Use of morc2 as a target in preparation of a drug for treating triple-negative breast cancer

Abstract

The application provides application of MORC2 as a target point in preparation of a drug for treating triple negative breast cancer, and through CRISPR-Cas9 gene knockout or antisense oligonucleotide targeted silencing of MORC2, transposon element transcription can be activated, a virus simulation effect is triggered, RIG-I / MDA5-MAVS and cGAS-STING nucleic acid perception pathways are activated, interferon signals and anti-tumor immune responses are enhanced, so that the response of TNBC to ICB treatment is sensitized. Meanwhile, MORC2 interacts with the N-terminal Tudor domain of SETDB1 through its ATPase domain, cooperatively deposits H3K9me3 inhibitory modification at the TEs site; SETDB1 enhances the protein stability of MORC2 by methylating the K234 and K643 sites of MORC2, forms a positive feedback regulation loop, and targeting the regulation axis can provide a new intervention strategy for TNBC immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy and epigenetics, specifically involving the application of MORC2 as a target in the preparation of drugs for treating triple-negative breast cancer. Background Technology

[0002] Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, accounting for approximately 15-20% of all breast cancer cases. It is characterized by negativity of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) (Foulkes WD et al., N Engl J Med, 2010, 363:1938-1948; Carey L et al., Nat Rev Clin Oncol, 2010, 7:683-692). TNBC commonly affects young women and is prone to early recurrence and distant metastasis. The median survival is only 9-12 months, and the five-year survival rate is less than 30%. Due to the lack of specific therapeutic targets such as ER, PR, and HER2, TNBC patients cannot benefit from endocrine therapy and HER2-targeted therapy. Chemotherapy remains the cornerstone of its systemic treatment, but its efficacy is not entirely satisfactory (Bianchini G et al., Nat Rev Clin Oncol, 2016, 13:674-690).

[0003] In recent years, immune checkpoint blockade (ICB) therapy targeting the programmed death receptor 1 / programmed death ligand 1 (PD1 / PD-L1) axis and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) has brought new breakthroughs to the treatment of TNBC. Multiple phase III randomized controlled clinical trials have confirmed that combining chemotherapy with anti-PD1 or PD-L1 monoclonal antibodies can significantly improve the prognosis of TNBC patients: the IMpassion031 trial (Mittendorf EA et al., Lancet, 2020, 396:1090-1100) and the IMpassion130 trial (Schmid P et al., N Engl J Med, 2018, 379:2108-2121) confirmed the efficacy of atezolizumab combined with chemotherapy; the KEYNOTE-355 trial (Cortes J et al., Lancet, 2020, 396:1817-1828) and the KEYNOTE-522 trial (Schmid P et al., N Engl J Med, 2020, 382:810-821; Schmid P et al., N Engl J Med, 2020, 382:810-821; Schmid P et al., N Engl J Med, 2020, 382:810-821) confirmed the efficacy of atezolizumab combined with chemotherapy; and the KEYNOTE-355 trial (Cortes J et al., Lancet, 2020, 396:1817-1828) confirmed the efficacy of atezolizumab combined with chemotherapy. The study *JMed*, 2024, 391:1981-1991, confirmed that pembrolizumab combined with chemotherapy significantly improves the pathological complete response rate and prolongs overall survival. Domestic trials such as the CamRelief trial (Chen L et al., *JAMA*, 2025, 333:673-681) and the TORCHLIGHT trial (Jiang Z et al., *Nat Med*, 2024, 30:249-256) also confirmed the clinical benefit of domestically produced PD1 / PD-L1 inhibitors combined with chemotherapy. However, these studies all indicate that only about 20-30% of TNBC patients benefit from ICB treatment (Wein L et al., *Br J Cancer*, 2018, 119:4-11; Liu Y et al., *Mol Cancer*, 2023, 22:145), and most patients have primary or secondary resistance. Therefore, it is urgent to elucidate the tumor immune escape mechanism and discover new therapeutic targets.

[0004] Epigenetic regulation is one of the important mechanisms of tumor immune escape. ATP-dependent chromatin remodeling factors affect DNA accessibility and gene transcription by regulating nucleosome localization and chromatin conformation (Clapier CR et al., Nat Rev Mol Cell Biol, 2017, 18:407-422; Eustermann S et al., Nat Rev Mol Cell Biol, 2024, 25:309-332). Increasing evidence suggests that aberrant regulation of chromatin remodeling factors not only drives tumorigenesis but also influences the efficacy of immunotherapy by shaping the tumor immune microenvironment (Malone HA et al., Nat Rev Drug Discov, 2024, 23:661-681). For example, the loss of the SWI / SNF complex subunit SMARCA4 can lead to resistance in tumor cells to T cell-mediated killing (Pan D et al., Science, 2018, 359:770-775), while epigenetic factors such as BATF affect tumor immunity by regulating the activation program of regulatory T cells (Itahashi K et al., Sci Immunol, 2022, 7:eabk0957). However, the specific functions and potential targets of chromatin remodeling factors in TNBC immune escape remain to be explored and investigated.

