PARP-mediated PARylation of MGMT is critical to promote repair of temozolomide-induced O6-methylguanine DNA damage in glioblastoma

Abstract

Background: Temozolomide (TMZ) resistance in glioblastoma multiforme (GBM) is mediated by the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT). MGMT promoter methylation (occurs in about 40% of patients) is associated with loss of MGMT expression (MGMT-) that compromises DNA repair, leading to a favorable response to TMZ therapy. The 60% of patients with unmethylated MGMT (MGMT+) GBM experience resistance to TMZ; in these patients, understanding the mechanism of MGMT-mediated repair and modulating MGMT activity may lead to enhanced TMZ activity. Here, we report a novel mode of regulation of MGMT protein activity by poly(ADP-ribose) polymerase (PARP).

Methods: MGMT-PARP interaction was detected by co-immunoprecipitation. PARylation of MGMT and PARP was detected by co-immunoprecipitation with anti-PAR antibody. O6-methylguanine (O6-MetG) adducts were quantified by immunofluorescence assay. In vivo studies were conducted in mice to determine the effectiveness of PARP inhibition in sensitizing GBM to TMZ.

Results: We demonstrated that PARP physically binds with MGMT and PARylates MGMT in response to TMZ treatment. In addition, PARylation of MGMT by PARP is required for MGMT binding to chromatin to enhance the removal of O6-MetG adducts from DNA after TMZ treatment. PARP inhibitors reduced PARP-MGMT binding and MGMT PARylation, silencing MGMT activity to repair O6-MetG. PARP inhibition restored TMZ sensitivity in vivo in MGMT-expressing GBM.

Conclusion: This study demonstrated that PARylation of MGMT by PARP is critical for repairing TMZ-induced O6-MetG, and inhibition of PARylation by PARP inhibitor reduces MGMT function rendering sensitization to TMZ, providing a rationale for combining PARP inhibitors to sensitize TMZ in MGMT-unmethylated GBM.

Keywords: DNA damage repair; MGMT PARylation; PARP; TMZ resistance.

Fig. 1

PARP inhibitor potentiated TMZ response in MGMT+ GSCs. A, MGMT expression was detected in 13 GSC cell lines by western blot, MGMT promoter methylation status determined by sequencing was shown in the bottom, U for unmethylated, M for methylated. B-D, MGMT+ and MGMT− GSCs were treated with serial diluted TMZ with or without talazoparib at indicated concentration. Dose-response curves were plotted (B), IC₅₀ of TMZ calculated by GraphPad (C), Graph shows cell proliferation inhibition by TMZ, talazoparib, and combinational treatment. Synergistic effect for each cell line as calculated by bliss model was shown in the bottom (D). E, Effect of combination of TMZ and talazoparib on sphere formation in MGMT+ and MGMT− GSCs. Synergistic effect for each cell line as calculated by bliss model was shown in the bottom. Abbreviations: GSCs, glioma sphere-forming cells; MGMT, O⁶-methylguanine DNA methyltransferase; PARP, poly(ADP-ribose) polymerase; TMZ, temozolomide.

Fig. 2

PARP physically binds with MGMT and PARylates MGMT. A-B, Endogenous physical interaction of PARP and MGMT was detected by PARP antibody IP in GSC23 (A) and GSC6-27 (B). C, GSC23 was treated with 100 μM TMZ, 100 nM talazoparib, or combination for 3 days, and MGMT-PARP interaction was detected by MGMT antibody IP. D, GSC23 was treated with TMZ, talazoparib or combination, IP was performed with anti-PAR antibody to purify all PARylated proteins and followed by western blot with PARP and MGMT antibodies to detect PARylated PARP and PARylated MGMT. Quantification of PARylated PARP and PARylated MGMT was shown in the bottom. E, GSC23 was treated with TMZ, olaparib, or combination, IP was performed with anti-PAR antibody to purify all PARylated proteins and followed by western blot with PARP and MGMT antibodies to detect PARylated PARP and PARylated MGMT. Quantification of PARylated PARP and PARylated MGMT was shown in the bottom. Abbreviations: GSCs, glioma sphere-forming cells; IP, immunoprecipitation; MGMT, O⁶-methylguanine DNA methyltransferase; PARP, poly(ADP-ribose) polymerase; TMZ, temozolomide.

