CN113564249B - Application of CXorf67 in judging sensitivity of tumor to DNA damage medicine - Google Patents

Abstract

The invention relates to an application of CXorf67 in judging the sensitivity of a tumor to a DNA damage drug. In particular, the invention provides an application of CXorf67 gene, mRNA, cDNA or protein or a detection reagent thereof in detecting sensitivity of tumor cells to DNA damage repair inhibitor drugs.

Description

Application of CXorf67 in judging sensitivity of tumor to DNA damage medicine

Technical Field

The present invention relates to the field of oncology and diagnostics. More particularly, the invention relates to the use of CXorf67 in determining the sensitivity of a tumor to a DNA damaging agent.

Background

Double strand breaks (double strand breaks, DSBs) of DNA in cells are one of the most serious DNA lesions faced by cells, and DNA double strand break repair is generally divided into two types depending on the presence or absence of homologous sequences. One is classical non-homologous end joining (cNHEJ), which is sequence independent and acts mainly in the G0/G1 phase of the cell cycle. The other is a recombinant (homologous recombination, HR) repair pathway, which is a fault-free repair that uses the homologous DNA inside the cell as a template to repair the fragmented DNA mainly in S/G2 phase of the cell cycle. Such homologous DNA may be sister chromatids, homologous chromosomes or sequences on ectopic chromosomes.

Poly (ADP-ribose) polymerase inhibitors (PARPi) is a recently discovered drug that can kill cancer cells using synthetic lethality. Poly (ADP-ribose) polymerase (PARP) has 17 family members, which are a class of nucleoproteins, of which PARP1 and PARP2 are the major proteins involved in the DNA damage response. PARP inhibitors were reported to specifically kill BRCA1 or BRCA2 mutated tumors in 2005 as early as possible. There are currently 5 PARP inhibitors, but there are great differences in their ability to PARP-tracking.

CXorf67 (chromosome X open reading frame 67) is located at Xp11.22 of the chromosome and has only one exon and no intron, and codes for 503 amino acids. CXorf67 is an unknown functional protein that is primarily localized in the nucleus, predicted by the website, without known domains, mostly disordered.

A tumor deficient in HR repair pathway is one that is very sensitive to PARP inhibitors. PARP inhibitors block DNA single strand break repair, which results in double strand DNA replication, leading to cell death if HR repair of tumor cells is defective; normal cells have intact HR repair and are therefore not killed.

Ependymoma (EPN) is a neuroepithelial malignancy that occurs in the central nervous system (central nervous system, CNS), occurring in both children and adults. EPN occurs mainly in three places: supratentorial (ST), posterior Fossa (PF), and Spinal (SP). Studies in 2015 further classified the molecular subtypes for ependymomas at these three locations by analysis of DNA methylation, ST including ST-SE, ST-EPN-YAP1 and ST-EPN-RELA; PF includes PF-SE, PFA and PFB; the SPs include SP-SE, SP-MPE and SP-EPN. PFA occurs mainly in infants and young children (average age 3 years, ranging from 0-51 years), with poor prognosis; PFB occurs mainly in adolescents (average age 30 years, ranging from 10-65 years), with good prognosis.

PFA subtype in ependymoma mainly occurs in the hindbrain part of children, has poor prognosis, mainly uses operation and radiotherapy at present, and lacks effective drug treatment.

Thus, there is an urgent need in the art to develop new targets that enhance the sensitivity of HR repair pathway deficient tumors to DNA damaging drugs, thereby more effectively treating HR repair pathway deficient tumors.

Disclosure of Invention

The invention aims to provide a novel target for enhancing sensitivity of a tumor with a defect of HR repair pathway to DNA damage drugs, so as to more effectively treat the tumor with the defect of HR repair pathway.

The first aspect of the present invention provides the use of a CXorf67 gene, mRNA, cDNA, or protein, or a detection reagent thereof, (i) as a marker for detecting sensitivity of a tumor cell to a DNA damage repair inhibitor drug; and/or (ii) for the preparation of a diagnostic reagent or kit for detecting the sensitivity of tumor cells to a DNA damage repair inhibitor drug.

In another preferred embodiment, the DNA damage repair inhibitor drug comprises a PARP inhibitor.

In another preferred embodiment, the PARP inhibitor is selected from the group consisting of: talazoparib, olaparib, veliparib, rucaparib, niraparib.

In another preferred embodiment, the diagnostic reagent comprises an antibody, a primer, a probe, a sequencing library, a nucleic acid chip (e.g., a DNA chip), or a protein chip.

In another preferred embodiment, the protein comprises a full-length protein or a protein fragment.

In another preferred embodiment, the protein contains a PALB2 binding motif (PALB 2-binding motif).

In another preferred embodiment, the PALB2 binding motif is located at positions 420-432 of the CXorf67 protein.

In another preferred embodiment, the CXorf67 gene, mRNA, cDNA, or protein is derived from a mammal, preferably a rodent (e.g., mouse, rat), primate, or human, and more preferably a patient diagnosed with a tumor deficient in the HR repair pathway.

In another preferred embodiment, the HR repair pathway deficient tumor is selected from the group consisting of: ependymoma (Ependymoma posterior fossa group A), renal clear cell carcinoma (kidney renal clear cell carcinoma, KIRC), renal papillary cell carcinoma (kidney renal papillary cell carcinoma, KIRP), or a combination thereof.

In another preferred embodiment, the ependymoma comprises PFA.

In another preferred embodiment, the tumor cell is a CXorf 67-expressing or high-expressing tumor cell.

In another preferred embodiment, the tumor cells comprise tumor cells deficient in HR repair pathway.

In another preferred embodiment, the tumor cells are selected from one or more of the following: ependymoma, renal clear cell carcinoma (kidney renal clear cell carcinoma, KIRC), renal papillary cell carcinoma.

In another preferred embodiment, the CXorf67 Gene has an accession number of Gene ID 340602.

In another preferred embodiment, the CXorf67 mRNA accession number is NM_203407.3.

In another preferred embodiment, the CXorf67 protein has an accession number of NP-981952.1.

In another preferred embodiment, the assay is a tissue sample assay.

In another preferred embodiment, the detection comprises immunohistochemistry, immunoblotting and fluorescent quantitative PCR method detection.

In another preferred embodiment, the detection is of tumor tissue.

In another preferred embodiment, the detection reagent comprises a specific antibody to CXorf67, a specific binding molecule to CXorf67, a specific amplification primer, a probe or a chip.

In another preferred embodiment, the CXorf67 protein or specific antibody or specific binding molecule thereof is conjugated or otherwise provided with a detectable label.

In another preferred embodiment, the detectable label is selected from the group consisting of: chromophores, chemiluminescent groups, fluorophores, isotopes or enzymes.

In another preferred embodiment, the antibody specific for CXorf67 is a monoclonal antibody or a polyclonal antibody.

In a second aspect, the present invention provides a diagnostic kit for detecting sensitivity of tumor cells to a DNA damage repair inhibitor drug, the kit comprising a container containing a detection reagent for detecting a CXorf67 gene, mRNA, cDNA, or protein; and a label or instruction stating that the kit is for detecting sensitivity of a tumor cell to a DNA damage repair inhibitor drug.

In another preferred embodiment, the detection reagent for detecting CXorf67 gene, mRNA, cDNA, or protein comprises:

(a) Antibodies specific for anti-CXorf 67 protein; and/or

(b) Specific primers for specific amplification of mRNA or cDNA of CXorf 67.

In another preferred embodiment, the assay is a tissue sample assay.

In a third aspect, the present invention provides a method of determining sensitivity to a DNA damage repair inhibitor drug, the method comprising:

a) Providing a test sample from a subject;

b) Detecting the expression level of CXorf67 protein in the test sample; and

c) And (c) judging the sensitivity to the DNA damage repair inhibitor drug based on the expression amount of CXorf67 protein measured in the step b).

In another preferred embodiment, sensitivity to a DNA damage repair inhibitor drug is determined when the CXorf67 protein is present in the test sample.

