Novel Cell Cycle Therapeutic Targets in Pancreatic Cancer

描述（由申请人提供）：胰腺癌是一种毁灭性的疾病，诊断后5年生存率为4%，目前的治疗方案基本上无法治愈，是癌症死亡的第四大原因。因此，非常需要在胰腺癌细胞中识别新的细胞靶点，以用于治疗益处。在包括胰腺癌在内的所有肿瘤细胞中，细胞周期检查点的过渡依赖于Cdk激活激酶（CAK）对细胞周期蛋白：Cdk复合物的激活。CAK代表了胰腺癌治疗干预治疗中一种潜在的新型阿喀里斯之踵类型的靶标，它会影响细胞周期中的许多位置。然而，CAK的身份仍不清楚。高分子量的CAK复合物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中的真正激酶，一种激活RNA聚合酶II启动转录的激酶。因此，所有破坏Cdk7假定的CAK活性的实验也导致Cdk7 TFIIH转录延伸的全球破坏，从而使解释存在问题。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作为CAK的作用。此前，我们已经检测到第二个完全独立于Cdk7的低分子量CAK活性。我们的中心假设是，低分子量复合物含有相关的Cdk激活激酶（CAK），该激酶磷酸化并激活细胞周期蛋白：驱动胰腺癌细胞通过细胞周期检查点所需的Cdk复合物。如果正确的话，这个CAK是一个潜在的新的治疗靶点

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.