WO2022184907A1 - Multiplex quantitative assay for dna double-strand break repair activities in a biological medium and its applications - Google Patents

Multiplex quantitative assay for dna double-strand break repair activities in a biological medium and its applications Download PDF

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WO2022184907A1
WO2022184907A1 PCT/EP2022/055587 EP2022055587W WO2022184907A1 WO 2022184907 A1 WO2022184907 A1 WO 2022184907A1 EP 2022055587 W EP2022055587 W EP 2022055587W WO 2022184907 A1 WO2022184907 A1 WO 2022184907A1
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repair
dsb
double
dna molecule
stranded dna
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French (fr)
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Giovanna MUGGIOLU
Sarah LIBERT
Thierry MAILLET
Sylvie Sauvaigo
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Lxrepair
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to a method and kit for determining the activities of the four DNA double-strand break (DSB) repair pathways in a biological medium, such as a biological sample from a patient, and their various uses for clinical and scientific applications, in particular for predicting the risk of late radiotoxicity before the beginning of radiotherapy.
  • a biological medium such as a biological sample from a patient
  • DNA lesions resulting from continuously endogenous or exogenous attacks can trigger, if not repaired, the appearance of several pathologies, including cancer.
  • the most life- threatening damages are lesions of the DNA double helix also named Double-Strand Breaks (DSBs).
  • DDR DNA damage Response
  • the mechanisms involved in DSB repair comprises two major pathways, a) homology-independent, as the non-homologous end joining (NHEJ) or b) homology-mediated as the homologous recombination (HR), and two minor alternative pathways, the single strand annealing (SSA) and the alternative end joining (alt- EJ) (Gupta R. et ah, Cell, 2018, 173:972-988).
  • NHEJ non-homologous end joining
  • HR homologous recombination
  • SSA single strand annealing
  • alt- EJ alternative end joining
  • the NHEJ is the most common mechanism accounting for approximately 75% of the double strand breaks repair events. It takes place within 30 minutes after the DSB is created and it repairs the damage by end ligation independently of sequence homology.
  • DSBs are firstly recognized by a heterodimer consisting of Ku70/Ku80 proteins. Then, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and the exonuclease Artemis are recruited to create DNA ends which are ligated by DNA module Ligase IV/XRCC4/XFL (Pannunzio N. et al, J. Biol. Chem., 2017, 293:10512-23).
  • DNA-PKcs DNA-dependent protein kinase catalytic subunit
  • Artemis the exonuclease Artemis
  • DSBs can be resected and one strand of the DNA duplex is degraded to produce 3’ single- stranded DNA (ssDNA) overhangs suitable for different repair pathways.
  • ssDNA single- stranded DNA
  • DSBs can be repaired by three possible mechanisms: HR, SSA end alt-EJ (Her J. and Bunting S., J. Biol. Chem., 2018, 293:10502-11).
  • end clipping The early stage of end resection, termed ‘end clipping’, is carried out by the structure- specific endonuclease Mrell which interacts with Rad50 to form the MRN complex.
  • the MRN complex linked with NBS1, ATM and CtIP stimulates resection.
  • helicases and exonucleases generate long stretches of ssDNA which can be processed by HR or SSA.
  • SSA like HR, is initiated by DNA resection. Then the two homologous DNA stretches are annealed by RAD52 and the generated tails are removed by the ERCC1/XPF nuclease. The resulting nicks are subsequently ligated by the DNA Ligase I.
  • SSA is a non-conservative process, causing DNA deletions of up to several hundred base-pairs long. (Mladenov E. et al., 2016, Seminars in Cancer Biology, 37-38:51-64).
  • Alt-EJ is distinct from NHEJ because it is Ku-independent and seems to be activated in cells lacking NHEJ by enzymes such as XRCC1/DNA Ligase III and PARP1 (Sallmyr A. and Tomkinson A., J. Biol. Chem., 2018, 293:10536-46).
  • PARP1 has been proposed to serve as a platform for recruiting alt-EJ repair factors such as polymerase theta (Pol 0), to microhomology sequences flanking the break (Wang M. et al., Nucleic Acids Res. 2006, 34:6170-6182).
  • repair pathways are involved in multiple health issues and they are critical for several biological mechanisms, such as in particular: the protection against the development of cancer; the defense against genotoxic exposure; the occurrence of adverse effects following radiation therapy; the occurrence of adverse effects following chemotherapy; the resistance to treatment in particular DNA damaging agents or treatments interacting with the DNA (radiotherapy, chemotherapy); synthetic lethality strategies for drug development; and genome editing methods where DSB repair mechanisms are involved.
  • BRCAl/2 are also implicated in the response to cancer therapies. Indeed, treating BRCA deficient cells with PARP inhibitors causes a significant cellular death. This mechanism, where depletions or inhibitions of two or more DNA repair proteins become lethal for a cell whereas a deficiency of only one is not, is identified with the term ‘synthetic lethality’ (Farmer H. et al., Nature, 2005, 434:917-921; Bryant H.et al., Nature, 2005, 434:913-917).
  • NBS1 and WRN proteins involved in DSB repair like ATM, NBS1 and WRN if defectives are the cause of hereditary diseases. Mutations of ATM protein are associated with Ataxia telangiectasia and these patients have high predisposition to leukemia, immunodeficiency, average life expectancy low and hypersensitivity to ionizing radiation (Mondello C. et al., Mutation Research, 2010, 704:29-37).
  • the Nijmegen breakage syndrome (NBS) is a DSB repair deficiency-related syndrome. NBS patients show a progressive microcephaly, growth retardation, sun sensitivity and immunodeficiency (Biau J. et al., Front. Oncology, 2019, 9:1- 10).
  • DNA repair is not only affected by alteration of the genetic information (mutations, chromosome rearrangements, epigenetic regulations), but also by post-transcriptional modifications (RNA splicing) and post-translational modifications (like phosphorylation, acetylation, methylation, ubiquitination, sumoylation and neddylation) (Oberle C. and Blattner C., Curr. Genomics, 2010, 11:184-198).
  • PCR polymerase chain reaction
  • SCGE Single Cell Gel Electrophoresis
  • PFGE Pulse-field gel electrophoresis
  • genomics gives information on the integrity of genes, it cannot predict what are the consequences at a functional level, and if compensatory pathways will balance the genomic defects. Many defects are the consequences of deregulations that may have been corrected, reversed or that do not exist anymore. Thus, all these methods quantify DNA lesions without determining directly the activity of DNA repair enzymes and the DNA DSB repair capacity in real time.
  • the amount of repair enzyme can be measured using different methods.
  • One type of methods determines the expression level of selected DNA repair proteins using specific antibody and colorimetric immunodetection assay such as ELISA or by detecting the foci number associated to the presence of DSB s using single molecule imaging (Tsvetkova A. et ah, Oncotarget, 2017, 38:64317-64329). However, they can, at the best, detect one single protein in one repair pathway.
  • the host cell reactivation (HRC) assay monitors the DNA repair activity of cells by measuring the capacity of cells to process one or more transfected DNA repair reporter vectors or DNA probes.
  • HRC host cell reactivation
  • These assays are commonly based on introducing multiple DNA repair reporter vectors or DNA probes into a cell and determining the capacity of the cell to process these multiple reporter vectors or DNA probes thereby determining multiple DNA repair capacities in the cell (US2014/0227701).
  • Examples of these in vivo assays include: (i) in vivo plasmid end joining (Wang M. et ah, Nucleic Acids Res.
  • DSB end joining assays employ DSB DNA substrates (generally linearized plasmids) to perform analysis of the repair of the DSBs through ligation. They address only one DSB repair pathway (NEHJ). These approaches allow one to overcome several technical challenges associated with other end joining assays requiring cultured cells (Pastwa E., Nucleic Acids Res., 2001, 29: e78; Datta K., Neumann R. and Winters T., Analytical Biochemistry, 2006, 358:155-157).
  • WO 2010/097584 discloses a method of measuring the DNA damage response, comprising: incubating nuclei with a cytosolic extract mixed with dNTP and a labeled nucleotide (biotinylated-UTP); adding streptavidin-FITC; and measuring the level of labelled nucleotide incorporated during in vitro DNA synthesis.
  • This approach requires the isolation of nuclei and cytosolic extract. It is not intended to characterize DNA repair pathways but only to investigate the DNA damaging properties of agents, through activation of DNA synthesis and elongation which is related to DNA damage response activation.
  • the level of H2AX phosphorylation (gamma-H2AX) which marks regions of DNA DSBs is also determined in parallel, to serve as control for DDR activation. No qualitative or quantitative information on specific repair pathways is provided by this method.
  • the inventors have designed an in vitro method which allows to precisely quantify the four double-strand break repair mechanisms simultaneously and their balance in any type of biological medium including biological samples from patients.
  • This method provides a comprehensive investigation of DSB repair capacities in any biological sample. This method enables to explore new features of the DSB repair network which have scientific and clinical applications.
  • the method of the invention allows to determine how the different double-strand break repair pathways are coordinated and how they interplay.
  • the method of the invention makes it possible to detect defects in any of the four double-strand break repair pathways, dysregulations or imbalance between the activities, and global down- or up-regulations.
  • One aspect of the invention relates to an in vitro method of determining the activities of at least two DNA double-strand break (DSB) repair pathways in a biological medium, comprising: a) Performing at least two DSB repair reactions, preferably simultaneously in a single reaction mixture: ai) a first repair reaction comprising a supercoiled circular double- stranded DNA molecule and a homologous linear double-stranded DNA molecule that are repaired by Homologous Recombination (HR) or Single-Strand Annealing (SSA) to form a repaired double- stranded DNA molecule; and a2) a second repair reaction comprising two linear double-stranded DNA molecules that may or may not be homologous and are repaired by Non-Homologous End- Joining (NHEJ) or Alternative-End Joining (alt-EJ) to form a repaired double- stranded DNA molecule; wherein said at least two repair reactions comprise DNA repair enzymes from said biological medium, and further comprise at least one label
  • the level of HR is determined by the level of labeled linear double- stranded DNA molecule incorporated into the repaired double- stranded DNA molecule from the first DSB repair reaction;
  • the level of SSA is determined by the level of labeled nucleotide incorporated into the repaired DNA molecule from the first DSB repair reaction;
  • the level of NHEJ is determined by the level of labeled linear double- stranded DNA molecule incorporated into the repaired double- stranded DNA molecule from the second DSB repair reaction;
  • the level of alt-EJ is determined by the level of labeled nucleotide incorporated into the repaired double-stranded DNA molecule from the second DSB repair reaction.
  • the supercoiled circular double-strand DNA molecule is a supercoiled plasmid and/or the linear double-stranded DNA molecule(s) are linearized plasmid(s), preferably from the same plasmid.
  • the supercoiled circular double-strand DNA molecule of the first DSB repair reaction and one of the linear double- stranded DNA molecules that is not labelled of the second DSB repair reaction are immobilized on a solid support.
  • the label is fluorescent and may be detected directly or indirectly; preferably a label chosen from Cy3 and Cy5; preferably wherein the method comprises two different labels.
  • the biological medium comprises cells or a mixture containing repair enzymes; preferably the biological medium is a sample obtained from a patient; more preferably the biological sample is selected from the group consisting of: plasma, whole-blood or PBMC fraction thereof; and tissue or tumor biopsy.
  • the DSB reaction is performed on a cell extract, preferably a nuclear extract; more preferably comprising a final protein concentration from 0.05 to 2 mg/mL; more preferably of 0.1 mg/mL or 0.2 mg/mL.
  • the DSB repair reactions are performed at 30°C; preferably for one hour.
  • the method comprises determining the activities of at least two, preferably four DSB repair pathways chosen from: Homologous Recombination (HR), Single- Strand Annealing (SSA), Non-Homologous End-Joining (NHEJ) and Alternative-End Joining (alt-EJ).
  • HR Homologous Recombination
  • SSA Single- Strand Annealing
  • NHEJ Non-Homologous End-Joining
  • alt-EJ Alternative-End Joining
  • Another aspect of the invention relates to a method of detecting a dysregulation of the DNA repair mechanisms in cells comprising: a) determining the activities of at least two DNA double-strand break (DSB) repair pathways in a cell sample according to the method of the present disclosure; b) detecting the presence of an altered activity of said DSB repair pathway(s).
  • DSB DNA double-strand break
  • said altered activity is a defect of said DSB repair pathway(s).
  • the detection of said defect is used to predict the risk of occurrence of cancer or immunological disease or the risk of toxicity following exposure to DNA damaging or DNA interacting agents in an individual.
  • the detection of said defect is used to predict the risk of late radiotoxicity before radiotherapy.
  • said cells are from a treated patient and said altered activity is used to predict the response to therapy, in particular cancer therapy in said patient.
  • Another aspect of the invention relates to a method of screening DSB repair modulators, comprising: a) contacting at least one candidate compound with a biological medium comprising DNA repair enzymes; b) determining the activities of at least two DNA double-strand break (DSB) repair pathways in said biological medium in the presence and absence of said at least one candidate compound, according to the method of the disclosure; c) identifying the compounds which modulate the activity of at least one of said DSB repair pathways.
  • DSB DNA double-strand break
  • kits for determining the activities of at least two DNA double-strand break (DSB) repair pathways in a biological medium comprising at least: a) a supercoiled circular double-strand DNA molecule, preferably a supercoiled plasmid; more preferably immobilized on a solid support; and b) at least one linear double-stranded DNA molecule; preferably linearized plasmid(s) with cohesive ends; more preferably from the same plasmid as in a); even more preferably immobilized on a solid support.
  • said kit further comprising at least one label, preferably fluorescent; more preferably a dNTP and a linear double-stranded DNA molecule with different labels.
  • said kit comprises a solid support comprising at least one pad, comprising at least one spot of the supercoiled circular double-strand DNA molecule and at least one spot of the linear double- stranded DNA molecule; preferably further comprising a reaction mixture comprising a dNTP and a linear double- stranded DNA molecule with different labels; more preferably further comprising a buffer appropriate for DNA repair enzymes.
  • the present invention relates to an in vitro method for determining simultaneously the activity of the four main DSB repair mechanisms in a biological medium.
  • One aspect of the invention relates to an in vitro method of determining the activities of at least two DNA double-strand break (DSB) repair pathways in a biological medium, comprising: a) Performing at least two DSB repair reactions, preferably simultaneously in a single reaction mixture: ai) a first repair reaction comprising a supercoiled circular double- stranded DNA molecule and a homologous linear double-stranded DNA molecule that are repaired by Homologous Recombination (HR) or Single-Strand Annealing (SSA) to form a double- stranded repaired DNA molecule; and ai) a second repair reaction comprising two linear double-stranded DNA molecules that may or may not be homologous and are repaired by Non-Homologous End- Joining (NHEJ) or altemative-End Joining (alt-EJ) to form a repaired double-stranded DNA molecule; wherein said at least two repair reactions comprise DNA repair enzymes from said biological medium, and further comprise at least
  • the level of HR is determined by the level of labeled linear double- stranded DNA molecule incorporated into the repaired double- stranded DNA molecule from the first DSB repair reaction;
  • the level of SSA is determined by the level of labeled nucleotide incorporated into the repaired double-stranded DNA molecule from the first DSB repair reaction;
  • the level of NHEJ is determined by the level of labeled linear double- stranded DNA molecule incorporated into the repaired double- stranded DNA molecule from the second DSB repair reaction;
  • the level of alt-EJ is determined by the level of labeled nucleotide incorporated into the repaired double-stranded DNA molecule from the second DSB repair reaction.
