Mutant IDH1-driven cellular transformation increases RAD51-mediated homologous recombination and temozolomide resistance

Abstract

Isocitrate dehydrogenase 1 (IDH1) mutations occur in most lower grade glioma and not only drive gliomagenesis but are also associated with longer patient survival and improved response to temozolomide. To investigate the possible causative relationship between these events, we introduced wild-type (WT) or mutant IDH1 into immortalized, untransformed human astrocytes, then monitored transformation status and temozolomide response. Temozolomide-sensitive parental cells exhibited DNA damage (γ-H2AX foci) and a prolonged G2 cell-cycle arrest beginning three days after temozolomide (100 μmol/L, 3 hours) exposure and persisting for more than four days. The same cells transformed by expression of mutant IDH1 exhibited a comparable degree of DNA damage and cell-cycle arrest, but both events resolved significantly faster in association with increased, rather than decreased, clonogenic survival. The increases in DNA damage processing, cell-cycle progression, and clonogenicity were unique to cells transformed by mutant IDH1, and were not noted in cells transformed by WT IDH1 or an oncogenic form (V12H) of Ras. Similarly, these effects were not noted following introduction of mutant IDH1 into Ras-transformed cells or established glioma cells. They were, however, associated with increased homologous recombination (HR) and could be reversed by the genetic or pharmacologic suppression of the HR DNA repair protein RAD51. These results show that mutant IDH1 drives a unique set of transformative events that indirectly enhance HR and facilitate repair of temozolomide-induced DNA damage and temozolomide resistance. The results also suggest that inhibitors of HR may be a viable means to enhance temozolomide response in IDH1-mutant glioma.

Figure 1

(A) Western blot validation of the identity of the cell lines used in this panel. 1, U87; 2, U87+MGMT; 3, E6/E7/hTERT (vector); 4, E6/E7/hTERT+WT IDH1; 5, E6/E7/hTERT+R132H mutant IDH1; 6, E6/E7/hTERT+RasV12. (B) FACS analysis of cell cycle distribution in various cell lines 1-7 days after TMZ (100μM, 3hr) exposure. Shading, cell populations exhibiting significant G2/M arrest. (C, right panel) Immunofluorescence images of gamma-H2AX foci (pink) in DAPI-stained (blue) control and drug-treated parental E6/E7/hTERT cells at various times after TMZ exposure (100 μM, 3 hrs) and quantitation of data (left panel) in all cells studied. (D) The ratio of cell numbers at 7 days post-TMZ exposure to that 4 days after TMZ exposure (100 μM, 3 hrs) in control and IDH1-modified cells. (E) colony formation efficiency of cells studied following TMZ exposure (0-100 μM, 3 hr). *, p<.05. Values derived are representative of, or derived from, ≥3 independent experiments.

Figure 2

(A) Western blot validation of the identity of the cell lines used in this panel. 1, E6/E7/hTERT+RasV12 (RasV12) ; 2, E6/E7/hTERT+RasV12+R132H mutant IDH1; 3, U87; 4, U87+ R132H mutant IDH1. (B) FACS analysis of cell cycle distribution in various cell lines 1-7 days after TMZ (100μM, 3hr) exposure. Shading, cell populations exhibiting significant G2/M arrest. (C) Quantitated data from immunofluorescence analysis of gamma-H2AX foci in control and drug-treated parental and IDH1-modified cells at various times after TMZ exposure (100 μM, 3 hrs). (D) Colony formation efficiency of parental and IDH1-modified cells following TMZ (100 μM, 3 hr) exposure. Values derived are representative of, or derived from, ≥3 independent experiments.

Figure 3

(A) Immunofluorescence analysis of the physical co-incidence of gamma-H2AX and RAD51 foci in E6/E7/hTERT parental cells three days following TMZ exposure (0 or 100 μM, 3 hrs). (B) Quantitated data from immunofluorescence analysis of gamma-H2AX foci in control and drug-treated parental (E6/E7/hTERT) and IDH1-modified cells at three and seven days after TMZ exposure (100 μM, 3 hrs). (C), Homologous recombination activity as determined by an in vivo plasmid-based recombination reporter assay. Value of the parental cells was set at 1. *, p<.05. Values derived are representative of, or derived from, ≥3 independent experiments.

Figure 4

(A) Western blot analysis of RAD51 and β-actin levels in E6/E7h/TERT+mutant IDH1 cells three days following exposure to scramble or RAD51-targeting siRNA. (B) Homologous recombination activity as determined by an in vivo plasmid-based recombination reporter assay. Value of the si-scramble cells was set at 1. (C) Quantitated data from immunofluorescence analysis of gamma-H2AX foci in control and drug-treated parental and RAD51-suppressed cells 3 and 7 days after TMZ exposure (100 μM, 3 hrs). (D) FACS analysis of cell cycle distribution in control and RAD51-suppressed TMZ-treated (100 μM, 3 hrs) cells at the time of arrest and two days later. Shading, cell populations exhibiting significant G2/M arrest. (E) Colony formation efficiency of control and RAD51-suppressed cells studied following TMZ exposure (0-100 μM, 3 hr). *, p<.05. Values derived are representative of, or derived from, ≥3 independent experiments.

Figure 5

(A) Homologous recombination activity in vehicle or drug-treated (10 μM B02, 48 hrs) E6/E7/hTERT+mutant IDH1 cells as determined by an in vivo plasmid-based recombination reporter assay. Value of the vehicle cells was set at 1. (B) Quantitated data from immunofluorescence analysis of RAD51 foci in the same vehicle and drug-treated (B02, 10 μM, 48 hrs) cells 7 days after TMZ exposure (100 μM, 3 hrs). (C) Colony formation efficiency of vehicle and drug-treated (B02, 10 μM) cells after TMZ exposure (0-100 uM, 3 hrs). *, p<.05. Values derived are representative of, or derived from, ≥3 independent experiments.