PEGylated talazoparib enhances therapeutic window of its combination with temozolomide in Ewing sarcoma

Abstract

Current therapy is ineffective for relapsed and metastatic Ewing sarcoma (EwS) owing to development of drug resistance. Macromolecular prodrugs potentially lead to lower drug exposure in normal tissues and reduced toxicity. We evaluated the efficacy of PEGylated talazoparib (PEG∼TLZ), a PARP1 inhibitor, alone or in combination with the DNA-alkylating agent temozolomide (TMZ) in EwS and other pediatric tumors using conventional testing or single-mouse trial (SMT). A single dose of PEG∼TLZ (10 μmol/kg on day 0) combined with 5 daily doses of TMZ (40 mg/kg starting on day 3/4) produced minimal toxicity, and the combination achieved maintained complete response in EwS and glioblastoma models. The SMT trial with the 3-day interval between PEG∼TLZ and TMZ resulted in objective responses in EwS and other xenografts. Thus, PEG∼TLZ + TMZ demonstrated a broad range of activity in pediatric solid tumor models. Furthermore, the therapeutic window of PEG∼TLZ + TMZ was enhanced compared with the free-TLZ combination.

Keywords: Molecular medicine; Oncology; Pharmacology.

Graphical abstract

Figure 1

Ewing sarcoma cell lines sensitivity to TMZ and PEG∼TLZ + TMZ Alamar Blue concentration-response curves.Cells were treated for 72 h with indicated concentrations of TMZ alone or in combination with a single concentration of PEG∼TLZ. The red line in each graph indicates the effect of PEG∼TLZ alone at the concentration used in combination with TMZ. Results represent mean ± SEM for n = 3 independent experiments, with each concentration tested in triplicate. Bottom, Table with the Bliss combination indices for PEG∼TLZ + TMZ. See also Figure S1 and Table S1.

Figure 2

Induction of apoptosis in Ewing sarcoma cells by PEG∼TLZ, TMZ, and PEG∼TLZ + TMZ (A and B) Ewing sarcoma cell lines were treated for 24 (A) or 48 h (B) with vehicle, PEG∼TLZ (IC₅₀), TMZ (IC₅₀), and PEG∼TLZ + TMZ (IC₅₀ of each).PARP1 cleavage (cPARP1; 89 kDa) was evaluated by immunoblotting with PARP1 antibody (89/116 kDa). GAPDH (37 kDa) was used as a loading control. See also Table S1.

Figure 3

PEG∼TLZ + TMZ tumor activity in Ewing sarcoma-bearing mice (A) PEG∼TLZ dose-response study: TC-71 tumor-bearing mice received various doses of PEG∼TLZ ± TMZ (30 mg/kg daily x 5) starting 3 days after single dose of PEG∼TLZ. Tumor volumes were measured for 56 days. The thinner lines represent individual mice and the bolder lines (with error bars) represent median tumor growth in each group. Error bars represent mean ± SEM. See also Figures S2 and S3. (B) Efficacy study of PEG∼TLZ + TMZ in TC-71 model: tumor-bearing mice received PEG∼TLZ (10 μmol/kg, single dose), TMZ (40 mg/kg daily x 5), and PEG∼TLZ + TMZ (in 2 schedules, p = 0.002). Tumor volumes were measured for 42 days. The thinner lines represent individual mice and the bolder lines (with error bars) represent median tumor growth in each group. Error bars represent mean ± SEM. P-values were calculated by two-sided log rank test. See also Figures S2 and S3. (C) Efficacy study of PEG∼TLZ + TMZ in ES-7 model: tumor-bearing mice received PEG∼TLZ, TMZ, and PEG∼TLZ + TMZ (in two schedules, p = 0.002). Tumor volumes were measured for 35 days. The thinner lines represent individual mice and the bolder lines (with error bars) represent median tumor growth in each group. Error bars represent mean ± SEM. P-values were calculated by two-sided log rank test. See also Figures S2 and S4. (D) Drug treatment schedules scheme for the in vivo experiments presented in this figure. See also Figure S2.

Figure 4

PEG∼TLZ + TMZ efficacy in glioblastoma xenograft model (A) GBM2 tumor-bearing mice received PEG∼TLZ (10 μmol/kg, single dose), TMZ (40 mg/kg daily x 5), and PEG∼TLZ + TMZ (in two schedules and two PEG∼TLZ doses, p ≥ 0.002). Tumor volumes were measured for 91 days. The thinner lines represent individual mice, and the bolder lines (with error bars) represent median tumor growth in each group. Error bars represent mean ± SEM. p-values were calculated by two-sided log rank test. See also Figures S2 and S4. (B) Drug treatment schedule scheme for the in vivo testing of PEG∼TLZ and TMZ in the GBM2 xenograft model. See also Figure S2.

Figure 5

Single-mouse testing of PEG∼TLZ + TMZ in pediatric xenograft tumor models (A) Antitumor activity to the combination of PEG∼TLZ + TMZ in 28 xenograft models. Tumor volumes were measured for up to 140 days. See also Figure S2 and Table S2. (B) Kaplan-Meier EFS curves for control versus treated groups. Tumor volumes were measured weekly for up to 100 days (p < 0.001). p-value was calculated by the two-sided log rank test. See also Figure S2 and Table S2. (C) Drug treatment schedule scheme for the in vivo single-mouse testing study with PEG∼TLZ (10 μmol/kg, single dose, on day 0) and TMZ (40 mg/kg x 5 days starting on day 3) in Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, malignant rhabdoid tumor, and synovial sarcoma xenograft models. X, final day varies depending on the model. See also Figure S2 and Table S2.