[0005] Transposable elements (TEs) are repetitive sequences in the genome, comprising approximately 45% of the human genome. In normal somatic cells, they are strictly repressed by epigenetic mechanisms, particularly histone H3 lysine trimethylation (H3K9me3) (Karimi MM et al., Cell Stem Cell, 2011, 8:676-687; Oleksiewicz U et al., Int J Mol Sci, 2024, 25). When TEs are activated, their transcripts can form double-stranded RNA (dsRNA) or be reverse transcribed to produce double-stranded DNA (dsDNA). dsRNA is recognized through the RIG-I / MDA5-MAVS (retinoic acid-induced gene I / melanoma differentiation-associated gene 5-mitochondrial antiviral signaling protein) pathway, while dsDNA is sensed through the cGAS-STING (cyclic guanosine monophosphate-adenosine monophosphate synthase-interferon gene stimulatory factor) axis. Both pathways converge to activate TBK1 (TANK-binding kinase 1) and IRF3 (interferon regulatory factor 3), inducing interferon responses, triggering viral mimicry effects similar to viral infection, and enhancing tumor immunogenicity (Chen R et al., Cancer Discov, 2021, 11:2707-2725; Kassiotis G et al., Nat Rev Immunol, 2016, 16:207-219). Recent studies have shown that targeting TE silencing mechanisms can effectively activate intratumoral interferon signaling pathways and enhance the efficacy of ICB: KDM5B promotes tumor immune escape by recruiting SETDB1 to silence retrotransposons (Zhang SM et al., Nature, 2021, 598:682-687), PHF8 deficiency can induce viral mimicry responses by activating endogenous retrotransposons (Liu Y et al., NatCommun, 2023, 14:4225), and SETDB1 itself has also been shown to inhibit endogenous tumor immunogenicity by silencing TEs (Griffin GK et al., Nature, 2021, 595:309-314). Therefore, recent high-impact studies demonstrate the therapeutic potential of TE-targeting.

[0006] MORC2 (MORC family CW-type zinc finger protein 2) is an ATP-dependent chromatin remodeling factor containing an N-terminal ATPase domain, a middle CW-type zinc finger (CW-ZF) domain, and a C-terminal chromatin-like (CL) domain (Li DQ et al., Epigenetics, 2013, 8:685-693; Tan W et al., Nat Commun, 2025, 16:5606). Structural studies have shown that MORC2 mediates chromatin compression and gene silencing through its ATPase domain (Tchasovnikarova IA et al., Nat Genet, 2017, 49:1035-1044; Douse CH et al., Nat Commun, 2018, 9:651). A series of studies by our research group and other research teams have confirmed that MORC2 is a novel oncoprotein that is significantly upregulated in breast cancer, promotes tumor progression, and confers resistance to endocrine therapy, chemotherapy, and radiotherapy (Zhang FL et al., Cancer Res, 2018, 78:5780-5792; Liu HY et al., Nucleic Acids Res, 2020, 48:3638-3656; Zhang FL et al., Theranostics, 2023, 13:973-990).

[0007] Therefore, MORC2, as an epigenetic regulator that is significantly upregulated in TNBC, has an expression level that is closely related to the tumor immunosuppressive microenvironment and ICB treatment resistance. It is expected to become a novel molecular target for TNBC diagnosis stratification, efficacy prediction and immunotherapy sensitization, and to provide a new intervention strategy for overcoming TNBC immunotherapy resistance. Summary of the Invention

[0008] This invention provides the application of MORC2 as a target in the preparation of drugs for treating triple-negative breast cancer. By using MORC2 as a target, MORC2 expression is inhibited through antisense oligonucleotides (ASO) and other means, transposon element (TE)-mediated viral mimicry reactions are activated, tumor immunogenicity is enhanced, and thus the efficacy of immune checkpoint blockade (ICB) therapy for triple-negative breast cancer is improved.

[0009] The technical solution adopted by the present invention to achieve the above objective is: the application of MORC2 as a target in the preparation of drugs for treating triple-negative breast cancer, wherein the nucleotide sequence of MORC2 is shown in SEQ ID NO:1.

[0010] Furthermore, the specific information of MORC2 is NM_001303256.2:399-3497 Homo sapiensMORC family CW-type zinc finger 2 (MORC2), transcript variant 1, mRNA.

[0011] Furthermore, the expression of the MORC2 gene or protein is suppressed by inhibitors and CRISPR-Cas9 gene knockout.

[0012] Furthermore, the inhibitors include antisense oligonucleotides, shRNA, siRNA, MORC2 antibodies, and small molecule inhibitors.

[0013] Furthermore, the inhibitor is an antisense oligonucleotide.

[0014] Furthermore, the antisense oligonucleotide is designed with a 5-10-5 gapmer and modified with a phosphorus thioyl backbone, and mediates the cleavage and degradation of Morc2a mRNA through an RNase H-dependent mechanism.

[0015] In some embodiments, the inhibitor is a small molecule inhibitor targeting the MORC2 ATPase domain.

[0016] In some embodiments, the inhibitor triggers viral mimicry effects by activating transposon element (TE) transcription, activating the RIG-I / MDA5-MAVS and cGAS-STING nucleic acid sensing pathways, upregulating interferon-stimulated gene (ISG) expression, enhancing MHC-I antigen presentation, and promoting CD8+ T cell infiltration and activation, thereby enhancing the anti-tumor immune response.

