RNA解旋酶DDX17与p53抑癌基因突变的协同作用及分子机制研究

Tumor suppressor gene TP53, the “Guardian of the Genome”, is the most frequently mutated gene in human cancer, yet no effective therapies are currently available that specifically target p53 mutant tumors. To identify genetic vulnerabilities of p53-mutated cancer cells, we employed a pooled shRNA screen across a panel of well-characterized p53 mutant and wildtype cell lines. We identified that p53 mutated cells are selectively dependent on the RNA helicase DDX17. The screen results were further validated in a subset of both previously RNAi-screened and non-screened additional cancer lines using 3 of potent doxycycline-inducible shRNAs against DDX17. Interestingly, in comparison to DDX17-sensitive p53R248W/- mutant knockin cell line, the isogenic line with complete deletion of p53 was not dependent on DDX17, suggesting that the presence of p53 missense mutations, rather than loss of p53 function per se determines sensitivity to DDX17 inhibition. Furthermore, immunofluorescence assay showed DDX17 co-localized with mutant p53 in the nucleus but not with wild type p53, which indicate DDX17 might directly interact with mutant p53 at protein level. Collectively, these preliminary findings led us to hypothesize that DDX17 may be synthetic lethal to p53 missense mutants. To test the hypothesis, this proposal aims to functionally validate the synthetic lethality of DDX17 to p53 missense mutants in vivo, and to define underlying molecular mechanisms of interaction of DDX17 with mutant p53. The findings of this proposal would present DDX17 as a potential therapeutic target for cancers with p53 mutations, which is a high-unmet medical need worldwide.

抑癌基因p53在正常细胞中应对外界各种应激，对于维护基因组稳定起着重要保护作用，而突变p53是诱发肿瘤及产生放化疗抵抗的关键机制。肿瘤谱显示超过50%以上存在p53突变，并以错义突变为主。可见，靶向突变p53是肿瘤防治的重大突破口，也是国际科研舞台攻关的焦点，但至今尚未发现靶向突变p53有效途径。前期，运用大规模RNAi库体外功能筛选平台，我们发现RNA解旋酶DDX17与突变型p53存在协同致死效应；进一步体外实验支持抑制DDX17可导致突变p53肿瘤细胞死亡的科学假设；此外，研究还发现DDX17与突变型p53在细胞内可能存在相互作用。为深入探讨DDX17与突变型p53的协同效应及分子机制，我们拟运用多种前沿生物技术并结合体内功能实验，系统深入展开研究。本项目成果有望为DDX17成为p53突变肿瘤治疗新靶点提供重要科学依据,为肿瘤靶向治疗开拓新的契机。

抑癌基因p53是人类肿瘤中最常见的突变基因。针对突变型P53进行药物开发一直是肿瘤靶向治疗领域的研究热点。我们前期通过在p53突变型和p53野生型细胞群中运用RNAi技术进行高通量功能筛选，我们发现当RNA解旋酶DDX17缺失时，p53突变型肿瘤细胞的生长受到显著抑制，而p53野生型细胞生长未见明显改变。随后在该基金支持下对DDX17的协同致死作用进行验证，并对DDX17与突变型p53相互作用的潜在分子机制进行系统研究。结果发现在突变型p53细胞上，敲低DDX17抑制细胞增殖并且诱导凋亡，而DDX17发挥协同致死作用依赖于突变型p53；DDX17与突变型p53共定位于核内，且其N端和解旋酶结构域与p53的N端发生相互作用。在体内，DDX17敲降选择性抑制突变型p53肿瘤细胞生长。本项目成果为p53突变肿瘤治疗新靶点提供重要科学依据，并且在PNAS、BMC Medicine、Antioxidants & Redox Signaling、PLoS Genetics等期刊发表系列重大原创性成果，获得专利授权一项，培养博士硕士研究生13人，博士后2人。