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Sup Figure S8. Time course analysis of pDC activation by CpG- Dbait. Representative experiments of time course analysis of pDC activation by flow cytometry analysis of cell membrane activation markers (A) and interferon release by ELISA assay (B) in presence of doses escalation of CpG-Dbait molecules.

Hematologic malignancies are rare cancers that develop refractory disease upon patient relapse, resulting in decreased life expectancy and quality of life. DNA repair inhibitors are a promising strategy to treat cancer but are limited by their hematologic toxicity in combination with conventional chemotherapies. Dbait are large molecules targeting the signaling of DNA damage and inhibiting all the double-strand DNA break pathways. Dbait have been shown to sensitize resistant solid tumors to radiotherapy and platinum salts. Here, we analyze the efficacy and lack of toxicity of AsiDNA, a cholesterol form of Dbait, in hematologic malignancies. We show that AsiDNA enters cells via LDL receptors and activates its molecular target, the DNA dependent protein kinase (DNA-PKcs) in 10 lymphoma and leukemia cell lines (Jurkat-E6.1, MT-4, MOLT-4, 174xCEM.T2, Sup-T1, HuT-78, Raji, IM-9, THP-1, and U-937) and in normal primary human PBMCs, resting or activated T cells, and CD34+ progenitors. The treatment with AsiDNA induced necrotic and mitotic cell death in most cancer cell lines and had no effect on blood or bone marrow cells, including immune activation, proliferation, or differentiation. Sensitivity to AsiDNA was independent of p53 status. Survival to combined treatment with conventional therapies (etoposide, cyclophosphamides, vincristine, or radiotherapy) was analyzed by isobolograms and combination index. AsiDNA synergized with all treatments, except vincristine, without increasing their toxicity to normal blood cells. AsiDNA is a novel, potent, and wide-range drug with the potential to specifically increase DNA-damaging treatment toxicity in tumor without adding toxicity in normal hematologic cells or inducing immune dysregulation. Mol Cancer Ther; 16(12); 2817–27. ©2017 AACR.

Supplementary figure S3. Estimation and functions of genes uniquely correlated with sensitivity to AsiDNA or olaparib. (A) Number of non-overlapping genes correlated with survival to AsiDNA and Ola. Relationship between the number of non-overlapping genes and the p-value from LOO Spearman correlation analysis of expression data from BC cell lines. (B) Atlas of Cancer Signaling Network (ACSN) global map. (C, D) Coverage of ACSN map with unique genes correlated with survival to AsiDNA or Ola in BC cell lines. Distribution of non-overlapping genes robustly correlated with survival to AsiDNA (C) or Ola (D). Red - Positive correlation with survival to treatment; Green - Negative correlation with survival to treatment. Functional modules as WNT-canonical, Cell-matrix adhesion, Cell polarity, MOMP are implicated in survival to both treatments, but regulated in an opposite manner.

Lack of a correlation between sensitivity of BC cells to AsiDNA and their proliferation rate. Survival (ordinate) was monitored by trypan blue staining and manual counting (10 days after treatment). Survival is expressed as the percentage of living cells relative to non-treated cells (NT). The doubling time (abscissa) was calculated by counting the cells during the 10 days of exponential growth. The correlation analysis between survival in the presence of AsiDNA and doubling time was performed using GraphPadPrism5 software and indicates that there is no significant correlation.

Supplementary Materials and Methods used to generate data represented in Figure S2.

Supplementary Table 2. ACSN functional modules enrichment by unique genes robustly correlated with survival to AsiDNA in BC cell lines. Red - positive correlation, green - negative correlation. (P value

Supplementary Table 3. ACSN functional modules enrichment by unique genes robustly correlated with survival to Olaparib in BC cell lines. Red - positive correlation, green - negative correlation, (P value

Purpose: Cancer treatments using tumor defects in DNA repair pathways have shown promising results but are restricted to small subpopulations of patients. The most advanced drugs in this field are PARP inhibitors (PARPi), which trigger synthetic lethality in tumors with homologous recombination (HR) deficiency. Using AsiDNA, an inhibitor of HR and nonhomologous end joining, together with PARPi should allow bypassing the genetic restriction for PARPi efficacy.Experimental Design: We characterized the DNA repair inhibition activity of PARPi (olaparib) and AsiDNA by monitoring repair foci formation and DNA damage. We analyzed the cell survival to standalone and combined treatments of 21 tumor cells and three nontumor cells. In 12 breast cancer (BC) cell lines, correlation with sensitivity to each drug and transcriptome were statistically analyzed to identify resistance pathways.Results: Molecular analyses demonstrate that olaparib and AsiDNA respectively prevent recruitment of XRCC1 and RAD51/53BP1 repair enzymes to damage sites. Combination of both drugs increases the accumulation of unrepaired damage resulting in an increase of cell death in all tumor cells. In contrast, nontumor cells do not show an increase of DNA damage nor lethality. Analysis of multilevel omics data from BC cells highlighted different DNA repair and cell-cycle molecular profiles associated with resistance to AsiDNA or olaparib, rationalizing combined treatment. Treatment synergy was also confirmed with six other PARPi in development.Conclusions: Our results highlight the therapeutic interest of combining AsiDNA and PARPi to recapitulate synthetic lethality in all tumors independently of their HR status. Clin Cancer Res; 23(4); 1001–11. ©2016 AACR.

Supplementary Table 1. Unique genes robustly correlated with survival to AsiDNA or Olaparib in BC cell lines. The values are correlation coefficients (Spearman r; P value

Micronuclei assessement in Breast Cancer patients'' biopsies. MN, Micronuclei; BC, Breast Cancer; C: canalar; L: lobular.

Therapeutic strategies targeting DNA repair pathway defects have been widely explored, but often only benefit small numbers of patients. Here we characterized potential predictive biomarkers for treatment with AsiDNA, a novel first-in-class DNA repair inhibitor. We evaluated genetic instability and DNA repair defects by direct and indirect assays in 12 breast cancer cell lines to estimate the spontaneous occurrence of single-strand and double-strand breaks (DSB). For each cell line, we monitored constitutive PARP activation, spontaneous DNA damage by alkaline comet assay, basal micronuclei levels, the number of large-scale chromosomal rearrangements (LST), and the status of several DNA repair pathways by transcriptome and genome analysis. Sensitivity to AsiDNA was associated with a high spontaneous frequency of cells with micronuclei and LST and specific alterations in DNA repair pathways that essentially monitor DSB repair defects. A high basal level of micronuclei as a predictive biomarker for AsiDNA treatment was validated in 43 tumor cell lines from various tissues and 15 models of cell- and patient-derived xenografts. Micronuclei quantification was also possible in patient biopsies. Overall, this study identified genetic instability as a predictive biomarker for sensitivity to AsiDNA treatment. That micronuclei frequency can be measured in biopsies and does not reveal the same genetic instability as conventional genome assays opens new perspectives for refining the classification of tumors with genetic instability. Cancer Res; 77(16); 4207–16. ©2017 AACR.

