TLK biomarker composition for predicting PARP inhibitor treatment responsiveness or prognosis of BRCA mutant cancer patients and use thereof

Abstract

The present invention relates to a TLK biomarker composition for predicting PARP inhibitor treatment responsiveness or prognosis of BRCA mutant cancer patients and a use thereof. In the present invention, it was confirmed that human Tousled-like Kinase (TLK) is essential for NHEJ-mediated DSB repair and PARP inhibitor sensitivity in cancer cells having BRCA1 mutations. TLK is essential for nuclear translocation of LC8 and 53BP1 focus formation at DNA double-strand breaks (DSBs); however, it was confirmed that TLK deficiency in BRCA1 mutations promotes resistance to PARP inhibitors, reduces 53BP1 recruitment, and restores homologous recombination (HR). Therefore, the TLK biomarker of the present invention can be usefully applied to predict treatment responsiveness or prognosis of PARP inhibitors in BRCA mutant cancer.

Description

TLK biomarker composition for predicting PARP inhibitor treatment response or prognosis in patients with BRCA mutant cancer and use thereof

[0001] The present invention relates to a TLK biomarker composition for predicting the treatment response or prognosis of a patient with BRCA mutant cancer to a PARP inhibitor and its use, and more particularly, to a TLK biomarker composition for predicting the treatment response or prognosis of a patient with BRCA mutant cancer to a PARP inhibitor and a method for providing information for predicting the treatment response or prognosis of a patient with BRCA mutant cancer using the same.

[0002]

[0003] DNA double-strand breaks (DSBs) are the most cytotoxic of DNA damage, and improper DSB repair often leads to several human diseases, including premature aging-like phenotypes, tumorigenesis, and genomic instability (Helleday, T., et al., DNA Repair (Amst), 6:923-935, 2007). To repair DSBs, cells mainly utilize two pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR) (Ceccaldi, R., et al., Trends Cell Biol., 26:52-64, 2015).

[0004] Because sister chromatids generated during DNA replication are required, HR occurs only during restricted phases of the cell cycle (S to G2 phases), whereas NHEJ is a relatively rapid process that is active throughout the cell cycle, and most DSBs are repaired by NHEJ. The choice between NHEJ and HR at a DSB depends largely on whether the BRCA1-associated complex can access the DSB and resect the ends to generate ssDNA. BRCA1 mutations produce HR deficiency (HRD), which increases the lifetime risk of developing certain types of cancer, particularly breast and ovarian cancer, but conversely, HR deficiency in tumor cells makes cancers susceptible to treatment with poly (ADP-ribose) polymerase (PARP) inhibitors and platinum compounds (Toh, M. and Ngeow, J, Oncologist, 26:e1526-e1537, 2021).

[0005] PARP inhibitors inhibit base excision repair (BER), which inhibits DNA damage repair in cancer cells and can enhance the effectiveness of cancer treatments such as radiation. They have shown promising results in the treatment of breast and ovarian cancer with BRCA mutations. Although PARP inhibitors have already been launched as new drugs on the market, follow-up research is actively underway to expand their indications. Currently, AstraZeneca's olaparib (active ingredient name: olaparib), Tesaro's niraparib, and Clovis Oncology's rucaparib are being used to treat ovarian cancer, and olaparib has recently received additional FDA approval for the indication of BRCA-mutated HER2-negative metastatic breast cancer.

[0006] Although PARP inhibitors have shown significant benefits in progression-free survival and overall response rate, not all patients with BRCA1 mutations respond to PARP inhibitor therapy.

[0007] A 2019 study published in the New England Journal of Medicine found that patients with BRCA1 / 2-mutant advanced breast cancer treated with the FDA-approved PARP inhibitor talazoparib had an objective response rate of 62.6% and a median progression-free survival of 8.6 months. However, not all patients benefited from the treatment, and the study reported that 37.4% of patients had advanced disease (Litton, JK, et al., N Engl J Med, 379:753-763, 2018).

[0008] Similarly, a study published in the Journal of Clinical Oncology in 2020 found that among patients with BRCA1 / 2-mutant ovarian cancer who received niraparib, another FDA-approved PARP inhibitor, the objective response rate was 73%, and the median progression-free survival was 12.9 months. However, the study also reported that 27% of patients did not respond to treatment (Turner, NC, et al., Clin Cancer Res, 27:5482-5491, 2021).

[0009]

[0010] Therefore, despite the significant clinical benefits of PARP inhibitors in breast and ovarian cancer patients with BRCA1 mutations, there is a need to develop new biomarkers to select patients eligible for PARP inhibitor therapy and to predict treatment response or prognosis to PARP inhibitor therapy.

[0011]

[0012] Meanwhile, Toulouse-like kinases (TLKs) are a family of serine / threonine kinases conserved across diverse eukaryotic organisms. In mammals, two homologs, TLK1 and TLK2, share a high degree of sequence similarity and are expressed throughout the cell cycle. TLKs play a role in various cellular processes, including DNA replication, chromatin remodeling, and DNA damage responses. TLK activity is highest during the S phase of the cell cycle and is essential for efficient DNA replication and proper chromosome segregation. TLKs phosphorylate ASF1 to promote histone chaperone activity during DNA replication (Sillje, HH, et al., EMBO J, 18, 5691-5702, 1999).

[0013] Although TLKs are transiently inhibited by ATM-dependent Chk1 phosphorylation during the DNA damage response (DDR), few reports have highlighted their role in the DNA damage response. TLKs also play a role in DNA double-strand break repair, and depletion of TLK1 delays S phase progression and impairs DSB repair efficiency in cells recovering from irradiation (Sunavala-Dossabhoy, G., et al., BMC Mol Biol 6:19, 2005).

[0014] Overexpression of TLK1 has been shown to confer radioresistance in mouse mammary fibroblasts, and pharmacological inhibition of TLK using specific phenothiazines impairs DSB repair, resulting in the accumulation of γH2AX. Similarly, TLKs have been implicated in several cancers and developmental disorders (Khalil, MI, et al., Cancer Drug Resist 5, 93-101, 2022).

[0015]

[0016] In the present invention, as a result of efforts to screen biomarkers that can determine the therapeutic responsiveness or resistance of BRCA mutant cancer patients to PARP inhibitors, a novel mechanism was discovered in which TLKs promote PARP inhibitor sensitivity by repairing DNA double-strand breaks (DSBs) in breast and ovarian cancer cells with BRCA1 deficiency. In the present invention, it was confirmed that TLK1 and TLK2 promote NHEJ repair by regulating the nuclear translocation of LC8, which is required for 53BP1 recruitment to DSBs. In addition, it was confirmed that when TLK1 or TLK2 is depleted, NHEJ is reduced and HR is increased despite BRCA1 deficiency, so that triple-negative breast cancer (TNBC) and ovarian cancer cells with BRCA1 deficiency further increase resistance to PARP inhibitors.

[0017]

[0018] Accordingly, the purpose of the present invention is to provide a TLK (Tousled-like kinase) biomarker composition for predicting the therapeutic response or prognosis of a PARP inhibitor in a patient with BRCA mutant cancer, a composition for predicting the therapeutic response or prognosis of a PARP inhibitor comprising the same, and a kit for predicting the therapeutic response or prognosis of a PARP inhibitor.

[0019] Another object of the present invention is to provide a method for providing information for predicting the therapeutic response or prognosis of a PARP inhibitor using the biomarker composition.

[0020] Another object of the present invention is to provide a composition for preventing or treating BRCA mutant cancer that is resistant to a PARP inhibitor containing a substance for promoting mRNA or protein expression of TLK (Tousled-like kinase), and a screening method therefor.

[0021]

[0022] To achieve the above-mentioned purpose,

[0023] The present invention provides a TLK (Tousled-like kinase) biomarker composition for predicting therapeutic response or prognosis of a PARP inhibitor in BRCA mutant cancer.

[0024] In a preferred embodiment of the present invention, the TLK may be TLK1 or TLK2.

[0025] In another preferred embodiment of the present invention, the PARP inhibitor may be olaparib, talazoparib, niraparib, rucaparib, veliparib, or pamiparib.

[0026]

[0027] In addition, the present invention provides a composition for predicting the therapeutic response or prognosis of a PARP inhibitor in BRCA mutant cancer, including a preparation for measuring the mRNA or protein expression level of the TLK (Tousled-like kinase), and a kit for predicting the therapeutic response or prognosis of a PARP inhibitor in BRCA mutant cancer, including the same.

[0028] In a preferred embodiment of the present invention, the TLK may be TLK1 or TLK2.

[0029] In another preferred embodiment of the present invention, the PARP inhibitor may be olaparib, talazoparib, niraparib, rucaparib, veliparib, or pamiparib.

[0030] In another preferred embodiment of the present invention, the agent for measuring the mRNA expression level may be a sense and antisense primer, or probe, that complementarily binds to the mRNA of TLK.