[0017] This invention discloses the application of MORC2 gene or protein expression inhibitors combined with immune checkpoint blockers in the preparation of drugs for treating triple-negative breast cancer.

[0018] Furthermore, the immune checkpoint blocker includes anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody.

[0019] In some embodiments, the MORC2 gene or protein expression inhibitor and the immune checkpoint blocker may be administered simultaneously or sequentially; preferably, when the MORC2 gene or protein expression inhibitor is an antisense oligonucleotide, it is administered via intratumoral injection (5 nmol, 3 times a week), and the anti-PD-1 antibody is administered via intraperitoneal injection (10 mg / kg).

[0020] This invention also discloses the application of the MORC2 / SETDB1 epigenetic regulatory axis as a target in the preparation of immunosensitizing drugs for triple-negative breast cancer.

[0021] In some embodiments, drugs targeting the MORC2 / SETDB1 epigenetic regulatory axis include SETDB1 inhibitors (such as mithramycin A, which indirectly reduces MORC2 protein stability), MORC2 K234 / K643 methylation site mutants, or combined inhibitors that simultaneously target MORC2 and SETDB1.

[0022] This invention also discloses the application of MORC2 as a biomarker in the preparation of reagents for the diagnosis of triple-negative breast cancer, prediction of immunotherapy efficacy, and patient stratification.

[0023] In some embodiments, the reagents used to detect the expression level of the MORC2 gene or protein are selected from those used for detection by quantitative PCR, RNA sequencing, Western blotting, immunohistochemistry (IHC), multiplex immunofluorescence (mIHC), or quantitative proteometry.

[0024] In some implementations, low MORC2 expression suggests that patients have a high level of CD8+ T cell infiltration, a strong anti-tumor immune response, and a good response to ICB therapy; high MORC2 expression suggests that patients have an immunosuppressive tumor microenvironment and a risk of ICB therapy resistance.

[0025] In some implementations, MORC2 can be used not only in the preparation of drugs for treating triple-negative breast cancer, but also in other solid tumors that highly express MORC2, including lung adenocarcinoma, gastric cancer, and bladder cancer.

