Glioblastoma cells containing mutations in the cohesin component STAG2 are sensitive to PARP inhibition

Abstract

Recent data have identified STAG2, a core subunit of the multifunctional cohesin complex, as a highly recurrently mutated gene in several types of cancer. We sought to identify a therapeutic strategy to selectively target cancer cells harboring inactivating mutations of STAG2 using two independent pairs of isogenic glioblastoma cell lines containing either an endogenous mutant STAG2 allele or a wild-type STAG2 allele restored by homologous recombination. We find that mutations in STAG2 are associated with significantly increased sensitivity to inhibitors of the DNA repair enzyme PARP. STAG2-mutated, PARP-inhibited cells accumulated in G2 phase and had a higher percentage of micronuclei, fragmented nuclei, and chromatin bridges compared with wild-type STAG2 cells. We also observed more 53BP1 foci in STAG2-mutated glioblastoma cells, suggesting that these cells have defects in DNA repair. Furthermore, cells with mutations in STAG2 were more sensitive than cells with wild-type STAG2 when PARP inhibitors were used in combination with DNA-damaging agents. These data suggest that PARP is a potential target for tumors harboring inactivating mutations in STAG2, and strongly recommend that STAG2 status be determined and correlated with therapeutic response to PARP inhibitors, both prospectively and retrospectively, in clinical trials.

Fig. 1

STAG2-mutated cell lines are more sensitive to PARP inhibitors. A) STAG2-mutated and STAG2 KI H4 glioblastoma cell lines were treated with increasing concentrations of olaparib in 96-well format and cell nuclei were counted after 4 days. B) 42MGBA glioblastoma cell lines were treated as in A) and cell nuclei were counted after 5 days. C) Clonogenic survival of H4 cells after olaparib treatment. The graph represents 3 technical replicates. D–E) STAG2-mutated and KI cell lines were treated with increasing concentrations of the PARP inhibitors D) veliparib and E) rucaparib as in A). ** p < 0.005, * p < 0.01

Fig. 2

Treatment of STAG2-mutated H4 cells with olaparib results in an accumulation of G2 cells. A) Flow cytometry profiles of untreated and olaparib-treated H4 cell lines stained with propidium iodide for DNA content and pS10 Histone H3 antibody as a mitotic marker. B) Cell cycle distribution of H4 cell populations from three independent experiments. * p < 0.05

Fig. 3

Cells treated with PARP inhibitor show more genome instability and DNA repair defects. A–C) H4 cells grown with or without olaparib for three days were scored for the presence of A) micronuclei (MN), B) chromatin bridges (CB) and C) fragmented nuclei (FN). Graphs represent cell counts from three independent experiments. D) Presence of 53BP1 foci in paired H4 cell lines untreated and treated with olaparib for three days. Error bars in D) are 95% confidence intervals. ** p < 0.005, * p < 0.05

Fig. 4

Combinations of PARP inhibitor and known chemotherapeutics are more effective in STAG2-mutated H4 cells. Paired H4 cell lines were treated with either A) camptothecin (CPT) and olaparib or B) temozolomide (TMZ) and olaparib and cell number was determined after 4 days using nuclei-counting. ** p < 0.005