Flavien Devun

Ubiquinone Analogs: A Mitochondrial Permeability Transition Pore-Dependent Pathway to Selective Cell Death

Background Prolonged opening of the mitochondrial permeability transition pore (PTP) leads to cell death. Various ubiquinone analogs have been shown to regulate PTP opening but the outcome of PTP regulation by ubiquinone analogs on cell fate has not been studied yet. Methodology/Principal Findings The effects of ubiquinone 0 (Ub0), ubiquinone 5 (Ub5), ubiquinone 10 (Ub10) and decyl-ubiquinone (DUb) were studied in freshly isolated rat hepatocytes, cultured rat liver Clone-9 cells and cancerous rat liver MH1C1 cells. PTP regulation by ubiquinones differed significantly in permeabilized Clone-9 and MH1C1 cells from that previously reported in liver mitochondria. Ub0 inhibited PTP opening in isolated hepatocytes and Clone-9 cells, whereas it induced PTP opening in MH1C1 cells. Ub5 did not affect PTP opening in isolated hepatocytes and MH1C1 cells, but it induced PTP opening in Clone-9 cells. Ub10 regulated PTP in isolated hepatocytes, whereas it did not affect PTP opening in Clone-9 and MH1C1 cells. Only DUb displayed the same effect on PTP regulation in the three hepatocyte lines tested. Despite such modifications in PTP regulation, competition between ubiquinones still occurred in Clone-9 and MH1C1 cells. As expected, Ub5 induced a PTP-dependent cell death in Clone-9, while it did not affect MH1C1 cell viability. Ub0 induced a PTP-dependent cell death in MH1C1 cells, but was also slightly cytotoxic in Clone-9 by an oxidative stress-dependent mechanism. Conclusions/Significance We found that various ubiquinone analogs regulate PTP in different ways depending on the cell studied. We took advantage of this unique property to develop a PTP opening-targeted strategy that leads to cell death specifically in cells where the ubiquinone analog used induces PTP opening, while sparing the cells in which it does not induce PTP opening.

Colorectal Cancer Metastasis: The DNA Repair Inhibitor Dbait Increases Sensitivity to Hyperthermia and Improves Efficacy of Radiofrequency Ablation

PURPOSE To assess the usefulness of combining hyperthermia with a DNA repair inhibitor (double-strand break bait [Dbait]) and its potential application to radiofrequency ablation (RFA) in a preclinical model of human colorectal cancer. MATERIALS AND METHODS The local ethics committee of animal experimentation approved all investigations. First, the relevance was assessed by studying the survival of four human colorectal adenocarcinoma cell cultures after 1 hour of hyperthermia at 41°C or 43°C with or without Dbait. Human colon adenocarcinoma cells (HT-29) were grafted subcutaneously into nude mice (n = 111). When tumors reached approximately 500 mm(3), mice were treated with Dbait alone (n = 20), sublethal RFA (n = 21), three different Dbait schemes and sublethal RFA (n = 52), or a sham treatment (n = 18). RFA was performed to ablate the tumor center alone. To elucidate antitumor mechanisms, 39 mice were sacrificed for blinded pathologic analysis, including assessment of DNA damage, cell proliferation, and tumor necrosis. Others were monitored for tumor growth and survival. Analyses of variance and log-rank tests were used to evaluate differences. RESULTS When associated with mild hyperthermia, Dbait induced cytotoxicity in all tested colon cancer cell lines. Sublethal RFA or Dbait treatment alone moderately improved survival (median, 40 days vs 28 days for control; P = .0005) but combination treatment significantly improved survival (median, 84 days vs 40 days for RFA alone, P = .0004), with approximately half of the animals showing complete tumor responses. Pathologic studies showed that the Dbait and RFA combination strongly enhances DNA damage and coagulation areas in tumors. CONCLUSION Combining Dbait with RFA sensitizes the tumor periphery to mild hyperthermia and increases RFA antitumor efficacy.

