Single-dose radiotherapy disables tumor cell homologous recombination via ischemia/reperfusion injury

Abstract

Tumor cure with conventional fractionated radiotherapy is 65%, dependent on tumor cell-autonomous gradual buildup of DNA double-strand break (DSB) misrepair. Here we report that single-dose radiotherapy (SDRT), a disruptive technique that ablates more than 90% of human cancers, operates a distinct dual-target mechanism, linking acid sphingomyelinase-mediated (ASMase-mediated) microvascular perfusion defects to DNA unrepair in tumor cells to confer tumor cell lethality. ASMase-mediated microcirculatory vasoconstriction after SDRT conferred an ischemic stress response within parenchymal tumor cells, with ROS triggering the evolutionarily conserved SUMO stress response, specifically depleting chromatin-associated free SUMO3. Whereas SUMO3, but not SUMO2, was indispensable for homology-directed repair (HDR) of DSBs, HDR loss of function after SDRT yielded DSB unrepair, chromosomal aberrations, and tumor clonogen demise. Vasoconstriction blockade with the endothelin-1 inhibitor BQ-123, or ROS scavenging after SDRT using peroxiredoxin-6 overexpression or the SOD mimetic tempol, prevented chromatin SUMO3 depletion, HDR loss of function, and SDRT tumor ablation. We also provide evidence of mouse-to-human translation of this biology in a randomized clinical trial, showing that 24 Gy SDRT, but not 3×9 Gy fractionation, coupled early tumor ischemia/reperfusion to human cancer ablation. The SDRT biology provides opportunities for mechanism-based selective tumor radiosensitization via accessing of SDRT/ASMase signaling, as current studies indicate that this pathway is tractable to pharmacologic intervention.

Trial registration: ClinicalTrials.gov NCT02570919 NCT01223248.

Keywords: Cancer; DNA repair; Oncology; Vascular Biology.

Conflict of interest statement

Conflict of interest: BE and HJH are members of a start-up company, O2M, to market the pO2 imaging technology. YY is a speaker for Varian Medical Systems, BrainLab, and Vision RT, and a volunteer for the Medical Advisory Board of the Chordoma Foundation. ES, RK, AHF, and ZF are inventors on a patent application related to this work (62/078,280). RK and ZF are cofounders of Ceramedix Holding LLC. The following authors are listed inventors on patents and/or patent applications: CC (US20170246098A1), DSH (62729321 and 62688350), HC (11/478033), YM (6,556,856 and WO 00/40990), BE (9,392,957), HJH (4,714,886; 5,431,901; 7,444,011; 8,644,955 B1; and 9,392,957 B1), AHF (15/525,856), RK (09/503,852; 10/217,259; 12/599,280; 13/974,405; 14/854,891; 14/162,494; 14/402,875; 15/502,162; 15/525,856; and 15/643,430), and ZF (15/525,856; 15/643,430; and PCT/US2017/049378).

Figures

Figure 1. SDRT induces I/R in tumor microvasculature.

(A) Representative fluorescent Hoechst 33342 accumulation in the interstitial space of fresh-frozen sections of B16F1 melanoma, injected at indicated times after 15 Gy SDRT; tumors were removed 2 minutes later. Blue regions are perfused and white regions are hypoxic. Scale bar: 1 mm. (B) Tumor perfusion reconstructed from whole-section mounts of tumors exposed to 15 Gy SDRT, quantified pixel by pixel by Hoechst 33342 fluorescence intensity. Data represent mean ± 95% CI collated from 2–3 mice per time point. *P < 0.05, **P < 0.01, ***P < 0.001, vs. unirradiated controls, Bonferroni correction (threshold: α = 0.05/4 = 0.0125). (C) Tumor perfusion assessed by DCE-MRI–derived Akep in B16F1 melanomas before and 30 minutes after 20 Gy SDRT. Color-coded Akep intensity heatmaps (top) and respective Akep histograms (bottom) are quantified pixel by pixel across a representative 1-mm DCE-MRI slice, expressed as median Akep values. Scale bar: 1 mm. (D) Each dot represents perfusion reduction from 1 mouse implanted with B16F1 melanoma, reconstructed from whole-tumor Akep histograms normalized to preradiation values (4 mice per group). Data represent mean ± SEM. *P < 0.05. (E) Effect of SDRT on MCA/129 fibrosarcoma pO2 quantified by the OxyLite method. Data represent mean ± 95% CI before versus 30 minutes after 15 Gy SDRT (5 mice per group). ****P < 0.0001 vs. unirradiated, paired 2-tailed t test. (F) DW-MRI–derived IVIM coefficients in a patient receiving 24 Gy SDRT for primary prostate cancer. Time-dependent changes of f and D* register I/R after SDRT. (G) Fold changes in fD* in oligometastatic bone lesions exposed to 24 Gy SDRT, 9 Gy or 3×9 Gy radiotherapy. Data represent mean ± SD of 16 repeat fD* values per point. **P < 0.01, Bonferroni correction (threshold: α = 0.05/2 = 0.025).

