Abstract 1121: Redox-mediated inactivation of MGMT DNA repair through thiolation or nitrosylation of the active site cysteine145 sensitizes human gliomas to alkylating agents in vitro and in xenograft models
Authors: Basak Debasish , Srivenugopal Kalkunte S. 
Published: July 2017

Abstract
O6-Methylguanine-DNA methyltransferase (MGMT) is a simple and unique antimutagenic DNA repair protein that plays a crucial role in conferring tumor resistance to various alkylating agents in brain tumor therapy. MGMT repairs O6-alkylguanines by transferring the alkyl groups to its active site cysteine (Cys145) in a stoichiometric/suicidal reaction thereby restoring the original nucleotide sequence in a single step. Although O6-benzylguanine (BG), a pseudosubstrate inhibitor, currently in clinical trials, potently inhibits MGMT and improves the efficacy of alkylating agents, unacceptable levels of bone marrow toxicity has been a great concern. For developing new MGMT inhibitors, we exploited the reactive nature of the active site Cys145 (pKa 4.5) and its susceptibility for thiolation and nitrosylation, both of which inactivate the MGMT. A new strategy involving inhibition of S-nitosoglutathione reductase (GSNOR) was also explored to increase the steady-state levels of nitrosylated MGMT. First, we designed a redox perturbing glutathione mimetic, a homoglutathione disulfide (hGTX) that has been stabilized with the addition of small amounts of cisplatin (1000:10). Spermine NONOate and S-nitroso penicillamine (SNAP) served as the protein-nitrosylating agents. N6022, a specific and reversible inhibitor of GSNOR was used with nitrosylators in some assays. Both the hGTX and spermine NONOate at 100-400 µM concentrations for 24 h inhibited the repair activity of MGMT &gt;75% in HT29, SF188, and T98G cells. The NONOate strongly curtailed the DNA repair activity of recombinant MGMT showing a direct interaction. The inactivated MGMT protein was promptly eliminated from glioma cells. MGMT disappeared from cells much faster in spermine NONOate +N6022 treated cells, suggesting an extended steady-state of nitrosylated MGMT and its subsequent degradation. MGMT depletion was accompanied by 4-6 fold increases in cell killing by BCNU or temozolomide (TMZ). Binding of the biotinylated-O6-benzylguanine to the MGMT protein was prevented by hGTX and spermine NONOate treatments indicating the Cys145 was indeed the site of modification. In the nude mice bearing T98G or HT29-luc2 xenografts, combinations of hGTX and TMZ or spermine NONOate +TMZ produced markedly synergistic tumor regressions compared with TMZ alone. A reduction of MGMT protein as by immunoblotting and diminished MGMT activity levels in tumor tissues was verified. Apoptosis regulatory proteins were upregulated in excised tumors and H&E staining confirmed no discernible host organ toxicities. Our studies highlight the options for redox-driven therapeutic strategies for MGMT [supported by CPRIT grants RP130266 and RP170207 to KSS].
Citation Format: Debasish Basak, Kalkunte S. Srivenugopal. Redox-mediated inactivation of MGMT DNA repair through thiolation or nitrosylation of the active site cysteine145 sensitizes human gliomas to alkylating agents in vitro and in xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1121. doi:10.1158/1538-7445.AM2017-1121