Novel Cell Cycle Therapeutic Targets in Pancreatic Cancer

描述(申请人提供)：胰腺癌在确诊后5年存活率为4%，是一种毁灭性的疾病，目前的治疗方案基本上仍无法治愈，是癌症死亡的第四大原因。因此，迫切需要在胰腺癌细胞中确定新的细胞靶点，以利用这些靶点进行治疗。在包括胰腺癌细胞在内的所有肿瘤细胞中，细胞周期检查点的转换依赖于CDK激活激酶(CAK)激活细胞周期蛋白：CDK复合体。CAK代表了一种潜在的新的阿喀琉斯跟腱类型的靶点，用于胰腺癌的治疗干预治疗，将影响细胞周期中的许多位置。然而，CAK的身份仍然不清楚。一个高相对分子质量的CAK复合体先前被鉴定为细胞周期蛋白H：CDK7；然而，细胞周期蛋白H：CDK7也是TFIIH中的真正的激酶，TFIIH是一种激活RNA聚合酶II以启动转录的激酶。因此，所有破坏CDK7可能的CAK活性的实验也导致了CDK7 TFIIH转录延长的整体中断，从而使解释成为问题。此外，我们发现细胞中CDK7的选择性失活导致体内CAK活性的维持，从而质疑CDK7的S作为CAK的作用。此前，我们已经检测到第二个低分子量CAK活性，该活性完全独立于CDK7。我们的中心假设是，低分子量复合体包含相关的CDK激活激酶(CAK)，它可以磷酸化并激活细胞周期蛋白：CDK复合体，这是驱动胰腺癌细胞通过细胞周期检查点所必需的。如果正确，这种CAK是一种潜在的新的治疗靶点 胰腺癌的治疗，其失活会同时影响多个细胞周期检查点。我们的R21探索性应用的目标是分离低分子量的CAK基因(S)，并确定CAK是否对周期蛋白：CDK的激活具有限速作用。目的：生化纯化和鉴定低分子量钙激活蛋白激酶基因(S)我们将从胰腺癌细胞中分离出低分子量钙激活蛋白激酶复合体，该复合体含有结构性活性的细胞周期蛋白依赖性蛋白激酶2，因此，根据定义，也含有结构性表达的钙激活蛋白激酶，并用质谱仪鉴定其序列，并通过RNA干扰进行确认。目的2.应用无偏倚的RNA干扰技术从胰腺细胞中鉴定S的钙调蛋白激酶基因。我们将在Panc1胰腺癌细胞中进行无偏见的Kinome RNAi筛选。在人类基因组中有大约448个已证实的和可能的Ser/Thr激酶，因此，这是一个使用高验证、高滴度的慢病毒shRNA文库进行的低通量、高信息量的RNAi筛选。

DESCRIPTION (provided by applicant): With a 4% survival rate at 5 years after diagnosis, pancreatic cancer is a devastating disease that remains essentially incurable with current therapeutic regimens and is the fourth leading cause of cancer mortality. Thus, there is a great need to identify novel cellular targets in pancreatic cancer cells that can be exploited for therapeutic benefit. In all tumor cells, including pancreatic cancer cells, transition across cell cycle checkpoints is dependent on activation of cyclin:Cdk complexes by Cdk Activating Kinase (CAK). CAK represents a potential novel Achilles' heel type of target for therapeutic intervention treatment in pancreatic cancer that would impact many positions in the cell cycle. However, the identity of CAK remains unclear. A high molecular weight CAK complex was previously identified as cyclin H:Cdk7; however, cyclin H:cdk7 is also the bona fide kinase in TFIIH, a kinase that activates RNA polymerase II for initiation of transcription. Consequently, all experiments that disrupted the putative CAK activity of Cdk7 also resulted in global disruption of transcriptional elongation by Cdk7 TFIIH, thereby making interpretation problematic. Moreover, we find that selective inactivation of Cdk7 in cells results in the maintenance of in vivo CAK activity and thereby questions Cdk7's role as a CAK. Previously, we have detected a second Low Molecular Weight CAK activity that is entirely independent of Cdk7. Our central hypothesis is that the low molecular weight complex contains the relevant Cdk Activating Kinase (CAK) that phosphorylates and activates cyclin:Cdk complexes required to drive pancreatic cancer cells across cell cycle checkpoints. If correct, this CAK is a potential novel therapeutic target for the treatment of pancreatic cancer whose inactivation would simultaneously affect multiple cell cycle checkpoints. Our goal for this R21 exploratory application is to isolate the Low Mw CAK gene(s) and determine if CAK is rate-limiting for cyclin:Cdk activation. Aim 1: Biochemically Purify and Identify the Low Mw CAK Gene(s) We will isolate the Low Mw CAK complex from Panc1 pancreatic cancer cells that contain constitutively active Cdk2, and hence, by definition, also contain constitutively express CAK, and identify it's sequence by mass spectrometry and confirm by RNAi. Aim 2. Unbiased RNAi Screen to Identify the CAK Gene(s) from Pancreatic Cells. We will perform an unbiased kinome RNAi screen in Panc1 pancreatic cancer cells. There are ~448 proven and putative Ser/Thr kinases in the human genome and therefore, this is manageable as a low throughput, but high information generating RNAi screen using a high validated, high titer lentiviral shRNA library.