[0026] Compared with the prior art, the beneficial effects that at least one technical solution adopted in the embodiments of this specification can achieve include at least: (1) Through systematic analysis of multiple public databases (CBCGA, TCGA, METABRIC, CPTAC) and internal cohorts (FUSCC-TNBC, 88 pairs of TNBC and adjacent normal tissues), this invention has confirmed that the mRNA and protein levels of MORC2 in TNBC tissues are significantly higher than those in normal breast tissues and non-TNBC subtypes. Moreover, its high expression is closely related to the immunosuppressive tumor microenvironment and ICB treatment resistance, suggesting that MORC2 has the potential to serve as a biomarker for predicting the efficacy of TNBC immunotherapy. (2) In this invention, Morc2a gene knockout models were constructed in three mouse breast cancer cell lines (4T1, 66cl-4, AT-3) using CRISPR-Cas9 technology. It was confirmed that Morc2a deletion significantly inhibited tumor growth in immune-normal mice (both tumor volume and weight were significantly reduced), while no such effect was observed in immunodeficient nude mice. This indicates that the tumor-promoting effect of MORC2 depends on the intact tumor immune microenvironment. (3) This invention reveals through multi-omics analysis such as single-cell RNA sequencing (scRNA-seq), flow cytometry and RNA sequencing that MORC2 deficiency can significantly reshape the tumor immune microenvironment: promote macrophage polarization to M1 type (increase the proportion of iNOS+ and decrease the proportion of CD206+), increase CD8+ T cell infiltration and the proportion of IFN-γ+ / GzmB+ cytotoxic T cells, enhance MHC-I antigen presentation capacity, thereby activating anti-tumor immune response; (4) This invention elucidates the molecular mechanism by which MORC2 inhibits tumor immunity: MORC2 interacts with the N-terminal Tudor domain of SETDB1 through its ATPase domain, and synergistically deposits H3K9me3 inhibitory modification at TE sites (LTR22C2, MER74C, etc.), inhibiting TE transcription, blocking viral mimicry effects, inhibiting the RIG-I / MDA5-MAVS and cGAS-STING nucleic acid sensing pathways and downstream interferon signaling, and maintaining the tumor immunosuppressive microenvironment; (5) This invention found that SETDB1 enhances the protein stability of MORC2 by methylating the K234 and K643 sites, forming a MORC2 / SETDB1 positive feedback regulatory loop, providing a new intervention target for targeting this regulatory axis; (6) This invention designed and screened antisense oligonucleotides targeting Morc2a (ASO-Morc2a). In the 4T1 mouse TNBC model, it was confirmed that ASO-Morc2a (5 nmol, intratumoral injection, 3 times a week) combined with anti-PD-1 therapy (10 mg / kg, intraperitoneal injection) can synergistically inhibit tumor growth, reshape the tumor immune microenvironment, and is well tolerated (no significant decrease in mouse weight). This provides a new combination therapy strategy for TNBC immunotherapy. (7) Through multiple immunofluorescence (mIHC) analysis of 143 clinical samples of TNBC, this invention confirms that low expression of MORC2 is significantly associated with increased CD8+ T cell infiltration and increased production of GZMB / IFN-γ, thus verifying the clinical value of MORC2 as a biomarker for predicting the efficacy of TNBC immunotherapy. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0028] Figure 1 Figures show the results of Examples 2 and 3. MORC2 is highly expressed in TNBC, and its absence inhibits tumor growth in an immune microenvironment-dependent manner. Figures (AC) show the analysis using CBCGA, TCGA, and METABRIC transcriptome datasets, showing that MORC2 mRNA levels in TNBC are significantly higher than in non-TNBC and normal breast tissues; (D) shows the results of the FUSCC-TNBC dataset (88 pairs of TNBC and adjacent normal tissues) verifying the significant upregulation of MORC2 mRNA in TNBC; (EF) shows the results of the CBCGA and CPTAC proteome datasets confirming the significant increase in MORC2 protein levels in TNBC; (GH) shows the results of Western blotting analysis detecting MORC2 protein expression levels in 14 pairs of TNBC and adjacent normal tissues; (IJ) shows the results of tissue microarray mIHC analysis of 143 TNBC cases and 25 normal breast tissue cases, showing significantly enhanced MORC2 staining in TNBC; (K) shows the effect of Morc2a knockout 4T1 cells on immune... Tumor volume changes in functionally normal BALB / c mice; (L) Tumor weight changes in immunocompetent BALB / c mice with Morc2a knockout 4T1 cells; (M) Tumor growth changes in the Morc2a knockout 66cl-4 syngeneic model; (N) Tumor weight changes in the Morc2a knockout 66cl-4 syngeneic model; (O) Tumor growth changes in the Morc2a knockout AT-3 syngeneic model; (P) Tumor weight changes in the Morc2a knockout AT-3 syngeneic model. Figure 2The results analysis diagram for Example 4 shows that (AB) are single-cell RNA sequencing (scRNA-seq) UMAP dimensionality reduction clustering diagrams of the control group and Morc2a-KO 4T1 tumors, respectively, identifying 9 major cell types; (C) is a diagram showing significant changes in the proportions of macrophages, T cells, and neutrophils in Morc2a-KO tumors; (D) is a GSEA analysis diagram showing significant enrichment of anti-tumor immune-related pathways such as TNF-α / NF-κB, IFN-γ, IFN-α, inflammatory response, and IL-6 / JAK / STAT3 in Morc2a-KO tumors; (E) is a diagram showing the upregulation of MHC-I molecules (H2-K1, H2-D1, H2-T23) expression in macrophages in Morc2a-KO tumors; and (F) is a diagram showing the upregulation of macrophage expression in Morc2a-KO tumors. (G) Flow cytometry analysis of the changes in the proportion of total macrophages (CD11b+F4 / 80+) in Morc2a-KO tumors; (H) Flow cytometry analysis of the increased proportion of M1 macrophages (iNOS+) in Morc2a-KO tumors; (I) Flow cytometry analysis of the decreased proportion of M2 macrophages (CD206+) in Morc2a-KO tumors; (J) scRNA-seq analysis showing significant upregulation of key CTL genes such as CD8a, Prf1, and Gzmb in Morc2a-KO tumors; (K) Flow cytometry analysis of CD8+ in Morc2a-KO tumors. (L) is a graph showing the change in the proportion of T cells; (M) is a graph showing the change in the frequency of IFN-γ+ cytotoxic T cell subsets in Morc2a-KO tumors analyzed by flow cytometry; (N) is a graph showing the change in the frequency of GzmB+ cytotoxic T cell subsets in Morc2a-KO tumors analyzed by flow cytometry; and (N) is a graph summarizing the proportions of various CD8+ T cell subsets in Morc2a-KO tumors analyzed by flow cytometry. Figure 3The results analysis diagrams for Example 5 are shown below. (A) is an RNA-seq GSEA analysis diagram, showing significant enrichment of the interferon response pathway in Morc2a-KO4T1 cells; (B) is an RT-qPCR analysis diagram, confirming that Morc2a deletion significantly upregulated classical interferon-stimulated genes (ISGs): Cxcl9, Ifit3, Ifi207, Ifitm1, Bst2, and Oas3; (C) is an RT-qPCR analysis diagram, showing significant upregulation of mouse MHC-I related genes (B2m, H2-K1, H2-D1, H2-Q1) in Morc2a-KO cells. (D) is the result of the Western blot experiment, confirming the upregulation of MHC-I related protein expression in Morc2a-KO cells; (E) is the result of flow cytometry, confirming the significant upregulation of H-2Kb / H-2Db of classical mouse MHC-I molecules in Morc2a-KO cells; (F) is the result of immunofluorescence staining, confirming the significant upregulation of H-2Kb / H-2Db of classical mouse MHC-I molecules in Morc2a-KO cells; (G) is the TETranscripts analysis diagram, showing that Morc2a deletion significantly alters… Expression profiles of transposable elements (TEs) in 4T1 cells; (H) RT-qPCR analysis confirms that Morc2a deletion significantly upregulates specific TEs (LTR53-int, RLTR26D, RLTR9A3B, RLTR6C); (I) Results of MORC2 knockout activating the interferon signaling pathway in human TNBC cells MDA-MB-231; (J) MORC2 knockout upregulates ISGs (CXCL9, CXCL10, BST2, ...) in human TNBC cells MDA-MB-231. (IFIT3) Results: (K) Results showing significant upregulation of key genes (B2M, HLA-A, HLA-C, TAP1) in the human MHC-I antigen presentation pathway due to MORC2 deletion; (L) Quantitative analysis of key genes in the human MHC-I antigen presentation pathway due to MORC2 deletion; (M) Results showing significant upregulation of human transposon elements (LTR06, LTR22C0, LTR22C2, MER74C) due to MORC2 deletion; (N) Quantitative analysis of human transposon element expression due to MORC2 deletion. Figure 4The following are the results analysis figures for Example 6, where (A) shows the results of MORC2 knockout in MDA-MB-231 and BT-549 cells. MORC2 knockout enhances the expression of MDA5 and cGAS proteins in MDA-MB-231 and BT-549 cells, as well as the phosphorylation activation of STING, TBK1, and IRF3; (B) shows the results of IFN-γ treatment further enhancing the activation of the nucleic acid sensing pathway induced by MORC2 deficiency; and (C) shows the results of IFN-γ treatment enhancing the activation of ISGs (CXCL) induced by MORC2 deficiency. 