  • the method involves the measurement of the four repair pathways (HR, NHEJ, SSA, alt-EJ) simultaneously from the same reaction medium.
  • the method provides a multiplexed quantitative assay for DNA DSB repair activities.
  • in vitro means “cell-free””. This means that the in vitro method of the invention is different from an in cellulo method and does not require living cells maintained in culture to determine DNA DSB repair activities. In particular, the in vitro method of the invention does not require introduction of the circular double-stranded and linear DNA molecules inside a cell to determine DNA DSB repair activities, nor does it require complex cell culture or manipulation.
  • supercoiled DNA refers to a supercoiled circular double-stranded DNA molecule, in particular a supercoiled plasmid.
  • DNA molecule refers to double-stranded DNA molecule
  • DSB repair or DNA DSB repair refers to DNA double-strand break repair
  • DSB repair enzyme, DNA DSB repair enzyme or DNA repair enzyme refers to DNA double-strand break repair enzyme.
  • the DNA which serves as repair matrix for the DNA DSB repair enzymes present in the biological sample is a double- stranded DNA whether linear or circular.
  • it involves long DNA fragments (over 100 bases) such as those described in the application WO 01/090408 or a supercoiled circular double- stranded DNA, in particular a plasmid such as described in the Application WO 2004/059004, Millau et al., Lab. Chip., 2008, 8, 1713-1722; Prunier et al., Mutation Research, 2012, 736, 48-55.
  • the method uses plasmid(s) for the DSB reactions; a supercoiled plasmid and at least one linear plasmid (e.g., linearized plasmid).
  • the supercoiled plasmid and the linear plasmid for the first DSB repair reaction have sufficient homology to engage into a Homologous Recombination (HR) reaction or a Single-Strand Annealing (SSA) reaction and generate a repaired double- stranded DNA molecule.
  • the linear plasmids for the second DSB repair reaction have advantageously at least a micro-homology to engage into an altemative-End Joining (alt-EJ) reaction and generate a repaired double- stranded DNA molecule.
  • a single plasmid in supercoiled and linear forms may be advantageously used for the four DSB repair reactions (HR, SSA, NHEJ and alt-EJ).
  • Linear plasmid(s) are obtained by linearization of a supercoiled plasmid using a restriction enzyme.
  • the restriction enzyme usually cleaves the plasmid at a unique restriction site and generates blunt or cohesive ends, advantageously cohesive ends.
  • the linear plasmid is also named DSB-plasmid.
  • the supercoiled and linear plasmids of the first DSB reaction and/or the two linear plasmids of the second DSB reaction are from the same plasmid.
  • the non-labelled DNA molecules from the first and second DSB repair reaction e.g., supercoiled circular double-strand DNA molecule and one of the linear double-stranded DNA molecules
  • the label e.g., labelled nucleotide and/or DNA molecule
  • the solid support is any appropriate support for the immobilization of nucleic acids and in particular supercoiled plasmid that are well-known in the art.
  • suitable support for immobilization include glass, polypropylene, polystyrene, silicone, metal, nitrocellulose or nylon; preferably coated with an agent that forms a 3D layer that binds DNA covalently or non-covalently.
  • coating agent include coating with porous film (3D layer), such as nylon or hydrogel film, in particular a multi-component polymer layer, activated with N-Hydroxysuccinimide (NHS) esters.
  • the support is advantageously a microchip type miniaturized support.
  • Suitable supports for DNA immobilization are well-known in the art and commercially available: 3-D Hydrogel coated slides (SCHOTT); silicone coated with a hydrogel (Nanochip TM; NANOGEN); glass or polymer slides coated with a hydrogel (3D-reactive slides, Poly An); low-fluorescence glass coated with multi-component polymer layer (Nexterion® Slide H).
  • the DNA immobilization method does not induce DNA breaks and therefore preserves the supercoiled structure of the plasmid.
  • the method usually comprises: depositing the DNA molecule on the surface of the solid support, such as 3D-coated slide, using appropriate spotting device suitable for the preparation of DNA microarrays, for example by automation with a robot such as a piezoelectric robot; and drying in controlled humidity environment to immobilize the DNA without inducing DNA breaks.
  • each spot of the microarray comprises from 0.01 ng to 1 ng of double-stranded DNA, preferably from 0.12 ng to 0.2 ng of double-stranded DNA.
  • the solid support advantageously further comprises at least one reaction chamber.
  • the reaction chamber comprises at least one spot of the supercoiled circular double-strand DNA molecule and at least one spot of the linear double- stranded DNA molecule.
  • the reaction chamber comprises two identical spots of the supercoiled circular double-strand DNA molecule and two identical spots of the linear double- stranded DNA molecule.
  • At least one of a dNTP and a linear double-stranded DNA molecule or both comprise a label (detectable moiety), which makes it possible to detect and quantify them in step (b) after they are incorporated into the repaired double- stranded DNA molecule following the DNA DSB repair reaction of step a).
  • the tracer or label which is used for labeling the nucleotide or DNA molecule is detected by measurement of the signal which is proportional to the quantity of nucleotide or linear DNA molecule incorporated into the repaired double- stranded DNA molecule.
  • a label is a moiety that can be detected directly or indirectly by the production of a detectable signal such as colorimetric, fluorescent, chemiluminescent, electrochemoluminescent, magnetic or radioactive signal.
  • Directly detectable labels include radioisotopes and luminescent compounds (e.g. radioluminescent, chemiluminescent, bioluminescent, fluorescent or phosphorescent).
  • Indirectly detectable labels have the ability to bind to or cleave another moiety, which itself may emit or absorb light of a particular wavelength, and include for example, biotin, avidin such as streptavidin, antibody, hapten, epitope tag such as the FLAGepitope, digoxigenin and enzyme tag such as horseradish peroxidase.
  • Preferred labels include fluorescent labels (fluorophores) such as without limitation, Cyanine 3 and Cyanine 5.
  • the means and techniques for labeling nucleotides and DNA molecules are well known to the person skilled in the art.
  • the labelled nucleotide or DNA molecule in particular fluorescent, radioactive, magnetic or colorimetric label is detectable by any technique known to the person skilled in the art such as, without limitation, fluorescence microscopy, flow cytometry, scintigraphy, magnetic resonance imaging and mass spectrometry.
  • step (a) makes it possible to perform the at least two DSB repair reactions of step (a) (steps (al), (a2) and others, (a3), (a4)) simultaneously in a single reaction but requires a specific detector for each label to measure the DNA DSB repair signal (step (b)).
  • step (a) steps (al), (a2) and others, (a3), (a4)
  • step (b) steps (al), (a2) and others, (a3), (a4)
  • the at least one label is fluorescent and may be detected directly or indirectly.
  • the label is chosen from Cy3 and Cy5.
  • Indirect label is advantageously biotin.
  • the method comprises two different labels, preferably Cy3 and Cy5.
  • the method comprises a biotinylated dNTP that is detected using a Streptavidin-Cy5 conjugate and a linear double- stranded DNA molecule labelled with Cy3, preferably a linearized plasmid labelled with Cy3.
  • the at least two DNA DSB repair reactions are performed under conditions allowing repair of double- stranded DNA by DSB repair enzymes present in said biological medium and thereby incorporation of label into repaired double-stranded DNA molecule.
  • These conditions which are well known to the person skilled in the art are disclosed for example by Wong and Mermod and colleagues (Wong B. et ah, J. Biol. Chem., 1998, 273:12120-12127; Kostyrko K. and Mermod N., Nucl. Acids Res., 2016, 44(6):e56).
  • the repair reaction is performed in the presence of ATP, an ATP regeneration system and any other agent necessary for the activity of DNA DSB repair enzymes present in the biological medium.
  • the repair buffer contains ATP ImM, MgCh lOmM, KC1 80mM, DTT 2mM, BSA O.lmg/mL, Tris-HCl 20mM pH 7.5, Creatin Phosphokinase 0.05mg/mL, Phosphocreatine lOmM and eventually, all or other dNTPs 0.25mM.
  • Other dNTPs refer to the three dNTPs other than the labelled one.
  • the repair reaction is generally done with a nuclear extract, preferably comprising a final protein concentration from 0.05 to 2 mg/mL; more preferably of 0.1 mg/mL or 0.2 mg/mL.
  • the reaction is performed at a temperature promoting the DNA DSB repair reaction, preferably 30°C, for sufficient time, generally included between fifteen minutes and five hours.
  • the reaction is advantageously performed in the microwells of a microchip type miniaturized support such as defined above.
  • the method is applicable to any biological medium comprising DNA repair enzymes including any cells, tissues, tumors or mixture of repair enzymes, artificially produced or isolated from cells, which are prepared in a way that preserves the repair enzyme activities.
  • biological medium comprising DNA repair enzymes including any cells, tissues, tumors or mixture of repair enzymes, artificially produced or isolated from cells, which are prepared in a way that preserves the repair enzyme activities.
  • samples include, and are not limited to, primary cells (freshly isolated or cultured), cell lines, patient’s biopsies, peripheral blood mononuclear cells (PBMCs), xenograft, preferably but not limited to eukaryotes.
  • the biological medium comprises cells or a mixture containing repair enzymes; preferably the biological medium is a biological sample obtained from a patient; more preferably the biological sample is selected from the group consisting of: plasma, whole-blood or PBMC fraction thereof; and tissue or tumor biopsy.
  • Biological medium comprising a mixture containing repair enzymes can be tested directly, without pre-treatment.
  • Biological medium comprising cells are pretreated so as to recover or extract nuclear proteins which contain the DNA repair enzymes.
  • the recovery or extraction of nuclear proteins from cells is done according to standard techniques well known to the person skilled in the art.
  • Cells are generally lysed for isolating the nuclei and then the nuclei are in turn lysed for extracting nuclear proteins.
  • the protocols described for in vitro transcription tests or in vitro DNA repair tests can be used, such as for example those described by Dignam JD et al. (Nucleic Acids Research, 1983, 11 ; 1475— 1489); Kaw L. et al.
  • the nuclear extracts can further be fractionated or dialyzed according to conventional methods. All the steps for preparation of the nuclear protein extract are done under conditions that do not denature the enzymatic activities and the proteins contained in the cells, such that the DNA repair activity initially present in the sample of cells is preserved in the cellular extract which is analyzed in the method of the invention.
  • the DSB repair reaction is performed on a cellular lysate or extract thereof, preferably a nuclear lysate or extract thereof, more preferably a nuclear protein extract.
  • the method comprises determining the activities of at least three, preferably four DSB repair pathways chosen from: Homologous Recombination (HR), Single-Strand Annealing (SSA), Non-Homologous End-Joining (NHEJ) and Alternative-End Joining (alt-EJ).
  • HR Homologous Recombination
  • SSA Single-Strand Annealing
  • NHEJ Non-Homologous End-Joining
  • alt-EJ Alternative-End Joining
  • the signal emitted by the DSB -plasmid and the signal emitted by the labeled nucleotide are measured on the 2 immobilized plasmids: the supercoiled plasmid and the DSB-plasmid, resulting in 2 different values, or four different values if the labelled dNTP is added during the repair reaction.
  • the repair reaction can be run at several different final protein concentrations of the same lysate, on different biochips.
  • the signal measured at the level of the immobilized supercoiled plasmid that comes from the labeled DSB -plasmid in solution, is attributed to components of the HR repair reaction which requires the strand invasion.
  • the signal measured at the level of the immobilized supercoiled plasmid that comes from the labeled dNTP is attributed to components of the SSA repair reaction, that involves nucleotide incorporation by specific polymerases.
  • the signal measured at the level of the immobilized DSB -plasmid that comes from the labeled DSB-plasmid in solution is attributed to components of the NHEJ repair reaction, able to ligate the plasmids ends.
  • the signal measured at the level of the immobilized DSB-plasmid that comes from the labeled dNTP is attributed to components of the alt-EJ repair reaction.
  • the biological medium is then characterized by 1, 2, 3, or 4 values.
  • repair reaction is run using a second final protein concentration
  • the same biological medium is then characterized by other 1, 2, 3, or 4 values.
  • the biological medium can be characterized by any combinations between these values.
  • the DSB repair defects can be identified:
  • kits for determining the activities of at least two DNA double-strand break (DSB) repair pathways in a biological sample comprising at least: a) a supercoiled circular double-strand DNA molecule, preferably a supercoiled plasmid; more preferably immobilized on a solid support; and b) at least one linear double- stranded DNA molecule; preferably linearized plasmid(s) with cohesive ends; more preferably from the same plasmid as in a); even more preferably immobilized on a solid support.
  • said kit further comprising at least one label, preferably fluorescent; more preferably a dNTP and a linear double-stranded DNA molecule with different labels.
  • said kit comprises a solid support, comprising at least one pad containing at least one spot of the supercoiled circular double-strand DNA molecule and at least one spot of the linear double-stranded DNA molecule.
  • the solid support is preferably a 3D-coated support according to the present disclosure and advantageously further comprises at least one reaction chamber.
  • the reaction chamber comprises two identical spots of the supercoiled circular double-strand DNA molecule and two identical spots of the linear double-stranded DNA molecule.
  • the kit further comprises a reaction mixture comprising a dNTP and a linear double- stranded DNA molecule with different labels; more preferably further comprising a buffer suitable for DNA repair enzymes.
  • the buffer comprises: ATP 1 mM, MgCk 10 mM, KC1 80 mM, DTT 2 mM, BSA 0.1 mg/mL, Tris-HCl 20 mM pH 7.5, Creatin Phosphokinase 0.05mg/mL, Phosphocreatine 10 mM and other dNTPs 0.25 mM.
  • the method according to the invention has a certain number of advantages over the prior art methods. It allows the mapping of a given biological medium according to its enzymatic activities for DSB repair. It measures the real DNA repair activities at a defined time point. It allows the identification of global or partial dysregulation of DSB repair mechanisms, thanks to the simultaneous evaluation of the activity of at least two DSB repair mechanisms. It is used to determine whether DSB repair mechanisms are deficient or partially deficient for certain activities. It makes it possible to compare various biological medium in term of DBS repair capability. It allows to develop specific inhibitors for DSB repair pathways. It allows to detect up-regulations, compensatory pathways, following treatments or not.
