Published OnlineFirst September 8, 2020; DOI: 10.1158/1078-0432.CCR-20-1729 CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY AsiDNA Is a Radiosensitizer with no Added Toxicity in Medulloblastoma Pediatric Models Sofia Ferreira1,2, Chloe Foray1,2, Alberto Gatto3,4, Magalie Larcher1,2, Sophie Heinrich1,2, Mihaela Lupu5,6, Joel Mispelter5,6, Francois¸ D. Boussin7,Celio Pouponnot1,2, and Marie Dutreix1,2 ABSTRACT ◥ Purpose: Medulloblastoma is an important cause of mortality of medulloblastoma cell lines from different molecular subgroups and morbidity in pediatric oncology. Here, we investigated and TP53 status. Role of TP53 on the AsiDNA-mediated radio- whether the DNA repair inhibitor, AsiDNA, could help address sensitization was analyzed by RNA-sequencing, DNA repair asignificant unmet clinical need in medulloblastoma care, by recruitment, and cell death assays. improving radiotherapy efficacy without increasing radiation- Results: Capable of penetrating young brain tissues, AsiDNA associated toxicity. showed no added toxicity to radiation. Combination of AsiDNA Experimental Design: To evaluate the brain permeability of with radiotherapy improved the survival of animal models more AsiDNA upon systemic delivery, we intraperitoneally injected a efficiently than increasing radiation doses. Medulloblastoma radio- fluorescence form of AsiDNA in models harboring brain tumors sensitization by AsiDNA was not restricted to a specific molecular and in models still in development. Studies evaluated toxicity group or status of TP53. Molecular mechanisms of AsiDNA, associated with combination of AsiDNA with radiation in the previously observed in adult malignancies, were conserved in treatment of young developing animals at subacute levels, related pediatric models and resembled dose increase when combined with to growth and development, and at chronic levels, related to brain irradiation. organization and cognitive skills. Efficacy of the combination of Conclusions: Our results suggest that AsiDNA is an attractive AsiDNA with radiation was tested in two different preclinical candidate to improve radiotherapy in medulloblastoma, with no xenografted models of high-risk medulloblastoma and in a panel indication of additional toxicity in developing brain tissues. Introduction reports of medulloblastoma survivors (6). Notwithstanding, CSI is accountable for the widespread adverse effects in patients with medul- Medulloblastoma (MB), an embryonal tumor of the cerebellum, is loblastoma (7, 8). Medulloblastoma survivors often experience irre- the most common form of childhood brain malignancy (1). Medul- versible and disabling adverse effects in their psychologic, endocrine, loblastoma comprises of four biologically distinct groups: WNT and neurocognitive functioning (9–12). Despite aggressive and highly (wingless), SHH (Sonic Hedgehog), group 3, and group 4 (2). Although toxic regimens, current protocols still fail to treat high-risk subsets of management is adapted to risk, prognosis and clinical outcome within the disease. Medulloblastoma remains an important cause of pediatric groups can vary greatly (3). Standard management includes surgery, morbidity and mortality (1, 13). It is crucial to reduce treatment- craniospinal irradiation (CSI), and chemotherapy (4, 5), and achieves related toxicity while maintaining or improving cure rates in medul- cure in approximately 75% of cases (1). Introduction of CSI in loblastoma care. medulloblastoma management was pivotal, resulting in the first In our laboratory, we developed a pioneering family of DNA repair inhibitors, Dbaits, to enhance the efficacy of genotoxic agents. Dbait molecules are composed of short double-stranded oligonucleotides, 1 Institut Curie, PSL Research University, CNRS UMR 3347, INSERM U1021, Orsay, mimicking DNA double-strand breaks (DSBs; Supplementary Fig. S1). Paris, France. 2Institut Curie, Universite Paris-Sud, Universite Paris-Saclay, CNRS UMR 3347, INSERM U1021, Orsay, Paris, France. 3Institut Curie, Paris Sciences et Upon uptake, cells perceive Dbait as damage and trigger an aberrant Lettres Research University, Centre National de la Recherche Scientifique, activation of the DNA damage signaling pathways (14). Cellular Dbait UMR3664, Equipe Labellisee Ligue contre le Cancer, Paris, France. 4Sorbonne activity is characterized by a pan-nuclear phosphorylation of H2AX Universites, Universite Pierre et Marie Curie Paris 06, Centre National de la (gH2AX) and sustained high levels of poly (ADP-ribose) mediated Recherche Scientifique, UMR3664, Paris, France. 