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.
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.
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|Abbas, Tarek; Dutta, Anindya (2017) Regulation of Mammalian DNA Replication via the Ubiquitin-Proteasome System. Adv Exp Med Biol 1042:421-454|
|Kumar, Pankaj; Dillon, Laura W; Shibata, Yoshiyuki et al. (2017) Normal and Cancerous Tissues Release Extrachromosomal Circular DNA (eccDNA) into the Circulation. Mol Cancer Res 15:1197-1205|
|Lee, Kyung Yong; Im, Jun-Sub; Shibata, Etsuko et al. (2017) ASF1a Promotes Non-homologous End Joining Repair by Facilitating Phosphorylation of MDC1 by ATM at Double-Strand Breaks. Mol Cell 68:61-75.e5|
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|Shibata, Etsuko; Kiran, Manjari; Shibata, Yoshiyuki et al. (2016) Two subunits of human ORC are dispensable for DNA replication and proliferation. Elife 5:|
|Dillon, Laura W; Kumar, Pankaj; Shibata, Yoshiyuki et al. (2015) Production of Extrachromosomal MicroDNAs Is Linked to Mismatch Repair Pathways and Transcriptional Activity. Cell Rep 11:1749-59|
|Lee, Kyung Yong; Dutta, Anindya (2015) Initiation of replication in Xenopus egg extracts at a spatially defined origin. Cell Cycle 14:2391|
|Lee, Kyung Yong; Im, Jun-Sub; Shibata, Etsuko et al. (2015) MCM8-9 complex promotes resection of double-strand break ends by MRE11-RAD50-NBS1 complex. Nat Commun 6:7744|
|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|
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