O6-Methylguanine-DNA methyltransferase (MGMT) is a highly promising target for improving the efficacy of alkylating agents in the treatment of brain tumors and other cancers. Recently, our laboratory provided the first data that human MGMT protein is phosphorylated at tyrosine and serine/threonines and that phosphorylation inhibits its activity. Two cellular protein kinases that phosphorylate these amino acids in the MGMT protein were also characterized. The overall objective of this proposal is to define the role of protein phosphorylation in MGMT function and to assess its significance to improve glioma therapy. We hypothesize that multisite phosphorylation of MGMT controls its catalytic activity, and protein stability in human gliomas. We postulate that the net phosphorylation state of tumor MGMT profoundly affects its function and, in turn the sensitivity of gliomas to alkylating agents.
The Specific Aims to be pursued in this 4-year project are (1) to identify the in vivo phosphorylation sites of human MGMT and define the contribution of each phosphorylation to MGMT's DNA repair activity, (2) to characterize the phosphorylation-dependent turn-over of the MGMT protein through the ubiquitin-proteolytic pathway, and (3) to generate phospho-specific antibodies against the MGMT protein, assess the levels of phospho-MGMT in glioma specimens, cell lines, and to correlate it with the catalytic activity of MGMT and drug sensitivity. The structural impact of phosphorylations on the DNA binding domain of MGMT, and the consequent alterations in MGMT activity towards O6-methylguanine, and O6-benzylguanine will also be examined as a part of the first Specific Aim. Changes in MGMT phosphorylation during the cell cycle progression will be determined by using the phospho-specific antibodies. These original studies promise to yield novel and critical information on the physiological regulation of MGMT, and are likely to provide new directions for improved glioma therapy and MGMT-targeted cancer therapies.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Experimental Therapeutics Subcommittee 1 (ET)
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Okano, Paul
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Texas Tech University
Schools of Pharmacy
United States
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Srivenugopal, Kalkunte S; Rawat, Amit; Niture, Suryakant K et al. (2016) Posttranslational Regulation of O(6)-Methylguanine-DNA Methyltransferase (MGMT) and New Opportunities for Treatment of Brain Cancers. Mini Rev Med Chem 16:455-64
Yusuf, Mohd A; Chuang, Trinette; Bhat, G Jayarama et al. (2010) Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines. Free Radic Biol Med 49:908-17
Velu, Chinavenmeni S; Niture, Suryakant K; Doneanu, Catalin E et al. (2007) Human p53 is inhibited by glutathionylation of cysteines present in the proximal DNA-binding domain during oxidative stress. Biochemistry 46:7765-80
Niture, Suryakant K; Velu, Chinavenmani S; Smith, Quentin R et al. (2007) Increased expression of the MGMT repair protein mediated by cysteine prodrugs and chemopreventative natural products in human lymphocytes and tumor cell lines. Carcinogenesis 28:378-89
Niture, Suryakant K; Rao, U Subrahmanyeswara; Srivenugopal, Kalkunte S (2006) Chemopreventative strategies targeting the MGMT repair protein: augmented expression in human lymphocytes and tumor cells by ethanolic and aqueous extracts of several Indian medicinal plants. Int J Oncol 29:1269-78
Niture, Suryakant K; Velu, Chinavenmeni S; Bailey, Nathan I et al. (2005) S-thiolation mimicry: quantitative and kinetic analysis of redox status of protein cysteines by glutathione-affinity chromatography. Arch Biochem Biophys 444:174-84
Niture, Suryakant K; Doneanu, Catalin E; Velu, Chinavenmeni S et al. (2005) Proteomic analysis of human O6-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry. Biochem Biophys Res Commun 337:1176-84