Adenovirus-Mediated Sensitization to the Cytotoxic Drugs Docetaxel and Mitoxantrone Is Dependent on Regulatory Domains in the E1ACR1 Gene-Region

Oncolytic adenoviruses have shown promising efficacy in clinical trials targeting prostate cancers that frequently develop resistance to all current therapies. The replication-selective mutants AdΔΔ and dl922–947, defective in pRb-binding, have been demonstrated to synergise with the current standard of care, mitoxantrone and docetaxel, in prostate cancer models. While expression of the early viral E1A gene is essential for the enhanced cell killing, the specific E1A-regions required for the effects are unknown. Here, we demonstrate that replicating mutants deleted in small E1A-domains, binding pRb (dl1108), p300/CBP (dl1104) and p400/TRRAP or p21 (dl1102) sensitize human prostate cancer cells (PC-3, DU145, 22Rv1) to mitoxantrone and docetaxel. Through generation of non-replicating mutants, we demonstrate that the small E1A12S protein is sufficient to potently sensitize all prostate cancer cells to the drugs even in the absence of viral replication and the E1A transactivating domain, conserved region (CR) 3. Furthermore, the p300/CBP-binding domain in E1ACR1 is essential for drug-sensitisation in the absence (AdE1A1104) but not in the presence of the E1ACR3 (dl1104) domain. AdE1A1104 also failed to increase apoptosis and accumulation of cells in G2/M. All E1AΔCR2 mutants (AdE1A1108, dl922–947) and AdE1A1102 or dl1102 enhance cell killing to the same degree as wild type virus. In PC-3 xenografts in vivo the dl1102 mutant significantly prolongs time to tumor progression that is further enhanced in combination with docetaxel. Neither dl1102 nor dl1104 replicates in normal human epithelial cells (NHBE). These findings suggest that additional E1A-deletions might be included when developing more potent replication-selective oncolytic viruses, such as the AdΔCR2-mutants, to further enhance potency through synergistic cell killing in combination with current chemotherapeutics.

溶瘤腺病毒（oncolytic adenoviruses）在针对对所有现有疗法均常产生耐药性的前列腺癌的临床试验中已展现出良好的疗效。缺失pRb结合功能的复制选择性突变株AdΔΔ与dl922–947，已被证实在前列腺癌模型中可与当前标准治疗药物米托蒽醌（mitoxantrone）、多西他赛（docetaxel）产生协同效应。尽管早期病毒E1A基因（E1A）的表达对于增强细胞杀伤效果至关重要，但介导该效应所需的具体E1A区域仍未明确。本研究证实，缺失小E1A结构域的复制型突变株——包括结合pRb的dl1108、结合p300/CBP的dl1104，以及结合p400/TRRAP或p21的dl1102——可使人前列腺癌细胞系（PC-3、DU145、22Rv1）对米托蒽醌与多西他赛增敏。通过构建非复制型突变株，本研究证实即便在病毒复制缺失且不存在E1A反式激活结构域保守区域（CR）3的情况下，小E1A12S蛋白仍足以强效增强所有前列腺癌细胞对上述药物的敏感性。此外，在缺失E1ACR3结构域（AdE1A1104）的背景下，E1ACR1中的p300/CBP结合结构域是药物增敏所必需的，但在存在E1ACR3结构域（dl1104）时则并非如此。AdE1A1104同样无法促进细胞凋亡与G2/M期细胞蓄积。所有缺失CR2的E1A突变株（AdE1A1108、dl922–947）以及AdE1A1102或dl1102均可达到与野生型病毒相当的细胞杀伤效果。在体内PC-3异种移植瘤模型中，dl1102突变株可显著延长肿瘤进展时间，且与多西他赛联合使用时该效果可进一步增强。dl1102与dl1104均无法在正常人上皮细胞（NHBE）中复制。上述研究结果表明，在开发更高效的复制选择性溶瘤腺病毒（如AdΔCR2突变株）时，可引入额外的E1A缺失突变，从而通过与当前化疗药物的协同细胞杀伤作用进一步提升其抗肿瘤效力。