Abstract Background: Targeting specific DNA Damage Response (DDR) proteins has been worldwide studied and developed, with the example of PARP inhibitors being the only approved treatments in this field. Here, we describe the antineoplastic and immunomodulatory effects of VIO-01, a first-in-class DNA decoy-cholesterol conjugate, that operates as a pan repair proteins decoy, resulting in constitutive exhaustion of the DNA damage response. Methods: VIO-01-induced DDR proteins trapping and cell cytotoxicity were examined in both homologous recombination proficient (HRP) and deficient (HRD) cancer cells. DNA repair efficacy was monitored by analyzing repair protein recruitment to damage sites. RNAseq analysis in HRP/HRD ovarian cancer cells was employed to uncover the molecular mechanisms underlying VIO-01 effects. Effects on the innate and adaptive immune responses were assessed by following T-cell mediated anti-tumor cytotoxicity. VIO-01 antitumor efficacy and biodistribution were also assessed. Additionally, the interest of cholesterol as a vector for VIO-01 was analyzed compared to other ligands like the folate receptor ligand. Results: VIO-01 bounds to several DDR proteins, such as PARP1, KU70/80, MRN complex and MSH2/MSH3 with high affinity, resulting in an abrogation of single- and double-strand break repair. In line with this, and through global transcriptome analysis, VIO-01 treatment resulted in notable downregulation of Base Excision Repair, NHEJ and Nucleotide Excision Repair pathways in both HRP/HRD cell lines. Additionally, VIO-01 elicited the activation of the immune system and inflammatory responses in ovarian cancer cells. Conjointly, VIO-01 treatment induced a significant differentiation of monocytes into non-classical monocytes, leading to a shaping of the tumor microenvironment via CD8+ T cell recruitment in early stage of activation. The activity of VIO-01 was specific to tumor cells, while sparing healthy and immune cells, at odds with PARP inhibitors. In line with in vitro results, VIO-01 mediated antitumor efficacy in vivo coupled to tumor-targeting T-cell responses. These interesting effects were driven by a favorable ADME/PK profile, showing a long-lasting VIO-01 residence time VIO-01 into tumors (at least 3 days post treatment), coupled to a clear hijacking from the liver and a rapid blood clearance, ensuring a minimal toxicity. Moreover, regulatory toxicology studies demonstrated a favorable safety profile of VIO-01 in non-human primate with the major findings being a transient increase in complement factors induced by the binding of VIO-01 to factor H, which was not observed in human serum, predicting favorable safety profile in humans. Conclusions: Our results provide a preclinical rationale for using VIO-01 to trigger DNA damage exhaustion and an antitumor immune response, paving the way for rapid clinical application in patients bearing HRD or HRP tumors. Citation Format: Vlada Zakharova, Chloe Doizelet, Nicolas Babault, Marie-Christine Lienafa, Fetta Mazed, Megane Debiais, Pierre Vilela, Emilie Perroud, Wael Jdey. VIO-01, a pan-DDR DNA decoy mediating DNA repair abrogation and unleashing the anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6013.

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.

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.

Abstract 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.