9. (D) The results of upregulation of IFIT3 and BST2; (E) The results of immunofluorescence staining with J2 antibody, showing significant accumulation of cytoplasmic dsRNA in MORC2-KO cells, which can be partially eliminated by RNase III treatment; (F) The results of treatment with the nucleotide reverse transcriptase inhibitor lamivudine (3TC) to reduce the accumulation of cytoplasmic dsDNA induced by MORC2 deletion; (G) The results of knockdown of nucleic acid sensing receptor MDA5 (dsRNA) or cGAS (dsDNA) to significantly reduce the upregulation of ISGs induced by MORC2 deletion. Figure 5The following are the results analysis diagrams for Example 7: (A) shows the results of the endogenous co-immunoprecipitation (co-IP) experiment in MDA-MB-231 cells confirming the physical interaction between MORC2 and SETDB1; (B) shows the results of the endogenous co-immunoprecipitation (co-IP) experiment in BT-549 cells confirming the physical interaction between MORC2 and SETDB1; (C) shows the results of immunofluorescence staining, indicating significant co-localization of MORC2 and SETDB1 in MDA-MB-231 and BT-549 cells; (D) shows the results of the co-IP experiment of the truncated MORC2 mutant (T1-T3), showing that the N-terminal ATPase domain of MORC2 mediates its interaction with SETDB1; (E) shows the quantitative analysis diagram of the interaction between the truncated MORC2 mutant and SETDB1; and (F) shows the results of the co-IP experiment of the truncated SETDB1 mutant (T1-T2), showing that SETDB1... The N-terminal region (containing the Tudor domain) mediates its interaction with MORC2; (G) is a quantitative analysis diagram of the interaction between the SETDB1 truncated mutant and MORC2; (H) is a diagram of the results of public ChIP-seq data analysis, showing that the levels of H3K9me3 at LTR22C2 and MER74C sites decreased after MORC2 knockout; (I) is a diagram of the results of SETDB1 knockdown reversing the transcriptional repression of LTR22C2 and MER74C induced by MORC2 overexpression; (J) is a diagram of SETDB1 knockdown reversing the transcriptional repression of downstream ISGs (CXCL9, IFIT3) induced by MORC2 overexpression; (K) is a diagram of the results of ChIP-qPCR experiments, showing that MORC2 overexpression enhances the levels of H3K9me3 at LTR22C2 and MER74C sites, and SETDB1 knockdown can eliminate this effect; Figure 6The results analysis diagrams for Example 8 are shown below. (A) shows the mIHC staining results indicating significantly high expression of SETDB1 in TNBC tissues; (B) shows the mIHC quantitative analysis results indicating SETDB1 expression levels in TNBC tissues; (C) shows the mIHC analysis results indicating a significant positive correlation between SETDB1 and MORC2 expression levels; (D) shows the statistical analysis diagram of the positive correlation between SETDB1 and MORC2 expression; (E) shows the CBCGA proteome dataset confirming a significant positive correlation between SETDB1 and MORC2 protein levels in TNBC; (F) shows the CPTAC proteome dataset confirming a significant positive correlation between SETDB1 and MORC2 protein levels in TNBC; (G) shows that SETDB1 knockdown significantly reduces MORC2 protein expression levels without affecting its mRNA levels; (H) shows the results of cyclohexylimide (… The CHX tracking experiment results show that SETDB1 knockdown significantly shortens the half-life of MORC2 protein; (I) shows the results of SETDB1 knockdown reducing MORC2 methylation levels; (J) shows the results of wild-type SETDB1 overexpression enhancing MORC2 methylation, while the catalytically inactive mutants (H1224K or C1226A) have no such effect; (K) shows the results of point mutation experiments identifying K234 as the major SETDB1 methylation site of MORC2; (L) shows the results of point mutation experiments identifying K643 as the major SETDB1 methylation site of MORC2 and the results of double mutant (2KR) significantly reducing methylation levels; (M) shows the results of SETDB1 inhibitor sclerotinib A (MMA) reducing MORC2 protein expression in a dose-dependent manner; (N) shows the results of the CHX tracking experiment, showing that the half-life of the 2KR mutant protein is significantly shorter than that of wild-type MORC2; Figure 7The following are the results analysis figures for Examples 9 and 10, where (A) is a schematic diagram of the experimental protocol for ASO-Morc2a combined with anti-PD-1 treatment of 4T1 tumor-bearing mice; (B) is a graph showing the change in tumor volume of 4T1 mice after ASO-Morc2a combined with anti-PD-1 treatment; (C) is a graph showing the change in tumor weight of 4T1 mice after ASO-Morc2a combined with anti-PD-1 treatment; (D) is a graph showing the change in the proportion of total macrophages in the tumor after combined treatment analyzed by flow cytometry; (E) is a graph showing the results of flow cytometry analysis of the promotion of macrophage polarization to M1 type in the tumor by combined treatment; (F) is a statistical graph showing the summary of the M1 / M2 macrophage ratio after combined treatment analyzed by flow cytometry; and (G) is a graph showing the results of flow cytometry analysis of CD8+ in the tumor after combined treatment. (H) Flow cytometry analysis of changes in T cell infiltration ratio; (I) Flow cytometry analysis of changes in the frequency of IFN-γ+ cytotoxic T cell subsets after combined treatment; (J) Tissue microarray mIHC analysis (143 TNBC cases) showing a significant increase in CD8+ T cell infiltration in tumors with low MORC2 expression; (K) Quantitative statistical analysis of low MORC2 expression and increased CD8+ T cell infiltration; (L) Significantly increased GZMB production in tumors with low MORC2 expression; (M) Significantly increased IFN-γ production in tumors with low MORC2 expression; (N) Significant negative correlation analysis between MORC2 expression level and GZMB level; (O) Significant negative correlation analysis between MORC2 expression level and IFN-γ level; (P) Significant negative correlation analysis between MORC2 expression level and CD8+ T cell infiltration. A comprehensive summary diagram of the negative correlation between T cell infiltration and effector function indicators; (R) is an overall schematic diagram of the present invention, illustrating that MORC2 is highly expressed in TNBC, and mediates H3K9me3 modification and transcriptional silencing of transposable elements (TEs) through interaction with SETDB1, inhibiting cytoplasmic nucleic acid accumulation and activation of the downstream MDA5 / cGAS-STING-interferon signaling pathway, thereby inhibiting MHC-I antigen presentation, reducing CD8+ T cell infiltration and macrophage M1 polarization, and promoting tumor immune escape; Targeted inhibition of MORC2 (ASO-Morc2a) can reactivate the interferon signaling pathway, reshape the immune microenvironment, and synergize with anti-PD-1 therapy, providing a new potential target for TNBC immunotherapy. Detailed Implementation

