HK1261357A1 - Homologous recombination factors - Google Patents

Description

Homologous recombination factor

Technical Field

The present invention relates to factors that affect or modulate homologous recombination, methods of monitoring these factors, uses of these factors to screen for agents that modulate homologous recombination, and methods of modulating homologous recombination.

Background

Breast and ovarian tumor suppressor factors BRCA1, PALB2, and BRCA2 promote DNA Double Strand Break (DSB) repair by Homologous Recombination (HR) [8-10 ]. BRCA1 functions in at least two discrete steps in the process. First, it facilitates DNA end excision [11,12], an initial step in HR involving nucleolytic processing of breaks to produce single-stranded (ss) DNA necessary for homology searching and strand invasion [1 ]. Second, BRCA1 interacts with PALB2[ 13-15] to direct recruitment of BRCA2[13] and RAD51[16,17] to the DSB site. Accumulation of BRCA1 flanking the DSB site on chromatin was significantly inhibited in G1 cells [18], reminiscent of effective inhibition of homologous recombination during this phase of the cell cycle. Inhibition of BRCA1 recruitment in G1 was dependent on 53BP1 and RIF1 proteins [18,19], which are two inhibitors of end excision [18-22 ]. BRCA1 is also implicated in promoting recruitment of BRCA2 by interacting with PALB2[ 13-15 ].

Tumors with impaired ability to repair double-stranded DNA breaks by HR, including tumors deficient in BRCA1 and BRCA2, have been shown to be highly sensitive to poly ADP-ribose polymerase (PARP) inhibitors. PARP inhibitors have also been proposed for the treatment of other conditions such as stroke, myocardial infarction, inflammatory bowel disease, head trauma and neurodegenerative diseases. Inhibition of ubiquitin-specific peptidase 11(USP11) has been shown to supersensitise cells to PARP inhibitors, and it has been suggested that USP11 status or other HR-protein status in tumors may provide biomarkers for the use of PARP inhibitors (Wiltshire et al, JBC285(19), 14565-.

It is desirable to identify and evaluate factors that affect or modulate homologous recombination repair proteins and to identify events necessary and sufficient for HR inhibition in G1 cells. Furthermore, identification and assessment of factors that affect or modulate USP11 may aid in the selection and detection of PARP inhibitor treatments, particularly in the selection of treatments that reverse or delay the emergence of PARP inhibitor resistance.

Summary of The Invention

The inventors have found that the cell cycle tightly controls the interaction of BRCA1 with PALB2-BRCA2 in order to restrict BRCA2 function to the S/G2 phase. The BRCA1 interaction site on PALB2 is targeted by an E3 ubiquitin ligase consisting of KEAP1(PALB2 interacting protein [6]) complexed with cullin 3(CUL3) -RBX 1[ 7 ]. PALB2 ubiquitination inhibits its interaction with BRCA1 and is counteracted by deubiquitinase USP11, which is itself under cell cycle control. The combination of restoration of BRCA1-PALB2 interaction and activation of DNA end excision is sufficient to induce HR in G1 phase cells as determined by RAD51 recruitment, extra-phase DNA synthesis, and CRISPR/Cas 9-based gene targeting. The mechanism of blocking HR in G1 consists of at least inhibition of DNA end excision linked to multi-step blockade of recruitment of BRCA2 to the site of DNA damage, which involves inhibition of BRCA1-PALB2-BRCA2 complex assembly. The ability to induce HR in G1 cells using defined factors can be used for gene targeting applications in non-dividing cells or in cells that are dormant in G1 phase. The discovery also provides a basis for targeting USP11 in combination with poly (ADP-ribose) polymerase (PARP) inhibitors.

The inventors also found that USP11 is regulated by cell cycle-CULLIN 4-RING-ligase (CRL4), and that DCAF10 serves as an adaptor for USP 11E 3 ligase.

The present invention provides methods for monitoring USP11 activity in a sample by determining the interaction of BRCA1 and PALB 2.

The present invention provides methods for monitoring USP11 activity in a sample by determining the interaction of BRCA1, PALB2 and BRCA 2.

The present invention provides methods for monitoring USP11 activity in a sample by determining the interaction of USP11 and PALB 2.

The present invention provides methods for monitoring USP11 activity in a sample by assaying DCAF 10.

The present invention provides methods for monitoring USP11 activity or expression in a sample by determining the following complex: (a) BRCA1 and PALB 2; (b) BRCA1, PALB2, and BRCA 2; (c) USP11 and PALB 2; and/or (d) USP11 and DCAF 10.

In one aspect, the present invention provides a method for monitoring the activity or expression of USP11 in a sample, comprising (i) isolating in said sample a complex of: (a) BRCA1 and PALB 2; (b) BRCA1, PALB2, and BRCA 2; (c) USP11 and PALB 2; and/or (d) USP11 and DCAF 10; (ii) measuring the level of the complex; and (iii) detecting an increase or decrease in activity or expression of the complex compared to a control as an indication of activity or expression of USP 11.

In one aspect, the invention provides a method for monitoring activity or expression of USP11 in a sample, comprising (i) isolating in said sample by immunopurification a complex of: (a) BRCA1 and PALB 2; (b) BRCA1, PALB2, and BRCA 2; (c) USP11 and PALB 2; and/or (d) USP11 and DCAF 10; (ii) measuring the level of the complex; and (iii) detecting an increase or decrease in activity or expression of the complex compared to a control as an indication of activity or expression of USP 11.

In one aspect, the present invention provides a method for monitoring the activity or expression of USP11 in a sample, comprising (i) isolating in said sample a complex of: (a) BRCA1 and PALB 2; (b) BRCA1, PALB2, and BRCA 2; (c) USP11 and PALB 2; and/or (d) USP11 and DCAF 10; (ii) preparing a peptide or peptide fragment from the isolated complex; and (iii) subjecting the peptide or peptide fragment to mass spectrometry, thereby monitoring the activity or expression of USP 11.

The present invention provides methods for monitoring activity or expression of USP11 in a sample by determining ubiquitination of PALB2, particularly the N-terminus of PALB 2.

In one aspect, the invention provides a method of monitoring activity or expression of USP11 in a sample by determining ubiquitination of PALB2, comprising measuring the amount of polyubiquitin bound to CRL3-KEAP 1E 3 ligase in the sample and detecting an increase or decrease in polyubiquitin bound to CRL3-KEAP 1E 3 ligase compared to a control as an indication of activity or expression of USP 11.

In another aspect, the present invention provides a method of monitoring activity or expression of USP11 in a sample by determining ubiquitination of PALB2, comprising measuring the activity of CRL3-KEAP 1E 3 ligase and detecting an increase or decrease in CRL3-KEAP 1E 3 ligase activity as an indication of activity or expression of USP11 compared to a control.

The methods of the invention may be performed in the presence or absence of a test compound or agent, and detection of an increase or decrease in activity or expression of one or more of USP11, DCAF10, PALB2, PALB2 ubiquitination, BRCA1-PALB2-BRCA2 complex, PALB2-USP11 complex, KEAP1, USP11-DCAF complex, CRL3-KEAP1 complex, CRL3-KEAP1-PALB2 complex, and KEAP1-PALB2 complex as compared to a control in the absence of a test compound or agent indicates that the test compound or agent may be used as a therapeutic, or to modulate homologous recombination.

In one aspect, the present invention provides a method of identifying or evaluating the ability of an agent to sensitize or reverse resistance or delay the onset of resistance to a PARP inhibitor by determining the effect of the agent on USP11 activity or expression using the method of the present invention.

In one aspect, the invention relates to a method of identifying or evaluating the ability of an agent to sensitize a cell to a PARP inhibitor or to reverse resistance to or delay the onset of resistance to a PARP inhibitor by determining the effect of the agent on KEAP1, CRL3-KEAP1, KEAP1-PALB2 or CRL3-KEAP 1.

In one aspect, the invention provides a method of detecting an anti-cancer agent comprising performing a test assay comprising contacting an immortalized cell with a test compound and determining USP11 activity or expression using the method of the invention.

The invention also provides a method for identifying or assessing the ability of an agent to modulate homologous recombination, comprising determining the effect of a test compound or agent on one or more of: USP11, DCAF10, PALB2, PALB2 ubiquitination, BRCA1-PALB2-BRCA2 complex, PALB2-USP11 complex, KEAP1, USP11-DCAF10 complex, CRL3-KEAP1 complex and CRL3-KEAP1-PALB2 complex.

The invention provides a method of screening for a therapeutic agent for treating a disease associated with HR deficiency (i.e. HR disease) comprising identifying an agent that disrupts or modulates one or more of: USP11, PALB2, PALB2 ubiquitination, DCAF10, BRCA1-PALB2-BRCA2 complex, PALB2-USP11 complex, KEAP1, USP11-DCAF10 complex, CRL3-KEAP1 or CRL3-KEAP1-PALB2 complex.

The screening method of the present invention may further comprise: characterizing the therapeutic profile of efficacy and toxicity of the identified agent or additional analog thereof in the animal; optionally formulating a pharmaceutical composition comprising one or more agents identified as having an acceptable therapeutic property; and optionally administering the agent to the subject or individual.

The invention provides a method of treating HR disease in an individual comprising identifying an agent that modulates HR according to the method of the invention and administering the agent to the individual.

In some embodiments, the present invention provides a method for sensitizing cells to a PARP inhibitor in an individual comprising identifying an agent sensitizing cells to a PARP inhibitor according to the methods of the present invention and administering the agent to the individual.

In some embodiments, the present invention provides a method for reversing or delaying the onset of resistance to a PARP inhibitor in an individual comprising identifying an agent that reverses resistance to or delays the onset of resistance to a PARP inhibitor according to the methods of the present invention and administering the agent to the individual.

In some embodiments, the present invention provides a method of treating cancer in an individual comprising identifying an anti-cancer agent identified according to the method of the present invention, and administering the agent to the individual.

The present invention also provides a method for predicting or classifying a response to a PARP inhibitor in a subject comprising determining in a sample from said subject one or more of: USP11, DCAF10, BRCA1, BRCA2, PALB2, KEAP1, CRL3, CRL3-KEAP1, USP11-DCAF10 complex, BRCA1-PALB2-BRCA2 complex, PALB2-USP11 complex and CRL3-KEAP1-PALB2 complex. In one aspect, there is provided a method for predicting or classifying a response to a PARP inhibitor in a subject comprising determining USP11 activity or expression in a sample from said subject using the method of the invention. In one aspect, there is provided a method for predicting or classifying a response to a PARP inhibitor in a subject comprising determining PALB2 activity or expression in a sample from the subject using the methods of the invention.

In one aspect, a subject is classified as having a response to a PARP inhibitor if one or more of the following is decreased as compared to a control: USP11, DCAF10, BRCA1, BRCA2, PALB2, KEAP1, CRL3, USP11-DCAF10, CRL3-KEAP1, BRCA1-PALB2 and BRCA1-PALB2-BRCA2 activity or expression or PALB2 ubiquitination. In one aspect, a subject is classified as having a response to a PARP inhibitor if one or more of the following is increased as compared to a control: USP11, DCAF10, BRCA1, BRCA2, PALB2, KEAP1, CRL3, USP11-DCAF10, CRL3-KEAP1, BRCA1-PALB2 and BRCA1-PALB2-BRCA2 activity or expression or PALB2 ubiquitination.

The method of predicting responsiveness to a PARP inhibitor may further comprise administering the PARP inhibitor to the individual.

The present invention provides a method for treating a patient in need of treatment with a PARP inhibitor, comprising (a) requesting a test that provides an assay result to determine whether the patient is sensitive to or has a response to the PARP inhibitor: determining whether the patient is sensitive or responsive to a PARP inhibitor by detecting one or more of USP11, DCAF10, BRCA1, BRCA2, PALB2, KEAP1, USP11-DCAF10, CRL3, CRL3-KEAP1, BRCA1-PALB2, and BRCA1-PALB2-BRCA2 in a sample from the subject and comparing to a control; and (b) administering a PARP inhibitor to the patient if the patient is sensitive to or responsive to the PARP inhibitor. In one aspect of the method of the invention, the patient has breast cancer. In one aspect of this method of the invention, the patient has ovarian cancer.

In one aspect, the present invention provides a method for treating a patient in need of treatment with a PARP inhibitor, comprising (a) requesting a test to provide an assay result to determine whether the patient is susceptible to the PARP inhibitor: determining whether the patient is sensitive to a PARP inhibitor by detecting USP11, DCAF10, BRCA1, BRCA2, PALB2, KEAP1, and/or CRL3 in a sample from the subject and comparing to a control; and (b) administering a PARP inhibitor to the patient if the patient is sensitive to the PARP inhibitor. In one aspect of the method of the invention, the patient has breast cancer. In one aspect of this method of the invention, the patient has ovarian cancer.

The present invention also provides a method for assigning an individual to one of a plurality of categories in a clinical trial of a PARP inhibitor, comprising determining in a sample from a subject using the method of the invention: USP11, DCAF10, PALB2, PALB2 ubiquitination, BRCA1-PALB2-BRCA2 complex, PALB2-USP11 complex, USP11-DCAF complex, KEAP1, CRL3-KEAP1 and/or CRL3-KEAP1-PALB2 complex.

The present invention also provides pharmacogenetic methods for determining an appropriate treatment regimen for a disease, particularly cancer, based on the selection of patients for PARP responsiveness, particularly USP11 activity, and methods of treating patients.

The methods of the invention, particularly the methods for determining USP11 activity or CRL3-KEAP1 activity, are useful as readouts in novel animal model-based therapeutic methods and screening methods for compounds. In one aspect, the methods of the invention are used to predict the efficacy of potential new therapies for disease states in animal models.

The present invention provides methods for activating or modulating (e.g., promoting) homologous recombination in a cell, comprising:

(a) promoting or stimulating the assembly or occurrence of BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex in a cell;

(b) activating or stimulating recruitment of BRCA 1to the site of DNA Double Strand Break (DSB);

(c) contacting the cell with BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex;

(d) inhibiting or removing KEAP1 or CRL3-KEAP 1;

(e) inhibit degradation of USP11 or promote USP11 activity; and/or

(f) Inhibiting or removing DCAF 10.

The present invention provides a method for activating or modulating homologous recombination in a cell, in particular in a cell of the G1 phase of the cell cycle (G1) or the G0 phase of the cell cycle, comprising administering a BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex in the cell, or stimulating the assembly of a BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex.

The invention also provides a method for activating or modulating homologous recombination in a cell, in particular in a cell in the G1 phase of the cell cycle (G1) or in the G0 phase of the cell cycle (G0), comprising promoting or stimulating the assembly or occurrence of a BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex in the cell.

The invention also provides a method for activating or modulating homologous recombination in a cell, in particular in a cell in the G1 phase of the cell cycle (G1) or in the G0 phase of the cell cycle (G0), comprising administering to the cell BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex or contacting the cell with BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex.

The invention also provides a method for repairing a DNA double strand break in a cell of the G1 phase of the cell cycle (G1) or the G0 phase of the cell cycle (G0), comprising promoting or stimulating the assembly or occurrence of a BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex in the cell.

In various aspects of the invention, assembly of the BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex is facilitated or stimulated by administering an agent that facilitates or stimulates such assembly or an agent identified using the methods of the invention that facilitates or stimulates such assembly. In one embodiment, the agent is an agonist of USP11 or USP 11. In one embodiment, the agent is an inhibitor of CRL-KEAP 1. In one embodiment, the agent is an inhibitor of KEAP 1. In one embodiment, the agent is a PALB2 mutant. In one embodiment, the agent is a PALB2 mutant that disrupts its interaction with KEAP 1. In one embodiment, the agent is PALB2 comprising mutations of its Lys20, Lys25, and Lys30 residues.

Methods for activating or modulating homologous recombination in a cell can be performed in a cell in which a single-stranded dna (ssdna) production pathway is activated. In one aspect, ssDNA production pathways in cells are activated by DNA end excision.

The invention also provides a method for activating or modulating homologous recombination in a cell, in particular in a cell of the G1 phase of the cell cycle (G1) or the G0 phase of the cell cycle (G0), wherein DNA end excision is activated or has been activated to produce single-stranded DNA, said method comprising promoting or stimulating the assembly or occurrence of a BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex in the cell.

The invention also provides a method for repairing a DNA double strand break in a cell of the G1 phase of the cell cycle (G1) or the G0 phase of the cell cycle (G0), wherein DNA end excision is activated or has been activated to produce single stranded DNA, comprising promoting or stimulating the assembly or occurrence of a BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex in the cell. In one embodiment, assembly of the complex is promoted or stimulated by administration of an agent that modulates HR. In one embodiment, the agent is an agent that modulates HR identified using the methods of the invention. In one embodiment, the agent is an agonist of USP11 or USP 11. In one embodiment, the agent is an inhibitor of CRL-KEAP 1. In one embodiment, the agent is an inhibitor of KEAP 1. In one embodiment, the agent is a PALB2 mutant. In one embodiment, the agent is an inhibitor of DCAF 10. In one embodiment, the agent is an inhibitor of the CULLIN4-RING ligase.

The invention also provides a method for repairing a DNA double strand break in a cell of the G1 phase of the cell cycle (G1) or the G0 phase of the cell cycle (G0), wherein DNA end excision is activated or has been activated to produce single stranded DNA, comprising contacting the cell with a BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex.

In one aspect, the invention provides a method for activating or modulating homologous recombination in a cell, in particular in a G1 or G0 cell, comprising the step of inhibiting KEAP1 or CRL3-KEAP1 or administering an inhibitor of KEAP1 or CRL3-KEAP 1. In one aspect, the invention provides a method for activating or modulating homologous recombination in a cell, particularly a G1 or G0 cell, comprising the step of blocking USP11 degradation or promoting or stimulating USP11 activity. In one embodiment, the method comprises administering USP11 or an agonist thereof. In one aspect, the invention provides a method for activating or modulating homologous recombination in a cell, in particular a G1 or G0 cell, comprising the steps of inhibiting CRL-KEAP1 or administering a KEAP1 or CRL3-KEAP1 inhibitor and blocking USP11 degradation or promoting or stimulating USP1 activity.

The invention also provides a method for repairing a DNA double strand break in G1 or G0 cells, wherein DNA end excision is activated or has been activated to produce single stranded DNA, the method comprising (a) inhibiting KEAP1 or CRL3-KEAP 1; (b) blocking the degradation of USP11 or promoting or stimulating USP11 activity; (c) administering USP11 or an agonist thereof; (d) administering an inhibitor of KEAP1 or CRL3-KEAP 1; (e) administering an inhibitor of DCAF 10; and/or (e) inhibits CRL-KEAP1 and blocks degradation of USP 11.

Methods for activating or modulating homologous recombination in a cell can also include activating or promoting a single-stranded dna (ssdna) production pathway. In one aspect, the ssDNA production pathway is activated by DNA end excision.

The method for activating or modulating homologous recombination in a cell may further comprise a gene editing system. In one aspect, the gene editing step comprises contacting the cell with a nuclease. In aspects of the invention, the gene editing system can correct for genome modification.

The invention also provides methods for inhibiting homologous recombination in a cell, particularly a G1 cell, comprising inhibiting the assembly of a BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex in the cell. In one embodiment, the interaction is inhibited by administering KEAP1 or CRL3-KEAP1 or an agonist thereof. In one embodiment, the interaction is inhibited by administration of a USP11 antagonist/inhibitor (e.g., mitoxantrone). In one embodiment, the interaction is inhibited by administering an agent that inhibits or prevents HR identified using the methods of the invention.

The invention also provides kits for performing the methods of the invention.

The present invention also provides a system comprising: an assay for determining the level of USP11 activity, complex or biomarker level in a sample obtained from the subject; a processor for processing the results; computer code instructions for comparing the results to a database; and a user display for providing the comparison results. The database may contain reference values for USP11 activity or biomarker levels.