BackgroundAsiDNA, a first-in-class oligonucleotide-mimicking double-stranded DNA breaks, acts as a decoy agonist to DNA damage response in tumour cells. It also activates DNA-dependent protein kinase and poly (adenosine diphosphate [ADP]-ribose) polymerase enzymes that induce phosphorylation of H2AX and protein PARylation.MethodsThe aim of this Phase 1 study was to determine dose-limiting toxicities (DLTs), maximum tolerated dose (MTD), safety and pharmacokinetics/pharmacodynamics of AsiDNA administered daily for 3 days in the first week then weekly thereafter. Twenty-two patients with advanced solid tumours were enrolled in 5 dose levels: 200, 400, 600, 900, and 1300 mg, using a 3 + 3 design.ResultsThe MTD was not reached. IV AsiDNA was safe. Two DLTs (grade 4 and grade 3 hepatic enzymes increased at 900 and 1300 mg), and two related SAE at 900 mg (grade 3 hypotension and grade 4 hepatic enzymes increased) were reported. AsiDNA PK increased proportionally with dose. A robust activation of DNA-PK by a significant posttreatment increase of γH2AX was evidenced in tumour biopsies.ConclusionThe dose of 600 mg was identified as the optimal dose for further clinical development.Clinical trial registrationClinical trial registration (NCT number): NCT03579628.

Background: Poly (ADP-ribose) polymerase inhibitors (PARPis) lead to synthetic lethality when used in cancers with homologous recombination deficiency (HRD). However, the development of resistance to PARPis is a recurrent problem thus limits the duration of response and hence the clinical utility of these agents. Here, we describe the antineoplastic and immunomodulatory effects of OX425, a first-in-class oligodeoxynucleotide that operates as a PARP1 decoy, resulting in constitutive PARP1 hyperactivation and consequent exhaustion of the DNA damage response. Methods: OX425-induced PARP trapping, hyperactivation and cell cytotoxicity were examined in vitro in HRD and homologous recombination proficient (HRP) human cancer cells, as well as in non-transformed cell lines. DNA repair efficacy was monitored by analyzing repair protein recruitment to damage sites. OX425 effects on the innate and adaptive immune responses were assessed by following STING activation and T-cell mediated anti-tumor cytotoxicity. RNAseq analysis in HRP/HRD tumor cells treated with OX425 or PARP inhibitors was employed to uncover the molecular mechanisms underlying OX425 effects. The anticancer efficacy of OX425 was assessed in vivo in different HRD and HRP tumor models. OX425-induced PARP activation and tumor infiltration by immune cells were analyzed by flow cytometry. Results: At odds with conventional PARP inhibitors, OX425 bound to and hyperactivated PARP1 with high affinity in a dose-dependent manner, resulting in elevated cytotoxicity to multiple cancer cells (breast, ovarian, prostate, colon, hematological, endometrial cancers) irrespective of HR status. Interestingly, long-term treatment with OX425 did not show any mutagenicity compared to PARPi. The activity of OX425 was specific to tumor cells, as no significant effect on cell viability was observed for normal cells, at odds with PARP inhibitors. In line with in vitro results, OX425 mediated considerable anticancer effects in vivo. Moreover, OX425 triggered activation of the STING pathway and CCL5 secretion in the EMT6 mouse mammary carcinoma model. The anticancer effect of OX425 was coupled with tumor-targeting T cell responses. In MPA/DMBA-driven mammary tumors, OX425 mediated considerable anticancer effects in monotherapy and synergistic effects in combination with PD1 inhibition. Moreover, OX425 treatment significantly delayed acquired resistance to olaparib in BRCA1 mutated MDA-MB-436 cell-derived xenografts. Conclusions: Our results provide preclinical rationale for using OX425 to trigger DNA damage exhaustion and STING activation in cancer cells and initiate inflammatory responses that can be actioned by immune checkpoint inhibitors in patients bearing HRD or HRP tumors Citation Format: Vlada Zakharova, Claudia Galassi, Chloé Doizelet, Vincent Hayes, Lorenzo Galluzzi, Wael Jdey. PARP1 hyperactivation by the decoy oligodeoxynucleotide OX425 mediates DNA repair abrogation and unleashes the anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6200.

Purpose: Conventional cytotoxic therapies are still the standard of care for the treatment of many cancers. However, the associated severe side effects, especially damage to normal proliferating cells like stem and progenitor cells, often lead to drug dose reduction, which limits treatment success. Indeed, chemotherapy-induced myelosuppression is manifested by neutropenia, lymphopenia, anemia, and thrombocytopenia. Although growth factors ameliorate this myelosuppression, their efficacy is still suboptimal and lineage specific. Pre-clinical and clinical studies have shown that AsiDNA, a double-stranded (DS) DNA molecule that mimics DS DNA breaks to interfere with DNA repair by over-activating a false DNA damage signaling through DNA-PK and PARP enzymes (decoy agonist), is extremely well tolerated in standalone in mammals. These observations led us to assess the potential of AsiDNA to protect healthy cells from toxicities of several anti-cancer treatments. Experimental design: In vivo, we analyzed the safety profile of AsiDNA during a recent clinical trial (DRIIV-1b/NCT03579628) in combination with platinum-based chemotherapy. In vitro, we used isolated blood cells from healthy donors, and epithelial and fibroblast cells as models to study if AsiDNA could protect healthy cells to chemo- and radiotherapy-induced toxicity. We monitored cell survival, DNA damage (comet assays) and repair (53BP1 and Rad51 foci), DNA-PK (HSP90 and H2AX phosphorylation) and PARP (PARylation) activation, cell cycle modulation and p53 dependency to identify mechanisms underlying the effects of AsiDNA on healthy cells. Results: In vivo, long-term treatment of several patients with carboplatine+/-paclitaxel + AsiDNA showed no increase of chemotherapies toxicities allowing longer periods of disease control and suggesting a protective effect of AsiDNA. In the in vitro models, we showed that AsiDNA enters non dividing and dividing healthy cells as revealed by intracellular PARylation but induces its nuclear target engagement (H2AX and HSP90 phosphorylation) only in dividing cells. Association of AsiDNA to antitumor treatments increased survival of healthy proliferative cells. Interestingly, AsiDNA displayed two distinct mechanisms of protection depending on the origin of the cells: p53-dependent G1/S cell cycle arrest in fibroblasts and epithelial cells, and DNA repair “doping” in hematological cells revealed by higher recruitment of Rad51 and 53BP1 at damage sites in those cells. Enzymatic inhibition and gene editing revealed that hyperactivation of DNA-PK/p53 pathway by AsiDNA might be required for healthy cells preservation. Conclusion: These findings suggest that the combination of AsiDNA with anticancer treatments should provide a means to attenuate therapy-induced toxicity, while showing the well-documented synergy in tumor cells, thus providing an opportunity to increase the therapeutic window. Citation Format: Wael Jdey, Anouk Sesink, Agathe Cohendet, Juliette Rieu, Chloe Doizelet, Vincent Hayes, Pierre-Marie Girard, Marie Dutreix, Judith Greciet. AsiDNA® treatment protects healthy cells from anticancer treatment toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2600.