Chronic oxidative stress promotes H2AX protein degradation and enhances chemosensitivity in breast cancer patients

Anti‐cancer drugs often increase reactive oxygen species (ROS) and cause DNA damage. Here, we highlight a new cross talk between chronic oxidative stress and the histone variant H2AX, a key player in DNA repair. We observe that persistent accumulation of ROS, due to a deficient JunD‐/Nrf2‐antioxidant response, reduces H2AX protein levels. This effect is mediated by an enhanced interaction of H2AX with the E3 ubiquitin ligase RNF168, which is associated with H2AX poly‐ubiquitination and promotes its degradation by the proteasome. ROS‐mediated H2AX decrease plays a crucial role in chemosensitivity. Indeed, cycles of chemotherapy that sustainably increase ROS reduce H2AX protein levels in Triple‐Negative breast cancer (TNBC) patients. H2AX decrease by such treatment is associated with an impaired NRF2‐antioxidant response and is indicative of the therapeutic efficiency and survival of TNBC patients. Thus, our data describe a novel ROS‐mediated regulation of H2AX turnover, which provides new insights into genetic instability and treatment efficacy in TNBC patients.

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.

Prerequisites for ubiquinone analogs to prevent mitochondrial permeability transition-induced cell death

The permeability transition pore (PTP) is a mitochondrial inner membrane channel involved in cell death. The inhibition of PTP opening has been proved to be an effective strategy to prevent cell death induced by oxidative stress. Several ubiquinone analogs are known to powerfully inhibit PTP opening with an effect depending on the studied cell line. Here, we have studied the effects of ubiquinone 0 (Ub0), ubiquinone 5 (Ub5) and ubiquinone 10 (Ub10) on PTP regulation, H2O2 production and cell viability in U937 cells. We found that Ub0 induced both PTP opening and H2O2 production. Ub5 did not regulate PTP opening yet induced H2O2 production. Ub10 potently inhibited PTP opening yet induced H2O2 production. Both Ub0 and Ub5 induced cell death, whereas Ub10 was not toxic. Moreover, Ub10 prevented tert-butyl hydroperoxide-induced PTP opening and subsequent cell death. We conclude that PTP-inhibitor ubiquinone analogs are able to prevent PTP opening-induced cell death only if they are not toxic per se, which is the case when they have no or low pro-oxidant activity.

First-in-human phase I study of the DNA-repair inhibitor DT01 in combination with radiotherapy in patients with skin metastases from melanoma.

Background:DT01 is a DNA-repair inhibitor preventing recruitment of DNA-repair enzymes at damage sites. Safety, pharmacokinetics and preliminary efficacy through intratumoural and peritumoural injections of DT01 were evaluated in combination with radiotherapy in a first-in-human phase I trial in patients with unresectable skin metastases from melanoma.Methods:Twenty-three patients were included and received radiotherapy (30 Gy in 10 sessions) on all selected tumour lesions, comprising of two lesions injected with DT01 three times a week during the 2 weeks of radiotherapy. DT01 dose levels of 16, 32, 48, 64 and 96 mg were used, in a 3+3 dose escalation design, with an expansion cohort at 96 mg.Results:The median follow-up was 180 days. All patients were evaluable for safety and pharmacokinetics. No dose-limiting toxicity was observed and the maximum-tolerated dose was not reached. Most frequent adverse events were reversible grades 1 and 2 injection site reactions. Pharmacokinetic analyses demonstrated a systemic passage of DT01. Twenty-one patients were evaluable for efficacy on 76 lesions. Objective response was observed in 45 lesions (59%), including 23 complete responses (30%).Conclusions:Intratumoural and peritumoural DT01 in combination with radiotherapy is safe and pharmacokinetic analyses suggest a systemic passage of DT01.

DNA repair inhibition by DT01 as an adjuvant therapy at each stage of hepatocellular cancer (HCC) treatment.