The invention also contemplates the use of the methods, kits and systems of the invention in genome modification or editing.

In one aspect, the invention also contemplates the use of the methods, compositions, kits and systems of the invention in genome modification or editing, provided that the use is not a method of treating the human or animal body by surgery or therapy, and provided that the use is not a method for modifying germline genetic identity in humans. Genomic modifications may include modifying a target polynucleotide sequence in a cell, modifying expression of a polynucleotide sequence in a cell, generating a model cell comprising a mutated disease gene, or knocking out a gene. The use of the invention may also comprise repair or editing of a cleaved target polynucleotide by insertion of an exogenous template polynucleotide, wherein the repair or editing results in one or more nucleotide mutations, including insertions, deletions or substitutions, of the target polynucleotide.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Description of the drawings

The invention will now be described with reference to the accompanying drawings, in which:

inhibition of the BRCA1-PALB2 interaction in FIG. 1, G1 is CRL3-KEAP1 dependent. a, photomicrographs of irradiated (2Gy) G1-synchronized U2OS cells for γ -H2AX, BRCA1, and BRCA2 immunofluorescence treatment. DAPI, 4', 6-diamidino-2-phenylindole; IR, ionizing radiation; WT, wild type. b, a and quantification of the experiments shown in FIG. 5 d. ASN, asynchronously dividing cells. WT, wild type (mean ± standard deviation (s.d.), N ═ 3). c, PALB2 was Immunoprecipitated (IP) from extracts prepared from mock-or X-irradiated S-phase or G1-synchronized 293T cells. Normal immunoglobulin (Ig) G immunoprecipitation was performed as a control. Cyclin a staining determines cell cycle synchronization. The left-hand number indicates kDa. The gel source data is shown in FIG. 5. d, quantification of the experiment shown in FIG. 7 a. 53BP 1. delta. U2OS cells transfected with the indicated GFP-PALB2 vector and short interfering (si) RNA were irradiated (20Gy) prior to treatment for microscopy. (average ± s.d., N ═ 3). e, immunoprecipitation of normal IgG and PALB2 from extracts prepared from synchronized and irradiated 293T cells of the indicated genotype. The left-hand number indicates kDa.

FIG. 2 ubiquitination of PALB2 prevents BRCA1-PALB2 interaction. a, the N-terminus of PALB2 and the sequence of the mutant. [ SEQ ID NO:1-3] b, GFP Immunoprecipitation (IP) from extracts of G1-or S-phase synchronized 293T cells expressing the indicated GFP-PALB2 protein. c, in vitro ubiquitination of the indicated HA-tagged PALB2 protein by CRL3-KEAP 1. d, pull-down assay of ubiquinated HA-PALB2(1-103) incubated with MBP or MBP-BRCA 1-CC. I, inputting; FT, flowing through; PD, pull down. Asterisks indicate fragments of HA-PALB2 capable of binding BRCA 1. b-d, left hand numbers indicate kDa.

FIG. 3.USP11 interferes with the activity of CRL3-KEAP 1. a, normal IgG or PALB2 Immunoprecipitates (IP) from extracts of 293T cells treated with Camptothecin (CPT) of the indicated genotype transfected with the GFP-USP11 construct. EV, empty vector; CS, C318S; WT, wild type. b, colony formation survival assay (mean. + -. s.d., N.gtoreq.3) of 293T cells of the indicated genotype treated with Olaparib (olaparib). c, normal IgG or PALB2 immunoprecipitates from extracts of CPT-treated 293T cells of the indicated genotype. d, immunoblot of deubiquitination reactions comprising ubiquitinated HA-tagged PALB2(1-103) and increasing concentrations of glutathione S-transferase (GST) -USP11 or its C270S (CS) mutant. USP2 was used as a control. DUB, deubiquitinase. e, cell cycle synchronized U2OS cells were irradiated (20Gy dose) and processed for immunoblotting. IR, ionizing radiation. f, immunoblotting of extracts from irradiated U2OS cells transfected with indicated sirnas. CTRL, control. G, fluorescence micrographs of G1 synchronized and irradiated (20Gy) 53BP 1. delta. U2OS cells transfected with the indicated siRNAs. The percentage of cells with more than 5 γ -H2 AX-co-localized BRCA2 foci is indicated (mean ± s.d., N ═ 3). Scale bar, 5 μm. a. c, d, f, left or right numbers indicate kDa.

Reactivation of the HR in the G1 phase. Quantification of Wild Type (WT) and 53BP1 Δ U2OS cells cotransfected with non-targeting (CTRL) or KEAP1siRNA and vectors expressing wild type CtIP or T847E (TE) mutants, synchronized in G1, irradiated (2Gy) and treated for γ -H2AX and RAD51 immunofluorescence (mean ± s.d., N ═ 3). b, representative micrograph from a. IR, ionizing radiation. c, schematic representation of gene targeting assay. d, gene targeting efficiency at LMNA locus in asynchronous division (ASN) and G1 arrested U2OS cells (mean ± s.d., N ═ 3). HR, homologous recombination; sgRNA, single guide rna (single guide rna). e, gene targeting at LMNA locus in G1-arrested cells transfected with the indicated siRNA or PALB2-KR expression vector (mean ± s.d., N ═ 3). f, model of cell cycle regulation by homologous recombination.

FIG. 5 inhibition of PALB2-BRCA2 accumulation at the DSB site in G153 BP1 Δ cells. a, schematic representation of human 53BP1 gene tissue and targeting sites for sgrnas used. Boxes represent exons (E: yellow, coding sequence; brown, untranslated region (UTR)). Indels introduced by CRISPR/Cas9 and their respective frequencies are indicated. b, Wild Type (WT) as well as 53BP1 Δ and U2OS cells were mock-or X-irradiated (10Gy) before treatment for 53BP1 fluorescence microscopy. DAPI was used to stain DNA and trace the contour of the nuclei. c, Wild Type (WT) and 53BP1 Δ U2OS cells were treated for 53BP1 immunoblotting. Tubulin was used as loading control. d, Wild Type (WT) and 53BP1 Δ U2OS cells that divide synchronously or Asynchronously (ASN) in G1 following double thymidine blockade and release were irradiated (2Gy) and treated for γ -H2AX, PALB2, BRCA2, and BRCA1 immunofluorescence. The micrographs for BRCA1 and BRCA2 staining in G1 are shown in FIG. 1 a. e, synchronized Wild Type (WT) and 3BP1 Δ U2OS cells in G1 after release from the double thymidine block were irradiated (20Gy) and treated for γ -H2AX, BRCA1 and BRCA2 immunofluorescence. Left is a representative micrograph of G1 arrested cells, right shows quantification of the complete experiment (mean ± s.d., N ═ 3).

FIG. 6 BRCA1-PALB2 interaction is cell cycle regulated. a schematic representation of a LacO/LacR chromatin targeting system. b, transfection of U2OS256 cells with the indicated mCherry-LacR and GFP fusions. GFP fluorescence was measured at the site of the lacO array-localized mCherry focus. Each circle represents one cell analyzed and the bar is located in the middle. Cells were also stained with cyclin a antibody to determine cell cycle position (N ═ 3). IR, ionizing radiation. c, representative photomicrographs of U2OS256 cells transfected with the indicated mCherry-LacR and GFP fusions; data were quantified in d. d, quantification of U2OS256 cells transfected with the indicated mCherry-LacR and GFP fusions to line BRCA1 or PALB2 to lacO arrays (N ═ 3). e, schematic representation of the PALB2 framework and its major interacting proteins. f, quantification of U2OS256 cells transfected with the indicated GFP-PALB2 mutant and mCherry-LacR-BRCA 1-CC. Cells were also stained with cyclin a antibody to determine cell cycle position (N ═ 3).

FIG. 7. inhibition of the BRCA1-PALB2 interaction in G1 was dependent on CRL3-KEAP 1. a, representative photograph of the experiment shown in FIG. 1 d. b, schematic representation of human KEAP1 gene tissue and targeting sites for sgrnas used as described in fig. 5 a. Indels introduced by CRISPR/Cas9 and their respective frequencies are indicated. c, PALB2 Immunoprecipitates (IP) from extracts prepared from irradiated 293T cells. IP of normal IgG was performed as a control. d, 293T cells with the indicated genotype were transfected with the indicated HA-KEAP1 construct, synchronized and irradiated in G1 or S phase. Cells were treated for PALB2 Immunoprecipitation (IP). EV, empty vector; WT, wild type. e, quantification of U2OS256 cells transfected with the indicated GFP-PALB2 mutant and mCherry-LacR-BRCA 1. Cells were also stained with cyclin a antibody to determine cell cycle position (N ═ 3). f, quantification of U2OS256 cells transfected with GFP-PALB2 and mCherry-LacR-BRCA1-CC (wild type or K1406R mutant). Cells were also stained with cyclin a antibody to determine cell cycle position. This figure shows that the only lysine in the PALB2 interaction motif of BRCA1 is not involved in cell cycle regulation of the PALB2-BRCA1 interaction. e. Each circle represents the cell analyzed, and the bar is located in the middle (N-3).

FIG. 8 PALB2 ubiquitinated by CRL3-KEAP 1. A, transfection of His expressing Doxycycline (DOX) inducibility with the indicated siRNA₆HEK293Flp-In T-REX cells from Ub. Cells were treated for Ni-NTA pulldown (IP). b, 293T cells transfected with siRNA targeting USP11 and Flag-PALB2 expression vector were treated for Flag immunoprecipitation, followed by Mass Spectrometry (MS). A schematic representation of the identified MS/MS profile of the tryptic diglycine (diG) -PALB2 peptide (K16, top; K43, bottom), c, lacO/LacR chromatin targeting system and in vivo quantification of ubiquitinated PALB2 is shown. d, representative micrograph of U2OS256 transfected with the designated mCherry-LacR-PALB2 vector. Cells were treated for FK2 immunofluorescence. EV, empty vector. Scale bar, 5 μm. e, quantification of U2OS256 cells transfected with the designated mCherry-LacR-PALB2 vector. Cells were treated to quantify FK2 fluorescence at the focus of LacO. Each circle represents the cell analyzed, and the bar is located in the middle (N-3). Cells were also stained with cyclin a antibody to determine cell cycle position. Statistical significance was determined by Kruskall-Wallis test<0.001；**P<0.01)。

FIG. 9 analysis of the dependence modulation of KEAP1 and USP11 by PALB2 and homologous recombination. a, site-specific chemical ubiquitination of HA-PALB2(1-103) at residues 20(PALB2-KC20-Ub) and 45(PALB2-KC45-Ub) by dichloroacetone ligation. The resulting ubiquinated PALB2 polypeptide and its modified counterpart were subjected to pull-down with MBP fused to the coiled-coil domain of BRCA1 (MBP-BRCA 1-CC). I, inputting; PD, pull down. Asterisks indicate nonspecific bands. b, wild type and KEAP1 Δ 293T cells were treated with Cycloheximide (CHX) for the indicated time and then treated for NRF2 and KEAP1 immunoblotting. Actin levels were also determined as loading controls. c, USP11 Immunoprecipitated (IP) from extracts prepared from 293T cells treated or not with camptothecin (CPT; 200 nM). Immunoprecipitation of normal IgG was performed as a control. d, U2OS DR-GFP cells were transfected with the indicated siRNAs. 24 hours post transfection, cells were further transfected with the indicated siRNA-resistant USP11 expression vector (WT, wild type; CS, C318S and CA, C318A catalytic death mutant) or Empty Vector (EV) with or without the I-SceI expression vector. The percentage of GFP positive cells for each condition was determined 48 hours after plasmid transfection and normalized to I-SceI plus non-targeting (siCTRL) conditions (mean ± s.d., N ═ 3). e, schematic representation of human USP11 (top) and KEAP1 (bottom) gene tissues and targeting sites for sgrnas (as shown in fig. 5a) for generating USP11 Δ and USP11 Δ/KEAP1 Δ 293T cells. Indels introduced by CRISPR/Cas9 and their respective frequencies are indicated. A USP11 knockout was first generated and subsequently used to make a USP11 Δ/KEAP1 Δ double mutant. f, PALB2 was immunoprecipitated from extracts prepared from 293T cells transfected with the indicated siRNA and treated with or without CPT (200 nM). Immunoprecipitation of normal IgG was performed as a control.

FIG. 10 USP11 antagonizes the effect of KEAP1 on PALB 2. a, transfection of U2OS DR-GFP cells with the indicated siRNA or no (-). 24 hours after transfection, cells were transfected with the I-SceI expression vector (round). For each condition, the percentage of GFP positive cells was determined 48 hours after plasmid transfection and normalized to I-SceI plus non-targeting (CTRL) conditions (mean ± range, N ═ 3). b, parental 293T cells (wild type (WT)) or USP11 Δ derivatives were transfected with the indicated GFP-PALB2 construct, treated with CPT and treated for GFP Immunoprecipitation (IP). c, transfection of parental 293T cells (wild type) or USP11 Δ derivatives with Empty Vector (EV) or the indicated PALB2 expression vector. The sensitivity of the cells to the PARP inhibitor olaparib was then determined by colony formation survival assay (mean ± s.d., N ═ 3).

FIG. 11 characterization of protein stability of USP 11. a, U2OS cells synchronized in G1 or S/G2 were treated with Cycloheximide (CHX) and treated at the indicated time points to monitor USP11 stability. b, PALB2 was Immunoprecipitated (IP) from extracts prepared from 293T cells synchronized in G1 or S phase and treated or untreated with IR (20 Gy). c, thinning U2OSCells were irradiated at a dose of 2 or 20Gy and treated at the indicated time after IR for USP11 immunoblotting. Actin was used as loading control. d, U2OS cells mock treated or incubated with ATM inhibitor KU55933(ATMi), ATR inhibitor VE-821(ATRi), or DNA-PKcs inhibitor NU7441(DNAPKi) were irradiated (20Gy) and treated for USP11 and actin (loading controls) immunoblots. e, experiment similar to d, except cells were exposed to Ultraviolet (UV) radiation (50 mJ/cm)^-2). f, U2OS cells mock treated or incubated with the proteasome inhibitor MG132 were irradiated (20Gy) and processed for USP11 and actin (loading control) immunoblots. g, U2OS cells mock treated or incubated with the Cullin inhibitor MLN4924 were irradiated (20Gy) and treated for USP11 and actin (loading control) immunoblots.

FIG. 12, G1 Loading of RAD51 and reactivation of extraterm DNA synthesis. a, 53BP1 Δ U2OS cells were transfected with the indicated sirnas, synchronized and irradiated (20Gy) in G1 or S/G2 by release from a double thymidine block, before treatment for fluorescence microscopy. DAPI was used to track nuclear boundaries and cyclin a staining was used to determine cell cycle position. The percentage of cells with more than 5 γ -H2 AX-co-localized PALB2 foci was expressed as mean ± s.d., N-3. Scale bar, 5 μm. Representative micrographs of irradiated G1 synchronized Wild Type (WT) and 53BP1 Δ U2OS cells transfected with the indicated sirnas and expressing wild type CtIP. c, representative photomicrographs of irradiated G1 synchronized wild-type U2OS cells transfected with the indicated sirnas and expressing CtIP (T847E). d, U2OS 53BP1 Δ cells were synchronized in G1, supplemented with BrdU, irradiated (2Gy) and processed for γ -H2AX and BrdU immunofluorescence. The percentage of cells with more than 5 γ -H2 AX-co-localized BrdU foci is indicated (mean ± s.d., N-3). e, photomicrographs of U2OS cells targeted with the CRISPR-mClover system, showing a typical perinuclear expression pattern of lamin a. f, micrographs of U2OS cells targeted with mClover system, showing the expression pattern characteristic of the sub-nuclear PML foci. g, timeline of gene targeting (LMNA) experiment shown in fig. 4 d. h, time lines for gene targeting (LMNA or PML) experiments shown in FIG. 4e and FIG. 13.

Analysis of homologous recombination in G1. a, quantification of gene targeting efficiency at LMNA locus in asynchronously dividing U2OS cells transfected with increasing amounts of donor template and with (grey) or without (white) sgRNA transfection. Gene targeting events were detected by flow cytometry (mean. + -. s.d., N.gtoreq.3). b, quantification of gene targeting efficiency at the LMNA locus in asynchronously dividing cells transfected with the indicated sirnas. Gene targeting events were detected by flow cytometry (mean ± s.d., N ═ 3). c, gene targeting efficiency at the PML locus measured by flow cytometry (mean ± s.d., N ═ 3) in G1 arrested 53BP1 Δ U2OS cells expressing CtIP (T847E) mutants and cotransfected with the indicated siRNA or PALB2-KR expression constructs. d, gene targeting efficiency at LMNA locus measured by flow cytometry in G1 arrested parent (wild type (WT)) and 53BP1 Δ U2OS cells transfected with KEAP1siRNA and expressing CtIP (T847E) mutant (mean ± s.d., N ═ 3). e, gene targeting efficiency at LMNA locus (mean ± s.d., N ═ 3) in G1 arrested parent (wild type (WT)) and 53BP1 Δ U2OS cells transfected with the indicated sirnas and expressing wild type or CtIP (T847E) mutants.

Figure 14 identifies DCAF10 as a modulator of USP11 stability in response to DNA damage. One. a, siRNA screening, in which U2OS cells were transfected with sirnas targeting DCAF and other CUL4 interacting proteins, which are known and predicted. Cells were irradiated with IR (20Gy) or UV (50J/m-2) for 3 hours and then treated for USP11 immunofluorescence. Each point plotted corresponds to the percentage of USP11 remaining after irradiation. The red dots correspond to siRNA non-targeted Control (CTRL) and targeted USP11, while the red dots correspond to the core CRL4 factor, including CUL4 itself. b, U2OS cells were transfected with the indicated sirnas and then irradiated at a dose of 20Gy and treated at the indicated times after ionizing radiation for USP11 immunoblotting. Actin was used as loading control.

FIG. 15 demonstrates DCAF10 as a modulator of USP 11. a, DCAF10 interacts with USP 11. Flag-USP11 was Immunoprecipitated (IP) from extracts prepared from 293 Flag-IN/T-Rex cells. Cells were probed with DCAF10 and DCAF15 antibodies. b, whole cell extracts of Mouse Embryonic Fibroblasts (MEFs) of the indicated genotype were treated for USP11 immunoblotting. Tubulin was used as loading control. c. U2OS DR-GFP cells were transfected with the indicated siRNA or expression vector. 24 hours after transfection, cells were transfected with the I-SceI expression vector. The percentage of GFP positive cells for each condition was determined 48 hours after plasmid transfection and normalized to I-SceI plus non-targeting (CTRL) + Empty Vector (EV) conditions.

Figure 16 KEAP1 inhibition can activate HR in G1 cells. Gene targeting at the LMNA locus in G1 arrested cells transfected with the indicated siRNA and a vector expressing the R1 KEAP1 inhibitor or its FN3 backbone control (mean ± s.d., N ═ 3).

Detailed Description

Unless otherwise indicated, the preparation and use of the disclosed agents and the practice of the methods employed herein employ, as known to those skilled in the art, conventional techniques of molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA and related fields. These techniques are fully disclosed in the literature. [ see, e.g., Sambrook et al Molecular Cloning A Laboratory Manual, second edition, Cold Spring harbor Laboratory Press,1989 and third edition, 2001; ausubel et al, Current Protocols in molecular biology, John Wiley & Sons, New York,1987 and periodic updates; the series Methods in enzymology, Academic Press, San Diego; wolffe, chromatography Structure and Function, third edition, Academic Press, San Diego, 1998; methods in Enzymology, volume 304, "Chromatin" (edited by p.m. wassarman and a.p. wolffe), Academic Press, San Diego, 1999; and Methods in Molecular Biology, Vol 119, "chromatography in Protocols" (edited by P.B. Becker) Humana Press, Totowa,1999 ].

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions supplement those in the art and are directed to the present application and are not intended to be used in any relevant or irrelevant context. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the specific materials and methods are described herein.