Purpose: Drug-tolerance has emerged as one of the major non-genetic mechanism that drives resistance to targeted therapies in lung cancers. Identification of drug-tolerant cells (DTC) vulnerabilities thus represents an attractive strategy to prevent relapse in patients. We observed that EGFR-TKI-derived DTC displayed an increased level of DNA Double Strand Breaks (DSB), raising the hypothesis that genomic instability in these cells could favor the onset of resistance mechanisms. We tested AsiDNA, a small molecule that interferes with the DNA repair machinery, to prevent the emergence of resistances Experimental design: AsiDNA is a double stranded DNA molecule (decoy oligonucleotide) that mimics DNA DSB to interfere with DNA repair, by over-activating a false signaling of DNA damage through DNA-PK and PARP enzymes. We used EGFR-mutated (PC9 and HCC827) or ALK-translocated (H3122) lung tumor cells as well as a PC9 xenograft model, to assess whether a long-term combination of AsiDNA and TKI (erlotinib or osimertinib for EGFR-mutated and alectinib for ALK-translocated cells) could prevent the emergence of resistances arising from DTC. We also performed RNAseq to identify molecular mechanisms underlying AsiDNA effect on TKI-derived DTC. Results:Concomitant treatment of TKI with AsiDNA strongly prevented the emergence of TKI-resistant clones in the three cell lines tested, whereas AsiDNA alone did not display cytotoxic activity. Interestingly, AsiDNA didn’t increase the apoptotic activity of TKI but rather prevented the re-entry of drug-tolerant “dormant” cells into a proliferative state, ultimately resulting in DTC death. RNAseq data revealed a strong downregulation of gene signatures associated with cell cycle, DNA repair but also several pathways known to promote DTC survival such as Epithelial-to-Mesenchymal Transition (EMT) or Interferon pathway in AsiDNA+osimertinib vs osimertinib alone. In vivo, AsiDNA strongly interfered with the emergence of resistances in a PC9 xenograft model treated with erlotinib, resulting in a delayed relapse for the majority of tumors and a complete tumor regression for 20% of them, with no evidence of toxicity for mice. Conclusion: Inhibition of the DNA repair machinery using AsiDNA efficiently prevented the emergence of resistances to tyrosine kinase inhibitors and point to the therapeutic opportunity of combining AsiDNA and TKI to overcome resistance in clinical situation. These findings should have immediate implications to design drug combination strategies to prevent TKI-induced resistance in lung cancers. Citation Format: Sarah Figarol, Sandra Pagano, Wael Jdey, Hugo Guillaume, Gilles Favre, Olivier Calvayrac. DNA repair-interfering molecule AsiDNA® overcomes resistance to tyrosine kinase inhibitors in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 656.

e15060 Background: Poly (ADP-ribose) polymerase (PARP) inhibitors mediate significant anticancer effects against homologous recombination defective (HRD) tumors but have limited efficacy against homologous recombination proficient (HRP) neoplasms. Moreover, HRD tumors often become resistant to PARP inhibitors during treatment, constituting a major limitation to the clinical use of these agents. Here, we describe the metabolic, antineoplastic and immunomodulatory effects of a first-in-class oligodeoxynucleotide that operates as a PARP1 decoy (OX413), resulting in constitutive hyperactivation of PARP1 and consequent exhaustion of the DNA damage response. Methods: OX413-induced PARP trapping and hyperactivation, NAD+ consumption and cell cytotoxicity were monitored using (HRP and HRD) breast, ovarian or prostate cancer cells as well as normal fibroblasts and leukocytes. DNA repair abrogation was monitored by analyzing repair protein recruitment to damage sites. Accumulation of cytoplasmic DNA fragments was monitored after DNA staining and microscopy analysis. Effects on the innate immune response was assessed by following STING pathway activation and T-cell mediated anti-tumor cytotoxicity. In a mouse xenograft model of breast cancer, OX413-induced PARP activation and tumor infiltration by immune cells were analyzed ex-vivo by flow cytometry. Antitumor efficacy of OX413 alone or combined to a PD1 blocker was assessed in MPA/DMBA-driven HR+ HER2- mouse mammary carcinomas. Results: At odds with conventional PARP inhibitors, OX413 bound to and hyperactivated PARP1 with high affinity and in a dose-dependent manner, resulting in elevated cytotoxicity in multiple cancer (but not normal) cells irrespective of HR status. OX413 impaired DNA repair due to PARP trapping and rapid NAD+ consumption, leading to the accumulation of cytoplasmic chromatin fragments (CCFs). Consistent with this, OX413 triggered activation of the CGAS/STING pathway, CCL5 secretion and potentiation of tumor-targeting T cell responses. PARP1 engagement and STING activation were corroborated in vivo in EMT6 mouse mammary carcinomas, correlating with anticancer effects coupled to tumor infiltration by innate and adaptive immune cells. In MPA/DMBA-driven tumors, OX413 mediated considerable anticancer effects that could be exacerbated by PD1 inhibition. Conclusions: Our results provide preclinical rationale for using OX413 to trigger metabolic exhaustion in cancer cells and initiate inflammatory responses that can be actioned by immune checkpoint inhibitors in patients bearing HRD as well as HRP tumors.

