Ubiquitin-dependent proteolysis plays a significant role in various physiological processes including cell cycle control and cellular proliferation and is frequently the target of oncogenic transfomation. The specficity of protein substrate ubiquitylation is dictated by the activity of one of many E3 ubiquitin ligases either directly or through substrate recognition through adaptor proteins. Among these, the Cullin 4-based E3 ubiquitin ligase (CRL4) is emerging as a master regulator of cellular proliferation and genomic stability and is involved in multiple DNA repair processes. Cdt2/DTL, a WD-repeat containing protein associates with CRL4 (CRL4-Cdt2) and functions as a substrate recognition factor for recuiting substrates to the rest of the CRL4 ubiquitin ligase. CRL4-Cdt2 has recently been shown to promote the ubiquitin-dependent destruction of the replication initiation protein Cdt1 and the cyclin-dependent kinase (CDK) inhibitor p21, both in S-phase of the cell cycle and in response to UV irradiation. Significantly, Cdt2 is frequently overexpressed in a variety of human tumors and its expression correlates with tumor grade, metastasis and poor survival. This study aims at understanding how CRL4-Cdt2 impacts on genomic stability and contributes to cancer development. Specifically, I will A) Identify and characterize novel substrates for the CRL4-Cdt2 E3 ubiquitin ligase complex. B) Identify the mechanism by which Cdt2 negatively regulates p21 transcription and C) Test whether Cdt2 exhibits oncogenic activity in vivo. Using tap-tandem purification and mass-spectrometry analysis of Cdt2-associated proteins from human cells with or without DNA damage, I will identify new CRL4-Cdt2 substrates that may be involved in cellular proliferation and/or DNA repair. Standard biochemical techniques, including in vivo and in vitro ubiquitylation assays using purfied CRL4-Cdt2 E3 ubiquitin ligase complexes, will verify whether the identified proteins are bona fide substrates. I will also generate transgenic mice overexpressing Cdt2 from Cre/LoxP constructs and test whether Cdt2 overexpression contributes to tumor development in animal model system. The results will advance our understanding of how to exploit the regulated process of protein ubiquitylation and proteolysis for cancer intervention purposes.

Public Health Relevance

One of the fundamental challenges in treating cancer is the lack of a comprehensive understanding of the various players that are important for the survival of cancer cells. This proposal aims at identifying proteins essential for cellular proliferation and DNA repair that are targeted for destruction by a critical enzyme that is involved in the regulation of these processes. It will also test the enzymes'role in tumor development.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Transition Award (R00)
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No Study Section (in-house review) (NSS)
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Hildesheim, Jeffrey
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University of Virginia
Schools of Medicine
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Olmez, Inan; Zhang, Ying; Manigat, Laryssa et al. (2018) Combined c-Met/Trk Inhibition Overcomes Resistance to CDK4/6 Inhibitors in Glioblastoma. Cancer Res 78:4360-4369
Vanderdys, Vanessa; Allak, Amir; Guessous, Fadila et al. (2018) The Neddylation Inhibitor Pevonedistat (MLN4924) Suppresses and Radiosensitizes Head and Neck Squamous Carcinoma Cells and Tumors. Mol Cancer Ther 17:368-380
Abbas, Tarek; Dutta, Anindya (2017) Regulation of Mammalian DNA Replication via the Ubiquitin-Proteasome System. Adv Exp Med Biol 1042:421-454
Benamar, Mouadh; Guessous, Fadila; Du, Kangping et al. (2016) Inactivation of the CRL4-CDT2-SET8/p21 ubiquitylation and degradation axis underlies the therapeutic efficacy of pevonedistat in melanoma. EBioMedicine 10:85-100
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Abbas, Tarek; Keaton, Mignon A; Dutta, Anindya (2013) Genomic instability in cancer. Cold Spring Harb Perspect Biol 5:a012914
Park, Jonghoon; Long, David T; Lee, Kyung Yong et al. (2013) The MCM8-MCM9 complex promotes RAD51 recruitment at DNA damage sites to facilitate homologous recombination. Mol Cell Biol 33:1632-44
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
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

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