Role of DDX3 in DR5-Mediated Apoptosis

描述(申请人提供)：死亡受体通过TRAIL或激动型抗体诱导肿瘤细胞的凋亡被认为是一种重要的癌症治疗策略。我们研制了一种激动型抗人DR5单抗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有效性和安全性，并计划进行I期临床试验。然而，肿瘤细胞对DR5介导的凋亡的预先存在和诱导的抵抗都是一个令人担忧的问题。我们已经发现了DEAD盒蛋白家族的一种RNA解旋酶DDX3，它是调节DR5信号转导的关键适配子蛋白，并在诱导DR5细胞凋亡抵抗中起着重要作用。这项建议的总体目标是研究DDX3在抵抗DR5介导的细胞凋亡中的作用。中心假说是，DDX3作为一个接头蛋白，通过每个分子中特定的结合基序与DR5结构性地联系在一起。在其N端附近，DDX3通过两个分子之间的卡片/卡片相互作用招募clAP1。因此，DR5/DDX3/clAP1的默认功能是负调控DR5介导的细胞凋亡。在DR5凋亡敏感细胞中，启动子caspase 8的激活导致DDX3在aa135处的裂解，从而使DDX3和clAP1的N末端卡片从DR5/DDX3/CLAP复合体中释放出来，从而使正反馈环路放大细胞凋亡信号。相反，在DR5耐药细胞中，clAP1的募集增加导致caspase8活性的抑制和DDX3的切割失败，从而形成一个负反馈环，阻止了最初的凋亡信号的放大。我们的目标是验证四个假设：1)DDX3与DR5的结合是必不可少的；2)DDX3卡片招募的clAP1是启动DR5介导的细胞凋亡的关键抑制分子；3)caspase介导的DDX3的裂解释放了DR5的N-末端卡片，从而逆转了耐药性；4)DR5/DDX3/clAP1复合体的阻断可能会提高TRA-8和其他DR5导向药物的治疗效果。这些研究将为DDX3在DR-5介导的细胞凋亡中的作用提供新的见解，也将对进一步开发干预措施以提高TRA-8和其他激动型DR5抗体和TRAIL的治疗效果具有指导意义。

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.