As used herein and in the appended claims, a noun that is not defined by a quantitative term includes a plural reference unless the context clearly dictates otherwise. As used herein, "comprising" (and any form of comprising, such as "comprises", "comprising" and "comprises", "having", "and any form of having, such as" has "and" has) "," including "(and any form of comprising, such as" includes "and" includes) ", or" containing "(and any form of comprising, such as" containing "and" containing "are inclusive or open-ended, and do not exclude additional unrecited elements or process steps.

A "gene editing system" is a system for targeting and editing a genome, including, but not limited to, TALEN (Transcription Activator-Like Effector Nucleases) systems, CRISPR (Clustered regularly Interspaced Short palindromic repeats) systems, and Zinc Finger Nucleases (ZFN) systems (see Nemadryi A. et al, Acta Naturae.2014 7-9; 6(3) reviews TALEN and CRISPR systems for 19-40; Gaj et al, Trends Biotechnol.2013 7; 31(7) 2012: 397; reviews, reviews and ZFN systems; U.S. published patent application No.20110145940, Bikoen et al, Biokok et al, 2002. 161, 2010, 161, 57; Nature H5778; Nature 5726, 103; Nature J. 201, 11, 103; Nature J. 201, 103; Nature 5726, 11, 103; Nature J. 201, 103; Nature 5726, 11, 103; Nature J. 201, 103; Nature 5726, 103; Nature J. 11, 103; US 5726, 11, J. 11, 103; Nature 5726; Nature J. 11, 103; Nature et al, J. 11, 103; Nature 5726, 103; Nature J. 11, 103; Nature J. 201, 103; Nature 5726, 103; Nature 5726, US published patent application No. 7, US; Nature J. 10, 103; Nat, etc., j.. nat.med.2012; 18(5) 807-; 8(10) 861-869, the ZFN system is described.

"CRISPR system" generally refers to transcripts and other elements involved in expression of or directing the activity of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -associated ("Cas") genes. CRISPR systems can include, but are not limited to, sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active portion tracrRNA), tracr-partner sequences, guide sequences, or other sequences, and transcripts from CRISPR loci. One or more elements of the CRISPR system may be derived from a type I, type II or type III CRISPR system. The CRISPR system facilitates the formation of a CRISPR complex (comprising a guide sequence that hybridizes to a target sequence and complexes with one or more Cas proteins) at a target sequence site. By "target sequence" or "target polynucleotide" is meant a sequence sufficiently complementary to a designed guide sequence such that the target sequence hybridizes to the guide sequence promoting CRISPR complex formation. The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide, and it may be located in the nucleus, cytoplasm, or organelle, such as mitochondria or chloroplasts. In the case of endogenous CRISPR systems, formation of a CRISPR complex in the endogenous CRISPR system results in cleavage of one or both strands in or near (e.g., within 1,2, 3, 4,5, 6,7, 8,9, 10,20, 50 or more base pairs) the target sequence.

CRISPR systems are described in the following: U.S. Pat. nos. 8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356, 8,889,418, and 8,895,308; U.S. patent publications US 2014-0310830, US 2014-0287938, US 2014-0273234, US2014-0273232, US 2014-0273231, US 2014-0246, US 2014-0248702), US 2014-0242700, US 2014-0242699, US 2014-0242664, US2014-0234972, US 2014-7702287, US 2014-01896, US 2014-0186958, US 2014-0186919, US 2014-0186843, US 2014-0179770, and US 2014-0179006, US 2014-70753 and US 20152883; european patent applications EP 2771468(EP13818570.7), EP 2764103(EP13824232.6) and EP2784162(EP 14170383.5); and PCT patent publications WO2014/093661(PCT/US2013/074743), WO2014/093694(PCT/US2013/074790), WO2014/093595(PCT/US2013/074611), WO2014/093718(PCT/US2013/074825), WO2014/093709(PCT/US2013/074812), WO2014/093622(PCT/US2013/074667), WO2014/093635(PCT/US2013/074691), WO2014/093655(PCT/US2013/074736), WO2014/093712(PCT/US2013/074819), WO2014/093701(PCT/US2013/074800), WO2014/018423(PCT/US2013/051418) and WO2014/093622(PCT/US 2013/074667). General information on CRISPR-Cas systems is also described in the following publications: cong, L., et al, Science, month 2, day 15; 339(6121) 819-23 (2013); jiang W., et al, Nat Biotechnol 3 months; 31(3) 233-9 (2013); wang h., et al, Cell 5 month 9 day; 153(4) 910-8 (2013); konermann S, et al, Nature.2013, 8.22 months; 500(7463) 472-6.doi 10.1038/Nature12466.Epub 2013, 8/23; ran, F a., et al, Cell 8 month 28. pi: S0092-8674(13)01015-5 (2013); hsu, P.et al, Nat Biotechnol doi:10.1038/nbt.2647 (2013); ran, F a., et al, Nature Protocols 11 months; 8(11) 2281-308 (2013); shalem, o., et al, Science 12.12.12 (2013) [ prior to printed electronic publication ]; nishimasu, H., et al, Cell 2.27.d. (2014).156(5) 935-49; wu X, et al, Nat Biotechnol (2014)4 months 20 days doi: 10.1038/nbt.2889; platt et al, Cell 159(2):440-455(2014) DOI 10.1016/j.cell.2014.09.014; hsu et al Cell 157, 1262-; wang et al, science.2014, 1 month, 3 days; 343(6166) 80-84.doi 10.1126/science.1246981; doench et al, Nature Biotechnology 2014, on-line, 9/3; doi: 10.1038/nbt.3026; storrs, The Scientist, Article No.39239,2014, month 3, day 1; and Swiech et al, Nature Biotechnology; online publication 10/19/2014; doi: 10.1038/nbt.3055). A variety of programs are available for designing guide sequences, such as MIT's CRISPR Design [ http:// CRISPR. MIT. edu ] and E-CRISP [ www.e-CRISP. org/E-CRISP ], developed by German cancer Research Center (German cancer Research Center). CRISPR systems also include systems developed by or available from: editas Medicine (Cambridge, MA), Caribou Biosciences (Berkeley, CA), CRIPRTherepeutics (Basel, Switzerland), Addgene (Cambridge, MA) and Intellia Therapeutics (Cambridge, MA).

"DNA end excision" generally refers to the nucleolytic degradation of the 5 'end strand of a double-stranded break in DNA, resulting in the formation of 3' -terminated single-stranded DNA. DNA end excision in eukaryotes involves two stages: a slow initial phase catalyzed by Mre11-Rad50-Nbs1(MRN) complex in mammals, and a second and faster phase catalyzed by exonucleolytic Exo1 or helicase Bloom Syndrome Protein (BLM). DNA end excision begins with a cell cycle activation step that involves phosphorylation of the accessory protein CtIP (also known as retinoblastoma binding protein 8). Pathways involved in DNA end excision can be activated by inhibiting TP53BP1(53BP1) or RIF to stimulate or activate recruitment of BRCA 1to the DNA double strand break or to block recruitment of 53BP1 or RIF to the DNA double strand break site. In one aspect, DNA end-excision can be activated by inhibiting 53BP1 (or RIF) expression and/or activity, as well as expressing mutant forms of CtIP that mimic constitutive phosphorylation (e.g., CtIP-Thr879 Glu). In one aspect, an inhibitor of 53BP1 and a mutant form of CtIP, particularly CtIP-Thr879Glu, that mimics constitutive phosphorylation are used to reconstitute or activate DNA end-excision. In one aspect, the following purified human proteins can be used to reconstitute or activate DNA end excision: bloom helicase (BLM); DNA2 helicase/nuclease; exonuclease 1(EXO 1); a complex (MRN) comprising MRE11, RAD50, and NBS 1; and Replication Protein A (RPA) (see Nimonkar A. V. et al, Genes & Development 25:350-362, 2011; Huertas, P, Nat Struct MolBiol,17(10:11-16, doi:10.1038/nsmb.1710, 2010; Jimeno S., et al, nucleic acids Res do: 101093/nar/gkui384,2015 for description of DNA end excision).

"homologous recombination" and "HR" refer to a type of genetic recombination in which DNA strands of similar or identical nucleotide sequences are exchanged. Cells can use HR to repair DNA Double Strand Breaks (DSBs) by the following general procedure. HR begins when the DSB is cleaved by nuclease and helicase, creating a 3' single stranded dna (ssdna) overhang on which RAD51 recombinase assembles into nucleoprotein filaments. This structure can invade homoduplex DNA, which serves as a template for repair DNA synthesis. The resulting intermediate can be metabolized to produce non-cross-products, thereby recovering the damaged DNA molecule as it existed prior to the double strand break (SanFilippo et al, annu. rev. biochem.2008.77: 229-57). The term also includes recombinations using single stranded donor oligonucleotides (ssodns), particularly recombinations using single stranded donor oligonucleotides (ssodns) that require excision and can be activated by 53BP1 inhibitors.

"HR disease" refers to any condition, disease, disorder, syndrome, or combination of manifestations or symptoms that is believed or diagnosed as a condition that may be associated with or characterized by a deficiency in HR. Exemplary diseases include, for example, cancer; cardiovascular diseases including heart failure, hypertension and atherosclerosis; respiratory diseases; renal disease; gastrointestinal diseases including inflammatory bowel disease, such as Crohn's disease and ulcerative colitis; liver, gall bladder and bile duct diseases including hepatitis and cirrhosis; hematological, metabolic, endocrine, and reproductive diseases, including diabetes; bone and bone mineral metabolism disorders; immune system disorders including autoimmune disorders such as rheumatoid arthritis, lupus erythematosus and other autoimmune disorders; musculoskeletal and connective tissue diseases including arthritis, infectious diseases of dysplasia cartilage; and neurological diseases such as Alzheimer's disease, Huntington's disease and Parkinson's disease.

The methods of the invention are useful for monitoring or treating diseases caused by defects in genes that mediate homologous recombination, such as BRCA1, BRCA2, PALB2, PARP-1, USP11, RAD51, and/or DCAF 10.

Embodiments of the invention provide methods for monitoring or treating various cancers, including but not limited to, carcinomas, melanomas, lymphomas, sarcomas, blastomas, leukemias, myelomas, osteosarcomas, neural tumors, and cancers of organs such as breast, ovary, and prostate.

In embodiments, the invention provides methods of monitoring or treating cancer having a BRCA-1 deficiency, a BRCA-2 deficiency, a dual BRCA-1/BRCA-2 deficiency, and Fanconi anemia. In an embodiment of the invention, the cancer is breast cancer, in particular invasive ductal carcinoma and invasive lobular carcinoma. In an embodiment of the invention, the cancer is ovarian cancer, in particular epithelial ovarian tumors, germ cell ovarian tumors, and sex cord stromal tumors.

The methods of the invention for activating or modulating homologous recombination are useful for genetically modifying polynucleotides associated with genetic disorders. In some embodiments, the genetic disorder is a monogenic disorder. In some embodiments, the genetic disorder is a polygenic disorder. In some embodiments, the genetic disorder is associated with one or more SNPs. In a specific embodiment of the invention, the genomic modification corrects for a point mutation.

Examples of genetic disorders and polynucleotide sequences associated with genetic disorders can be found on the world Wide Web (see, e.g., the National Center for Biotechnology Information, National Library of Medicine (Bethesda, MA) or the Mkusco-Neisson Institute of genetic Medicine, University of John Hopkins (McKumock-Nathans Institute of genetic Medicine, Johns Hopkins University) (Bamore, Md)), as set forth in published patents and applications (see, e.g., U.S. published application No.2015/0247150) and publications (see, e.g., Turitz CoxD.B. et al, Nature Medicine 21,2006,2015; and O' Connor T.P. and R.G.Crystal, Natureview/research, pp.7, 276, supplementary Information included in the publication No. 276, Vol.276, and Vol.7, Vol.276).

In one aspect, the genetic disorder is a muscle genetic disorder. In one aspect, the genetic disorder is myotonic dystrophy type 1. In one aspect, the genetic disorder is myotonic dystrophy type 2. In one aspect, the genetic disorder is Duchenne Muscular Dystrophy (DMD). In one aspect, the genetic disorder is Becker muscular dystrophy (Becker musculoskystrophy).

In one aspect, the genetic disorder is a genetic disorder of the liver, such as α -1 antitrypsin deficiency, Wilson Disease, hereditary hemochromatosis, tyrosinemia type I, glycogen storage Disease type IV, argininosuccinate lyase deficiency, limonin deficiency, cholesteryl ester storage Disease, and hereditary fructose intolerance.

In one aspect, the genetic disorder is α -1 antitrypsin deficiency, an autosomal recessive (co-dominant) disease caused by a mutation in the SERPINA1 gene encoding the serine protease inhibitor AAT.

In one aspect, the genetic disorder is wilson's disease, which depends on mutations in the gene encoding ATP7B Cu translocase, which is expressed primarily by hepatocytes, regulating copper content in the liver.

In one aspect, the genetic disorder is a pulmonary genetic disorder.

In one aspect, the genetic disorder is cystic fibrosis, an autosomal recessive disease resulting from mutation of a Cystic Fibrosis Transmembrane Regulator (CFTR) protein that is a member of the ATP-binding cassette superfamily of transmembrane proteins.

In other aspects of the invention, the genetic disorder can be hemophilia, α 1-antitrypsin deficiency, Canavan disease, adenosine deaminase deficiency, X-linked severe combined immunodeficiency, familial amyloidosis polyneuropathy, thalassemia, Tay-Sachs disease, late baby waxy lipid brown deposition disease (late infantile lipofuscinosis), mucopolysaccharidosis, Niemann-pick disease (Niemann-pick disease), chondrodysplasia, huntington's disease, spinocerebellar ataxia, fredriechitaxia, amyotrophic lateral sclerosis, monogenic hypercholesterolemia, and other monogenic diseases.

In aspects of the invention, the genetic disorder is sickle cell anemia, and the methods of the invention include correcting the mutated HBB hemoglobin gene by gene conversion with its paralogous HBD.

An "effective amount" refers to an amount of a compound or composition as described herein that is effective to achieve a particular biological result. Such results include, but are not limited to, treatment of the diseases or conditions disclosed herein as determined by any method appropriate in the art.

"PARP inhibitors" refer to inhibitors of the ribozymes poly (adenosine 5' -diphosphate-ribose) polymerase [ "poly (ADP-ribose) polymerase" or "PARP"), which are also known as ADPRT (NAD: protein (ADP-ribosyltransferase (polymerization)) and PARS (poly (ADP-ribose) synthetase). PARP inhibitors have been described in the following: banasik et al, "Specific inhibition of Poly (ADP-Ribose) Synthesis and Mono (ADP-Ribosyl) -transfer enzyme", J.biol.chem.,267:3,1569-75(1992), and Banasik et al, "inhibition and Activators of ADP-Ribosyl Reactions", Molec.cell.biochem.,138,185-97 (1994). PARP inhibitors have been disclosed and described in the following patents and patent applications: WO 00/42040; WO 00/39070; WO 00/39104; WO 99/11623; WO 99/11628; WO 99/11622; WO 99/59975; WO 99/11644; WO 99/11945; WO 99/11649; and WO 99/59973; U.S. patent No.8,894,989, U.S. patent No.8,946,221; 8,778,966, respectively; 8,669,249, respectively; 8,623,884, respectively; 8,592,416, respectively; 8,546,368, respectively; 8,541,417, respectively; 8,541,403, respectively; 8,420,650, respectively; 8,362,030, respectively; 8,236,802, respectively; 8,217,070, respectively; 8,188,103, respectively; 8,188,084, respectively; 8,183,250, respectively; 8,173,682, respectively; 8,129,382, respectively; 8,088,760, respectively; 8,080,557, respectively; 8,071,623, respectively; 8,058,275, respectively; 8,012,976, respectively; 8,008,491, respectively; 7,999,117, respectively; 7,956,064, respectively; 7,875,621, respectively; 7,820,668, respectively; 7,750,008, respectively; 7,732,491, respectively; 7,728,026, respectively; 7,652,014, respectively; 7,601,719, respectively; 7,462,724, respectively; 7,087,637, respectively; 7,041,675, respectively; 6,977,298, respectively; 6,924,284, respectively; 6,737,421, respectively; 6,635,642, respectively; 6,495,541, respectively; 6,444,676, respectively; 6,395,749, respectively; 6,380,211, respectively; 6,380,193, respectively; 6,346,536, respectively; 6,197,785, respectively; 5,756,510, respectively; and re.36,397.

In aspects of the invention, the PARP inhibitor is Olaparib (Olaparib) (AstraZeneca). In various aspects of the invention, the PARP inhibitor is Veliparib (Veliparib) (AbbVie Inc, Chicago, IL). In various aspects of the invention, the PARP inhibitor is rucapaib (Rucaparib) (Clovis Oncology, inc., Boulder, CO). In aspects of the invention, the PARP inhibitor is INO-1001(Inotek Pharmaceuticals Corp, Lexington, Mass.). In various aspects of the invention, the PARP inhibitor is MK-4827 (nilapanib) (Tesaro, Waltham, MA, see also Montoni et al, Frontiers in pharmacy, [4], Article 18, pages 1-7). In aspects of the invention, the PARP inhibitor is talazoparib (Medivation, Inc, san francisco CA).

A "sample" is a sample derived from any biological source, e.g., a tissue, extract, or cell culture, including cells (e.g., tumor cells), cell lines, cell lysates, and physiological fluids such as, for example, blood or subsets thereof (e.g., white blood cells, red blood cells), plasma, serum, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, feces, tears, bronchial lavage, swab, milk, ascites, nipple aspirate, needle aspirate, synovial fluid, peritoneal fluid, lavage, and the like. The sample may be obtained from an animal, preferably a mammal, most preferably a human. Samples can be from a single individual or pooled prior to analysis. The sample may be processed prior to use, for example to prepare plasma from blood, dilute viscous fluids, and the like. Methods of processing the sample may include filtration, distillation, extraction, centrifugation, concentration, inactivation of interfering components, addition of reagents, and the like.

In an embodiment of the method of the invention, the sample is a mammalian tissue sample. In another embodiment, the sample is a cell lysate. In another embodiment, the sample is a cell. In another embodiment, the sample is a human physiological fluid. In a specific embodiment, the sample is human serum. In another embodiment, the sample is a white blood cell or a red blood cell.

The terms "subject", "individual" or "patient" refer interchangeably to a warm-blooded animal such as a mammal. In particular, the term refers to a human. The subject, person or patient may be suffering from or suspected of suffering from or susceptible to a disease as described herein. The term also includes animals raised for food, as pets, or for research, including horses, cattle, sheep, poultry, fish, pigs, cats, dogs, and zoo goats, apes (e.g., gorilla or chimpanzee), and rodents such as rats and mice.

NCBI accession numbers for USP11, PALB2, BRCA1, BRCA2, KEAP1, 53BP1, DCAF10, RBX1, CUL3 and CtIP are in table 1, and the human sequences for USP11, PALB2, BRCA1, BRCA2, KEAP1, 53BP1, DCAF10, RBX1, CUL3 and CtIP are in the sequence listing.

Screening and monitoring assays

The present invention provides methods for monitoring activity or expression of USP11 by determining the interaction of BRCA1 and PALB2, the interaction of BRCA1, PALB2 and BRCA2, the interaction of USP11 and DCAF10, and/or the interaction of USP11 and PALB 2. Conventional methods known to those skilled in the art can be used to determine protein interactions in a sample. For example, a BRCA1-PALB2, BRCA1-PALB2-BRCA2, USP11-DCAF10, or USP11-PALB2 complex may be isolated using affinity techniques such as immune-based purification (e.g., immunoaffinity chromatography), peptides may be prepared from the isolated complexes using conventional methods (e.g., gel electrophoresis, liquid chromatography, capillary electrophoresis, nano-reversed phase liquid chromatography, high performance liquid chromatography, or reversed phase high performance liquid chromatography), and mass spectrometry (e.g., quantitative mass spectrometry such as selected reaction monitoring mass spectrometry (sMRM), non-high resolution data-dependent analysis (SWATH), high resolution multi-reaction monitoring (MRM)^HR) Or based on quantification of MS 1).

