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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA109821-10
Application #
8677736
Study Section
Basic Mechanisms of Cancer Therapeutics Study Section (BMCT)
Program Officer
Arya, Suresh
Project Start
2004-08-01
Project End
2015-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
10
Fiscal Year
2014
Total Cost
$220,391
Indirect Cost
$66,762
Name
University of Arkansas for Medical Sciences
Department
Biochemistry
Type
Schools of Medicine
DUNS #
122452563
City
Little Rock
State
AR
Country
United States
Zip Code
72205
Sakurikar, Nandini; Eichhorn, Joshua M; Alford, Sarah E et al. (2014) Identification of a mitotic death signature in cancer cell lines. Cancer Lett 343:232-8
Eichhorn, Joshua M; Alford, Sarah E; Sakurikar, Nandini et al. (2014) Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival. Exp Cell Res 322:415-24
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
Terrano, David T; Upreti, Meenakshi; Chambers, Timothy C (2010) Cyclin-dependent kinase 1-mediated Bcl-xL/Bcl-2 phosphorylation acts as a functional link coupling mitotic arrest and apoptosis. Mol Cell Biol 30:640-56
Bene, Anca; Chambers, Timothy C (2009) p21 functions in a post-mitotic block checkpoint in the apoptotic response to vinblastine. Biochem Biophys Res Commun 380:211-7
Bateman, T David; Joshi, Aarti L; Moon, Kwangyul et al. (2009) Synthesis and anticancer activity of sclerophytin-inspired hydroisobenzofurans. Bioorg Med Chem Lett 19:6898-901
Chu, Rong; Upreti, Meenakshi; Ding, Wen-Xing et al. (2009) Regulation of Bax by c-Jun NH2-terminal kinase and Bcl-xL in vinblastine-induced apoptosis. Biochem Pharmacol 78:241-8
Upreti, Meenakshi; Chu, Rong; Galitovskaya, Elena et al. (2008) Key role for Bak activation and Bak-Bax interaction in the apoptotic response to vinblastine. Mol Cancer Ther 7:2224-32

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