p21介导的细胞有丝分裂压力在KRAS突变肿瘤生长中的作用与分子机制研究

Oncogenic mutations in the GTPase protein RAS family occur in up to a third of all human cancers. KRAS is the principal isoform of RAS, and somatic mutations in KRAS are associated with over 20% of all cancers; a particularly high frequency of KRAS mutations is observed in pancreatic, colorectal, and lung cancers, three of the top four leading causes of new cancer deaths. Although RAS gene has been investigated for over 30 years, no effective targeted therapies exist for cancers with somatic KRAS mutations. In our previous work, we developed a synthetic lethal chemical screen in isogenic KRAS-mutant and wild-type cells to identify clinical drug pairs. Our results showed that dual inhibition of polo-like kinase 1 (Plk1) and RhoA/Rho kinase (ROCK) led to synergistic effects in KRAS-mutant cancers. This combined therapy-mediated overexpression of p21 preferentially impairs the growth of KRAS-mutant cells, suggesting a druggable synthetic lethal interaction between KRAS and CDKN1A (encoding p21 protein). In addition, we found that p21 overexpression specifically disrupted the formation of mitotic centrosomes and spindle assembly checkpoint in KRAS mutant cancer cells. Based on these interesting findings, we hypothesize that the growth of KRAS-mutant cancers are specifically dependent on p21-mediated mitotic stress. In this project, we will further pursue the role and mechanism of p21-mediated mitotic regulation in KRAS-muant cancers and elucidate its clinical significance. Ou study will reveal the novel signaling transduction for the KRAS dependency, and provide potential avenue for therapeutic treatment of KRAS-mutant cancers.

RAS 作为最恶性的癌基因促发人类三分之一肿瘤的发生，其家族成员KRAS在人类常见致死性肿瘤中频发突变。由于KRAS突变肿瘤的异质性，临床至今无有效的治疗靶向。我们课题组前期建立了协同致死化学筛选体系，将靶向药物作为探针研究KRAS突变肿瘤生长所依赖的分子机制，发现联合抑制Plk1激酶和ROCK激酶可显著激活KRAS突变细胞中周期调节蛋白p21，p21表达改变可特异性杀伤KRAS突变肿瘤，揭示CDKN1A（编码p21蛋白）是KRAS崭新的协同致死基因。后续研究发现，相比KRAS野生型细胞，p21显著影响KRAS突变细胞中心体分布和纺锤体形成，由此我们推测p21介导有丝分裂压力并直接参与了KRAS突变肿瘤的生长。本项目计划在前期工作基础之上，多角度、多层次地研究p21在KRAS突变肿瘤生长中的作用、分子机制和临床意义。本研究为阐明KRAS突变肿瘤的分子特征以及其临床诊治提供理论基础。

癌基因KRAS驱动了人类近20%恶性肿瘤的发生发展。虽然目前针对KRAS（G12C）突变体的小分子药物研发取得了突破性进展，但是在临床上伴随KRAS其他突变体的人类肿瘤仍然无药可医。我们前期研究揭示，细胞周期调节蛋白p21的表达对于KRAS突变肿瘤细胞的存活至关重要，编码p21的基因CDKN1A与癌基因KRAS具有合成致死关系。本项目以p21为研究切入点，探究KRAS突变肿瘤中p21的表达和调控特征，以及不同靶向策略对KRAS突变肿瘤生长的作用。我们首先构建了一系列含有KRAS不同突变位点的等基因细胞株。在等基因细胞株中，过表达p21能特异性地引起KRAS突变细胞周期阻滞，诱导有丝分裂异常和细胞死亡。我们通过分析KRAS等基因细胞株的转录组后发现，转录抑制因子BCL6在KRAS突变细胞中显著高表达，其作为KRAS信号通路的下游分子，可抑制p21的转录。基于此，我们的研究提示了p21受KRAS信号通路调控的新机制。在体内外肿瘤模型中，BCL6小分子抑制剂能显著提升KRAS突变肿瘤细胞中p21的表达，进而有效且特异性地抑制KRAS突变肿瘤细胞的克隆形成以及实体肿瘤的生长。此外，为了进一步开发针对p21且临床可转化的肿瘤靶向策略，我们选择并研究了p21诱导剂—临床组蛋白去乙酰化酶抑制剂（HDACi）对肿瘤生长的抑制作用。实验结果表明，在体外肿瘤水平上HDACi可有效抑制肿瘤细胞存活，但是在体内肿瘤模型中HDACi抑瘤药效并未达到预期，树突状细胞分泌的吲哚胺2,3-双加氧酶1的激活是肿瘤细胞抵抗HDACi治疗的潜在分子基础。总之，本项目的研究为KRAS突变肿瘤的治疗提供机制新视角和潜在新手段。