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.
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.
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.
Our results highlight the therapeutic interest of combining AsiDNA and PARPi to recapitulate synthetic lethality in all tumors independently of their HR status.
.
Abstract 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).
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.
The effects of ubiquinone 0 (Ub(0)), ubiquinone 5 (Ub(5)), ubiquinone 10 (Ub(10)) 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. Ub(0) inhibited PTP opening in isolated hepatocytes and Clone-9 cells, whereas it induced PTP opening in MH1C1 cells. Ub(5) did not affect PTP opening in isolated hepatocytes and MH1C1 cells, but it induced PTP opening in Clone-9 cells. Ub(10) 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, Ub(5) induced a PTP-dependent cell death in Clone-9, while it did not affect MH1C1 cell viability. Ub(0) induced a PTP-dependent cell death in MH1C1 cells, but was also slightly cytotoxic in Clone-9 by an oxidative stress-dependent mechanism.
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.
Background
Dbait molecules are a new class of DNA repair inhibitors triggering false DNA damage signaling in cancer cells. Dbait has already been shown to be effective in combination with ...radiotherapy. The aim of this study was to assess the adjuvant impact of Dbait on chemotherapy in vitro and in mouse models of colorectal cancer.
Methods
We assessed DNA repair efficiency over time, in vitro, in human colon adenocarcinoma HT-29 (wild-type
KRAS
) and HCT-116 (mutated
KRAS
) cell lines treated with Dbait in combination with 5-fluorouracil and/or camptothecin. Genetically engineered mice spontaneously developing colorectal tumors in the intestines were selected for the evaluation of treatment efficacy.
Results
Dbait delayed the repair of DNA damage induced by chemotherapy in vitro. In
APC
+/1638N
mutant mice, the combination of Dbait and chemotherapy decreased tumor size more effectively than chemotherapy alone (median size: 3.6 vs. 10.85 mm
2
,
P
< 0.05). In
APC
+/1638N
/
KRAS
V12G
mutant mice, animals treated with a combination of Dbait and chemotherapy survived significantly longer than animals treated by chemotherapy alone (median survival: 210 vs. 194 days,
P
< 0.05). A quarter of all the animals treated by chemotherapy alone died as rapidly as untreated animals, whereas the first death was delayed by 29 days by the addition of Dbait. No increase in toxicity due to Dbait was observed in either mouse model.
Conclusions
The use of Dbait to inhibit DNA repair may be an effective additional treatment for increasing the efficacy of chemotherapy in colon or rectal cancer, independently of KRAS status.
Abstract Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our ...goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery.
Increased DNA repair activity in cancer cells is one of their primary mechanisms of resistance to current radio- and chemotherapies. The molecule coDbait is the first candidate in a new class of ...drugs that target the double-strand DNA break repair pathways with the aim of overcoming these resistances. coDbait is a 32-base pair (bp) double-stranded DNA molecule with a cholesterol moiety covalently attached to its 5′-end to facilitate its cellular uptake. We report here the preclinical pharmacokinetic and toxicology studies of subcutaneous coDbait administration in rodents and monkeys. Maximum plasma concentration occurred between 2 to 4 hours in rats and at 4 hours in monkeys. Increase in mean AUC0–24h was linear with dose reaching 0.5 mg·h/ml for the highest dose injected (32 mg) for both rats and monkeys. No sex-related differences in maximum concentration (Cmax) nor AUC0–24h were observed. We extrapolated these pharmacokinetic results to humans as the subcutaneous route has been selected for evaluation in clinical trials. Tri-weekly administration of coDbait (from 8 to 32 mg per dose) for 4 weeks was overall well tolerated in rats and monkeys as no morbidity/mortality nor changes in clinical chemistry and histopathology parameters considered to be adverse effects have been observed.
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.
Synopsis
This work gives new insights into the regulation of H2AX protein turnover under chronic oxidative stress that affects DNA damage response. H2AX degradation upon chronic stress sensitizes tumour cells to chemotherapy and is indicative of better survival in triple‐negative breast cancer patients.
Physiological conditions of chronic oxidative stress, mediated by the loss of JunD or Nrf2 transcription factors, are associated with a reduced protein level of the histone variant H2AX.
