Delineating ARF negative regulation of PAF1C-dependent oncogenic programs

项目摘要 肿瘤抑制因子（TS）通过在多个水平上发挥作用来调节细胞生长和死亡的平衡。放松管制 这些层次中的任何一个都可以促进肿瘤的发生，影响癌症患者的生存和对当前癌症的反应。 可用的治疗。因此，更好地了解这些过程和识别新的 在TS丧失后的治疗靶点是癌症生物学领域的优先事项。几十年的研究已经确定了 p14 ARF（替代阅读框架）TS以p53依赖性方式起作用以防止肿瘤发生。 然而，ARF也以不依赖于p53的方式运作，以潜在地“备份p53”，但其机制 仍然知之甚少。在该领域有一种重新获得的想法，即深入研究肿瘤抑制机制， 利用基础生物学来治疗疾病。在此背景下，本研究提案的主要目标 目的是确定ARF（p53缺失后重新激活）如何发挥作用以抑制肿瘤启动子程序。我们未发表 研究已经确定了以前被忽视的ARF肿瘤抑制机制。首先，在p53缺失时， 重新激活的ARF选择性靶向聚合酶相关因子1复合物（PAF 1C）的基因编码 促生长GDF和BMP配体抑制异常细胞生长。第二，原发性p53-/- 细胞去抑制GDF/BMP程序，导致SMAD 1/5磷酸化和靶基因激活 （“致癌GDF/SMAD轴”），表明在双TS（p53和ARF）后致癌脆弱性增加 损失作为替代治疗目标出现。然而，ARF如何使PAF 1C失活仍然是未知的。 GDF/SMAD轴是否具有诊断和治疗价值。 这项小额拨款提案旨在解决ARF如何以基因特异性方式使PAF 1C失活 抑制肿瘤启动子程序，并评估靶向致癌基因的治疗价值， GDF/SMAD程序。本提案中的工作以这些先前的调查结果和知识方面的关键差距为指导 检验ARF结合Paf 1和基因特异性因子RUNX 1阻断PAF 1C的中心假设 以基因特异性方式组装，从而抑制肿瘤启动子程序。为了测试潜在的 假设，我们将利用生物化学和遗传学方法以及临床和分子注释 临床前模型。具体来说，我们将首先研究ARF如何结合Paf 1和RUNX 1来阻断PAF 1C 以基因特异性方式组装（目的1），然后探测肿瘤是否携带双TS（p53和ARF）缺失 显示致癌GDF/SMAD轴的再激活，并检查其治疗和诊断潜力（Aim 2）。总之，阐明了ARF负性调节PAF 1C依赖性肿瘤促进因子的机制， 转录程序将提高我们对一个重要的生物过程的理解， 在具有双重ARF和p53失活的癌症中靶向TS丢失的机会（如肉瘤、胰腺和 肺腺癌），这与NCI的使命一致，即确定新的靶点， 把长凳搬到诊所

PROJECT SUMMARY Tumor suppressors (TS) tune the balance of cell growth and death by acting at multiple levels. Deregulation of any of these hierarchies can promote tumorigenesis, impact cancer patient’s survival and response to currently available treatments. Therefore, having a better understanding of these processes and the identification of novel therapeutic targets upon TS loss is a priority in the field of cancer biology. Decades of work have defined how the p14ARF (Alternative Reading Frame) TS operates in a p53-dependent manner to prevent tumorigenesis. However, ARF also operates in a p53-independent manner to potentially “back-up p53”, but the mechanisms remain poorly understood. There is a regained idea in the field to delve into tumor suppressive mechanisms and to leverage the basic biology to therapeutic opportunities. In this context, the major goal of this research proposal is to define how ARF (reactivated upon p53 loss) functions to restrain tumor promoter programs. Our unpublished studies have defined a previously overlooked mechanism of ARF tumor suppression. First, upon p53 loss, reactivated ARF selectively targets the Polymerase Associated Factor 1 complex (PAF1C) at genes encoding the pro-growth GDF and BMP ligands to restrain abnormal cell growth. Second, loss of ARF in primary p53-/- cells de-repress GDF/BMP programs leading to SMAD1/5 phosphorylation and target gene activation (“oncogenic GDF/SMAD axis”), suggesting oncogenic vulnerabilities accrued upon double TS (p53 and ARF) loss emerge as alternative therapeutic targets. However, it remains unknown how ARF inactivates PAF1C in a gene-specific manner and whether the oncogenic GDF/SMAD axis has any diagnostic and therapeutic value. This small grant proposal aims at addressing how ARF inactivates PAF1C in a gene-specific manner to restrain tumor promoter programs and to evaluate the therapeutic value of targeting the oncogenic GDF/SMAD program. Work in this proposal is guided by these previous findings and critical gaps in knowledge to test the central hypothesis that ARF binds Paf1 and the gene-specific factor RUNX1 to block PAF1C assembly in a gene-specific manner thereby restraining tumor promoter programs. To test the underlying hypothesis, we will leverage biochemical and genetic approaches as well as clinically and molecularly annotated preclinical models. Specifically, we will first investigate how ARF binds Paf1 and RUNX1 to block PAF1C assembly in a gene-specific manner (Aim 1), and then probe if tumors bearing double TS (p53 and ARF) loss exhibit reactivation of the oncogenic GDF/SMAD axis and examine its therapeutic and diagnostic potential (Aim 2). Together, elucidating the mechanisms by which ARF negatively regulates PAF1C-dependent tumor promoter transcriptional programs will improve our understanding of an important biological process and offer alternative opportunities to target TS loss in cancers with dual ARF and p53 inactivation (such as sarcoma, pancreas, and lung adenocarcinoma), which is in-line with NCI’s mission to identify novel targets to move the discoveries from the bench to the clinics.