5Institut Curie, Research by DNA-PK and PARP, respectively (15, 16). This aberrant activation Center, PSL Research University, CNRS UMR 9187, INSERM U 1196, Orsay, Paris, of the DNA damage signaling pathways “blinds” cells to real chro- 6 France. Institut Curie, Universite Paris-Sud, Universite Paris-Saclay, CNRS UMR mosomal damage created by genotoxic agents and fails to signal it to 9187, INSERM U1196, Orsay, Paris, France. 7Universite de Paris and Universite Paris-Saclay, Inserm, LRP/iRCM/IBFJ CEA, UMR StabiliteG en etique Cellules downstream DNA repair factors. Dbait-treated cells do not form DNA fi Souches et Radiations, F-92265, Fontenay-aux-Roses, France. damage foci after radiation, signi cantly hampering the recruitment and activity of the repair proteins (14). As a result, both main DSBs Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). repair pathways, homologous recombination and nonhomologous end joining, are inhibited. Rather than targeting a single enzyme, Corresponding Author: Marie Dutreix, Institute Curie, Centre Universitaire, Dbaits deregulate major pathways involved in the repair of DSBs. This Batiment 112, Orsay, Paris 91405, France. Phone: 331-6986-7186; Fax: 331- 6986-3141; E-mail: [email protected] broad mechanism of action makes it an effective radio- and chemo- sensitizer in a wide range of malignancies (17–26) including brain Clin Cancer Res 2020;XX:XX–XX tumors, such as glioblastoma (27, 28). Preclinical studies using Dbait doi: 10.1158/1078-0432.CCR-20-1729 or its clinical form, AsiDNA, did not report additional toxicity of these Ó2020 American Association for Cancer Research. drugs alone or in association with genotoxic agents. Moreover, recent AACRJournals.org | OF1 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst September 8, 2020; DOI: 10.1158/1078-0432.CCR-20-1729 Ferreira et al. attached, were harvested 7 days after AsiDNA treatments, stained with Translational Relevance 0.4% Trypan Blue (Sigma-Aldrich), and counted with LUNA Cell Medulloblastoma is an important cause of mortality and mor- Automated Counter (Logos Biosystems). Half-maximal dose survival bidity in pediatric oncology. Here, we propose the use the brain- (D50) was calculated using a linear regression model. penetrant, DNA repair inhibitor, AsiDNA, to enhance radiother- apy efficacy in medulloblastoma. Our work provides robust evi- Repair factors recruitment assay dence on the well-tolerated radiosensitizing properties of AsiDNA HDMB03 cells were treated with 7 mmol/L of AsiDNA and irra- and how it could help address a significant unmet clinical need in diated with 6 Gy. Fixation, preparation for immunofluorescence (IF), medulloblastoma care by improving clinical outcomes in high-risk and analysis were performed as described in (19), 2 hours after subsets and allowing radiation deescalation in standard risk to radiation. One nucleus in nontreated (NT) conditions was disregarded mitigate the dose-dependent adverse effects. Robustness of proto- from analysis of BRCA1 foci due to a high number of foci (n ¼ 152). cols used throughout this study could help translate the therapeutic Antibodies used were: 53BP1 (Cell Signaling Technology, 4937, 1:100), benefits of AsiDNA into other pediatric cancer models. The BRCA1 (Santa Cruz Biotechnology, sc-6954, 1:100), and gH2AX combination of AsiDNA with the conventionally used treatment (Millipore, 05-636, 1:500). agent, such as radiotherapy, brings this study closer to the reality of clinic practice. Potential for clinical translation exists as AsiDNA Western blotting and antibodies has already been tested in clinical trials, showing to be well- Protein extracts and signal revelation were obtained as described tolerated and safe in combination with radiotherapy. in (35). Membranes were hybridized overnight at 4C with anti-p53 (Cell Signaling Technology, #9282, 1:1,000), anti-p21 (Santa Cruz Biotechnology, sc-6246, 1:500), and anti-b-actin (Sigma-Aldrich, a-1978, 1:5,000). results from the DRIIM phase I/II clinical trial demonstrated that local AsiDNA administration improved radiation response of melanoma RNA extraction skin metastases, without additional skin toxicity (29). Cells were treated with 7 mmol/L of AsiDNA and irradiated with 1.8 The sensitizing properties of AsiDNA have been extensively studied or 6 Gy. Total RNA was harvested using RNeasy Kit (Qiagen), 24 hours in adult cancers. However, these properties have never been tested in after radiation treatments. RNA-sequencing data analysis is described young models still in development or in pediatric tumor models. in Supplementary Materials and Methods. Therefore, we evaluated for the first time whether AsiDNA could be a good candidate to improve treatment outcomes in a pediatric cancer Annexin V/propidium iodide assay model, such as medulloblastoma.