Abstract 296: Interplay of human MGMT DNA repair protein with PCNA / p21cip1 and MGMT’s novel role as an S-phase checkpoint

Abstract

Abstract O6-Methylguanine-DNA methyltransferase (MGMT) is a critical antimutagenic DNA repair protein that protects the genome and an established target for improving the efficacy of alkylating agents. In contrast with the stoichiometric repair reaction performed by a single small MW protein, we show for the first time that MGMT in human glioblastoma cells specifically associates with PCNA, p21cip1, and undergoes selective degradation at mid-S-phase along with replication-licensing components to maintain genomic integrity. First, we identified a PCNA-Interacting Protein (PIP box) motif between amino acids 61-70, QCTAWLNAYF in the MGMT protein. PCNA encircles the DNA and functions as a sliding clamp by interacting with DNA metabolic proteins having a PIP-box to make the replication processive. In p53-null H1299 lung cancer cells engineered to express the p21cip1, either by Tet-off conditional or lentiviral stable transfections, a reciprocal immunoprecipitation/western blot analyses using antibodies to PCNA or MGMT confirmed the specific association of MGMT and PCNA proteins. Expression of the CDK inhibitor p21 disrupted the interaction between PCNA and MGMT in cells, indicating its regulatory role in DNA repair during cell cycle blockade. Confocal immunofluorescence imaging in glioblastoma cells and isogenic HCT116 cells with and without p21cip1 expression, showed a co-localization of MGMT and PCNA proteins in glioma cells; when cells were subjected to alkylation DNA damage, the co-localization pattern was punctate and more prominent, suggesting that PCNA functions to recruit the repair protein to the damage sites. To probe the cell-cycle dependent regulation of MGMT, we used synchronized human GBM cells at the G1/S boundary using double thymidine-block or single thymidine-mimosine blocks. Cells released into the S-phase showed that the MGMT disassociates from PCNA during the late S-phase and undergoes specific degradation before re-accumulating in G2/M. The replication-licensing protein Cdt1 and p21cip1 were also observed to undergo co-degradation in S-phase, which has been established as a key step in marking the replication origin sites. MGMT inhibition, either by O6-benzylguanine or specific shRNAs greatly impeded the progression of cells into the S-phase in synchronized cells. DNA synthesis measured by 3H-thymidine or BrdU incorporation was also curtailed significantly by MGMT inhibition. Furthermore, enforced expression of MGMT in two GBM cell lines led to a moderate endo-reduplication of the genome. Collectively, we show new non-repair functions for MGMT, its requirement for cell cycle progression and timed elimination to maintain genomic stability. On the clinical front, the observations provide a clear biochemical rationale for combining MGMT inhibitors (apart from the alkylators) with antimetabolites [supported by CPRIT grants RP130266 & RP170207 to KSS]. Citation Format: AGM Mostofa, Kalkunte S. Srivenugopal. Interplay of human MGMT DNA repair protein with PCNA / p21cip1 and MGMT’s novel role as an S-phase checkpoint [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 296. doi:10.1158/1538-7445.AM2017-296