In another preferred embodiment, sensitivity to DNA damage repair inhibitor drugs is determined when the CXorf67 protein expression level in the test sample is > 0.5, preferably > 1.5, more preferably > 2.

In another preferred embodiment, the subject is a human or non-human mammal.

In another preferred embodiment, the test sample is a tumor cell or tissue that expresses or is highly expressed by CXorf 67.

In another preferred embodiment, the test sample is a tumor cell or tissue deficient in HR repair pathway.

In another preferred embodiment, the detecting step (b) comprises detecting the amount of CXorf67 mRNA, or the amount of CXorf67 cDNA; and/or detecting the amount of CXorf67 protein.

In another preferred embodiment, the expression level of CXorf67 protein in the sample is detected by fluorescent quantitative PCR or immunohistochemistry.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In a fourth aspect, the invention provides a method of determining a treatment regimen comprising:

a) Providing a test sample from a subject;

b) Detecting the expression level of CXorf67 protein in the test sample; and

c) Determining a treatment regimen based on the expression level of the CXorf67 protein in the sample.

In another preferred embodiment, the subject is a human or non-human mammal.

In another preferred embodiment, when CXorf67 protein (preferably CXorf67 protein is expressed in an amount > 0.5, preferably > 1.5, more preferably, > 2) is present in the sample, the treatment regimen comprises CXorf67 inhibitor therapy, DNA damage repair inhibitor drug therapy, and a therapy in which a CXorf67 inhibitor is combined with a DNA damage repair inhibitor drug.

In another preferred embodiment, the CXorf67 inhibitor therapy and DNA damage repair inhibitor drug therapy are selected from the group consisting of:

CXorf67 inhibitor therapy: an antibody, a small molecule compound, microRNA, siRNA, shRNA, or a combination thereof;

DNA damage repair inhibitor drug therapy: PARP inhibitors.

In another preferred embodiment, when the subject has a higher sensitivity to the DNA damage repair inhibitor drug than the general population (control population), the treatment regimen further comprises a CXorf67 inhibitor therapy, a DNA damage repair inhibitor drug therapy, a CXorf67 inhibitor in combination with a DNA damage repair inhibitor drug.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows CXorf67 expression levels and drug sensitivity. (a) The relationship between CXorf67 expression level and pharmaceutical activity in brain cell lines in the GDSC database was analyzed. 93 medicines are screened out according to the correlation coefficient larger than 0.1 and the p value smaller than 0.05. (b) And carrying out targeted path analysis on 60 medicines with classified information in 93 medicines.

FIG. 2 shows CXorf67 is a DNA damage response protein. (a) U2OS cells were plated into glass-bottomed dishes and transfected with EGFP-CXorf67 plasmid after 24 hours. Hoechst dye was added after 24 hours for 15 minutes, and then washed three times with a culture solution. A picture was taken every 20 seconds on a live cell workstation with 405nm laser damage. (b) U2OS was stimulated with 0.1. Mu.M CPT, then cells were collected at different time points and then chromatin isolation was performed. (c) U2OS cells were plated on 24-Well slides and then stimulated with IR or CPT at 0.1. Mu.M, fixed with 4% paraformaldehyde after 1 hour, washed with PBST overnight with CXorf67 antibody, blocked with fluorescent secondary antibody and DAPI the next day for 1 hour, and then photographed.

FIG. 3 shows CXorf67 can inhibit DNA damage repair. (a) CXorf67 WT and KO U2OS cells were seeded in 24-well and then stimulated with IR (10 Gy), samples were collected at different time points and WB detected the levels of r-H2 AX. (b) CXorf67 was reverted to expression in U2OS of CXorf67 KO, then IR stimulated, WB detected the level of r-H2 AX. (c) In CXorf67 WT, KO and U2OS reverting to CXorf67 expression, stimulation with IR (10 Gy) was followed by collection of samples for 0.5H and 24H, immunofluorescent staining was performed and formation of r-H2AX foci was detected. (d) CXorf67 WT and KO U2OS after IR (10 Gy) stimulation, cells were collected and tested for tailing of nuclear DNA by comet electrophoresis experiments. Figure 4 shows that CXorf67 inhibited HR without affecting NHEJ. (a) Two concentrations of CXorf67 plasmid (0.1. Mu.g and 0.5. Mu.g) and I-SceI plasmid (3. Mu.g) were transfected into 6-well U2OS DR-GFP cells, and cells were harvested 48 hours later for flow analysis of GFP and RFP positive cell numbers. the t-test assay analyzes p-values. (b) CXorf67 plasmid (0.5. Mu.g) and I-SceI plasmid (3. Mu.g) were transfected into 6-well U2OS EJ5-GFP cells, and cells were collected after 48 hours for flow analysis of GFP and RFP positive cell numbers.

FIG. 5 shows CXorf67 affects Rad51 foci, but not RPA2 and BRCA1 foci. (a) After KO cells of U2OS CXorf67-WT, KO and gyratory CXorf67 were subjected to CPT for 4 hours at 0.1. Mu.M, the cells were fixed and the Rad51 foci was observed by immunofluorescence assay using Rad51 antibody. And the number of foci per cell was counted using Image-pro plus software. the t-test assay analyzes p-values. (b, c) after subjecting U2OS CXorf67-WT and KO cells to CPT 0.1. Mu.M for 4 hours, the cells were fixed and immunofluorescence was performed using the RPA2, BRCA1 antibody to observe the corresponding foci. (d) KO cells of Daoy CXorf67-WT, KO and gyratory CXorf67 were subjected to CPT for 4 hours at 0.1. Mu.M, and then the cells were fixed, and the Rad51 foci was observed by immunofluorescence assay using Rad51 antibody. (e, f) after treatment of Daoy CXorf67-WT and KO cells with CPT 0.1. Mu.M for 4 hours, the cells were fixed and immunofluorescence was performed with the RPA2, BRCA1 antibody to observe the corresponding foci.

FIG. 6 shows CXorf67 binding to PALB 2. (a) In Daoy cells, CXorf67 was immunoprecipitated and binding of PALB2, BRCA1, RPA2 and Rad51 was then detected. (b) CXorf67-HA, PALB2-Flag or BRCA1-Flag was transfected in 293T cells, then CXorf67 was immunoprecipitated with HA antibody, followed by Western blot analysis of PALB2 and BRCA1. (c) PALB2-HA plasmid was transfected in U2OS cells and then co-localization of endogenous CXorf67 with PALB2 was observed.

FIG. 7 shows CXorf67 binding to WD40 domain of PALB 2. (a) Three PALB2 fragment plasmids were constructed from the reported PALB2 domains. (b) In 293T cells, the PALB2 plasmid and CXorf67 plasmid, which are different forms of fragments, were transfected and Flag immunoprecipitated, and then CXorf67 was detected. (c) The His-tagged CXorf67 protein and GST-tagged WD40 protein were purified separately in E.coli and then GST pull-down experiments were performed.

FIG. 8 shows that CXorf67 was highly similar to the 26-38 amino acid sequence of BRCA2 by binding of binding motif to PALB2 (a) sequence alignment at 420-432 amino acid sequence of CXorf67. Biotin modified CXorf67 WT and W425C mutant polypeptides (420-432) were synthesized and then subjected to streptavidin pull-down experiments with GST-WD40 protein. (b) The CXorf67 WT and W425C mutant plasmids were transfected in 293T and then co-immunoprecipitated to detect binding of PALB 2. (c) I-SceI and WT CXorf67 or W425C mutants were transfected into U2OS DR-GFP cells, and after 48h the cells were collected and FACS analyzed for the proportion of positive cells occupied by GFP, RFP. The p-value was analyzed using t-test detection. (d) The WT CXorf67 or W425C mutant was complemented back in cells of Daoy CXorf67-KO, respectively, and then CPT stimulation was applied followed by fixation of the dye Rad51 foci, t-test assay p-values.

