Mechanistic validation of SCF E3 ligase as a cancer and radiosensitizing target

描述（由申请人提供）：靶向癌症治疗依赖于对癌症靶点的彻底验证。我们的长期目标是发现一类新的抗癌药物，选择性地靶向一种在人类癌症中被激活的E3泛素连接酶。选择性靶向一种泛素连接酶途径将降低与蛋白质降解的总体抑制相关的正常细胞毒性，如在万珂（也称为Bortezelatin或PS-341）中所见，万珂是FDA批准用于治疗复发性/难治性多发性骨髓瘤和套细胞淋巴瘤的第一类（也是唯一一类）一般蛋白酶体抑制剂。为此，我们重点关注SCF（Skp 1-Cullin-F-box蛋白）E3泛素连接酶，也称为CRL（Cullin-RING泛素连接酶）。SCF E3连接酶是最大的E3连接酶家族，由Skp 1、cullin、F-box蛋白和RING蛋白、ROC或RBX组成，其促进关键调节蛋白亚组的泛素化以进行靶向降解，从而控制重要的生物学过程，包括细胞周期进展、信号转导和DNA复制。虽然cullin-ROC构成核心连接酶组分，但cullin需要通过Nedd 8-活化酶（NAE）进行neddylated以获得其酶活性。我们强有力的初步数据表明，ROC 1在许多人类癌症中过表达，通过ROC 1 siRNA沉默或NAE抑制剂MLN 4924使SCF E3泛素连接酶活性失活，触发DNA双链断裂（DSB）和DNA损伤反应（DDR），并通过自噬、衰老和凋亡诱导肿瘤细胞杀伤。SCF E3连接酶的抑制也增强辐射诱导的DDR，导致放射增敏。本研究的目的是阐明ROC 1-SCF E3连接酶失活引发这些生化和生物学变化的潜在机制，导致各种类型的细胞死亡，并验证ROC 1-SCF E3连接酶作为抗癌和放射增敏靶点。我们的中心假设是ROC 1-SCF E3泛素连接酶的失活导致几种关键底物的积累，这触发了DSB和DDR，并通过自噬、衰老和凋亡以顺序或平行的顺序诱导细胞死亡。这些触发的细胞杀伤机制也可以增强辐射效应，导致癌细胞的放射增敏。提出了三个具体目标：1）确定ROC 1-SCF E3连接酶的失活如何触发DNA损伤和DDR，并通过不同的机制诱导细胞死亡; 2）确定ROC 1-SCF E3连接酶的失活如何阻断mTOR诱导自噬; 3）验证ROC 1-SCF E3连接酶作为新的放射增敏靶点。影响：这项工作具有高度创新性，通过验证SCF E3连接酶作为抗癌靶点并为MLN 4924或其类似物作为新型放射增敏剂的未来开发奠定基础，具有重大的转化价值。

DESCRIPTION (provided by applicant): Targeted cancer therapy relies on a thorough validation of cancer targets. Our long-range goal is to discover a novel class of anticancer drugs that selectively target one type of E3 ubiquitin ligase, which is activated in human cancer. Selective targeting one ubiquitin ligase pathway will reduce normal cell toxicity associated with overall inhibition of protein degradation, as seen in Velcade (also known as Bortezomib or PS-341), the first (and only) class of general proteasome inhibitor, approved by FDA for the treatment of relapsed/refractory multiple myeloma and mantle cell lymphoma. To this end, we have focused on SCF (Skp1-Cullin-F-box proteins) E3 ubiquitin ligases, also known as CRLs (Cullin-RING ubiquitin ligases). SCF E3 ligases, the largest E3 ligase family consisting of Skp1, cullins, F-box proteins, and a RING protein, ROC or RBX, promotes the ubiquitination of a subset of key regulatory proteins for targeted degradation, thus governing important biological processes, including cell cycle progression, signal transduction and DNA replication. While cullin-ROC constitutes the core ligase component, cullin needs to be neddylated via Nedd8-Activating Enzyme (NAE) for its enzymatic activity. Our strong preliminary data showed that ROC1 is over-expressed in a number of human cancers, and inactivation of SCF E3 ubiquitin ligase activity via ROC1 siRNA silencing or by an NAE inhibitor, MLN4924, triggers DNA double strand breaks (DSB) and the DNA damage response (DDR), and induces tumor cell killing via autophagy, senescence and apoptosis. Inhibition of SCF E3 ligase also enhances radiation-induced DDR, leading to radiosensitization. The objective of this proposed study is to elucidate the underlying mechanisms by which inactivation of ROC1-SCF E3 ligase triggers these biochemical and biological changes, leading to various types of cell death, and to validate ROC1-SCF E3 ligase as an anticancer and radiosensitizing target. Our central hypothesis is that inactivation of ROC1-SCF E3 ubiquitin ligase causes accumulation of several key substrates, which triggers DSB and DDR and induces cell death via autophagy, senescence and apoptosis in a sequential or parallel order. These triggered cell killing mechanisms could also enhance radiation effects, leading to radiosensitization of cancer cells. Three specific aims are proposed 1) to determine how inactivation of ROC1-SCF E3 ligase triggers DNA damage and DDR, and induces cell death via different mechanisms; 2) to determine how inactivation of ROC1-SCF E3 ligase blocks mTOR to induce autophagy; and 3) to validate ROC1-SCF E3 ligase as a novel radiosensitizing target. IMPACT: This work is highly innovative and of significant impact with translational value by validating SCF E3 ligase as an anticancer target and by paving the ground for future development of MLN4924 or its analogues as a novel class of radiosensitizers.