International audience; It is increasingly suggested that ecological and evolutionary sciences could inspire novel therapies against cancer but medical evidence of this remains scarce at the moment. The Achilles heel of conventional and targeted anticancer treatments is intrinsic or acquired resistance following Darwinian selection, i.e. treatment toxicity places the surviving cells under intense evolutionary selective pressure to develop resistance. Here, we review a set of data that demonstrate that Darwinian principles derived from the “smoke detector” principle can instead drive the evolution of malignant cells toward a different trajectory. Specifically, long term exposure of cancer cells to a strong alarm signal, generated by the DNA repair inhibitor AsiDNA, induces a stable new state characterized by a down‐regulation of the targeted pathways and does not generate resistant clones. This property is due to the original mechanism of action of AsiDNA, which acts by over‐activating a “false” signaling of DNA damage through DNA‐PK and PARP enzymes, and is not observed with classical DNA repair inhibitors such as the PARP inhibitors. Long‐term treatment with AsiDNA induces a new “alarm down” state in the tumor cells with decrease of NAD level and reactiveness to it. These results suggest that agonist drugs such as AsiDNA could promote a state‐dependent tumor cell evolution by lowering their ability to respond to high “danger” signal. This analysis provides a compelling argument that evolutionary ecology could help drug design development in overcoming fundamental limitation of novel therapies against cancer due to the modification of the targeted tumor cell population during treatment.

Purpose: Mutated KRAS represents one of the most prevalent tumor-driving events in a variety of human cancers. Although there is compelling evidence that oncogenic KRAS drives tumorigenesis, efforts to target mutant KRAS have been stalled for many years. Recently, progress has been made in the development of therapeutics against KRASG12C mutation, which is present in ~15% of lung adenocarcinoma and 0-8% of other cancers. However, therapeutic resistance to KRASG12C inhibition (KRASG12Ci) is still a clinical hurdle. Here, we describe that resistance to KRASG12Ci emerges from Drug Tolerant Cells (DTC) preventing KRASG12Ci from achieving long-term efficacy. AsiDNA is able to target specifically DTC and therefore represents a therapeutic opportunity to impede tumor progression or relapse. Experimental design: AsiDNA is a double-stranded (DS) DNA molecule that mimics DS DNA breaks to interfere with DNA repair, by over-activating a false DNA damage signaling through DNA-PK and PARP enzymes (decoy agonist). We used continuous treatment protocols, to select resistance to KRASG12Ci Sotorasib and MRTX849 and assessed the impact of AsiDNA addition on resistance prevention and abrogation. We also addressed the mechanisms underlying the survival and evolution of the residual DTC under KRASG12Ci treatment, and how AsiDNA could impede their outgrowth. Results: Long-term treatment of NCI-H23, Calu-1 or MIA PACA 2 cells harboring KRASG12C mutation led to acquired resistance in all independently treated populations. Addition of AsiDNA to Sotorasib or MRTX849 completely and irreversibly abolished resistance emergence. Cloning assays showed that acquired resistance to KRASG12Ci does not occur through the selection of a pre-existing KRAS-wt or KRAS-independent sub-clones, but rather via de novo evolution of tumor cells toward a drug resistant state. We hypothesized that resistance to KRASG12Ci could occur through the evolution of remaining tumor cells through drug-tolerant dormant cells, as it's described for other targeted therapies like tyrosine kinase inhibitors. DTC are characterized by a transient senescent phenotype and evolve progressively into proliferative state. We showed that continuous treatment with KRASG12Ci lead to the emergence of a DTC-like population displaying a senescent phenotype evolving over time into proliferative cells. The co-treatment with AsiDNA and KRASG12Ci prevents DTC regrowth. This resistance prevention was also observed when adding AsiDNA during the DTC state (three to five weeks after KRASG12Ci start), indicating a DTC-dependent resistance abrogation by AsiDNA. In line with this, we showed that DTC are hyper-sensitive to AsiDNA compared to parental cells. Conclusion: Our results provide the evidence that resistance to KRASG12Ci can evolve from DTC, and point to the therapeutic opportunity of combining AsiDNA and KRASG12Ci to overcome tumor progression or relapse. Citation Format: Wael Jdey, Aurelie Dubois, Loane Ratel, Marie-Christine Lienafa, Veronique Trochon-Joseph, Francoise Bono. Acquired resistance to KRASG12C inhibitors evolves from drug tolerant cells vulnerable to AsiDNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1433.

Purpose: Therapeutic strategies targeting DNA repair defects have been widely explored, but often restricted to a specific population of patients and tackled with resistance issues. We pioneered a new approach of anti-cancer treatment to tackle emergence of resistance: the decoy agonist mechanism of action. Drugs based on this mechanism hijack and hyperactivate therapeutic targets leading to an impairment of the repair signaling. This breakthrough decoy agonist action has already shown, using our lead compound AsiDNA™, target engagement, excellent safety profile in humans, and importantly lack of acquired resistance. Here, we describe mechanistically the immunomodulatory properties and metabolic effects of a new generation product OX401, generated using the proprietary PlatON™ platform of oligonucleotides designed to trap PARP proteins. Experimental design: OX401-induced PARP activation, NAD+ consumption and cell cytotoxicity were monitored using tumor and non-tumor cells. DNA repair abrogation was monitored by analyzing repair protein recruitment to damage sites. Accumulation of cytoplasmic DNA fragments was monitored after DNA staining and microscopy analysis. OX401 effect on the innate immune response was assessed by following STING pathway activation and T-cell mediated anti-tumor cytotoxicity. OX413, a “super” OX401 was designed to be more stable and less prone to intracellular enzymatic degradation. In a cell-derived xenograft model of mouse breast cancer, OX413-induced PARP activation and percentages of tumor-infiltrating leucocytes (CD45+: CD3+, CD8+, NK, DCs) were analyzed ex-vivo by flow cytometry. Results: Using different tumor models, we showed that OX401 binds and hyper-activates PARP1 with a high affinity and in a dose-dependent manner. As a consequence, OX401 impaired DNA repair due to PARP sequestration leading to an accumulation of cytoplasmic chromatin fragments (CCFs). In line with this, we demonstrated that OX401 displayed cell-autonomous immunomodulatory properties. Mechanistically, the generated CCFs triggered an innate immunity activation through cGAS/STING pathway, downstream CCL5 secretion and potentiation of the anti-tumor T-cell dependent immune response. Through PARP hyper-activation, OX401 also induced a rapid NAD+ consumption (below the viability threshold). This metabolic exhaustion caused selective tumor cell death while sparing healthy cells. These effects were enhanced using the optimized OX413 molecule, and at doses approximately 10 to 100-fold lower compared to OX401. In-vivo, OX413 triggered PARP1 target engagement and STING pathway activation, correlated to an increased innate and adaptive immune cells infiltration. Conclusions: Our results provide a preclinical rationale for using OX413 as an immunomodulatory and “metabolic exhauster” agent, especially in appropriately molecularly selected patients with tumors showing metabolic deficiencies. Citation Format: Wael Jdey, Christelle Zandanel, Véronique Trochon-Joseph, Chloé Doizelet, Vincent Hayes, Marie-Christine Lienafa, Richard Tripelon, Françoise Bono. A new generation of PARP interfering drug candidates for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 527.