FIG. 9 shows CXorf67 competes with BRCA2 for binding to PALB2. (a) PALB2-Myc, BRCA2-N-GFP (1-200 amino acids) and gradient CXorf67-HA plasmid were transfected in 293T cells and PALB2 was immunoprecipitated. (b) PALB2-GFP, BRCA2-N-Myc were transfected in U2OS cells, while co-localization of PALB2 and BRCA2 was observed with or without CXorf 67. (c) CXorf67-GFP and Myc-LacR or Myc-LacR-PALB2 plasmids were transfected into U2OS-LacO cells and immunofluorescence experiments were performed. (d) GFP-LacR, GFP-LacR-PALB2, myc-BRCA2-N (1-200) or CXorf67-HA plasmids were transfected into U2OS-LacO cells and immunofluorescence experiments were performed. Fluorescent quantitative analysis was performed using ImageJ software.

Figure 10 shows that Daoy cells highly expressing CXorf67 are more sensitive to PARP inhibitors. (a) Daoy CXorf67 WT and KO cells were plated in 96-well and treated with varying concentrations of Talazoparib for 5 days, after which the viability of the cells was checked using CellTiter-Glo kit and analyzed for p-value by Two-way ANOVA. (b) Cells that were back expressed CXorf67 KO in Daoy CXorf67 KO were plated in 96-well for 5 days with varying concentrations of Talazoparb or Olaparib and then assayed for cell viability. (c) Daoy CXorf67-KO cells and cells reverting CXorf67 expression in KO and Matrigel 1:1 were mixed and injected subcutaneously in 5 week old BALB/c nude mice, 5 in each group, when tumor volume reached 100mm ³ In this case, talazoparib (0.33 mg/kg) was administered for gastric lavage treatment, and tumor volumes were measured twice a week. (d) CXorf67 was overexpressed in U251 and U87 cells, and then treated with varying concentrations of Talazoparib for 5 days, and cell viability was measured using the CellTiter-Glo kit.

FIG. 11 shows that CXorf67 is highly expressed in the PFA subtype of ependymoma (a) analysis of the mRNA levels of CXorf67 in GSE64415 (PFA, 72 cases; non-PFA,137 cases) and GSE94349 (PFA, 12 cases; non-PFA,179 cases) data. (b) Protein was extracted from the collected 28 cases (numbered 1-28 in order) of the ependymoma specimens, and the protein expression level of CXorf67 and the level of gamma-H2 AX were detected by western blotting, followed by a Pearson correlation analysis.

Figure 12 shows that PFA tumors that highly express CXorf67 are more sensitive to PARP inhibitors. (a) Fresh samples of 5 children's hindbrain tumors, PFA 1-4 and MB-1, were collected and then tested for CXorf67 and gamma-H2 AX expression levels using Western blotting. (b) Primary cells derived from PFA-1 and PFA-2 tumors were established, then treated with Talazoparib at various concentrations for 5 days, and then the viability of the cells was examined using CellTiter-Glo kit. Two-way ANOVA analysis p-values. (c) PDXs derived from PFA-3, PFA-4 and MB-1 tumors were established. Each of which The group PDX was inoculated with 10 nude mice each and then divided into two groups, i.e., a medicated group and a non-medicated group. When the tumor volume reaches 50mm ³ At this time, talazoparib (0.33 mg/kg) was lavage treated, 5 days per week, and tumor volumes were measured. When the tumor volume reaches 1000mm ³ When the mice survived, the mice survival curves were drawn. The p-value was analyzed by Log rank (Mantel-Cox) detection. (d) Tumor volumes of the dosed and non-dosed groups PFA-3, PFA-4 and MB-1 were measured and p-values were analyzed by Two-way ANOVA.

Fig. 13 shows analysis of CXorf67 expression in other cancers. (a) mRNA expression levels of CXorf67 in 16 cancers in TCGA database were analyzed. (b) Analysis of the relationship between CXorf67 expression levels and patient prognosis in both KIRC (higher, n=55; lower, n=53), KIRP (higher, n=59; lower, n=57), log-rank detection analysis of p-values.

Detailed Description

The present inventors have made extensive and intensive studies and have unexpectedly found that a tumor cell line or a tumor tissue which highly expresses CXorf67 is more sensitive to a DNA damage repair inhibitor drug (such as a PARP inhibitor) for the first time. Thus, the CXorf67 gene or protein thereof can be used as a marker for detecting the sensitivity of tumor cells to DNA damage repair inhibitor drugs. On this basis, the present inventors have completed the present invention.

Tumors with defective HR repair pathway

The homologous recombination (homologous recombination, HR) repair pathway is an error-free repair that repairs broken DNA mainly during the S/G2 phase of the cell cycle using the homologous DNA inside the cell as a template. Defects or deficiencies in HR can lead to genomic instability, which is accompanied by the development and progression of tumors, where genes such as BRCA1, PLAB2, and BRCA2 in the HR pathway are found to have mutations in many tumors. Mutations in these genes can result in DNA double strand breaks that cannot be repaired effectively, causing alterations, rearrangements and mutations in the cell gene copy number, and thus causing the occurrence of tumors and diseases such as breast, pancreatic and prostate cancer, etc.

In a preferred embodiment, the HR repair pathway deficient tumor is a CXorf67 highly expressed tumor selected from the group consisting of: ependymoma (Ependymoma posterior fossa group A), renal clear cell carcinoma (kidney renal clear cell carcinoma, KIRC), renal papillary cell carcinoma (kidney renal papillary cell carcinoma, KIRP), ependymoma, renal clear cell carcinoma (kidney renal clear cell carcinoma, KIRC), renal papillary cell carcinoma, or a combination thereof.

Sample of

The term "sample" or "specimen" as used herein refers to a material that is specifically associated with a subject from which particular information about the subject can be determined, calculated, or inferred. The sample may be composed in whole or in part of biological material from the subject. The sample may also be a material that has been contacted with the subject in a manner that allows the test performed on the sample to provide information about the subject. The sample may also be a material that has been contacted with another material that is not the subject, but that enables the first material to be subsequently tested to determine information about the subject, e.g., the sample may be a cleaning solution for a probe or scalpel. The sample may be a source of biological material other than that contacting the subject, so long as one skilled in the art is still able to determine information about the subject from the sample.

Expression of

As used herein, the term "expression" includes the production of mRNA from a gene or gene portion, and includes the production of a protein encoded by RNA or gene portion, and also includes the presence of a detection substance associated with expression. For example, cDNA, binding of a binding ligand (e.g., an antibody) to a gene or other oligonucleotide, protein or protein fragment, and chromogenic portions of the binding ligand are included within the term "expressed". Thus, an increase in half-pel density in immunoblots, such as western blots, is also within the term "expression" based on biological molecules.

Reference value

As used herein, the term "reference value" refers to a value that is statistically relevant to a particular result when compared to the result of an analysis. In a preferred embodiment, the reference value is determined from a statistical analysis performed to compare the expression of CXorf67 with studies of known clinical outcomes. Some of these studies are shown in the examples section herein. However, the studies from the literature and the user experience of the methods disclosed herein can also be used to produce or adjust the reference value. Reference values may also be determined by considering conditions and results that are particularly relevant to the patient's medical history, genetics, age and other factors.

In the present invention, the reference value refers to a cut-off value, which refers to the expression level of CXorf67 in a tumor cell or tissue deficient in HR repair pathway, preferably the expression level of CXorf67 is > 0.5, preferably > 1.5, more preferably > 2.

Samples of non-tumor cells

As used herein, the term "sample of non-tumor cells" includes, but is not limited to, a population of tumors that do not have defects in HR repair pathways.

CXorf67 proteins and polynucleotides

In the present invention, the terms "protein of the present invention", "CXorf67 protein" and "CXorf67 polypeptide" are used interchangeably and refer to a protein or polypeptide having the amino acid sequence of CXorf 67. They include CXorf67 protein with or without an initiating methionine. In addition, the term also includes full length CXorf67 and fragments thereof. The CXorf67 protein referred to in the invention includes the complete amino acid sequence, the secretory protein, the mutant and the functional active fragment thereof.

