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, 9 (3), 829-41

Targeting the DNA Repair Pathway in Ewing Sarcoma

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Targeting the DNA Repair Pathway in Ewing Sarcoma

Elizabeth Stewart et al. Cell Rep.

Abstract

Ewing sarcoma (EWS) is a tumor of the bone and soft tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using three different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice.

Figures

Figure 1
Figure 1. EWS cell lines are defective in dsDNA-break repair
(A) Representative image of single-cell alkali electrophoresis with genomic DNA is shown in red; the tail moment is indicated. Comet data for U2OS cells (B) or ES-8 cells (C) prior to exposure to 10-Gy IR (untreated), 30 minutes after treatment, or 11 hours after treatment. The red line indicates the mean. (D-G) Comet data for cells treated with 1 or 10 μM PARPi before and 12 hours after exposure to 10-Gy IR. (H) Micrographs of U2OS and ES-8 nuclei (blue) stained for γ-H2AX (red). Scale bars: 1 μm. The proportion of γ-H2AX+ cells (>20 foci/nucleus) are shown in the histograms to the right of the micrographs. Each bar represents the mean ± SD of duplicate scoring.
Figure 2
Figure 2. Potentiation of PARPi cytotoxicity with IRN and TMZ
(A) Model of DNA damage and synthetic lethality for the combination of PARPis with TMZ or IRN. (B-D) Dose response for EW-8, ES-6, and SAOS cells 72 hours after exposure to each indicated PARPi. Curves fit using data pooled from two biological replicates, each with at least three technical replicates. (E) A similar experiment was performed at 144 hours. (F) Immunoblot with quantification (G) of the knock down of PARP1 in EW-8 cells transfected with a PARP1 siRNA. (H-J) Dose response for BMN-673, olaparib, and veliparib in EW-8 cells at 72 hours with (solid line) and without (dashed line) knock down of PARP1. Each data point is the mean ± SD of triplicate wells. (K-P) Potentiation of PARPi in the presence of increasing concentrations of TMZ and SN-38. Curves were generated by taking horizontal slices through the efficacy surface estimated using the response surface model (RSM) approach.
Figure 3
Figure 3. Development and characterization of an orthotopic EWS tumor model
(A) Diagram of the injection procedure. (B-D) X-ray images of a mouse leg showing the injection procedure before, during, and after injection. (E, F) Hematoxylin and eosin staining of mouse femur and bone marrow after injection of EWS cells in Matrigel (yellow dashed line). Arrows indicates the injection site. (G) X-ray image of an orthotopic tumor with bony extensions (arrow). (H) Transverse view of the soft-tissue and bony component of the orthotopic EWS xenograft in an MR image. (I-M) Micro-PET/CT scans using 11C-methionine. The tumor (arrow) shows accumulation of the radiotracer. (N) Photograph of the femur removed from a mouse with a large mass from the orthotopic xenograft. (O, P) High- and low-power images of the orthotopic tumor showing its extension from the bone to the surrounding soft tissue.
Figure 4
Figure 4. Preclinical phase I/II studies
(A) Drug-combination schedules. Yellow circles represent the PARPi, green bars represent the TMZ and red stars represent daily IP dosing of IRN. (B-D) Survival of mice in the preclinical phase I trial of each PARPi combined with IRN (I) or TMZ (T). In some groups, TMZ was reduced by 50% (T50), 64% (T36), or 70% (T30). For BMN-673, the dose was reduced by 20% in 1 group (P80). (E-G) Preclinical phase II data for IRN+TMZ alone or in combination with veliparib (blue), olaparib (red), or BMN-673 (green). Tumor burden was monitored by Xenogen imaging. (H-L) Representative Xenogen images for each treatment group and photographs of the tumors (arrows) or femurs at the end of the study.
Figure 5
Figure 5. Preclinical phase III study
(A-D) Survival curves for each of the 15 treatment groups. (E-G) Tumor response for individual mice in the TMZ+IRN group and the triple-drug combinations for veliparib (blue), olaparib (red), and BMN-67e (green). The cutoffs for progressive disease (PD), stable disease (SD), partial response (PR), and complete response (CR) are indicated by gray shading. (H) Histogram of the proportion of CRs seen in each triple-drug treatment group. (I) Representative Xenogen images of single-agent PARPis and corresponding triple-drug treatment groups. (J) Representative photographs of tumors from placebo, IRN+TMZ, single-agent PARPi groups and corresponding triple-drug treatment groups. (K,L) Representative micrographs of H&E stained tissue sections from placebo, IRN+TMZ, single-agent PARPi groups and corresponding triple-drug treatment groups. Scale bars K, 500μm; L, 100μm.

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