Onxeo (France)

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: 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.

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.

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.

Abstract 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 (<0.07%), indicating that the drug acts through multiple targets. In the MDA-MB-231 xenograft model, whereas Olaparib failed to prevent tumor growth and AsiDNA provided only a 62% increase in tumor growth delay, the combination of Olaparib and AsiDNA increase 215% days the mean tumor growth delay. 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

Belinostat, a potent pan-inhibitor of histone deacetylase (HDAC) enzymes, is approved in the United States (US) for relapsed/refractory peripheral T-cell lymphoma. In nonclinical studies, bile and feces were identified as the predominant elimination routes (50-70%), with renal excretion accounting for ~30-50%. A Phase 1 human mass balance study was conducted to identify species-dependent variations in belinostat metabolism and elimination. Patients received a single 30-min intravenous (i.v.) infusion of (14)C-labeled belinostat (1500 mg). Venous blood samples and pooled urine and fecal samples were evaluated using liquid chromatography-tandem mass spectroscopy for belinostat and metabolite concentrations pre-infusion through 7 days post-infusion. Total radioactivity was determined using liquid scintillation counting. Continued treatment with nonradiolabled belinostat (1000 mg/m(2) on Days 1-5 every 21 days) was permitted. Belinostat was extensively metabolized and mostly cleared from plasma within 8 h (N = 6), indicating that metabolism is the primary route of elimination. Systemic exposure for the 5 major metabolites was >20% of parent, with belinostat glucuronide the predominant metabolite. Mean recovery of radioactive belinostat was 94.5% ± 4.0%, with the majority excreted within 48 and 96 h in urine and feces, respectively. Renal elimination was the principal excretion route (mean 84.8% ± 9.8% of total dose); fecal excretion accounted for 9.7% ± 6.5%. Belinostat was well tolerated, with mostly mild to moderate adverse events and no treatment-related severe/serious events. Mass balance was achieved (~95% mean recovery), with metabolism identified as the primary route of elimination. Radioactivity was predominantly excreted renally as belinostat metabolites.