CXorf67 (chromosome X open reading frame 67) is located at Xp11.22 of the chromosome and has only one exon and no intron, and codes for 503 amino acids. CXorf67 is an unknown functional protein that is primarily localized in the nucleus, predicted by the website, without known domains, mostly disordered.

The human CXorf67 protein is 503 amino acids in full length (accession number NP-981952.1). The murine CXorf67 protein is 589 amino acids in full length (accession number NP-001159905.1).

In the present invention, the terms "CXorf67 gene", "CXorf67 polynucleotide" are used interchangeably and refer to a nucleic acid sequence having a CXorf67 nucleotide sequence.

The genome of human CXorf67 Gene is 1896bp (NCBI GenBank accession number is Gene ID: 340602), and the mRNA sequence of the transcription product is 1512bp (NCBI GenBank accession number is NM-203407.3).

The genome of the mouse CXorf67 Gene is 2203bp (NCBI GenBank accession number Gene ID: 102991) in total length and the mRNA sequence of the transcription product is 1770bp (NCBI GenBank accession number NM-001166433.1).

Human and murine CXorf67 showed 39% similarity at the DNA level and 39% protein sequence similarity.

It is understood that substitution of nucleotides in the codon is acceptable when encoding the same amino acid. It is further understood that nucleotide substitutions are also acceptable when conservative amino acid substitutions are made by the nucleotide substitutions.

In the case where an amino acid fragment of CXorf67 is obtained, a nucleic acid sequence encoding it can be constructed therefrom, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis. For the PCR amplification method, primers can be designed based on the CXorf67 nucleotide sequences disclosed in the present invention, particularly the open reading frame sequences, and amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template to obtain the relevant sequences. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, it is entirely possible to obtain the DNA sequences encoding the proteins of the invention (or fragments, derivatives thereof) by chemical synthesis. The DNA sequence may then be introduced into a variety of existing DNA molecules (e.g., vectors) and cells known in the art.

The polynucleotide sequences of the invention can be used to express or produce recombinant CXorf67 polypeptides by conventional recombinant DNA techniques. Generally, there are the following steps:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a human CXorf67 polypeptide of the invention, or with a recombinant expression vector containing the polynucleotide;

(2) Host cells cultured in a suitable medium;

(3) Isolating and purifying the protein from the culture medium or the cells.

In the present invention, CXorf67 polynucleotide sequences can be inserted into a recombinant expression vector. In general, any plasmid or vector can be used as long as it replicates and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing CXorf67 encoding DNA sequences and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; insect cells; animal cells, and the like.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl ₂ The process is carried out using procedures well known in the art. Another approach is to use MgCl ₂ . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

Specific antibodies

In the present invention, the terms "antibody of the present invention" and "specific antibody against CXorf 67" are used interchangeably.

The invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the human CXorf67 polypeptide. Here, "specific" refers to an antibody that binds to a human CXorf67 gene product or fragment. Preferably, those antibodies that bind to the human CXorf67 gene product or fragment but do not recognize and bind to other unrelated antigenic molecules. Antibodies of the invention include those molecules that bind to and inhibit the human CXorf67 protein, as well as those that do not affect the function of the human CXorf67 protein. The invention also includes antibodies that bind to the modified or unmodified form of the human CXorf67 gene product.

The invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ Fragments; antibody heavy chain; an antibody light chain; genetically engineered single chain Fv molecules (Ladner et al, U.S. Pat. No.4,946,778); or chimeric antibodies, such as antibodies having murine antibody binding specificity but retaining antibody portions derived from humans.

Antibodies of the invention may be prepared by various techniques known to those skilled in the art. For example, a purified human CXorf67 gene product, or an antigenic fragment thereof, can be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing the human CXorf67 protein or antigenic fragment thereof can be used to immunize animals to produce antibodies. The antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al,Nature256;495,1975; the composition of Kohler et al,Eur.J.Immunol.6:511,1976; the composition of Kohler et al,Eur.J.Immunol.6:292,1976; hammerling et al,In Monoclonal Antibodies and T Cell Hybridomaselsevier, n.y., 1981). The antibody of the present invention includes an antibody that blocks the function of human CXorf67 protein and an antibody that does not affect the function of human CXorf67 protein. The various antibodies of the invention can be obtained by conventional immunization techniques using fragments or functional regions of the human CXorf67 gene product. These fragments or functional regions may be prepared by recombinant methods or synthesized by a polypeptide synthesizer. Antibodies that bind to unmodified forms of the human CXorf67 gene product can be used in prokaryotic cells (e.g., E.Coli) by immunization of animals; antibodies (e.g., glycosylated or phosphorylated proteins or polypeptides) that bind to post-translational modifications can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (e.g., a yeast or insect cell).

Antibodies against human CXorf67 protein can be used in immunohistochemical techniques to detect the presence of human CXorf67 protein in a sample, particularly a tissue sample or serum sample. Since the CXorf67 protein has an extracellular region, these soluble CXorf67 extracellular regions can be targets for serum detection when the extracellular region is shed and enters the blood.

Detection method

The invention also provides a method for detecting the sensitivity of tumor cells to DNA damage repair inhibitor drugs by utilizing the characteristic that CXorf67 exists in cells or tissues of tumors (such as tumors with defects of HR repair channels, preferably ependymoma) and is closely related to the sensitivity of the DNA damage repair inhibitor drugs.

In a preferred embodiment of the invention, the invention provides a high throughput second generation sequencing method for detecting CXorf67 as well as Sanger sequencing, fluorescent quantitative PCR (qPCR), in situ immunofluorescence (FISH), immunohistochemistry and the like.

Detection kit

Based on the correlation between the sensitivity of CXorf67 high-expressing tumor cells and DNA damage repair inhibitor drugs, that is, CXorf67 high-expressing tumor cells are more sensitive to DNA damage repair inhibitor drugs, CXorf67 can be used as a biomarker for guiding the use of DNA damage repair inhibitor drugs.

The invention also provides a diagnostic kit for detecting the sensitivity of tumor cells to DNA damage repair inhibitor drugs, which contains a detection reagent for detecting CXorf67 gene, mRNA, cDNA or protein; and a label or instruction stating that the kit is for detecting sensitivity of a tumor cell to a DNA damage repair inhibitor drug.

Detection method and kit

The present invention relates to diagnostic assays for the quantitative and positional detection of human CXorf67 protein levels or mRNA levels. Such tests are well known in the art. The human CXorf67 protein level detected in the assay can be used to detect the sensitivity of tumor cells to DNA damaging drugs.

One method of detecting the presence or absence of CXorf67 protein in a sample is by using an antibody specific for CXorf67 protein comprising: contacting the sample with a antibody specific for CXorf67 protein; observing whether an antibody complex is formed, the formation of which indicates the presence of CXorf67 protein in the sample.

The CXorf67 protein or the polynucleotide thereof can be used for diagnosing and treating CXorf67 protein related diseases. A part or all of the polynucleotides of the present invention can be immobilized as probes on a microarray or DNA chip for analysis of differential expression of genes in tissues and diagnosis of genes. Antibodies against CXorf67 can be immobilized on a protein chip for detecting CXorf67 protein in a sample.

The main advantages of the invention include:

(1) The present invention discovers for the first time that CXorf67 is a DNA damage response protein that can be recruited to the DNA cleavage site. CXorf67 and can inhibit DNA homologous recombination (homologous recombination, HR) repair.

(2) The invention discovers for the first time that tumor cells with high CXorf67 expression are more sensitive to DNA damage repair inhibitor drugs (such as PARP inhibitors).

(3) The invention discovers for the first time that CXorf67 can be used as a biomarker for guiding the use of DNA damage repair inhibitor drugs (such as PARP inhibitors).

(4) The invention discovers for the first time that CXorf67 can be used as a marker for detecting the sensitivity of tumor cells to DNA damage repair inhibitor drugs.

(5) The invention discovers for the first time that the expression quantity of CXorf67 is related to the sensitivity of DNA damage repair inhibitor drugs.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Unless otherwise specified, materials and reagents used in the examples of the present invention are commercially available products.

