Mechanism-based approach to combine histone deacetylase inhibitors with PARP inhibitors for therapy in AML

项目摘要 组蛋白去乙酰酶抑制剂(HDACi)与以下药物联合用于白血病的临床治疗，例如， 化疗。阐明它们的作用机制将导致基于机制的联合治疗 战略。 我们最近发表的数据表明，HDACi可以通过一种新的机制来介导细胞毒作用。我们 证明HDAC不仅能差异乙酰化NHEJ蛋白Ku70/Ku80，还能乙酰化多聚ADP-核糖 聚合酶-1(PARP1)，已知与Ku蛋白竞争结合DSB。一个重要的发现是 PARP1与染色质的结合随着HDACis暴露时间的延长而增加，类似于PARP的“陷阱” 最近用PARP抑制剂证实了这一点。PARP1基因敲除抑制染色质捕获并缓解 高密度脂蛋白对NHEJ的影响。此外，HDACis与有效的PARP抑制剂(PARPI)BMN673联合诱导 PARP捕获率呈剂量依赖性增加。这些结果为HDAC提供了一种新的机制 乙酰化PARP1并增加PARP1与DSB的结合，导致C-NHEJ因子与DSB的结合减少 DNA损伤部位，减少白血病细胞毒性双链断裂的修复。对这一新概念的验证 通过我们提出的研究，将提供一种令人信服的基于机制的方法来结合HDACI 用PARPis增强急性白血病的细胞毒性。 在这项提案中，我们将重点关注需要新疗法治疗的急性髓系白血病(AML)，以进行测试 临床相关的HDACis可通过乙酰化引起细胞毒性DSB的中心假设 PARP1，导致PARP在染色质中捕获。HDACis与PARPis的结合将增强 在AML细胞中捕获PARP导致细胞毒性增加。 在具体目标1中，我们将确定HDACis诱导的PARP1乙酰化在多大程度上有助于 PARP被捕获，NHEJ活性降低。阐明导致PARP的精确分子步骤 染色质和NHEJ活性下降，我们将使用最先进的分析方法并进行临床研究- 稳定表达PARP1的AML细胞系中相关的HDACI及过表达PARP1的影响 乙酰化的3个关键赖氨酸残基靶点的突变体。 在特定目标2中，我们将测定HDACis和PARPis在原代AML细胞中的敏感性 在体外和体内。我们将使用“最先进”的初级小鼠模型来研究这些抑制剂的效果。 这些研究将阐明涉及HDACI的新机制，这将导致未来基于机制的 将HDACis和PARPis结合起来作为白血病和其他癌症的治疗方法的临床策略。

Project Summary Histone deacetylase inhibitors (HDACi) are in clinical use in leukemias in combination with agents, such as, chemotherapies. Elucidating their mechanisms of action will lead to mechanism-based combination therapy strategies. Our recently published data suggests a novel mechanism through which HDACi could mediate cytotoxicity. We demonstrate that HDACis differentially acetylate not only NHEJ proteins Ku70/Ku80, but also poly ADP-ribose polymerase-1 (PARP1), known to compete with Ku proteins for binding DSBs. An important finding is that PARP1 binding to chromatin increases with duration of HDACis exposure, resembling PARP “trapping” recently demonstrated with PARP inhibitors. PARP1 knockdown inhibits chromatin trapping and mitigates HDACis effect on NHEJ. Moreover, HDACis combined with the potent PARP inhibitor (PARPi) BMN673 induce a dose-dependent increase in PARP trapping. These results provide a new mechanism by which HDACis both acetylate PARP1 and increase binding of PARP1 to DSBs, leading to decreased access of C-NHEJ factors to DNA damage sites, decreasing repair of cytotoxic DSBs in leukemia cells. Validation of this novel concept through the studies we propose, will provide a compelling mechanism-based approach for combining HDACis with PARPis to enhance cell cytotoxicity in acute leukemias. In this proposal we will focus on acute myeloid leukemia (AML) for which novel therapies are needed, to test the central hypothesis that clinically relevant HDACis can cause cytotoxic DSBs through acetylation of PARP1, leading to PARP trapping in chromatin. HDACis in combination with PARPis will enhance PARP trapping in AML cells leading to increased cell cytotoxicity. In specific aim 1, we will determine the extent to which acetylation of PARP1 induced by HDACis contributes to PARP trapping and decreased NHEJ activity. To elucidate the precise molecular steps leading to PARP trapping in chromatin and decreased NHEJ activity, we will use “state of the art” assays and study clinically- relevant HDACis in AML cell lines stably knocked down for PARP1 and the effects of over-expressing PARP1 mutants of 3 key lysine residue targets of acetylation. In specific aim 2, we will determine the sensitivity of HDACis in combination with PARPis in primary AML cells in vitro and in vivo. We will use “state of the art” primary mouse models to study the effects of these inhibitors. These studies will elucidate new mechanisms involving HDACis that will lead to future mechanism-based clinical strategies to combine HDACis with PARPis as a therapy approach in leukemias and other cancers.