Under conditions of chronic stress due to junD or Nrf2 deficiency, H2AX protein is targeted for degradation by the proteasome.
ROS‐dependent H2AX degradation is mediated by enhanced interaction of H2AX protein with the E3 ubiquitin ligase RNF168.
H2AX decrease by chronic oxidative stress increases tumour cell genomic instability and death.
Chemosensitivity and survival of triple‐negative breast cancer patients are improved by stress‐mediated H2AX degradation following successive cycles of chemotherapy.
This work gives new insights into the regulation of H2AX protein turnover under chronic oxidative stress that affects DNA damage response. H2AX degradation upon chronic stress sensitizes tumour cells to chemotherapy and is indicative of better survival in triple‐negative breast cancer patients.
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.
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.
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.
Combining Dbait with RFA sensitizes the tumor periphery to mild hyperthermia and increases RFA antitumor efficacy.
The ability of cancer cells to recognize damage and initiate DNA repair is an important mechanism for therapeutic resistance. The use of inhibitors of DNA damage repair or signaling pathways appears ...to provide a unique opportunity for targeting genetic differences between tumor and normal cells. In this review, we firstly describe the main DNA lesions induced by the different treatments and the pathways involved in their repair. Then we review the mechanism of action and applications of an innovative DNA repair inhibitor: Dbait (and its clinical form DT01). Dbait/DT01 consists of 32 bp deoxyribonucleotides forming an intramolecular DNA double helix that mimics DNA lesions. They act as a bait for DNA damage signaling enzymes, the polyadenyl-ribose polymerase (PARP), and the DNA-dependent kinase (DNA-PK), inducing a "false" DNA damage signal and ultimately inhibiting recruitment at the damage site of many proteins involved in double-strand break and single-strand break repair pathways. Preclinical studies have demonstrated the capacity of Dbait/DT01 to improve the efficiency of (i) chemotherapy in colorectal cancer or hepatocellular carcinoma models, (ii) radiofrequency ablative in colorectal cancer liver metastases models, and (iii) radiotherapy in xenografted mice with head & neck squamous cell carcinoma, glioblastoma and melanoma. Following this good preclinical results, we performed a first-in-human phase 1-2a study evaluating the safety and efficacy of the combination of DT01 with radiotherapy for the treatment of skin metastases of melanoma. Twenty-three patients were included. No dose-limiting toxicity was observed. An objective response was observed in 59% lesions, including 30% complete responses. This first promising clinical efficacy provides future potential interesting clinical development of Dbait/DT01 with various anticancer treatments.
Objective
This study aimed to explore the antitumour effect of the DNA repair inhibitor, DT01 (the cholesterol conjugated form of Dbait), as an adjunct treatment to enhance the therapeutic efficacy ...of transarterial chemoembolization (TACE) in pre-clinical models of hepatocellular carcinoma (HCC).
Methods
A rabbit model bearing liver tumours was either left untreated or treated with TACE or with a combination of TACE+DT01. Tumour growth was monitored by ultrasound. These results were further confirmed in mice grafted with an intrahepatic human HCC model treated with doxorubicin (DOX) alone or DOX+DT01.
Results
The combination of DT01 with TACE in a rabbit liver model led to a significant decrease in tumour volume (p=0.03). Colour Doppler and immunohistochemical staining revealed a strong decrease in vascularization in the DT01+TACE-treated group preventing the tumour growth restart observed after TACE alone. Similarly, the DT01 combination with DOX led to significant anti-tumour efficacy compared to DOX alone (p=0.02) in the human HCC model. In addition, a significant decrease in vascularization in the group receiving combination DT01 and DOX treatment was observed.
Conclusions
DT01 is well tolerated and may potentiate HCC treatment by enhancing the DNA-damaging and anti-vascularization effect of TACE with doxorubicin.
Key point
s
• DT01 combined with TACE leads to significant anti-tumour efficacy without additional toxicity.
• A potential anti-angiogenic role of DT01 was identified in preclinical models.
• DT01 may potentiate HCC treatment by enhancing the efficacy of TACE.
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