Search

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

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

Centrosome amplification has been described as a common feature of human cancers and it is known to promote tumorigenesis when induced in animals. However, little is known about the real status of centrosome numbers in human cancers and whether numerical alterations are solely associated with poor prognosis. To address this question, we have analyzed a large cohort of human epithelial ovarian cancers (EOCs) from 100 patients using state-of-the-art microscopy to determine the Centrosome-Nucleus Index (CNI) of each tumor. We found that EOCs are highly heterogeneous, with infrequent but strong centrosome amplifications leading to higher CNI than in healthy tissues. Strikingly, while a correlation between CNI and genomic alterations, such as aneuploidy or chromosome rearrangements could not be established, we found that high CNI correlates with increased patient survival and sensitivity to chemotherapy. Using ovarian cancer cellular models to manipulate centrosome numbers and Patient-Derived Xenografts (PDXs), we found that higher CNIs can positively impact the response to chemotherapy and inhibit cell dissemination. Our findings highlight a novel paradigm linking centrosome amplification to the inhibition of tumor progression.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. The most common subtype of EOC is high-grade serous (HGSOC), which responds at least initially to chemotherapy but has a worse overall prognosis. Genomic, transcriptomic, and proteogenomic profiling of HGSOC suggested a whole spectrum of molecular diversity, including homologous recombination pathway deficiencies (HRD). The centrosome is the main microtubule (MT)-organizing center of animal cells. It facilitates the accuracy of chromosome segregation during mitosis and influences cell polarity and migration. The presence of more than two centrosomes in a cell, centrosome amplification, has long been associated with tumorigenesis. However, little is known about the real status of centrosome numbers in human cancers and whether numerical alterations are solely associated with poor prognosis. We screened 100 samples of primary EOCs including 88 HGSOC, using immunofluorescence and state-of-the-art microscopy, to determine the centrosome-nucleus index (CNI). We integrated these data with genomic alterations, HRD status, and patient outcome. We found that EOCs are highly heterogeneous, with infrequent but strong centrosome amplifications leading to higher CNI than in healthy tissues. Strikingly, while a correlation between CNI and genomic alterations, such as aneuploidy or chromosome rearrangements, could not be established, we found that high CNI correlates with increased patient survival and sensitivity to chemotherapy, independently of HRD status. Using ovarian cancer cellular models to manipulate centrosome numbers and patient-derived xenografts (PDXs), we found that higher CNIs can positively impact the response to chemotherapy and inhibit peritoneal cell dissemination. Citation Format: Jean-Philippe Morretton, Aurelie Herbette, Camille Cosson, Bassirou Mboup, Aurelien Latouche, Pierre Gestraud, Tatiana Popova, Marc-Henri Stern, Fariba Nemati, Didier Decaudin, Guillaume Bataillon, Veronique Becette, Didier Meseure, Andre Nicolas, Odette Mariani, Claire Bonneau, Jorge Barbazan, Anne Vincent-Salomon, Fatima Mechta-Grigoriou, Sergio Roman-Roman, Roman Rouzier, Xavier Sastre-Garau, Oumou Goundiam, Renata Basto. Centrosome amplification favors survival and impairs ovarian cancer progression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A43.

// Claire Viallard 1, 2 , Jean-Michel Chezal 1, 2 , Florence Mishellany 3 , Isabelle Ranchon-Cole 4 , Bruno Pereira 5 , Aurelie Herbette 6 , Sophie Besse 1, 2 , Zied Boudhraa 1, 2 , Nathalie Jacquemot 4 , Anne Cayre 3 , Elisabeth Miot-Noirault 1, 2 , Jian-Sheng Sun 7 , Marie Dutreix 6 , Francoise Degoul 1, 2 1 Clermont Universite, Universite d’Auvergne, Imagerie Moleculaire et Therapie Vectorisee, BP 10448, F-63000 Clermont-Ferrand, France 2 Inserm, U 990, F-63000 Clermont-Ferrand, France 3 Anatomopathology Department, Centre Jean Perrin, Comprehensive Cancer Center, 63011 Clermont-Ferrand, France 4 Clermont Universite, Universite d’Auvergne, UFR Pharmacie Laboratoire de Biophysique Neurosensorielle, Inserm U 1107, F-63001 Clermont-Ferrand, France 5 DRCI, CHU, 63003 Clermont-Ferrand, France 6 CNRS-UMR3347, INSERMU1021, Institut Curie, Universite Paris Sud, Bat 110, Centre Universitaire 91405 Orsay, Cedex, France 7 DNA Therapeutics, SA, 91058 Evry Cedex, France Correspondence to: Francoise Degoul, e-mail: francoise.degoul@inserm.fr Keywords: targeted radionuclide therapy, melanoma, coDbait, DNA repair Received: October 16, 2015 Accepted: January 24, 2016 Published: February 12, 2016 ABSTRACT 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 [ 131 I]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 [ 131 I]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.

