Abstract 4473: The NO-releasing aspirin inactivates and degrades human MGMT more efficiently than O6-benzylguanine and greatly sensitizes brain tumor cells to alkylating agents.
Authors: Srivenugopal Kalkunte S. , Paranjpe Ameya S. 
Published: August 2014

The DNA repair protein MGMT (O6-methylguanine-DNA methyltransferase) is a major determinant of tumor resistance to alkylating agents. Current clinical strategies involving O6-benzylguanine (BG) to inhibit MGMT and increase the drug efficacy have not been successful due to cumulative and unmanageable bone marrow toxicity. Although the gene therapy of hematopoietic stem cells with BG-resistant MGMT has been proposed, such practices are likely expensive and unpractical for routine clinical use. Therefore, there is an urgent need for finding new and transient inhibitors of MGMT. In a new approach, we exploited the highly reactive nature of Cysteine 145 which accepts the alkyl groups for novel drug design. Cys145 has a pKa of 4.8 and is susceptible for thiolation and nitrosylation, both of which inactivate the MGMT. In line with this, nitroaspirin (NCX-4016, NA), a cardioprotective drug was found to be an extremely potent inhibitor of MGMT, far superior to BG. Nitroaspirin is degraded by plasma and tissue esterases to release NO and aspirin in tissues. NA at pharmacologically achievable concentrations of 10-20 μM caused 100% inhibition of MGMT within 1 h in a large panel of human cancer cell lines including gliomas. The nitrosylated MGMT protein disappeared completely within 2 h. These data are highly comparable and superior to those elicited by BG. Using mutant MGMT proteins, we showed the active site cysteine (Cys145) and a neighboring Tyr114, a residue critical for DNA repair, were both modified by nitroaspirin. The ubiquitin-proteasome pathway mediated the degradation of nitrosylated MGMT. In cultured cells, MGMT suppression by NA was less prolonged than BG (80% regeneration at 40 h post NA exposure compared to 20% that by BG in different cell lines). However, pre-exposure of tumor cells to NA followed by BCNU resulted in (i) 2.5-fold greater levels of DNA interstrand crosslinks, (ii) a 4-fold increased G2/M cell cycle arrest, and (iii) 2-4 fold increased cell killing in various tumor cell lines. A single injection of NA (100 mg/kg) caused 50-60% inhibition of MGMT activity in mouse brain and liver. Because NA is a very nontoxic (IC50 &gt;500 μM for most cell lines), is lipophilic enough to cross the BBB, and its pleiotropic actions actually benefit chemotherapy, the creation of an MGMT-deficient state by NA holds unequivocal promise for improving the therapy of primary and metastatic brain tumors (supported by RO3 CA125872 and the Association for Research of Childhood Cancer).

Citation Format: Kalkunte S. Srivenugopal, Ameya S. Paranjpe. The NO-releasing aspirin inactivates and degrades human MGMT more efficiently than O6-benzylguanine and greatly sensitizes brain tumor cells to alkylating agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4473. doi:10.1158/1538-7445.AM2013-4473