Purpose: Cancer treatments using tumor defects in DNA repair pathways have shown promising results but are restricted to small subpopulations of patients. The most advanced drugs in this field are PARP inhibitors (PARPi), which trigger synthetic lethality in tumors with homologous recombination (HR) deficiency. Using AsiDNA, an inhibitor of HR and nonhomologous end joining, together with PARPi should allow bypassing the genetic restriction for PARPi efficacy. Experimental Design: We characterized the DNA repair inhibition activity of PARPi (olaparib) and AsiDNA by monitoring repair foci formation and DNA damage. We analyzed the cell survival to standalone and combined treatments of 21 tumor cells and three nontumor cells. In 12 breast cancer (BC) cell lines, correlation with sensitivity to each drug and transcriptome were statistically analyzed to identify resistance pathways. Results: Molecular analyses demonstrate that olaparib and AsiDNA respectively prevent recruitment of XRCC1 and RAD51/53BP1 repair enzymes to damage sites. Combination of both drugs increases the accumulation of unrepaired damage resulting in an increase of cell death in all tumor cells. In contrast, nontumor cells do not show an increase of DNA damage nor lethality. Analysis of multilevel omics data from BC cells highlighted different DNA repair and cell-cycle molecular profiles associated with resistance to AsiDNA or olaparib, rationalizing combined treatment. Treatment synergy was also confirmed with six other PARPi in development. Conclusions: Our results highlight the therapeutic interest of combining AsiDNA and PARPi to recapitulate synthetic lethality in all tumors independently of their HR status. Clin Cancer Res; 23(4); 1001–11. ©2016 AACR.

Purpose: PARP inhibitors (PARPi) resistance remains a major clinical hurdle as virtually all patients treated with PARPi will eventually relapse. We already demonstrated that co treatment with AsiDNA abrogates and reverses PARPi-acquired resistance. However, the mechanisms by which AsiDNA prevents genetic adaptations driving resistance and allows long-term efficacy of PARPi remain unknown. Here, we describe that PARPi resistance occurs through emergence of drug-tolerant cells (DTC) preventing PARPi from achieving long-term efficacy. Emerging evidence implicates a survival of residual DTC that constitutes a reservoir from which drug-resistant proliferative cells may emerge. We demonstrate in this study that AsiDNA is able to target specifically DTC and therefore represents a therapeutic opportunity to impede tumor relapse under PARPi treatment. Experimental design: AsiDNA is a double stranded (DS) DNA molecule (decoy oligonucleotide) that mimics DS DNA breaks to interfere with DNA repair, by over-activating a false DNA damage signaling through DNA-PK and PARP enzymes. We used different treatment protocols, cyclic or continuous, to select resistance to the PARPi Olaparib and Talazoparib and assessed the impact of AsiDNA addition on resistance abrogation, durability and irreversibility. We also addressed the mechanisms underlying the survival and evolution of the residual DTC under PARPi treatment, and how AsiDNA could impede their outgrowth. Results: Long-term treatment of BRCA2-mutated cells initially highly sensitive to PARPi leads to acquired resistance in all independently treated populations. Addition of AsiDNA to PARPi completely and irreversibly abolishes resistance emergence. This AsiDNA-dependent resistance abrogation is maintained for at least twelve weeks after AsiDNA removal (nine weeks after treatment start), indicating that AsiDNA maintains PARPi efficacy. PARPi-acquired resistance does not occur through selection of pre-existing BRCA2-wt and/or HR proficient sub-clones, but rather via (epi)-genetic evolution toward an active HR pathway and high genetic instability. We hypothesize that PARPi resistance occurs through a DTC stage as it is described for other targeted therapies like tyrosine kinase inhibitors (TKi). DTC are characterized by a transient senescent phenotype and evolve progressively into proliferative state. We showed that repeated treatments with PARPi lead to the apparition of a DTC-like population displaying a senescent phenotype evolving over time into proliferative cells. The co-treatment with AsiDNA and PARPi prevents DTC regrowth. Conclusion: Several groups have demonstrated that resistance to TKi can emerge from DTC. Our findings provide for the first time the evidence that PARPi-resistant cells can evolve from DTC, and point to the therapeutic opportunity of combining AsiDNA and PARPi to prevent or overcome resistance in clinical situation. Citation Format: Wael Jdey, Olivier Calvayrac, Orlane Maloudi, Florent Ubelmann, Gilles Favre, Françoise Bono. Acquired resistance to PARP inhibitors evolves from drug-tolerant cells vulnerable to AsiDNA [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4078.

Mitotic arrest deficient 2 (Mad2) belongs to the spindle assembly checkpoint (SAC), a mechanism that blocks progression of the cell cycle until microtubule attachment to kinetochores is complete. It has been found to be involved in the resistance of cancer cells to “anti-mitotic” drugs such as paclitaxel. Mad2 controls meiotic progression, but its role during sea urchin development had never been investigated. Furthermore, the existence of a SAC in this species had never been proved. The present data show that a Mad2 protein, highly homologous to that of humans, is expressed in this species. Mad2 expression increases during development, becoming confined to the endomesoderm at gastrula stages. The level of Mad2 expression is enhanced in embryos that do not gastrulate after treatment with anti-mitotic drugs, lithium or inhibition of the ERK pathway. Mis-aligned and lagging chromosomes were induced after injection of an anti-Mad2 antibody or a Mad2 morpholino. Our results point to the role of a non-canonical SAC involving Mad2 in the control of mitotic divisions of the sea urchin embryo.