  • the method of the invention identifies DSB repair defects responsible for radiotoxicity syndromes whereas methods developed up to now show little performances and are not widely used in the clinics.
  • An example of method to predict the radiotoxicity is the radiation-induced CD8 T-lymphocyte Apoptosis (RILA) as disclosed in WO 2014/154854. Results of RILA assessment in breast cancer patients showed a sensitivity of 70% which is not sufficient for incorporation into routine analysis.
  • Another method comprising the detection of nucleo- shuttling of the ATM Protein in the healthy tissues of patients in response to radiation therapy (Ganzotto A. et al., Int. J. Radiation Oncol.
  • Genomics approaches have also been developed for the radiotoxicity risk prediction. Consortium identified common genetic variants affecting radiotoxicity but the identified SNP- toxicity associations were not significant (Kems S. et al., J. National Cane. Inst., 2020, 112:179-190).
  • the invention can be widely applied for diagnostic purposes, it requires only several mL of blood, it is fast, user friendly and it can be performed before the treatment begins. Importantly, it displays the best performances known today for late severe (grade >3) radiotoxicity prediction.
  • the method and kit of the invention can be used for multiple applications.
  • the method of the invention can be integrated into the clinical guidelines to screen all patients before they receive radiotherapy.
  • the method and kit of the invention can be used to identify DSB repair defects leading to cancer predisposition.
  • the method and kit invention can be used to identify DSB repair defects responsible for resistance to cancer treatments.
  • the method and kit of the invention can be used to identify DSB repair defects allowing to develop synthetic lethality approaches.
  • the method and kit of the invention can be used to evaluate the balance between various DSB repair pathways, allowing to specifically modulate one or several pathways.
  • the method and kit of the invention can be used to quantify the various DSB repair pathways, allowing to identify imbalanced activities.
  • the method and kit of the invention can be used to evaluate the specificity and efficacy of various DNA repair inhibitors.
  • the method and kit according to the invention are used to detect DNA repair defect(s) in individuals to predict cancer risk occurrence.
  • the method and kit according to the invention are used to detect DNA repair defect(s) in individuals to predict or understand immunological diseases.
  • the method and kit according to the invention are used to detect DNA repair defect(s) in individuals to predict toxicity occurrence following exposure to DNA damaging agents or DNA interacting agents.
  • the detection of said defect is used to predict the risk of late radiotoxicity before radiotherapy.
  • the method and kit according to the invention are used to identify specific biomarkers in cells, tumors, or in patient cells, treated or not, that predict response to cancer therapies.
  • one aspect of the invention relates to a method of detecting a dysregulation of the DNA repair mechanisms in cells comprising: a) determining the activities of at least two DNA double-strand break (DSB) repair pathways in a cell sample according to the method of the present disclosure; b) detecting the presence of an altered activity of said DSB repair pathway(s).
  • DSB DNA double-strand break
  • said altered activity is a defect of said DSB repair pathway(s).
  • the detection of said defect is used to predict the risk of occurrence of cancer or immunological disease or the risk of toxicity following exposure to DNA damaging or DNA interacting agents in an individual.
  • said cells are from a treated patient and said altered activity is used to predict the response to therapy, in particular cancer therapy in said patient.
  • the method and kit according to the invention are used to develop specific DNA DSB repair inhibitors.
  • one aspect of the invention relates to a method of screening DSB repair modulators, comprising: a) contacting at least one candidate compound with a biological sample comprising DNA repair enzymes; b) determining the activities of at least two DNA double-strand break (DSB) repair pathways in said biological sample in the presence and absence of said at least one candidate compound, according to the method of the present disclosure; c) identifying the compounds which modulate the activity of at least one of said DSB repair pathways.
  • the method is used to screen for activators and inhibitors of the four DNA double strand break (DSB) repair pathways which either activate or inhibit one of said pathways.
  • the method and kit according to the invention are used to control small molecules or inhibitors or compounds, mechanisms of action and off-target mechanisms of action.
  • the method and kit according to the invention are used to control DNA repair inhibitors impact on different DSB repair pathways.
  • the method and kit according to the invention are used to assess the chemical or biological modulation of certain DSB repair pathways for genome editing strategies.
  • the practice of the present invention will employ, unless otherwise indicated, conventional techniques, which are within the skill of the art. Such techniques are explained fully in the literature.
  • FIG. 1 Schematic representation of Next-SPOT assay according to the invention. Two identical spots containing a supercoiled plasmid and two identical spots containing a linear plasmid are printed on specific areas of the biochip (a). Once added the repair enzymes, a Cy 3 -labelled linear plasmid and a Cy5-labelled dNTP, on each printed spot two reactions can happen: the HR and SSA on the supercoiled plasmid and the NHEJ and alt- EJ on the linear plasmid. Two wavelengths, 532 and 635 nm, are used to quantify the fluorescence in each spot and to obtain the DSB profile of the analysed sample (b).
  • Figure 2 The relative contributions of all four DSB repair pathways to DSB repair as a percentage, for each sample are shown. Each bar corresponds to one sample. All bars are constituted by four rectangles representing from the bottom to the top HR, NHEJ, SSA and alt-EJ, respectively. The data obtained at 0.1 mg/L final protein concentration are shown.
  • Figure 7 The relative contribution of each DSB repair pathway to total DSB repair was assessed for the three cell lines at basal level, at 0.1 mg/mL (A) and at 0.2 mg/mL (B) protein concentration.
  • FIG. 8 Impact of doxorubicin, B02, NU7026, Olaparib and their combinations on the four DSB repair mechanisms is shown for M059K (A), M059J (B) and Hela (C) cells at 0.1 mg/mL protein concentration, determined using the ratio of the fluorescence intensity obtained for each pathway, of treated cells to non-treated cells (FI T/FI NT).
  • Figure 9 Impact of doxorubicin, B02, NU7026, Olaparib and their combinations on the four DSB repair mechanisms was determined for M059K (A), M059J (B) and Hela (C) cells at 0.2 mg/mL protein concentration, using the ratio of the fluorescence intensity obtained for each repair pathway, for treated cells to non-treated cells (FI T/FI NT).
  • Nuclear cell extracts were obtained from PBMCs or from cell lines as described in Candeias et al, with some adaptations (Candeias S et al., Mutation Research, 2010, 694:53- 59). Thawed PBMCs and cell lines were washed in ice-cold PBS. The cell pellet was suspended and incubated 8 or 20 min in 3mL of ice-cold buffer A (lOmM HEPES pH 7.9, 1.5mM MgC12, lOmM KC1, 0.02% TritonX-100, 0.5mM DTT, 0.5mM PMSF).
  • ice-cold buffer A lOmM HEPES pH 7.9, 1.5mM MgC12, lOmM KC1, 0.02% TritonX-100, 0.5mM DTT, 0.5mM PMSF.
  • nuclei were suspended in 25, 50 or 75pl of ice-cold Buffer B depending on the cell number (lOmM HEPES pH 7.9, 1.5mM MgC12, 400mM KC1, 0.2mM EDTA pH 8.0, 25% Glycerol, 0.5mM DTT, 0.5mM PMSF and complete-mini antiproteases (Roche)). Nuclei were lysed by incubation 30 min at 4°C and two cycles of freezing-thawing in liquid nitrogen alternated with 30 sec of vortex. The nuclear extracts were then collected after 15 min of centrifugation at 16000g and stored at -80°C. The protein concentration for each sample was determined using the Pierce BCA Protein Assay Kit (Interchim). Production of plasmids and biochips
  • the biochips were constituted by 14 or 21 identical areas.
  • the plasmid containing DSB (DSB-plasmid) was obtained by digestion of pBlueScript plasmid using the restriction enzyme Afllll which creates cohesive ends.
  • One hundred pg of supercoiled pBLueScript plasmid were incubated with 100 units of enzyme in IX NEBuffer 3.1 during 1 hour at 37°C. Then, the DSB-plasmid was precipitated and finally suspended in molecular biological-grade water at the desired concentration.
  • Each sample was tested at 0.1 and 0.2 mg/mL final protein concentrations.
  • the repair reaction was carried out during 1 hour at 30°C.
  • the slide was then rinsed twice with Milli-Q water.
  • an incubation with 0.1 pg/mL Streptavidin-Cy5 was performed for 30 minutes at 30°C.
  • the slide was rinsed twice with Milli-Q water and dried out.
  • Each sample was tested on two pads (areas).
  • the labeling of the DSB-plasmid with Cy3 was obtained using the Label IT® TrackerTM Kit (Mims). Briefly, 100pg of DSB-plasmid were incubated with 50pl of Label IT® Reagent and IOOmI of Labeling Buffer 3.1 during 2 hours at 37°C. labeled DSB-plasmid was then diluted in molecular biological-grade water and stored at -80°C.
  • Fluorescent signals were quantified using a scanner (Innoscan 710AL from Innopsys, and the Mapix software) at two wavelengths, 532 and 635nm.
  • the enzymes contained in the extracts performed specific repair reactions leading to the incorporation of labeled dNTP and the integration/ligation of labeled DSB plasmid on the immobilized plasmids according to their proficiency to repair DSBs.
  • EXAMPLE 1 Multiplex DSB repair assay to predict radiation-induced late toxic effects due to defects in double-strand break repair (Next-SPOT assay and kit)
  • PBMCs peripheral blood mononuclear cells
  • the raw data (R) corresponded to the average of the fluorescence intensity of all replicates.
  • the Dose-Response (DR) was calculated by dividing the values obtained at the highest protein concentration by the values obtained at the lowest protein concentration for each repair mechanism.
  • the dose-response values were calculated using the formula: R_HR_0.2/ R_HR_0.1 for each pathway.
  • CTCAE Common Terminology Criteria for Adverse Events
  • HR and NHEJ repair pathways characterized using the invention were significantly different after the fifth sessions of treatment (J8), for patients who developed early grade >3 events compared to patients who did not (grade ⁇ 3).
  • the HR normalized data, the HR dose-response and the NHEJ normalized data were lower in patients presenting early grade >3 events compared to patients who did not show these events.
  • the test identifies differences in the DSB repair pathways of patients associated with an increased risk of early radiotoxicity after radiotherapy.
  • Late adverse events >6 months after the beginning of RT were recorded in breast and prostate cancer patients. Patients were divided in two groups according to the presence or not of late grade >2 adverse events. Significant results are showed as Box-plots
  • the assay that enabled to detect simultaneously defects in all DSB repair pathways identified patients with an increased late radiotoxicity risk following radiotherapy.
  • EXAMPLE 2 Characterization of DSB repair activities in cancer cell lines. Analysis of impact of DNA damaging agents, DNA repair inhibitors and their combination on DSB repair activities. Detection of DSB repair pathway defects and deregulations Cell lines
  • the DSB repair signature was established for 3 cell lines before any treatment at, 0.1 and 0.2 mg/mL final protein concentration.
  • the relative contribution of each pathway to the total DSB repair capacity, was calculated as a percentage for each pathway, at the two final protein concentrations, for each cell line ( Figure 7, A and B).
  • M059J cell line is described as being defective for NHEJ. With, this assay it is clearly shown that M059J cell line differs from M059K regarding NHEJ pathway regulation and that a defective gene in the DSB repair pathway does not mean that the repair reaction cannot occur. All data regarding the different DSB repair pathways, obtained and considered together, revealed that mutation in the PRKDC gene affected only NHEJ pathway, which can only be determined using the invention. Using the NEXT-SPOT assay, it is shown that despite this truncated protein, these cells are able to perform the end ligation reaction.
  • NEXT-SPOT assay allows one to understand what are the possible consequences of a treatment on DSB repair pathways in terms of induction or inhibition, and enables one to identify strategies to modulate them.
  • a DNA damaging agent or more generally a genotoxic
  • a DDR modulator for example, one can determine if a certain pathway is induced by a DNA damaging agent (or more generally a genotoxic), or a DDR modulator, and identify an inhibitor specifically targeting the induced repair pathway, as a sensitization strategy.
  • Cells were treated by doxorubicin and the DSB repair profile of each cell line was determined. The cells were also treated by several DSB repair inhibitors (B02, NU7026 and Olaparib), alone or in combination with doxorubicin (Dox+B02, Dox+NU7029 and Dox+Olap).
  • each treatment induces a different modulation of the four DSB repair pathways in the cell lines tested.
  • the ratios of the fluorescence intensity obtained for treated cells (T) to non-treated cells (NT) were calculated at the 2 protein concentrations.
  • repair reactions run with the 2 protein concentrations give complementary results, and inform on the balance between repair activities contained in the same extract.
  • a repair protein might be present in the extract when used at 0.2 mg/mL and absent in the extract at 0.1 mg/mL because it exists in the cell in a limited concentration.
  • the inhibitors have a synergic inhibitory effect with the DNA damaging agent as seen at 0.2 mg/mL. That can be an important information gained form this finding as sometimes the DNA damage response must be activated in cells by a DNA damaging agent, to be effectively inhibited by the DNA repair inhibitors.
  • HeLa cells DSB repair activities are slightly modulated when the repair reaction is conducted using 0.1 mg/mL protein concentration, except with Olaparib, alone or in combination with doxorubicin.
  • B02 alone and in combination with doxorubicin, effectively inhibits HR
  • NU7026 slightly inhibits NHEJ and HR only when combined with doxorubicin
  • the third inhibitor, Olaparib affects considerably HR and NHEJ and slightly SSA and alt-EJ pathways, only when combined with doxorubicin.
  • EXAMPLE 3 Functional DSB repair are associated with defects in gene involved in DSB repair
  • DSB repair ability was quantified in two cancer cell lines, Control Hela SilenciX ® , and BRCA2 HeLa SilenciX ® silenced for BRCA2 (tebu-bio, France).
  • Cells were grown following the supplier instructions with DMEM+GlutaMAXTM containing 4.5 g/L D-Glucose supplemented with 125pg/mL hygromycin B, 1% penicillin- streptomycin solution and 10% fetal bovine serum (Gibco, USA) at 37°C under 5% CO2.

Abstract

The invention relates to a method and kit for determining the activities of the four DNA double-strand break (DSB) repair pathways in a biological medium, such as a biological sample from a patient, and their various uses for clinical and scientific applications, in particular for predicting the risk of late radiotoxicity before the beginning of radiotherapy.

Description

MULTIPLEX QUANTITATIVE ASSAY FOR DNA DOUBLE-STRAND BREAK REPAIR ACTIVITIES IN A BIOLOGICAL MEDIUM AND ITS APPLICATIONS
FIELD OF THE INVENTION
[0001] The invention relates to a method and kit for determining the activities of the four DNA double-strand break (DSB) repair pathways in a biological medium, such as a biological sample from a patient, and their various uses for clinical and scientific applications, in particular for predicting the risk of late radiotoxicity before the beginning of radiotherapy.