Figure 2. I/R, not endothelial apoptosis, impairs DSB repair.

(A) Time-dependent (after 15 Gy) and dose effects (registered at 6 hours after radiation) on γH2AX focus resolution after SDRT in MCA/129 fibrosarcomas (left panel) and B16F1 melanomas (right panel). Each data point represents mean ± SEM (2–4 tumors) with high-power microscopic fields scanned for each time/dose focus count. (B) Kinetics of γH2AX, MDC1, and 53BP1 focus resolution in HCT116 xenografts after 15 Gy SDRT. (A and B) Data represent mean ± SEM. *P < 0.05 asmase+/+ (designated WT) vs. asmase–/– (designated KO) tumor host. (C) Impact of mechanical I/R on focus resolution in HCT116 xenografts at 6 hours after 15 Gy SDRT. Mechanical percutaneous clamping (designated C) of large tumor-feeding vessels was applied immediately after SDRT in I/R-inert BQ-123–inhibited tumors in asmase+/+ (WT) hosts or tumors in asmase–/– (KO) hosts. BQ-123 (designated B) was injected i.p. (2 mg/kg) at 30 minutes before SDRT. Data represent median ± IQR. ****P < 0.0001, B and KO vs. WT, Bonferroni correction (threshold: α = 0.05/4 = 0.0125). (D) Tumor pretreated with BQ-123 fails to impact SDRT-induced endothelial apoptosis. Representative apoptotic endothelial cells (arrows) double-stained with pan-endothelial MECA-32 (blue) and TUNEL (brown) in irradiated MCA/129 fibrosarcoma (top), quantified (bottom) in HCT116 tumor xenografts in asmase+/+ hosts. Data represent mean ± 95% CI, P = 0.7. Scale bar: 20 μm. Data are collated from 2 × 103 to 4 × 103 nuclei per point using 3–5 mice per group in A–C, and from 6 mice per group in D.

Figure 3. I/R confers epigenetic HDR loss of function.

Foci were scored as in Figure 2 in asmase+/+ (WT) or asmase–/– (KO) hosts. (A) Canonical NHEJ is insensitive to SDRT-I/R. Left: Representative DNA-PKcs foci in MCA/129 fibrosarcomas 1 hour after 15 Gy SDRT. Right: Time-dependent change in DNA-PKcs and XRCC4 foci in SDRT-treated B16F1 melanomas. Scale bar: 20 μm. Data represent mean ± SEM collated from 2–4 independent experiments per panel of 3 mice per group. P > 0.05, WT vs. KO. (B) Time course of SUMO2/3, PIAS1, and BRCA1 focus accrual/resolution after 15 Gy SDRT in HCT116 xenografts. *P < 0.05, WT vs. KO unpaired t test. Data represent mean ± SEM collated from 2–4 independent experiments per panel of 3 mice per group. *P < 0.05, WT vs. KO. (C) Effects of SDRT-I/R injury (WT, WT+B+C, KO+C) versus SDRT-inert (WT+B, KO) settings on SUMO2/3 foci formation in HCT116 tumor xenografts at 6 hours after 15 Gy SDRT. BQ-123 (designated B), when used, was injected i.p. 30 minutes before SDRT, while mechanical percutaneous clamp (designated C) of large tumor-feeding vessels was used immediately after SDRT. Data represent median ± IQR percent foci-positive nuclei in tumor-derived histological specimens from 2–4 mice each, scoring a total of 2 × 103 to 7 × 103 HCT116 cells. ***P < 0.001, ****P < 0.0001 vs. WT, with Bonferroni correction (threshold: α = 0.05/4 = 0.0125). Inset shows representative SUMO2/3 focus images in respective SDRT-I/R–conditioned and I/R-inert. Scale bar: 20 μm.