Hematologic malignancies are rare cancers that develop refractory disease upon patient relapse, resulting in decreased life expectancy and quality of life. DNA repair inhibitors are a promising strategy to treat cancer but are limited by their hematologic toxicity in combination with conventional chemotherapies. Dbait are large molecules targeting the signaling of DNA damage and inhibiting all the double-strand DNA break pathways. Dbait have been shown to sensitize resistant solid tumors to radiotherapy and platinum salts. Here, we analyze the efficacy and lack of toxicity of AsiDNA, a cholesterol form of Dbait, in hematologic malignancies. We show that AsiDNA enters cells via LDL receptors and activates its molecular target, the DNA dependent protein kinase (DNA-PKcs) in 10 lymphoma and leukemia cell lines (Jurkat-E6.1, MT-4, MOLT-4, 174xCEM.T2, Sup-T1, HuT-78, Raji, IM-9, THP-1, and U-937) and in normal primary human PBMCs, resting or activated T cells, and CD34+ progenitors. The treatment with AsiDNA induced necrotic and mitotic cell death in most cancer cell lines and had no effect on blood or bone marrow cells, including immune activation, proliferation, or differentiation. Sensitivity to AsiDNA was independent of p53 status. Survival to combined treatment with conventional therapies (etoposide, cyclophosphamides, vincristine, or radiotherapy) was analyzed by isobolograms and combination index. AsiDNA synergized with all treatments, except vincristine, without increasing their toxicity to normal blood cells. AsiDNA is a novel, potent, and wide-range drug with the potential to specifically increase DNA-damaging treatment toxicity in tumor without adding toxicity in normal hematologic cells or inducing immune dysregulation. Mol Cancer Ther; 16(12); 2817–27. ©2017 AACR.

Melanomas are highly radioresistant tumors, mainly due to efficient DNA double-strand break (DSB) repair. Dbait (which stands for DNA strand break bait) molecules mimic DSBs and trap DNA repair proteins, thereby inhibiting repair of DNA damage induced by radiation therapy (RT). First, the cytotoxic efficacy of Dbait in combination with RT was evaluated in vitro in SK28 and 501mel human melanoma cell lines. Though the extent of RT-induced damage was not increased by Dbait, it persisted for longer revealing a repair defect. Dbait enhanced RT efficacy independently of RT doses. We further assayed the capacity of DT01 (clinical form of Dbait) to enhance efficacy of "palliative" RT (10 × 3 Gy) or "radical" RT (20 × 3 Gy), in an SK28 xenografted model. Inhibition of repair of RT-induced DSB by DT01 was revealed by the significant increase of micronuclei in tumors treated with combined treatment. Mice treated with DT01 and RT combination had significantly better tumor growth control and longer survival compared to RT alone with the "palliative" protocol [tumor growth delay (TGD) by 5.7-fold; median survival: 119 vs 67days] or the "radical" protocol (TGD by 3.2-fold; median survival: 221 vs 109days). Only animals that received the combined treatment showed complete responses. No additional toxicity was observed in any DT01-treated groups. This preclinical study provides encouraging results for a combination of a new DNA repair inhibitor, DT01, with RT, in the absence of toxicity. A first-in-human phase I study is currently under way in the palliative management of melanoma in-transit metastases (DRIIM trial).

Purpose: PARP inhibitors (PARPi) are approved for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, drug resistance remain a clinical hurdle, and several attempts failed to decrease this resistance. In the current study, we propose a novel therapeutic strategy, based on drug combination to abolish the onset of resistance to PARPi. Experimental design: We performed a set of experiments to induce resistance to PARPi and assess the impact of AsiDNA addition to decrease this resistance occurrence. AsiDNA is a double stranded DNA molecule (decoy oligo-nucleotide) that mimics double stranded DNA breaks (DSBs) to interfere with DNA repair, by over-activating a “false” signaling of DNA damage through DNA-PK and PARP enzymes. We characterized the effects of long term treatment with the PARPi olaparib and talazoparib on resistance selection in two different cell lines, BC227 and NCI-H446, and we further analyzed the effect of low doses of AsiDNA on the occurrence of acquired resistance to these PARPi. Finally, we addressed the mechanisms underlying resistance emergence and abrogation. Results: Long term exposure of cancer cells to PARPi induces the emergence of resistance in all the tested independent populations (11 BC227 independent populations and 3 NCI-H446 for talazoparib, 5 BC227 independent populations for olaparib), raising the question of the clinical benefit of long-term maintenance monotherapy with PARPi. Interestingly, double-treated populations with AsiDNA (2.5µM - low non cytotoxic dose) and talazoparib or olaparib showed a significant lower probability of resistance occurrence. In fact, 9/11 (or 5/5) BC227 populations treated with AsiDNA and talazoparib (or AsiDNA and olaparib) remained sensitive up to 5 cycles of treatment, and 3/3 NCI-H446 populations treated with AsiDNA and talazoparib. Interestingly, AsiDNA is also able to partially revert talazoparib resistance in BC227 and NCI-H446 resistant populations. As described, PARP1, EZH2 and/or 53BP1 protein expression loss or reduction are important mechanisms leading to PARPi resistance. We confirmed in these experiments that treatments with PARPi significantly modify the expression and activity of these proteins and we demonstrated that co-treatment with AsiDNA inhibits these changes. These results indicate that AsiDNA may abrogate and reverse PARPi acquired resistance by the normalization of involved protein expression and activity. Conclusion: Our results highlight the therapeutic interest of combining AsiDNA and PARPi to recapitulate drug-driven PARPi resistance abrogation. As PARPi have obtained FDA approval, and AsiDNA is being currently tested in a clinical trial, a potential exists for a rapid clinical confirmation of AsiDNA-induced PARPi resistance abrogation and reversion. Citation Format: Wael Jdey, Pauline Lascaux, Françoise Bono. AsiDNA abrogates acquired resistance to PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3797.