BACKGROUND OF THE INVENTION
[0002] DNA lesions resulting from continuously endogenous or exogenous attacks, can trigger, if not repaired, the appearance of several pathologies, including cancer. The most life- threatening damages are lesions of the DNA double helix also named Double-Strand Breaks (DSBs). To be protected from genomic instability, cells have evolved elaborate defense strategies, collectively termed the DNA damage Response (DDR) (Ciccia A. and Elledge S., Molecular Cell, 2010, 40: 179-204). Among the DDR, the mechanisms involved in DSB repair comprises two major pathways, a) homology-independent, as the non-homologous end joining (NHEJ) or b) homology-mediated as the homologous recombination (HR), and two minor alternative pathways, the single strand annealing (SSA) and the alternative end joining (alt- EJ) (Gupta R. et ah, Cell, 2018, 173:972-988).
[0003] The NHEJ is the most common mechanism accounting for approximately 75% of the double strand breaks repair events. It takes place within 30 minutes after the DSB is created and it repairs the damage by end ligation independently of sequence homology. To process the damage, DSBs are firstly recognized by a heterodimer consisting of Ku70/Ku80 proteins. Then, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and the exonuclease Artemis are recruited to create DNA ends which are ligated by DNA module Ligase IV/XRCC4/XFL (Pannunzio N. et al, J. Biol. Chem., 2017, 293:10512-23).
[0004] Alternatively, DSBs can be resected and one strand of the DNA duplex is degraded to produce 3’ single- stranded DNA (ssDNA) overhangs suitable for different repair pathways. In this case DSBs can be repaired by three possible mechanisms: HR, SSA end alt-EJ (Her J. and Bunting S., J. Biol. Chem., 2018, 293:10502-11). [0005] The early stage of end resection, termed ‘end clipping’, is carried out by the structure- specific endonuclease Mrell which interacts with Rad50 to form the MRN complex. The MRN complex, linked with NBS1, ATM and CtIP stimulates resection. In the second phase, called ‘extensive resection’, helicases and exonucleases generate long stretches of ssDNA which can be processed by HR or SSA.
[0006] HR predominates in the mid-S and mid-G2 cell cycle phases, when the sister template is available. During this mechanism, the ssDNA is coated with RPA which recruits recombinases such as Dmcl and RAD51. BRCA1 and BRCA2 stabilize the RAD51 nucleoprotein filament and the recombinase RAD51 mediates strand invasion of the homologous template (Ceccaldi R. et al., Trends in Cell Biology, 2016, 26:52-64).
[0007] SSA, like HR, is initiated by DNA resection. Then the two homologous DNA stretches are annealed by RAD52 and the generated tails are removed by the ERCC1/XPF nuclease. The resulting nicks are subsequently ligated by the DNA Ligase I. SSA is a non-conservative process, causing DNA deletions of up to several hundred base-pairs long. (Mladenov E. et al., 2016, Seminars in Cancer Biology, 37-38:51-64).
[0008] If short stretches of ssDNA are available they are processed by the alt-EJ. This pathway has been described in various cellular contexts but the mechanisms details remain unclear. Alt-EJ is distinct from NHEJ because it is Ku-independent and seems to be activated in cells lacking NHEJ by enzymes such as XRCC1/DNA Ligase III and PARP1 (Sallmyr A. and Tomkinson A., J. Biol. Chem., 2018, 293:10536-46). PARP1 has been proposed to serve as a platform for recruiting alt-EJ repair factors such as polymerase theta (Pol 0), to microhomology sequences flanking the break (Wang M. et al., Nucleic Acids Res. 2006, 34:6170-6182).
[0009] All these pathways cooperate and sometimes compete for the repair of DSBs. Many regulators, like DSB-type, the cell type, the cell cycle phase, and availability of accessory and repair factors, define the pathway choice. For example, a central step for choosing the repair pathway is controlled by the interaction between BRCA1 and 53BP1. While BRCA1 supports resection, 53BP1 suppresses resection (Her J. and Bunting S., J. Biol. Chem., 2018, 293:10502-11).
[0010] These repair pathways are involved in multiple health issues and they are critical for several biological mechanisms, such as in particular: the protection against the development of cancer; the defense against genotoxic exposure; the occurrence of adverse effects following radiation therapy; the occurrence of adverse effects following chemotherapy; the resistance to treatment in particular DNA damaging agents or treatments interacting with the DNA (radiotherapy, chemotherapy); synthetic lethality strategies for drug development; and genome editing methods where DSB repair mechanisms are involved.
[0011] DNA repair capacities vary among individuals, and deficiencies in DSB repair are associated with a large number of diseases. Many evidences show the association between mutations in BRCA1 or BRCA2 genes and ovarian and breast cancers (Chen S. et al., J. Clin. Oncol., 2006, 24:863-71).
[0012] BRCAl/2 are also implicated in the response to cancer therapies. Indeed, treating BRCA deficient cells with PARP inhibitors causes a significant cellular death. This mechanism, where depletions or inhibitions of two or more DNA repair proteins become lethal for a cell whereas a deficiency of only one is not, is identified with the term ‘synthetic lethality’ (Farmer H. et al., Nature, 2005, 434:917-921; Bryant H.et al., Nature, 2005, 434:913-917).
[0013] Many other proteins involved in DSB repair like ATM, NBS1 and WRN if defectives are the cause of hereditary diseases. Mutations of ATM protein are associated with Ataxia telangiectasia and these patients have high predisposition to leukemia, immunodeficiency, average life expectancy low and hypersensitivity to ionizing radiation (Mondello C. et al., Mutation Research, 2010, 704:29-37). The Nijmegen breakage syndrome (NBS) is a DSB repair deficiency-related syndrome. NBS patients show a progressive microcephaly, growth retardation, sun sensitivity and immunodeficiency (Biau J. et al., Front. Oncology, 2019, 9:1- 10). Another pathology related to defects in DSB repair is the Werner syndrome. Patients affected with Wemer syndrome have mutations in the WRN gene and they present skin ulceration, osteoporosis, cataracts and elevated cancer risks (Knoch J. et al., Eur. J. Dermatol., 2012, 22:443-455).
[0014] More generally, individuals with defective repair pathways are increasingly recognized with radiosensitivity and also immunodeficiency (A R Gennery, Br Med Bull, 2006; 77-78:71-85) because of the role of repair proteins in the development of adaptive immunity by VDJ recombination, antibody isotype class switching and affinity maturation by somatic hypermutations. [0015] DNA repair is not only affected by alteration of the genetic information (mutations, chromosome rearrangements, epigenetic regulations), but also by post-transcriptional modifications (RNA splicing) and post-translational modifications (like phosphorylation, acetylation, methylation, ubiquitination, sumoylation and neddylation) (Oberle C. and Blattner C., Curr. Genomics, 2010, 11:184-198).
[0016] There are very few assays allowing the direct measurement of DSB repair capacities. Many assays measure DNA damage and only indirectly the DNA repair capabilities. Among the molecular strategies to quantify DNA breaks, polymerase chain reaction (PCR), Single Cell Gel Electrophoresis (SCGE, also known as the Comet assay) and Pulse-field gel electrophoresis (PFGE) are the most frequently methods used (Figueroa-Gonzalez G. and Perez-Plasencia C., Oncol. Letters, 2017, 13:3982-88; Olive P.L., Wlodek D., Banath J.P., Cancer Res., 1991, 51:4671-4676; Wang M. et ah, Nucleic Acids Res. 2006, 34:6170-6182).
[0017] Indeed, genomics gives information on the integrity of genes, it cannot predict what are the consequences at a functional level, and if compensatory pathways will balance the genomic defects. Many defects are the consequences of deregulations that may have been corrected, reversed or that do not exist anymore. Thus, all these methods quantify DNA lesions without determining directly the activity of DNA repair enzymes and the DNA DSB repair capacity in real time.
[0018] The amount of repair enzyme can be measured using different methods.
[0019] One type of methods determines the expression level of selected DNA repair proteins using specific antibody and colorimetric immunodetection assay such as ELISA or by detecting the foci number associated to the presence of DSB s using single molecule imaging (Tsvetkova A. et ah, Oncotarget, 2017, 38:64317-64329). However, they can, at the best, detect one single protein in one repair pathway.
[0020] In vivo, the host cell reactivation (HRC) assay monitors the DNA repair activity of cells by measuring the capacity of cells to process one or more transfected DNA repair reporter vectors or DNA probes. These assays are commonly based on introducing multiple DNA repair reporter vectors or DNA probes into a cell and determining the capacity of the cell to process these multiple reporter vectors or DNA probes thereby determining multiple DNA repair capacities in the cell (US2014/0227701). Examples of these in vivo assays include: (i) in vivo plasmid end joining (Wang M. et ah, Nucleic Acids Res. 2006, 34:6170-6182); (ii) real-time in vivo monitoring of HR and NHEJ using chromosomally integrated fluorescent reporter substrates (Her J. and Bunting S., J. Biol. Chem., 2018, 293:10502-11; Mao et al., DNA repair (2008), 10: 1765-1771); and (iii) in vivo oligonucleotide probe retrieval assay (US 2013/0115598). Some cell lines need to be immortalized using exogen agents and this can alter their original DNA repair capacity and thus can lead to misinterpretations regarding DSB repair capacity (Johnson J. and Latimer J., Methods Mol. Biol., 2005, 291:321-335; Kostyrko K. and Mermod N., Nucleic Acids Res., 2016, 44: e56). They are not easily applicable to tumors and cells from patients. In addition, these methods are cumbersome and require a certain expertise so they cannot be largely extended in routine.
[0021] In vitro, DSB end joining assays employ DSB DNA substrates (generally linearized plasmids) to perform analysis of the repair of the DSBs through ligation. They address only one DSB repair pathway (NEHJ). These approaches allow one to overcome several technical challenges associated with other end joining assays requiring cultured cells (Pastwa E., Nucleic Acids Res., 2001, 29: e78; Datta K., Neumann R. and Winters T., Analytical Biochemistry, 2006, 358:155-157).
[0022] WO 2010/097584 discloses a method of measuring the DNA damage response, comprising: incubating nuclei with a cytosolic extract mixed with dNTP and a labeled nucleotide (biotinylated-UTP); adding streptavidin-FITC; and measuring the level of labelled nucleotide incorporated during in vitro DNA synthesis. This approach requires the isolation of nuclei and cytosolic extract. It is not intended to characterize DNA repair pathways but only to investigate the DNA damaging properties of agents, through activation of DNA synthesis and elongation which is related to DNA damage response activation. The level of H2AX phosphorylation (gamma-H2AX) which marks regions of DNA DSBs is also determined in parallel, to serve as control for DDR activation. No qualitative or quantitative information on specific repair pathways is provided by this method.
[0023] Although tests for evaluating DSB DNA damage and repair activity have been developed, they only provide a partial view of the DSB repair network functionality and cannot be performed directly on any type of biological medium including biological samples from patients.
[0024] As the different DSB repair pathways are interconnected, there is a need for methods providing a more complete overview of the DSB repair pathways and the balance between the different DSB repair pathways that are adapted to any type of biological medium including biological samples from patients.
SUMMARY OF THE INVENTION
[0025] The inventors have designed an in vitro method which allows to precisely quantify the four double-strand break repair mechanisms simultaneously and their balance in any type of biological medium including biological samples from patients.
[0026] This method provides a comprehensive investigation of DSB repair capacities in any biological sample. This method enables to explore new features of the DSB repair network which have scientific and clinical applications. The method of the invention allows to determine how the different double-strand break repair pathways are coordinated and how they interplay. The method of the invention makes it possible to detect defects in any of the four double-strand break repair pathways, dysregulations or imbalance between the activities, and global down- or up-regulations.
[0027] One aspect of the invention relates to an in vitro method of determining the activities of at least two DNA double-strand break (DSB) repair pathways in a biological medium, comprising: a) Performing at least two DSB repair reactions, preferably simultaneously in a single reaction mixture: ai) a first repair reaction comprising a supercoiled circular double- stranded DNA molecule and a homologous linear double-stranded DNA molecule that are repaired by Homologous Recombination (HR) or Single-Strand Annealing (SSA) to form a repaired double- stranded DNA molecule; and a2) a second repair reaction comprising two linear double-stranded DNA molecules that may or may not be homologous and are repaired by Non-Homologous End- Joining (NHEJ) or Alternative-End Joining (alt-EJ) to form a repaired double- stranded DNA molecule; wherein said at least two repair reactions comprise DNA repair enzymes from said biological medium, and further comprise at least one label that is incorporated into the repaired double- stranded DNA molecule, and wherein said at least one label is on the linear double-stranded DNA molecule of the first repair reaction or one of said molecules of the second repair reaction, on a deoxy nucleotide, or on both; b) Measuring the level of label incorporated into the repaired double-stranded DNA molecule in said at least first and second DSB repair reactions; and c) Determining the activities of at least two DSB repair pathways, a first pathway chosen from: Homologous Recombination (HR) and Single-Strand Annealing (SSA); and a second pathway chosen from Non-Homologous End-Joining (NHEJ) and Alternative- End Joining (alt-EJ) in said biological medium, wherein:
The level of HR is determined by the level of labeled linear double- stranded DNA molecule incorporated into the repaired double- stranded DNA molecule from the first DSB repair reaction;
The level of SSA is determined by the level of labeled nucleotide incorporated into the repaired DNA molecule from the first DSB repair reaction;
The level of NHEJ is determined by the level of labeled linear double- stranded DNA molecule incorporated into the repaired double- stranded DNA molecule from the second DSB repair reaction; and
The level of alt-EJ is determined by the level of labeled nucleotide incorporated into the repaired double-stranded DNA molecule from the second DSB repair reaction.
[0028] In some embodiments, the supercoiled circular double-strand DNA molecule is a supercoiled plasmid and/or the linear double-stranded DNA molecule(s) are linearized plasmid(s), preferably from the same plasmid.
[0029] In some embodiments, the supercoiled circular double-strand DNA molecule of the first DSB repair reaction and one of the linear double- stranded DNA molecules that is not labelled of the second DSB repair reaction are immobilized on a solid support.
[0030] In some embodiments, the label is fluorescent and may be detected directly or indirectly; preferably a label chosen from Cy3 and Cy5; preferably wherein the method comprises two different labels.
[0031] In some embodiments, the biological medium comprises cells or a mixture containing repair enzymes; preferably the biological medium is a sample obtained from a patient; more preferably the biological sample is selected from the group consisting of: plasma, whole-blood or PBMC fraction thereof; and tissue or tumor biopsy. [0032] In some embodiments, the DSB reaction is performed on a cell extract, preferably a nuclear extract; more preferably comprising a final protein concentration from 0.05 to 2 mg/mL; more preferably of 0.1 mg/mL or 0.2 mg/mL.
[0033] In some embodiments, the DSB repair reactions are performed at 30°C; preferably for one hour.