General method

1. Cell culture

Human U2OS cells and HEK293T cells were purchased from ATCC; daoy cells were purchased from the chinese academy of sciences stem cell bank; u2OS DR-GFP cells and U2OS EJ5-GFP cells were given benefit by university of Zhejiang Huang Jun; u2OS LacO cells are given benefit from university of Zhejiang Wang Fang. All cell lines were cultured in a 5% carbon dioxide incubator at 37 ℃. All media were purchased from Gibco plus 10% FBS (fetal bovine serum). HEK293T used DMEM medium, U2OS used 1640 medium, daoy used MEM medium.

The primary cell culture of ependymoma uses Neurobasal medium plus B27, N2, glutaMAX, EGF, FGF, heparin. Poly-D (100 mg, 10 mg/ml) was filtered. 10 μl was added at 10cm dish.

FGF was prepared with PBS containing 0.1% BSA (0.22 μm filtration) at 5. Mu.g/ml. EGF was formulated with PBS containing 0.1% BSA (0.22 μm filtration) at 10. Mu.g/ml. Hepatrin was formulated with PBS at 250. Mu.g/ml and filtered. DNase I was prepared at 1mg/ml (10X) with DMEM stock solution without FBS, filtered, and stored in-20℃aliquots at a working concentration of 0.1mg/ml.

2. Antibodies to

An antibody:

rad51 (ab 88572,1:500 IF), PALB2 (ab 220861,1:1000 WB), H2A.X (ab 11175,1:5000 WB) were purchased from abcam corporation. Phosphor-H2A.X (Ser 139) (05-636, 1:1000IF,1:2000 WB) was purchased from Merck Millipore. HA-tag (3724, 1:1000IF), flag-tag (2368, 1:1000WB), myc-tag (2276, 1:1000WB), H3 (4620, 1:5000 WB) were purchased from CST company. BRCA1 (sc-6954, 1:500 IF), LOC340602 (CXorf 67) (sc-515296, 1:500IF, 1:1000WB), ig-G (sc-2025), beta-actin (sc 47778,1:2000 WB) are purchased from Santa Cruz corporation. Flag-tag (F1804, 1:1000IF), GST-tag (G1166, 1:1000WB), his-tag (H10294, 1:1000WB) were purchased from Sigma company. HA-tag (901515, 1:1000WB) was purchased from Biolegend corporation. GFP-tag (11814460001, 1:1000WB) was purchased from Roche Inc.

And (2) secondary antibody:

donkey anti-rabbit IgG coupled to Alexa Fluor 488, sheep anti-rabbit IgG coupled to Alexa Fluor 647, sheep anti-mouse IgG coupled to HRP and sheep anti-rabbit IgG coupled to HRP were purchased from Invitrogen. Goat anti-mouse IgG conjugated with Cy3 was purchased at Jackson ImmunoResearch.

3. Reagent(s)

CXorf67 Biotin peptide WT: BIOTIN-GGPIPQQWDESSSSS (SEQ ID NO.: 1); mut: BIOTIN-GGPIPQQCDESSSSS (SEQ ID NO: 2) (purity greater than 90%) was synthesized by Abclonal corporation. Olaparib, talazoparib, DMAC (dimethylacetamide) and Solutol (polyethylene glycol-15 hydroxystearate) were purchased from MCE company. DAPI (D8417) and Paraformaldehyde (P6148) were purchased from Sigma. Capper VECTASHIELD (H-1400) was purchased at Vector Laboratories. Hoechst 33343 was purchased at Thermo Scientific. Lipofectamine 3000 transfection reagent and Lipofectamine RNAiMAX transfection reagent are purchased from Invitrogen.

4. Chromatin separation

First, U2OS cells were prepared, 60mm dish (. About.2X10) ⁶ cells). Pancreatin digestion 15ml centrifuge tube centrifugation, transfer to 1.5ml centrifuge tube, 5000rpm/5min/4 ℃, centrifugation. Washed one time with PBS, washed one time with 200. Mu.l SA buffer (10mM HEPES PH 7.9, 10mM KCl,1.5mM MgCl) ₂ ，0.34M Sucrose，10％glycerol，1mM DTT，10mM NaF，1mM Na ₂ VO ₃ 1mM PPi,1 XPI) was resuspended. Mu.l of 10% Triton-X100 was added to give a final concentration of 0.1%. Placed on ice for 5min (Total leave-on can be taken at this time). Then centrifuging 1300g/5min

The supernatant is cytoplasmic fraction and the pellet is nuclear fraction. (Cyto sample is taken at this time). The core fraction was washed once with SA buffer, then 200. Mu.l SB buffer (3mM EDTA,0.2mM EGTA,1mM DTT,10mM NaF,1mM Na) ₂ VO ₃ 1mM PPi,1 XPI), on ice for 10min. Then centrifuging1700g/5min/4 ℃, the supernatant is a soluble nuclear component, and the precipitate is chromatin. (at this time, nuc is sampled and loaded with 6 XSDS). The Chromatin fraction was washed once with SB buffer and then centrifuged at 13500rpm/5min/4℃at which time the supernatant was removed with a gun head and the viscosity was too high. Chromain was resuspended with 2X SDS loading and sonicated for 10min (on: 20s, off:40 s).

5. Comet electrophoresis

And (3) tabletting: soaking in 0.7% of common agarose (TAE buffer), and air drying. Preparing glue: 1% agarose gel with low melting point, melting in water bath at 100deg.C, and cooling to 37deg.C. Cells were digested with pancreatin, centrifuged at 3000rpm/5min and washed once with PBS. Mu.l of the cell suspension was mixed with 200. Mu.l of glue and spread on a Comet slide and flattened with a cover slip. Standing at 4deg.C for 30min, and solidifying. Cover glass was removed and the slide was lysed overnight at 4 ℃ (lysate formulation: 2.5M NaCl,100mM Na) ₂ EDTA,1% sarcosyl, 10mM Tris, pH 7.5). (1% Triton/10% DMSO was added before use) neutral electrophoresis solution (1 Xneutral electrophoresis solution pH 7.5: tris,89mM boric acid, 2mM EDTA) was placed for 30min,25V, and electrophoresis was performed for 30min. DAPI (2.5 mg/ml) 1:100 was diluted with water, 40. Mu.l was dropped onto the slide, covered with glass, and observed. And adding 5min before observation.

6. Preparation of CXorf67 knockout cell line Using CRISPR/Cas9

2 sgrnas for the CXorf67 gene were designed using the CRISPR online design website (http:// CRISPR. Mit. Edu). CXorf67#1 ACGGCTCAGGCGGGTGTTGCG (SEQ ID NO.: 3); CXorf67#2: ATCTTTGATTCCCGGGTCCGC (SEQ ID NO: 4). The sgRNA was annealed forward and reverse to form a double strand and phosphate was added (using T4 ligation buffer, T4 PNK formulation system. In PCR apparatus, 37 ℃,30min, 95 ℃,5min. Gradually reduced to 25 ℃). After dilution of sgRNA 1:200, PCR reactions (program: 37 ℃,5min;21 ℃,5min;8 cycles) were performed in the ligation system (pSpCas 9 (BB) -2A-GFP (PX 458) all-in-one plasmid, diluted ol igo, tango buffer,100mM DTT,10mM ATP,FastDigest BbSI,T4 ligase). The constructed expression plasmid is transferred into U2OS or Daoy cells, and single cells with GFP are subjected to flow sorting after 24 hours. The single cells obtained were plated in 96-well plates, and finally, monoclonal cells derived from one cell were identified.

7. HR and NHEJ reporting systems

Day 1, plating U2OS DR-GFP or U2OS EJ5-GFP cells, and plating 20-30 ten thousand cells per well in 6 wells. Day 2, I-SceI plasmid (3. Mu.g/well) and CXorf67 plasmid were transfected with lipo 3000. (I-SceI plasmid fused with an RFP, which can be used to analyze transfection efficiency) Day 4, cells were digested with pancreatin and subjected to flow analysis (at least 5 ten thousand cells were analyzed).

