Abstract 3627: Generation and characterization of phospho-specific antibodies against human O6-methylguanine-DNA methyltransferase (MGMT) DNA repair protein

The DNA repair protein MGMT (O6-methylguanine-DNA methyltransferase) is overexpressed in human brain cancers and its ability to reverse the O6-guanine alkylation damage induced by many alkylating agents is a major reason for resistance development and therapy failure. Studies in our laboratory have shown that phosphorylation is a negative regulatory mechanism for MGMT (Cancer Res 60, 282, 2000) and that Tyr114 is a major site for this modification (Proc AACR 46, Abst. 5426, 2005). Phosphorylated MGMT is inactive and therefore, the net content of the inactive phospho-MGMT in gliomas and other cancers has the potential to determine patient response to alkylating agents. To determine the (i) functional inactivation of MGMT due to Tyr114 phosphorylation, (ii) the consequent increases in the tumor sensitivity to alkylating agents and (iii) to assess the cellular significance of this event, we developed phospho-specific polyclonal antibodies against the peptide 106-120 (CKFGEVIS-pY-QQLAAL) encompassing the modified tyr114 site. The rabbit antibodies were affinity-purified by chromatography on phospho- and non-phospho peptide columns and further characterized. Specificity studies involving ELISA and western blotting of phosphorylated and non-phosphorylated protein fractions revealed that the antibody recognized only the phosphorylated MGMT, but not the recombinant or unmodified proteins. Immunoblot, immunocytochemical and flow cytometric analyses showed the presence of varied but significant amounts of phosphorylated MGMT (10-20% of cellular MGMT in phosphorylated state) in human tumor cell lines. Other experiments showed that phosphorylated MGMT was incapable of binding the DNA substrate or the biotinylated O6-benzylguanine. Molecular modeling showed that phospho-tyr114 fills up the substrate binding pocket, makes new salt bridges and prevents the flipping of O6-alkylated guanine from the base stack into the MGMT active site. Studies are ongoing to determine (i) the cell cycle phase-dependent phosphorylation of MGMT and its relationship with protein turnover, (ii) whether the abundance of phospho-MGMT in human cancers will predict improved drug-efficacy and (iii) the disposition of phospho vs. unphosphorylated MGMT. In conclusion, just similar to the silencing of MGMT expression through promoter methylation, the phosphorylated MGMT content (determined by the balance of kinase and phosphatase activities) in tumors may increase the alkylator efficacy and predict improved responses to therapy. The antibodies described here should aid efforts in this direction (supported by CPRIT RP130266 and NIH RO3CA125872 grants to KSS). Citation Format: Amit Rawat, Upendra Gumaste, Mohammed A. Yusuf, Suryakant K. Niture, Kalkunte S. Srivenugopal. Generation and characterization of phospho-specific antibodies against human O6-methylguanine-DNA methyltransferase (MGMT) DNA repair protein. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3627. doi:10.1158/1538-7445.AM2015-3627