[0029] The embodiments of this application will now be described in detail with reference to the accompanying drawings.

[0030] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. This application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0031] Example 1: Experimental Materials and Methods 1.1 Cell lines and reagents Human TNBC cell lines (BT-549 and MDA-MB-231), mouse breast cancer cell lines (4T1, 66cl-4, and AT-3), and human embryonic kidney 293T (HEK293T) cell line were all derived from the Cell Bank of the Chinese Academy of Sciences (Shanghai) and Fudan University Cancer Center (FUSCC). All cell lines were identified by short tandem repeat (STR) mapping and were periodically tested for mycoplasma contamination. Cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0032] 1.2 Multi-omics datasets and clinical samples Transcriptome and proteome datasets were obtained from the Chinese Breast Cancer Genome Atlas (CBCGA) cohort, the FUSCC-TNBC cohort, the Cancer Genome Atlas (TCGA), the Breast Cancer International Molecular Taxonomy Consortium (METABRIC), and the Clinical Proteomics Tumor Analysis Consortium (CPTAC). TNBC specimens and matched adjacent normal breast tissue were obtained from patients who underwent FUSCC surgery. The research protocol was approved by the FUSCC Ethics Committee (#NSFC-25-0303-92), and all participants signed informed consent forms.

[0033] 1.3 CRISPR-Cas9 gene knockout and stable shRNA knockdown Stable knockout cell lines were constructed using lentiviral-mediated CRISPR-Cas9 technology. Annealed gRNA oligonucleotides were cloned into the BsmB1-digested lentiCRISPR-v2 vector (Addgene, #52961). After transfection, cells were selected with puromycin for 7-10 days, and knockout efficiency was verified by Western blotting. shRNA was cloned into the pLKO.1 lentiviral vector (Addgene, #10878), and stable cell lines were established through selection with puromycin or neomycin.

[0034] 1.4 Antisense Oligonucleotide (ASO) Design and In Vivo Dosage The antisense oligonucleotide (ASO-Morc2a) targeting Morc2a (NM_001159288.2 Mus musculus microrchidia 2A (Morc2a), transcript variant 1, mRNA, nucleotide sequence as shown in SEQ ID NO:2) was designed and synthesized by Ribobio (Guangzhou). It utilizes a 5-10-5 gapmer design, contains a phosphorus thioyl backbone modification, and mediates mRNA cleavage via an RNase H-dependent mechanism. After in vitro screening, the most potent ASO#3 was selected for in vivo experiments. ASO was administered via intratumoral injection (5 nmol, dissolved in PBS) three times weekly; the anti-PD-1 antibody (10 mg / kg) was administered via intraperitoneal injection.