The present invention also provides a method for monitoring activity of USP11 by determining ubiquitination of the N-terminus of PALB 2. Conventional methods known to those skilled in the art can be used to determine ubiquitination in a sample. For example, ubiquitination or PALB2 can be determined by measuring changes in PALB2 (e.g., weight, see U.S. patent No.6,413,725), the amount of polyubiquitin bound to CRL3-KEAP 1E 3 ligase (see, e.g., EP 1268847), and/or the activity of CRL3-KEAP 1E 3 ligase (see, e.g., U.S. publication No. 2013/0116152). Mass spectrometry techniques can also be used, such as selective reaction monitoring mass spectrometry (sMRM), high resolution data-independent analysis (SWATH), high resolution Multiple Reaction Monitoring (MRM)^HR) Or based on quantification of MS 1) to monitor ubiquitin residues on peptides from PALB2N after protease digestion. In a more specific example, isotopically labeled synthetic peptides corresponding to tryptic digests of ubiquinated PALB2, particularly those corresponding to ubiquitination on Lys14, Ly16, Lys20, Lys25, Lys30, Lys43, or Lys45, can be used as internal standards to quantify the degree of ubiquitination of PALB 2.

In one aspect, the invention provides a method for determining ubiquitination of a PALB2 polypeptide in a sample, the method comprising digesting ubiquitinated PALB2 polypeptide in the sample with a protease, thereby producing a plurality of test peptides; determining the number of ubiquitination sites by mass spectrometry for the test peptide to determine the number of ubiquitination sites by the presence of at least one isopeptide bond between ubiquitin and lysine residues, thereby determining the number of ubiquitination of the PALB2 polypeptide in the sample. In one embodiment, the test peptide is from the end of PALB 2N. In one embodiment, the lysine residue corresponds to Lys14, Lys16, Lys20, Lys25, Lys30, Lys43, or Lys 45. The method may utilize peptide internal standards corresponding to different peptide subsequences of PALB2 to provide controls in the quantitative assay. In one aspect, different synthetic peptide internal standards corresponding to PALB2 were generated and differentially labeled.

Proximity Ligation Assays (PLA) can also be used to determine USP11 activity by using DNA-based assays to determine the interaction between BRCA1 and PALB2 and/or PALB 2-interacting proteins (e.g., BRCA 2). For example, a primary antibody directed against an interacting binding partner (e.g., PALB2 and BRCA1) is added to the cell lysate. A second set of antibodies, called PLA probes or proximity probes (proximity probes), recognizes the first set of primary antibodies. PLA probes contain DNA strands that assemble into assay-specific DNA molecules when in close proximity. Such DNA molecules can then be used and detected using, for example, fluorescent probes. [ see, e.g., Soderberg O. et al, nat. methods, 12 months 2006; 3(12) 995-1000; jarvius m. et al, mol.cell.proteomics, 9 months 2007; 6(9):1500-9)].

In one aspect, the invention provides a method for determining the interaction of BRCA1-PALB2 or BRCA1-PALB2-BRCA2 in a sample, comprising: contacting the sample with a primary antibody for each binding partner in the interaction; contacting the sample with proximity probes comprising secondary antibodies that bind to respective primary antibodies, wherein each proximity probe has an oligonucleotide conjugated thereto; wherein when the oligonucleotides of the proximity probes are sufficiently adjacent to each other, the oligonucleotides of the proximity probes interact to form a circular product, which is amplified by rolling circle replication to produce an amplification product; and measuring the amplification product, thereby determining or measuring the interaction.

Assays to monitor the in situ co-localization of PALB2 (or a related protein such as BRCA2) to BRCA1 also provide a means to monitor USP11 activity (see examples herein). For example, the location of PALB2 at the site of DNA damage (marked by BRCA1 or other markers such as γ -H2 AX) is dependent on USP11 activity. In such assays, cells are fixed, permeabilized, and then incubated with an antibody that detects PALB2, BRCA2, or a protein related thereto (e.g., BRCA 1). The addition of labeled secondary antibodies enables visualization of protein accumulation in situ at sites of DNA damage in a sub-nuclear structure called foci. The addition of genotoxic lesions (such as ionizing radiation or other treatments that cause chromosome breakage) increases the number of "foci" and can be included to increase the dynamic range of the assay.

It is to be understood that proximity ligation assays and in situ co-localization assays can be used to determine any of the interactions disclosed herein.

The methods of the invention can be performed in the presence or absence of a test compound or agent, and in the absence of a test compound or agent, detecting an increase or decrease in activity or expression of one or more of USP11, DCAF10, PALB2, PALB2 ubiquitination, BRCA1-PALB2-BRCA2 complex, PALB2-USP11 complex, KEAP1, USP11-DCAF10 complex, CRL3-KEAP1 complex, CRL3-KEAP1-PALB2 complex, and KEAP1-PALB2 complex, as compared to a control, indicates that the test compound or agent can be used as a therapeutic, or to modulate homologous recombination.

In one aspect, the present invention provides a method of identifying or evaluating the ability of an agent to sensitize or reverse resistance or delay the onset of resistance to a PARP inhibitor by determining the effect of the agent on the activity of USP11 using the method of the present invention. In one aspect, the negative impact on USP11 indicates that the agent is a cell sensitizer for PARP inhibitors, or may reverse or delay the development of resistance to PARP inhibitors. On the one hand, the positive effect on USP11 indicates that this agent is a poor cell sensitizer for PARP inhibitors. In one aspect, the ability of an agent to sensitize or reverse resistance or delay the onset of resistance to a PARP inhibitor is determined by the reduction in the level of USP11 activity when compared to such levels obtained from a control. In one aspect, the ability of an agent to sensitize or reverse resistance or delay the onset of resistance to a PARP inhibitor is determined by an increase in the level of USP11 activity when compared to such levels obtained from a control.

The present invention also relates to a method of identifying or evaluating the ability of an agent to sensitize a cell to a PARP inhibitor or to reverse resistance to or delay the onset of resistance to a PARP inhibitor by determining the effect of the agent on KEAP1, CRL3-KEAP1 or KEAP1-PALB 2. On the one hand, the negative impact on KEAP1 (loss of KEAP1 or CRL3-KEAP1 activity) indicates that this agent is a poor cell sensitizer for PARP inhibitors. In one aspect, a positive effect on KEAP1 or CRL3-KEAP1 activity indicates that the agent is a cell sensitizer for PARP inhibitors or may reverse or delay the development of resistance to PARP inhibitors. In one aspect, the ability of an agent to sensitize to or reverse resistance to or delay the onset of resistance to a PARP inhibitor is determined by an increase in the level of KEAP1, CRL3-KEAP1, KEAP1-PALB2 or CRL3-KEAP1 activity or expression when compared to such levels obtained from a control. In one aspect, the ability of an agent to sensitize to or reverse resistance to or delay the onset of resistance to a PARP inhibitor is determined by a decrease in the level of KEAP1, CRL3-KEAP1, KEAP1-PALB2 or CRL3-KEAP1 activity or expression when compared to such levels obtained from a control.

The invention also contemplates a method of detecting an anti-cancer agent comprising performing a test assay comprising contacting an immortalized cell with a test compound and measuring USP11 activity or CRL3-KEAP1 using the method of the invention and comparing to a control test assay in the absence of the test compound. In one aspect, USP11 activity is determined by measuring USP11, PALB2, DCAF10, PALB2 ubiquitination, BRCA1-PALB2-BRCA2 complex, PALB2-USP11 complex, KEAP1, USP11-DCAF10 complex, CRL3-KEAP1 and/or CRL3-KEAP1-PALB2 complex in a cell. In one aspect, detection of a negative effect of an agent on USP11 activity or expression compared to a control is indicative of a potential anti-cancer agent or PARP inhibitor sensitizer. In one aspect, detection of a negative effect of an agent on the activity or expression of BRCA1-PALB2-BRCA2 complex and/or PALB2-USP11 complex as compared to a control is indicative of a potential anti-cancer agent or PARP inhibitor sensitizer. In one aspect, detection of a positive effect of an agent on KEAP1, CRL3-KEAP1 and/or CRL3-KEAP1-PALB2 complex activity or expression compared to a control is indicative of a potential anti-cancer agent or PARP inhibitor sensitizer. In one aspect, a decrease in the level of activity of USP11 when compared to such levels obtained from a control is indicative that the agent has anti-cancer activity or is a PARP inhibitor sensitizer.

The invention provides a method of identifying or assessing the ability of an agent to modulate homologous recombination, comprising determining the effect of a test compound or agent on one or more of: USP11, DCAF10, PALB2, PALB2 ubiquitination, BRCA1-PALB2-BRCA2 complex, PALB2-USP11 complex, KEAP1, USP11-DCAF10 complex, CRL3-KEAP1 complex and CRL3-KEAP1-PALB2 complex. In one aspect, the invention provides a method of identifying or evaluating the ability of an agent to modulate homologous recombination in a cell, comprising: (i) determining in the sample USP11, DCAF10, PALB2, PALB2 ubiquitination, BRCA1-PALB2-BRCA2 complex, PALB2-USP11 complex, KEAP1, USP11-DCAF10 complex, CRL3-KEAP1 and/or CRL3-KEAP1-PALB2 complex in the cells in the presence or absence of said reagent; and (ii) detecting an increase or decrease in USP11, DCAF10, PALB2, PALB2 ubiquitination, BRCA1-PALB2-BRCA2 complex, PALB2-USP11 complex, KEAP1, USP11-DCAF10 complex, CRL3-KEAP1 complex and/or CRL3-KEAP1-PALB2 complex in the sample as compared to a control, as an indication of the ability of the agent to modulate homologous recombination.

The invention provides a method of screening for a therapeutic agent for treating a disease associated with HR deficiency (i.e. HR disease) comprising identifying an agent that disrupts or modulates one or more of: USP11, PALB2, PALB2 ubiquitination, DCAF10, BRCA1-PALB2-BRCA2 complex, PALB2-USP11 complex, KEAP1, USP11-DCAF10 complex, CRL3-KEAP1 or CRL3-KEAP1-PALB2 complex. In one aspect, detection of a positive effect of an agent on activity or expression of USP11, BRCA1-PALB2-BRCA2 complex and/or PALB2-USP11 complex as compared to a control is indicative of a potential therapeutic for the treatment of HR disease. In one aspect, detection of a negative effect of an agent on the activity or expression of DCFA10, KEAP1, CRL3-KEAP1 or CRL3-KEAP1-PALB2 complex as compared to a control is indicative of a potential therapeutic for the treatment of HR disease.

The test compounds used in the methods of the invention may be in isolated form or any product in a mixture. The test compound may be defined by structure or function, or may be undefined. Examples of non-limiting test compounds include, but are not limited to, tissue samples, biological fluids, cell supernatants, plant preparations, and the like. The test compound can be a peptide, e.g., a soluble peptide, including Ig-tail fusion peptides, members of random peptide libraries, and combinatorial chemistry derived molecular libraries made of D-and/or L-configuration amino acids, carbohydrates, nucleic acids, antisense molecules, phosphopeptides (including members of random or partially degenerate directed phosphopeptide libraries), antibodies [ e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, single chain antibodies, fragments (e.g., Fab, F (ab))₂And Fab expression library fragments and epitope binding fragments thereof]Small organic or inorganic molecules or compound libraries. The test compound may be an endogenous physiological compound or a natural or synthetic compound.

In embodiments, the methods of the invention for identifying agents, particularly anti-cancer agents, comprise contacting more than one test compound in parallel. In some embodiments, the method comprises contacting 2,3, 4,5, 6,7, 8,9, 10, 15, 20, 25, 30, 40, 50, 100, 1000, at least 2, at least 5, at least 10, at least 50, at least 100, or at least 1000 test compounds in parallel. In some embodiments, high throughput screening of compounds and complete combinatorial libraries is assayed. Methods for performing high throughput screening are well known in the art. These methods may also be automated so that the robot can perform the experiments.

In embodiments, the methods of the invention for identifying an agent, particularly an anti-cancer agent, comprise the step of contacting a cell in the presence of a test compound. The cells can then be observed to determine whether the test compound affects USP11 activity, DCAF10, PALB2, ubiquitination of PALB2, KEAP1, CRL-KEAP1 activity, interaction of BRCA1 and PALB2, interaction of USP11 and PALB2, interaction of USP11 and DCAF10, and/or interaction of BRCA1, PALB2 and BRCA 2. Positive and negative controls can be performed in which known amounts of test compound and no compound are added to the assay, respectively. One skilled in the art can select and make appropriate controls.

The activity of the test compound may be unknown, and the methods of the invention may be used to identify compounds exhibiting selected properties (e.g., PARP inhibitor sensitizers). In some embodiments, the activity or type of activity of a test compound is known or expected, and the methods of the invention can be used to further characterize or optimize the activity (e.g., specificity, potency, etc.).

The methods of the present invention may further comprise measuring PARP activity in the presence of a test compound. PARP activity can be determined by measuring changes in poly (ADP-ribose) Polymers (PAR) and measuring NAD levels and/or ATP levels using routine methods by one of ordinary skill in the art. In some embodiments, the level of NAD is depleted in the presence of the test compound. In some embodiments, the level of ATP is depleted in the presence of the test compound. In some embodiments, the level of NAD is elevated in the presence of the test compound. In some embodiments, the level of ATP is increased in the presence of the test compound.

The method of the invention may comprise the step of determining whether the cells undergo necrosis after administration of the test compound. The physical properties of the cells can be analyzed to determine whether the cells undergo necrosis using conventional methods known to those skilled in the art. For example, necrosis can be determined by measuring organelle swelling, plasma membrane breakdown, intracellular vacuole formation, and nuclear degradation without coagulation.

The screening methods of the invention can also include therapeutic profiling of the efficacy and toxicity of the identified agent or other analog thereof in an animal; optionally formulating a pharmaceutical composition comprising one or more agents identified as having an acceptable therapeutic property; and optionally administering the agent to the subject or individual.

In one aspect, the therapeutic activity of agents and compositions identified using the methods of the invention can be evaluated in vivo using a suitable animal model. Thus, the screening methods of the invention may also include performing an in vivo study that includes administering the agent to a suitable animal model.

The present invention also provides a method of predicting or classifying a response to a PARP inhibitor in a subject comprising determining in a sample from the subject one or more of: USP11, DCAF10, BRCA1, BRCA2, PALB2, KEAP1, CRL3, CRL3-KEAP1, BRCA1-PALB2-BRCA2 complex, PALB2-USP11 complex and CRL3-KEAP1-PALB2 complex or PALB2 ubiquitination. Significantly different levels of one or more of USP11, DCAF10, BRCA1, BRCA2, PALB2, KEAP1, CRL3, CRL3-KEAP1, BRCA1-PALB2-BRCA2 complex, PALB2-USP11 complex, and CRL3-KEAP1-PALB2 complex or PALB2 ubiquitination, as compared to controls, are indicative of responsiveness (e.g., sensitivity) to a PARP inhibitor.

In one aspect, the present invention provides a method of predicting or classifying a response to a PARP inhibitor in a subject comprising detecting USP11, BRCA1, BRCA2, PALB2 and KEAP1 in a sample from the subject using the method of the invention. In one embodiment, significantly different levels of USP11, BRCA1, BRCA2, PALB2, and KEAP1 as compared to controls are indicative of responsiveness (e.g., sensitivity) to PARP inhibitors.

In one aspect, the present invention provides a method of predicting or classifying a response to a PARP inhibitor in a subject comprising detecting USP11, DCAF10, BRCA1, BRCA2, PALB, KEAP1 and CRL3 in a sample from said subject using the method of the invention. In one embodiment, significantly different levels of USP11, DCAF10, BRCA1, BRCA2, PALB, KEAP1 and CRL3, as compared to a control, are indicative of responsiveness (e.g., sensitivity) to PARP inhibitors.

In one aspect, a subject is classified as responsive to a PARP inhibitor if one or more of USP11, DCAF10, BRCA1, BRCA2, PALB2, KEAP1, CRL3, CRL3-KEAP1, BRCA1-PALB2, and BRCA1-PALB2-BRCA2 activity or expression, or PALB2 ubiquitination, is reduced as compared to a control. In one aspect, a subject is classified as responsive to a PARP inhibitor if one or more of USP11, DCAF10, BRCA1, BRCA2, PALB2, KEAP1, CRL3, CRL3-KEAP1, BRCA1-PALB2, and BRCA1-PALB2-BRCA2 activity or expression, or PALB2 ubiquitination, is increased as compared to a control. In one embodiment, a significantly different level (e.g., a lower level) of USP11 activity compared to a control is indicative of sensitivity to PARP inhibitors.

In one aspect, the present invention provides a method of predicting response to or classifying response to a PARP inhibitor in a subject, comprising detecting one or more of USP11, DCAF10, BRCA1, BRCA2, PALB2, KEAP1, CRL3 and CRL3-KEAP1 activity or expression or PALB2 ubiquitination in a sample from the subject and comparing to a control to determine whether the subject has response (e.g. is sensitive) to a PARP inhibitor.

In one aspect, the present invention provides a method of predicting response to or classifying response to a PARP inhibitor in a subject comprising detecting USP11, BRCA1, BRCA2, PALB2 and KEAP1 activity or expression in a sample from the subject and comparing to a control to determine whether the subject is responsive (e.g. sensitive) to a PARP inhibitor.

In one aspect, the present invention provides a method of predicting response to or classifying response to a PARP inhibitor in a subject comprising detecting USP11, DCAF10, BRCA1, BRCA2, PALB2, KEAP1 and CRL3 activity in a sample from the subject and comparing to a control to determine whether the subject is responsive (e.g., sensitive) to a PARP inhibitor.

In one aspect, the present invention provides a method of predicting or classifying a response to a PARP inhibitor in an individual comprising determining USP11 activity or expression in a sample from said individual using the method of the invention. In one embodiment, a significantly different level (e.g., a lower level) of USP11 activity or expression compared to a control is indicative of sensitivity to a PARP inhibitor.

In one aspect, the present invention provides a method of predicting or classifying a response to a PARP inhibitor in an individual comprising detecting KEAP1 in a sample from said individual and comparing to a control to determine whether said subject is sensitive to a PARP inhibitor. In one embodiment, a significantly different level (e.g., a higher level) of KEAP1 compared to a control is indicative of sensitivity to a PARP inhibitor.

The present invention also provides a method of predicting or classifying a response to a PARP inhibitor in an individual comprising detecting CRL3-KEAP1 activity in a sample from the individual and comparing to a control to determine whether the subject is sensitive to a PARP inhibitor. In one embodiment, a significantly different level (e.g., a higher level) of CRL3-KEAP1 as compared to a control is indicative of sensitivity to a PARP inhibitor.

The present invention also provides a method of predicting or classifying a response to a PARP inhibitor in an individual comprising detecting PALB2 ubiquitination in a sample from the individual and comparing to a control to determine whether the individual is sensitive to the PARP inhibitor. In one embodiment, a significantly different level of PALB2 ubiquitination compared to a control is indicative of sensitivity to a PARP inhibitor.

The present invention also provides a method of predicting or classifying a response to a PARP inhibitor in an individual comprising detecting a complex of BRCA1, PALB2 and BRCA2 in a sample from the individual and comparing to a control to determine whether the individual is responsive (e.g., sensitive) to the PARP inhibitor. In one embodiment, significantly different levels (e.g., absence or low levels) of the complex of BRCA1, PALB2, and BRCA2 are indicative of sensitivity to PARP inhibitors.