Fig. 3

PARylated MGMT binds to chromatin and PARP inhibitors suppressed MGMT-Chromatin DNA binding. A, GSC23 was treated with 100 μM TMZ for 3 days. Cytoplasmic, nuclear soluble, and chromatin-bound fraction were analyzed for MGMT expression by western blot. Actin, Lamin B, and histone H3 were used as makers for cytoplasm, nuclear and chromatin-bound fraction, respectively. B, Nuclear soluble and chromatin-bound fractions from TMZ-treated GSC23 were analyzed by IP with anti-MGMT antibody, followed by western blot with PARP antibody to detect MGMT-PARP interaction, and PAR antibody to detect PARylated MGMT. C, GSC23 was treated with TMZ and talazoaprib, western blot to show MGMT expression in subcellular fraction. D, GSC23 was treated with TMZ and different PARP inhibitors, Chromain-bound subcellular fraction was purified and MGMT expression was analyzed by western blot. Abbreviations: IP, immunoprecipitation; MGMT, O⁶-methylguanine DNA methyltransferase; PARP, poly(ADP-ribose) polymerase; TMZ, temozolomide.

Fig. 4

PARP inhibition potentiated TMZ-induced O⁶-MetG accumulation in MGMT+ cells. GSC23 (A) and GSC272 (B) were treated with 100 µM TMZ, 100 nM talazoparib, or combination for 3 days, cells were stained with O⁶-MetG antibody and DAPI. C, GSC23 cells were treated with TMZ, pamiparib, veliparib, olaparib, or combination, cells were stained with O⁶-MetG antibody and DAPI. Representative fields were shown. and O⁶-MetG-positive cells were quantified by counting 3 fields of each treatment. Scale bars, 50 microns. D, GSC23 and GSC272 cells were treated with 100 μM TMZ, 100 nM talazoparib, or combination for 3 days. Cells were stained with FITC annexin V and 7-amino-actinomycin D and cell death was measured by flow cytometry. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GSCs, glioma sphere-forming cells; MGMT, O⁶-methylguanine DNA methyltransferase; PARP, poly(ADP-ribose) polymerase; TMZ, temozolomide.

Fig. 5

PARP inhibition restored TMZ sensitivity in MGMT+ tumor in vivo. A, A graphic scheme for sequential combination of TMZ with PARP inhibitors, TMZ 50 mg/kg/day; talazoparib 0.33 mg/kg/day, olaparib 25 mg/kg/day. B and C, MGMT+ GSC23 (B) and MGMT− GSC272 (C) were implanted in nude mice to establish subcutaneous models. Mice were administrated with TMZ, talazoparib, or sequential combination. Tumor size was monitored by bioluminescence imaging. D-G, Mice bearing GSC23 (D-E) and GSC272 (F-G) intracranial tumors were administrated with TMZ, talazoparib, or sequential combination. Tumor growth was evaluated by bioluminescence imaging (D and F), survival curves were compared using Kaplan-Meier survival plots (E and G). H-J, Mice bearing GSC23 intracranial tumors were administrated with TMZ, olaparib, or sequential combination. Tumor growth was evaluated by bioluminescence imaging (H), survival curves were compared using Kaplan-Meier survival plots (I). The tissue sections were incubated with antibodies against O⁶-MetG and quantified by Image-Pro Plus (J). Scale bars: 50 microns. Abbreviations: GSCs, glioma sphere-forming cells; MGMT, O⁶-methylguanine DNA methyltransferase; PARP, poly(ADP-ribose) polymerase; TMZ, temozolomide.

Fig. 6

Model summarizing the proposed mechanism. PARP physically interacts with and PARylates MGMT to remove O⁶-MetG adducts in damaged DNA, independent of base excision repair, in response to TMZ treatment. In addition, PARP acts as a sensor to elicit response pathways in base excision repair. PARP inhibitors suppress PARP-MGMT binding and abolish MGMT function. Therefore, treatment with TMZ and PARP inhibitors inhibits both BER- and MGMT-mediated repair and results in an increased antitumor effect. Abbreviations: BER, base excision repair; MGMT, O⁶-methylguanine DNA methyltransferase; PARP, poly(ADP-ribose) polymerase; TMZ, temozolomide.