[0035] 1.5 RNA Sequencing and Transposon Element Analysis RNA-seq library preparation and sequencing were performed on the Illumina NovaSeq platform. Sequencing reads were aligned to a reference genome using STAR (v2.7.11a), and gene and transposon element (TE) expression was quantitatively analyzed using TEtranscripts (v2.0.3). TE analysis utilized species-specific GTF annotation files (GRCm38_Ensembl_rmsk_TE.gtf and GRCh38_Ensembl_rmsk_TE.gtf). Differential gene expression analysis was performed using the DESeq2 program, and gene set enrichment analysis (GSEA) was used to elucidate biological significance.

[0036] 1.6 Single-cell RNA sequencing (scRNA-seq) Mouse tumor samples were dissociated using a mouse tumor dissociation kit (Miltenyi Biotec, #130-096-730), and scRNA-seq was performed using the 10× Genomics Chromium single-cell platform. A total of 18,095 cells were filtered, and 18 cell clusters were identified using UMAP dimensionality reduction clustering, annotated as 9 major cell types.

[0037] 1.7 Multiplex immunofluorescence (mIHC) staining and tissue microarray analysis Human breast cancer tissue microarrays were purchased from Shanghai Chip-Super Biotechnology Co., Ltd. (#HBreD180Bc01-2), containing 143 TNBC specimens and 25 normal breast tissues. mIHC analysis was performed using the following antibodies: MORC2 (Affinity, #AF6501), SETDB1 (Proteintech, #11231-1-AP), IFN-γ (Abcam, #abs119966), and GZMB (Abcam, #ab255598). Whole-section fluorescence images were acquired using a 3DHISTECH Pannoramic SCAN scanner and analyzed using SlideViewer software. Patients were divided into high-expression and low-expression groups based on the median fluorescence intensity in the cohort.

[0038] 1.8 Mass spectrometry analysis and identification of methylation sites Mass spectrometry analysis of MORC2 protein after immunoprecipitation in HEK293T cells identified SETDB1-dependent methylation sites. Prediction using the PhosphoSitePlus database and GPS-MCP calculation tool confirmed K234 and K643 as the major methylation sites of MORC2. Candidate sites were validated by lysine (K) → arginine (R) point mutations; the double mutants (K234R / K643R, 2KR) showed significantly reduced methylation levels.

[0039] Example 2: MORC2 is highly expressed in TNBC and is associated with the immunosuppressive tumor microenvironment. Using the CBCGA, TCGA, and METABRIC transcriptome datasets and the CBCGA and CPTAC proteome datasets, the expression levels of MORC2 in normal breast tissue, non-TNBC, and TNBC samples were systematically analyzed. The results showed ( Figure 1 MORC2 mRNA and protein levels were significantly higher in TNBC than in non-TNBC and normal breast tissue. Figure 1 AF). The FUSCC-TNBC dataset (88 pairs of TNBC and adjacent normal tissues) further validated the significant upregulation of MORC2 mRNA in TNBC. Figure 1 D). Immunoblotting assays detected MORC2 protein expression in 14 pairs of TNBC tissues and adjacent normal tissues, confirming that MORC2 was significantly upregulated in TNBC tissues. Figure 1 GH). Tissue microarray mIHC analysis (143 cases of TNBC + 25 cases of normal breast tissue) showed that MORC2 staining was significantly enhanced in TNBC. Figure 1 IJ).

[0040] In the ICBatlas database's NSCLC, gastric cancer, and melanoma cohorts, low MORC2 expression was significantly associated with improved response to ICB treatment. In the FUSCC-TNBC dataset, high MORC2 expression was significantly associated with a low proportion of immunomodulatory (IM) subtypes and a reduction in tumor-infiltrating lymphocytes (TILs). In the immunotherapy TNBC cohorts (GSE225078, GSE260989, GSE241876), low MORC2 expression was associated with higher objective response rates.

[0041] Example 3: MORC2 deficiency inhibits TNBC tumor growth in an immune microenvironment-dependent manner. Morc2a gene knockout (KO) cell lines were constructed in three mouse breast cancer cell lines (4T1, 66cl-4, and AT-3) using CRISPR-Cas9 technology, and Morc2a was reintroduced into the KO cells to verify target specificity. Cell proliferation and colony formation experiments confirmed that Morc2a deficiency did not significantly affect in vitro cell growth.

[0042] Morc2a-KO and control cells were inoculated into the mammary fat pads of immunodeficient BALB / c nude mice and immunocompetent BALB / c mice, respectively. The results showed ( Figure 1 Morc2a deficiency did not affect tumor growth in immunodeficient nude mice, but significantly inhibited tumor growth in immunocompetent mice, with both tumor volume and weight being significantly reduced. Figure 1 Reintroduction of Morc2a into Morc2a-KO cells partially restored tumor growth, confirming the targeting specificity of this phenotype. These results indicate that the pro-tumor effect of MORC2 depends on an intact tumor immune microenvironment.

