Role of DDX3 in DR5-Mediated Apoptosis

描述（由申请人提供）：通过肿瘤坏死因子相关凋亡配体（TRAIL）或激动性抗体诱导死亡受体诱导肿瘤细胞凋亡被认为是癌症治疗的一种重要新兴策略。我们已经开发了激动性抗人DR 5单克隆抗体TRA-8。我们的临床前研究已经在动物模型中证明了其强大的抗肿瘤疗效和安全性，并计划进行I期临床试验。然而，肿瘤细胞对DR 5介导的凋亡的预先存在的和诱导的抗性是一个问题。我们已经确定了一个RNA解旋酶的DEAD盒蛋白家族，DDX 3，作为一个关键的衔接蛋白调节DR 5信号转导，并发挥了致病作用，诱导DR 5凋亡抗性。该提案的总体目标是检查DDX 3在DR 5介导的细胞凋亡抗性发展中的作用。核心假设是DDX 3作为衔接蛋白，通过每个分子中的特异性结合基序与DR 5组成性相关。在其N末端附近，DDX 3通过两个分子之间的CARD/CARD相互作用募集cIAP 1。因此，DR 5/DDX 3/cIAP 1的默认功能是负调节DR 5介导的细胞凋亡。在DR 5凋亡敏感性细胞中，引发剂半胱天冬酶8的活化导致DDX 3在aa 135处裂解，这从DR 5/DDX 3/cIAP复合物释放DDX 3和cIAP 1的N-末端CARD，从而使得能够形成正反馈环以放大凋亡信号。相比之下，在DR 5凋亡抗性细胞中，cIAP 1募集的增加导致胱天蛋白酶8活性的抑制和DDX 3裂解的失败，从而形成负反馈环以防止初始凋亡信号的放大。本研究的目的是验证以下四个假设：1）DDX 3与DR 5的结合是必需的; 2）DDX 3的CARD募集的cIAP 1是启动DR 5介导的凋亡的关键抑制分子; 3）半胱天冬酶介导的DDX 3切割从DR 5释放N-末端CARD，从而逆转抗性;和4）阻断DR 5/DDX 3/cIAP 1复合物可提高TRA-8和其它DR 5导向剂的治疗功效。拟议的研究将为DDX 3在DR-5介导的细胞凋亡中的作用提供新的见解，并且还将对进一步开发干预措施以增强TRA-8和其他激动性DR 5抗体和TRAIL的治疗效果具有影响。

DESCRIPTION (provided by applicant): The death receptor-induced apoptosis of tumor cells by TRAIL or agonistic antibodies is thought to be an important emerging strategy for cancer therapy. We have developed an agonistic anti-human DR5 monoclonal antibody, TRA-8. Our pre-clinical studies have demonstrated its strong anti-tumor efficacy and safety in animal models, and Phase I clinical trials are planned. However, both pre-existing and induced resistance of tumor cells to DR5-mediated apoptosis is a concern. We have identified a RNA helicase of the DEAD box protein family, DDX3, which serves as a critical adaptor protein in regulation of DR5 signaling transduction, and plays a causative role in induction of DR5 apoptosis resistance. The overall goal of this proposal is to examine the role of DDX3 in the development of resistance to DR5-mediated apoptosis. The central hypothesis is that DDX3, functioning as an adaptor protein, is constitutively associated with DR5 via a specific binding motif in each molecule. Near its N-terminus, DDX3 recruits clAP1 via a CARD/CARD interaction between the two molecules. Thus, a default function of the DR5/DDX3/clAP1 is to negatively regulate DR5-mediated apoptosis. In DR5 apoptosis sensitive cells, activation of the initiator caspase 8 leads to cleavage of DDX3 at aa135, which releases the N-terminal CARD of DDX3 and clAP1 from DR5/DDX3/clAP complex, thereby enabling a positive feedback loop to amplify apoptosis signal. In contrast, in DR5 apoptosis resistant cells, increased recruitment of clAP1 leads to inhibition of caspase 8 activity and failure of DDX3 cleavage, thereby forming a negative feedback loop to prevent amplification of the initial apoptosis signal. The Aims to test four hypotheses are: 1) that the association of DDX3 with DR5 is essential; 2) that the clAP1 recruited by a CARD of DDX3 is a key inhibitory molecule in the initiation of DR5-mediated apoptosis; 3) that the caspase-mediated cleavage of DDX3 releases the N-terminal CARD from DR5 thereby reversing the resistance; and 4) that the interruption of the DR5/DDX3/clAP1 complex may improve the therapeutic efficacy of TRA-8 and other DR5-directed agents. The proposed studies will provide novel insights into the role of DDX3 in DR-5 mediated apoptosis, and also will have implications for the further development of interventions to enhance the therapeutic efficacy of TRA-8 and other agonistic DR5 antibodies and TRAIL.