[0031] In another preferred embodiment of the present invention, the agent for measuring the protein expression level may be an antibody, an interacting protein, a ligand, a nanoparticle, or an aptamer that specifically binds to a TLK protein or a peptide fragment.

[0032] In another preferred embodiment of the present invention, the BRCA mutant cancer may be BRCA mutant breast cancer, ovarian cancer, or uterine cancer.

[0033]

[0034] To achieve other purposes,

[0035] The present invention provides a method for providing information on the therapeutic responsiveness or prognosis of a PARP inhibitor in BRCA mutant cancer, comprising the step of measuring the mRNA or protein expression level of TLK from a sample of a BRCA mutant cancer patient.

[0036] In a preferred embodiment of the present invention, when the mRNA or protein expression level of TLK is lower than that of a normal control group, it may provide information that the treatment response to a PARP inhibitor is low or the prognosis is poor.

[0037]

[0038] In addition, the present invention provides a method for providing information on the administration of a PARP inhibitor in BRCA mutant cancer, comprising the step of measuring the mRNA or protein expression level of TLK from a sample of a BRCA mutant cancer patient.

[0039] In a preferred embodiment of the present invention, if the mRNA or protein expression level of TLK is lower than that of a normal control group, information may be provided that a PARP inhibitor was not administered.

[0040]

[0041] To achieve another purpose,

[0042] The present invention provides a pharmaceutical composition for preventing or treating BRCA mutant cancer resistant to PARP inhibitors, comprising a substance for promoting mRNA or protein expression level of TLK.

[0043] In a preferred embodiment of the present invention, the composition may further comprise a PARP inhibitor, and the PARP inhibitor may be olaparib, talazoparib, niraparib, rucaparib, veliparib, or pamiparib.

[0044]

[0045] In addition, the present invention provides a method for screening a substance for prevention or treatment of BRCA mutant cancer cells that are resistant to PARP inhibitors, comprising the step of treating BRCA mutant cancer cells with a test substance and then measuring the mRNA or protein expression level of TLK.

[0046] In a preferred embodiment of the present invention, if the mRNA or protein expression level of TLK is increased by the test substance compared to the control group, the substance can be screened as a preventive or therapeutic substance for BRCA mutant cancer that is resistant to PARP inhibitors.

[0047]

[0048] In the present invention, we confirmed that human Tousled-like Kinase (TLK) is essential for NHEJ-mediated DSB repair and PARP inhibitor sensitivity in cancer cells harboring BRCA1 mutations. While TLK is independent of kinase activity, we confirmed that TLK1 is essential for nuclear translocation of LC8 and 53BP1 foci formation at DNA double-strand breaks (DSBs). Furthermore, we confirmed that TLK deficiency promotes resistance to PARP inhibitors in TNBC and OVCA cell lines with BRCA1 deficiency, reducing 53BP1 recruitment and restoring homologous recombination (HR). Therefore, the TLK biomarker of the present invention can be usefully applied to predict treatment response or prognosis of BRCA mutant cancers with PARP inhibitors.

[0049]

[0050] Figure 1 is a diagram showing the results that TKL promotes NHEJ-mediated DSB repair in a kinase-independent manner.

[0051] Figure 1a is a schematic diagram of a GFP reporter assay method using U2OS EJ5-GFP to measure NHEJ efficiency.

[0052] Figure 1b is immunoblot data confirming the level of expression of related proteins when cells were transfected with siRNA for TLK1 and / or TLK2, and Figure 1c is the result of measuring NHEJ efficiency thereof.

[0053] Figure 1d is immunoblot data showing the level of expression of related proteins to confirm the degree of restoration of NHEJ by siRNA-resistant wild-type TLK1 (WT) in cells depleted of endogenous TLK1, and Figure 1e is the result of measuring the NHEJ efficiency thereof.

[0054] Figure 1f shows the results of measuring NHEJ efficiency to confirm the extent of NHEJ restoration by transient expression of siRNA-resistant TLK2 WT in siTLK2-transfected cells.

[0055] Figure 1g shows data confirming cell sensitivity to a DSB inducer (Bleomycin) in H1299 cells depleted of TLK.

[0056] Figure 1h shows immunoblot data confirming the degree of delayed γH2AX ablation following bleomycin in TLK-depleted U2OS cells. The amount of each protein in the figure was quantified using ImageJ software, and the ratio was calculated and displayed.

[0057] Figures 1i and 1j show data confirming that TLK promotes NHEJ independent of kinase activity. Cells stably expressing HA-TLK1-WT or HA-TLK1-KD (kinase-dead) were transfected with HA-I-SceI with or without siRNA TLK transfection, and then NHEJ efficiency was measured.

[0058] Figure 1k shows data confirming the degree of decrease in H1299 cell sensitivity to bleomycin in the expression of HA-TLK1-WT or HA-TLK1-KD.

[0059]

[0060] Figure 2 is data confirming that TLK is required for DSB localization of 53BP1, a NHEJ promoting factor.

[0061] Figures 2a and 2b are images (scale bar 10 μm) and numerical data showing 53BP1 focal positive cells observed when cisplatin was treated in A549 cells transfected with siRNA TLK.

[0062] Figures 2c and 2d are images (scale bar 10 μm) and numerical data showing RPA32 foci-positive cells observed when cisplatin was treated in A549 cells transfected with siRNA TLK.

[0063] Figure 2e is a schematic diagram of U2OS ER-mCherry-LacI-FokI-DD cells used to monitor recruitment of a protein of interest at an artificial DSB cleaved by FokI endonuclease. Translated ER-mCherry-LacI-FokI-DD is stabilized by co-treatment with Shield-1 and 4-OHT and translocates to the nucleus, where it generates a DSB at the LacO repeat site.

[0064] Figures 2f and 2g are data confirming that the localization of 53BP1 at the FokI cleavage site was reduced upon depletion of TLK1 or TLK2. These are images and numerical data showing 53BP1 foci-positive cells observed after transfecting U2OS ER-mCherry-LacI-FokI-DD cells with siRNA TLK.

[0065]

[0066] Figure 3 shows data confirming the effect of TLK on DSB localization of LC8 and its interaction with 53BP1.

[0067] Figure 3a is an image (scale bar 10 μm) showing DSB localization of Flag-LC8 when Flag-LC8-expressing U2OS cells were treated with bleomycin.

[0068] Figures 3b to 3d are images (scale bar 10 μm) and numerical data showing the formation of Flag-LC8 and γH2AX foci when U2OS cells stably expressing Flag-LC8 were transfected with siRNA TLK and then treated with bleomycin. It was confirmed that depletion of TLK1 or TLK2 reduced Flag-LC8 foci formation at DSBs.

[0069] Figure 3e shows immunoblot data demonstrating the reduction of chromatin-bound LC8 following TLK depletion in U2OS cells stably expressing Flag-LC8, transfected with siRNA TLK, and then treated with bleomycin. Immunoblots of γH2AX and histone H3 indicate DNA damage responses induced by bleomycin and chromatin enrichment in the extract, respectively. TCE represents total cell extract, and Chr represents chromatin-rich fraction.

[0070] Figure 3f shows data confirming restoration of LC8 localization at the FokI cleavage site by siRNA-resistant TLK1 in TLK1-depleted cells, using qChIP analysis on U2OS ER-mCherry-LacI-FokI-DD cells using primers near the FokI-cut.

[0071] Figure 3g shows data confirming the degree of reduction in the interaction between 53BP1 and LC8 upon TLK1 or TLK2 deficiency. H1299 cells were transfected with the indicated siRNA TLK, treated with bleomycin, and then immunoblotted.

[0072]

[0073] Figure 4 shows data confirming that the nuclear localization signal (NLS) of TLK is essential for the nuclear movement of LC8 and NHEJ.

[0074] Figure 4a shows immunoblotting data obtained after transfection of U2OS cells with siRNA TLK and subsequent treatment with bleomycin. Cyt represents the cytoplasmic fraction; Nuc represents the nucleosolic fraction. NBS1 and GAPDH immunoblots were used as representative markers for the nucleosolic and cytosolic fractions, respectively. It was confirmed that nuclear LC8 was reduced in TLK-depleted cells.

[0075] Figure 4b is an image showing the highly conserved NLS sequences of TLK1 and TLK2 across various species. The putative NLS sequences were predicted using the cNLS Mapper program available at http / nls-mapper.iab.keio.ac.jp.

[0076] Figure 4c confirms the failure of nuclear localization of HA-TLK1 NLS mutants, showing a schematic diagram of the functional motif of TLK1 including NLS (upper panel) and images of cells observed when amino acids that inactivate the NLS function of TLK1 and cytoplasmic localization of HA-TLK1 NLSm were substituted (lower panel).

[0077] Figure 4d shows data from IP (mmunoprecipitation) performed when HA-TLK1-WT or NLSm was overexpressed in HEK293T cells, showing that the interaction between TLK1 and LC8 was not affected by the NLS mutation.