303 Background: Hepatocellular carcinoma (HCC) is the most common liver cancer. Radiofrequency ablation (RFA), transarterial chemoembolization (TACE) or chemotherapy (CT) can be considered at each stage of HCC. The efficacy of these DNA damage inducing treatments could be enhanced by DNA damage repair inhibition. DT01 inhibits the complete DNA double-strand break repair machinery. Here, we assess the combination of DT01 with RFA, TACE or CT in preclinical models. Methods: For association with RFA, mice bearing flank-grafted tumors were sham treated (n=18), treated by DT01 (n=22), RFA (n=21) or a combination of DT01 and RFA (n=19). Mice were either sacrificed for pathological study or followed for survival. For association with TACE, rabbits bearing VX2 hepatocellular carcinoma in liver were untreated (n=9), treated with TACE (n=13) or treated with TACE and DT01 (n=14). Tumor growth, vascularization, necrosis and metastases were assessed with ultrasound scanning, color Doppler, pathology and autopsy respec...

Abstract 4483: Preclinical study of Dbait, an inhibitor of three DNA repair pathways, in breast cancer treatment.

Background: Five main types of therapies are available for the treatment of Breast Cancer (BC): surgery, radiation therapy, chemotherapy, hormone therapy, and most recently, targeted therapy. Most patients are treated with surgery in combination with one or more additional therapies for optimal cancer management. The limit of targeted therapy is that they are usually directed at specific targets in the body (extracellular receptors, kinases and enzymes) and therefore apply for limited number of patients. Herceptin and more recently trastuzumab have been shown to increase survival of patients over-expressing the HER2/neu receptor (20-30% of BC). PARP inhibitors that act as synthetic lethal with BRCA deficiency have shown promising results. These inhibitors that target the PARP-dependent DNA repair pathway seem however less efficient in patient with active homologous recombination repair. We have recently developed a new strategy (DNA repair bait, Dbait in short) that inhibits three central repair pathways (Homologous Recombination, Non Homologous End Joining and Single Strand Break Repair). DT01 is the 1st drug candidate of this family of DNA repair inhibitor already in a phase 1 trial in local metastatic melanoma. This study demonstrates the efficiency of DT01 in BC treatment. Materials and methods: Twelve BC cell lines were characterized for DNA repair gene expression and activation of the DNA damage response DDR (pH2AX and PAR detection) by western blot analysis of the proteins and classified according to their BRCA status. Clonal survival to Dbait (the DT01 in vitro variant) was analyzed 10 days after treatment. Four xenografted models derived from cell lines or patient samples were assayed for sensitivity to DT01. DT01 (5mg) was subcutaneousely administered every day for a week (5 sessions) or four days per week for three weeks (12 sessions of 2mg). In parallel, the PARP inhibitor ABT-888 was orally administered at the dose of 25 mg/day/kg during one week to compare efficiencies. Tumor growth and survival of untreated and treated animals were monitored during 150 days. Tumors were analyzed for different repair and stress markers by immunocytology. Results: This study provides the evidences that: i) Dbait induces death in vitro in BRCA-/- cells but also in BRCA+/+ cells having a high level of spontaneous DDR; ii) DT01 controls tumor growth in all the model tested with or without BRCA mutation; iii) DT01 cured 30% BRCA-/- tumors; iv) DT01 was more efficient that ABT-888 which did not allow any cure and showed a 73% increase of survival. At the end of the study 80% of the animals treated by DT01 were still alive. Conclusion: DT01 is a promising investigational medicinal product to treat Breast Cancer. Its association with radiotherapy is under investigation. Citation Format: Marie Dutreix, Youlia M. Kirova, Amelie Croset, Flavien Devun, Jian-Sheng Sun. Preclinical study of Dbait, an inhibitor of three DNA repair pathways, in breast cancer treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4483. doi:10.1158/1538-7445.AM2013-4483