The present invention also provides a method for assigning an individual to one of a plurality of categories in a clinical trial of a PARP inhibitor, comprising determining USP11, DCAF10, PALB2, PALB2 ubiquitination, BRCA1-PALB2-BRCA2 complex, PALB2-USP11 complex, USP11-DCAF complex, KEAP1, CRL3-KEAP1 or CRL3-KEAP1-PALB2 complex in a sample from the individual using the method of the invention.

The present invention also provides a method for assigning an individual to one of a plurality of categories in a clinical trial of a PARP inhibitor comprising determining USP11 activity in a sample from the individual using the method of the invention.

The present invention also provides a method for assigning an individual to one of a plurality of categories in a clinical trial of a PARP inhibitor comprising determining CRL3-KEAP activity in a sample from the individual.

The present invention also provides a method for assigning an individual to one of a plurality of categories in a clinical trial of PARP inhibitors comprising detecting or quantifying USP11, BRCA1, BRCA2, PALB2 and KEAP1 in a sample from the individual.

The present invention also provides a method for assigning an individual to one of a plurality of categories in a clinical trial of a PARP inhibitor comprising detecting or quantifying USP11, DCAF10, BRCA1, BRCA2, PALB2 and KEAP1 in a sample from said individual.

The present invention also provides a method for assigning an individual to one of a plurality of categories in a clinical trial of a PARP inhibitor, comprising detecting or quantifying the BRCA1-PALB2-BRCA2 complex in a sample from the individual.

The present invention also provides a method for assigning an individual to one of a plurality of classes in a clinical trial of PARP inhibitors comprising detecting or quantifying PALB2 ubiquitination in a sample from said individual.

In one aspect, individuals are assigned to a category for clinical trials of PARP inhibitors based on a reduction in one or more of USP11, DCAF10, BRCA1, BRCA2, PALB2, KEAP1, CRL3, CRL3-KEAP1, BRCA1-PALB2, and BRCA1-PALB2-BRCA2 activity or expression, or PALB2 ubiquitination, as compared to controls. In one aspect, individuals are assigned to a category for clinical trials of PARP inhibitors based on an increase in one or more of USP11, DCAF10, BRCA1, BRCA2, PALB2, KEAP1, CRL3, CRL3-KEAP1, BRCA1-PALB2, and BRCA1-PALB2-BRCA2 activity or expression, or PALB2 ubiquitination, as compared to controls.

Various conventional methods known to those skilled in the art can be used to detect or determine the biomarkers USP11, DCAF10, BRCA1, BRCA2, PALB2, KEAP1, CRL3 and/or complexes thereof in a sample. The level of a biomarker present in a sample may be determined by any suitable assay, which may include the use of any one of the following, including or consisting of: immunoassay, spectroscopy, mass spectrometry, matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, microscopy, northern blotting, isoelectric focusing, SDS-PAGE, PCR, quantitative RT-PCR, gel electrophoresis, DNA microarrays, and antibody microarrays, or a combination thereof.

The present invention also provides a system comprising: an assay for determining the level of USP11 activity, complex or biomarker level in a sample obtained from the subject; a processor for processing the results; computer code instructions for comparing the results to a database; and a user display for providing the comparison results. The database may contain reference values for USP11 activity or biomarker levels.

Method of treatment

The methods of the invention for predicting or characterizing responsiveness to a PARP inhibitor may further comprise administering a PARP inhibitor to an individual or subject. In one aspect, the present invention relates to a method of treating a subject with a PARP inhibitor comprising:

a) determining the responsiveness or sensitivity of a sample from a subject to one or more PARP inhibitors using the methods of the invention;

b) identifying a PARP inhibitor to which a subject is effectively responsive or sensitive; and

c) administering the PARP inhibitor to a subject.

In one aspect, the present invention provides a method for treating a patient in need of treatment with a PARP inhibitor, comprising (a) requesting a test that provides an assay result to determine whether the patient is sensitive to or has a response to the PARP inhibitor: determining whether the patient is sensitive to a PARP inhibitor by detecting USP11, DCAF10, BRCA1, BRCA2, PALB2, KEAP1, and/or CRL3 in a sample from the subject and comparing to a control; and (b) administering a PARP inhibitor to the patient if the patient is sensitive to the PARP inhibitor. In one aspect of the method of the invention, the patient has breast cancer. In one aspect of this method of the invention, the patient has ovarian cancer. In one aspect of the invention, the assay detects USP11 expression or activity using the methods disclosed herein.

The present invention also provides a method for treating cancer in a subject, comprising: (i) selecting a subject having a response to a PARP inhibitor using the methods of the invention, and (ii) administering to said subject an effective amount of a PARP inhibitor to treat cancer. In one embodiment, the cancer is breast cancer. In one embodiment, the cancer is ovarian cancer.

The agents identified using the methods of the invention have a number of therapeutic applications relating to, for example, cancer, ischemia reperfusion injury, inflammatory disease, degenerative disease, protection from adverse effects of cytotoxic compounds, and enhanced cytotoxic cancer therapy. Agents identified using the methods of the invention can be used to enhance radiation and chemotherapy by reducing apoptosis of cancer cells, limiting tumor growth, reducing metastasis, and prolonging survival of tumor-bearing subjects. In various aspects of the invention, the agents may be used to treat leukemia, colon cancer, glioblastoma, lymphoma, melanoma, breast cancer, ovarian cancer, and cervical cancer.

In other aspects of the invention, the agents are useful for treating, but not limited to, retroviral infections, arthritis, gout, inflammatory bowel disease, CNS inflammation, multiple sclerosis, allergic encephalitis, sepsis, septic shock, hemorrhagic shock, pulmonary fibrosis, uveitis, diabetes, parkinson's disease, myocardial infarction, stroke, other nerve trauma, organ transplantation, eye reperfusion, kidney reperfusion, intestinal reperfusion, skeletal muscle reperfusion, hepatic toxicity following acetaminophen overdose, toxicity of cardiac and renal doxorubicin and platinum-based antineoplastic drugs, and skin injury secondary to sulfur mustard.

In some embodiments, the present invention provides a method for sensitizing an individual to treatment with a PARP inhibitor comprising identifying an agent that sensitizes a cell to a PARP inhibitor according to the methods of the present invention and administering the agent to the individual.

In some embodiments, the present invention provides methods for treating an individual treated with a PARP inhibitor comprising administering to the individual an agent that sensitizes cells to the PARP inhibitor identified using the methods of the present invention.

In some embodiments, the present invention provides a method for reversing resistance or delaying the onset of resistance to a PARP inhibitor in an individual comprising identifying an agent that reverses resistance or delays the onset of resistance to a PARP inhibitor according to the methods of the present invention and administering the agent to the individual.

In some embodiments, the present invention provides a method of treating cancer in an individual comprising identifying an anti-cancer agent according to the method of the present invention, and administering the agent to the individual.

In another embodiment, the invention provides a method of treating leukemia, colon cancer, glioblastoma, lymphoma, melanoma, breast cancer, ovarian cancer, or cervical cancer in a mammal in need of such treatment comprising administering to the mammal a therapeutically acceptable amount of an agent identified using the methods of the invention, or a therapeutically acceptable salt thereof.

In another embodiment, the present invention provides a method of enhancing a cytotoxic cancer therapy in a mammal in need of such treatment comprising administering to the mammal a therapeutically acceptable amount of an agent identified using the methods of the present invention that enhances a cytotoxic cancer therapy, or a therapeutically acceptable salt thereof.

In one aspect, the invention provides a method of treating a disease associated with HR deficiency (i.e. HR disease) in an individual comprising identifying an agent that modulates HR according to the method of the invention and administering the agent to the individual.

In another embodiment, the invention provides the use of an agent identified using the methods of the invention in the manufacture of a medicament for the treatment of HR disease in a mammal in need of such treatment. In another embodiment, the invention provides the use of an agent identified using the methods of the invention in the manufacture of a medicament for inhibiting tumor growth in a mammal in need of such treatment. In another embodiment, the invention provides the use of an agent identified using the methods of the invention in the manufacture of a medicament for treating cancer in a mammal in need of such treatment. In another embodiment, the invention provides the use of an agent identified using the methods of the invention in the manufacture of a medicament for treating leukemia, colon cancer, glioblastoma, lymphoma, melanoma, breast cancer, ovarian cancer, or cervical cancer in a mammal in need of such treatment. In another embodiment, the invention provides the use of an agent identified using the methods of the invention in the manufacture of a medicament for potentiating a cytotoxic cancer therapy in a mammal in need of such treatment, comprising administering to the mammal a therapeutically acceptable amount of the agent.

In one embodiment, the invention provides a pharmaceutical composition comprising an agent identified using the methods of the invention, or a therapeutically acceptable salt thereof, in combination with a therapeutically acceptable carrier. The pharmaceutical compositions can be prepared by methods known per se for the preparation of pharmaceutically acceptable compositions administrable to a subject, such that an effective amount of the active substance is combined with a pharmaceutically acceptable vehicle. Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the compositions include, but are not limited to, solutions of the active agent in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in a buffer solution having a suitable pH and being isotonic with physiological fluids.

Pharmaceutical compositions are defined as therapeutic agents alone or in combination with other therapeutic agents or other forms of treatment. The compositions of the present invention may be administered simultaneously, separately or sequentially with other therapeutic agents or therapies (e.g., PARP inhibitors).

Homologous recombination method

The present invention provides a method for activating or modulating homologous recombination in a cell, comprising:

(a) promoting or stimulating the assembly or occurrence of BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex in a cell;

(b) promoting or stimulating recruitment of BRCA 1to the site of DNA Double Strand Break (DSB);

(c) contacting a cell with BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex;

(d) suppression KEAP1 or CRL3-KEAP 1;

(e) inhibit degradation of USP11 or promote USP11 activity; and/or

(f) Inhibiting DCAF 10.

In one aspect, the cell is in the G1 phase of the cell cycle (G1). In one aspect, the cell is a non-dividing cell or a quiescent cell in G1. In one aspect, the cell is in the G0 phase of the cell cycle (G0). In one aspect, the methods of the invention are used in vitro to activate or modulate homologous recombination in a cell.

In one aspect, the invention provides a method for activating or modulating homologous recombination in a cell, particularly a cell in the G1 phase (G1) or the G0 phase (G0) of the cell cycle, comprising (a) promoting or stimulating the assembly or occurrence of a BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex in the cell; and/or (b) contacting the cell with BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex.

In embodiments, assembly of the BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex is promoted or stimulated by administration of an agent that promotes or stimulates the BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex identified using the methods of the invention.

In one aspect, the invention provides a method for activating or modulating homologous recombination in a cell, in particular in a G1 or G0 cell, comprising the step of inhibiting KEAP1 or CRL3-KEAP 1. In one aspect, the invention provides a method for activating or modulating homologous recombination in a cell, particularly a G1 or G0 cell, comprising the step of blocking USP11 degradation or promoting USP11 activity. In one embodiment, the method comprises administering USP11 or an agonist thereof. In one aspect, the invention provides a method for activating homologous recombination in a cell, in particular a G1 or G0 cell, comprising the steps of inhibiting KEAP1 and blocking USP11 degradation. In one aspect, the invention provides a method for activating homologous recombination in a cell, particularly a G1 or G0 cell, comprising the steps of inhibiting CRL3-KEAP1 and blocking USP11 degradation. In one aspect, the invention provides a method for activating homologous recombination in a cell, particularly a G1 or G0 cell, comprising the steps of inhibiting CRL3 and blocking USP11 degradation.

The method of the invention may be carried out in a cell, in particular in a cell in the G1 phase of the cell cycle (G1) or in the G0 phase of the cell cycle (G0), in which DNA end excision is activated or has been activated to produce single-stranded DNA.

The present invention provides a method for activating or modulating homologous recombination in a cell, in particular in a cell in the G1 phase of the cell cycle (G1) or in the G0 phase of the cell cycle (G0), wherein DNA end excision is activated or has been activated to produce single stranded DNA, said method comprising promoting or stimulating the assembly or occurrence of a BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex in the cell. In one embodiment, assembly of the complex is promoted or stimulated by administering an agent that promotes or stimulates assembly or occurrence of the BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex identified using the methods of the invention.

The invention also provides a method for repairing a DNA double strand break in a cell of the G1 phase of the cell cycle (G1) or the G0 phase of the cell cycle (G0), comprising promoting or stimulating the assembly or occurrence of a BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex in the cell.

The invention also provides a method for repairing a DNA double strand break in a cell of the G1 phase of the cell cycle (G1) or the G0 phase of the cell cycle (G0), wherein DNA end excision is activated or has been activated to produce single stranded DNA, comprising promoting or stimulating the assembly or occurrence of a BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex in said cell. In one embodiment, assembly of the complex is promoted or stimulated by administering an agent that promotes or stimulates assembly or occurrence of the BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex identified using the methods of the invention.

The invention also provides a method for repairing a DNA double strand break in a cell of the G1 phase of the cell cycle (G1) or the G0 phase of the cell cycle (G0), wherein DNA end excision is activated or has been activated to produce single stranded DNA, comprising contacting the cell with a BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex.

The invention also provides a method for repairing a DNA double strand break in G1 or G0 cells, wherein DNA end excision is activated or has been activated to produce single stranded DNA, the method comprising (a) inhibiting KEAP1 and/or CRL3-KEAP 1; (b) blocking the degradation of USP11 or promoting or stimulating USP11 activity; (c) administering USP11 or an agonist thereof; and/or (d) inhibits CRL3-KEAP1 and blocks degradation of USP 11.

Methods for activating homologous recombination in a cell may further comprise activating or promoting a single-stranded dna (ssdna) production pathway. In one aspect, the ssDNA production pathway is activated by DNA end excision. In one embodiment, the method for activating homologous recombination in a cell further comprises activating DNA end excision.

In one embodiment, DNA end excision is activated or promoted by inhibiting 53BP1 (or RIF1) expression or activity (e.g., recruitment of 53BP 1to the DSB site) and/or up-regulating or expressing CtIP. In one embodiment, DNA end excision is activated or promoted by inhibiting 53BP1 (or RIF1) expression or activity (e.g., recruitment of 53BP 1to the DSB site) and upregulating or expressing CtIP. In one embodiment, the method comprises inhibiting 53BP1 with antagonists, including but not limited to short interfering (si) RNA, short hairpin (sh) RNA, and microrna (mirna), or inhibitors of Histone Deacetylase (HDAC) family enzymes (e.g., trichostatin a), and using analogs of CtIP that mimic constitutive phosphorylation, such as CtIP-Thr879 Glu.

Methods for activating homologous recombination in a cell can include activating recruitment of BRCA 1to a DNA Double Strand Break (DSB) site. In one embodiment, BRCA1 recruitment is activated by inhibiting expression of 53BP1(TP53BP1) or RIF1, or inhibiting recruitment of 53BP1 or RIF 1to the DSB site. Antagonists including, but not limited to, short interfering (si) RNA, short hairpin (sh) RNA, and microrna (mirna) can be used to inhibit 53BP1 or RIF 1. In one embodiment, 53BP1(Fukuda T. et al, Cancer Sci.2015 August; 106(8):1050-6.doi:10.1111/cas.12717.2015, 14. 7.2015, electronic publication) is inhibited using an inhibitor of a Histone Deacetylase (HDAC) family of enzymes, in particular a histone deacetylase inhibitor (HDACI), preferably trichostatin.

In one aspect, the method for activating or stimulating homologous recombination in a cell further comprises a gene editing system. In one aspect, the gene editing system comprises contacting the cell with a nuclease. Examples of nucleases include, but are not limited to, Zinc Finger Nucleases (ZFNs), engineered meganucleases, transcription activator-like effector nucleases (TALENS), megaendonucleases or homing endonucleases, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -associated (Cas) proteins, fusions between Ttago nucleases and nucleases, such as megatals and compact TALENS.

In one aspect, the gene editing step comprises a TALEN system.

In one aspect, the gene editing step comprises a ZFN system.

In one aspect, the gene editing step comprises the CRISPR/Cas9 system.

In aspects of the invention, the gene editing system can correct for genome modification. The genetic modification may comprise at least one mutation in a polynucleotide sequence having a locus associated with a genetic disorder. In one aspect, the genomic modification is selected from the group consisting of an insertion, a deletion, and combinations thereof. In some embodiments, the genetic disorder is a monogenic disorder. In some embodiments, the disorder is a polygenic disorder. In some embodiments, the disorder is associated with one or more SNPs. In a specific embodiment of the invention, the genomic modification corrects for a point mutation.

In one aspect of the methods of the invention to correct genome modification, a gene editing system comprises contacting a cell with a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids direct the Cas protein to and hybridize to a selected motif of a target polynucleotide sequence associated with a genetic disorder, wherein the target polynucleotide sequence is cleaved.

In one aspect, the invention provides a method for altering a genetic disorder associated with a target polynucleotide sequence in a cell, comprising: (1) contacting said cell with a system that activates homologous recombination in said cell, wherein said system comprises BRCA1-PALB2 or BRCA1-PALB2-BRCA2 or an agent that maintains BRCA1-PALB2 or BRCA1-PALB2-BRCA2 interactions throughout the cell cycle; and (2) contacting the target polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids direct the Cas protein to and hybridize to selected motifs of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved. The methods can reduce expression of, knock out, or correct a target polynucleotide sequence from an undesired sequence to a desired sequence.

In one aspect, the invention provides a method for altering a genetic disorder associated with a target polynucleotide sequence in a cell, comprising: (1) contacting the cell with a system that activates homologous recombination in the cell, wherein the system comprises a kit, vector or composition of the invention, in particular the system comprises a 53BP1 inhibitor, a KEAP1 inhibitor or a DCAF10 inhibitor, and a CtIP analog that mimics constitutive phosphorylation, preferably the system comprises a KEAP1 inhibitor, a 53BP1 inhibitor selected from short interfering (si) RNA, short hairpin (sh) RNA and microrna (mirna), and CtIP-Thr879 Glu; and (2) contacting the target polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids direct and hybridize the Cas protein to selected motifs of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved. The methods can reduce expression of, knock out, or correct a target polynucleotide sequence from an undesired sequence to a desired sequence.

The present invention contemplates a method for treating or preventing a genetic disorder in a subject, the method comprising altering a target polynucleotide sequence associated with the genetic disorder in a cell by contacting the cell with a system that activates homologous recombination in the cell, wherein the system comprises BRCA1-PALB2 or BRCA1-PALB2-BRCA2 or an agent that maintains BRCA1-PALB2 or BRCA1-PALB2-BRCA2 interactions throughout the cell cycle; and contacting the target polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids direct the Cas protein to and hybridize to selected motifs of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, thereby treating or preventing the genetic disorder.

In one aspect, there is provided a method for treating or preventing a genetic disorder in a subject, the method comprising: (a) altering a target polynucleotide sequence associated with a genetic disorder in a cell by contacting the cell with a system that activates homologous recombination in the cell, wherein the system comprises a kit, vector or composition of the invention, in particular the system comprises a 53BP1 inhibitor, a KEAP1 inhibitor or a DCAF10 inhibitor, and a CtIP analog that mimics constitutive phosphorylation, preferably the system comprises a KEAP1 inhibitor, a 53BP1 inhibitor selected from short interfering (si) RNA, short hairpin (sh) RNA and microrna (mirna), and CtIP-Thr879 Glu; and (b) contacting the target polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids direct the Cas protein to and hybridize to selected motifs of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved, thereby treating or preventing the genetic disorder.

Methods for treating or preventing a genetic disorder include introducing a cell into a subject, thereby treating or preventing a genetic disorder associated with a target polynucleotide sequence. The method may comprise repairing the cleaved target polynucleotide sequence by inserting an exogenous template polynucleotide, wherein the repair results in a mutation comprising an insertion, deletion or substitution of one or more nucleotides of the target polynucleotide sequence.