[0034] In some embodiments, the method comprises determining the activities of at least two, preferably four DSB repair pathways chosen from: Homologous Recombination (HR), Single- Strand Annealing (SSA), Non-Homologous End-Joining (NHEJ) and Alternative-End Joining (alt-EJ).
[0035] Another aspect of the invention relates to a method of detecting a dysregulation of the DNA repair mechanisms in cells comprising: a) determining the activities of at least two DNA double-strand break (DSB) repair pathways in a cell sample according to the method of the present disclosure; b) detecting the presence of an altered activity of said DSB repair pathway(s).
[0036] In some embodiments, said altered activity is a defect of said DSB repair pathway(s). Preferably, wherein the detection of said defect is used to predict the risk of occurrence of cancer or immunological disease or the risk of toxicity following exposure to DNA damaging or DNA interacting agents in an individual. In some preferred embodiments, the detection of said defect is used to predict the risk of late radiotoxicity before radiotherapy.
[0037] In some embodiments, said cells are from a treated patient and said altered activity is used to predict the response to therapy, in particular cancer therapy in said patient.
[0038] Another aspect of the invention relates to a method of screening DSB repair modulators, comprising: a) contacting at least one candidate compound with a biological medium comprising DNA repair enzymes; b) determining the activities of at least two DNA double-strand break (DSB) repair pathways in said biological medium in the presence and absence of said at least one candidate compound, according to the method of the disclosure; c) identifying the compounds which modulate the activity of at least one of said DSB repair pathways. [0039] Another aspect of the invention relates to a kit for determining the activities of at least two DNA double-strand break (DSB) repair pathways in a biological medium, comprising at least: a) a supercoiled circular double-strand DNA molecule, preferably a supercoiled plasmid; more preferably immobilized on a solid support; and b) at least one linear double-stranded DNA molecule; preferably linearized plasmid(s) with cohesive ends; more preferably from the same plasmid as in a); even more preferably immobilized on a solid support.
[0040] In some embodiments, said kit further comprising at least one label, preferably fluorescent; more preferably a dNTP and a linear double-stranded DNA molecule with different labels.
[0041] In some embodiments, said kit comprises a solid support comprising at least one pad, comprising at least one spot of the supercoiled circular double-strand DNA molecule and at least one spot of the linear double- stranded DNA molecule; preferably further comprising a reaction mixture comprising a dNTP and a linear double- stranded DNA molecule with different labels; more preferably further comprising a buffer appropriate for DNA repair enzymes.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The present invention relates to an in vitro method for determining simultaneously the activity of the four main DSB repair mechanisms in a biological medium.
[0043] One aspect of the invention relates to an in vitro method of determining the activities of at least two DNA double-strand break (DSB) repair pathways in a biological medium, comprising: a) Performing at least two DSB repair reactions, preferably simultaneously in a single reaction mixture: ai) a first repair reaction comprising a supercoiled circular double- stranded DNA molecule and a homologous linear double-stranded DNA molecule that are repaired by Homologous Recombination (HR) or Single-Strand Annealing (SSA) to form a double- stranded repaired DNA molecule; and ai) a second repair reaction comprising two linear double-stranded DNA molecules that may or may not be homologous and are repaired by Non-Homologous End- Joining (NHEJ) or altemative-End Joining (alt-EJ) to form a repaired double-stranded DNA molecule; wherein said at least two repair reactions comprise DNA repair enzymes from said biological medium, and further comprise at least one label that is incorporated into the repaired double-stranded DNA molecule, and wherein the at least one label is on the linear double-stranded DNA molecule of the first repair reaction or one of said molecules of the second repair reaction, on a deoxynucleotide (dNTP), or on both; b) Measuring the level of label incorporated into the repaired double-stranded DNA molecule in said at least first and second DSB repair reactions; and c) Determining the activities of at least two DSB repair pathways, a first pathway chosen from: Homologous Recombination (HR) and Single-Strand Annealing (SSA); and a second pathway chosen from Non-Homologous End-Joining (NHEJ) and Altemative- End Joining (alt-EJ) in said biological sample, wherein:
The level of HR is determined by the level of labeled linear double- stranded DNA molecule incorporated into the repaired double- stranded DNA molecule from the first DSB repair reaction;
The level of SSA is determined by the level of labeled nucleotide incorporated into the repaired double-stranded DNA molecule from the first DSB repair reaction;
The level of NHEJ is determined by the level of labeled linear double- stranded DNA molecule incorporated into the repaired double- stranded DNA molecule from the second DSB repair reaction; and
The level of alt-EJ is determined by the level of labeled nucleotide incorporated into the repaired double-stranded DNA molecule from the second DSB repair reaction.
[0044] The method involves the measurement of the four repair pathways (HR, NHEJ, SSA, alt-EJ) simultaneously from the same reaction medium.
[0045] The method provides a multiplexed quantitative assay for DNA DSB repair activities.
[0046] A used herein, “in vitro” means “cell-free””. This means that the in vitro method of the invention is different from an in cellulo method and does not require living cells maintained in culture to determine DNA DSB repair activities. In particular, the in vitro method of the invention does not require introduction of the circular double-stranded and linear DNA molecules inside a cell to determine DNA DSB repair activities, nor does it require complex cell culture or manipulation.
[0047] As used herein, “supercoiled DNA” refers to a supercoiled circular double-stranded DNA molecule, in particular a supercoiled plasmid.
[0048] As used herein, “DNA molecule” refers to double-stranded DNA molecule; DSB repair or DNA DSB repair, refers to DNA double-strand break repair; DSB repair enzyme, DNA DSB repair enzyme or DNA repair enzyme, refers to DNA double-strand break repair enzyme.
[0049] According to the method of the invention, the DNA which serves as repair matrix for the DNA DSB repair enzymes present in the biological sample is a double- stranded DNA whether linear or circular. In particular, it involves long DNA fragments (over 100 bases) such as those described in the application WO 01/090408 or a supercoiled circular double- stranded DNA, in particular a plasmid such as described in the Application WO 2004/059004, Millau et al., Lab. Chip., 2008, 8, 1713-1722; Prunier et al., Mutation Research, 2012, 736, 48-55.
[0050] In some preferred embodiments, the method uses plasmid(s) for the DSB reactions; a supercoiled plasmid and at least one linear plasmid (e.g., linearized plasmid). The supercoiled plasmid and the linear plasmid for the first DSB repair reaction have sufficient homology to engage into a Homologous Recombination (HR) reaction or a Single-Strand Annealing (SSA) reaction and generate a repaired double- stranded DNA molecule. The linear plasmids for the second DSB repair reaction have advantageously at least a micro-homology to engage into an altemative-End Joining (alt-EJ) reaction and generate a repaired double- stranded DNA molecule. A single plasmid in supercoiled and linear forms may be advantageously used for the four DSB repair reactions (HR, SSA, NHEJ and alt-EJ). Linear plasmid(s) are obtained by linearization of a supercoiled plasmid using a restriction enzyme. The restriction enzyme usually cleaves the plasmid at a unique restriction site and generates blunt or cohesive ends, advantageously cohesive ends. The linear plasmid is also named DSB-plasmid.
[0051] Preferably, the supercoiled and linear plasmids of the first DSB reaction and/or the two linear plasmids of the second DSB reaction are from the same plasmid.
[0052] In some preferred embodiments, the non-labelled DNA molecules from the first and second DSB repair reaction (e.g., supercoiled circular double-strand DNA molecule and one of the linear double-stranded DNA molecules) are immobilized on a solid support to easily eliminate the label (e.g., labelled nucleotide and/or DNA molecule) not incorporated in step a), by a washing step, before step b) of measuring incorporation of the label into the repaired double-stranded DNA molecule.
[0053] The solid support is any appropriate support for the immobilization of nucleic acids and in particular supercoiled plasmid that are well-known in the art. Non-limiting examples of suitable support for immobilization include glass, polypropylene, polystyrene, silicone, metal, nitrocellulose or nylon; preferably coated with an agent that forms a 3D layer that binds DNA covalently or non-covalently. Non-limiting examples of coating agent include coating with porous film (3D layer), such as nylon or hydrogel film, in particular a multi-component polymer layer, activated with N-Hydroxysuccinimide (NHS) esters. 3D coating functionalized by agents such as N-Hydroxysuccinimide (NHS) esters immobilizes DNA by covalent binding through reactive groups such as amine reactive chemistry. The support is advantageously a microchip type miniaturized support. Suitable supports for DNA immobilization are well-known in the art and commercially available: 3-D Hydrogel coated slides (SCHOTT); silicone coated with a hydrogel (Nanochip ™; NANOGEN); glass or polymer slides coated with a hydrogel (3D-reactive slides, Poly An); low-fluorescence glass coated with multi-component polymer layer (Nexterion® Slide H). The DNA immobilization method does not induce DNA breaks and therefore preserves the supercoiled structure of the plasmid. The method usually comprises: depositing the DNA molecule on the surface of the solid support, such as 3D-coated slide, using appropriate spotting device suitable for the preparation of DNA microarrays, for example by automation with a robot such as a piezoelectric robot; and drying in controlled humidity environment to immobilize the DNA without inducing DNA breaks. In particular embodiments, each spot of the microarray comprises from 0.01 ng to 1 ng of double-stranded DNA, preferably from 0.12 ng to 0.2 ng of double-stranded DNA. The solid support advantageously further comprises at least one reaction chamber. Preferably, the reaction chamber comprises at least one spot of the supercoiled circular double-strand DNA molecule and at least one spot of the linear double- stranded DNA molecule. In some preferred embodiments, the reaction chamber comprises two identical spots of the supercoiled circular double-strand DNA molecule and two identical spots of the linear double- stranded DNA molecule. [0054] At least one of a dNTP and a linear double-stranded DNA molecule or both, comprise a label (detectable moiety), which makes it possible to detect and quantify them in step (b) after they are incorporated into the repaired double- stranded DNA molecule following the DNA DSB repair reaction of step a). The tracer or label which is used for labeling the nucleotide or DNA molecule is detected by measurement of the signal which is proportional to the quantity of nucleotide or linear DNA molecule incorporated into the repaired double- stranded DNA molecule.
[0055] A label is a moiety that can be detected directly or indirectly by the production of a detectable signal such as colorimetric, fluorescent, chemiluminescent, electrochemoluminescent, magnetic or radioactive signal. Directly detectable labels include radioisotopes and luminescent compounds (e.g. radioluminescent, chemiluminescent, bioluminescent, fluorescent or phosphorescent). Indirectly detectable labels have the ability to bind to or cleave another moiety, which itself may emit or absorb light of a particular wavelength, and include for example, biotin, avidin such as streptavidin, antibody, hapten, epitope tag such as the FLAGepitope, digoxigenin and enzyme tag such as horseradish peroxidase. Preferred labels include fluorescent labels (fluorophores) such as without limitation, Cyanine 3 and Cyanine 5.
[0056] The means and techniques for labeling nucleotides and DNA molecules are well known to the person skilled in the art. The labelled nucleotide or DNA molecule, in particular fluorescent, radioactive, magnetic or colorimetric label is detectable by any technique known to the person skilled in the art such as, without limitation, fluorescence microscopy, flow cytometry, scintigraphy, magnetic resonance imaging and mass spectrometry.
[0057] The use of different labels makes it possible to perform the at least two DSB repair reactions of step (a) (steps (al), (a2) and others, (a3), (a4)) simultaneously in a single reaction but requires a specific detector for each label to measure the DNA DSB repair signal (step (b)). The use of a single label requires performing the different DSB repair reactions of step (a) (steps (al), (a2) and others, (a3), (a4)) in separate reactions but makes it possible to use a single detector for measuring the DNA DSB repair signal (step (b)).
[0058] In some particular embodiments, the at least one label is fluorescent and may be detected directly or indirectly. In some preferred embodiments, the label is chosen from Cy3 and Cy5. Indirect label is advantageously biotin. In some preferred embodiments, the method comprises two different labels, preferably Cy3 and Cy5. For example, the method comprises a biotinylated dNTP that is detected using a Streptavidin-Cy5 conjugate and a linear double- stranded DNA molecule labelled with Cy3, preferably a linearized plasmid labelled with Cy3.
[0059] The at least two DNA DSB repair reactions (step a) are performed under conditions allowing repair of double- stranded DNA by DSB repair enzymes present in said biological medium and thereby incorporation of label into repaired double-stranded DNA molecule. These conditions which are well known to the person skilled in the art are disclosed for example by Wong and Mermod and colleagues (Wong B. et ah, J. Biol. Chem., 1998, 273:12120-12127; Kostyrko K. and Mermod N., Nucl. Acids Res., 2016, 44(6):e56). The repair reaction is performed in the presence of ATP, an ATP regeneration system and any other agent necessary for the activity of DNA DSB repair enzymes present in the biological medium. All nucleotides triphosphate (dATP, dCTP, dGTP, dTTP) can be present in the repair medium to evaluate the SSA and alt-EJ reactions but they are not absolutely required. For example, the repair buffer contains ATP ImM, MgCh lOmM, KC1 80mM, DTT 2mM, BSA O.lmg/mL, Tris-HCl 20mM pH 7.5, Creatin Phosphokinase 0.05mg/mL, Phosphocreatine lOmM and eventually, all or other dNTPs 0.25mM. Other dNTPs refer to the three dNTPs other than the labelled one. The repair reaction is generally done with a nuclear extract, preferably comprising a final protein concentration from 0.05 to 2 mg/mL; more preferably of 0.1 mg/mL or 0.2 mg/mL. The reaction is performed at a temperature promoting the DNA DSB repair reaction, preferably 30°C, for sufficient time, generally included between fifteen minutes and five hours. The reaction is advantageously performed in the microwells of a microchip type miniaturized support such as defined above.
[0060] The method is applicable to any biological medium comprising DNA repair enzymes including any cells, tissues, tumors or mixture of repair enzymes, artificially produced or isolated from cells, which are prepared in a way that preserves the repair enzyme activities. Such samples include, and are not limited to, primary cells (freshly isolated or cultured), cell lines, patient’s biopsies, peripheral blood mononuclear cells (PBMCs), xenograft, preferably but not limited to eukaryotes.
[0061] In some particular embodiments, the biological medium comprises cells or a mixture containing repair enzymes; preferably the biological medium is a biological sample obtained from a patient; more preferably the biological sample is selected from the group consisting of: plasma, whole-blood or PBMC fraction thereof; and tissue or tumor biopsy.
