Avasopasem manganese synergizes with hypofractionated radiation to ablate tumors through the generation of hydrogen peroxide

Abstract

Avasopasem manganese (AVA or GC4419), a selective superoxide dismutase mimetic, is in a phase 3 clinical trial (NCT03689712) as a mitigator of radiation-induced mucositis in head and neck cancer based on its superoxide scavenging activity. We tested whether AVA synergized with radiation via the generation of hydrogen peroxide, the product of superoxide dismutation, to target tumor cells in preclinical xenograft models of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma, and pancreatic ductal adenocarcinoma. Treatment synergy with AVA and high dose per fraction radiation occurred when mice were given AVA once before tumor irradiation and further increased when AVA was given before and for 4 days after radiation, supporting a role for oxidative metabolism. This synergy was abrogated by conditional overexpression of catalase in the tumors. In addition, in vitro NSCLC and mammary adenocarcinoma models showed that AVA increased intracellular hydrogen peroxide concentrations and buthionine sulfoximine- and auranofin-induced inhibition of glutathione- and thioredoxin-dependent hydrogen peroxide metabolism selectively enhanced AVA-induced killing of cancer cells compared to normal cells. Gene expression in irradiated tumors treated with AVA suggested that increased inflammatory, TNFα, and apoptosis signaling also contributed to treatment synergy. These results support the hypothesis that AVA, although reducing radiotherapy damage to normal tissues, acts synergistically only with high dose per fraction radiation regimens analogous to stereotactic ablative body radiotherapy against tumors by a hydrogen peroxide-dependent mechanism. This tumoricidal synergy is now being tested in a phase I-II clinical trial in humans (NCT03340974).

Conflict of interest statement

Competing interests: D.P.R., J.L.K., and R.A.B. are employed by and hold equity interests in Galera Therapeutics Inc., which provided the Mn-pentaazamacrocyclic dismutase mimetic AVA used in this study. D.R.S. and M.D.S. have Sponsored Research Agreements supported by Galera Therapeutics Inc. in preclinical studies of AVA in cancer therapy. D.R.S. is a consultant/advisory board member for Galera Therapeutics. M.A.F., D.R.S., R.A.B., D.P.R., and J.L.K. are inventors on patent application PCT/US2017/030871 submitted by Galera Therapeutics LLC that relates to the combination of pentaazamacrocyclic dismutase mimetics with high dose per fraction radiation and with pharmacologic inhibitors of hydrogen peroxide metabolism for cancer treatment. R.A.B., D.P.R., and J.L.K. are inventors on patent application PCT/US2018/027588 submitted by Galera Therapeutics LLC that relates to the combination of pentaazamacrocyclic dismutase mimetics with high dose per fraction radiation, with immune checkpoint inhibitors, and with both checkpoint inhibitors and high dose per fraction radiation for cancer treatment. All other authors declare that they have no competing interests.

Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Figures

Fig. 1.. Pretreatment of NSCLC cell lines with AVA does not protect against IR-induced cancer cell killing.

(A) Chemical structure of AVA. (B toD) NSCLC tumor cell lines (B) H1299, (C) A549, and (D) H460 were treated with 24 or 48 μM concentrations of AVA 30 min before irradiation with 0 to 8 Gy of γ-rays. Data shown as means ± SEM of three replicates in four independent experiments.

Fig. 2.. AVA enhances TGD in H1299, A549, and HCC827 ectopic xenografts after IR.

Averaged and individual mouse xenograft tumor volumes comparing the effects of AVA alone or combined with a single dose of 18 Gy. H1299 (top two rows), A549 (middle two rows), and HCC827 (bottom two rows) were all evaluated with a single dose of AVA 30 to 60 min before irradiation (left column) and AVA delivered once per day on an additional 4 days after irradiation (right column). All animal cohorts contained n = 8 to 10 animals per group.

Fig. 3.. AVA enhancement of radiation-induced TGD increases with increasing radiation dose per fraction.

