<p>Supplementary figure S4. Molecular portraits of resistance to AsiDNA or olaparib in BC cell lines on Atlas of Cancer Signaling Networks. Molecular portraits generated for two groups of BC cell lines that demonstrate resistance to (A) AsiDNA or (B) Ola using expression, copy number variations and mutation frequency, projected on ACSN global map (Supplementary Figure S3-B). Red and green background colors respectively represent high and low mRNA expression levels across genes of a same pathway; intensity of color shows the level of the change to the mean value of the BC cell lines group. Copy number variations are represented as glyphs where yellow squares indicated copy number gains and blue indicate copy number losses. Mutations are represented using cyan diamonds. The data is displayed at the top level zoom demonstrating a pattern of data distribution across biological processes.</p>

Supplementary Data from Small-Molecule Drugs Mimicking DNA Damage: A New Strategy for Sensitizing Tumors to Radiotherapy

Table S1: Treatment group allocation of mice post intrahepatic grafting; Figure S1: DT01 in combination with single agent chemotherapy; Figure S2: Proportion of apoptotic nuclei in HES sections; Figure S3: Average body weight (g) of mice during treatment; Figure S4: Liver function assessment; Figure S5: Average body and liver weights (g) of mice during treatment with escalating doses of DT01 in association with OXA or 5-FU; Table S2: Summary of main findings

<p>Supplementary Table 1. Unique genes robustly correlated with survival to AsiDNA or Olaparib in BC cell lines. The values are correlation coefficients (Spearman r; P value <0.005) between gene expression and survival to AsiDNA or Olaparib. Blue: genes involved in DNA repair or cell cycle. (+): positive correlation; (-): negative correlation.</p>

<p>Supplementary figure S4. Molecular portraits of resistance to AsiDNA or olaparib in BC cell lines on Atlas of Cancer Signaling Networks. Molecular portraits generated for two groups of BC cell lines that demonstrate resistance to (A) AsiDNA or (B) Ola using expression, copy number variations and mutation frequency, projected on ACSN global map (Supplementary Figure S3-B). Red and green background colors respectively represent high and low mRNA expression levels across genes of a same pathway; intensity of color shows the level of the change to the mean value of the BC cell lines group. Copy number variations are represented as glyphs where yellow squares indicated copy number gains and blue indicate copy number losses. Mutations are represented using cyan diamonds. The data is displayed at the top level zoom demonstrating a pattern of data distribution across biological processes.</p>

<p>Supplementary Table 4. IC20 and IC50 of different PARP inhibitors in MDAMB231 cell line. IC20 and IC50 were calculated according to the dose-response curves of the different PARP inhibitors using GraphPadPrism software.</p>

<p>Supplementary Table 1. Unique genes robustly correlated with survival to AsiDNA or Olaparib in BC cell lines. The values are correlation coefficients (Spearman r; P value <0.005) between gene expression and survival to AsiDNA or Olaparib. Blue: genes involved in DNA repair or cell cycle. (+): positive correlation; (-): negative correlation.</p>

Supplementary figure S1. Synergy between AsiDNA and PARP defect in DT40 cells. (A) Cytotoxicity of AsiDNA toward various isogenic DT40 cell lines. (B) Comparison of cell survival to AsiDNA in DT40 cells wild-type (WT; black) or PARP KO (red) alone (continuous line) or in combination with veliparib 1µM (blue discontinuous line). Survivals were monitored by ATPlite 1-step kit (72 hours after treatment) in various mutant DT40 cells as described in (36). Survival is expressed as % of non-treated cells. Results are represented as mean survival {plus minus} SEM for three independent experiments.

<p>Supplementary Table 4. IC20 and IC50 of different PARP inhibitors in MDAMB231 cell line. IC20 and IC50 were calculated according to the dose-response curves of the different PARP inhibitors using GraphPadPrism software.</p>

<p>Supplementary figure S2. Correlation between sensitivity of tumor cells to AsiDNA and olaparib. Survival was moniored by trypan blue staining and manual counting (6 days after treatment). Survivals are expressed as % of living non treated cells. Correlation analysis between survival to AsiDNA and olaparib was performed using GraphPadPrism5 software and indicates a non significant correlation.</p>

OBJECTIVE:This study was initiated and conducted by several laboratories, 3 of the main cosmetic ingredient suppliers and 4 brands of cosmetics in France. Its objective is to show the interest and robustness of coupling chemical and genetic analyses in the identification of plant species. In this study, the Lavandula genus was used. METHODS:In this study, we used two analytical methods. Chemical analysis from UHPLC (ultra-high-performance liquid chromatography) and genetic analysis from barcoding with genetic markers. RESULTS:Eleven lavender species were selected (botanically authenticated) and analysed. The results show that three chemical compounds (coumaric acid hexoside, ferulic acid hexoside and rosmarinic acid) and three genetic markers (RbcL, trnH-psbA and ITS) are of interest for the differentiation of species of the genus lavandula. CONCLUSION:The results show that the combination of complementary analytical methods is a relevant system to prove the botanical identification of lavender species. This first study, carried out on a plant of interest for cosmetics, demonstrates the need for authentication using a tool combining genetic and chemical analysis as an advance over traditional investigation methods used alone, in terms of identification and authentication reliability.