[0078] Figures 4e to 4g show immunoblot data according to cell fractions obtained after transfection with siRNA TLK into U2OS EJ5 reporter cells stably expressing siRNA-resistant HA-TLK1-WT, HA-TLK1-KD, or -NLSm, followed by treatment with bleomycin, confirming that the TLK1 NLS mutation failed to restore the nuclear localization of LC8 and NHEJ. The ratio of Nuc:Cyt in the figure is indicated at the bottom of 4e, and the quantification of NHEJ efficiency and the corresponding immunoblots are shown in Figures 4f and 4g, respectively.

[0079] Figure 4h shows immunoblot data according to cell fractionation after transfection with siRNA TLK into U2OS EJ5 reporter cells stably expressing siRNA-resistant HA-TLK1-WT or HA-TLK1-KD (kinase-dead) and treatment with bleomycin, confirming that the role of TLK in regulating nuclear translocation of LC8 is functionally independent of its kinase activity.

[0080] Figure 4i shows data confirming the effect of ectopic expression of HA-TLK1 NLSm on cell sensitivity to bleomycin in TLK1 depletion, when H1299 cells stably expressing siTLK1-resistant WT or NLSm were transfected with siRNA TLK and then treated with bleomycin.

[0081] Figure 4j is a schematic diagram showing the functional role of TLK in mediating nuclear localization of LC8 to promote 53BP1-dependent NHEJ repair at DSBs.

[0082]

[0083] Figure 5 is data confirming that PARP inhibitor resistance increased in BRCA1 mutant cancer due to TLK deficiency.

[0084] Figures 5a and 5b are immunoblot data for siRNA TLK transfection in BRCA1-proficient MDA-MB-468 cells and data for confirming cell viability to observe PARP inhibitor (Olaparib) sensitivity according to TLK loss.

[0085] Figures 5c and 5e are immunoblot data for siRNA TLK transfection in BRCA1 mutant MDA-MB-436 cells and data for confirming cell viability to observe whether PARP inhibitor (Olaparib and Veliparib) resistance is induced by TLK loss.

[0086] Figures 5f and 5g are immunoblot data for siRNA TLK transfection in BRCA1 normal MDA-MB-468 cells transfected with siBRCA1 and data for confirming cell viability to observe whether PARP inhibitor (Olaparib) resistance is induced by TLK loss.

[0087] Figures 5h and 5i are immunoblot data for siRNA TLK transfection in BRCA1 mutant UWB1.289 ovarian cancer cells and data for confirming cell viability to observe whether PARP inhibitor (Olaparib) resistance is induced by TLK loss.

[0088]

[0089] Figure 6 shows data confirming that HR is restored in BRCA1-deficient cells by TLK loss.

[0090] Figure 6a is a schematic diagram illustrating the measurement of HR recovery following BRCA1 depletion in U2OS DR-GFP cells.

[0091] Figures 6b and 6c are data confirming the expression level of each protein by immunoblotting and data confirming HR efficiency when U2OS DR-GFP cells were transfected with siBRCA1 and siRNA TLK.

[0092] Figures 6d and 6e are images (scale bar 10 μm) and quantification data of 53BP1 foci-positive cells observed when A549 cells were transfected with siBRCA1 and siRNA TLK and then treated with bleomycin.

[0093] Figures 6f to 6h are images (scale bar 10 μm) and quantification data of 53BP1 focus-positive cells observed when A549 cells were transfected with siBRCA1 and siRNA TLK and then treated with cisplatin. In the absence of BRCA1, simultaneous depletion of TLK1 and TLK2 resulted in a decrease in 53BP1 focus formation at DSBs.

[0094] Figures 6i and 6j are data showing the expression levels of each protein confirmed by immunoblotting to analyze NHEJ efficiency after transfecting NHEJ / DsRed293B cells with siBRCA1 and siRNA TLK, and data showing NHEJ efficiency analyzed by calculating the number of DsRed-positive cells in FACS analysis.

[0095] Figure 6k shows data from HR analysis performed using the method of Figure 6a to confirm whether TLK and LC8 act in the same DSB repair pathway upon BRCA1 inactivation. After transfecting U2OS DR-GFP cells with siBRCA1, siLC8, or siRNA TLK, HR efficiency was analyzed.

[0096]

[0097] Figure 7 shows data observing the prognosis according to TLK loss in patients with BRCA1 deficiency.

[0098] Figures 7a to 7c are patient survival plots generated by comparing patient groups with high and low expression of TLK1 or TLK2. Breast invasive carcinoma (BRCA), ovarian serous cystadenocarcinoma (OVCA), and uterine corpus endometrial carcinoma (UCEC) were selected from the TCGA database and classified based on the corresponding TLK expression in patients with BRCA1 WT or deficiency.

[0099] Figure 7d shows data analyzing the risk profile across various cancers (pan-cancer) in a patient cohort with low TLK1 or TLK2 expression. Cox coefficients for the low TLK1 / TLK2 cohort were analyzed across various cancers, with red indicating a higher risk (poor prognosis) for the group with low TLK1 or TLK2 expression, and blue indicating a lower risk (good prognosis). Patient groups were categorized by BRCA1 mutation status and then by TLK expression level.

[0100] Figure 7e is a boxplot of TLK1 or TLK2 expression (z-score) in BRCA1 WT and deficient cohorts.

[0101] Figure 7f is a schematic diagram showing a proposed model for the role of TLK in NHEJ and PARP inhibitor sensitivity under BRCA mutation conditions.

[0102]

[0103] Hereinafter, the present invention will be described in detail.

[0104]

[0105] Biomarker composition for predicting therapeutic response or prognosis of PARP inhibitors in BRCA mutant cancer

[0106] The present invention relates, from a consistent perspective, to a TLK (Tousled-like kinase) biomarker composition for predicting therapeutic response or prognosis of a PARP inhibitor in BRCA mutant cancer.

[0107] In the present invention, the TLK may be TLK1 or TLK2.

[0108] In the present invention, the PARP inhibitor may be olaparib, talazoparib, niraparib, rucaparib, veliparib, or pamiparib, but is not limited thereto, and any substance having a PARP inhibitory effect known in the art, such as a next-generation PARP inhibitor approved by the FDA or EMA for clinical use, may be applied without limitation.

[0109]

[0110] In a specific embodiment of the present invention, it was confirmed that TLK promotes 53BP1-dependent NHEJ activity, and the NLS (nuclear translocation signal) of TLK is essential for nuclear translocation of LC8 and 53BP1 focus formation at DSBs rather than kinase activity.

[0111] In BRCA1 mutant cancers, TLK depletion was shown to decrease NHEJ activity. Furthermore, TLK depletion was found to decrease 53BP1 recruitment and restore homologous recombination (HR), promoting resistance to PARP inhibitors.

[0112] Therefore, the TLK biomarker of the present invention can be usefully applied to predicting the therapeutic response or prognosis of PARP inhibitors in BRCA mutant cancer.

[0113]

[0114] Composition for predicting therapeutic response or prognosis of PARP inhibitors in BRCA mutant cancer

[0115] In another aspect, the present invention relates to a composition for predicting therapeutic response or prognosis of a PARP inhibitor in BRCA mutant cancer, comprising a preparation for measuring the mRNA or protein expression level of the TLK (Tousled-like kinase).

[0116] The composition for predicting the therapeutic response or prognosis of a PARP inhibitor in BRCA mutant cancer according to the present invention applies the <biomarker composition> described above.

[0117]

[0118] In the present invention, the BRCA mutation cancer may be breast cancer, ovarian cancer, or uterine cancer in which a BRCA mutation exists.

[0119] In the present invention, the agent for measuring the mRNA expression level may be a sense and antisense primer or probe that complementarily binds to the mRNA of TLK.

[0120] Measurement of mRNA expression levels is the process of determining the presence and expression level of mRNA for biomarkers for chronic liver disease diagnosis in biological samples. This involves measuring the amount of mRNA. Analytical methods used for this purpose include, but are not limited to, reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, and DNA chips.

[0121] The preparation for measuring the mRNA level of the above-mentioned complex marker is characterized by being a primer pair, probe or antisense nucleotide that specifically binds to the gene of the above-mentioned complex marker, and since the nucleic acid information of the above-mentioned genes is known in GeneBank, etc., a person skilled in the art can design these primer pairs, probes or antisense nucleotides based on the above-mentioned sequence.

[0122] The term "primer" used in the present invention refers to a fragment that recognizes a target gene sequence, and includes a pair of forward and reverse primers, but is preferably a pair of primers that provides analysis results with specificity and sensitivity.

[0123] The term "probe" used in the present invention refers to a substance that can specifically bind to a target substance to be detected in a sample, and through said binding, refers to a substance that can specifically confirm the presence of the target substance in the sample. The type of probe is not limited to a substance commonly used in the art, but is preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA, or DNA, and most preferably PNA.