8. Protein purification

100 μl of glycerol bacteria is transferred to 10ml of LB (K+/Amp+), 12h later transferred to 1L of TB medium for about 3h, and OD value is measured to be 0.6-0.8. And simultaneously cooling, and taking 1ml of fungus reserved sample for detection. IPTG (1M, 1:1000 use) was added. Inducing expression at 18 deg.c for 24 hr and detecting 1ml of bacteria. The rest is collected by centrifugation and stored at-80 ℃. The bacteria-destroying instrument is precooled, washed 3 times with water and washed 2 times with buffer. And (5) precooling by a centrifugal machine. 500ml of bacteria were resuspended in 20ml buffer and were not pelleted. PMSF (final concentration 0.15 mM) may be added simultaneously. Pouring into a bacteria breaker, slowly pressurizing to 800Pa, clarifying for about 3min, and collecting under reduced pressure. 13000rpm/1h/4 ℃. Meanwhile, preparing the beads, washing with single steam for three times, and washing with buffer for two times. 500 μl of each sample. 1750rpm/2min, and the deceleration was set to 2.HIS: the supernatant was poured into 50ml tubes and spun for 2h with glycerol (final concentration 5%) + imidazole (final concentration 10 mM) +beads (28 ml supernatant+1.4 g glycerol+70ul 4M imidazole+500. Mu.lbeads). GST: the supernatant was poured into a 50ml tube and 500. Mu.l beads were added directly. The supernatant was directly applied to the column, washed three times with 5 times the volume of beads, and then eluted (His-tag protein washed with 20, 30, 40mM imidazole concentration, 400mM imidazole, 500. Mu.l with 6-7 tubes, added and collected in portions; GST eluted with GSH).

9、GST Pull Down

NETN buffer (100mM NaCl,20mM Tris-HCl,0.5mM EDTA and 0.5% NP-40) was prepared. To 500. Mu.l of the reaction system, 0.25. Mu.g of His-CXorf67 protein and GST-WD40 protein or GST protein were added, and the mixture was spun at 4℃for 20 minutes. Then 25. Mu.l of GST beads was added and the mixture was spun at 4℃for 30min. The beads were then washed 3 times with NETN buffer. Add 25. Mu.l of 2XSDS loading cook.

10、Biotin-Peptide Pull Down

200 mu l streptavidin agarose beads was taken and washed 3 times with Binding buffer (20mM HEPES pH 7.5, 100mM NaCl,1mM DTT,1mM EDTA,0.01% IGEPAL CA-630). The beads were divided into 3 parts, one without addition and two with 10. Mu. g C67-WT, C67-Mut peptide, respectively. Rotate at 4℃for 1h. The beads were washed 3 times with buffer and 1. Mu.g of GST-WD40 protein was added, respectively. Rotating at 4 ℃ for 2h. The beads were washed 3 times with buffer and cooked by adding 30ul 2XSDS loading.

11. Immunofluorescence

The cover glass is soaked in alcohol, quickly baked on the flame of an alcohol lamp for a few days, and then spread into a 24-well plate. Adding the culture solution for soaking, and then washing off the culture solution. After cell digestion counts, cells were split into 24-well plates. The culture broth was aspirated the third day and 200 μl of 4% PFA was added for fixation for 15min. Washed 3 times with PBST (0.1% Triton-X100) and blocked with 5% BSA for 1h. The mixture was washed 3 times with PBST, and the primary antibody solution was added thereto and left overnight at 4 ℃. After washing the primary antibody with PBST every other day, adding the corresponding fluorescent secondary antibody and DAPI, and sealing for 1h in a dark place. The secondary antibody was washed off with PBST, and then coverslips were blocked on slides with a blocking agent and stored in the dark at 4 ℃.

12. Co-immunoprecipitation

Cells were split into 6-well plates (exogenous) or 10cm dish (endogenous). Preparation of lysis buffer (1 XTriton lysis (1% Triton,5mM EDTA,150mM NaCl,50mM Tris-HCl, pH 7.4), 10m NaF,1XPI,1mM PPi,2mM Na ₃ VO ₄ ). The cell culture solution was aspirated and placed on ice, followed by addition of lysis buffer. Placing on a shaker for 10min. The cell lysate was transferred to a cold 1.5ml EP tube and centrifuged at 13000rpm/15min/4 ℃. The supernatant was pipetted into a new EP tube and 30. Mu.l was boiled with 30ul 2XSDS loading as input. The remainder was added with 0.25% BSA and corresponding antibody. Rotate at 4℃for 2h or overnight. 30 μl protein A/Gagarose beads were added to the tip of the gun. Rotating at 4 ℃ for 2h. After centrifugation to remove the supernatant, the supernatant was washed 3 times with lysis buffer. Add 30. Mu.l of 2XSDS loading cook.

13. Primary cell culture of ependymoma

Freshly resected tumor tissue from the hospital was placed in a 50ml centrifuge tube containing Neurobasal culture fluid and sent to the laboratory within 1 h. Tumor tissue was sent to a laboratory clean bench and washed once with PBS (Penicillin-Streptomycin), and blood was washed. Samples were cut into small pieces (run in 60mm dish) in PBS using sterilized forceps and scissors (pre-sterilization). There are also shears in the digestive juice, but some blood is mixed, preferably in PBS, which can be washed once. The minced sample and PBS were transferred to a 15ml centrifuge tube at 1000rpm/3min. Ackutase digestion enzyme was added and the tube placed in a shaker at 37 ℃. DNaseI (0.1 mg/ml) was added thereto and the mixture was further treated for 10 minutes. After the tissue was digested into single cells, the single cells were filtered into 50ml centrifuge tubes using a 70 μm cell strainer, and transferred into 15ml centrifuge tubes for centrifugation (1700 rpm/5 min). Cell counts were re-selected and 60mm or 10cm dishes were selected depending on density (dishes were coated with Laminin prior).

14. Mouse tumor transplantation experiment

BALB/c Nude immunodeficient male mice 5 weeks old were ordered and acclimatized in SPF class mice room for one week. Will be 3X10 ⁶ Daoy cells were mixed with Matrigel 1:1 and then injected into the right flank of nude mice. PDX-derived cells: firstly taking out the PDX which is modeled successfully, then cutting up the PDX, adding Ackutase digestive enzyme, digesting into single cells, and counting 3X10 ⁶ Individual cells were mixed with Matrigel 1:1 and then injected into the right flank of nude mice. Until the tumor volume reaches 50-100mm ³ When the dose is left or right, administration can be started. Talazoparib was dissolved in 10% DMAC, 6% Solutol and 84% PBS (mother liquor was dissolved in DMSO, 10 mg/ml). Then, the medicine was administered by lavage at 0.33 mg/kg. The administration is carried out for 5 days in a week. Mice were observed daily and body weights and tumor sizes were recorded twice a week. (tumor volume= (length x width)/2). When the maximum tumor volume reaches 1000mm ³ At this time, mice were euthanized.

Example 1 CXorf67 expression level is related to sensitivity to DNA damaging drugs

Since CXorf67 is an unknown functional protein, laboratory has found that the expression level of CXorf67 correlates with the sensitivity of DNA damaging drugs by analysis of the GDSC database. Cell lines from the central nervous system were analyzed in the GDSC database to screen for drugs whose sensitivity correlates with CXorf67 expression levels (p <0.05,Pearson correlation>0.1). 93 drugs were found and are indicated in red in fig. 1 a. After enrichment of the target pathway, the most drugs associated with DNA damage were found, e.g., camptothecin, etoposide, doxorubicin and bleomycin et al (FIG. 1 b)

Example 2 CXorf67 is a DNA damage response protein

To investigate whether CXorf67 plays a role in DNA damage repair. First we constructed a plasmid of pEGFP-CXorf67, and transfection of the plasmid into U2OS cells using laser microdamage method observed recruitment of CXorf67 protein at the site of DNA damage (fig. 2 a). For further validation, subsequent chromatin separation experiments were performed to find that CXorf67 increased in chromatin content after CPT 0.1. Mu.M treatment (FIG. 2 b). The following immunofluorescence experiments found that: after treatment with IR or CPT, CXorf67 was seen to form Foci in some cells (FIG. 2 c).