In one aspect, the target polynucleotide sequence is associated with a genetic disorder of the lung. In one embodiment, the target polynucleotide sequence is associated with cystic fibrosis, in particular the polynucleotide sequence is a cystic fibrosis transmembrane conductor receptor (CFTR) locus. Mutations in CFTR (e.g., a deletion of phenylalanine at position 508 in exon 11) cause cystic fibrosis.

In one aspect, the target polynucleotide sequence is associated with a genetic disorder of muscle. In one aspect, the target polynucleotide sequence is associated with muscular dystrophy. In one aspect, the target polynucleotide sequence is associated with Duchenne Muscular Dystrophy (DMD), a mutation in the dystrophin gene. In one aspect, the target polynucleotide sequence is associated with becker muscular dystrophy (a mutation in the dystrophin gene). In one aspect, the target polynucleotide is associated with myotonic dystrophy type 1 (mutation in DMPK gene) or myotonic dystrophy type 2 (CNBP gene mutation). In one aspect, the target polynucleotide sequence is associated with sickle cell anemia (mutant HBB hemoglobin).

In various aspects of the invention, the targeting polynucleotide sequence is associated with a liver genetic disorder in one aspect, the targeting polynucleotide sequence is associated with α -1 antitrypsin deficiency (a mutation in the SERPINA1 gene) in one aspect, the targeting polynucleotide sequence is associated with wilson's disease (a mutation in the gene encoding ATP7B Cu translocase).

In one aspect, the methods of the invention further comprise providing a functional protein having enhanced properties compared to its naturally occurring counterpart, in particular a functional protein lacking or deficient in a subject, e.g., for use in the treatment of a genetic disorder. In an embodiment of the invention, the method comprises integrating a sequence encoding a functional protein in a cell of a subject in need thereof by sequential administration of a gene editing system and one or more transgenes encoding a non-naturally occurring protein having enhanced properties compared to its naturally occurring counterpart. In other embodiments, the method comprises administering to the subject a genetically modified cell that expresses a functional form of one or more proteins aberrantly expressed in the subject. Thus, isolated cells may be introduced into a subject (ex vivo cell therapy), or cells may be modified when they are part of a subject (in vivo). In certain embodiments, the transgene is delivered using a viral vector, a non-viral vector, and/or a combination thereof.

The invention also provides a method of inhibiting homologous recombination in a cell, particularly a G1 cell, comprising inhibiting the assembly of the BRCA1-PALB2-BRCA2 complex in the cell. In one embodiment, the interaction is inhibited by administering KEAP1 or CRL3-KEAP1 or an agonist thereof. In one embodiment, the interaction is inhibited by administration of a USP11 antagonist/inhibitor (e.g., mitoxantrone). In one embodiment, the interaction is inhibited by administering an agent that inhibits homologous recombination identified using the methods of the invention.

The components of the methods of the invention may be delivered by delivery systems known in the art, including but not limited to viral-based systems or non-viral-based systems. Conventional virus-based systems may comprise, for example, retroviral, lentiviral, adenoviral, adeno-associated viral and herpes simplex viral vectors for gene transfer. In one aspect, the expression vector is selected from the group consisting of a plasmid vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. In one embodiment, the virus-based system is an adenoviral vector or an adeno-associated viral vector. Examples of non-virus based systems include lipofection, nuclear transfection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycations or lipids nucleic acid conjugates, naked DNA, artificial viral particles, and agents that enhance DNA uptake.

In one aspect, the invention provides vectors comprising an activator or modulator of homologous recombination, and optionally an activator or modulator of DNA end-excision. In one aspect, the invention provides a vector (e.g., a viral vector) comprising one or more of the following components encoded in the vector: 1) activators of DNA end-excision, such as inhibitors of 53BP1 (or RIF) expression or activity and/or CtIP compounds that mimic constitutive phosphorylation; 2) factors that activate homologous recombination, such as factors that maintain the interaction of BRCA1-PALB2 or BRCA1-PALB2-BRCA2 in the cell cycle; and optionally 3) components of a gene editing system, in particular components of a CRISPR system, TALEN system or zinc finger nuclease system. In one embodiment, the components of the gene editing system are encoded in one or more separate expression vectors.

In another aspect, the invention provides compositions comprising an activator or modulator of homologous recombination and optionally an activator or modulator of DNA end-excision. In one aspect, the present invention provides a composition comprising one or more of the following components: 1) activators of DNA end-excision, such as inhibitors of 53BP1 (or RIF) expression or activity and/or CtIP compounds that mimic constitutive phosphorylation; 2) factors that activate homologous recombination, such as factors that maintain the interaction of BRCA1-PALB2 or BRCA1-PALB2-BRCA2 in the cell cycle; and optionally 3) components of a gene editing system, in particular components of a CRISPR system, TALEN system or zinc finger nuclease system. In one embodiment, the components of the gene editing system are in one or more separate compositions.

Examples of activators of DNA end-excision include, but are not limited to, the coding sequence of CtIP-Thr847Glu, shRNA against TP53BP1 mRNA, and shRNA against KEAP 1. shRNA directed to TP53BP1 may be replaced with shRNA directed to RIF1 or an agent that blocks recruitment of 53BP 1to the DSB site, including the dominant-negative 53BP1 protein. shRNA directed against KEAP1 may be substituted with the coding sequence of a PALB2 mutant containing mutations at its Lys20, Lys25 and Lys30 residues or containing mutations interfering with its interaction with KEAP 1.

Examples of factors that maintain a BRCA1-PALB2 or BRCA1-PALB2-BRCA2 interaction during the cell cycle include, but are not limited to, inhibitors of KEAP1, inhibitors of DCAF10, RNA interference agents that maintain USP11 expression in G0 and G1 cells, or PALB2 in a mutated form insensitive to ubiquitination of KEAP1-CUL3-RBX1 involving mutation of one or more of Lys20, Lys25, or Lys30 residues. An example of a KEAP1 inhibitor is an antibody analogue (monobody) of a potent competitive inhibitor of the KEAP1-NRF2 interaction disclosed in Guntas, g, et al, [44 ]. KEAP1 inhibitors are also described in, for example, Canning p. et al, Acta Pharm Sin b.,2015(4):285-99 and Wells, g., Biochem soctrans.2015,43(4): 674-9.

In one embodiment, the vector of the invention comprises sequences encoding a 53BP1 inhibitor, a KEAP1 inhibitor or a DCAF10 inhibitor and a CtIP analog that mimics constitutive phosphorylation. In a specific embodiment, the vectors of the invention comprise sequences encoding a KEAP1 inhibitor (e.g., R1 KEAP1 inhibitor; see example 3), a 53BP1 inhibitor, and CtIP-Thr879 Glu. In one embodiment, the vector of the invention comprises a sequence encoding: KEAP1 inhibitors (e.g., R1 KEAP1 inhibitors; see example 3), 53BP1 inhibitors selected from short interfering (si) RNA, short hairpin (sh) RNA and microRNA (miRNA), and CtIP-Thr879 Glu.

In one embodiment, the composition of the invention comprises a 53BP1 inhibitor, a KEAP1 inhibitor or a DCAF10 inhibitor and a CtIP analog that mimics constitutive phosphorylation. In a particular embodiment, the compositions of the invention comprise a KEAP1 inhibitor (e.g., R1 KEAP1 inhibitor; see example 3), a 53BP1 inhibitor, and CtIP-Thr879 Glu. In a specific embodiment, the compositions of the invention comprise a KEAP1 inhibitor (e.g., R1 KEAP1 inhibitor; see example 3) selected from short interfering (si) RNA, short hairpin (sh) RNA, and microrna (mirna)53BP1 inhibitor and CtIP-Thr879 Glu.

Reagent kit

The invention also provides kits for performing the assays or methods disclosed herein or comprising the compositions or vectors disclosed herein. In one embodiment, the kit of the invention comprises at least one reagent for determining USP11 activity in a sample. In another embodiment, the kit of the invention comprises at least one reagent for determining BRCA1-PALB2-BRCA2, PALB2-KEAP1, BRCA1-PALB2, USP11-DCAF10, or USP11-PALB2 complex in a sample. In another embodiment, the kit of the invention comprises at least one reagent for determining BRCA1-PALB2-BRCA2, PALB2-KEAP1, or USP11-PALB2 complex in a sample. In another embodiment, the kit of the invention comprises reagents for determining the level of BRCA1, BRCA2, PALB2, USP11, DCAF10 and KEAP1 in a sample. In another embodiment, the kit of the present invention comprises at least one reagent for determining ubiquitination of PALB2, in particular at the N-terminus of PALB2, in a sample. In some embodiments, the reagent is an antibody or a nucleic acid or primer for use in a PCR reaction.

The kit may also contain instructions for appropriate operating parameters in the form of inserts. The instructions may inform the consumer how to collect the sample. The kit may contain a sample to be used as a standard for calibration and comparison. The kit may further comprise instructions for comparing the level of activity or biomarker detected in the sample to a calibration sample or chart. The kit may further comprise instructions for indicating the level of activity or biomarker to diagnose a disease disclosed herein.

In one aspect, the invention provides a kit comprising one or more components of the method of the invention for activating homologous recombination and optionally components of a gene editing system. The kits of the invention may also comprise or be used in combination with a CRISPR system, a TALEN system, or a zinc finger nuclease system. In one embodiment, the kits of the invention comprise or are used in combination with a CRISPR system. In one embodiment, the kits of the invention comprise or are used in combination with a TALEN system. In one embodiment, the kit of the invention comprises or is used in combination with a zinc finger nuclease system.

In some embodiments, the kits of the invention comprise a carrier system and instructions for using the kit. In one aspect, the kit comprises a vector comprising an activator of DNA end excision and an activator of homologous recombination as discussed herein. In one aspect, the kit comprises one or more vectors (e.g., viral vectors) comprising one or more of the following components: 1) activators of DNA end-excision, such as inhibitors of 53BP1 (or RIF) expression or activity and/or CtIP compounds that mimic constitutive phosphorylation; 2) factors that activate homologous recombination, such as factors that maintain the BRCA1-PALB2 interaction during the cell cycle; and optionally 3) components of a gene editing system, in particular components of a CRISPR system, TALEN system or zinc finger nuclease system. Examples of factors that maintain the BRCA1-PALB2 interaction during the cell cycle are described herein, and include, but are not limited to, inhibitors of KEAP1, such as RNA interference agents that maintain USP11 expression in G0 and G1 cells or mutant forms of PALB2 that are insensitive to ubiquitination of KEAP1-CUL3-RBX1, involving mutation of one or more of Lys20, Lys25 or Lys30 residues. Examples of activators of DNA end-excision include, but are not limited to, the coding sequence of CtIP-Thr847Glu, shRNA against TP53BP1 mRNA, and shRNA against KEAP 1. shRNA directed to TP53BP1 may be replaced with shRNA directed to RIF1 or an agent that blocks recruitment of 53BP 1to the DSB site, including the dominant-negative 53BP1 protein. shRNA directed against KEAP1 may be substituted with the coding sequence of a PALB2 mutant containing mutations at its Lys20, Lys25 and Lys30 residues or containing mutations disrupting its interaction with KEAP 1.

In one embodiment, the kit of the invention comprises one or more vectors comprising sequences encoding 53BP1 inhibitor, KEAP1 inhibitor or DCAF10 inhibitor and a CtIP analog that mimics constitutive phosphorylation. In a specific embodiment, the kits of the invention comprise one or more vectors comprising sequences encoding a KEAP1 inhibitor (e.g., R1 KEAP1 inhibitor; see example 3), a 53BP1 inhibitor, and CtIP-Thr879 Glu. In one embodiment, the invention comprises one or more vectors comprising a sequence encoding: KEAP1 inhibitors (e.g., R1 KEAP1 inhibitors; see example 3), 53BP1 inhibitors selected from short interfering (si) RNA, short hairpin (sh) RNA, and microrna (mirna)), and CtIP-Thr879 Glu.

In some embodiments, a kit of the invention comprises a composition of the invention and instructions for using the kit. In one aspect, the kit comprises a composition comprising an activator or modulator of DNA end excision as discussed herein and an activator or modulator of homologous recombination. In one aspect, a kit comprises a composition comprising one or more of the following components: 1) activators of DNA end-excision, such as inhibitors of 53BP1 (or RIF) expression or activity and/or CtIP compounds that mimic constitutive phosphorylation; 2) factors that activate homologous recombination, such as factors that maintain the interaction of BRCA1-PALB2 or BRCA1-PALB2-BRCA2 in the cell cycle; and optionally 3) components of a gene editing system, in particular components of a CRISPR system, TALEN system or zinc finger nuclease system. In one embodiment, the components of the gene editing system are in separate kits.

In one embodiment, the kit of the invention comprises a composition comprising a 53BP1 inhibitor, a KEAP1 inhibitor or a DCAF10 inhibitor, and a CtIP analog that mimics constitutive phosphorylation. In a particular embodiment, the kit of the invention comprises a composition comprising a KEAP1 inhibitor (e.g., R1 KEAP1 inhibitor; see example 3), a 53BP1 inhibitor, and CtIP-Thr879 Glu. In a particular embodiment, the kits of the invention comprise a composition comprising a KEAP1 inhibitor (e.g., R1 KEAP1 inhibitor; see example 3), a 53BP1 inhibitor selected from short interfering (si) RNA, short hairpin (sh) RNA, and microrna (mirna)), and CtIP-Thr879 Glu.

In various aspects, the kits of the invention are used in combination with gene editing kits, in particular kits for CRISPR systems, TALEN systems, or zinc finger nuclease systems. Gene editing kits are commercially available from, for example, Addgene (Cambridge, MA), ThermoFisher Scientific, System Biosciences Inc. and OriGene technologies (MD), Clontech.

The following non-limiting examples are illustrative of the present invention:

example 1

The following materials and methods were used in the studies described in the examples.

Plasmids

The cDNA of PALB2 was obtained from a Mammalian Gene Collection (MGC). Full-length PALB2 and BRCA1 were amplified by PCR, subcloned into pDONR221, and delivered into pDEST-GFP, pDEST-Flag, and mCherry-LacR vectors using Gateway cloning technology (Invitrogen). Similarly, the coiled-coil domain (residues 1363-1437) of BRCA1 was connectedWas amplified by PCR, subcloned into the pDONR221 vector and delivered to the mCherryLacR and pDEST-GFP vectors. The N-terminal domain of PALB2 was amplified by PCR and introduced into the GST expression vector pET30-2-His-GST-TEV [31 ] using EcoRI/XhoI sites]. The coiled-coil domain of BRCA1 was cloned into pMAL-c2 using a BamHI/SalI site. Truncated forms of PALB2 were obtained by introducing stop codons or deletions by site-directed mutagenesis. The full-length CtIP was amplified by PCR, subcloned into pDONR221, and delivered to the lentiviral construct pCW57.1 (Dr. David Root gift; Addgene plasmid #41393) using Gateway cloning technology (Invitrogen). USP11cDNA was donated by David cortex and amplified by PCR and cloned into the pDsRed2-C1 vector using EcoRI/SalI sites. The bacterial codon optimized coding sequence of porcine USP11(USP11) was subcloned into the 6 XHis-GST vector pETM-30-Htb using the BamHI/EcoRI site. anti-siRNA forms of the PALB2, BRCA1 and USP11 constructs were generated as described previously [14 ]]. Full-length CUL3 and RBX1 were amplified by PCR from a human pancreatic cDNA library (Invitrogen) and cloned into the dual expression pFBDM vector using NheI/XmaI and bshii/NotI, respectively, as previously described. The NEDD8 cDNA was gifted by Dmitris Xiriodamas and fused at its C-terminus to a double StrepII tag in pET17b vector (Millipore). Human DEN1 was amplified from the vector supplied by Aude Echalier and fused by PCR to a non-cleavable N-terminal StrepII2x tag and inserted into the pET17b vector. The pCOOL-mKEAP1 plasmid was gifted by Feng Shao doctor. pcDNA3-HA2-KEAP1 and pcDNA3-HA2-KEAP 1. delta. BTB were gifted by the Yue Xiong doctor (Addgene plasmids #21556 and 21593). gRNAs were synthesized and processed as previously described [33 ]]. The annealed gRNAs were cloned into the Cas9 expression vector pSpCas9(BB) -2A-Puro (PX459) or pX330-U6-Chimeric _ BB-CBh-hSpCas9(Addge plasmid #48139 and 42230) as fed by Feng Zhang. gRNAs targeting the LMNA or PML locus and mClover-labeled LMNA or PML were as previously described [45]. Lentiviral packaging vector psPAX2 and envelope vector VSV-G were gifted by Didier Trono (Addgene plasmids #12260 and 12259). His coupling Using XhoI/HindIII sites₆Ubiquitin was cloned into pcDNA5-FRT/TO backbone. All mutations were introduced by site-directed mutagenesis using quikchange (stratagene) and all plasmids were sequence verified.

Cell culture and plasmid transfection

All media were supplemented with 10% Fetal Bovine Serum (FBS). U-2-OS (U2OS) cells were cultured in McCoy medium (Gibco). 293T cells were cultured in DMEM (Gibco). Parental cells were tested for mycoplasma contamination and verified by STR DNA profiling. Plasmid transfection was performed using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's protocol. Lentiviral infection was performed as described previously [18 ]. U2OS and 293T cells were purchased from atcc.u2os 256 cells and donated by r.greenberg.

Antibodies

The following antibodies were used: rabbit anti-BP 1(A300-273A, Bethyyl), rabbit anti-53 BP1(sc-22760, Santa Cruz), mouse anti-53 BP1(#612523, BD Biosciences), mouse anti-gamma-H2 AX (clone JBW301, Millipore), rabbit anti-gamma-H2 AX (#2577, Cell Signaling Technologies), rabbit anti-KEAP 1(ab66620, Abcam), rabbit anti-NRF 2(ab62352, Abcam), mouse anti-Flag (clone M2, Sigma), mouse anti-tubulin (CP06, Calbiochem), mouse anti-GFP (#11814460001, Roche), mouse anti-CCNA (MONX10262, Monosan), rabbit anti-BRCA 2(ab9143, Abcam), mouse anti-BRCA 2 OP (84, Calbiochem), rabbit anti-BRBiotechnologies (# 58613, Rabbit anti-rabbit (Bruch) and mouse anti-rabbit anti-mouse (Bruch 4670, Bruch) and mouse anti-mouse Ab 4670, mouse anti-rabbit LPE 4670 (RPE) and mouse anti-rabbit MRAB 33, mouse anti-rabbit MRAB 33, rabbit anti-rabbit MRAB 11, rabbit anti-rabbit MRB 4670, rabbit anti-rabbit MRAB, mouse (RPE) and mouse RPE 7, MRAB 33, MRAB 4670, MRSA 3, MR, bethyl), mouse anti-MBP (E8032, NEB), mouse anti-HA (clone 12CA5, gift from m.tyrs), rabbit anti-ubiquitin (Z0458, Dako), and mouse anti-actin (CP01, Calbiochem). The following antibodies were used as secondary antibodies in immunofluorescence microscopy: alexa Fluor 488 donkey anti-rabbit IgG, Alexa Fluor 488 donkey anti-goat IgG, Alexa Fluor 555 donkey anti-mouse IgG, Alexa Fluor 555 donkey anti-rabbit IgG, Alexa Fluor 647 donkey anti-mouse IgG, Alexa Fluor 647 donkey anti-human IgG, Alexa Fluor 647 donkey anti-goat IgG (molecular probes).

RNA interference

All sirnas used in this study were single duplex sirnas purchased from ThermoFisher. RNA interference (RNAi) transfection was performed using Lipofectamine RNAiMax (Invitrogen) in forward transfection mode. The individual siRNA duplexes used were BRCA1(D-003461-05), PALB2(D-012928-04), USP11(D-006063-01), CUL1(M-004086-01), CUL2(M-007277-00), CUL3(M-010224-02), CUL4A (M-012610-01), CUL4B (M-017965-01), CUL5(M-019553-01), KEAP1(D-12453-02), RAD51(M-003530-04), IP/RBBP8(M-001376-00), BRCA2(D-003462-04), 53BP1(D-003549-01) and non-targeted control siRNA (D-001210-02). Unless otherwise stated, siRNA was transfected 48 hours prior to cell treatment.

