Therapeutic targeting of PGBD5-induced DNA repair dependency in pediatric solid tumors

Abstract

Despite intense efforts, the cure rates of childhood and adult solid tumors are not satisfactory. Resistance to intensive chemotherapy is common, and targets for molecular therapies are largely undefined. We have found that the majority of childhood solid tumors, including rhabdoid tumors, neuroblastoma, medulloblastoma, and Ewing sarcoma, express an active DNA transposase, PGBD5, that can promote site-specific genomic rearrangements in human cells. Using functional genetic approaches, we discovered that mouse and human cells deficient in nonhomologous end joining (NHEJ) DNA repair cannot tolerate the expression of PGBD5. In a chemical screen of DNA damage signaling inhibitors, we identified AZD6738 as a specific sensitizer of PGBD5-dependent DNA damage and apoptosis. We found that expression of PGBD5, but not its nuclease activity-deficient mutant, was sufficient to induce sensitivity to AZD6738. Depletion of endogenous PGBD5 conferred resistance to AZD6738 in human tumor cells. PGBD5-expressing tumor cells accumulated unrepaired DNA damage in response to AZD6738 treatment and underwent apoptosis in both dividing and G₁-phase cells in the absence of immediate DNA replication stress. Accordingly, AZD6738 exhibited nanomolar potency against most neuroblastoma, medulloblastoma, Ewing sarcoma, and rhabdoid tumor cells tested while sparing nontransformed human and mouse embryonic fibroblasts in vitro. Finally, treatment with AZD6738 induced apoptosis and regression of human neuroblastoma and medulloblastoma tumors engrafted in immunodeficient mice in vivo. This effect was potentiated by combined treatment with cisplatin, including substantial antitumor activity against patient-derived primary neuroblastoma xenografts. These findings delineate a therapeutically actionable synthetic dependency induced in PGBD5-expressing solid tumors.

Figure 1. PGBD5-expressing cells do not tolerate deficiency of non-homologous end-joining DNA repair

(A) Western blot of PGBD5 protein expression after induction with doxycycline (500 ng/ml for 24 hours) of SV40 large T antigen-immortalized mouse embryonic fibroblasts deficient for Atm^−/−, Atr^S/S or Ku80^−/−. Actin serves as loading control. (B) Induction of apoptosis as measured by cleaved caspase-3 staining of mouse embryonic fibroblasts deficient DNA repair factors upon doxycycline-induced (48 hours) expression of PGBD5 (red) as compared to control PBS-treated cells (blue). *p = 0.010, 0.008, and 0.0010 for Atm^−/−, Atr^S/S, and Ku80^−/− of doxycycline vs. control, respectively. (C) Representative photomicrographs of mouse embryonic fibroblasts upon doxycycline-induced PGBD5 expression for 48 hours (+) as compared to PBS-treated controls (−), as stained for DAPI (blue) and cleaved caspase-3 (red). Scale bar = 100 μm. (D–E) Induction of DNA DSB as measured by TUNEL staining (D) and H2AX (E) of mouse embryonic fibroblasts deficient DNA repair factors upon doxycycline-induced (48 hours) expression of PGBD5 (red) as compared to control PBS-treated cells (blue). *p = 0.0010 and 0.0020 for Atm^−/− and Atr^S/S of doxycycline vs. control, respectively (D). *p = 0.0030 and 0.020 for Atm^−/− and Atr^S/S of doxycycline vs. control, respectively (E). Error bars represent standard deviations of three biologic replicates.

Figure 2. PGBD5 expression is sufficient to confer sensitivity to DNA damage signaling inhibition