[0062] Biological medium comprising a mixture containing repair enzymes can be tested directly, without pre-treatment. Biological medium comprising cells are pretreated so as to recover or extract nuclear proteins which contain the DNA repair enzymes. The recovery or extraction of nuclear proteins from cells is done according to standard techniques well known to the person skilled in the art. Cells are generally lysed for isolating the nuclei and then the nuclei are in turn lysed for extracting nuclear proteins. In order to prepare nuclear protein extracts, the protocols described for in vitro transcription tests or in vitro DNA repair tests can be used, such as for example those described by Dignam JD et al. (Nucleic Acids Research, 1983, 11 ; 1475— 1489); Kaw L. et al. (Gene Analysis Techniques, 1988, 5, 22-31); Iliakis G. et al. (Methods Mol. Biol., 2006, 314, 123-31); Luo Y et al. (BMC Immunol., 2014, 15, 586- ). The nuclear extracts can further be fractionated or dialyzed according to conventional methods. All the steps for preparation of the nuclear protein extract are done under conditions that do not denature the enzymatic activities and the proteins contained in the cells, such that the DNA repair activity initially present in the sample of cells is preserved in the cellular extract which is analyzed in the method of the invention.
[0063] In some particular embodiments, the DSB repair reaction is performed on a cellular lysate or extract thereof, preferably a nuclear lysate or extract thereof, more preferably a nuclear protein extract.
[0064] In some particular embodiments, the method comprises determining the activities of at least three, preferably four DSB repair pathways chosen from: Homologous Recombination (HR), Single-Strand Annealing (SSA), Non-Homologous End-Joining (NHEJ) and Alternative-End Joining (alt-EJ).
[0065] After a washing step, and if necessary a step to reveal the labels, the signal emitted by the DSB -plasmid and the signal emitted by the labeled nucleotide are measured on the 2 immobilized plasmids: the supercoiled plasmid and the DSB-plasmid, resulting in 2 different values, or four different values if the labelled dNTP is added during the repair reaction.
[0066] The repair reaction can be run at several different final protein concentrations of the same lysate, on different biochips. [0067] The signal measured at the level of the immobilized supercoiled plasmid that comes from the labeled DSB -plasmid in solution, is attributed to components of the HR repair reaction which requires the strand invasion.
[0068] The signal measured at the level of the immobilized supercoiled plasmid that comes from the labeled dNTP is attributed to components of the SSA repair reaction, that involves nucleotide incorporation by specific polymerases.
[0069] The signal measured at the level of the immobilized DSB -plasmid that comes from the labeled DSB-plasmid in solution is attributed to components of the NHEJ repair reaction, able to ligate the plasmids ends. [0070] The signal measured at the level of the immobilized DSB-plasmid that comes from the labeled dNTP is attributed to components of the alt-EJ repair reaction.
[0071] For each repair reaction that is run using one final protein concentration, the biological medium is then characterized by 1, 2, 3, or 4 values.
[0072] If the repair reaction is run using a second final protein concentration, the same biological medium is then characterized by other 1, 2, 3, or 4 values.
[0073] The biological medium can be characterized by any combinations between these values.
[0074] The DSB repair defects can be identified:
- from the signal of any of the four measured pathways, with any of the protein concentration,
- from a combination between the signal of at least 2 pathways, obtained with one protein concentration, or
- from a combination of signal from one pathway, measured at the 2 protein concentrations. [0075] Another aspect of the invention relates to a kit for determining the activities of at least two DNA double-strand break (DSB) repair pathways in a biological sample, comprising at least: a) a supercoiled circular double-strand DNA molecule, preferably a supercoiled plasmid; more preferably immobilized on a solid support; and b) at least one linear double- stranded DNA molecule; preferably linearized plasmid(s) with cohesive ends; more preferably from the same plasmid as in a); even more preferably immobilized on a solid support.
[0076] In some embodiments, said kit further comprising at least one label, preferably fluorescent; more preferably a dNTP and a linear double-stranded DNA molecule with different labels.
[0077] In some embodiments, said kit comprises a solid support, comprising at least one pad containing at least one spot of the supercoiled circular double-strand DNA molecule and at least one spot of the linear double-stranded DNA molecule. The solid support is preferably a 3D-coated support according to the present disclosure and advantageously further comprises at least one reaction chamber. Preferably, the reaction chamber comprises two identical spots of the supercoiled circular double-strand DNA molecule and two identical spots of the linear double-stranded DNA molecule.
[0078] In some preferred embodiments, the kit further comprises a reaction mixture comprising a dNTP and a linear double- stranded DNA molecule with different labels; more preferably further comprising a buffer suitable for DNA repair enzymes. In some preferred embodiments, the buffer comprises: ATP 1 mM, MgCk 10 mM, KC1 80 mM, DTT 2 mM, BSA 0.1 mg/mL, Tris-HCl 20 mM pH 7.5, Creatin Phosphokinase 0.05mg/mL, Phosphocreatine 10 mM and other dNTPs 0.25 mM.
[0079] The method according to the invention has a certain number of advantages over the prior art methods. It allows the mapping of a given biological medium according to its enzymatic activities for DSB repair. It measures the real DNA repair activities at a defined time point. It allows the identification of global or partial dysregulation of DSB repair mechanisms, thanks to the simultaneous evaluation of the activity of at least two DSB repair mechanisms. It is used to determine whether DSB repair mechanisms are deficient or partially deficient for certain activities. It makes it possible to compare various biological medium in term of DBS repair capability. It allows to develop specific inhibitors for DSB repair pathways. It allows to detect up-regulations, compensatory pathways, following treatments or not. Since it is miniaturized, it makes it possible to obtain many information using small amounts of biological material in a short time. [0080] The method of the invention identifies DSB repair defects responsible for radiotoxicity syndromes whereas methods developed up to now show little performances and are not widely used in the clinics. An example of method to predict the radiotoxicity is the radiation-induced CD8 T-lymphocyte Apoptosis (RILA) as disclosed in WO 2014/154854. Results of RILA assessment in breast cancer patients showed a sensitivity of 70% which is not sufficient for incorporation into routine analysis. Another method comprising the detection of nucleo- shuttling of the ATM Protein in the healthy tissues of patients in response to radiation therapy (Ganzotto A. et al., Int. J. Radiation Oncol. Biol. Phys., 2016, 94:450-460) cannot discriminate patients with severe adverse effects (grade >3) from patients with moderate adverse effects (grade 2). Considering that most patients with grade 2 adverse events, will spontaneously recover, the assay is not clinically useful. In addition, the latter two methods require the in vitro irradiation of samples, which is a major drawback, preventing widespread use of the methods. Indeed, the biomarkers that are measured need to be induced by exposure to ionizing radiations.
[0081] Genomics approaches have also been developed for the radiotoxicity risk prediction. Consortium identified common genetic variants affecting radiotoxicity but the identified SNP- toxicity associations were not significant (Kems S. et al., J. National Cane. Inst., 2020, 112:179-190). For radiotoxicity risk prediction, the invention can be widely applied for diagnostic purposes, it requires only several mL of blood, it is fast, user friendly and it can be performed before the treatment begins. Importantly, it displays the best performances known today for late severe (grade >3) radiotoxicity prediction.
Use of the method and kit
[0082] The method and kit of the invention can be used for multiple applications.
[0083] The method of the invention can be integrated into the clinical guidelines to screen all patients before they receive radiotherapy. The method and kit of the invention can be used to identify DSB repair defects leading to cancer predisposition. The method and kit invention can be used to identify DSB repair defects responsible for resistance to cancer treatments. The method and kit of the invention can be used to identify DSB repair defects allowing to develop synthetic lethality approaches. The method and kit of the invention can be used to evaluate the balance between various DSB repair pathways, allowing to specifically modulate one or several pathways. The method and kit of the invention can be used to quantify the various DSB repair pathways, allowing to identify imbalanced activities. The method and kit of the invention can be used to evaluate the specificity and efficacy of various DNA repair inhibitors.
[0084] In one embodiment, the method and kit according to the invention are used to detect DNA repair defect(s) in individuals to predict cancer risk occurrence.
[0085] In another embodiment, the method and kit according to the invention are used to detect DNA repair defect(s) in individuals to predict or understand immunological diseases.
[0086] In another embodiment, the method and kit according to the invention are used to detect DNA repair defect(s) in individuals to predict toxicity occurrence following exposure to DNA damaging agents or DNA interacting agents. In some preferred embodiments, the detection of said defect is used to predict the risk of late radiotoxicity before radiotherapy.
[0087] In another embodiment, the method and kit according to the invention are used to identify specific biomarkers in cells, tumors, or in patient cells, treated or not, that predict response to cancer therapies.
[0088] In connection with the above uses, one aspect of the invention relates to a method of detecting a dysregulation of the DNA repair mechanisms in cells comprising: a) determining the activities of at least two DNA double-strand break (DSB) repair pathways in a cell sample according to the method of the present disclosure; b) detecting the presence of an altered activity of said DSB repair pathway(s).
[0089] In some embodiments, said altered activity is a defect of said DSB repair pathway(s). Preferably, wherein the detection of said defect is used to predict the risk of occurrence of cancer or immunological disease or the risk of toxicity following exposure to DNA damaging or DNA interacting agents in an individual.
[0090] In some embodiments, said cells are from a treated patient and said altered activity is used to predict the response to therapy, in particular cancer therapy in said patient.
[0091] In another embodiment, the method and kit according to the invention are used to develop specific DNA DSB repair inhibitors.
[0092] In another embodiment, the method and kit according to the invention are used to develop and optimize specific genome editing strategies involving DSB repair. [0093] In connection with the above use, one aspect of the invention relates to a method of screening DSB repair modulators, comprising: a) contacting at least one candidate compound with a biological sample comprising DNA repair enzymes; b) determining the activities of at least two DNA double-strand break (DSB) repair pathways in said biological sample in the presence and absence of said at least one candidate compound, according to the method of the present disclosure; c) identifying the compounds which modulate the activity of at least one of said DSB repair pathways. [0094] The method is used to screen for activators and inhibitors of the four DNA double strand break (DSB) repair pathways which either activate or inhibit one of said pathways.
[0095] In another embodiment, the method and kit according to the invention are used to control small molecules or inhibitors or compounds, mechanisms of action and off-target mechanisms of action. [0096] In another embodiment, the method and kit according to the invention are used to control DNA repair inhibitors impact on different DSB repair pathways.
[0097] In another embodiment, the method and kit according to the invention are used to assess the chemical or biological modulation of certain DSB repair pathways for genome editing strategies. [0098] The practice of the present invention will employ, unless otherwise indicated, conventional techniques, which are within the skill of the art. Such techniques are explained fully in the literature.
[0099] The invention will now be exemplified with the following examples, which are not limitative, with reference to the attached drawings in which: FIGURE LEGENDS
[0100] Figure 1: Schematic representation of Next-SPOT assay according to the invention. Two identical spots containing a supercoiled plasmid and two identical spots containing a linear plasmid are printed on specific areas of the biochip (a). Once added the repair enzymes, a Cy 3 -labelled linear plasmid and a Cy5-labelled dNTP, on each printed spot two reactions can happen: the HR and SSA on the supercoiled plasmid and the NHEJ and alt- EJ on the linear plasmid. Two wavelengths, 532 and 635 nm, are used to quantify the fluorescence in each spot and to obtain the DSB profile of the analysed sample (b).
[0101] Figure 2: The relative contributions of all four DSB repair pathways to DSB repair as a percentage, for each sample are shown. Each bar corresponds to one sample. All bars are constituted by four rectangles representing from the bottom to the top HR, NHEJ, SSA and alt-EJ, respectively. The data obtained at 0.1 mg/L final protein concentration are shown.
[0102] Figure 3: Box-plots of the DSB repair activities at J1 (before radiotherapy) for patients with radiation-induced early grade >2 adverse effects (Yes category, grade >2, N=73) and the others (No category, grade <2, N=108). Only significant differences are shown.
[0103] Figure 4: Box-plots show the DSB repair activities, measured after the fifth session (J8) of radiotherapy, for which significant differences between breast and prostate cancer patients suffering from radiation-induced early grade >3 adverse effects (Yes category, grade >3, N=13) and the others (No category, grade <3, N=162) were detected.
[0104] Figure 5: Box-plots showing the results of the DSB repair activities determination, for which significant differences between breast and prostate cancer patients suffering from radiation-induced late grade >2 adverse effects (Yes category, grade >2, 36) and the others (No category, grade <2, N=137) at different treatment time points (J 1 , J2 and J8) are shown.
[0105] Figure 6: Box-plots of the DSB repair activities for which significant differences between breast and prostate cancer patients suffering from radiation-induced late grade >3 adverse effects (Yes category, grade >3, N=6) and the others (No category, grade <3, N=167) are shown at different treatment time points (J 1 , J2 and J8).
[0106] Figure 7: The relative contribution of each DSB repair pathway to total DSB repair was assessed for the three cell lines at basal level, at 0.1 mg/mL (A) and at 0.2 mg/mL (B) protein concentration.
[0107] Figure 8: Impact of doxorubicin, B02, NU7026, Olaparib and their combinations on the four DSB repair mechanisms is shown for M059K (A), M059J (B) and Hela (C) cells at 0.1 mg/mL protein concentration, determined using the ratio of the fluorescence intensity obtained for each pathway, of treated cells to non-treated cells (FI T/FI NT).
[0108] Figure 9: Impact of doxorubicin, B02, NU7026, Olaparib and their combinations on the four DSB repair mechanisms was determined for M059K (A), M059J (B) and Hela (C) cells at 0.2 mg/mL protein concentration, using the ratio of the fluorescence intensity obtained for each repair pathway, for treated cells to non-treated cells (FI T/FI NT).
[0109] Figure 10: The impact of BRCA2 silencing on the four DSB repair mechanisms was determined using the ratio of fluorescence intensity obtained for each pathway between BRCA2 wild-type and BRCA2-silenced cells. [0110] EXAMPLES
Materials and methods
Preparation of the nuclear cell extracts
[0111] Nuclear cell extracts were obtained from PBMCs or from cell lines as described in Candeias et al, with some adaptations (Candeias S et al., Mutation Research, 2010, 694:53- 59). Thawed PBMCs and cell lines were washed in ice-cold PBS. The cell pellet was suspended and incubated 8 or 20 min in 3mL of ice-cold buffer A (lOmM HEPES pH 7.9, 1.5mM MgC12, lOmM KC1, 0.02% TritonX-100, 0.5mM DTT, 0.5mM PMSF). After 5 min of centrifugation at 2300g, nuclei were suspended in 25, 50 or 75pl of ice-cold Buffer B depending on the cell number (lOmM HEPES pH 7.9, 1.5mM MgC12, 400mM KC1, 0.2mM EDTA pH 8.0, 25% Glycerol, 0.5mM DTT, 0.5mM PMSF and complete-mini antiproteases (Roche)). Nuclei were lysed by incubation 30 min at 4°C and two cycles of freezing-thawing in liquid nitrogen alternated with 30 sec of vortex. The nuclear extracts were then collected after 15 min of centrifugation at 16000g and stored at -80°C. The protein concentration for each sample was determined using the Pierce BCA Protein Assay Kit (Interchim). Production of plasmids and biochips
[0112] The biochips were constituted by 14 or 21 identical areas. The plasmid containing DSB (DSB-plasmid) was obtained by digestion of pBlueScript plasmid using the restriction enzyme Afllll which creates cohesive ends. One hundred pg of supercoiled pBLueScript plasmid were incubated with 100 units of enzyme in IX NEBuffer 3.1 during 1 hour at 37°C. Then, the DSB-plasmid was precipitated and finally suspended in molecular biological-grade water at the desired concentration.