The Achilles heel of conventional and targeted anticancer treatments is intrinsic or acquired resistance following Darwinian selection, i.e. treatment toxicity place the surviving cells under intense evolutionary selective pressure to develop resistance. Here, we demonstrate that AsiDNA, the first antitumor drug with an agonist activity, can instead drive the evolution of malignant cells toward a different trajectory. Specifically, long term exposure of cancer cells to the strong alarm signal, generated by the AsiDNATM DNA repair inhibitors, did not promote resistance emergence. It induces a stable new state characterized by the down regulation of the targeted pathways that persists for months after treatments. This property is due to the original mechanism of action of AsiDNATM, which acts by over-activating a “false” signaling of DNA damage through DNA-PK and PARP enzymes, and is not observed with other DNA repair inhibitors such as the PARP inhibitors olaparib and talazoparib. Transcriptomic analysis of AsiDNATM treated independent populations derived from lung and breast cancers revealed the common evolution of independent AsiDNA-treated samples, resulting in a general down-regulation of enzymes involved in DNA repair and oncogenesis, and a specific down regulation of DNA-PK targets. Similar down regulation of DNA-PK targets was observed in xenografted tumor in animal models after three cycles of AsiDNA treatment. Chromatin analysis revealed a major change in AsiDNATM treated populations with an increased nucleosome stability that could be responsible for the epigenetic modification observed. Long term treatment with AsiDNATM induces a new “alarm down” state in the tumor cells that increase its efficacy. These results suggest that agonist drugs such as AsiDNATM could promote a state-dependent tumor cell evolution by lowering their ability to respond to damage signal as predicted by the ecological and evolutionary “smoke detector principle”. Citation Format: Wael Jdey, Maria Kozlac, Sergey Alekseev, Danielle Johnson, françoise Bono, Srividya bhaskara, frederic thomas, Marie Dutreix. AsiDNATM, a targeted therapy with no acquired resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2095.

Purpose: Accurate evaluation and prediction of response to anti-cancer treatment remain a great challenge. Stratification biomarkers are of great value to identify responders or non-responders to a specific drug, or even to distinguish between early and delayed responses. In this study, we identified a gene signature to predict AsiDNA treatment efficacy in patients. Experimental design: The first step of this study consisted on the analysis of gene expression profile in a set of 12 Breast cancer cell lines. Genes retrieved from correlation analysis between sensitivity to AsiDNA and gene expression were used to create a gene signature predicated on the upregulation of positively correlated genes and the down regulation of negatively correlated genes (the AsiDNA sensitivity signature - AsiSS) using the UCSC Xena platform for data visualization. The AsiSS was used to sort the TCGA Breast Cancer (BRCA) patient cohort, DNA repair gene expression and cell lines from the CCLE (1100 cell lines) into rank orders, in order to retrieve a gene set highly predictive of response to AsiDNA. Results: A list of genes the most strongly correlated with survival to AsiDNA was compiled. 39 genes showed expression profiles positively correlated with survival to AsiDNA treatment (Spearman r > 0.67, P -0.63, P Conclusion: Overall, our results highlight the interest of retrospective analysis based on patient databases to retrieve gene biomarkers predictive of drug outcome. As AsiDNA is being currently tested in a clinical trial, a potential exist for a rapid validation of our gene set in the aim to develop a Biomarker-driven patient selection strategy for AsiDNA treatment. Citation Format: Wael Jdey, Donogh O'Brien, Richard Tripelon, Séverine Rochas, Marie Dutreix, Françoise Bono. Development of a biomarker-driven patient selection strategy for AsiDNA treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2130.

The anti-tumor potential of oncolytic adenoviruses (CRAds) has been demonstrated in preclinical and clinical studies. While these agents failed to eradicate tumors when used as a monotherapy, they may be more effective if combined with conventional treatments such as radiotherapy or chemotherapy. This study seeks to evaluate the combination of a CRAd bearing a ∆24 deletion in E1A with valproic acid (VPA), a histone deacetylase inhibitor, for the treatment of human colon carcinomas. This combination led to a strong inhibition of cell growth both in vitro and in vivo compared to treatment with CRAd or VPA alone. This effect did not stem from a better CRAd replication and production in the presence of VPA. Inhibition of cell proliferation and cell death were induced by the combined treatment. Moreover, whereas cells treated only with CRAd displayed a polyploidy (> 4N population), this phenotype was increased in cells treated with both CRAd and VPA. In addition, the increase in polyploidy triggered by combined treatment with CRAd and VPA was associated with the enhancement of H2AX phosphorylation (γH2AX), a hallmark of DNA damage, but also with a decrease of several DNA repair proteins. Finally, viral replication (or E1A expression) was shown to play a key role in the observed effects since no enhancement of polyploidy nor increase in γH2AX were found following cell treatment with a replication-deficient Ad and VPA. Taken together, our results suggest that CRAd and VPA could be used in combination for the treatment of colon carcinomas.

Therapeutic strategies targeting DNA repair pathway defects have been widely explored, but often only benefit small numbers of patients. Here we characterized potential predictive biomarkers for treatment with AsiDNA, a novel first-in-class DNA repair inhibitor. We evaluated genetic instability and DNA repair defects by direct and indirect assays in 12 breast cancer cell lines to estimate the spontaneous occurrence of single-strand and double-strand breaks (DSB). For each cell line, we monitored constitutive PARP activation, spontaneous DNA damage by alkaline comet assay, basal micronuclei levels, the number of large-scale chromosomal rearrangements (LST), and the status of several DNA repair pathways by transcriptome and genome analysis. Sensitivity to AsiDNA was associated with a high spontaneous frequency of cells with micronuclei and LST and specific alterations in DNA repair pathways that essentially monitor DSB repair defects. A high basal level of micronuclei as a predictive biomarker for AsiDNA treatment was validated in 43 tumor cell lines from various tissues and 15 models of cell- and patient-derived xenografts. Micronuclei quantification was also possible in patient biopsies. Overall, this study identified genetic instability as a predictive biomarker for sensitivity to AsiDNA treatment. That micronuclei frequency can be measured in biopsies and does not reveal the same genetic instability as conventional genome assays opens new perspectives for refining the classification of tumors with genetic instability. Cancer Res; 77(16); 4207–16. ©2017 AACR.