Figure 4. I/R disrupts SUMO2/3 function in tumors exposed to SDRT.

Western blot (WB) analysis of tumor extracts using rabbit polyclonal anti-SUMO2/3 antibody, quantified by densitometry relative to loading controls. (A) Whole-cell extracts from MCA/129 fibrosarcoma in asmase+/+ and asmase–/– hosts after 20 Gy SDRT. Top panels show representative WBs, and bottom panels quantify high-MW SUMO2/3 conjugates (>75 kDa) and free SUMO2/3. (B) Representative WBs of high-MW SUMO2/3 conjugates (>75 kDa) and free SUMO2/3 in cytoplasmic (left) and chromatin-bound (right) fractions isolated from MCA/129 fibrosarcomas in asmase+/+ and asmase–/– hosts at 3 hours after 20 Gy SDRT. Bottom panels show quantitative analysis of specimens at different times after 20 Gy. (A and B) Data represent mean ± SEM of at least 3 independent experiments of 2 mice per group. **P < 0.01 vs. 0min, Bonferroni correction (threshold: α = 0.05/3 = 0.017) .

Figure 5. SUMO3, not SUMO2, is required for conjugation-dependent activation of HDR mediators.

(A) Growth curves of shScramble-, shSUMO2-, and shSUMO3-transduced HCT116 cells after 5 Gy. Data represent mean ± SEM from 2 independent experiments performed in triplicate. (B) Clonogenic survival assay of transduced cells exposed to escalating SDRT doses. The inset provides the mathematically derived D0 and Dq coefficients of the dose-survival curves, defining the relative radiosensitivity of each cell line and the capacity to repair potentially lethal damage, respectively. Data represent mean ± SEM collated from 3 independent experiments performed in triplicate. (C and D) γH2AX focus resolution at 3–8 hours (C) and RAP80, BRCA1, and RAD51 accrual into repair foci at 6 hours (D) after 5 Gy in shSUMO2- or shSUMO3-expressing HCT116 cells. (C and D) Data represent median ± IQR from 2 independent experiments performed in triplicate. (A and C) *P < 0.05 vs. shScramble. (D) *P < 0.05, ***P < 0.001 vs. shScramble, with Bonferroni correction (threshold: α = 0.05/2 = 0.025).

Figure 6. ROS mediate SUMO dysfunction and synthetic tumor lethality via the reproductive death pathway.

(A–E) Experiments were performed using MCA/129 fibrosarcoma. Data were collated from 2–4 independent experiments using 2–4 mice per experiment. (A) Representative images (left; scale bar: 20 μm) and quantitation (right; data represent mean ± 95% CI) of dihydroethidium staining (DHE, red) and DAPI counterstaining (blue) of tumors in asmase+/+ mice before and 1 hour after SDRT, with or without BQ-123 (designated B, 30 minutes before SDRT) or tempol (designated T, 30 minutes after SDRT). *P < 0.05, Bonferroni correction (threshold: α = 0.05/3 = 0.017). (B) Prdx6 overexpression or tempol abrogates the SSR induced by SDRT. Representative WBs of whole-cell lysates from tumors in asmase+/+ (WT) mice. (C) Tempol abrogates chromatin-associated SUMO3 depletion. Representative WB of chromatin-enriched extract from tumors in asmase+/+ mice. (D and E) Prdx6 overexpression or tempol abrogates delayed γH2AX focus resolution (D) and restores BRCA1 and RAD51 loading into repair foci (E) in SDRT-I/R–competent (WT or KO+C) tumors. Data represent median ± IQR. **P < 0.01, ****P < 0.0001 for all focus types vs. WT, Bonferroni correction (threshold: α = 0.05/8 = 0.00625). (F) Left: Representative images of micronuclei (MN; arrows) in H&E-stained tumor sections. Scale bar: 5 μm. Right: MN quantitation; data represent mean ± 95% CI from 3 independent experiments using 3 mice per group, 2,000 cells per tumor. *P < 0.05, **P < 0.01 for 15 Gy and 15 Gy + C vs. unirradiated WT, Bonferroni correction (threshold: α = 0.05/4 = 0.0125). (G) Ablation of MCA/129 fibrosarcomas in sv129/BL6 mice is aborted by pre-SDRT treatment with BQ-123 or post-SDRT treatment with tempol. Each line represents an individual tumor volume. Arrows indicate day of SDRT. Tumors undetectable at 120 days are considered cured.