Abstract 3069: The S-phase checkpoint function of MGMT provides an unexpected rational route to profoundly increase the efficacy of anticancer antimetabolites in cell culture and xenograft models
Authors: Mostofa AGM , Basak Debasish , Madala Hanumantha , Punganuru Surendra R. , Srivenugopal Kalkunte S. 
Published: July 2017

Abstract
MGMT is an anti-mutagenic DNA repair protein highly expressed not only in brain tumors, but also breast cancers, melanoma and colon cancers, and confers drug resistance. Recently, we showed that human MGMT has non-repair functions with vital roles in cell cycle; MGMT underwent PCNA-dependent degradation in the S-phase along with p21cip1 and CDT1 proteins. MGMT inactivation by O6-benzylguanine (BG) or a pool of shRNAs in asynchronous or G1/S-phase synchronized cells led to the activation of a checkpoint resulting in a significant inhibition of DNA synthesis and greatly diminished progression of cells into S-phase. We hypothesized that the replication stress caused by MGMT inactivation will provide a means to combine the S-phase specific drugs for synergistic cytotoxicity. We screened the sensitivity of a panel of colon cancer and glioblastoma cell lines with this combination using MTT and clonogenic growth assays. Annexin V/PI and acridine orange/ EtBr cell stainings were used to quantify apoptosis. MGMT inhibition/depletion significantly reduced the IC50 (5 to 10-fold) for different antimetabolites (5-FU, gemcitabine, Ara-C) and enhanced the overall apoptotic cell death (&gt;5-fold). Nevertheless, the synergistic effects strongly depended on time and administration schedule of these two agents. Thus, a pretreatment of BG or MGMT shRNA followed by 5-FU for or a simultaneous treatment of the two were far less cytotoxic than the antimetabolite first followed by BG or shRNA as in the post-treatment mode. While the pre or simultaneous MGMT inhibition significantly prolonged the antimetabolite-induced S-phase arrest, the BG-post-treatments resulted in the abrogation of antimetabolite-induced cell cycle block and rapid progression into premature mitosis and apoptosis. We observed an increased activation of checkpoint kinases (pChk1 or pChk2) during pre or simultaneous treatments. In contrast, the post-MGMT inhibition interfered with S-phase checkpoint activity and had an abnormal CDK2 activation. Finally, we verified the therapeutic potentiation of antimetabolites by BG using the HT29-luc2 subcutaneous xenografts developed in nude mice. BG (60mg/Kg), 5-FU (30 mg/Kg), gemcitabine (100 mg/Kg) and Ara-C (40 mg/Kg/dose) were administered through i.p. route once a week for 7 weeks. Tumor growth revealed a 50 to 70% more reduction in combination groups (antimetabolites+BG) compared with drugs alone. No significant changes in mouse body weight, liver functions, and whole blood counts were noted. A marked downregulation of ki-67 and increased levels of apoptotic markers (cleaved caspase-3, cleaved PARP) in was evident in tumor tissues. Collectively, our data suggest that MGMT inhibition is a rational strategy not only for alkylating agents but also for antimetabolites as well (supported by CPRIT RP130266 & RP170207 grants to KSS).
Citation Format: AGM Mostofa, Debasish Basak, Hanumantha Madala, Surendra R. Punganuru, Kalkunte S. Srivenugopal. The S-phase checkpoint function of MGMT provides an unexpected rational route to profoundly increase the efficacy of anticancer antimetabolites in cell culture and 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 3069. doi:10.1158/1538-7445.AM2017-3069