Inhibitors and fine chemicals

The following drugs were used at the indicated concentrations: 100ng/mL^-1Cycloheximide (CHX; Sigma), 0.2. mu.M camptothecin (CPT; Sigma), 10. mu.M ATM inhibitor (KU 55933; Selleck Chemicals), 10. mu.M ATR inhibitor (VE-821; given by Philip Reaper), 10. mu.M DNA-PKcs inhibitor (NU 7441; Genetex), 2. mu.M proteasome inhibitor MG132(Sigma), 40. mu.M lovastatin (S2061; Selleck Chemicals), doxycycline (# 8634-1; Clontech), 5. mu.M Nedd8 activator inhibitor (MLN 4929; Active Biochem), and the indicated concentrations of Olaparib (Selleck).

Immunofluorescence microscopy

In most cases, cells were grown on glass coverslips, blocked with 2% (w/v) paraformaldehyde in PBS for 20 minutes at room temperature, permeabilized with 0.3% (v/v) Triton X-100 for 20 minutes at room temperature, and blocked with 5% BSA in PBS for 30 minutes at room temperature. Alternatively, cells were fixed with 100% cold methanol at-20 ℃ for 10 min and then washed with PBS at room temperature for 5 min before PBS-BSA blocking. Cells were then incubated with primary antibody diluted in PBS-BSA for 2 hours at room temperature. The cells were then washed with PBS and then incubated with a secondary antibody diluted in PBS-BSA supplemented with 0.8 μ g/ml DAPI at room temperature to stain DNA1 hours. The coverslips were mounted on slides using Prolong Gold mounting medium (Invitrogen). Confocal images were taken using a Zeiss LSM780 laser scanning microscope. For the G1 versus S/G2 analysis of the BRCA1-PALB2-BRCA2 axis, cells were first synchronized with a double thymidine block, released to allow for S phase entry and exposed to 2 or 20Gy of X-ray radiation 5 and 12 hours after release, and fixed (in the case specified) 1to 5 hours after treatment. To check for DNA replication, cells were preincubated with 30 μ M BrdU for 30 minutes prior to irradiation and treatment as described previously.

CRISPR/Cas9 genome editing of USP11/KEAP1

293T and U2OS cells were transiently transfected with 3 different sgRNAs targeted to 53BP1, USP11 or KEAP1 and expressed from a pX459 vector containing a Cas9 followed by a 2A-puromycin cassette. The next day, cells were selected with puromycin for 2 days and subcloned to form single colonies or subpopulations. Colonies were screened by immunoblotting and/or immunofluorescence to verify loss of 53BP1, USP11 or KEAP1 expression, followed by PCR and sequencing. The genomic region targeted by CRISPR/Cas9 was amplified by PCR using Turbo Pfu polymerase (Agilent) and the PCR product was cloned into PCR2.1topo vector (Invitrogen) prior to sequencing.

Olaparib clonogenic assay

293T cells were incubated with the indicated dose of Olaparib (Selleck Chemicals) for 24 hours, washed once with PBS, and counted by trypan blue staining. Then, for each condition, 500 cells were plated in duplicate. Cell survival assays were performed as previously described [35 ].

Recombinant protein production

GST and MBP fusion proteins were generated as described previously [36,37 ]]. Briefly, the MBP protein expressed in E.coli was purified on amylose resin (New England Biolabs) according to the batch method described by the manufacturer and stored in 1 × PBS, 5% glycerol. GST protein expressed in E.coli was purified in 50mM Tris HCl pH 7.5, 300mM NaCl, 2mM Dithiothreitol (DTT), 1mM EDTA, 15. mu.g/mL^-1Glutathione sepharose 4b (ge healthcare) resin from a mixture of AEBSF and 1 × holoprotease inhibitors (Roche). After elution from the resin with 50mM glutathione in 50mM Tris HCl pH 8,2mM DTT,his-tagged TEV protease (supplied by F.Sicheri) was cleaved using 50mM Tris HCl pH 7.5, 150mM NaCl, 10mM glutathione, 10% glycerol, 2mM sodium citrate, and 2mM β -mercaptoethanol₆His depletion Using 50mM Tris HCl pH 7.5, 300mM NaCl, 20mM imidazole, 5mM glutathione, 10% glycerol, 1mM sodium citrate and Ni-NTA agarose beads in 2mM β -mercaptoethanol (Qiagen)_6-Labeled proteins, then concentrated by centrifugation (Amicon centrifugal filter, Millipore). GST-mKEAP1[38 ] was purified as described previously]An additional anion exchange step was performed on a HiTrap Q HP column (GE Healthcare). The GST tag was left on the protein for in vitro experiments. Purification of CUL3 and RBX 1[ 32 ] was performed as described previously]Nedd8 and Den1 were grown in minimal broth medium (Terrific broth medium) and induced to express in Escherichia coli BL21 overnight at 16 ℃ with 0.5mM isopropyl- β -D-thiogalactoside (IPTG). cells were collected and resuspended in wash buffer (400mM NaCl, 50mM Tris-HCl, pH 8, 5% glycerol, 2mM DTT) supplemented with lysozyme, Universal nuclease (Pierce), benzamidine, leupeptin, pepstatin, PMSF and a complete protease inhibitor cocktail (Roche) except that the protease inhibitor was omitted for DEN1 expressing cells^-1The flow rate of (2) was such that the soluble supernatant was bound to 5ml strep30 Tactin Superflow Cartridge. The column was washed with 20 Column Volumes (CV) of wash buffer and eluted with 5CV of wash buffer, diluted 1:2 in water to reduce the final salt concentration, and supplemented with 2.5mM desthiobiotin. The eluted fractions were pooled and concentrated to a total volume of 4ml using an Amicon concentrator with a 3kDa cut-off. DEN1 was further purified by Superdex 75 size exclusion column buffer exchanged for 150mM NaCl, HEPES, pH 7.6, 2% glycerol and 1mM DTT. The C-terminal propeptide and StrepII2x tag were removed by incubation with StrepII2x-DEN1 at a 1:20 molar ratio for 1 hour at room temperature. The DEN1 cleavage reaction was buffer exchanged on a Zeba MWCO desalting column (Pierce) to remove desthiobiotin and passed through a Strep-Tactin card retaining the C-terminal propeptide and DEN 1. As described in Escherichia coliGST-tagged wild boar (Sus scrofa) (pig) USP11 protein [39]. At 50mM Tris pH 7.5, 300mM NaCl, 1mM EDTA, 1mM AEBSF, 1 XProtease inhibitor cocktail (284ng/ml leupeptin, 1.37. mu.g/ml^-1Pepstatin A, 170 μ g/ml^{- 1}PMSF and 330. mu.g/ml^-1Benzamidine) and 5% glycerol, cells were lysed by lysozyme treatment and sonication. The clear lysate was applied to a column (GE Healthcare) packed with glutathione sepharose 4B, washed extensively with lysis buffer, and then eluted in 50mM Tris pH 7.5, 150mM NaCl, 5% glycerol, and 25mM reduced glutathione. DUB activity was determined on fluorescent ubiquitin-AMC (Enzo life sciences) and measured using a Synergy Neo microtiter plate reader (Biotek). TEV-ubiquitin-G76C was purified on a chelating HiTrap resin according to the manufacturer' S instructions and then size exclusion chromatographed on an S-75 column (GE healthcare). The protein was extensively dialyzed against 1mM acetic acid and lyophilized.

In vitro ubiquitination and deubiquitination of PALB2

In a medium containing 50mM Tris HCl pH 7.5, 20mM NaCl, 10mM MgCl₂And 0.5mM DTT, HA-tagged N-terminal fragment of PALB2(1-103) (1. mu.M) was ubiquitinated in vitro using 50. mu.M wild-type ubiquitin (Ubi WT, Boston Biochem) or lysine-free ubiquitin (Ubi K0, Boston Biochem), 100nM human UBA1(E1), 500nM CDC34 (supplied by F.Sicheri and D.Ceccarelli), 250nM ubiquitinated CUL3/RBX1, 375nM GST-mKEAP1, and 1.5mM ATP. The ubiquitination reaction was carried out at 37 ℃ for 1 hour, unless otherwise stated. For USP 11-mediated deubiquitination assay, first at 37 ℃ in the presence of 25mM HEPES pH 8, 150mM NaCl, 10mM MgCl₂HA-PALB2(1-103) was ubiquitinated in a buffer of 0.5mM DTT and 1.5mM ATP for 1.5 hours in a 50. mu.L reaction using lysine-free ubiquitin with the enzyme concentration as described above. The reaction was stopped by the addition of 1 unit of apyrase (NewEngland Biolabs). The reaction products were mixed at a 1:1 ratio with wild type or catalytically inactive (C270S) USP11 or USP2 (supplied by Rooicheri and E.Zeqiraj) at final concentrations of 100nM, 500nM and 2500nM (USP11) and 500nM (USP2) and in a medium containing 25mMHEPES pH 8、150mM NaCl、2mM DTT、0.1mg/mL BSA、0.03％Brij-35、5mM MgCl₂0.375mM ATP, for 2 hours at 30 ℃.

Pull-down experiment between purified PALB2 and BRCA1

The product of the in vitro ubiquitination reaction of PALB2 was incubated at a final concentration of 50mM Tris-HCl pH 7.5, 150mM NaCl, 5mM MgCl₂0.25mM DTT and 0.1% NP-40. Mu.g of MBP or MBP-BRCA1-CC were coupled to amylose resin (New England Biolabs) in the above buffer supplemented with 0.1% BSA before the ubiquitination product was added. The pull-down reaction was carried out at 4 ℃ for 2 hours, followed by extensive washing.

Co-immunoprecipitation

Cells were collected by trypsinization, washed once with PBS, and lysed on ice in 500 μ L lysis buffer (20mM Tris-HCl pH 8.0, 150mM NaCl, 10% glycerol, 2mM EDTA, 1% NP-40, complete protease inhibitor cocktail (Roche), phosphatase inhibition cocktail (Sigma), and N-ethylmaleimide to inhibit deubiquitination). The lysate was centrifuged at 15000g for 10 min at 4 ℃ and the protein concentration was assessed using the absorbance at 280 nm. Equal amounts of protein (. about.0.5-1 mg) were incubated with 2. mu.g rabbit anti-PALB 2, rabbit anti-USP 11 antibody, rabbit anti-GFP antibody or normal rabbit IgG for 5 hours at 4 ℃. A mixture of protein A/protein G-Sepharose beads (Thermo Scientific) was added for 1 hour. The beads were collected by centrifugation, washed twice with lysis buffer and once with PBS, eluted by boiling in 2XLaemmli buffer, and then analyzed by SDS-PAGE and immunoblotting. For MS analysis of Flag-PALB2, 150X 10⁶Transiently transfected HEK293T cells in high salt lysis buffer (50mM Tris-HCl pH 7.5, 300mM NaCl, 1mM EDTA, 1% Triton X100, 3mM MgCl)₂、3mM CaCl₂) Lysis buffer supplemented with a complete protease inhibitor cocktail (Roche), 4mM 1, 10-phenanthroline, 50U benzonase and 50U Micrococcus nuclease. Clear lysates were incubated with Flag-M2 agarose (Sigma), then in lysis buffer andbulk wash in 50mM ammonium bicarbonate.

Mass spectrometry

Following immunoprecipitation of transiently transfected Flag-PALB2 from sirtrl-transfected or USP11 siRNA-depleted 293T cells, cysteine residues were reduced and alkylated on beads using 10mM DTT (30 min at 56 ℃) and 15mM 2-chloroacetamide (1 h at room temperature), respectively. The protein was digested using limited trypsin digestion on beads (1. mu.g trypsin (Worthington, NJ, USA) per sample, 20 min at 37 ℃) and dried to completion. For LCMS/MS analysis, peptides were redissolved in 5% formic acid and loaded onto a 12cm column of fused silica having drawn tips internally packed with 3.5 μm Zorbax C18(Agilent Technologies, Calif., USA). Samples were analyzed using Orbitrap Velos (Thermo Scientific, MA, USA) coupled to an Eksigent nano LCultra (AB SCIEX, CA). Peptides were eluted from the column using a 90 min linear gradient of 2% to 35% acetonitrile in 0.1% formic acid. Tandem MS spectra were obtained in a data-dependent fashion for the first two most abundant multiply-charged peptides, and included targeted scans of five specific N-terminal PALB2 trypsin digested peptides (charge states 1+, 2+, 3+) in unmodified form or containing diglycine-ubiquitin trypsin digestion residues. Tandem MS spectra were obtained using collision-induced dissociation. The spectra were searched against the human Refseq _ V53 database using Mascot, allowing up to 4 false cleavages and including ureylmethyl (C), deamidation (NQ), oxidation (M), glygly (k), and leuargglygly (k) [ SEQ ID NO:4] as variable modifications.

In vitro ubiquitinated HA-PALB2(1-103) (50. mu.L total reaction mix) was run briefly on SDS-PAGE gels, followed by full lane cutting, in-gel reduction using 10mM DTT (30 min at 56 ℃), alkylation using 50mM 2-chloroacetamide, and trypsinization for 16 h at 37 ℃. Mu.l of a mixture of digested peptides with 20. mu.l of 10 unique heavy-isotope labeled N-terminal PALB2(AQUA) peptides (complete or partial tryptic digest of the surrounding region comprising Lys16, 25, 30 or 43 in unmodified or diG modified form; 80-1,200 fmol. mu.l of each peptide^-1Based on single peptide sensitivity testing) mixAfter that, 6. mu.L was loaded onto a 12cm column of fused silica having a drawing tip internally filled with 3.5 μm Zorbax C18. Samples were measured on orbitrap (Thermo Scientific, MA, USA) coupled to eksingent nanoLC ultra (AB SCIEX, CA, USA). Peptides were eluted from the column using a 180 min linear gradient of 2% to 35% acetonitrile in 0.1% formic acid. Tandem MS spectra were obtained in a data-dependent fashion for the first two most abundant multiply-charged peptides, and included targeted scans of ten specific N-terminal PALB2 trypsin digested peptides (charge states 1+, 2+, 3+) in light or heavy isotopically labeled form. Tandem MS spectra were obtained using collision-induced dissociation. Searching the human Refseq _ V53 database for spectra using Mascot, allows up to 2 false cleavages, including ureidomethyl (C), deamidation (NQ), oxidation (M), GlyGly (K), and LeuArgGlyGly (K) [ SEQ ID NO: 4)]As a variable modification, the spectra were then verified manually.

His-ubiquitin pull-down

Will stably express His₆293FLIP-IN cells of Ub transfected with the indicated siRNA and treated with Doxycycline (DOX) for 24 hours to induce His₆-Ub expression. Cells were pretreated with 10mM N-ethylmaleimide for 30 min and in denaturing lysis buffer (6M guanidine hydrochloride, 0.1M Na)₂HPO₄/NaH₂PO₄10mM Tris-HCl,5mM imidazole, 0.01M β -mercaptoethanol, complete protease inhibitor mixture.) lysate is sonicated on ice for 10 seconds, stopped for 1 minute, and centrifuged at 15000g for 10 minutes at 4 deg.C, the supernatant is incubated with Ni-NTA-agarose beads (Qiagen) for 4 hours at 4 deg.C, the beads are collected by centrifugation, washed once with denaturing lysis buffer, and washed with washing buffer (8M Urea, 0.1M Na₂HPO₄/NaH₂PO₄10mM tris-HCl,5mM imidazole, 0.01M β -mercaptoethanol, whole protease inhibitor cocktail) and twice with wash buffer supplemented with 0.1% Triton X-100 and eluted in elution buffer (0.2M imidazole, 0.15M tris-HCl, 30% glycerol, 0.72M β -mercaptoethanol, 5% SDS) before analysis by SDS-PAGE and immunoblot.

HR-based repair assay

Parental U2OS cells and U2OS cells stably expressing wild-type CtIP or a CtIP (T847E) mutant were transfected with the indicated siRNA and PALB2-KR construct, synchronized with a single thymidine block, treated with doxycycline to induce CtIP expression, and subsequently blocked at G1 by the addition of 40 μ M lovastatin. Cells were collected by trypsinization, washed once with PBS, and electroporated with 2.5 μ g of sgRNA plasmid and 2.5 μ g of donor template using the Nucleofector technique (Lonza; protocol X-001). Cells were plated in medium supplemented with 40 μ M lovastatin and grown for 24 hours before flow cytometry analysis.

PALB2 chemical ubiquitination

As described previously [40,41]PALB2(1-103) polypeptides engineered to have only one cross-linkable cysteine were ubiquitinated by cross-linking alkylation with the following modifications. Purified PALB2 cysteine mutant (final concentration 600. mu.M) was mixed with 300mM Tris pH 8.8, 120mM NaCl and His in 5% glycerol₆TEV-ubiquitin G76C (350. mu.M) mix.Tris (2-carboxyethyl) phosphine (TCEP) (Sigma-Aldrich) reducing agent was added to the mixture to a final concentration of 6mM and incubated at room temperature for 30 minutes the dual reactive cysteine cross-linker 1, 3-dichloroacetone (Sigma-Aldrich) was dissolved in dimethylformamide and added to the protein mixture to a final concentration of 5.25 mM. to allow the reaction to proceed on ice for 1 hour, then quenched by addition of 5mM β -mercaptoethanol His enrichment by passing through Ni-NTA-agarose beads (Qiagen)₆TEV-ubiquitin conjugated PALB 2.

Studies and results of the studies are discussed below.

DNA repair by Homologous Recombination (HR) [1] is highly inhibited in G1 cells [2,3] to ensure that mitotic recombination occurs only between sister chromatids [4,5 ]. Although many HR factors are cell cycle regulated, the nature of the events necessary and sufficient to inhibit recombination in G1 cells remains unknown. The present study found that the cell cycle tightly controls the interaction of BRCA1 with PALB2-BRCA2 in order to constrain BRCA2 function to the S/G2 phase. The BRCA1 interaction site on PALB2 is targeted by E3 ubiquitin ligase consisting of KEAP1 complexed with CUL3-RBX 1(PALB2 interacting protein [6]) [7 ]. PALB2 ubiquitination inhibits its interaction with BRCA1 and is counteracted by deubiquitinase USP11, which is itself under cell cycle control. The combination of restoration of BRCA1-PALB2 interaction and activation of DNA end excision is sufficient to induce HR in G1 as determined by RAD51 recruitment, extra-phase DNA synthesis, and CRISPR/Cas 9-based gene targeting. The mechanism of inhibition of HR in G1 consists of at least a DNA end excision inhibition linked to a multi-step blockade of recruitment of BRCA2 to the site of DNA damage, which involves inhibition of BRCA1-PALB2-BRCA2 complex assembly. The ability to induce HR in G1 cells using defined factors can be used for gene targeting applications in non-dividing cells.

Breast and ovarian tumor suppressor factors BRCA1, PALB2, and BRCA2 promote DNA Double Strand Break (DSB) repair by HR [8-10 ]. BRCA1 facilitates DNA end excision to generate single-stranded (ss) DNA [1] necessary for homology searching and strand invasion, and interacts with PALB2[ 13-15] to direct recruitment of BRCA2[13] and RAD51[16,17] to the DSB site. Accumulation of BRCA1 flanking the DSB site on chromatin is inhibited in G1 cells [18], reminiscent of effective inhibition of HR at this stage of the cell cycle. Since inhibition of BRCA1 recruitment in G1 was dependent on 53BP1 and RIF1 proteins [18,19], which are two inhibitors of end excision [18-22], this regulation of BRCA1 was originally observed from its function of stimulating DNA end processing.