[0113] By using an ultra-low volume dispensing system, four spots with 0.12 ng of DNA were deposited in each of the 14 areas. The supercoiled plasmid and the linear DSB-plasmid were printed at two positions for each plasmid on a coated slide (Nexterion® H, Schott). The slides were stored under vacuum at -20°C.
[0114] An HybriWell™ Sealing System (Grace Bio-Labs) was applied on the microarray. Each nuclear extract was incubated on the biochip in the presence of mixture containing (i) 2 ng/pl of DSB plasmid labeled with a fluorophore (DSB plasmid*), (ii) one Biotin-labeled dNTP, (iii) all reagents necessary for the DSBs repair reaction (ATP ImM, MgC12 lOmM, KC1 80mM, DTT 2mM, BSA O.lmg/mL, Tris-HCl 20mM pH 7.5, Creatin Phosphokinase 0.05mg/mL, Phosphocreatine lOmM, dNTPs 0.25mM) in a total reaction volume of 12pl per well. Each sample was tested at 0.1 and 0.2 mg/mL final protein concentrations. The repair reaction was carried out during 1 hour at 30°C. The slide was then rinsed twice with Milli-Q water. To detect the dNTP biotin-labeled, an incubation with 0.1 pg/mL Streptavidin-Cy5 was performed for 30 minutes at 30°C. Then, the slide was rinsed twice with Milli-Q water and dried out. Each sample was tested on two pads (areas).
[0115] The labeling of the DSB-plasmid with Cy3 was obtained using the Label IT® Tracker™ Kit (Mims). Briefly, 100pg of DSB-plasmid were incubated with 50pl of Label IT® Reagent and IOOmI of Labeling Buffer 3.1 during 2 hours at 37°C. labeled DSB-plasmid was then diluted in molecular biological-grade water and stored at -80°C.
[0116] Fluorescent signals were quantified using a scanner (Innoscan 710AL from Innopsys, and the Mapix software) at two wavelengths, 532 and 635nm.
[0117] The total fluorescence intensity of each spot was quantified and the mean of the replicates was calculated for each fluorophore and for each plasmid on the slide. Results were expressed as Fluorescence Intensity (Arbitrary Units). Four values were eventually obtained that characterized each sample at each final protein concentration: 2 values characterized the immobilized supercoiled plasmid and 2 values characterized the immobilize DSB plasmid. Test design for the assessment of the four DSBs repair mechanisms
[0118] The enzymes contained in the extracts performed specific repair reactions leading to the incorporation of labeled dNTP and the integration/ligation of labeled DSB plasmid on the immobilized plasmids according to their proficiency to repair DSBs.
[0119] Four different signals were therefore obtained, that characterized each sample, at each protein concentration:
1) the homology between the labelled DSB-plasmid in solution and the immobilized supercoiled plasmid promoted the strand invasion of the supercoiled plasmid by the labelled DSB-plasmid. This signal reflected the HR component of DSB repair;
2) the cohesive ends of labelled DSB-plasmid and immobilized DSB-plasmid were recognized and ligated by enzymes specifically involved in the NHEJ;
3) the incorporation of labelled nucleotides into the supercoiled plasmid was attributed to SSA repair reaction;
4) the incorporation of labelled nucleotides into the immobilized DSB-plasmid was attributed to alt-EJ repair reaction.
[0120] The diagrams in Figure 1, illustrates the DSB repair reaction occurring on the biochip.
EXAMPLE 1: Multiplex DSB repair assay to predict radiation-induced late toxic effects due to defects in double-strand break repair (Next-SPOT assay and kit)
Patients and samples
[0121] 208 prostate or breast cancer patients were enrolled at the Institute de Cancer Lucien Neuwirth (Saint-Priest-en-Jarez, France). All patients received radiotherapy (RT) as a cancer treatment. The National Ethical Committee approved the study and all patients dated and signed written informed consent.
[0122] For each patient, three blood samples were collected in BD vacutainer CPT ™ tubes (reference BD362782; sodium citrate): one before any treatment (J 1), one after the first session of radiotherapy (J2, corresponding generally to a dose of 2 Gy) and one after the fifth session of radiotherapy (J8, corresponding generally to a dose of 10 Gy). Following the manufacturer protocol, the peripheral blood mononuclear cells (PBMCs) were recovered from 8 mL of blood and stored till use at -80°C in fetal bovine serum/DMSO (v/v 90/10%). Nuclear cell extracts were obtained from PBMCs as described in materials and methods.
Data analysis
[0123] The data obtained with the assay were expressed in different ways corresponding to different calculation methods, for each sample and each pathway: 1) raw data, 2) normalized data, 3) relative contribution and 4) Dose-Response.
[0124] The raw data (R) corresponded to the average of the fluorescence intensity of all replicates.
[0125] The normalized data (N) were obtained by considering the homologous recombination signal at the lowest protein concentration (0.1 mg/mL) as the reference. All values, for each pathway, were divided by the reference following the formula: R_HR (or R_HNEJ or R_SSA or R_alt-EJ)/R_HR_0.1.
[0126] This formula was applied also to data obtained with the highest protein concentration (0.2 mg/mL) by using the same reference (R_HR_0.1). [0127] The relative contribution (C) represented the contribution of each repair pathway to total repair and was expressed as a percentage of the total fluorescence for each pathway at a given protein concentration. The relative contribution was calculated as follow: 100 x R_HR (or R_HNEJ or R_SSA or R_alt-EJ)/ R_HR+ R_NHEJ+ R_SSA+ R_alt-EJ.
[0128] The Dose-Response (DR) was calculated by dividing the values obtained at the highest protein concentration by the values obtained at the lowest protein concentration for each repair mechanism. The dose-response values were calculated using the formula: R_HR_0.2/ R_HR_0.1 for each pathway.
[0129] Indeed, other calculations can be considered such as slope of the curves for each repair pathway, each repair pathway kinetic if more than 2 concentrations are being tested, etc. Selection of adverse events in breast and prostate cancer patients
[0130] All radiation-induced adverse events up to at least 6 months after the treatment were recorded and graded according to the Common Terminology Criteria for Adverse Events (CTCAE) scale published by the National Cancer Institute of the United States of America. The CTCAE provides 5 severity grades (from 1 to 5) with unique clinical severity descriptions for each adverse event and a specific tissue reaction for each grade.
[0131] Only cutaneous toxicities were considered for breast cancer patients and toxicities concerning digestive and urogenital systems were considered for prostate cancer patients. Toxicities were classified, as a function of the appearance date, in early (appeared during RT) and late (appeared after 6 months from the beginning of RT).
[0132] Several analyses were performed by dividing the same pool of patients in two groups according to the presence or not of adverse events. Differences in the DSB repair capacities evaluated using any of the calculation described above, between patients that show (i) early grade >2 events, (ii) early grade >3 events, (iii) late grade >2 events, (iv) late grade >3 events and patients who did not show these events, were evaluated. Differences between groups were evaluated using the Mann-Whitney U test; p-value inferior to 0.05 were considered significant.
[0133] Results issued from samples collected at different treatment time points (J 1 , J2, J8) were represented as box-plots, by day.
[0134] In addition, based on the same data set, a Random Forest algorithm was run to identify the best biomarkers combination, for samples tested prior RT (J 1), able to predict the risk of developing late grade >3 adverse events. It was completed by the Boruta algorithm (with 100.000 trees and a maximum of 1.000 iterations). The performances of the algorithm were visualized in confusion matrix that allow to calculate the accuracy of the test by giving both false and true positives and false and true negatives rates.
RESULTS
1. Characterization of DSB repair profiles in breast and prostate cancer patients using the DSB repair profiling NEXT-SPOT test [0135] To facilitate the understanding of results a series of contractions were used. Each pathway is preceded by “R_” or “N_” or “C_” or “DR_” which indicate the calculation method applied. In addition, each pathway is followed by “_1” or ”_2” indicating the final protein concentration, 0.1 and 0.2 mg/mL, respectively; then, the suffixes “_J1” or “_J2” or “_J8” indicate the blood sampling day. [0136] An example of the patients’ DSB repair activities measured with the assay described in the invention and expressed as relative contribution, are presented in Figure 2. It concerns the blood samples taken prior RT and characterized for their DSB repair capacities at 0.1 mg/mL final protein concentrations.
2. Use of the DSB repair profiling test in breast and prostate cancer patients, to distinguish patients who developed early grade >2 adverse events from those who did not (grade <2)
[0137] Patients were clustered in two groups according to the presence or not of grade >2 adverse effects appeared during the radiotherapy (early adverse events). The DSB repair activities of patients who developed early grade >2 adverse events and those who did not (grade <2) were compared. Significant results were displayed as Box-Plots in Figure 3 (p value <0.005).
[0138] The HR relative contribution at 0.2 mg/mL final protein concentration was lower in patients suffering from early grade >2 adverse events compared to patients who had early grade <2 adverse effects. Conversely, the NHEJ relative contribution, as the normalized NHEJ, were higher in patients with early grade >2 adverse effects than in the other patients (grade <2). The differences were detected before the radiotherapy (using the J1 samples) indicating a possible dysfunction of the balance between these mechanisms for these patients.
[0139] Hence, it was demonstrated that the functional quantification of the DSB repair pathways brings valuable information regarding radiotoxicity risk among patients suffering from breast or prostate cancer that will be treated by radiotherapy.
3. Use of the DSB repair profiling test to distinguish breast and prostate cancer patients who developed early grade >3 adverse events from those who did not (grade <3)
[0140] Patients were clustered in two groups according to the presence or not of grade >3 adverse effects appeared during the radiotherapy (early adverse events). DSB repair activities were compared between patients who developed grade >3 adverse events during RT and patients who did not (grade <3). Significant results were represented as Box-plots (Figure 4).
[0141] HR and NHEJ repair pathways characterized using the invention, were significantly different after the fifth sessions of treatment (J8), for patients who developed early grade >3 events compared to patients who did not (grade <3). After the fifth session of RT (J8), the HR normalized data, the HR dose-response and the NHEJ normalized data were lower in patients presenting early grade >3 events compared to patients who did not show these events.
[0142] Patients susceptible to be affected by early grade >2 and grade >3 adverse effects induced by radiotherapy can be distinguished from the other ones, by the analysis of their DSB repair pathway profile before RT (Jl) or after the fifth session (J8) of radiotherapy, respectively. These patients showed a down-regulation of HR pathway before and after five sessions of RT; the NHEJ pathway was up-regulated before RT and down-regulated at J8 (after the fifth session of RT) compared to patients that did not developed adverse events.
[0143] Thus, the test identifies differences in the DSB repair pathways of patients associated with an increased risk of early radiotoxicity after radiotherapy.
4. Use of the DSB profiling test in breast and prostate cancer patients to distinguish patients who developed late grade >2 adverse events from those who did not (grade <2)
[0144] Late adverse events (>6 months after the beginning of RT) of grade >2 were recorded in breast and prostate cancer patients. Patients were divided in two groups according to the presence or not of late grade >2 adverse events. Significant results are showed as Box-plots
(Figure 5).
[0145] Before (Jl) and after (J2 and J8) RT, patients who developed late grade >2 adverse events showed a DSB repair profile different from patients who did not (grade <2). Before RT (Jl), the relative contribution and the raw data for NHEJ mechanism were increased in patients who developed late grade >2 events compare to the others (grade <2). By opposition, the relative contribution of SSA pathway was decreased in these patients. After the first session of RT (J2), the alternative repair mechanisms (SSA and alt-EJ raw data) were significantly up-regulated in patients who showed late grade >2 adverse events compared to patients who did not (grade <2). After the fifth session of RT (J8), the patients who developed late events of grade >2 showed a down-regulation of NHEJ normalized data compared to the other ones (grade <2). 5. Use of the DSB profiling test identifies defects in DSB repair in breast and prostate cancer patients, leading to the development of late grade >3 adverse events from those who did not (grade <3)
[0146] Patients were divided in two groups according to the presence or not of grade >3 adverse events developed >6 months after the beginning of RT (late adverse events). Significant differences between the DSB activities in patients who developed late severe effects (grade >3) and the others (grade <3), were determined. Significant results are represented as Box-plots (Figure 6).
[0147] Before RT (Jl) and after the first session of RT (J2), the main DSB repair mechanisms, in particular the HR and NHEJ (expressed as raw data), were upregulated in patients who developed late grade >3 events compared to the other patients (grade <3). On the contrary, the alternative mechanisms, in particular expressed as normalized data and relative contributions, SSA and alt-EJ, were down-regulated in these patients. After the fifth session of RT (J8), the HR normalized data and dose-response for HR and NHEJ pathways, by opposition to what was found at Jl and J2, were down-regulated in patients with late grade >3 adverse effects compared to the other ones (grade <3).
[0148] Significant differences between patients presenting early radiation-induced grade >2 and grade >3 adverse events and the other patients were identified exclusively in HR and NHEJ pathways. Patients who developed late radiation-induced grade>2 and grade >3 adverse events show a deregulation of all DSB pathways.
[0149] Hence, the assay that enabled to detect simultaneously defects in all DSB repair pathways identified patients with an increased late radiotoxicity risk following radiotherapy.
6. Use of the biomarkers determined by NEXT-SPOT test to predict late grade >3 radiation-induced toxicity in breast and prostate cancer patients
[0150] To determine, the best biomarker combination able to predict individual susceptibility to suffer from late severe (grade >3) radiotoxicities the data set obtained before the beginning of treatment (Jl) was used to run the Random Forest algorithm to construct a predictor that distinguishes patients who will develop late grade >3 adverse events from those who will not (grade <3). Then, the Boruta algorithm was run to select the most significant variables. Eight variables were selected by Boruta: N_NHEJ_1, N_SSA_1, N_altEJ_l, N_SSA_2, N_altEJ_2, C_HR_1 , C_SS A_1 and C_altEJ_l . As shown, the best combination that predicted severe late adverse events involved calculations performed with N and C data obtained at a protein concentration of 0.1 or 0.2 mg/mL.
[0151] These selected variables were able to predict the appearance of late grade >3 adverse events with a sensibility of 100% and a specificity of 77.17%. The calculated negative predictive value was 100%; the positive predictive value was 13.6%.
EXAMPLE 2: Characterization of DSB repair activities in cancer cell lines. Analysis of impact of DNA damaging agents, DNA repair inhibitors and their combination on DSB repair activities. Detection of DSB repair pathway defects and deregulations Cell lines
[0152] Impact of DNA damaging agents and DNA repair inhibitors was determined in three cancer cell lines: HeLa, M059J and M059K. As opposed to the M059K cell line, M059J cells have a frameshift mutation in the PRKDC gene coding for the catalytic subunit of DNA-PK, resulting in an inactive truncated protein (Anderson C.W. et al., Radiat. Res., 2001, 156(1):2- 9).
