A Phase 1 dose-escalation study to evaluate safety, pharmacokinetics and pharmacodynamics of AsiDNA, a first-in-class DNA repair inhibitor, administered intravenously in patients with advanced solid tumours.

BACKGROUND: AsiDNA, 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. METHODS: The 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. RESULTS: The 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. CONCLUSION: The dose of 600 mg was identified as the optimal dose for further clinical development. CLINICAL TRIAL REGISTRATION: Clinical trial registration (NCT number): NCT03579628.

The AsiDNA™ decoy mimicking DSBs protects the normal tissue from radiation toxicity through a DNA-PK/p53/p21-dependent G1/S arrest.

AsiDNA™, a cholesterol-coupled oligonucleotide mimicking double-stranded DNA breaks, was developed to sensitize tumour cells to radio- and chemotherapy. This drug acts as a decoy hijacking the DNA damage response. Previous studies have demonstrated that standalone AsiDNA™ administration is well tolerated with no additional adverse effects when combined with chemo- and/or radiotherapy. The lack of normal tissue complication encouraged further examination into the role of AsiDNA™ in normal cells. This research demonstrates the radioprotective properties of AsiDNA™. In vitro, AsiDNA™ induces a DNA-PK/p53/p21-dependent G1/S arrest in normal epithelial cells and fibroblasts that is absent in p53 deficient and proficient tumour cells. This cell cycle arrest improved survival after irradiation only in p53 proficient normal cells. Combined administration of AsiDNA™ with conventional radiotherapy in mouse models of late and early radiation toxicity resulted in decreased onset of lung fibrosis and increased intestinal crypt survival. Similar results were observed following FLASH radiotherapy in standalone or combined with AsiDNA™. Mechanisms comparable to those identified in vitro were detected both in vivo, in the intestine and ex vivo, in precision cut lung slices. Collectively, the results suggest that AsiDNA™ can partially protect healthy tissues from radiation toxicity by triggering a G1/S arrest in normal cells.

Evolution of tumor cells during AsiDNA treatment results in energy exhaustion, decrease in responsiveness to signal, and higher sensitivity to the drug.

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; that is, 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 overactivating 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 in 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.

AsiDNA Treatment Induces Cumulative Antitumor Efficacy with a Low Probability of Acquired Resistance.

The Achilles heel of anticancer treatments is intrinsic or acquired resistance. Among many targeted therapies, the DNA repair inhibitors show limited efficacy due to rapid emergence of resistance. We examined evolution of cancer cells and tumors treated with AsiDNA, a new DNA repair inhibitor targeting all DNA break repair pathways. Effects of AsiDNA or Olaparib were analyzed in various cell lines. Frequency of AsiDNA- and olaparib-resistant clones was measured after 2 weeks of continuous treatment in KBM7 haploid cells. Cell survivals were also measured after one to six cycles of 1-week treatment and 1-week recovery in MDA-MB-231 and NCI-H446. Transcriptomes of cell populations recovering from cyclic treatments or mock treatment were compared. MDA-MB-231 xenografted models were treated with three cycles of AsiDNA to monitor the effects of treatment on tumor growth and transcriptional modifications. No resistant clones were selected after AsiDNA treatment (frequency < 3x10-8) in treatment conditions that generate resistance to olaparib at a frequency of 7.2x10-7 resistant clones per treated cell. Cyclic treatments promote cumulative sensitivity characterized by a higher mortality of cells having undergone previous treatment cycles. This sensitization was stable, and transcriptome analysis revealed a major gene downregulation with a specific overrepresentation of genes coding for targets of DNA-PK. Such changes were also detected in tumor models which showed impaired growth after cycles of AsiDNA treatment.

Preclinical Studies Comparing Efficacy and Toxicity of DNA Repair Inhibitors, Olaparib, and AsiDNA, in the Treatment of Carboplatin-Resistant Tumors.

Purpose: Carboplatin is used to treat many cancers, but occurrence of drug resistance and its high toxicity remain a clinical hurdle limiting its efficacy. We compared the efficacy and toxicity of DNA repair inhibitors olaparib or AsiDNA administered alone or in combination with carboplatin. Olaparib acts by inhibiting PARP-dependent repair pathways whereas AsiDNA inhibits double-strand break repair by preventing recruitment of enzymes involved in homologous recombination and non-homologous end joining. Experimental Design: Mice with MDA-MB-231 tumors were treated with carboplatin or/and olaparib or AsiDNA for three treatment cycles. Survival and tumor growth were monitored. Toxicities of treatments were assayed in C57BL/6 immunocompetent mice. Circulating blood hematocrits, bone marrow cells, and organs were analyzed 10 and 21 days after end of treatment using flow cytometry and microscopy analysis. Resistance occurrence was monitored after cycles of treatments with combination of AsiDNA and carboplatin in independent BC227 cell cultures. Results: Olaparib or AsiDNA monotherapies decreased tumor growth and increased mean survival of grafted animals. The combination with carboplatin further increased survival. Carboplatin toxicity resulted in a decrease of most blood cells, platelets, thymus, and spleen lymphocytes. Olaparib or AsiDNA monotherapies had no toxicity, and their combination with carboplatin did not increase toxicity in the bone marrow or thrombocytopenia. All animals receiving carboplatin combined with olaparib developed high liver toxicity with acute hepatitis at 21 days. In vitro, carboplatin resistance occurs after three cycles of treatment in all six tested cultures, whereas only one became resistant (1/5) after five cycles when carboplatin was associated to low doses of AsiDNA. All selected carboplatin-resistant clones retain sensitivity to AsiDNA. Conclusion: DNA repair inhibitor treatments are efficient in the platinum resistant model, MDA-MB-231. The combination with carboplatin improves survival. The association of carboplatin with olaparib is associated with high liver toxicity, which is not observed with AsiDNA. AsiDNA could delay resistance to carboplatin without increasing its toxicity.

Role of Mad2 expression during the early development of the sea urchin.

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.

Micronuclei Frequency in Tumors Is a Predictive Biomarker for Genetic Instability and Sensitivity to the DNA Repair Inhibitor AsiDNA.

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.

The DNA Repair Inhibitor Dbait Is Specific for Malignant Hematologic Cells in Blood.

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.

Combined therapy of colon carcinomas with an oncolytic adenovirus and valproic acid.

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.

Drug-Driven Synthetic Lethality: Bypassing Tumor Cell Genetics with a Combination of AsiDNA and PARP Inhibitors.

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.

A preclinical study combining the DNA repair inhibitor Dbait with radiotherapy for the treatment of melanoma.

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 67 days] or the "radical" protocol (TGD by 3.2-fold; median survival: 221 vs 109 days). 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).