[0124] The term "antisense" as used herein refers to an oligomer having a sequence of nucleotide bases and an intersubunit backbone that hybridizes with a target sequence within RNA by Watson-Crick base pairing, typically allowing the formation of an mRNA and RNA:oligomer heteroduplex within the target sequence. The oligomer may have exact sequence complementarity or approximate sequence complementarity to the target sequence.

[0125]

[0126] In the present invention, the agent for measuring the protein expression level may be an antibody, an interacting protein, a ligand, a nanoparticle, or an aptamer that specifically binds to a complementary TLK protein or a peptide fragment.

[0127] Protein expression level measurement is the process of determining the presence and level of protein expression from a biomarker for the diagnosis of chronic liver disease in biological samples. Protein levels can be determined using antibodies, interacting proteins, ligands, nanoparticles, or aptamers that specifically bind to the protein or peptide fragment of the gene.

[0128] Methods for measuring or comparing the above protein expression levels include, but are not limited to, protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, aukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry), Western blot, and ELISA (enzyme linked immunosorbent assay).

[0129] The term "antibody" as used herein refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For the purposes of the present invention, "antibody" refers to an antibody that specifically binds to the biomarker of the present invention. Antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

[0130]

[0131] Kit for predicting treatment response or prognosis of PARP inhibitors in BRCA mutant cancers

[0132] In another aspect, the present invention relates to a kit for predicting therapeutic response or prognosis of a PARP inhibitor in BRCA mutant cancer, comprising a composition for predicting therapeutic response or prognosis of a PARP inhibitor in BRCA mutant cancer.

[0133] The kit for predicting the therapeutic response or prognosis of a PARP inhibitor in BRCA mutant cancer according to the present invention comprises the above-described <biomarker composition> or<PARP 저해제의 치료 반응성 또는 예후 예측용 조성물> Apply.

[0134]

[0135] The above kit can be manufactured using conventional manufacturing methods known in the art. The kit may include, for example, a lyophilized antibody, a buffer, a stabilizer, an inactive protein, etc.

[0136] The kit may further comprise a detectable label. The term "detectable label" refers to an atom or molecule that allows for the specific detection of a molecule containing the label among identical molecules without a label. The detectable label may be attached to an antibody, interacting protein, ligand, nanoparticle, or aptamer that specifically binds to the protein or a fragment thereof. The detectable label may include a radionuclide, a fluorophore, or an enzyme.

[0137] The above kit can be used according to various immunoassays or immunostaining methods known in the art. The immunoassays or immunostaining methods may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA, capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining, and immunoaffinity purification. Preferably, the kit may be a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or an MRM (multiple reaction monitoring) kit.

[0138]

[0139] Methods for providing information on therapeutic responsiveness or prognosis of PARP inhibitors in BRCA-mutant cancers

[0140] In another aspect, the present invention relates to a method for providing information on the therapeutic responsiveness or prognosis of a PARP inhibitor in BRCA mutant cancer, comprising the step of measuring the mRNA or protein expression level of TLK from a sample of a BRCA mutant cancer patient.

[0141] In the present invention, if the mRNA or protein expression level of TLK is lower than that of a normal control group, it may provide information that the treatment response to a PARP inhibitor is low or the prognosis is poor.

[0142]

[0143] In another aspect, the present invention relates to a method for providing information on the administration of a PARP inhibitor in BRCA mutant cancer, comprising the step of measuring the mRNA or protein expression level of TLK from a sample of a BRCA mutant cancer patient.

[0144] In the present invention, if the mRNA or protein expression level of TLK is lower than that of the normal control group, it can provide information that the PARP inhibitor was not administered.

[0145]

[0146] In the above method, “biological sample” means a sample such as tissue, cell, blood, serum, plasma, saliva, cerebrospinal fluid or urine, and preferably means blood, plasma or serum.

[0147] In the above information provision method, the method for measuring the expression level of the biomarker mRNA or its protein is as described above.

[0148]

[0149] Composition for prevention or treatment of BRCA mutant cancer resistant to PARP inhibitor and screening method thereof

[0150] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating BRCA mutant cancer resistant to PARP inhibitors, comprising a substance for promoting mRNA or protein expression level of TLK.

[0151] The above composition may be administered in combination with a PARP inhibitor, and the PARP inhibitor may be olaparib, talazoparib, niraparib, rucaparib, veliparib, or pamiparib.

[0152]

[0153] In another aspect, the present invention relates to a method for screening a substance for prevention or treatment of BRCA mutant cancer cells that are resistant to PARP inhibitors, comprising the step of treating BRCA mutant cancer cells with a test substance and then measuring the mRNA or protein expression level of TLK.

[0154] In the present invention, if the mRNA or protein expression level of TLK is increased by the test substance compared to the control group, the substance can be screened as a preventive or therapeutic substance for BRCA mutant cancer that is resistant to PARP inhibitors.

[0155]

[0156] Hereinafter, the present invention will be described in more detail through examples.

[0157] These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

[0158]

[0159] Example 1: Confirmation of the efficacy of TKL in promoting NHEJ-mediated DSB repair.

[0160] 1-1: Investigation of NHEJ activity following TLK depletion

[0161] In the present invention, to investigate the unknown function of TLK in DNA double-strand break (DSB) repair induced by the DNA damage response (DDR), an NHEJ reporter assay was performed using the U2OS EJ5-GFP cell line. As shown in Fig. 1a, in the U2OS EJ5-GFP cell line, intact GFP is produced when the I-SceI cleavage site of the mutant GFP gene is successfully repaired by intracellular NHEJ.

[0162] U2OS EJ5-GFP cell line was prepared by a previously reported method (Gunn, A., and Stark, JM, Methods Mol Biol, 920:379-391, 2012) and cultured at 37°C in DMEM (Dulbecco's Modified Eagle's Medium, Gibco) containing 10% FBS and penicillin / streptomycin (1%, Thermo Scientific).

[0163] Then, 3 x 10 5 Dog cells were seeded in 6-well plates, and siRNA was used for depletion of TLK1 or TLK2, and cells were transfected using Lipofectamine RNAiMAX for siRNA (13778150, Invitrogen) according to the manufacturer's instructions.

[0164] siGL2: 5'-CGUACGCGGAAUACUUCGA-3' (SEQ ID NO: 1)

[0165] siTLK1: 5'-GAAGCUCGGUCUAUUGUAA-3' (SEQ ID NO: 2)

[0166] siTLK2: 5'-GAUAGAAAGACAACGGAAA-3' (SEQ ID NO: 3)

[0167] siBRCA1: 5'- CCUGUCUCCACAAAGUGUG-3' (SEQ ID NO: 4)

[0168] siLC8: 5'-AGCCUAAAUUCCAAAUAC-3' (SEQ ID NO: 5)

[0169]

[0170] After 24 h, 1.5 or 2 μg of the I-SceI expression vector pCβASce was transfected using Lipofectamine 2000 (Invitrogen). Cells were harvested 72 h after siRNA transfection and used for subsequent analytical experiments.

[0171]

[0172] For immunoblotting, U2OS EJ5-GFP cells transfected with the above siRNAs were lysed in RIPA lysis buffer (GenDEPOT) containing protease inhibitors on ice for 15 min, followed by brief sonication. Approximately 30–50 μg of the lysate was loaded onto an SDS-PAGE gel and transferred to a nitrocellulose membrane (Amersham Protran). The membrane was then blocked with 3% BSA in trisodium bromide-based saline (TBS-T) (0.1% Tween 20 in TBS) for 30 min at room temperature and incubated with primary antibodies overnight at 4°C or for 1 h at room temperature. The primary antibodies were detected with secondary antibodies for 1 h at room temperature and visualized using ECL solution (Millipore). The primary antibodies used in the present invention are as follows: TLK1 (4125S, Cell Signaling Technology), TLK2 (sc-393506, Santacruz), HA (sc-7392, Santacruz), GAPDH (6004-1-Ig, Proteintech). The ratio of HA-I-SceI to GAPDH was then quantified from the immunoblot and normalized to the percentage of GFP-positive cells in each sample.

[0173]

[0174] As a result, depletion of either TLK1 or TLK2 reduced NHEJ, but co-depletion of TLK1 and TLK2 caused a further decrease in NHEJ compared to single depletion, confirming that TLK1 and TLK2 play redundant roles in promoting NHEJ repair at DSBs. Furthermore, NHEJ efficiency was reduced by TLK depletion without a decrease in I-SceI expression, a marker of DSBs cleaved from the GFP cassette (Fig. 1b and 1c).

