In the last cycle of this project we discovered how easy it is for mammalian cells to bypass the regulatory pathways that ensure that origins of replication are licensed only once per cell-cycle and that the chromosomes are replicated once and only once per cell-cycle. Different manipulations that increased the ratio of the replication initiator, Cdt1, relative to its inhibitor, geminin, led to re-replication. Intriguingly, the extensive re-replication was accompanied by DNA damage and activation of checkpoint pathways that led to cell-cycle arrest and eventually cell-death. The checkpoint pathways used several genes whose mutations predispose individuals to genomic instability and cancer: p53, BrCa1 and the Fanconi Anemia proteins. These results lead us to the hypothesis that will be tested in the first Aim: small degrees of re- replication that are tolerated and are compatible with cancer cell viability lead to the deletions and gene amplifications that are the hallmark of cancer cell chromosomes. This will be the first model linking disorders in replication initiation in mammalian cells to chromosomal structural variation and the genomic instability of cancer. A "first-in-its-class" new drug has been developed by a pharmaceutical company and will be given to us. This drug, MLN4924, inhibits the ubiquitin ligases that help degrade Cdt1 in S phase, and as a result stabilizes Cdt1 and causes extensive re-replication, DNA damage and cytotoxicity.
Aim 2 will test the hypothesis that the cytotoxic effect of this drug is through the re-replication and activation of checkpoint pathways. If this hypothesis is true, we will provide a rationale for the development of other anti- cancer drugs that produce extensive re-replication and successfully predict whether the clinical trial of MLN4924 will benefit from a measurement of the status of p53, BrCa1 or Fanconi Anemia proteins in the tumors being treated. Although we have discovered two independent ubiquitin ligases that promote the polyubiquitinylation of Cdt1 and its degradation, preliminary results indicate that a third ubiquitin ligase of an entirely different class from the ones already implicated, has a critical role in destabilizing Cdt1. In the third Aim we will test the hypothesis that inhibition of this third ubiquitin ligases will also cause re-replication and cytotoxicity, determine the mechanism by which this ligase destabilizes Cdt1 and assess whether inhibition of this ligase will synergize with MLN4924 in cancer chemotherapy.

Public Health Relevance

Although genetic lesions like chromosomal deletions, amplifications and translocations are common in cancers, the mechanism by which these lesions arise is completely unclear. Our experiments will test a hypothesis about how these lesions arise and may suggest strategies to minimize the creation of these lesions during the treatment of cancers. New drugs for cancer chemotherapy are very important, and our experiments will elucidate how a new drug about to enter clinical trials hijacks the replication initiator process to cause extensive DNA damage in proliferating cells. Finally, the project is expected to yield another drug target whose inhibition will lead to cytotoxicity through this same pathway and so will be a great candidate for future drug discovery efforts.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Hildesheim, Jeffrey
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University of Virginia
Schools of Medicine
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Im, Jun-Sub; Keaton, Mignon; Lee, Kyung Yong et al. (2014) ATR checkpoint kinase and CRL1?TRCP collaborate to degrade ASF1a and thus repress genes overlapping with clusters of stalled replication forks. Genes Dev 28:875-87
Shibata, Etsuko; Dar, Ashraf; Dutta, Anindya (2014) CRL4Cdt2 E3 ubiquitin ligase and proliferating cell nuclear antigen (PCNA) cooperate to degrade thymine DNA glycosylase in S phase. J Biol Chem 289:23056-64
Dar, Ashraf; Wu, David; Lee, Nicholas et al. (2014) 14-3-3 proteins play a role in the cell cycle by shielding cdt2 from ubiquitin-mediated degradation. Mol Cell Biol 34:4049-61
Abbas, Tarek; Keaton, Mignon; Dutta, Anindya (2013) Regulation of TGF-* signaling, exit from the cell cycle, and cellular migration through cullin cross-regulation: SCF-FBXO11 turns off CRL4-Cdt2. Cell Cycle 12:2175-82
Abbas, Tarek; Keaton, Mignon A; Dutta, Anindya (2013) Genomic instability in cancer. Cold Spring Harb Perspect Biol 5:a012914
Jazaeri, Amir A; Shibata, Etsuko; Park, Jonghoon et al. (2013) Overcoming platinum resistance in preclinical models of ovarian cancer using the neddylation inhibitor MLN4924. Mol Cancer Ther 12:1958-67
Terai, Kenta; Shibata, Etsuko; Abbas, Tarek et al. (2013) Degradation of p12 subunit by CRL4Cdt2 E3 ligase inhibits fork progression after DNA damage. J Biol Chem 288:30509-14
Abbas, Tarek; Mueller, Adam C; Shibata, Etsuko et al. (2013) CRL1-FBXO11 promotes Cdt2 ubiquitylation and degradation and regulates Pr-Set7/Set8-mediated cellular migration. Mol Cell 49:1147-58
Dar, Ashraf; Shibata, Etsuko; Dutta, Anindya (2013) Deubiquitination of Tip60 by USP7 determines the activity of the p53-dependent apoptotic pathway. Mol Cell Biol 33:3309-20
Abbas, Tarek; Keaton, Mignon; Dutta, Anindya (2013) Regulation of TGF-* signaling, exit from the cell cycle, and cellular migration through cullin cross-regulation: SCF-FBXO11 turns off CRL4-Cdt2. Cell Cycle 12:

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