DNA-PK Inhibition Shows Differential Radiosensitization in Orthotopic GBM PDX Models Based on DDR Pathway Deficits

Abstract

Glioblastoma (GBM) remains one of the most therapy-resistant malignancies with frequent local failures despite aggressive surgery, chemotherapy, and ionizing radiation (IR). Small molecule inhibitors of DNA-dependent protein kinase (DNA-PKi) are potent radiosensitizers currently in clinical trials. Determining which patients may benefit from radiosensitization with DNA-PKi is critical to avoid unnecessary increased risk of normal tissue toxicity. In this study, we used GBM patient-derived xenografts (PDX) in orthotopic murine models to study the relationship between molecular features, pharmacokinetics, and the radiosensitizing potential of the DNA-PKi peposertib. We show that peposertib radiosensitizes established and PDX GBM lines in vitro at 300 nmol/L and above, with a significant increase in radiosensitization by maintaining post-IR exposure for >12 hours. Radiosensitization by peposertib is mediated by catalytic inhibition of DNA-PK, and knockdown of DNA-PK by short hairpin RNA (shRNA) largely abolished the radiosensitizing effect. Peposertib decreased auto-phosphorylation of DNA-PKcs after IR in a dose-dependent manner with a delay in resolution of γH2AX foci at 24 hours. The addition of peposertib to IR significantly increased survival in GBM120 orthotopic xenografts, but not in GBM10. There was no difference in plasma or average tumor concentrations of peposertib in the two cohorts. Although the mechanism underpinning this discordant effect in vitro versus in vivo is not clear, there was an association for greater sensitization in TP53 mutant lines. Transfection of a dominant-negative TP53 mutant in baseline TP53 wild-type GBM lines significantly delayed growth and decreased nonhomologous end joining efficiency (but not homologous recombination), after peposertib exposure. See related commentary by Buchsbaum, p. 840.

Figure 1.

Peposertib radiosensitization in GBM dependent on DNA-PKcs expression. A, Clonogenic survival after increasing doses of peposertib (0–1,000 nmol/L) in U251 cells transduced with control vector (U251V) or to overexpress MGMT (U251MGMT) with peposertib alone and (B) in combination with 5 Gy IR. C, Clonogenic survival of U251V with various exposure times of peposertib (300 nmol/L) post-IR (2.5 Gy) compared with IR alone (2.5 Gy). D, Clonogenic survival at various doses of IR ± peposertib (300 nmol/L) after short hairpin knockdown of control (U251 shNT) and E, DNA-PKcs (U251 shPRKDC). F, Immunofluorescence of γH2AX foci per cell at 0.5 and 24 hours after IR (2.5 Gy) ± peposertib (300 nmol/L). G, Western blot of DNA-PKcs pathway phosphorylation at various times and concentrations of peposertib ± IR (5 Gy) in U251MGMT and U251V. H, ELDA after IR (5 Gy) ± peposertib (300 nmol/L) in the PDX lines GBM120 and GBM10. I, Estimates of clonogenic cell frequency (%) from ELDA analysis for GBM120 and GBM10. J, Immunofluorescence of γH2AX foci per cell at 1 and 24 hours after IR (5 Gy) ± peposertib (300 nmol/L) in GBM120 and GBM10. *, P < 0.05; error bars, SEM of triplicate data unless otherwise specified.

Figure 2.

Peposertib radiosensitizes orthotopic murine PDX models of GBM. A, Treatment regimen, athymic nude mice were inoculated with the noted PDX lines and followed for survival and weight loss after IR (3.5 Gy × 5 daily fractions) ± peposertib (125 mg/kg p.o. BID during IR). B, Representative image of radiotherapy treatment plan/setup. Kaplan–Meier survival curves of (C) GBM120, (D) GBM10, and (E) GBM46. Acute weight loss during treatment in the (F) GBM120, (G) GBM10, and (H) GBM46 cohorts.

Figure 3.

Concentration and drug distribution of peposertib in orthotopic PDX models. A, Average plasma concentrations of peposertib 7 hours after 125 mg/kg in GBM10 and GBM120 cohorts. Representative H&E slides and matched colorized images demonstrating spatial distribution and concentration of peposertib as measured by MALDI for orthotopic PDX models of (B) GBM10 and (C) GBM120 in normal athymic nude mice. MALDI average concentrations in tumor, normal brain, and whole brain in (D) GBM10 and (E) GBM120. F, Percent of tumor tissue with peposertib concentrations greater than 1, 2, 5, and 10 μmol/L in indicated PDX GBM lines.

Figure 4.

TP53 mutant status, DNA-PK function predicts radiosensitization with peposertib. A, Survival in orthotopic brain tumors of U251 shNT and U251 shPRKDC ± IR (4Gy × 5 fractions). B, CTG cell viability assay in normal cell line SVG, U251 shNT, and U251 shPRKDC 14 days after IR (5 Gy) ± peposertib (300 nmol/L) relative to IR alone and C, in indicated GBM PDX lines, SVG, and U251 separated by TP53 mutant status. D, PRKDC mRNA z-scores in indicated GBM PDX lines, and E, correlation of decreased viability of IR + peposertib relative to IR alone and PRKDC mRNA z-score in the same PDX lines. F, Somatic mutations in key DDR proteins in indicated GBM PDX lines and U251 separated by TP53 mutant status. G, Gene set enrichment of DDR pathways based on RNA-seq data from indicated PDX lines separated by TP53 mutant status. H, Confluence in TP53 wild-type (TP53wt) vs. dominant-negative TP53 mutant transfected (ddTP53) established and GBM PDX lines after indicated treatments. I, NHEJ, and J, HR activity in GBM10 TP53wt and ddTP53 after peposertib (300 nmol/L) as measured by FM-HCR assay.