[0175]

[0176] 1-2: Investigation of NHEJ activity following restoration of TLK depletion

[0177] To restore TLK depletion, TLK1 or TLK2 sequences were PCR amplified and cloned into pcDNA3-HA vector, and siRNA-resistant TLK1 or TLK2 wild-type (WT) or kinase-dead (KD) were generated using the following primers:

[0178] <siRNA-resistant TLK1-wild type; HA-TLK1-WT>

[0179] F: 5'-CAAGTTAATGTCAGAGAAAGAGGCCAGGTCTATTGTAATGCAGATTG-3' (SEQ ID NO: 6)

[0180] R:5'-CAATCTGCATTACAATAGACCTGGCCTCTTTCTCTGACATTAACTTG-3' (SEQ ID NO: 7)

[0181] <siRNA-resistant TLK2-wild type; HA-TLK2-WT>

[0182] F: 5'-CAGAGGGAAGAGATCGAAAGGCAACGGAAAATGTTAGCAAAGCGG-3' (SEQ ID NO: 8)

[0183] R: 5'-CCGCTTTGCTAACATTTTCCGTTGCCTTTCGATCTCTTCCTCTG-3' (SEQ ID NO: 9)

[0184] <TLK1 KD; HA-TLK1-KD>

[0185] F: 5'-GATTTCCAAC AAGGTTGCTGTATGGTCGGTTGGAG-3' (SEQ ID NO: 10)

[0186] R: 5'-CTCCAACCGACCATACAGCAACCTTGTTGGAAATC-3' (SEQ ID NO: 11)

[0187]

[0188] To generate stable cell lines, plasmids were transfected into U2OS or U2OS EJ5 cells using Lipofectamine 2000 (11668019, Invitrogen), and 48 h after transfection, cells were treated with 500 μg / mL G418 (Invivogen) for drug selection for more than 1 week.

[0189]

[0190] As a result, the reduced NHEJ upon TLK1 or TLK2 depletion was completely restored by siRNA-resistant versions of wild-type TLK1 or TLK2, respectively, confirming that the reduction in NHEJ upon TLK loss was not due to off-target activity of siRNA (Fig. 1d to 1f).

[0191]

[0192] 1-3: Determination of cellular sensitivity to DSB-inducing agents in TLK-depleted cells.

[0193] When TLK-depleted cells were treated with bleomycin, a DSB inducer, cell sensitivity was examined. H1299 cells or U2OS cells were transfected using the same method as in <Example 1-1> and <Example 1-2>, and then treated with 2 μg / mL bleomycin (13877, Cayman Chemical) for 96 hours. Immunoblotting, NHEJ activity, and cell viability were then measured.

[0194] Cell viability was measured using the Cell Titer 96 AQueous One Solution Cell Proliferation Assay kit (G3581, Promega) according to the manufacturer's instructions. Briefly, each siRNA-transfected cell was treated with bleomycin, and then a mixture of Cell Titer 96 aqueous reagent and medium was added to the plate at a 1:5 ratio. The readings were obtained using a BioTek Power Wave HT 340 microplate reader. Viability analysis was normalized to the values ​​of the untreated control group.

[0195] H2AX and γH2AX (pS139-H2AX) were used as primary antibodies in immunoblots, and the amount of each protein was quantified using ImageJ software, and the ratio was calculated.

[0196]

[0197] Defects in NHEJ can sensitize cells to DSBs, and indeed, depletion of TLK1 or TLK2 rendered cells more sensitive to the DSB-inducing agent bleomycin (Fig. 1g). After pulse treatment with bleomycin, γH2AX (phospho-H2AX) decreased over time. Depletion of either TLK1 or TLK2 delayed γH2AX decay by approximately 2.5 h, and this delay was further increased by co-depletion of TLK1 and TLK2, indicating that both proteins are independently required for NHEJ (Fig. 1h).

[0198]

[0199] 1-4: Kinase-independent regulation of NHEJ activity by TLKs

[0200] To investigate the requirement for TLK kinase activity, we generated U2OS cell lines stably expressing an HA-tagged TLK1 kinase-dead mutant (D608A) and performed NHEJ and cell survival assays.

[0201]

[0202] As a result, TLK1 knockdown (KD) (kinase-dead) was able to restore the NHEJ activity reduced by TLK depletion, and overexpression also increased NHEJ activity similar to that of WT (Fig. 1i and 1j). Furthermore, bleomycin-induced cell sensitivity upon TLK1 depletion was alleviated by ectopic expression of TLK1 WT (Fig. 1k), but not by TLK1 knockdown (Fig. 1k).

[0203] Thus, we demonstrate that TLK1 and TLK2 play independent roles in promoting NHEJ repair at DSBs and suppressing cellular sensitivity to DSBs, and that at least for TLK1, kinase activity is not required for induction of NHEJ activity.

[0204]

[0205] Example 2: Determination of DSB localization of 53BP1 following TLK depletion.

[0206] At DNA double-strand breaks (DSBs), the DSB localization of 53BP1 promotes NHEJ by antagonizing ssDNA formation at DSBs coated with RPA and Rad51 (Xu, Y., and Xu, D., Essays Biochem, 64:765-724, 2020). Therefore, in the present invention, the DSB localization of 53BP1, an NHEJ promoting factor, was confirmed in TLK-depleted cells through immunostaining.

[0207] First, A549 cells transfected with siRNA TLK were plated on glass coverslips in 6-well plates, and then the cells were treated with DMSO, cisplatin, or bleomycin and extracted with extraction buffer (25 mM HEPES, pH 7.5, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 300 mM sucrose, and 0.5% Triton X-100). After incubation on ice for 5 min, the cells were fixed with 4% paraformaldehyde (PC2031-050-00, Biosesang) for 10 min. After washing three times with PBST (containing 0.1% Triton X-100 in PBS), the plates were blocked with a solution containing 10% FBS in PBST for at least 30 min at room temperature.

[0208] Primary antibody (anti-53BP1 antibody) was added and incubated with cells for 2 hours, followed by detection with secondary antibodies labeled with Alexa-555 (A21429; Life Technologies) or Alexa-488 (A11029; Life Technologies) for 40 minutes. Intracellular nuclei were stained with VECTASHIELD® Antifade Mounting Medium containing DAPI (H-1200-10; Vector LABORATORIES). Images were captured using an Axio Imager.M2 microscope (Zeiss) and adjusted using ZEN3.4 (Zeiss, blue edition) and Photoshop 7.0 (Adobe).

[0209]

[0210] As expected, the number of 53BP1 foci-positive cells was reduced in cells depleted of either TLK1 or TLK2, and co-depletion of TLK1 and TLK2 resulted in a statistically significant additional decrease (Fig. 2a and b). Notably, RPA32 foci, an indicator of ssDNA formation, were not reduced by depletion of either TLK1 or TLK2, while interestingly, co-depletion increased the number of anti-RPA32-positive cells (Fig. 2c).

[0211]

[0212] To confirm this, as shown in the schematic diagram in Figure 2e, a single DSB was introduced at a defined site using U2OS ER-mCherry-FokI-DD cells treated with Shield-1 and 4-OHT (Shanbhag, NM, et al., Cell, 141:970-981, 2010; Tang, J., et al., Nat Struct Mol Biol, 20:317-325, 2013). Shield-1 binds to the degron and stabilizes FokI, whereas 4-OHT binds to the ER fused to FokI, allowing the protein to translocate to the nucleus. Treatment of cells with 4-OHT and Shield-1 allows the mCherry-linked FokI nuclease to bind to specific FokI cleavage sites, and the mCherry signal allows visualization of the location of the protein of interest at the FokI cleavage site.

[0213] Depletion of TLK1 or TLK2 significantly reduced the number of cells harboring 53BP1 at the FokI site, but depletion of BRCA1, as a negative control, did not reduce this number (Fig. 2f and 2g). Thus, through multiple experimental systems, the present invention confirmed that TLK1 and TLK2 are essential for proper localization of 53BP1 at DSBs.

[0214]

[0215] Example 3: Confirmation of DSB localization of LC8 and its interaction with 53BP1 following TLK depletion.

[0216] 3-1: Confirmation of LC8 localization following TLK depletion

[0217] TLKs are known to promote NHEJ by enhancing DSB localization of 53BP1 by interacting with several proteins, including LC8 (also known as dynein light chain 1, DYNLL1), which acts as a hub protein mediating the oligomerization of target proteins, including 53BP1 (Kim, JA, et al., Mol Cancer Res, 14:920-927, 2016). Since depletion of LC8 or TLK reduces 53BP1 localization at DSBs, we aimed to determine whether the role of TLK proteins in 53BP1 activation for NHEJ is mediated through LC8.

[0218] To visualize the DSB location of LC8, we generated a U2OS cell line stably expressing Flag-LC8. LC8 cDNA was synthesized (Bioneer) and cloned into the pCAG-F-BSD (pCAG-FLAG-blasticidin) vector, which expresses an N-terminal FLAG tag. The plasmid was transfected into U2OS or U2OS EJ5 cells using Lipofectamine 2000 (11668019, Invitrogen) to generate stable cell lines. After 48 hours, the cells were treated with 500 μg / mL G418 (Invivogen) for at least one week for drug selection.