Example 3 CXorf67 inhibits DNA damage repair

From the previous experiments and bioinformatics analysis, it was speculated that CXorf67 might be involved in DNA damage repair. Both IR and CPT can cause DNA double strand breaks, which then cause rapid phosphorylation of histone H2AX at S139 (where phosphorylated H2AX is called γ -H2 AX), with the signal of γ -H2AX gradually decreasing to background levels as DNA repairs. First, U2OS WT and KO cells were subjected to IR stimulation, and then cells at different time points were collected for Western blot and immunofluorescence analysis of gamma-H2 AX. In WB experiments it was found that: there was no difference in the initial gamma-H2 AX signal for 15min and 1H, but the gamma-H2 AX signal in KO cells decreased more rapidly after 4H (FIG. 3 a). At the same time we supplemented CXorf67 in U2OS KO found an increase in the duration of the level of gamma-H2 AX, suggesting a slow repair of DNA damage (FIG. 3 b). The initial 0.5H gamma-H2 AX was also found to be no difference in immunofluorescence experiments, but the signal was decreased more rapidly at 24H KO and WT, and the return CXorf67 was restored in KO cells (FIG. 3 c). In order to determine more further, we performed comet electrophoresis experiments, also called single cell gel electrophoresis analysis, whose rationale is that undamaged cells, the more complete the nuclear DNA, and no tailing in electrophoresis. After the cells are damaged, the DNA breaks to produce fragments that migrate during electrophoresis to form a tail. Our experimental results showed that there was no significant difference in tailing produced by WT and KO at 0.5h after IR treatment, but KO cells were found to have shorter tailing than WT at 4h (fig. 3 d). The experimental results above show that: CXorf67 may be a factor inhibiting DNA damage repair and the ability of the cell to repair DNA damage increases after the absence of CXorf 67.

Example 4 CXorf67 inhibited HR without affecting NHEJ

The previous experiments used IR and CPT treatments, both forms of DNA damage were mainly DNA double strand break damage. There are two main ways of repair of double strand breaks: one is homologous recombination repair (homologous recombination, HR), which is a error-free repair, using homologous sister chromatids as template repair in S and G2 phases; another repair is non-homologous end joining (non-homologous end joining, NHEJ), which is a mismatch repair, where DNA ends can be directly joined by a ligase to create DNA insertion or deletion mutations.

To explore in which repair pathway CXorf67 plays a role, U2OS DR-GFP and EJ5-GFP reporter cell lines (Lou et al, 2017) were used. The cleavage plasmid I-SceI was transferred into both reporter cell lines and later used to indicate the repair efficiency of HR or NHEJ in cells by flow-through analysis. Overexpression of CXorf67 in the cell line was found to inhibit HR only and not NHEJ (FIGS. 4a and 4 b).

Example 5 CXorf67 affects Rad51 foci formation

In HR repair, one key protein is Rad51, which can be recruited to the site of DNA damage repair by the BRCA1-PALB2-BRCA2 complex. The effect on HR can be judged by observing the DNA damage induced Rad51 foci. It was found that under CPT treatment, cells of U2OS KO formed more Rad51 foci than WT, and this phenomenon could be reverted by transfection of CXorf67 (FIG. 5 a). After looking at RPA2 and BRCA1 upstream of Rad51, it was found that RPA2, BRCA1 foci did not change (fig. 5b and 5 c). To verify if there are the same phenomena in different cell lines, we performed the same experiments in Daoy cells later, and also found that KO increased compared to WT in the case of CPT treatment (fig. 5 d). RPA2, BRCA1 foci did not change (FIGS. 5e and 5 f). These results indicate that CXorf67 may play a role between BRCA1 and Rad 51.

EXAMPLE 6 CXorf67 binding to PALB2

According to the previous experimental results: CXorf67 affects Rad51 foci but not BRCA1 foci, presumably CXorf67 would play a role between the two. PALB2, BRCA1 could be detected in Daoy cells with IP CXorf67, but no RPA2 and Rad51 were detected in immunoprecipitation (fig. 6 a). In 293T cells, CXorf67-HA, PALB2-Flag or BRCA1-Flag was transfected and CXorf67 was immunoprecipitated, and it was seen that CXorf67 bound mainly to PALB2, but was weakly bound to BRCA 1. In the presence of PALB2, binding of CXorf67 to BRCA1 was enhanced, most likely with both being indirectly bound by PALB2 (fig. 6 b). We later found that CXorf67 co-localized with PALB2 in the presence of intact stimuli using immunofluorescence (FIG. 6 c). We speculate that CXorf67 may play a role by binding PALB 2.

EXAMPLE 7 CXorf67 WD40 Domain binding to PALB2

Later, to find the region where CXorf67 binds to PALB2, it was divided into 3 fragments according to the known PALB2 domain (FIG. 7 a). CXorf67 was found to bind predominantly to WD40 domain of PALB2 by exogenous co-immunoprecipitation (fig. 7 b). To verify whether binding of CXorf67 to the WD40 domain is a direct interaction, we purified the full length His-CXorf67 protein and GST-WD40 (853-1186 aa) protein. It was found by GST pull-down experiments that WD40 did interact directly with CXorf67 (FIG. 7 c).

EXAMPLE 8 CXorf67 binding to PALB2 by PALB2-binding motif

WD40 is known to be critical to PALB2 function, and BRCA2 also binds primarily to WD40 of PALB2, recruiting Rad51. Very interesting, we found that CXorf67 (420-432 aa) had strong homology to PALB2-binding motif (26-38 aa) of BRCA2 by protein sequence alignment (FIG. 8 a). This small peptide of BRCA2 has been reported to bind to the WD40 domain of PALB2 and tryptophan at position 31 plays a key role, just as tryptophan at position 425 of CXorf 67. To verify that amino acids 420-432 of CXorf67 do bind directly to the WD40 domain, a biotin-labeled peptide was synthesized. This small peptide was found to bind to the WD40 domain in a pull-down experiment with streptavidin. However, the W425C mutated peptide fragment was not able to bind (fig. 8 a). In the CO-IP experiments, it was also found that after the mutation of W425 of full length CXorf67 to cysteine, the binding of CXorf67 to PALB2 was reduced (fig. 8 b).

To examine whether there is an HR-inhibiting function after CXorf 67W 425 mutation, it was found that the inhibition of HR was significantly reduced after transfection of W425C mutant CXorf67 using the U2OS DR-GFP reporter system (FIG. 8C). At the same time, we complemented WT or W425C mutant in Daoy cells of CXorf67 KO and then stimulated CPT, found that W425 mutant showed a significant decrease in ability to inhibit Rad51 foci (FIG. 8 d)

Example 9 CXorf67 inhibits PALB2-BRCA2 binding

It is presumed that CXorf67 acts most likely to mimic the PALB2-binding motif of BRCA2, competing for binding to PALB2 to disrupt BRCA2-PALB2 binding. It was found by CO-IP experiments that binding of PALB2 and BRCA2 was decreased with increasing CXorf67 transfection (fig. 9 a). Furthermore, fluorescence co-localization experiments also found that co-localization of PALB2 and BRCA2 was reduced in the case of CXorf67 transfected (fig. 9 b) in order to verify our hypothesis more recently, a U2OS-LacO cell line was used. This cell line stably transformed LacO-containing plasmids into U2OS cells, and LacR-tagged proteins bind LacO sequences (Wang et al, 2010), and this system allows for the observation of protein co-localization at the single cell molecule level. First, a plasmid of Myc-LacR-PALB2 was constructed, and after transfection into a cell line, specific co-localization at PALB2 was observed by co-transfection of GFP-CXorf67 (FIG. 9 c). Next, in order to verify the relationship among CXorf67, PALB2 and BRCA2, GFP-LacR-PALB2, myc-BRCA2-N (1-200) and HA-CXorf67 plasmids were constructed. With CXorf67, the enrichment of the fluorescent signal of BRCA2 on PALB2 was found to decrease (FIG. 9 d). The previous experimental results were further verified by this single molecule cell level co-localization experiment: CXorf67 and PALB2 are combined with each other, and the combination of the CXorf67 and the PALB2 can inhibit the combination of BRCA2 and PALB 2.

