Cyclin-Dependent Kinase Inhibition During S Phase

描述(申请人提供)：细胞周期蛋白依赖性蛋白激酶(CDK)调节细胞周期进程和RNA转录。这项工作将集中在CDK与DNA损伤途径的相互作用。CDK是ATM(共济失调-毛细血管扩张突变)和ATR(ATM和RAD3相关)检查点级联反应DNA损伤的主要靶点，它们的抑制促进了细胞周期的停滞和修复。然而，越来越多的证据表明，在最终下调之前，CDK调控着DNA损伤反应的许多上游元件。初步数据表明，CDK1参与了DNA损伤后依赖BRCA1的S期检查点的调控。在shRNA介导的CDK1耗尽或小分子介导的CDK1抑制后，检查点控制无效，从而使癌细胞对DNA损伤治疗敏感，包括顺铂、γ-射线和核苷类似物。在第一个特定目标中，将评估CDK1介导的BRCA1在S1497处的磷酸化的作用，以确定这一事件是否对肺癌和乳腺癌细胞中的检查点控制是必要的。由于CDK1的缺失损害了BRCA1的功能，CDK1是否参与同源重组修复，或者它的缺失是否使细胞对PARP-1抑制敏感，也将被研究。将使用未转化的细胞系和成对的转化衍生物来研究癌细胞对CDK1耗尽所造成的DNA损伤的选择性敏化。CDK1缺失细胞对顺铂和PARP-1抑制的敏感性也将在体内通过异种移植模型得到证实。最后，RNA干扰筛选将被用来定义DNA损伤和其他途径中的新靶点，这些途径的耗尽可能会增强对CDK1耗尽或抑制的反应。为了获得与临床使用的抑制多个CDK家族成员的CDK抑制剂相关的结果，第二个特定目标将集中在CDK2、CDK1和CDK9联合缺失的细胞上。这一CDKs亚群的耗尽或抑制可诱导细胞凋亡。CDK2/1依赖的导致细胞死亡的事件将被描述为包括S期减慢，检查点激酶1耗尽，诱导DNA损伤反应，以及检查点控制受损促进内复制和重新进入S期，而S期是细胞最脆弱的阶段。此外，还将研究CDK9介导的转录抑制cdk在调节细胞凋亡阈值中的作用。还将评估CDK2、CDK1和CDK9抑制剂与DNA损伤剂和PARP-1抑制剂的相互作用。RNA干扰筛选将被用来询问联合CDK耗尽或抑制与DNA损伤和其他途径中的新靶点的交集。在第三个具体目标中，药效学终点将纳入相关临床试验。其中包括具有纳摩尔效力的CDKs 2、1和9的抑制剂SCH727965的1期试验，以及核苷类似物沙巴滨与CDK抑制剂seliclib的联合试验，后者有望增强DNA损伤反应。

DESCRIPTION (provided by applicant): Cyclin-dependent kinases (cdks) regulate cell cycle progression and RNA transcription. This work will focus on the interaction of cdks with DNA damage pathways. Cdks are major targets of ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) checkpoint cascades in response to DNA damage, and their inhibition promotes cell cycle arrest and repair. However, accumulating evidence suggests that cdks regulate many upstream elements of the DNA damage response, prior to their ultimate downregulation. Preliminary data indicate that cdk1 participates in BRCA1-dependent S phase checkpoint control following DNA damage. After shRNA-mediated cdk1 depletion or small molecule-mediated cdk1 inhibition, checkpoint control is ineffective, so that cancer cells are sensitized to DNA damaging treatments, including cisplatin, ?-irradiation and nucleoside analogs. In the first specific aim, the role of cdk1-mediated phosphorylation of BRCA1 at S1497 will be assessed to determine if this event is necessary for checkpoint control in lung and breast cancer cells. Since cdk1 depletion compromises BRCA1 function, whether cdk1 participates in homologous recombination repair or whether its depletion sensitizes cells to PARP-1 inhibition will also be studied. The selective sensitization of cancer cells to DNA damage by cdk1 depletion will be investigated using non-transformed cell lines and paired transformed derivatives. The sensitivity of cdk1-depleted cells to both cisplatin and PARP-1 inhibition will also be confirmed in vivo using xenograft models. Finally, RNA interference screens will be used to define novel targets in DNA damage and other pathways, depletion of which may augment the response to cdk1 depletion or inhibition. In order to derive results relevant to cdk inhibitors in clinical use, which inhibit multiple cdk family members, the second specific aim will focus on cells in which there is combined depletion of cdk2, cdk1 and cdk9. Depletion or inhibition of this subset of cdks induces apoptosis. The cdk2/1-dependent events leading to cell death will be characterized, including S-phase slowing, depletion of checkpoint kinase 1, induction of a DNA damage response, and impaired checkpoint control facilitating endoreduplication and re-entrance into S phase, where cells are the most vulnerable. Additionally, the role of cdk9-mediated transcriptional cdk inhibition in modulating the apoptotic threshold will be investigated. The interaction of inhibitors of cdk2, cdk1 and cdk9 with DNA damaging agents and PARP-1 inhibitors will also be assessed. RNA interference screens will be used to interrogate the intersection of combined cdk depletion or inhibition with novel targets in DNA damage and other pathways. In the third specific aim, pharmacodynamic endpoints will be incorporated into relevant clinical trials. These include a Phase 1 trial of SCH727965, an inhibitor of cdks 2, 1, and 9 with nanomolar potency, as well as a combination trial of the nucleoside analog sapacitabine with the cdk inhibitor seliciclib, the latter expected to augment the DNA damage response.