[0219] Next, the cells were transfected with siRNA using the same method as in <Example 1> above, and then treated with 2 μg / mL bleomycin (13877, Cayman Chemical) for 96 hours. Thereafter, immunostaining and immunoblotting were performed.

[0220]

[0221] As a result, we confirmed that bleomycin-induced Flag-LC8 foci almost overlapped with γH2AX foci, indicating that Flag-LC8 was located at DSBs (Fig. 3a). Depletion of TLK1 and / or TLK2 did not decrease the protein amount and foci formation of γH2AX in bleomycin-treated cells, but bleomycin-induced Flag-LC8 foci formation was significantly inhibited in TLK1- and TLK2-depleted cells (Figs. 3b-3d).

[0222]

[0223] 3-2: Confirmation of LC8 through intracellular fraction analysis

[0224] To confirm the above results, the present invention performed a subcellular fractionation analysis to identify chromatin-associated LC8 in bleomycin-treated cells.

[0225] Subcellular fractionation assays were performed using a cultured cell subcellular protein fractionation kit (78840, Thermo Scientific). U2OS cells were transfected with siRNA using the same method as in <Example 1>, and then treated with bleomycin.

[0226] After harvesting and washing the cells with cold PBS, half of the cells were lysed in 50–100 μl of RIPA buffer containing protease inhibitors to obtain whole cell extracts. The remaining half of the cells underwent sequential fractionation.

[0227] Specifically, cytoplasmic extracts were obtained by incubating 50–100 μl of CEB buffer containing protease inhibitors on ice for 10 min, followed by centrifugation at 500 g for 5 min. Nuclear extracts were obtained by incubating the remaining pellet with 25–50 μl of NEB buffer containing protease inhibitors on ice for 10 min.

[0228] For chromatin extracts, 25–50 μl of NEB containing MN enzyme supplemented with CaCl2 and protease inhibitors was incubated at 37°C for 5 min, followed by centrifugation at 16,000 g for 5 min. Proteins were then isolated from each fraction and subjected to immunoblotting. Immunoblots for histone H3, NBS1, and GAPDH served as differential markers for chromatin, nucleosol, and cytoplasm, respectively. Immunoblots for γH2AX and histone H3 indicate DNA damage responses induced by bleomycin and chromatin enrichment, respectively, in the extracts. TCE indicates total cell extract, and Chr indicates chromatin-enriched fraction.

[0229]

[0230] As a result, we confirmed that endogenous LC8 in the chromatin fraction (Chr) was removed after TLK depletion (Fig. 3e).

[0231]

[0232] 3-3: Confirmation of LC8 localization through qChIP analysis

[0233] In addition, since chromatin association of LC8 may be at sites without DSBs, in the present invention, ChIP analysis was performed to measure LC8 recruitment to DSBs generated by FokI nuclease in U2OS ER-mCherry-FokI-DD cells.

[0234] In addition, to confirm the degree of restoration of LC8 localization at the FokI cleavage site by siRNA-resistant TLK1 in TLK1-depleted cells, cells were transfected using the siRNA-resistant TLK1-wild type (HA-TLK1-WT) of <Example 1-2> above.

[0235]

[0236] For chromatin immunoprecipitation (ChIP) assay, U2OS ER-mCherry-LacI-FolkI-DD cells transfected with HA-TLK1-WT or siRNA TLK were fixed with 1% formaldehyde for 20 min at room temperature and quenched with 1.5 M glycine for 5 min. Fixed cells were lysed in SDS lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, pH 8.0, 0.1% SDS, and protease inhibitor cocktail (Roche)) on ice for 15 min or sonicated using a Covaris sonicator (P, 140; DF, 10; cycles, 200; treatment, 80 s at 4°C).

[0237] The resulting lysate was diluted with dilution buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and protease inhibitor cocktail) and incubated overnight with Protein G beads conjugated to dyna-beads conjugated with anti-LC8 antibody. The beads were washed sequentially with RIPA-150 buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, pH 8.0, 1% Triton X-100, 0.1% SDS, and 0.1% sodium deoxycholate), LiCl buffer (10 mM Tris-HCl, pH 8.0, 0.25 M LiCl, 1 mM EDTA, pH 8.0, 0.5% NP40, and 0.5% sodium deoxycholate), and TE buffer.

[0238] The beads were then incubated with ChIP elution buffer (10 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5 mM EDTA, pH 8.0, and 0.5% SDS) at 65°C for 8 h. IPed DNA was recovered using the QIAquick PCR purification kit (Qiagen), and qPCR analysis was performed using a LightCycler 96 (Roche). The primer sequences used for qPCR analysis are as follows:

[0239] Primer sequence

[0240] F: 5'-GGCATTTCAGTCAGTTGCTCAA-3' (SEQ ID NO: 12)

[0241] R: 5'-TTGGCCGATTCATTAATGCA-3' (SEQ ID NO: 13)

[0242]

[0243] As a result, LC8 enrichment at DSBs was suppressed in TLK1-depleted cells, but was partially restored by siRNA-resistant HA-TLK1 WT (Fig. 3f). These data specifically demonstrate that chromatin binding of LC8 at DSBs depends on the cellular presence of TLKs, and that LC8 plays a role in DSB localization and oligomerization of 53BP1 through physical interactions.

[0244]

[0245] 3-4: Confirmation of 53BP1 and LC8 interaction upon TLK depletion

[0246] In the present invention, we verified whether TLK is required for the physical interaction between LC8 and 53BP1.

[0247] H1299 cells were cultured in RPMI 1640 (Corning) supplemented with 10% FBS and 1% penicillin / streptomycin, transfected with siRNA using the same method as in <Example 1>, and then treated with bleomycin.

[0248] For immunoprecipitation, H1299 cells were lysed on ice for 15 min in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 2 mM EDTA, phosphatase inhibitors, and protease inhibitors). The lysate was then briefly sonicated and centrifuged at 15,000 rpm for 30 min at 4°C. Approximately 1–1.5 mg of the lysate was incubated with anti-53BP1 antibody or EZview Red Anti HA Affinity Gel (E6779, Sigma), and the immunoprecipitated 53BP1 was pulled down using protein A Sepharose beads (GE Healthcare). The beads or gel were washed five times with lysis buffer before immunoblotting.

[0249]

[0250] As a result, 53BP1 immunoprecipitates contained LC8 in normal cells (siGL2), but when TLK1 or TLK2 was depleted, the amount of co-immunoprecipitated LC8 was reduced upon bleomycin treatment (Fig. 3g), suggesting that TLKs are required for the 53BP1-LC8 interaction. In other words, these results support the notion that TLK1 and TLK2 promote DSB localization of LC8 by facilitating the interaction with 53BP1.

[0251]

[0252] Example 4: Confirmation of nuclear trafficking of LC8 and NHEJ by the nuclear localization signal (NLS) of TLK.

[0253] 4-1: Confirmation of LC8 distribution according to TLK depletion

[0254] In this study, we investigated how TLK induces DSB localization of LC8. The physical interaction between TLK and LC8 requires the coiled-coil domain (Segura-Bayona, S., et al., Cell Death Differ, 24:1872-1885, 2017). Despite the absence of a nuclear localization signal (NLS) sequence within LC8, LC8 functions to regulate 53BP1 multimerization in the nucleus. Therefore, we hypothesized that the nuclear translocation of LC8 is mediated by its interaction with TLK.

[0255] Therefore, in the present invention, U2OS cells were transfected with siRNA, and then treated with bleomycin. Then, the nuclear and cytoplasmic distribution of LC8 was investigated when TLK was depleted using a subcellular fractionation analysis using the same method as in <Example 3-2>.

[0256]

[0257] As a result, depletion of TLK1 or TLK2 decreased the amount of LC8 protein in the nucleus and increased its cytoplasmic distribution. Furthermore, we confirmed that simultaneous depletion of TLK1 and TLK2 maximized LC8 depletion in the nuclear fraction (Fig. 4a). These results support the notion that TLKs are required for LC8 nuclear transport.

[0258]

[0259] 4-2: Analysis of the nuclear localization signal (NLS) of TLK

[0260] In the present invention, we aimed to determine whether the NLS of TLK was used for nuclear translocation of LC8. The putative NLS sequence was predicted using the cNLS mapper program available at http / nls-mapper.iab.keio.ac.jp.

[0261]

[0262] Putative NLS sequences were predicted using the cNLS mapper program available at http / nls-mapper.iab.keio.ac.jp, and COBALT (Constraint-based alignment tool) was used for alignment (https: / www.ncbi.nlm.nih.gov / tools / cobalt / cobalt.cgi?CMD=Web).

[0263]

[0264] As a result, we identified a single NLS motif in TLK1 and found that it was highly conserved in various species as well as in TLK2 (Fig. 4b, SEQ ID NOs: 17 to 27).

