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. 2017 Apr 1;77(7):1709-1718.
doi: 10.1158/0008-5472.CAN-16-2773. Epub 2017 Feb 15.

Chemosensitivity of IDH1-Mutated Gliomas Due to an Impairment in PARP1-Mediated DNA Repair

Yanxin Lu  1 , Jakub Kwintkiewicz  2   3 , Yang Liu  1 , Katherine Tech  4 , Lauren N Frady  2   3 , Yu-Ting Su  1 , Wendy Bautista  1 , Seog In Moon  1 , Jeffrey MacDonald  4 , Matthew G Ewend  2   3 , Mark R Gilbert  1 , Chunzhang Yang  5 , Jing Wu  1   2   3
Affiliations

Chemosensitivity of IDH1-Mutated Gliomas Due to an Impairment in PARP1-Mediated DNA Repair

Yanxin Lu et al. Cancer Res. .

Abstract

Mutations in isocitrate dehydrogenase (IDH) are the most prevalent genetic abnormalities in lower grade gliomas. The presence of these mutations in glioma is prognostic for better clinical outcomes with longer patient survival. In the present study, we found that defects in oxidative metabolism and 2-HG production confer chemosensitization in IDH1-mutated glioma cells. In addition, temozolomide (TMZ) treatment induced greater DNA damage and apoptotic changes in mutant glioma cells. The PARP1-associated DNA repair pathway was extensively compromised in mutant cells due to decreased NAD+ availability. Targeting the PARP DNA repair pathway extensively sensitized IDH1-mutated glioma cells to TMZ. Our findings demonstrate a novel molecular mechanism that defines chemosensitivity in IDH-mutated gliomas. Targeting PARP-associated DNA repair may represent a novel therapeutic strategy for gliomas. Cancer Res; 77(7); 1709-18. ©2017 AACR.

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Figures

Figure 1
Figure 1. IDH1 mutant glioma cells are sensitive to TMZ
A. TMZ dose-response curves of U87 MG and U251 MG cells with IDH1 mutation for 96 hr (n=3). B. Annexin-V apoptosis analysis of IDH1 mutant cell with TMZ treatment. C. Cell cycle analysis of IDH1 mutant cells with TMZ treatment (left panel). The overview of cell cycle progression with clear G2/M arrest (right panel). D. DNA fragmentation test for IDH1 mutant cells treated with TMZ.
Figure 2
Figure 2. Enhanced DNA damage in IDH1 mutant glioma cells
A. Western blotting for γH2A.X expression in IDH1 mutant cells. Protein expression level was determined by densitometry analysis. B. Immunostaining for γH2A.X puncta in IDH1 mutant cells. C. Comet assay demonstrates DNA fragmentation in IDH1 mutant cells. D. Quantitative analysis showed enhancement in DNA fragmentation in IDH1-mutated cells.
Figure 3
Figure 3. Metabolic reprogramming results in chemo-sensitivity in IDH1 mutant cells
A. Seahorse XF Cell Mito Stress analysis showed alteration in oxidative metabolism in IDH1 mutant cells (n=4). B. Quantification of baseline oxidative metabolism in IDH1 mutant cells (n=4, **p<0.01). C. Quantification of maximum oxidative metabolism in IDH1 mutant cells (n=4, **p<0.01). D. Quantification of D-2-HG in IDH1 mutant cells. AGI-5198 treatment (1 µM) was used to determine specific activity from IDH1 mutant enzyme (n=3, **p<0.01) E. Cell viability assessment of U87 MG cells in the presence of TMZ and dimethyl-2-HG (n=3). F. Cell viability measurement of HT-1080 cells with the presence of TMZ and dimethyl-α-KG (n=3, *p<0.05 as compared with DMSO group, #p<0.05 as compared to TMZ group). G. Cell viability assessment of HT-1080 cells with stable expression of IDH1 shRNA (n=3, *p<0.05 as compared to 0 µM TMZ, #p<0.05, ##p<0.01 as compared to scramble RNA) H. Quantitative PCR (upper panel) and western blot analysis (lower panel) for IDH1 expression in HT-1080 cells (n=3).
Figure 4
Figure 4. Dysfunction of the PARP1 DNA repair pathway in IDH1 mutant cells
A. Immunostaining of γH2A.X in IDH1 mutant cells treated with TMZ and Ola (Bar=10 µm). B. Quantification of γH2A.X puncta in IDH1 mutant cells treated with TMZ and Ola (n=3, *p<0.05, **p<0.01). C. Comet assay measuring DNA fragmentation in IDH1 mutant cells treated with TMZ and Ola. D. Quantification of DNA fragmentation in IDH1 mutant cells. E. Western blot analysis of pADPR, PARP and β-actin expression in IDH1 mutant cells treated with TMZ and Ola. F. Quantification of NAD in IDH1 mutant cells treated with TMZ (n=3, *p<0.05, **p<0.01).
Figure 5
Figure 5. Targeting the PARP DNA repair pathway enhanced cytotoxicity induced by chemotherapy
A. Dose-response curves for IDH1 mutant cells treated with TMZ and Ola (n=3). B. Annexin-V apoptosis analysis demonstrated the potentiation effect of Ola for TMZ chemotherapy. C. Quantification of apoptotic cells in IDH1 mutant cells treated with TMZ and/or Ola.
Figure 6
Figure 6. Impaired PARP1 DNA repair defines chemo-sensitivity
Chemotherapy induces damages in chromosomal DNA in cancer cells. PARP serves an important role in maintaining genomic integrity and counteract with chemo-agent. In tumors with IDH mutation, cells are less capable of maintaining DNA repair mechanism, as deficiency in NAD+ metabolism deprives substrates for the BER pathway. Targeting PARP by olaparib significantly potentiates the therapeutic effect of TMZ in IDH mutant cells.
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