However, since BRCA1 is also involved in promoting recruitment of BRCA2 by interacting with PALB2[ 13-15], this study investigated whether inducing recruitment of BRCA 1to the DSB site in G1 by mutation (53BP1 Δ; fig. 5a-c) of 53BP1 (also known as TP53BP1) by genome editing also resulted in accumulation of BRCA2 into Ionizing Radiation (IR) -induced foci. In contrast to BRCA1, at IR doses ranging from 2 to 20Gy, neither BRCA2 nor PALB2 recruit to the G1DSB site in U-2-OS (U2OS) cells (FIG. 1ab and FIGS. 5d, e). Due to the direct interaction of BRCA1 and PALB2[ 13,14], this result suggests that G1 cells may block BRCA2 recruitment by inhibiting BRCA1-PALB2 interaction. Indeed, although PALB2 interacted with BRCA2 regardless of cell cycle position, it only interacted efficiently with BRCA1 in S phase (fig. 1 c). The presence of DNA damage resulted in loss of the residual PALB2-BRCA1 interaction in G1, while it had little effect on the assembly of BRCA1-PALB2-BRCA2 complex in the S phase (FIG. 1 c). Since all proteins are expressed at G1 (fig. 1c), the results indicate that the assembly of the BRCA1-PALB2-BRCA2 complex is controlled in the cell cycle, possibly limiting BRCA2 accumulation at the DSB site to S/G2 phase.

These results were confirmed using a single cell assay that evaluated the co-localization of mCherry-labeled LacR-BRCA1 fusion protein with GFP-labeled PALB2 at the integrated LacO array [23] (fig. 6 b). The LacR/LacO system outlines the cell cycle-dependent and DNA damage-sensitive BRCA1-PALB2 interactions (fig. 6a), and enables the finding that the sequence on PALB2 (residues 1-50) located outside its amino-terminal BRCA1 interaction domain is responsible for its cell cycle-dependent regulation of association with BRCA1 (fig. 6c, d). Further deletion mutagenesis identified a single region contained within residues 46-103 of PALB2 (fig. 6e, f) responsible for the cell cycle dependent regulation of BRCA1-PALB2 interaction. This region corresponds to the interaction site of KEAP1[6], identifying this protein as a candidate modulator of BRCA1-PALB2 interaction.

KEAP1 is a substrate adaptor for CULLIN 3-RING ubiquitin (Ub) ligase (CRL3) that targets the antioxidant modulator NRF2 for proteasomal degradation [24] and recognizes the "ETGE" motif on both PALB2 and NRF2 through its KELCH domain [6 ]. Depletion of KEAP1 from 53BP1 Δ cells or deletion of the ETGE motif in full-length PALB2 (PALB2 Δ ETGE) induced PALB2 IR-induced focal formation in G1 cells (fig. 1d and fig. 7 a). Furthermore, in cells with inactivation of KEAP1 by genome editing (KEAP1 Δ, fig. 7b), a stable BRCA1-PALB2-BRCA2 complex was detected both in G1 phase and S phase (fig. 1 e). Thus, KEAP1 is an inhibitor of BRCA1-PALB2 interaction.

CUL3 also interacted with PALB2 (fig. 7c), and its depletion in 53BP1 Δ U2OS cells de-inhibited the PALB2 IR-induced focal aggregation in G1 (fig. 1d and fig. 7 a). Furthermore, in G1 synchronized KEAP1 Δ cells, unlike their wild type counterpart, expression of CUL3 binding deficient KEAP1 protein lacking its BTB domain (Δ BTB) failed to inhibit BRCA1-PALB2 interaction (fig. 7 d). These results indicate that KEAP1 recruits CUL3 to PALB2 to inhibit its interaction with BRCA 1.

Using the LacR/LacO system and co-immunoprecipitation assays, it was found that the PALB2 mutant (PALB 2-KR; FIG. 2a), which lacks all 8 lysine residues in the BRCA1 interaction domain, interacted with BRCA1 regardless of cell cycle position (FIG. 2b and FIG. 7e, f). Further mutagenesis identified residues 20, 25 and 30 in PALB2 as being critical for inhibiting BRCA1-PALB2 interaction, as reintroduction of these lysines in the context of PALB2-KR (resulting in PALB 2-KR/K3; fig. 2a) resulted in inhibition of BRCA1-PALB2-BRCA2 complex assembly in G1 cells (fig. 2b and fig. 7 e). Taken together, these results suggest a model for PALB 2-bound KEAP 1to form an active CRL3 complex that ubiquitinates the PALB2N terminus to inhibit its interaction with BRCA 1.

Although ubiquitination of PALB2 could be detected in cells (fig. 8a), the lysine-rich nature of the end of PALB2N has so far prevented the unambiguous mapping of the ubiquitination site on Lys20, 25 or 30 in vivo. However, ubiquitination at Lys16 and Lys43 could be detected by mass spectrometry, indicating that the PALB2N terminus is ubiquitinated (fig. 8 b). In a complementary set of experiments, PALB2 targeting LacO arrays induced immunoreactivity to conjugated ubiquitin (fig. 8 c-e). Co-localization of Ub with PALB2 was highest in G1 and was dependent on the presence of KEAP1 interaction motif and Lys20/25/30 residues (fig. 8d-e), consistent with a model of PALB2 ubiquitination at those sites in G1 cells. Indeed, ubiquitination of the N-terminus of PALB2 (residues 1-103; fusion to Hemagglutinin (HA) epitope tag) can be readily reconstructed by recombinant CRL3-KEAP1 in a manner dependent on the KEAP1 interaction domain of PALB2 (FIG. 2c), and Lys25 and Lys30 were unambiguously identified in vitro by mass spectrometry as ubiquitination by KEAP 1.

Ubiquitination of PALB2 by CRL3-KEAP1 inhibited its interaction with BRCA1 fragment containing residues 1363-1437 (BRCA 1)CC), this inhibition was more pronounced for the highly modified form of PALB2 due to the presence of ubiquitinated lysine outside of the BRCA1 interaction domain (fig. 2 d). To specifically test whether ubiquitination of a single one of the three lysine residues identified as critical inhibited interaction with BRCA1, a single ubiquitination moiety placed at position 20 or 45 was used with chemical cross-linking (resulting in PALB 2-K)_C20-Ub and PALB2-K_C45-Ub). Ubiquitination of PALB2 at position 20 completely inhibited its interaction with BRCA1, while modification of residue 45 had no effect on the interaction (fig. 9a), similar to the in vivo data (fig. 7 e). Taken together, these results indicate that ubiquitination of PALB2 at a specific site at its N-terminus prevents its interaction with BRCA 1.

Since neither the activity of the CRL3-KEAP 1E 3 ligase (fig. 9b) nor the interaction of CRL3-KEAP1 with PALB2 (fig. 7c) is regulated by the cell cycle, it is possible to modulate the deubiquitylation of PALB2 in a cell cycle dependent manner. KEAP1 physically interacts with USP11 [25], USP11 is a deubiquitinating enzyme that also interacts with BRCA2[26] and PALB2 (fig. 9 c). USP11 depletion impairs gene conversion [27] (fig. 9d) and results in hypersensitivity to PARP inhibition [27], identifying USP11 as an HR modulator of unknown function. Co-immunoprecipitation experiments demonstrated that USP11 and its catalytic activity were essential for the formation of a stable BRCA1-PALB2-BRCA2 complex, particularly in the presence of DNA damage (fig. 3a and 9e, f).

Removal of KEAP1 (or CUL3) should reverse the phenotype conferred by USP11 loss if USP11 antagonizes ubiquitination of PALB2 by CRL3-KEAP 1. Indeed, deletion of KEAP1 restored resistance to PARP inhibitor (PARPi) and BRCA1-PALB2 interaction in USP11 knockout cells prepared by genome editing (USP11 Δ) (fig. 3b, c and fig. 9 e). Likewise, CUL3 or KEAP1 depletion also reversed the defect in gene transformation of USP 11-depleted cells (fig. 10 a). Introduction of the PALB2-KR mutant restored its interaction with BRCA1 in a Lys20/25/30 dependent manner and reversed PARPi sensitivity in USP11 delta cells. Since recombinant USP11 could directly de-ubiquitinate PALB2(1-103) (fig. 3d), these results indicate that USP11 promotes the assembly of BRCA1-PALB2-BRCA2 complex by reversing the inhibitory ubiquitination at the PALB2Lys20/25/30 residues.

USP11 was observed to circulate rapidly in G1 cells and interact poorly with PALB2 at this stage of the cell cycle (fig. 11a, b). Furthermore, upon induction of DNA damage, particularly stage G1, USP11 is rapidly lost (fig. 3e and fig. 11b, c). Instability of USP11 after IR treatment was ATM signaling dependent, whereas it was ATR dependent after UV irradiation (fig. 11d, e). The decrease in USP11 steady state levels in G1 was the result of proteasomal degradation (fig. 11 f). CRL4E3Ub ligase was most likely responsible for controlling the stability of USP11, as treatment of the pan-CRL inhibitor MLN4924 [28 (fig. 11g) or depletion of CUL4 (fig. 3f) protected USP11 from DNA damage induced degradation. CUL4 depletion resulted in BRCA2 and PALB2 IR-induced focal formation in G153 BP1 Δ cells (fig. 3G and fig. 12a), consistent with USP11 modulation of the CRL4 complex used as an upstream signal for final control of BRCA1-PALB2-BRCA2 complex assembly.

Although the deletion of 53BP1 produced low levels of ssDNA in G1 cells [29], the combination of the 53BP1 Δ mutation with KEAP1 depletion produced no aggregation sites induced by RAD51 IR that were resistant to extraction, indicating that almost no RAD51 nuclear filaments were formed (fig. 12 b). ssDNA formation in those cells is still insufficient, so the mock phosphorylated T847E mutant of CtIP that promotes excision by G1 cells was used [30 ]. Unlike wild-type CtIP, introduction of CtIP-T847E into KEAP 1-depleted 53BP1 Δ cells induced RAD51 IR-induced aggregation site formation in G1 cells (fig. 4a, b and 12b, c) and extra-phase DNA synthesis (fig. 12 d). These results indicate that downstream steps of RAD51 nuclear filament formation, i.e., strand invasion, D-loop formation and DNA synthesis, can be activated in G1.

To test whether productive HR can also be activated in G1, a CRISPR/Cas9 stimulated gene targeting assay (Pinder j. et al, nucleic Acids res.43,9379-9392,2015) was used, in which the insertion of the coding sequence for the mClover fluorescent protein 5' of the lamin a (lmna) or PML gene was monitored by microscopy or flow cytometry (fig. 4c and 12e, f), the latter method being able to gate cells with defined DNA content (such as G1 cells). A synchronization protocol was also established in which G1 cells obtained after thymidine block release were arrested at G1 by lovastatin treatment [2]24 hours (FIG. 12G, h). Using this system, donor template concentrations within the linear range of the assay were determined and it was determined that gene targeting at the LMNA locus was dependent on BRCA1-PALB2-BRCA2 complex assembly (fig. 13a, b). It was also demonstrated that gene targeting by HR was highly inhibited in G1 (fig. 4 d).

The combined activation of excision and recruitment of BRCA 1to the DSB site (i.e. in 53BP1 Δ cells expressing CtIP (T847E)) was insufficient to stimulate gene targeting at the LMNA or PML locus in G1 cells (fig. 4e and 13 c). However, when the BRCA1-PALB2 interaction was restored in resectable G1 cells using KEAP1 depletion or expression of PALB2-KR mutant, a strong increase of gene targeting events at both loci was detected (fig. 4e and fig. 13 c). However, the gene targeting frequency of G1 cells was still lower than that of asynchronously dividing cells, indicating incomplete activation of HR. Both 53BP1 inactivation and expression of CtIP (T847E) were required for G1HR (fig. 13d, e), indicating that simultaneous activation of terminal excision and recruitment of BRCA2 to the DSB site is necessary and sufficient for recombination outside the activation phase in this phase of the cell cycle.

In conclusion, the regulation of the assembly of the BRCA1-PALB2-BRCA2 complex is a key point in the cell cycle control of DSB repair by HR. This regulation is focused on the BRCA1 interaction site on PALB2 and is achieved by the opposite activities of the E3 ligase CRL3-KEAP1 and the deubiquitinase USP11, which is antagonized by the CRL4 complex in G1 (fig. 4 f). In this model, the stability of USP11 at the S phase allows for the recruitment and subsequent loading of RAD51 of PALB2-BRCA2 at the DSB site. Studies also showed that inhibition of HR in G1 cells was largely reversible and involved combined inhibition of terminal excision and recruitment of BRCA2 to the DSB site (fig. 4 f). Since most cells in humans are not actively circulating and therefore difficult to HR, the procedures described herein have led to the development of genome editing methods that enable therapeutic gene targeting in a variety of tissues.

Example 2

DCAF10 was identified as a substrate adaptor for USP11 degradation.

The CUL 4-RING-ligase (CRL4) complex is composed of CULLIN4(CUL4), RBX1, DDB1, DDA1 and a substrate adaptor called DCAF [42]. To search for substrate adaptors that mediate USP11 ubiquitination, a focused siRNA library (focused siRNA library) depleted of known and predicted DCAF and other CUL4 interacting proteins was assembled. The library was screened in a high content microscopy assay in which USP11 levels were assessed by immunofluorescence microscopy. Cells were treated with Ultraviolet (UV) light or Ionizing Radiation (IR) to induce USP11 degradation. Data were normalized to non-irradiated conditions and the average of two independent experiments was used to plot the values after UV-and IR-treatment. The data shown in figure 14a shows that in addition to the expected stabilization of USP11 following exhaustion of CUL4, exhaustion of DCAF10, DCAF15 and DCAF17 also resulted in stabilization of USP 11. Since siRNA-mediated knockdown is prone to off-target effects, whether knockdown of DCAF10, DCAF15, or DCAF17 by two independent sirnas could stabilize USP11 was evaluated in immunoblot experiments. It was found that while stabilization of USP11 was observed with only a single siRNA (fig. 14b), depletion of DCAF10 with both sirnas resulted in robust stabilization of USP11 (fig. 14 b). Since the CRL4 substrate adaptor binds to its substrate [42]Next, whether DCAF10 or DCAF15 could interact with USP11 was evaluated in a co-immunoprecipitation assay. It was found that when Flag-labeled USP11 was immunoprecipitated from HEK293 cell extracts, it interacted with DCAF10 instead of DCAF15 (fig. 15a), strongly indicating that DCAF10 is a true substrate adaptor targeting USP11 for degradation. To further assess whether DCAF10 was indeed involved in the regulation of USP11, wild type from the homologous line (DCAF 10)^+/+) Heterozygotes (Dcaf 10)^-/+) And Dcaf10^-/-Mice produced Mouse Embryonic Fibroblasts (MEFs) and were immunoblotted with USP 11. Loss of DCAF10 resulted in higher steady state levels of USP11 in mouse cells (fig. 15b), consistent with DCAF10 being an adaptor targeting the CRL4 complex of USP 11. Finally, using the Direct Repeat (DR) -GFP assay, it was assessed whether DCAF10 overexpression could be suppressed in a dominant mannerPreparation of homologous recombination [43]. Overexpression of DCAF10 but not DCAF15 resulted in HR reduction of the same magnitude as USP11 and other core HR factors depletion (fig. 15 c). Taken together, these data indicate that DCAF10 regulates HR by controlling USP 11.

Example 3

Genetically encoded inhibitors of KEAP1 promote homologous recombination in G1 cells.

Activation of gene targeting in G1 cells required removal of 53BP1, introduction of CtIP-T847E, and interaction between PALB2 and BRCA1, which could be achieved by removal of KEAP 1. To develop a system capable of activating HR in G1 and non-dividing cells, it was determined whether KEAP1siRNA could be replaced by inhibitors of KEAP 1. A recently described high affinity genetically encoded KEAP1 inhibitor named R1 was selected, which is based on the fibronectin-3 (FN3) scaffold [44 ]. LMNA gene targeting assays were performed in synchronized 53BP1 Δ U2OS cells at stage G1 [45], and it was found that transfection of R1 KEAP1 inhibitor instead of its FN3 control resulted in strong, although less, activation of gene targeting, and KEAP1 depletion (fig. 16). Inhibition of KEAP1 may be an advantageous pathway for activation of HR in non-dividing cells.

The scope of the invention is not limited to the specific embodiments described herein, as these embodiments are intended only as illustrations of one aspect of the invention, and any functionally equivalent embodiments are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

All publications, patents, and patent applications cited herein are incorporated by reference in their entirety as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference in its entirety. All publications, patents and patent applications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the methodologies, reagents and the like that are reported herein and that might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

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Claims (16)

1. A method for activating or modulating homologous recombination in a cell, comprising:

(a) promoting or stimulating the assembly or occurrence of BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex in said cell;

(b) contacting the cell with BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex;

(c) activating or stimulating recruitment of BRCA 1to the site of DNA Double Strand Break (DSB);

(d) inhibiting or removing KEAP1 or CRL3-KEAP 1;

(e) inhibit degradation of USP11 or promote USP11 activity; and/or

(f) Inhibiting DCAF 10.

2. The method of claim 1, comprising suppressing KEAP1 or DCAF 10.

3. The method of claim 1 or 2, further comprising reconstituting or activating DNA end excision.

4. A method for activating homologous recombination in a cell, wherein DNA end excision is activated or reconstituted to produce single-stranded DNA, comprising promoting or stimulating assembly or occurrence of a BRCA1-PALB2 or BRCA1-PALB2-BRCA2 complex in said cell.

5. The method of claim 3 or 4, wherein DNA end excision is activated by inhibiting 53BP1 expression or activity and up-regulating or expressing CtIP.

6. The method of claim 5, wherein CtIP is up-regulated or expressed by administering CtIP or an analog of CtIP.

7. The method of claim 3, comprising inhibiting or removing KEAP1 and 53BP1, and an analog that upregulates or expresses CtIP or CtIP.

8. The method of claim 5,6, or 7, wherein 53BP1 is inhibited by administering 53BP1 short interfering (si) RNA, short hairpin (sh) RNA, or microrna (mirna).

9. The method of any one of claims 1to 8, further comprising introducing a gene editing system into the cell.

10. The method of claim 9, wherein the gene editing system is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -CRISPR associated (Cas) system, a transcription activator-like effector nuclease (TALEN) system, or a zinc finger nuclease system.

11. A method for altering a genetic disorder associated with a target polynucleotide sequence in a cell, comprising: (1) activating homologous recombination in the cell according to the method of any one of claims 1to 8; and (2) contacting the target polynucleotide sequence with a CRISPR-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids direct and hybridize Cas protein to selected motifs of the target polynucleotide sequence, wherein the target polynucleotide sequence is cleaved such that expression of the target polynucleotide sequence is reduced, knocked out, or corrected from an undesired sequence to a desired sequence.

12. The method of any one of claims 1to 11, wherein the cell is in the G1 phase of the cell cycle (G1) or the G0 phase of the cell cycle (G0).

13. The method of claim 12, wherein the cell is in the G1 phase of the cell cycle (G1).

14. A method for monitoring the activity or expression of USP11 in a sample, comprising (i) isolating in said sample a complex of: (a) BRCA1 and PALB 2; (b) BRCA1, PALB2, and BRCA 2; (c) USP11 and PALB 2; and/or (d) USP11 and DCAF 10; (ii) measuring the level of the complex; and (iii) detecting an increase or decrease in activity or expression of the complex compared to a control as an indication of activity or expression of USP 11.

15. A method of predicting or classifying a response to a PARP inhibitor in an individual comprising determining a complex of BRCA1, PALB2 and BRCA2 in a sample from a subject and comparing to a control to determine whether the individual is sensitive to the PARP inhibitor.

16. A kit for stimulating or activating homologous recombination or gene editing in a cell, comprising: (a) inhibitors of 53BP 1; (b) inhibitors of KEAP 1; and (c) CtIP or an analog of CtIP.