(A) Ratios of 50% inhibitory concentrations (IC₅₀) upon 120 hours of drug treatment with the DNA damage signaling inhibitors AZD6738, KU60019, AZ20, VE822, and AZD7762 of RPE cells expressing GFP-PGBD5 or GFP control. Ratios of 1 indicate equal susceptibility of GFP-PGBD5 as compared to control GFP-expressing cells. (B) Dose response cell viability curves of RPE cells (blue) expressing GFP-PGBD5 (red) as compared to GFP control (green) or catalytically inactive mutant (D168A/D194A/D386A, black) GFP-PGBD5 treated with AZD6738 for 120 hours. (C) Dose response cell viability curves of wild-type MEFs immortalized with SV40 large T antigen (red), as compared to Atm^−/− (brown) and Atr^S/S (blue) treated with AZD6738 for 120 hours. (D) Representative photomicrographs of RPE cells upon treatment with 500 nM AZD6738 for 72 hours (+) as compared to DMSO-treated controls (−), and expression of GFP, GFP-PGBD5, or inactive mutant (D168A/D194A/D386A) GFP-PGBD5, as stained for DAPI (blue) and γH2AX (red). Scale bar = 100 μm. (E) Flow cytometric analysis of TUNEL and propidium iodide staining of RPE cells expression GFP-PGBD5 as compared to control GFP or inactive mutant (D168A/D194A/D386A) GFP-PGBD5, and treated with 500 nM AZD6738, as compared to DMSO control for 48 hours. Percentages of TUNEL-positive cells are labeled as indicated. (F–H) Induction of apoptosis and DNA damage, as measured by cleaved caspase 3 (F), γH2AX (G), and TUNEL staining (H) of RPE cells treated with 500 nM AZD6738 for 48 hours (red), as compared to DMSO control (blue). Expression of GFP-PGBD5 as compared to control GFP leads to significant induction of caspase 3 cleavage, γH2AX, and TUNEL *p = 0.00030, 0.040, and 5.6 x 10⁻⁶, respectively. Mutation of the catalytic D168A/D194A/D386A (inactive mutant) GFP-PGBD5 rescues this effect **p = 0.030, 0.010, and 1.5 x 10⁻⁵, respectively. Expression of GFP-PGBD5 but not its inactive mutant or control GFP causes significant increase in TUNEL staining in the absence of AZD6738 treatment ***p = 1.2 x 10⁻⁶. Error bars represent standard deviations of three biologic replicates.

Figure 3. Treatment with AZD6738 induces PGBD5-dependent DNA damage

Western immunoblot of RPE cells expressing GFP-PGBD5 or GFP and treated with 500 nM AZD6738 for 6 hours, as indicated. PGBD5 expression increases levels of H2AX and TP53 pS15 upon combination with AZD6738 treatment, but not RPA32 pT21 or pS4/8. Actin serves as loading control, and treatment with 1.5 μM camptothecin for 2 hours serves as positive control for replication stress-mediated induction of RPA32 phosphorylation. Arrow head marks the specific RPA32 pT21 band.

Figure 4. PGBD5-expressing rhabdoid tumor, medulloblastoma, neuroblastoma, and Ewing sarcoma cells exhibit enhanced susceptibility to AZD6738

(A) Susceptibility to AZD6738, as shown by its 50% inhibitory concentration (IC₅₀) upon 120 hour treatment of wild-type MEFs and BJ fibroblasts (Normal), rhabdoid tumor (RT), medulloblastoma (MB), neuroblastoma (NB), and Ewing sarcoma (ES) cell lines. Complete list of cell lines and their dose-response growth curves are shown in Figs. S1 & S3. (B) PGBD5 protein expression as measured by Western immunoblotting (Fig. S4) is significantly associated with the susceptibility to AZD6738 in pediatric tumor cell lines. Lines denote the 95% confidence interval of linear regression (p = 0.0044). Points are labeled according to the color scheme in (A). (C–F) Induction of apoptosis and DNA damage, as measured by caspase 3 cleavage (C–D), and TUNEL (E–F) of IMR5 neuroblastoma, G401 rhabdoid tumor, HD-MB03 medulloblastoma, and TC71 Ewing sarcoma cells with 500 nM AZD6738 for 72 hours (red) as compared to DMSO control (blue). *p = 6.1 x 10⁻⁴ and 3.9 x 10⁻⁴ for AZD6738 versus DMSO, of IMR5 and HD-MB03, respectively. (D) Representative photomicrographs of IMR5 neuroblastoma cells after treatment with 500 nM AZD6738 (+) or DMSO control (−) for 72 hours, stained for cleaved caspase-3 (red) and DAPI (blue). Scale bar = 100 μm. (E) Representative flow cytometric profile of TUNEL and propidium iodide incorporation into HD-MB03 cells after treatment with 500 nM AZD6738 or DMSO control for 48 hours. (F) Induction of TUNEL upon treatment with 500 nM AZD6738 (red) versus DMSO control (blue) for 48 hours. *p = 0.042, 0.025, 5.17 x 10⁻⁹, and 1.98 x 10⁻⁵ for IMR5, G401, HDMB03 and TC71, respectively. Error bars represent standard deviations of three biologic replicates.

