Published OnlineFirst August 24, 2016; DOI: 10.1158/1078-0432.CCR-16-1193 Cancer Therapy: Preclinical Clinical Cancer Research Drug-Driven Synthetic Lethality: Bypassing Tumor Cell Genetics with a Combination of AsiDNA and PARP Inhibitors Wael Jdey1,2,3, Sylvain Thierry1,2, Christophe Russo4, Flavien Devun3, Muthana Al Abo5, Patricia Noguiez-Hellin3, Jian-Sheng Sun3, Emmanuel Barillot4, Andrei Zinovyev4, Inna Kuperstein4, Yves Pommier5, and Marie Dutreix1,2 Abstract Purpose: Cancer treatments using tumor defects in DNA repair Results: Molecular analyses demonstrate that olaparib and pathways have shown promising results but are restricted to small AsiDNA respectively prevent recruitment of XRCC1 and subpopulations of patients. The most advanced drugs in this field RAD51/53BP1 repair enzymes to damage sites. Combination of are PARP inhibitors (PARPi), which trigger synthetic lethality in both drugs increases the accumulation of unrepaired damage tumors with homologous recombination (HR) deficiency. Using resulting in an increase of cell death in all tumor cells. In contrast, AsiDNA, an inhibitor of HR and nonhomologous end joining, nontumor cells do not show an increase of DNA damage nor together with PARPi should allow bypassing the genetic restric- lethality. Analysis of multilevel omics data from BC cells tion for PARPi efficacy. highlighted different DNA repair and cell-cycle molecular profiles Experimental Design: We characterized the DNA repair inhi- associated with resistance to AsiDNA or olaparib, rationalizing bition activity of PARPi (olaparib) and AsiDNA by monitoring combined treatment. Treatment synergy was also confirmed with repair foci formation and DNA damage. We analyzed the cell six other PARPi in development. survival to standalone and combined treatments of 21 tumor cells Conclusions: Our results highlight the therapeutic interest of and three nontumor cells. In 12 breast cancer (BC) cell lines, combining AsiDNA and PARPi to recapitulate synthetic lethal- correlation with sensitivity to each drug and transcriptome were ity in all tumors independently of their HR status. Clin Cancer statistically analyzed to identify resistance pathways. Res; 23(4); 1001–11. Ó2016 AACR. Introduction PARP is rapidly recruited at the site of damage where it strongly auto-modified. The polymers of poly(adenosine diphosphate DNA double-strand breaks (DSBs) are the most lethal of the [ADP]-ribose) formed by PARP are used as a platform for the DNA insults and, if left unrepaired, result in genomic instability recruitment of many enzymes involved in Base Excision Repair and ultimately cell death (1). Therefore, targeted therapies (BER; ref. 5) and in Microhomology Mediated End Joining increasing the frequency or the persistence of spontaneous DSBs (MMEJ) repair of DSBs (6). PARP inhibition prevents BER repair or DSBs induced by treatments such as radiotherapy or chemo- enzymes from being recruited at damage sites (7) and leads to therapy have been extensively studied during the last 2 decades. the accumulation of DNA single-strand breaks (SSB) that result The most advanced drugs in this field are the poly(adenosine in unrepaired stalled replication forks and consequent DSBs. diphosphate [ADP]-ribose) polymerase (PARP) inhibitors, with These DSBs are mainly repaired by the homologous recombi- clinical trials showing significant benefits in patients with BRCA- nation (HR) repair pathway. Cells with BRCA1/2 mutations are mutated ovarian cancer (2). Essentially, cells deficient in BRCA1 defective in HR (so-called BRCAness) and die directly or indi- or 2 are 100- to 1,000-fold more sensitive to PARP inhibitors rectly from unrepaired DSBs (1). Cells with functional HR (PARPi) than BRCA1/2 heterozygote or wild-type cell lines (3, 4). accurately and efficiently repair DSBs and are not sensitive to PARP inhibition. Although PARPi monotherapy showed prom- 1Institut Curie, PSL Research University, CNRS, INSERM, Orsay, France. 2Uni- ising efficacy and safety profiles in the clinic (8, 9), their major versite Paris Sud, Universite Paris-Saclay, CNRS, INSERM, Orsay, France. 3DNA limitations are the necessity of HR deficiency (HRD) and the Therapeutics, Genopole, Evry, France. 4Institut Curie, PSL Research University, 5 rapid emergence of resistance. Many tumors that initially INSERM, Paris, France. National Institute of Health, National Cancer Institute, responded to PARPi treatments finally relapsed through com- Bethesda, Maryland. pensatory mutations restoring the HR activity or stimulating the Note: Supplementary data for this article are available at Clinical Cancer activity of alternative repair pathways such as the nonhomol- Research Online (http://clincancerres.aacrjournals.org/). ogous end joining (NHEJ) pathway (10, 11). Corresponding Author: Marie Dutreix, Institut Curie, Universite Paris-sud, We have recently developed an original class of DNA repair Building 112. 