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). 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. 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. DT01 is well tolerated and may potentiate HCC treatment by enhancing the DNA-damaging and anti-vascularization effect of TACE with doxorubicin. • 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.

International audience; Introducing small DNA molecules (Dbait) impairs the repair of damaged chromosomes and provides a new method for enhancing the efficiency of radiotherapy in radio-resistant tumors. The radiosensitizing activity is dependent upon the efficient delivery of Dbait molecules into the tumor cells. Different strategies have been compared, to improve this key step. We developed a pipeline of assays to select the most efficient nanoparticles and administration protocols before preclinical assays: (i) molecular analyses of complexes formed with Dbait molecules, (ii) cellular tests for Dbait uptake and activity, (iii) live zebrafish embryo confocal microscopy monitoring for in vivo distribution and biological activity of the nanoparticles and (iv) tumor growth and survival measurement on mice with xenografted tumors. Two classes of nanoparticles were compared, polycationic polymers with linear or branched polyethylenimine (PEI) and covalently attached cholesterol (coDbait). The most efficient Dbait transfection was observed with linear PEI complexes, in vitro and in vivo. Doses of coDbait ten-fold higher than PEI/Dbait nanoparticles, and pretreatment with chloroquine, were required to obtain the same antitumoral effect on xenografted melanoma. However, with a 22-fold lower 'efficacy dose/toxicity dose' ratio as compared with Dbait/PEI, coDbait was selected for clinical trials.

Purpose: Enhanced DNA repair activity is often associated with tumor resistance to radiotherapy. We hypothesized that inhibiting DNA damage repair would sensitize tumors to radiation-induced DNA damage. Experimental Design: A novel strategy for inhibiting DNA repair was tested. We designed small DNA molecules that mimic DNA double-strand breaks (called Dbait) and act by disorganizing damage signaling and DNA repair. We analyzed the effects of Dbait in cultured cells and on xenografted tumors growth and performed preliminary studies of their mechanism(s) of action. Results: The selected Dbait molecules activate H2AX phosphorylation in cell culture and in xenografted tumors. In vitro, this activation correlates with the reduction of Nijmegen breakage syndrome 1 and p53-binding protein 1 repair foci formation after irradiation. Cells are sensitized to irradiation and do not efficiently repair DNA damage. In vivo, Dbait induces regression of radioresistant head and neck squamous cell carcinoma (Hep2) and melanoma (SK28 and LU1205) tumors. The combination of Dbait32Hc treatment and fractionated radiotherapy significantly enhanced the therapeutic effect. Tumor growth control by Dbait molecules depended directly on the dose and was observed with various irradiation protocols. The induction of H2AX phosphorylation in tumors treated with Dbait suggests that it acts in vivo through the induction of “false” DNA damage signaling and repair inhibition. Conclusions: These data validate the concept of introducing small DNA molecules, which mimic DNA damage, to trigger “false” signaling of DNA damage and impair DNA repair of damaged chromosomes. This new strategy could provide a new method for enhancing radiotherapy efficiency in radioresistant tumors.

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.

DNA damage triggers a complex signaling cascade involving a multitude of phosphorylation events. We found that the threonine 7 (Thr-7) residue of heat shock protein 90α (Hsp90α) was phosphorylated immediately after DNA damage. The phosphorylated Hsp90α then accumulated at sites of DNA double strand breaks and formed repair foci with slow kinetics, matching the repair kinetics of complex DNA damage. The phosphorylation of Hsp90α was dependent on phosphatidylinositol 3-kinase-like kinases, including the DNA-dependent protein kinase (DNA-PK) in particular. DNA-PK plays an essential role in the repair of DNA double strand breaks by nonhomologous end-joining and in the signaling of DNA damage. It is also present in the cytoplasm of the cell and has been suggested to play a role in cytoplasmic signaling pathways. Using stabilized double-stranded DNA molecules to activate DNA-PK, we showed that an active DNA-PK complex could be assembled in the cytoplasm, resulting in phosphorylation of the cytoplasmic pool of Hsp90α. In vivo, reverse phase protein array data for tumors revealed that basal levels of Thr-7-phosphorylated Hsp90α were correlated with phosphorylated histone H2AX levels. The Thr-7 phosphorylation of the ubiquitously produced and secreted Hsp90α may therefore serve as a surrogate biomarker of DNA damage. These findings shed light on the interplay between central DNA repair enzymes and an essential molecular chaperone.