H1299 xenografts were treated with IR schedules of 18 Gy × 1 fraction, 9.9 Gy × 2 fractions, 7.3 Gy × 3 fractions, 5 Gy × 5 fractions, or 2 Gy × 16 fractions alone (biologically equivalent schedules) and in combination with AVA given once 30 to 60 min before irradiation and once per day for the next 4 days. Average tumor volumes (left column), individual tumor volumes (middle column), and Kaplan-Meier analysis using the IACUC threshold of 1000-mm3 tumor volume as a proxy for survival are shown. All animal cohorts contained n = 8 to 10 animals per group.

Fig. 4.. AVA decreases the TCD50 dose for H1299 xenografts exposed to IR.

H1299 tumor xenografts were treated with single doses of radiation alone or with either 10 or 24 mg/kg of AVA given 30 to 60 min before irradiation and once daily for the next 4 days. Tumor growth was followed for 120 days after radiation exposure, and tumor cure was determined by necropsy. The horizontal bars reflect the 95% confidence interval for each curve at a relative tumor cure value of 0.50 (TCD50). All animal cohorts containedn = 10 to 12 animals per group.

Fig. 5.. AVA increases the steady-state concentration of H2O2 leaving cells susceptible to modulation of redox metabolism in vitro AVA.

(A) H1299 cells were grown exponentially in vitro for 2 days and then assayed for intracellular steady-state concentrations of H2O2 for 60 min in the presence of 20 μM AVA. H2O2 concentration (picomolars per cell) was normalized to the untreated control in each experiment (n = 3, *P = 0.006, one-tailed t test). (B) HBEpC and H1299 cells were grown exponentially for 2 days and then treated with BSO (100 μM) and AVA (20 μM) for 48 hours with Au (500 nM) added for the last 15 min immediately before the clonogenic assay (N = 3, **P < 0.0001 compared to HBEpC). (C) H1299 cells were grown exponentially 2 days then treated with AVA (20 μM), BSO (100 μM), or NAC (10 mM) for 48 hours with Au (500 nM) added for the last 15 min before clonogenic assay (α = P < 0.0001, one-tailed ttest). (D) The doxycycline induction of catalase activity in H1299-CAT tumors harvested after 3 days of doxycycline treatment (2.5 mg/ml in drinking water). (E) Growth delay of H1299-CAT xenografts exposed to AVA alone, a single 18-Gy IR dose, or 18-Gy IR plus AVA before IR and for 4 days after IR, without (left column) or with doxycycline (right column) in the water (n = 8 animals per group).

Fig. 6.. AVA enhancement of tumor response to ablative radiation doses is not specific to NSCLC xenografts.

Growth delay of xenografts exposed to a single 12-Gy IR fraction with or without AVA using human cancer cell lines SqCC/Y1 (HNSCC), Panc 02.03, SW1990, and PANC-1 (all PDAC).

Fig. 7.. Hedgehog and apoptosis signaling pathways are differentially regulated after irradiation of H1299 xenograft tumors depending on AVA treatment.

Tumors were treated with a single fraction of 18-Gy IR alone or 18 Gy + AVA. (A) Hedgehog signaling and (B) Apoptosis signaling, are described by activation z scores, heatmaps, and leading edge analysis, from the top to bottom of each panel. Boxplots of activation z scores for each treatment cohort (median, 75th, and 99th percentiles) are plotted over their respective column of the heatmap of log gene expression for each gene associated with the hallmark pathway. The results of leading edge analysis for samples at day 1 after irradiation confirmed the enrichment direction for each pathway including the nominalP value and FDR at that time.

Fig. 8.. GSEA identified inflammatory and TNFα/NFκB pathways as differentially regulated in irradiated H1299 tumors treated with AVA over time after irradiation.

(A) Inflammatory signaling, and (B) TNFα via NFKβ signaling, are described by activationz scores, heatmaps and leading edge analysis, from the top to bottom of each panel. Boxplots of activation z scores for each treatment cohort (median, 75th and 99th percentiles) are plotted over their respective column of the heatmap of log gene expression for each gene associated with the hallmark pathway. The results of leading edge analysis for samples at day 1 after irradiation confirm the enrichment direction for each pathway including the nominal P value and FDR at that time. Tumors were treated with a single fraction of 18-Gy IR alone or 18 Gy + AVA.