15, rue Georges Clemenceau, F-91405 Orsay, France. Phone: 33- pathway inhibitor, Dbait (12). AsiDNA, a molecule of Dbait 1-69-86-71-86; Fax: 33-1-69-86-31-41; E-mail: [email protected] family, consists of a 32-base pair oligonucleotide forming a doi: 10.1158/1078-0432.CCR-16-1193 double helix that mimics a DSB. AsiDNA acts by hijacking and Ó2016 American Association for Cancer Research. hyperactivating PARP1 (13) and the DNA-dependent protein www.aacrjournals.org 1001 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst August 24, 2016; DOI: 10.1158/1078-0432.CCR-16-1193 Jdey et al. MCF7, MDAMB231, and MDAMB468 from the ATCC), three Translational Relevance nontumor mammary cell lines (184B5, MCF10A, and MCF12A PARP inhibitors have shown significant benefits in cancer from the ATCC), five human cervical cancer HeLa cell lines silenced patients with BRCA mutations. However, they show no effi- for BRCA1 (HelaBRCA1SX, Tebu-Bio referenced as 00301-00041), cacy in tumors with active homologous recombination repair. for BRCA2 (HelaBRCA2SX, Tebu-Bio referenced as 00301–00028), In the current study, we propose a novel therapeutic strategy, for PARP1 (HeLaPARP1KD, a kind gift of Vincent Pennanaech, based on drug combination to achieve synthetic lethality Institut Curie, France) and controls (HeLaCTLSX, Tebu-Bio01- independently of the tumor genetics. We use AsiDNA, a DNA 00001, and HeLaCTLKD a kind gift of Vincent Pennanaech, Institut repair pathways antagonist (Dbait molecules family), to Curie, France), human glioblastoma cell lines MO59K and MO59J deplete double-strand break repair activities (homologous (DNA-PKcs deficient), human melanoma cell lines SK28LshCTL recombination and nonhomologous end joining) and pro- and SK28 LshDNA-PKcs, human colorectal cancer cell lines þ mote sensitivity to olaparib. The drug-driven synthetic lethal- HCT116 WT and HCT116 KU70 /- (heterozygote for KU70 gene), ity is specific to tumor cells and is not observed in nontumor human head and neck cancer cell line Hep2, hematologic cancer cells predicting a good safety of the association. As olaparib has cell lines Hut78, IM9, and Jurkat. Cells were grown according to obtained FDA approval, and AsiDNA have already been tested the supplier's instructions. Cell lines were maintained at 37Cin fi in a rst-in-man clinical trial, a potential exists for a rapid a humidified atmosphere at 5% CO2. clinical translation. DT40 Burkitt lymphoma cells are chicken cells that have been knocked out for different genes as previously described in ref. 23. For this study, we used DT40 wild-type cells as control (DT40WT), and four cell lines respectively knocked out for KO kinase (DNA-PK; ref. 14) which modify the chromatin and BRCA1, KU70, TDP1, and PARP1 genes (DT40BRCA1 , DT40KU70KO, DT40TDP1KO, and DT40PARP1KO). The DT40 consequently inhibit the recruitment of many proteins involved in the HR and NHEJ pathways at the damage sites (14). This cells were cultured at 37 C with 5% CO2 in Roswell Park Memorial Institute (RPMI-1640) medium supplemented with strategy sensitizes tumors to DNA-damaging therapies such as À5 radiotherapy and chemotherapy (15–18). The first-in-human 1% chicken serum (Life Technologies), 10 mol/L b-mercapto- phase I trial, combining AsiDNA to radiotherapy, to treat patients ethanol, penicillin, streptomycin, and 10% FBS. Reagents for cell with skin metastases from melanoma showed encouraging cultivation were obtained from Gibco Invitrogen. results, with 30% of complete responses (19). We anticipated All PARPi, AZD-2281 (olaparib), AZD-2461, ABT888 (veli- that AsiDNA could potentiate PARPi activity in BRCA-proficient parib), MK-4827 (niraparib), BSI-201 (iniparib), BMN673 (tala- cells by inhibiting HR and establishing a transient state of BRCA- zoparib), and AG-014699 (rucaparib), were purchased from ness. However, as both drugs act differently on DNA damage Medchem express and diluted on DMSO to a stock concentration response, the inhibitory activities and the efficacy of the associ- of 10 mmol/L. ation had to be demonstrated. AsiDNA is a new chemical entity, a 64-nucleotide (nt) oligo- To test this combined treatment, we first analyzed the effects in deoxyribonucleotide consisting of two 32-nt strands of comple- bis DNA repair of the PARPi olaparib (Ola) and AsiDNA, to check that mentary sequence connected through a 1,19- (phospho)-8- each drug does not interfere with the DNA repair inhibition hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane linker with a cho- activity of the other drug. These analyses were performed in the lesterol