任何一个都会导致小鼠的胚胎死亡。其次，我们证明了在PARPI处理后复制相关DSB的修复缺陷，以H_2AX基因剂量依赖的方式。最后，我们证明了在11q23缺失和单等位基因H2 AX表达的NB细胞中，H_2AX也限制了DSB的修复。为了验证我们的假设，我们将在小鼠的原代细胞和转化细胞以及人类NB细胞上使用生化、分子和细胞遗传学分析的组合。具体地说，Aim 1中的实验将使用小鼠细胞来测试H_2AX对于HR介导的PARPI诱导的损伤修复的限制这一假设，表征潜在的遗传途径，并确定PARP1和PARP2对这些表型的相对贡献。在目标2中，我们将评估11q23缺失的NB细胞中PARPI的敏感性作为H2AX基因剂量的函数，并确定PARP1和PARP2在H2 AX表达降低的人癌细胞DSB修复中的作用。在目标3中，我们将使用一种新的小鼠模型来检查ATM和H2AX在PARPI诱导的损伤修复中的不重叠功能，并对共同删除这两个因子的人类癌症子集进行建模。从长远来看，从这些探索性研究中获得的知识将增加我们对PARPI如何干扰复制过程中正常保护基因组的机制的理解，并促进未来针对具有11q异常的各种人类恶性肿瘤的生物标记物开发的临床试验的发展。

 DESCRIPTION (provided by applicant): Deletions at 11q are common in human malignancies and associate with poor clinical outcomes. In this context, deletions at 11q22, containing the Ataxia-Telangiectasia Mutated (ATM) locus, are frequently observed in B cell lymphomas (BCL) and, combined with mutation of the second allele, render these tumors ATM null. In contrast, deletions at 11q23, containing the histone H2AX locus, result in monoallelic H2AX expression in a subset of neuroblastomas (NB). Given the prominent roles for the ATM kinase and its substrate histone H2AX in the signaling and repair of DNA double-strand breaks (DSB), alterations in their copy number may result in differential responses to DNA damaging agents in cancer cells. In this context, PARP inhibitors (PARPi) are a novel class of DNA damaging chemotherapeutic agents that preferentially eliminate cells with defects in replication-associated DSBs. Based on these previous observations, we propose here to test the hypothesis that monoallelic expression of H2AX in 11q-deleted malignancies sensitizes them to PARPi, by conferring a defect in Homologous Recombination (HR)-mediated repair of PARPi-induced DSBs in human cancer cells. In support of our hypothesis, we provide strong genetic evidence that H2AX deficiency is synthetic lethal with PARP inhibition. First, we find that combined loss of H2AX and either of the two main PARPi targets, PARP1 or PARP2, results in embryonic lethality in the mouse. Secondly, we demonstrate a defect in the repair of replication-associated DSBs after treatment with PARPi, in an H2AX gene dose- dependent manner. Lastly, we demonstrate that H2AX is also limiting for DSB repair in NB cells with 11q23 deletion and monoallelic H2AX expression. To test our hypotheses, we will employ a combination of biochemical, molecular and cytogenetic assays on mouse primary and transformed cells and in human NB cells. Specifically, experiments in Aim 1 will employ murine cells to test the hypothesis that H2AX becomes limiting for HR-mediated repair of PARPi-induced lesions, characterize the underlying genetic pathway and define the relative contribution of PARP1 and PARP2 to these phenotypes. In Aim 2, we will assess PARPi sensitivity in 11q23-deleted NB cells as a function of H2AX gene dose and define roles for PARP1 and PARP2 in DSB repair in human cancer cells with reduced H2AX expression. In Aim 3, we will employ a novel murine model to examine nonoverlapping functions for ATM and H2AX in the repair of PARPi-induced lesions, modeling the subset of human cancers that co-delete the two factors. In the longer term, knowledge gained from these exploratory studies will increase our understanding of how PARPi interfere with the mechanisms that normally protect the genome during replication and facilitate the development of future clinical trials for biomarker development in a variety of human malignancies with 11q abnormalities.