Telomere fusions lead to a state of genomic instability, and are thought to drive clonal evolution and tumorigenesis. Telomere fusions occur via both Classical and Alternative Non-Homologous End Joining repair pathways. AsiDNA is a DNA repair inhibitor that acts by mimicking a DNA double strand break (DSB) and hijacking the recruitment of proteins involved in various DNA repair pathways. In this study, we investigated whether the inhibition of DSB-repair pathways by AsiDNA could prevent telomere fusions during crisis. The present study showed that AsiDNA decreased the frequency of telomere fusions without affecting the rate of telomere erosion. Further, it indicated that AsiDNA does not impact the choice of the repair pathway used for the fusion of short dysfunctional telomeres. AsiDNA is thought to prevent short telomeres from fusing by inhibiting DNA repair. An alternative, non-mutually exclusive possibility is that cells harbouring fusions preferentially die in the presence of AsiDNA, thus resulting in a reduction in fusion frequency. This important work could open the way for investigating the use of AsiDNA in the treatment of tumours that have short dysfunctional telomeres and/or are experiencing genomic instability.

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.

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.

Abstract 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.

Radiolabelled melanin ligands offer an interesting strategy for the treatment of disseminated pigmented melanoma. One of these molecules, ICF01012 labelled with iodine 131, induced a significant slowing of melanoma growth. Here, we have explored the combination of [I-131]ICF01012 with coDbait, a DNA repair inhibitor, to overcome melanoma radioresistance and increase targeted radionuclide therapy (TRT) efficacy. In human SK-Mel 3 melanoma xenograft, the addition of coDbait had a synergistic effect on tumor growth and median survival. The anti-tumor effect was additive in murine syngeneic B16Bl6 model whereas coDbait combination with [I-131]ICF01012 did not increase TRT side effects in secondary pigmented tissues (e.g. hair follicles, eyes). Our results confirm that DNA lesions induced by TRT were not enhanced with coDbait association but, the presence of micronuclei and cell cycle blockade in tumor shows that coDbait acts by interrupting or delaying DNA repair. In this study, we demonstrate for the first time, the usefulness of DNA repair traps in the context of targeted radionuclide therapy.

Abstract Metastatic liver disease from colorectal cancer is a significant clinical problem. This is mainly attributed to nonresectable metastases that frequently display low sensitivities to available chemotherapies and develop drug resistance partly via hyperactivation of some DNA repair functions. Combined therapies have shown some disease control; however, there is still a need for more efficient chemotherapies to achieve eradication of colorectal cancer liver metastasis. We investigated the tolerance and efficacy of a novel class of DNA repair inhibitors, Dbait, in association with conventional chemotherapy. Dbait mimics double-strand breaks and activates damage signaling, consequently inhibiting single- and double-stranded DNA repair enzyme recruitment. In vitro, Dbait treatment increases sensitivity of HT29 and HCT116 colorectal cancer cell lines. In vivo, the pharmacokinetics, biodistribution and the efficacy of the cholesterol-conjugated clinical form of Dbait, DT01, were assessed. The chemosensitizing abilities of DT01 were evaluated in association with oxaliplatin and 5-fluorouracil in intrahepatic HT29 xenografted mice used as a model for colorectal cancer liver metastasis. The high uptake of DT01 indicates that the liver is a specific target. We demonstrate significant antitumor efficacy in a liver metastasis model with DT01 treatment in combination with oxaliplatin and 5-fluorouracil (mean: 501 vs. 872 mm2, P = 0.02) compared to chemotherapy alone. The decrease in tumor volume is further associated with significant histologic changes in necrosis, proliferation, angiogenesis and apoptosis. Repeated cycles of DT01 do not increase chemotherapy toxicity. Combining DT01 with conventional chemotherapy may prove to be a safe and effective therapeutic strategy in the treatment of metastatic liver cancer. Mol Cancer Ther; 15(1); 15–22. ©2015 AACR.

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.