Cell treatments
[0153] DNA repair inhibitors and DNA damaging agents were solubilized in DMSO (Table
1).
Figure imgf000031_0001
Table 1: DNA repair inhibitors and DNA damaging agents [0154] Cells were treated with the different chemicals for 48h: Doxorubicin was used at IC10 and DNA repair inhibitors were used at IC50 as determined using MTT assay. Compounds were used, either alone or in combination. After the treatment, the cells were frozen and stored at -80°C. Nuclear cell extracts were obtained as previously described. 1. Specific DSB repair profiles distinguished the different cell lines at basal level (absence of treatment)
[0155] The DSB repair signature was established for 3 cell lines before any treatment at, 0.1 and 0.2 mg/mL final protein concentration. The relative contribution of each pathway to the total DSB repair capacity, was calculated as a percentage for each pathway, at the two final protein concentrations, for each cell line (Figure 7, A and B).
[0156] M059J cell line is described as being defective for NHEJ. With, this assay it is clearly shown that M059J cell line differs from M059K regarding NHEJ pathway regulation and that a defective gene in the DSB repair pathway does not mean that the repair reaction cannot occur. All data regarding the different DSB repair pathways, obtained and considered together, revealed that mutation in the PRKDC gene affected only NHEJ pathway, which can only be determined using the invention. Using the NEXT-SPOT assay, it is shown that despite this truncated protein, these cells are able to perform the end ligation reaction.
[0157] In parallel, by comparison, it was shown that HeLa cells exhibited marked different functional DSB repair pathways, which is neither predictable using all other methods that investigate DSB repair, not predictable through genes analysis.
[0158] The analysis of the relative contribution for each DSB repair pathway to total DSB repair, obtained at 0.1 and 0.2 mg/mL, revealed other features related to the repair reaction kinetics of each cell line. The profiles differed between 0.1 and 0.2 mg/mL.
[0159] For example, for M059K, as determined at 0.1 mg/mL, the relative contribution of the alternative DSB repair pathways, SSA and alt-EJ, represents almost 50% of the total DSB repair reaction compared to the other cell lines, M059J and Hela, which show 26% and 10% relative contribution of the alternative DSB repair pathways, respectively. Therefore these latter DSB repair pathways could determine strongly the overall ability of these cells to repair DSB compared to the other cell lines.
[0160] Interestingly, the difference between the profiles obtained at 0.1 and 0.2 mg/mL informed about the relative abundance of the different proteins involved in each DSB repair pathway. For example, for M059K, this relative abundance differed between the two protein concentrations, leading to different profiles at the two protein concentrations tested: HR and NHEJ capacity was more prevalent at 0.2 mg/mL than at 0.1 mg/mL. [0161] Only the simultaneous determination of the repair capacity of at least two different pathways with an in vitro approach such as provided by the NEXT-SPOT assay, at two protein concentrations allows such a determination.
2. Specific impact of different treatments on the DSB repair profiles in different cell lines
[0162] NEXT-SPOT assay allows one to understand what are the possible consequences of a treatment on DSB repair pathways in terms of induction or inhibition, and enables one to identify strategies to modulate them.
[0163] For example, using the invention, one can determine if a certain pathway is induced by a DNA damaging agent (or more generally a genotoxic), or a DDR modulator, and identify an inhibitor specifically targeting the induced repair pathway, as a sensitization strategy.
[0164] Cells were treated by doxorubicin and the DSB repair profile of each cell line was determined. The cells were also treated by several DSB repair inhibitors (B02, NU7026 and Olaparib), alone or in combination with doxorubicin (Dox+B02, Dox+NU7029 and Dox+Olap).
[0165] Each treatment induces a different modulation of the four DSB repair pathways in the cell lines tested. To visualize the impact of each treatment, the ratios of the fluorescence intensity obtained for treated cells (T) to non-treated cells (NT) were calculated at the 2 protein concentrations.
• If ratio <1, that means that the treatment inhibited the considered repair activity
• If ratio >1, that means that the treatment induced the considered repair activity
[0166] Functional impact of the DNA damaging agent (doxorubicin), of the DNA Repair inhibitors (B02, NU7026, Olaparib) and of their combinations, on the four DSB repair pathways, were determined at 0.1 mg/mL (Figures 8, A, B and C) and at 0.2 mg/mL (Figures 9, A, B and C) final protein concentration.
[0167] Important information is gained from these calculations at 0.1 mg/mL and 0.2 mg/mL.
[0168] At 0.1 mg/mL significant differences are shown between the M059K and M059J cell lines in DSB repair activities modulation. Both cell lines were sensitives to B02 inhibitor showing an inhibition of HR and NHEJ pathways. When cells were treated with the inhibitor NU7026, M059J cells did not show any change in the regulation of DSB repair pathways, whereas in M059K cells the HR and NHEJ mechanisms were hugely increased, probably to compensate the inhibitor effect. Hence, not only the impact of the treatment NU7026 is not specific for one pathway but seems to stimulate the HR and NHEJ in non-defective cells for these mechanisms.
[0169] In M059J cells, at 0.2 mg/mL the presence of DNA-PK inhibitor (NU7026), alone or in combination with doxorubicin, is able to induce HR. NHEJ despite supposed to be defective, is also slightly induced. This paradoxical activation of DSB repair, if occurring in tumors, could be responsible for failure to DNA repair inhibitor treatments. Here HR induction could be a consequence or a compensation for NHEJ deficiencies. This gives an important complementary information on the regulation of the major DSB repair pathway for this cell line. This information is critical to develop effective DNA repair inhibitors as compensatory mechanisms are known to be responsible for failure to treatments.
[0170] The repair reactions run with the 2 protein concentrations give complementary results, and inform on the balance between repair activities contained in the same extract. A repair protein might be present in the extract when used at 0.2 mg/mL and absent in the extract at 0.1 mg/mL because it exists in the cell in a limited concentration.
[0171] The main DSB repair pathways of M059J cell line were inhibited by the 2 other inhibitors, alone (Olaparib) or in combination with doxorubicin (B02 and Olaparib).
[0172] For M059K, despite an activation of HR and NHEJ detected at 0.1 mg/mL in response to NU7026 alone, one can see at 0.2 mg/mL that all DNA repair inhibitors affect both major DSB repair pathways, i.e. HR and NHEJ, whereas the 2 alternative pathways are not affected by the treatments. It is worth to note that when the doxorubicin treatment was applied, NU7026 led to an effective inhibition of both HR and NHEJ pathways.
[0173] Most of the time the inhibitors have a synergic inhibitory effect with the DNA damaging agent as seen at 0.2 mg/mL. That can be an important information gained form this finding as sometimes the DNA damage response must be activated in cells by a DNA damaging agent, to be effectively inhibited by the DNA repair inhibitors.
[0174] HeLa cells DSB repair activities are slightly modulated when the repair reaction is conducted using 0.1 mg/mL protein concentration, except with Olaparib, alone or in combination with doxorubicin. [0175]When the repair reaction is conducted using 0.2 mg/mL protein in repair reaction, one can note that B02, alone and in combination with doxorubicin, effectively inhibits HR; NU7026 slightly inhibits NHEJ and HR only when combined with doxorubicin; the third inhibitor, Olaparib, affects considerably HR and NHEJ and slightly SSA and alt-EJ pathways, only when combined with doxorubicin.
[0176]Here again, inhibition of NHEJ by NU7026 leads to a marked stimulation of HR, and to a slight induction of all other DSB repair pathways. In a tumor that paradoxical induction could be associated with a failure when a treatment by a DNA repair inhibitor alone is applied.
[0177] The alternative DSB repair pathways might be affected too, but to a lesser extent. However, that can be an important information, as alternative DSB repair pathways can be at the origin to resistance to treatment.
[0178] All the information detailed here cannot be obtained by measuring only one DSB repair pathway.
[0179] Moreover, they cannot be deduced from genomic analysis that inform about possible defects in certain genes but cannot account for the complexity of the DSB regulation from a functional point of view.
EXAMPLE 3: Functional DSB repair are associated with defects in gene involved in DSB repair
Materials and Methods
[0180] DSB repair ability was quantified in two cancer cell lines, Control Hela SilenciX®, and BRCA2 HeLa SilenciX® silenced for BRCA2 (tebu-bio, France). Cells were grown following the supplier instructions with DMEM+GlutaMAX™ containing 4.5 g/L D-Glucose supplemented with 125pg/mL hygromycin B, 1% penicillin- streptomycin solution and 10% fetal bovine serum (Gibco, USA) at 37°C under 5% CO2.
Results
[0181] The signature for DSB repair was established with the NEXT-SPOT assay for these two cell lines at basal level, at a final protein concentration of 0.2 mg/mL. Results were expressed as fluorescence intensity ratio compared to WT cell line, for all repair pathways (HR, NHEJ, SSA, alt-EJ). See Figure 10. [0182] All repair pathways were affected in the BRCA2-silenced cell line; HR was the repair pathway most affected by BRCA2 gene silencing.

Claims

1. An in vitro method of determining the activities of at least two DNA double-strand break (DSB) repair pathways in a biological medium, comprising: a) Performing at least two DSB repair reactions, preferably simultaneously in a single reaction mixture: ai) a first repair reaction comprising a supercoiled circular double-strand DNA molecule and a homologous linear double-stranded DNA molecule that are repaired by Homologous Recombination (HR) or Single-Strand Annealing (SSA) to form a repaired double-stranded DNA molecule; and a2) a second repair reaction comprising two linear double- stranded DNA molecules that may or may not be homologous and are repaired by Non- Homologous End-Joining (NHEJ) or alternative-End Joining (alt-EJ) to form a repaired double- stranded DNA molecule; wherein said at least two repair reactions comprise DNA repair enzymes from said biological medium, and further comprise at least one label that is incorporated into the repaired double-stranded DNA molecule, and wherein the at least one label is on the linear double-stranded DNA molecule of the first repair reaction or one of said molecules of the second repair reaction, on a deoxynucleotide or on both; b) Measuring the level of label incorporated into the repaired double-stranded DNA molecule in said at least first and second DSB repair reactions; and c) Determining the activities of at least two DSB repair pathways, a first pathway chosen from: Homologous Recombination (HR) and Single-Strand Annealing (SSA); and a second pathway chosen from Non-Homologous End-Joining (NHEJ) and Alternative-End Joining (alt-EJ) in said biological medium, wherein:
The level of HR is determined by the level of labeled linear double-stranded DNA molecule incorporated into the repaired double- stranded DNA molecule from the first DSB repair reaction;
The level of SSA is determined by the level of labeled nucleotide incorporated into the repaired double- stranded DNA molecule from the first DSB repair reaction; The level of NHEJ is determined by the level of labeled linear double- stranded DNA molecule incorporated into the repaired double- stranded DNA molecule from the second DSB repair reaction; and
The level of alt-EJ is determined by the level of labeled nucleotide incorporated into the repaired double- stranded DNA molecule from the second DSB repair reaction.
2. The method according to claim 1, wherein the supercoiled circular double-strand DNA molecule is a supercoiled plasmid and/or the linear double- stranded DNA molecule(s) are linearized plasmid(s), preferably from the same plasmid.
3. The method according to claim 1 or 2, wherein the supercoiled circular double-strand DNA molecule from the first DSB repair reaction and one of the linear double-stranded DNA molecules that is not labelled from the second DSB repair reaction are immobilized on a solid support.
4. The method according to any one of claims 1 to 3, wherein the label is fluorescent and may be detected directly or indirectly; preferably a label chosen from Cy3 and Cy5; preferably wherein the method comprises two different labels.
5. The method according to any one of claims 1 to 4, wherein the biological medium comprises cells or a mixture containing repair enzymes; preferably the biological sample is obtained from a patient; more preferably the biological sample is selected from the group consisting of: plasma, whole-blood or PBMC fraction thereof; and tissue or tumor biopsy.
6. The method according to any one of claims 1 to 5, wherein the DBS reaction is performed on a cell extract, preferably a nuclear extract; more preferably comprising a final protein concentration from 0.05 to 2 mg/mL; more preferably of 0.1 mg/mL or 0.2 mg/mL.
7. The method according to any one of claims 1 to 6, wherein the DSB repair reactions are performed preferably at 30°C; preferably for one hour.
8. The method according to any one of claims 1 to 7, which comprises determining the activities of at least three, preferably four DSB repair pathways chosen from: Homologous Recombination (HR), Single-Strand Annealing (SSA), Non-Homologous End-Joining (NHEJ) and Alternative-End Joining (alt-EJ).
9. A method of detecting a dysregulation of the DNA repair mechanisms in cells comprising: a) determining the activities of at least two DNA double-strand break (DSB) repair pathways in a cell sample according to the method of any one of steps 1 to 8; b) detecting the presence of an altered activity of said DSB repair pathway(s).
10. The method according to claim 9, wherein said altered activity is a defect of said DSB repair pathway(s). Preferably, wherein the detection of said defect is used to predict the risk of occurrence of cancer or immunological disease or the risk of toxicity following exposure to DNA damaging or DNA interacting agents in an individual, in particular the risk of late toxicity before the beginning of radiotherapy.
11. The method according to claim 9 or 10, wherein said cells are from a treated patient and said altered activity is used to predict the response to therapy, in particular cancer therapy in said patient.
12. A method of screening DSB repair modulators, comprising: a) contacting at least one candidate compound with a biological sample comprising DNA repair enzymes; b) determining the activities of at least two DNA double-strand break (DSB) repair pathways in said biological sample in the presence and absence of said at least one candidate compound, according to the method of any one of steps 1 to 9; c) identifying the compounds which modulate the activity of at least one of said DSB repair pathways.
13. A Kit for determining the activities of at least two DNA double-strand break (DSB) repair pathways in a biological sample, comprising at least: a) a supercoiled circular double-strand DNA molecule, preferably a supercoiled plasmid; more preferably immobilized on a solid support; and b) at least one linear double- stranded DNA molecule; preferably linearized plasmid(s) with cohesive ends; more preferably from the same plasmid as in a); even more preferably immobilized on a solid support.
14. The kit according to claim 13, further comprising at least one label, preferably fluorescent; more preferably a dNTP and a linear double-stranded DNA molecule with different labels.
15. The kit according to claim 13 or 14, comprising a solid support comprising at least one pad, comprising at least one spot of the supercoiled circular double-strand DNA molecule and at least one spot of the linear double-stranded DNA molecule; preferably further comprising a reaction mixture comprising a dNTP and a linear double-stranded DNA molecule with different labels ; more preferably further comprising a buffer appropriate for DNA repair enzymes.
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