[0265]

[0266] 4-3: Confirmation of interaction with LC8 according to NLS mutation of TLK

[0267] In addition, in the present invention, a nuclear localization defective mutant (RGRAAKAENQ, SEQ ID NO: 16) of HA-TLK1 (NLSm) was produced by substituting KR (RGRKRKAENQ, SEQ ID NO: 15) with AA in the NLS 9 motif.

[0268] HA-TLK1(NLSm) was generated using the same method as in <Example 1-2> above, using the following primers:

[0269] TLK1 NLSm - F: 5'-GGAATCATCCAGGGGAAGAGCGGCAAAAGCAGAAAACCAGAATG-3' (SEQ ID NO: 14)

[0270] Next, HA-TLK1-WT or NLSm was overexpressed in HEK293T cells, followed by immunostaining and immunoprecipitation using anti-HA antibody.

[0271]

[0272] Figure 4c is a schematic diagram of the functional motif of TLK1 including NLS. Amino acid substitutions that inactivate the NLS function of TLK1 and cytoplasmic localization of HA-TLK1 NLSm resulted in failure of nuclear localization of HA-TLK1 NLS mutants, resulting in cytoplasmic localization of TLK1 NLSm.

[0273] Furthermore, we found that HA-TLK1 NLSm immunoprecipitates contained the same LC8 as HA-TLK1 WT, indicating that TLK1 interaction with LC8 is independent of its subcellular location (Fig. 4d).

[0274] In the present invention, we confirmed that HA-TLK1 NLSm does not interact with endogenous TLK2, suggesting that nuclear localization of TLKs functions differently by interacting with LC8 and TLK oligomerization.

[0275]

[0276] 4-4: Confirmation of 53BP1-dependent NHEJ promotion through nuclear localization of TLK.

[0277] Next, in the present invention, we evaluated whether nuclear localization of TLK is important for nuclear translocation of LC8 and confirmed whether it contributes to promotion of East Sea 53BP1-dependent NHEJ.

[0278] U2OS EJ5 reporter cells stably expressing siRNA-resistant HA-TLK1-WT, HA-TLK1-KD, or -NLSm were transfected with siRNA using the same method as in <Example 1>, and then treated with bleomycin. Subsequently, intracellular fractionation analysis and NHEJ analysis were performed.

[0279]

[0280] As a result, we confirmed that the decrease in LC8 in the nuclear fraction after TLK1 and TLK2 depletion was successfully restored by siRNA-resistant HA-TLK1 WT, but not by NLSm (Fig. 4e). Since LC8 binding to NLSm was not reduced compared to that of WT, this result suggests that most of the LC8 interacting with NLSm exists in the cytoplasm. Furthermore, the decrease in NHEJ efficiency upon TLK1 depletion was restored by WT, but not by NLSm (Figs. 4f and 4g).

[0281] Furthermore, TLK1-KD restored the nuclear translocation of LC8 in TLK depletion, indicating that TLK promotes the nuclear translocation of LC8 in a kinase-independent manner (Fig. 4h).

[0282] Finally, we found that NLSm did not alleviate the cell sensitivity to bleomycin observed upon TLK depletion (Fig. 4i).

[0283] Collectively, the above results support the conclusion that the NLS of TLK promotes 53BP1-dependent NHEJ by mediating the nuclear translocation of LC8, as shown in the schematic diagram in Fig. 4j.

[0284]

[0285] Example 5: Confirmation of PARP inhibitor resistance in BRCA1 mutant cancers following TLK depletion.

[0286] Loss of NHEJ is known to render mutant cells resistant to PARP inhibitors by restoring a certain level of HR in BRCA1. Therefore, the present invention investigated whether TLK depletion could confer resistance to PARP inhibitors.

[0287] BRCA1-proficient MDA-MB-468 breast cancer cells (BRCA1 WT), BRCA1 mutant MDA-MB-436 cells (BRCA1 m ) Immunoblot analysis was performed on each cell, and PARP inhibitors Olaparib or Veliparib were treated at different concentrations, and then cell viability analysis was performed.

[0288] The same siRNA as in <Example 1-1> was used for depletion of TLK1 or TLK2, and siBRCA1 was used to confirm the effect of BRCA1 depletion in BRCA1 normal MDA-MB-468 cells.

[0289]

[0290] As a result, depletion of TLK1 or TLK2 in MDA-MB-468 cells, which are BRCA1-proficient triple-negative breast cancer (TNBC) cells, did not cause any change in PARP inhibitor sensitivity in cell survival assays using olaparib (Fig. 5a and Fig. 5b).

[0291] However, when TLK1 or TLK2 was depleted in MDA-MB-436 cells with BRCA1 mutation, resistance to the PARP inhibitors olaparib and veliparib increased (Figures 5c to 5e).

[0292] As expected, depletion of BRCA1 by siRNA BRCA1 in BRCA1-normal triple-negative breast cancer cells, MDA-MB-468 cells, became sensitive to olaparib, but simultaneous depletion of BRCA1 and TLK resulted in resistance to olaparib (Fig. 5f and 5g).

[0293] Additionally, the BRCA1 mutant ovarian cancer cell line UWB1.289 showed reduced sensitivity to olaparib upon TLK1 or TLK2 depletion (Fig. 5h and 5i).

[0294] Our results imply that TLK expression is functionally proportional to PARP inhibitor sensitivity in BRCA1 mutant cells.

[0295]

[0296] Example 6: Confirmation of HR restoration in BRCA1 mutant cells following TLK depletion.

[0297] 6-1: Confirmation of HR restoration following TLK depletion

[0298] BRCA1 is essential for HR, and HR deficiency promotes PARP inhibitor toxicity, so the efficacy of PARP inhibitors is maximized in BRCA1-mutated, HR-deficient cancers. However, when NHEJ is inactivated in HR deficiency (HRD), nucleases required for HR can partially restore HR, which is an important mechanism for resistance to PARP inhibitors (Lord, CJ, Tutt, AN, and Ashworth, A, Annu Rev Med 66, 455-470, 2015; Jaspers, JE, et al., Cancer Discov 3, 68-81, 2013).

[0299] Since the present invention confirmed that TLK depletion promotes resistance to PARP inhibitors, it was confirmed whether HR was restored in BRCA1-deficient cells when TLK1 was depleted, thereby increasing resistance to PARP inhibitors.

[0300]

[0301] Figure 6a is a schematic diagram illustrating the measurement of HR efficiency according to BRCA1 depletion in U2OS DR-GFP cells. When U2OS DR-GFP cells were transfected with siBRCA1 and siRNA TLK, immunoblot analysis and HR efficiency were confirmed.

[0302] Analysis of HR efficiency using the U2OS DR-GFP cell line revealed that BRCA1 depletion reduced HR efficiency to 25%. However, co-depletion of TLK1 or TLK2 in BRCA1-depleted cells resulted in a significant recovery of HR, with HR efficiency reaching approximately 55% of that in wild-type cells (Figs. 6b and 6c). In other words, we confirmed that HR was restored by TLK1 or TLK2 depletion, not by BRCA1 depletion.

[0303]

[0304] 6-2: Observation of 53BP1 foci-positive cells following TLK depletion

[0305] Next, we investigated whether HR restoration upon TLK depletion was due to NHEJ impairment as 53BP1 did not localize to DSBs.

[0306] After transfecting A549 cells with siBRCA1 and siRNA TLK, 53BP1 focal positive cells were observed by immunostaining when treated with bleomycin or cisplatin.

[0307]

[0308] As a result, 53BP1 locus-positive cells were reduced after co-depletion of TLK1 or TLK2 with BRCA1, indicating that TLK is essential for 53BP1 binding to DSBs even in BRCA1-deficient cells (Fig. 6d-f).

[0309] Furthermore, the reduction of 53BP1 locus-positive cells after TLK1 and TLK2 depletion was the same regardless of whether cells were BRCA1-mutant or -deficient, indicating that the importance of TLKs in 53BP1 binding to DSBs does not depend on the cellular capacity for HR (Fig. 6g and h).

[0310] In the present invention, NHEJ efficiency was measured to determine whether NHEJ was inactivated by TLK depletion in BRCA1-depleted cells (Figures 6F and 6G).

[0311] As a result, BRCA1 depletion slightly increased NHEJ efficiency, but the increase in NHEJ in BRCA1-deficient cells was reversed by TLK1 and / or TLK2 depletion, demonstrating that TLKs are required for NHEJ repair even in HR-deficient cells (Figures 6F and 6G).

[0312]

[0313] 6-3: Analysis of NHEJ efficiency following TLK depletion

[0314] In the present invention, to determine whether NHEJ was inactivated by TLK depletion in BRCA1-depleted cells, NHEJ / DsRed293B cells were transfected with siBRCA1 and siRNA TLK, and then NHEJ efficiency was measured.