Article Tools Developmental Therapeutics—Clinical Pharmacology and Experimental Therapeutics Article Tools OPTIONS & TOOLS Export Citation Track Citation Add To Favorites Rights & Permissions COMPANION ARTICLES No companion articles ARTICLE CITATION DOI: 10.1200/jco.2015.33.15_suppl.2555 Journal of Clinical Oncology - published online before print May 20, 2015 First-in-human phase I study of the DNA repair inhibitor DT01 in combination with radiotherapy in patients with in transit melanoma. Christophe Le TourneauxChristophe Le TourneauSearch for articles by this author , Brigitte DrenoxBrigitte DrenoSearch for articles by this author , Youlia KirovaxYoulia KirovaSearch for articles by this author , Jean Jacques GrobxJean Jacques GrobSearch for articles by this author , Thomas JouaryxThomas JouarySearch for articles by this author , Caroline DutriauxxCaroline DutriauxSearch for articles by this author , Luc ThomasxLuc ThomasSearch for articles by this author , Celeste LebbexCeleste LebbeSearch for articles by this author , Laurent MortierxLaurent MortierSearch for articles by this author , Philippe SaiagxPhilippe SaiagSearch for articles by this author , Marie-Françoise AvrilxMarie-Françoise AvrilSearch for articles by this author , Eve MaubecxEve MaubecSearch for articles by this author , Pierre BeyxPierre BeySearch for articles by this author , Jean-Marc CossetxJean-Marc CossetSearch for articles by this author , Jian-sheng SunxJian-sheng SunSearch for articles by this author , Bernard AsselainxBernard AsselainSearch for articles by this author , Flavien DevunxFlavien DevunSearch for articles by this author , Michel E. MartyxMichel E. MartySearch for articles by this author , Marie DutreixxMarie DutreixSearch for articles by this author Show More Institut Curie, Department of Medical Oncology, Paris, France; CHU de Nantes - Hôtel Dieu, Nantes, France; Institut Curie, Radiotherapy Department, Paris, France; CHU Timone APHM, Aix-Marseille University, Marseille, France; Saint André Hospital CHU de Bordeaux, Dermatology, Bordeaux, France; Hôpital Saint-André,CHU Bordeaux, Bordeaux, France; Lyon 1 University Centre Hospitalier Lyon Sud, Pierre Benite, France; Dermatology Department, Hôpital Saint-Louis, Assistance-Publique-Hôpitaux de Paris, Paris, France; CHRU de Lille, Lille, France; Hospital Ambroise Pare, Boulogne-Billancourt, France; Assistance Publique–Hôpitaux de Paris, Hôpital Cochin, Paris, France; Hopital Bichat, Paris, France; CUrie Institute, Paris, France; Institut Curie, Paris, France; DNA Therapeutics, Evry, France; Institut Curie, Orsay, France; Hopital Saint Louis, Paris Cedex, France; Institut Curie, Université Paris-Sud, Orsay, France Abstract Disclosures https://doi.org/10.1200/jco.2015.33.15_suppl.2555 Abstract Abstract 2555 Background: DT01 is a double stranded DNA oligonucleotide conjugated to cholesterol, which mimics "false" double strand breaks (DSB). DT01 triggers DNA repair proteins trapping, thereby inhibiting their repair activity. In non-clinical studies, DT01 showed antitumor activity as a single agent and in combination with radiotherapy (RT) in several tumor types including melanoma. No NOAEL could be defined. Methods: Subcutaneous peritumoral and intratumoral injections of DT01 were evaluated in combination with RT in a first-in-human phase I trial in patients (pts) with unresectable in-transit metastases of melanoma. Each pt received RT at a standard dose (3Gy/d 5 days/week for 2 weeks) on all selected tumor lesions, while only one or two tumor lesions were treated with DT01 3 times a week during both weeks of RT. A 3+3 dose escalation design was used. DT01 dose levels explored were 16, 32, 48, 64, and 96 mg. RT was given using a rectangular field by a linear accelerator. Tolerance was assessed using NCI CTCAE v4.3 and efficacy using modified RECIST criteria 1.1. Results: Seventeen pts (16 eligible) were included in the trial, of whom received RT alone in 31 and RT + DT01 in 31 tumor target lesions. As no dose-limiting toxicity was observed, the maximum tolerated dose was not reached. Most frequent adverse events were reversible grade 1 and 2 injection site reactions. PK analyses suggested a systemic passage of DT01 in a non-linear fashion. Overall response rates (ORR) were 68% and 48% in tumor lesions treated with RT + DT01 and RT alone, respectively. Notably, ORR correlated with blood AUC (p = 0.01), suggesting 1) an anti-tumor activity of DT01, and 2) a systemic effect of DT01. Conclusions: DT01 in combination with RT is safe in patients with metastatic in-transit melanoma, and provides antitumor activity in DT01-treated and non-treated lesions possibly explained by a systemic distribution of the drug. Clinical trial information: NCT01469455. © 2015 by American Society of Clinical Oncology

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 respectively. For association with CT, mice bearing orthotopic liver tumors were administered NaCl (n=7), CT (doxorubicin) (n=10), systemic DT01 treatment (n=7) or an association of DT01 and CT (n=10). Tumor growth and pathological studies were assessed. Results: Mice treated by RFA and DT01 have longer survival compared to RFA alone (median survival: 57 vs 40 days) with 54% of complete responses while RFA alone improved survival moderately (median survival: 40 vs 28 days for control). Rabbits treated with TACE and DT01, in comparison to CT alone, show efficient tumor growth control, an increase of tumor necrosis (61% vs 40%), a decrease of metastases (21 vs 54%) and an absence of neoangiogenesis rebound. Mice treated with CT in combination with DT01 show a significant decrease in tumor volume and tumor necrosis, compared to the mice treated with CT alone. In all models, DT01 addition to treatments did not add any toxicity. Conclusions: Our results show that the addition of DT01 to RFA, TACE or CT enhances their antitumor activities and provide an experimental basis for the use of DT01 as an adjuvant therapy at each stage of HCC treatment.