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

 描述（由申请方提供）：11 q缺失在人类恶性肿瘤中很常见，并与不良临床结局相关。在这种情况下，在B细胞淋巴瘤（BCL）中经常观察到11 q22（包含共济失调-毛细血管扩张突变（ATM）基因座）的缺失，并结合第二等位基因的突变，使这些肿瘤ATM无效。相反，在11 q23，包含组蛋白H2 AX基因座的缺失，导致单等位基因H2 AX表达的神经母细胞瘤（NB）的子集。鉴于ATM激酶及其底物组蛋白H2 AX在DNA双链断裂（DSB）的信号传导和修复中的突出作用，其拷贝数的改变可能导致癌细胞对DNA损伤剂的不同反应。在这种情况下，PARP抑制剂（PARPi）是一类新型的DNA损伤化疗剂，其优先消除具有复制相关DSB缺陷的细胞。基于这些先前的观察结果，我们在此提出通过在人类癌细胞中赋予同源重组（HR）介导的PARPi诱导的DSB修复缺陷来检验H2 AX在11 q缺失的恶性肿瘤中的单等位基因表达使它们对PARPi敏感的假设。为了支持我们的假设，我们提供了强有力的遗传证据，即H2 AX缺乏是PARP抑制的合成致死。首先，我们发现， H2 AX和两个主要PARPi靶标PARP 1或PARP 2中的任一个导致小鼠中的胚胎致死。其次，我们证明了在用PARPi处理后，以H2 AX基因剂量依赖性方式修复复制相关DSB的缺陷。最后，我们证明了H2 AX也限制了具有11 q23缺失和单等位基因H2 AX表达的NB细胞中的DSB修复。为了验证我们的假设，我们将采用生物化学，分子和细胞遗传学的小鼠原代细胞和转化细胞和人NB细胞的组合测定。具体地，目的1中的实验将采用鼠细胞来测试H2 AX变得限制HR介导的PARPi诱导的病变的修复的假设，表征潜在的遗传途径并定义PARP 1和PARP 2对这些表型的相对贡献。在目标2中，我们将评估PARPi在11 q23缺失的NB细胞中的敏感性作为H2 AX基因剂量的函数，并确定PARP 1和PARP 2在H2 AX表达降低的人癌细胞中DSB修复中的作用。在目标3中，我们将采用一种新的小鼠模型来检查ATM和H2 AX在PARPi诱导的病变修复中的非重叠功能，对共同缺失这两种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通常保护基因组的机制的理解，并促进未来临床试验的发展，以在各种11 q异常的人类恶性肿瘤中开发生物标志物。

 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.