Purpose: The Achilles' heel of all conventional and targeted anticancer treatments is intrinsic or acquired resistance. The era of precision medicine where tumors are selected for specific treatments based on their genetic alterations is revolutionary, but unfortunately prolonged responses are rarely observed due to rapid emergence of resistant clones. We recently developed a new concept of DNA repair inhibitor (Dbait) acting by activating enzymes involved in DNA damage signaling. We tested how such agonist activity would be prone to induce resistance to Dbait treatment. Experimental design: We performed repeated cycles of treatment with various targeted therapy agents (Imatinib, Olaparib, 6-thioguanine and the clinical form of Dbait, AsiDNATM). We analyzed the specific sensitivity of independent cultures after each treatment cycle. The study was performed in different tumor cell lines ((MDAMB231, MDAMB468, HCC1143, THP1, U937, KBM7) and in two non-malignant breast cell lines (MCF10A, MCF12A). Extensive transcriptome and genome studies were performed on MDAMB231 breast cancer cell lines. Results: Resistant clones appeared at a frequency of 1.45% for Olaparib, 0.62% for imatinib, and 1.66% for 6-thioguanine. Similar protocol did not select for resistance to AsiDNA. Unexpectendly, tumor cells became more sensitive to AsiDNA after each cycle of treatment. This behavior was specific of AsiDNA and was not observed with other treatments. The six tumor cell lines tested developed AsiDNA autosensitisation and no resistance. Non tumoral cells were not affected by repeated treatments. The acquired sensitivity of the treated tumor populations was conserved for months after end of treatment Transcriptional and genetic analysis of independently treated MDAMB-231 populations reveals that all evolved similarly. They display a few conserved genome modifications and a large deregulation of 1160 genes resulting in an overexpression of luminal associated genes and a decrease in mesenchymal associated genes. Conclusion: Our results indicate a phenomenon of “autosensitisation”, along treatment which has never been described for anticancer treatment and could prevent development of resistance during treatment. Citation Format: Maria Kozlac, Wael Jdey, Pierre-Marie Girard, Françoise Bono, Marie Dutreix. The evolution of tumor cells under AsiDNA treatment results in “autosensitization” [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2851.

Purpose: while being registered and used in restricted subset of T-cell lymphoma, HDAC inhibitors (HDACi) have shown limited antitumor effect as single agents. Recent studies have shown an effect of HDACi on DNA damage accumulation, rationalizing their combination with DNA repair inhibitors. In the current study, we propose a novel therapeutic strategy, based on drug combination of HDACi with the pan-DNA break repair inhibitor AsiDNA to promote their antitumor activity. Experimental design: AsiDNATM is a double stranded DNA molecule (decoy oligo-nucleotide) that mimics double stranded DNA breaks (DSBs) to interfere with DNA repair, redirecting repair enzymes away from sites of tumor DNA damage. Belinostat is a pan-HDACi displaying a better safety profile compared to other HDACi. We characterized the effects of each drug on DNA break accumulation and genetic instability by γH2AX analysis, COMET assay and micronuclei detection. We further studied the antitumor efficacy of the combination of the two drugs. Finally, we assessed the effect of AsiDNA on the occurrence of acquired resistance after long term treatment with belinostat. Results: Molecular analyses of DNA damage after treatment demonstrate that belinostat paves the way for AsiDNA efficacy by inducing DNA DSBs as measured by γH2AX accumulation and tail moment increase on COMET assay. Moreover, continuous treatment with belinostat induced an increase of basal genetic instability in tumor cells measured by micronuclei accumulation, a prerequisite for AsiDNA antitumor efficacy1. On the other hand, AsiDNA enhances the effects of belinostat on histone acetylation, demonstrating a high potentiation of belinostat activity on its targets by AsiDNA. This mechanism-based cross-potentiation between AsiDNA and belinostat results in a high synergistic antitumor efficacy of the combined treatment in different tumor models. This synergistic effect was further confirmed with several HDACi belonging to different classes. Importantly, the combined treatment do not induce any DNA damage increase and/or lethality in non-tumor cells. Finally, repeated treatments allowed the emergence of resistance to belinostat, which is abrogated in presence of AsiDNA, indicating an unlikely tumor escape to this combined therapy. Conclusion: Altogether these results indicate a cross potentiation between AsiDNA and belinostat, and support the rational to investigate the clinical activity of this novel synergistic combination in different tumor types. As belinostat has obtained FDA conditional approval, and AsiDNA is already tested in a first-in-man clinical trial, a potential exists for a rapid clinical confirmation of the interest of this new combination. Citation Format: Wael Jdey, Hélène Toussaint, Coraline Cassegrain, Laura Bellassen, Richard Tripelon, Vincent Hayes, Perrine Pivette, Véronique Trochon-Joseph, Jean-Louis Labernardière, Marie Dutreix, Françoise Bono Colombie. AsiDNA and HDAC inhibitors: A cross-potentiation team working to kill tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2818.

Purpose: PARP inhibitors (PARPi) have shown significant benefits in cancer patients with BRCA mutations. However, their major limitations are the necessity of homologous recombination (HR) deficiency and the rapid emergence of resistance. In the current study, we propose a novel therapeutic strategy, based on drug combination to promote sensitivity to PARPi independently of the tumor genetics. Experimental design: We used AsiDNA, a DNA repair pathways agonist (Dbait concept), consisting in small molecules mimicking double-strand DNA breaks to activate ectopic signaling of DNA damage and prevent recruitment at damage sites of HR repair enzymes. We characterized the DNA repair inhibition activity of AsiDNA by monitoring repair foci formation and DNA damage and analyzed the cell survival to AsiDNA monotherapy and combination with the PARPi Olaparib of 21 tumor cell lines, and 3 non-tumor cell lines. Efficacy of the combination treatment was analyzed in tumor xenografts derived from the MDA-MB-231 cell line showing reduced sensitivity to AsiDNA and Olaparib in vitro. We screened for the frequency of clones resistant to two weeks treatment with AsiDNA or Olaparib in the 4NQO-mutagenized haploid KBM7 cells. Results: Molecular analyses of repair foci formed after irradiation demonstrate that AsiDNA inhibits recruitment of RAD51 and 53BP1 whereas Olaparib prevents XRCC1. Combination of both drugs increases the accumulation of unrepaired spontaneous damage resulting in an increase of cell death in all tumor cell lines. The synergy of the association of AsiDNA with PARPi was also confirmed with 6 other PARPi. AsiDNA do not induce any increase in DNA damage or lethality in non-tumor cells when used in monotherapy as well as in association with PARPi. Multi-omics analysis of tumor cells highlighted a correlation between similarity to normal cells profile and AsiDNA resistance. Selection of resistant clones allowed the emergence of resistance to Olaparib (1.45%), Imatinib (0.6%) and 6-Thioguanine (1.6%) but not to AsiDNA ( Conclusion: Our results highlight the therapeutic interest of combining AsiDNA and PARPi to recapitulate synthetic lethality in all tumors independently of their HR status. Moreover, the low frequency of appearance of resistant clones to AsiDNA suggests a sustained efficacy during treatment unlike most targeted therapies. Citation Format: marie DUTREIX, Wael Jdey, sylvain Thierry, Inna Kuperstein, Graham Dixon. AsiDNA induce tumor sensitivity to PARP inhibitors in homologous recombination proficient breast cancer AsiDNA induce tumor sensitivity to PARP inhibitors in homologous recombination proficient breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1110. doi:10.1158/1538-7445.AM2017-1110