PMLRARalpha and PML directly regulate Fas-mediated apoptosis in vivo

描述（由申请方提供）：早幼粒细胞白血病（PML）/维甲酸受体（RAR）α融合蛋白（PMLRAR？）是调节受体信号传导、核转录和癌症发展的中心。PMLRAR？通过对核转录的显性负效应参与调节死亡受体信号传导和细胞凋亡。PML是一种已知刺激细胞凋亡的肿瘤抑制因子，是癌症中最常见的缺陷之一（> 60%）。因此，在癌症中恢复PML功能将是一种有效的治疗方法。如果PMLRAR的直接影响？和PML对Fas死亡受体介导的细胞凋亡的影响，这将是理解细胞凋亡调控的一个突破，并可能为癌症治疗开辟新的可能性。我们筛选了造血癌细胞中潜在的Fas结合抑制剂，并确定PML为这样的蛋白质。鉴于PML的促凋亡作用和PMLRAR？的促存活作用，我们测试是否PMLRAR？阻断Fas介导的凋亡。在初步研究中，PMLRAR？阻断Fas配体（FasL）诱导的细胞凋亡。PMLRAR？在直接从患者分离的急性早幼粒细胞白血病细胞中，PMLRAR？- 表达U937/PR9细胞结合cFLIP并排除死亡诱导信号复合物（DISC）中的半胱天冬酶8;因此，半胱天冬酶8的活化被阻断。此外，击倒PMLRAR？在NB4细胞中，这些细胞对Fas介导的凋亡敏感。我们的假设是，PMLRAR？将Fas信号传导转移至非凋亡终点。拟议的研究结果将对所有癌症产生重大影响，因为PMLRAR？对抗PML在细胞凋亡调节中的作用。通过证明PML和PMLRAR的直接相互作用？我们将揭示一个新的凋亡调控靶点，可用于预防和治疗许多具有PML功能缺陷的癌症。 公共卫生相关性：Fas系统是一种重要的细胞清除系统，具有几种既定的功能，包括清除自身反应性淋巴细胞、感染和缺陷细胞以及由化疗和辐射损伤的细胞。我们已经确定了早幼粒细胞白血病蛋白-视黄酸受体作为Fas结合蛋白和Fas信号转导的潜在关键调节因子。该项目将表征PML与Fas的结合，并证明Fas在急性早幼粒细胞白血病细胞和小鼠中是如何调节的;因此，通过显示调节的关键部位，我们将能够确定绝大多数具有缺陷的早幼粒细胞白血病蛋白并恢复凋亡信号的癌症的治疗方法。

DESCRIPTION (provided by applicant): The promyelocytic leukemia (PML)/retinoic acid receptor (RAR) alpha fusion protein (PMLRAR?) is at the center of regulation of receptor signaling, nuclear transcription, and cancer development. PMLRAR? has been implicated in regulating death receptor signaling and apoptosis through dominant-negative effects on nuclear transcription. PML is a tumor suppressor known to stimulate apoptosis and is one of the most commonly defective (>60%) in cancers. Thus, restoration of PML function in cancer would be an effective therapy. If direct effects of PMLRAR? and PML on Fas death receptor-mediated apoptosis were to be established, this would represent a breakthrough in understanding apoptosis regulation and may open new possibilities for cancer therapy. We screened hematopoietic cancer cells for potential Fas binding inhibitors and identified PML as such a protein. Given the pro-apoptotic role of PML and pro-survival role of PMLRAR?, we tested whether PMLRAR? blocked Fas-mediated apoptosis. In preliminary studies, PMLRAR? blocked apoptosis induced by Fas ligand (FasL). PMLRAR? interacted with Fas in acute promyelocytic leukemia cells isolated directly from patients. PMLRAR? -expressing U937/PR9 cells bound cFLIP and excluded procaspase 8 in the death-induced signaling complex (DISC); thus, activation of caspase 8 was blocked. Also, knockdown of PMLRAR? in NB4 cells sensitized these cells to Fas-mediated apoptosis. Our hypothesis is that PMLRAR? diverts Fas signaling to a non-apoptosis endpoint. The results of the proposed studies will have significant implications for all cancers because PMLRAR? counters the effects of PML in apoptosis regulation. By demonstrating a direct interaction of PML and PMLRAR? with Fas, we will reveal a new target for apoptosis regulation that can be exploited for preventing and treating the many cancers that have defective PML function. PUBLIC HEALTH RELEVANCE: The Fas system is an important cell elimination system that has several established functions, including the elimination of autoreactive lymphocytes, infected and defective cells, and cells damaged by chemotherapy and irradiation. We have identified promyelocytic leukemia protein-retinoic acid receptor as a Fas-binding protein and potential key regulator of Fas signaling. This project will characterize the binding of PML with Fas and demonstrate how Fas is regulated in acute promyelocytic leukemia cells and in mice; thus, by showing the pivotal site of regulation, we will be able to identify a therapeutic approach for the vast majority of cancers that have defective promyelocytic leukemia protein and restore apoptosis signaling.