International audience; BACKGROUND: Glioma is the most aggressive tumor of the brain and the most efficient treatments are based on radiotherapy. However, tumors are often resistant to radiotherapy due to an enhanced DNA repair activity. Short and stabilized DNA molecules (Dbait) have recently been proposed as an efficient strategy to inhibit DNA repair in tumor. METHODOLOGY/PRINCIPAL FINDINGS: The distribution of three formulations of Dbait, (i) Dbait alone, (ii) Dbait associated with polyethylenimine, and (iii) Dbait linked with cholesterol (coDbait), was evaluated one day after intratumoral delivery in an RG2 rat glioma model. Dbait molecule distribution was assessed in the whole organ with 2D-FRI and in brain sections. CoDbait was chosen for further studies given its good retention in the brain, cellular localization, and efficacy in inducing the activation of DNA repair effectors. The radiosensitizing effect of coDbait was studied in four groups of rats bearing RG2-glioma: no treatment, radiotherapy only, coDbait alone, and CoDbait with radiotherapy. Treatment started 7 days after tumor inoculation and consisted of two series of treatment in two weeks: coDbait injection followed by a selective 6-Gy irradiation of the head. We evaluated the radiosensitizing effect using animal survival, tumor volume, cell proliferation, and vasculature characteristics with multiparametric MRI. CoDbait with radiotherapy improved the survival of rats bearing RG2-glioma by reducing tumor growth and cell proliferation without altering tumor vasculature. CONCLUSION/SIGNIFICANCE: coDbait is therefore a promising molecular therapy to sensitize glioma to radiotherapy.

We aim to evaluate environmental and genetic effects on the expansion/proliferation of committed single cells during embryonic development, using melanoblasts as a paradigm to model this phenomenon. Melanoblasts are a specific type of cell that display extensive cellular proliferation during development. However, the events controlling melanoblast expansion are still poorly understood due to insufficient knowledge concerning their number and distribution in the various skin compartments. We show that melanoblast expansion is tightly controlled both spatially and temporally, with little variation between embryos. We established a mathematical model reflecting the main cellular mechanisms involved in melanoblast expansion, including proliferation and migration from the dermis to epidermis. In association with biological information, the model allows the calculation of doubling times for melanoblasts, revealing that dermal and epidermal melanoblasts have short but different doubling times. Moreover, the number of trunk founder melanoblasts at E8.5 was estimated to be 16, a population impossible to count by classical biological approaches. We also assessed the importance of the genetic background by studying gain- and loss-of-function β-catenin mutants in the melanocyte lineage. We found that any alteration of β-catenin activity, whether positive or negative, reduced both dermal and epidermal melanoblast proliferation. Finally, we determined that the pool of dermal melanoblasts remains constant in wild-type and mutant embryos during development, implying that specific control mechanisms associated with cell division ensure half of the cells at each cell division to migrate from the dermis to the epidermis. Modeling melanoblast expansion revealed novel links between cell division, cell localization within the embryo and appropriate feedback control through β-catenin.

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.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.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), P0.05). 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, P0.05). 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.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.

Background Peritoneal carcinomatosis is an unmet medical need. Laparoscopy offers a unique opportunity to control and to steer the operating environment during surgery by loading carbon dioxide with a therapeutic substance and creating the so-called therapeutic capnoperitoneum. We have treated a human sample of peritoneal carcinomatosis from an endometrial adenocarcinoma ex vivo just after surgery. Methods A nontoxic therapeutic agent (Dbait) was aerosolized into a box containing diseased human peritoneum under a pressure of 12 mmHg CO2. Dbait (noncoding DNA fragments) acts through jamming DNA damage sensing and signaling, ultimately inhibiting DNA repair system of cancer cells. Dbait were coupled to cholesterol molecules to facilitate intracellular uptake, and to Cyanine (Cy5) to allow detection by fluorescence. In a control experiment, the same solution was applied to the other half of the sample using conventional lavage. Results Physical results revealed fluorescence within the tumor up to 1 mm depth in the therapeutic capnoperitoneum sample and no uptake in the lavage sample. Biological results showed intranuclear phosphorylation of H2AX in the nebulized sample and no activity in the lavage sample. Importantly, tumor nodules showed more activity than the neighbor, normal peritoneum. Detection of histone gamma-H2AX (phosphorylated H2AX) reveals activation of DNA-dependent protein kinase (DNA-PK) by Dbait, which has been shown to be the key step for sensitization to genotoxic therapy. Conclusions Dbait are taken up by cancer cells and have a biological activity up to 1 mm depth. Nebulization of the molecule is significantly more effective than conventional lavage. This proof of principle supports the need for clinical studies applying therapeutic capnoperitoneum together with Dbait for treating peritoneal carcinomatosis.