[0315]

[0316] As a result, although BRCA1 depletion slightly increased NHEJ efficiency, the increase in NHEJ in BRCA1-deficient cells was reversed by TLK1 and / or TLK2 depletion, demonstrating that TLKs are required for NHEJ repair even in HR-deficient cells (Fig. 6i and 6j).

[0317]

[0318] 6-3: Analysis of NHEJ efficiency following TLK depletion

[0319] Depletion of LC8 is known to increase HR efficiency in BRCA1-deficient cells, leading to resistance to PARP inhibitors. Because LC8 is also required for the formation of 53BP1 foci, a step dependent on TLK1, we expected that co-depletion of TLK and LC8 would no longer increase HR efficiency compared to single depletion of LC8.

[0320] In the present invention, to confirm whether TLK and LC8 act in the same DSB repair pathway upon BRCA1 inactivation, HR analysis was performed using the method of Fig. 6a, and after transfecting U2OS DR-GFP cells with siBRCA1, siLC8, or siRNA TLK of <Example 1-1>, HR efficiency was analyzed.

[0321]

[0322] As a result, we confirmed that TLK and LC8 act in the same pathway for DSB repair in BRCA1 deficiency (Fig. 6k).

[0323]

[0324] Example 7: Observation of prognosis according to TLK depletion in patients with BRCA1 deficiency.

[0325] LC8 expression confers a better prognosis and sensitivity to PARP inhibitors in BRCA1-mutant cancers. In the present invention, we confirmed that TLK depletion confers resistance to PARP inhibitors in BRCA1-deficient cells. Some studies have shown that TLK is upregulated in certain cancers with poor prognoses. Contrary to our results showing that TLK depletion confers PARP inhibitor resistance in cancers, previous studies have shown that TLK loss predicts a better prognosis. Of course, survival studies on TLK were conducted on patients who had not been treated with PARP inhibitors.

[0326] However, another possibility arises due to the interaction between BRCA1 status and TLK levels in the present invention, whereby the survival effects of TLK levels may differ in BRCA1-normal and BRCA1-deficient tumors, even when the tumors are not treated with PARP inhibitors.

[0327] To confirm this, in the present invention, tumors from the TCGA database were analyzed to investigate whether the correlation between TLK expression and patient survival was affected by BRCA1 status.

[0328]

[0329] Because sufficient numbers of BRCA1-deficient patients were not available for some cancers, nine cancers were analyzed: human neural stem cells (HNSC), colon adenocarcinoma (COAD), kidney renal clear cell carcinoma (KIRC), uterine corpus endometrial carcinoma (UCEC), ovarian cancer (OVCA), breast invasive carcinoma (BRCA), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), bladder urothelial carcinoma (BLCA), and lung adenocarcinoma (LUAD).

[0330] All TCGA data were downloaded via the TCGA biolinks package in R and prepared according to the package manual. BRCA1 was considered a 'mutant' if:

[0331] a) Recorded as containing one or more single nucleotide variants (SNVs).

[0332] b) The copy number is less than 2.

[0333] c) Expression z-score is less than 2.

[0334] Additionally, for BRCA and OVCA, patients with germline mutations in BRCA1 were also considered mutated. Each cancer was initially divided into two cohorts based on the mutation status of BRCA1, and further analysis was performed only if both cohorts included more than 20 cases. Each cohort was then divided into two groups based on the expression level of TLK using a minimum sample size of 10, using the threshold that yielded the lowest p-value in the log-rank test of the Kaplan-Meyer analysis. Nine cancers met the aforementioned criteria: BLCA, BRCA, CESC, COAD, HNSC, KIRC, LUAD, OVCA, and UCEC.

[0335] Cox proportional hazards regression analysis was performed to calculate regression coefficients, which represent the likelihood of risk in the low-expression group compared to the high-expression group. Positive values ​​indicate a higher risk, and negative values ​​indicate a lower risk. All data analyses were performed using R version 4.2.1.

[0336]

[0337] As a result, patients with BRCA1 normal cancers, such as BRCA, OVCA, and UCEC, had poor outcomes when the level of TLK1 or TLK2 expression was elevated, whereas patients with BRCA1 mutant cancers had better prognoses when the level of TLK1 or TLK2 expression was elevated (Fig. 7a to Fig. 7c).

[0338] In a comprehensive survey of several cancers, high expression of TLK1 or TLK2 was significantly associated with poor prognosis in most cancer types with wild-type BRCA1 (or low TLK1 associated with good outcome), but this phenomenon was not observed in tumors with BRCA1 deficiency (Fig. 7d).

[0339]

[0340] Furthermore, TLK1 expression was significantly lower in BRCA1-mutant cancers, BRCA and UCEC, whereas TLK2 expression was lower in BRCA1-mutant cancers, OVCA and UCEC, compared to BRCA1-wild-type cancers (Fig. 7e). These results suggest that tumors can select for reduced TLK expression even before treatment with PARP inhibitors if they are BRCA1-deficient. In other words, low TLK levels, regardless of PARP inhibitor treatment, may promote tumorigenesis in BRCA1-mutant cancers.

[0341]

[0342] In the present invention, it was confirmed that human TLK (Tousled-like Kinase) is essential for NHEJ-mediated DSB repair and PARP inhibitor sensitivity in cancer cells with BRCA1 mutations. Therefore, the TLK biomarker of the present invention can be usefully applied to predict the therapeutic response or prognosis of PARP inhibitors in BRCA mutant cancers.

Claims

1. A TLK (Tousled-like kinase) biomarker composition for predicting therapeutic response or prognosis of PARP inhibitors in BRCA mutant cancer.

2. In paragraph 1, The above TLK biomarker is TLK1 or TLK2, A biomarker composition, characterized in that the PARP inhibitor is olaparib, talazoparib, niraparib, rucaparib, veliparib, or pamiparib.

3. In paragraph 1, A biomarker composition, characterized in that the above BRCA mutant cancer is breast cancer, ovarian cancer, or uterine cancer in which a BRCA mutation exists.

4. A composition for predicting therapeutic response or prognosis of a PARP inhibitor in BRCA mutant cancer, comprising a preparation for measuring the mRNA or protein expression level of TLK (Tousled-like kinase).

5. In paragraph 4, The above TLK biomarker is TLK1 or TLK2, A composition for predicting therapeutic response or prognosis of a PARP inhibitor in BRCA mutant cancer, characterized in that the PARP inhibitor is olaparib, talazoparib, niraparib, rucaparib, veliparib, or pamiparib.

6. In paragraph 4, The agent for measuring the mRNA expression level is a sense and antisense primer, or probe, that binds complementarily to the mRNA of TLK. A composition for predicting therapeutic response or prognosis of a PARP inhibitor in BRCA mutant cancer, characterized in that the agent for measuring the protein expression level is an antibody, interacting protein, ligand, nanoparticle or aptamer that specifically binds to a complement TLK protein or peptide fragment.

7. In paragraph 4, A composition for predicting the therapeutic response or prognosis of a PARP inhibitor in BRCA mutant cancer, wherein the BRCA mutant cancer is breast cancer, ovarian cancer, or uterine cancer in which a BRCA mutation exists.

8. A method for providing information on the therapeutic responsiveness or prognosis of a PARP inhibitor in BRCA mutant cancer, comprising the step of measuring the mRNA or protein expression level of TLK from a sample of a BRCA mutant cancer patient.

9. In paragraph 8, A method for providing information on the therapeutic responsiveness or prognosis of a PARP inhibitor in BRCA mutant cancer, characterized in that when the mRNA or protein expression level of the above TLK is lower than that of a normal control group, information is provided that the therapeutic responsiveness to the PARP inhibitor is low or the prognosis is poor.

10. A method for providing information on the administration of a PARP inhibitor in BRCA mutant cancer, comprising the step of measuring the mRNA or protein expression level of TLK from a sample of a BRCA mutant cancer patient.

11. In paragraph 10, A method for providing information on administration of a PARP inhibitor in BRCA mutant cancer, characterized in that when the mRNA or protein expression level of the above TLK is lower than that of a normal control group, information is provided that the PARP inhibitor was not administered.

12. A pharmaceutical composition for preventing or treating BRCA mutant cancer resistant to PARP inhibitors, comprising a substance for promoting mRNA or protein expression level of TLK.

13. In paragraph 12, A pharmaceutical composition for the prevention or treatment of BRCA mutant cancer that is resistant to a PARP inhibitor, characterized in that the composition further comprises a PARP inhibitor.

14. In paragraph 12, A pharmaceutical composition for preventing or treating BRCA mutant cancer that is resistant to a PARP inhibitor, characterized in that the PARP inhibitor is olaparib, talazoparib, niraparib, rucaparib, veliparib, or pamiparib.

15. A method for screening a substance for prevention or treatment of BRCA mutant cancer cells resistant to a PARP inhibitor, comprising the step of treating BRCA mutant cancer cells with a test substance and then measuring the mRNA or protein expression level of TLK.

Images