Example 10 CXorf 67-expressing cancer cells are more sensitive to PARP inhibitors

Daoy cells highly expressing CXorf67The difference in sensitivity of the Daoy cells of C67-WT and KO to PARP inhibitors was compared to the cell survival experimental test, and it was found that the Daoy cells lacking CXorf67 had reduced sensitivity to inhibitors (fig. 10 a). Reversion of CXorf67 expression in KO cells significantly increased the sensitivity of the cells to PARP inhibitors (tazoparib and olaharib) compared to KO cells (10 b). Simultaneously, the constructed stable transgenic cell line is injected into the BALB/c nude mice of 6 weeks to construct a transplantation tumor model subcutaneously until the tumor grows to 100mm ³ At this time, mice were perfused with talazoparb (0.33 mg/kg,5 days/week), the tumor volume was measured twice a week, and the experiment was terminated after 28 days. Experimental results showed that the transplanted tumors reverting CXorf67 expression were more sensitive to the drug, and the tumor volume was significantly reduced after the treatment with the mice (fig. 10 c). Taken together, it was found that Daoy cells highly expressing CXorf67 were very sensitive to PARP inhibitors. Then, if CXorf67 is overexpressed in tumor cells that do not express CXorf67, there is the same phenomenon. Two strains of human glioblastomas U251 and U87, which do not express CXorf67, were collected, and we found that overexpression of CXorf67 in these two strains of cells made the tumor cells more sensitive to PARP inhibitors (fig. 10 d).

EXAMPLE 11 CXorf67 was highly expressed in PFA and caused accumulation of DNA damage

Analysis of GEO data also confirmed that CXorf67 was indeed highly expressed in PFA (fig. 11 a), and in cooperation with the affiliated children's hospital at the double denier university, 28 ventricular tube tumor samples (and numbered sequentially) from children, with an average age of 3 years, were collected from the frozen stock of hospitals at 2013-2018. After Western blot analysis of frozen samples, CXorf67 expression was found in most tumor samples, and the expression differences between samples were large. Furthermore, we also examined protein levels that reflect the DNA damage repair index γ -H2 AX. Interestingly, it was found that there was a positive correlation between the expression level of CXorf67 and the protein level of γ -H2AX (r=0.698, p=0.0075), which also validated our previous findings at the tissue level of ependymoma, i.e. high expression of CXorf67 could inhibit DNA damage repair (fig. 11 b).

EXAMPLE 12 CXorf67 high expression ependymoma is more sensitive to PARP inhibitors

A sample of surgically excised fresh ventricular tube tumor was collected to establish some study system. 5 fresh ventricular membrane samples (4 ventricular membranous tumor samples, PFA 1-4 and 1 medulloblastoma, MB-1) were collected, and the expression level of CXorf67 in these 5 tumor samples was examined, and it was found that the expression level of CXorf67 in PFA-1 and PFA-4 was higher than that of PFA-2 and PFA-3, and that no CXorf67 expression was detected in MB-1 (FIG. 12 a). We performed primary cell culture and drug testing on PFA-1 and PFA-2, and found that PFA-1 that highly expressed CXorf67 was more sensitive to taazoparib drugs than PFA-2 that lowly expressed CXorf67 (12 b). It was later found that the number of passages of primary cells was limited and that in vitro culture may have changed the original state of the tumor. Thus, we constructed a human tumor xenograft model (PDX) from fresh tumor samples collected later. Compared with a tumor cell line, the PDX model better reserves the tumor microenvironment, is closer to the original tumor in histopathological morphology, and can better evaluate medicines. We planted fresh tumor samples of PFA-3, PFA-4 and MB-1 subcutaneously in NOD-SCID mice, grown to first generation PDX over a period of 3-6 months, after which we removed PDX to digest it into single cells, and then 3X10 ⁶ Cells were injected subcutaneously into nude mice until tumors grew to 50mm ³ At this time, mice were gavaged with talazoparb (0.33 mg/kg,5 days/week) and tumor volumes were measured for one week. At 1000mm ³ Survival curves were plotted for the end-point of death in mice, and experimental results showed that both PFA-3 and PFA-4 dosed groups had an extended survival time compared to the non-dosed group, but the extended survival time of PFA-4 highly expressing CXorf67 was more pronounced, while MB-1 not expressing CXorf67 had substantially no difference in survival time between the dosed and non-dosed groups (FIG. 12 c). It was also found that the tumor volume of the PFA-4 dosed mice was significantly reduced (12 d). These results indicate that PARP inhibitors are also sensitive to ependymomas that highly express CXorf 67.

Example 13 CXorf67 expression in other cancers

Further analysis by TCGA data revealed that CXorf67 was highly expressed in cancers such as renal clear cell carcinoma (kidney renal clear cell carcinoma, KIRC) and renal papillary cell carcinoma (kidney renal papillary cell carcinoma, KIRP) (fig. 13 a). And survival analysis was performed on both KIRC and KIRP patients, and found that the prognosis was worse for patients with high CXorf67 expression (fig. 13 b). These results indicate that CXorf67 as an oncogene may play a role in the development of tumorigenesis.

Discussion of the invention

CXorf67 is not expressed in most tumor cell lines, but it is very interesting that CXorf67 is highly expressed in the PFA subtype of ependymoma, which is very specific. PFA occurs mainly in the posterior brain area of children and little progress has been made in its treatment modalities in the past few decades. At present, surgical excision is mainly adopted, radiotherapy is adopted as an auxiliary material, and no consistent conclusion is obtained on chemotherapy. The invention shows that the higher the expression quantity of CXorf67 is, the more sensitive the inhibitor of PAPR is through Daoy cell line and established PFA patient-derived primary cell and PDX model. All of the above studies indicate that CXorf67 is a biomarker for guiding the administration of PFA patients. Furthermore, CXorf67 specific expression in PFA patients can also be used as an index of molecular classification of ependymoma.

Reference to the literature

1.Lou,J.,Chen,H.,Han,J.,He,H.,Huen,M.S.Y.,Feng,X.H.,Liu,T.,and Huang,J.(2017).AUNIP/C1orf135 directs DNA double-strand breaks towards the homologous recombination repair pathway.Nat Commun 8,985.

2.Wang,F.,Dai,J.,Daum,J.R.,Niedzialkowska,E.,Banerjee,B.,Stukenberg,P.T.,Gorbsky,G.J.,and Higgins,J.M.(2010).Histone H3 Thr-3phosphorylation by Haspin positions Aurora B at centromeres in mitosis.Science 330,231-235.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

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Claims (7)

1. The application of CXorf67 gene, mRNA, cDNA or protein or detection reagent thereof is characterized in that the application is used for preparing a diagnostic reagent or a kit for detecting the sensitivity of tumor cells to DNA damage repair inhibitor drugs, wherein the DNA damage repair inhibitor drugs are PARP inhibitors, and the tumor cells are CXorf67 expression or high expression tumor cells.

2. The use according to claim 1, wherein the PARP inhibitor is selected from the group consisting of: talazoparib, olaparib, veliparib, rucaparib, niraparib.

3. The use according to claim 1, wherein the protein comprises a PALB2 binding motif (PALB 2-binding motif).

4. The use according to claim 3, wherein the PALB2 binding motif is located at positions 420-432 of the CXorf67 protein.

5. The use according to claim 1, wherein the tumour cell is a tumour cell deficient in the HR repair pathway.

6. The use according to claim 5, wherein the HR repair pathway deficient tumor is selected from the group consisting of: ependymoma (Ependymoma posterior fossa group A), renal clear cell carcinoma (kidney renal clear cell carcinoma, KIRC), renal papillary cell carcinoma (kidney renal papillary cell carcinoma, KIRP), or a combination thereof.

7. The use of claim 6, wherein the ependymoma comprises PFA.

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