Understanding the molecular mechanisms of action of antitumor agents is significant for advancing fundamental knowledge and for improvement in cancer treatment. By defining these mechanisms, new drug targets can be uncovered;points of vulnerability in tumor cells versus normal cells exploited;novel drug combinations tested;and determinants of drug sensitivity revealed. Thus a major long-term goal is to use this information to more accurately predict clinical response, such that patients most likely to benefit can be identified, and treatment and attendant adverse side-effects avoided for those unlikely to benefit. Microtubule inhibiting agents (MIAs) including the vinca alkaloids and taxanes are important drugs in the arsenal of cancer chemotherapeutics. These drugs typically induce mitotic arrest leading to sustained activation of the spindle checkpoint and subsequent apoptotic cell death. Remarkably however, despite their widespread use, a solid molecular explanation of how cells die after spindle checkpoint activation has yet to emerge. The most prominent effect elicited by microtubule inhibitors is the phosphorylation of the antiapoptotic proteins, Bcl-2 and Bcl-xL. In the previous funding cycle, studies were undertaken to test our main hypothesis, that Bcl-2/Bcl-xL phosphorylation is a key event controlling apoptosis induction by anti-mitotic drugs and is catalyzed by a novel or unsuspected kinase. We were successful in largely completing the aims and validating our hypothesis, and excitingly, obtained evidence implicating CDK1/cyclin B as the kinase responsible for MIA-induced Bcl-2/Bcl-xL phosphorylation. Such a redirection in CDK1 function, from pro- proliferative during normal mitosis to pro-apoptotic after MIA-induced mitotic arrest, is conceptually and mechanistically appealing. Further, we showed that CDK1 partially and transiently phosphorylates Bcl-2/Bcl-xL during normal mitosis. The results provide compelling evidence that CDK1-mediated Bcl-2/Bcl-xL phosphorylation acts as a functional link coupling mitotic arrest and apoptosis, and suggest the possibility that anti-apoptotic Bcl-2 proteins act as sensors for CDK1 signal duration. In this proposal we will draw on these advances and propose the following Specific Aims.
Specific Aim 1 will test the hypothesis that Bcl-2/Bcl-xL phosphorylation regulates protein:protein interaction, in particular the binding/release of pro-apoptotic Bcl-2 proteins and especially activator and/or sensitizer BH3-only proteins.
In Specific Aim 2, we will examine the role of Mcl-1 phosphorylation in MIA-induced apoptosis, based on preliminary data that CDK1 mediates Mcl-1 degradation.
Specific Aim 3 will investigate the mechanisms of pro-apoptotic signaling by CDK1 and anti- apoptotic Bcl-2 proteins.
Specific Aim 4 will use blasts derived from pediatric patients with acute lymphoblastic leukemia to establish the role of CDK1 activation in vincristine sensitivity in a clinically relevant setting. At the completion of these experiments, we hope to have gained considerable new insight into the molecular mechanisms of action of this important class of cancer drugs.
Bcl-2 Proteins in Mechanism of Anti-mitotic Drug Action Narrative: Anti-tumor drugs are widely used in cancer treatment. However, we do not know exactly how they function, because after they cause initial damage to a cell, the damage is interpreted and acted upon through complex intracellular signaling pathways. We are investigating these damage sensing pathways for drugs such as Taxol. Understanding these pathways is important because the knowledge gained provides a basis for the appropriate use of existing agents and a theoretical basis for the discovery of new agents and targets. Most anti-tumor agents are toxic to normal cells and produce undesirable side-effects. Knowledge of the mechanism of these drugs is vital for the proper management of cancer patients especially when drug combinations are used.
|Kothari, Anisha; Hittelman, Walter N; Chambers, Timothy C (2016) Cell Cycle-Dependent Mechanisms Underlie Vincristine-Induced Death of Primary Acute Lymphoblastic Leukemia Cells. Cancer Res 76:3553-61|
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|Eichhorn, Joshua M; Kothari, Anisha; Chambers, Timothy C (2014) Cyclin B1 overexpression induces cell death independent of mitotic arrest. PLoS One 9:e113283|
|Eichhorn, J M; Alford, S E; Hughes, C C et al. (2013) Purported Mcl-1 inhibitor marinopyrrole A fails to show selective cytotoxicity for Mcl-1-dependent cell lines. Cell Death Dis 4:e880|
|Pawlowska, Monika; Chu, Rong; Fedejko-Kap, Barbara et al. (2013) Metabolic transformation of antitumor acridinone C-1305 but not C-1311 via selective cellular expression of UGT1A10 increases cytotoxic response: implications for clinical use. Drug Metab Dispos 41:414-21|
|Eichhorn, J M; Sakurikar, N; Alford, S E et al. (2013) Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death. Cell Death Dis 4:e834|
|Chu, Rong; Terrano, David T; Chambers, Timothy C (2012) Cdk1/cyclin B plays a key role in mitotic arrest-induced apoptosis by phosphorylation of Mcl-1, promoting its degradation and freeing Bak from sequestration. Biochem Pharmacol 83:199-206|
|Sakurikar, Nandini; Eichhorn, Joshua M; Chambers, Timothy C (2012) Cyclin-dependent kinase-1 (Cdk1)/cyclin B1 dictates cell fate after mitotic arrest via phosphoregulation of antiapoptotic Bcl-2 proteins. J Biol Chem 287:39193-204|
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