Figure 5. PGBD5 expression is necessary for tumor cell susceptibility to AZD6738

(A) PGBD5 mRNA expression in cells transduced with shRNA against PGBD5, as compared to shGFP and untransduced control cells. (B–D) Dose-response of childhood tumor cell lines HDMB-03 (B), IMR5 (C) and G401 (D) treated with varying concentrations of AZD6738 for 120 hours upon PGBD5 depletion. Error bars represent standard deviations of three replicates.

Figure 6. AZD6738 induces DNA damage, apoptosis, and exhibits anti-tumor efficacy in xenograft models of high-risk medulloblastoma and neuroblastoma in vivo

(A–B) Tumor volumes over time of nude mice harboring IMR5 (A) and HD-MB03 (B) subcutaneously xenografted tumors treated with AZD6738 by oral gavage at 50 mg/kg body weight/day (red) or as compared to vehicle control (blue). Asterisks denote p < 0.05. Error bars represent standard deviations of 10 individual xenograft mice per group. Arrows denote start of treatment. (C) Representative photomicrographs of sections from IMR5 (top) or HD-MB03 (bottom) tumors upon completion of treatment with AZD6738 (50 mg/kg/day) or vehicle control in vivo, and stained for hematoxylin and eosin H&E, Ki67, cleaved caspase-3, and H2AX, as indicated. Scale bar = 100 μm. (D–F) Quantification of the number of cells positively stained for Ki67 (D), cleaved caspase-3 (E) and H2AX (F) in IMR5 or HD-MB03 xenograft tumors upon completion of treatment with AZD6738 (50 mg/kg/day, red) or vehicle control (blue) in vivo. *p = 3.1 x 10⁻⁵ and 0.001 for Ki67 in AZD6738 versus vehicle-treated IMR5 and HD-MB03, respectively. *p = 0.001 and 4.9 x 10⁻⁴ for cleaved caspase-3 in AZD6738 versus vehicle-treated IMR5 and HD-MB03, respectively. *p = 0.014 and 4.3 x 10⁻⁴ for H2AX in AZD6738 versus vehicle-treated IMR5 and HD-MB03, respectively. Error bars represent standard deviations of 3 independent fields analyzed.

Figure 7. Synergistic targeting of PGBD5-induced DNA repair dependency in primary patient derived high-risk neuroblastoma xenografts in vivo

(A) Combination indices (CI) for IMR5, HDMB-03 and TC71 cells treated with cisplatin and AZD6738. (B) PGBD5 mRNA expression in two patient derived high-risk neuroblastoma xenografts; complete demographic and molecular features are described in Table S4 (C) Tumor volumes over time of mice engrafted with PDX1 and treated with 50 mg/kg AZD6738 by daily oral gavage (red), as compared to vehicle control (blue), 2 mg/kg cisplatin by weekly IP (black), or combination of AZD6738 and cisplatin (violet). (D) Tumor volumes over time of mice engrafted with PDX2 and treated with 50 mg/kg AZD6738 by daily oral gavage (red), as compared to vehicle control (blue), 2 mg/kg cisplatin by weekly IP (black), or combination of AZD6738 and cisplatin (violet). Asterisks denote p < 0.05. Error bars represent standard deviations of 4 individual xenograft mice per group.

Figure 8. Model of therapeutic targeting of PGBD5-induced DNA damage signaling synthetic dependency

Tumors with increasing levels of PGBD5 expression and DNA recombinase activity accumulate DNA damage, in concert with other intrinsic sources of cellular DNA damage such as replication stress. PGBD5-dependent DNA damage leads to the generation of various DNA damage structure and DNA damage signals, which in turn activate distinct arms of the DNA damage signaling. Consequently, PGBD5-dependent DNA damage signaling can be inhibited using selective pharmacologic inhibitors, inducing accumulation of DNA damage, impairing DNA repair, and leading to cell death.