[0043] Example 4: MORC2 deficiency reshapes the tumor immune microenvironment and activates anti-tumor immunity. scRNA-seq analysis was performed on the control group and Morc2a-KO 4T1 tumors. UMAP dimensionality reduction clustering identified 18 cell clusters, which were annotated as 9 major cell types. Figure 2 AB). The proportions of macrophages, T cells, and neutrophils in Morc2a-KO tumors changed significantly ( Figure 2 C). GSEA analysis showed significant enrichment of anti-tumor immune-related pathways such as TNF-α / NF-κB, IFN-γ, IFN-α, inflammatory response, and IL-6 / JAK / STAT3 in Morc2a-KO tumors. Figure 2 D).

[0044] Flow cytometry analysis confirmed an increased proportion of M1 macrophages (iNOS+) and a decreased proportion of M2 macrophages (CD206+) in Morc2a-KO tumors. Figure 2 GI). Both scRNA-seq and flow cytometry analyses showed an increased proportion of CD8+ T cells and elevated frequencies of IFN-γ+ and GzmB+ cytotoxic T cell subsets in Morc2a-KO tumors. Figure 2 (JN). In the TCGA and METABRIC cohorts, MORC2 expression was significantly negatively correlated with activated CD8+ T cell markers such as CD8A / B, PRF1, GZMB, and CD247.

[0045] Example 5: MORC2 deletion activates transposon transcription and induces interferon response. RNA-seq GSEA analysis showed that the interferon response pathway was significantly enriched in Morc2a-KO 4T1 cells. Figure 3 A). RT-qPCR confirmed that Morc2a deletion significantly upregulated classical ISGs (Cxcl9, Ifit3, Ifi207, Ifitm1, Bst2, Oas3) and mouse MHC-I related genes (B2m, H2-K1, H2-D1, H2-Q1). Figure 3 BD). Flow cytometry and immunofluorescence staining confirmed that the ratio of H-2Kb / H-2Db of classical mouse MHC-I molecules was significantly upregulated in Morc2a-KO cells. Figure 3 EF).

[0046] TEtranscripts analysis showed that Morc2a deletion significantly altered the expression profile of TEs in 4T1 cells. Figure 3 G), RT-qPCR confirmed that specific TEs (LTR53-int, RLTR26D, RLTR9A3B, RLTR6C) were significantly upregulated. Figure 3 H). In human TNBC cells MDA-MB-231, MORC2 knockout also activated the interferon signaling pathway and ISGs (CXCL9, CXCL10, BST2, IFIT3), upregulated key genes in the MHC-I antigen presentation pathway (B2M, HLA-A, HLA-C, TAP1), and significantly upregulated human TEs (LTR06, LTR22C0, LTR22C2, MER74C). Figure 3 Subfamily level analysis showed that ERVK subfamily members were the most significantly upregulated TE class after MORC2 deletion, and were the main driving factor of viral mimicry effects.

[0047] Example 6: MORC2 deletion triggers interferon response by activating the nucleic acid sensing pathway. In MDA-MB-231 and BT-549 cells, MORC2 knockout enhanced the expression of MDA5 and cGAS proteins, as well as the phosphorylation activation of STING, TBK1, and IRF3. Figure 4 A). IFN-γ treatment further enhances the activation of the above signaling pathways and the upregulation of ISGs ( Figure 4 BC). J2 antibody immunofluorescence staining showed significant accumulation of cytoplasmic dsRNA in MORC2-KO cells, and RNase III treatment could partially eliminate this signal (BC). Figure 4 D). Treatment with the nucleotide reverse transcriptase inhibitor lamivudine (3TC) effectively reduced MORC2 deletion-induced accumulation of cytoplasmic dsDNA. Figure 4 E). Knocking down nucleic acid sensing receptors MDA5 or cGAS significantly attenuated MORC2 loss-induced upregulation of ISGs ( Figure 4 F). The above results confirm that MORC2 deficiency activates the dsRNA and dsDNA sensing pathways by releasing TE-derived nucleic acids, triggering an interferon response.

[0048] Example 7: MORC2 and SETDB1 synergistically deposit H3K9me3 inhibitory modification at TEs sites Endogenous co-IP experiments confirmed the physical interaction between MORC2 and SETDB1 in MDA-MB-231 and BT-549 cells. Figure 5 AB), immunofluorescence staining showed significant colocalization between the two ( Figure 5 C). Co-IP experiments with MORC2 truncated mutants (T1-T3) showed that the N-terminal ATPase domain of MORC2 mediates its interaction with SETDB1. Figure 5 DE); SETDB1 truncated mutant (T1-T2) co-IP experiments showed that the N-terminal region of SETDB1 (containing the Tudor domain) mediates its interaction with MORC2 ( Figure 5 FG).

[0049] Public ChIP-seq data analysis showed that the levels of H3K9me3 at LTR22C2 and MER74C sites decreased after MORC2 knockout. Figure 5 H). SETDB1 knockdown reverses the transcriptional repression induced by MORC2 overexpression in LTR22C2, MER74C, and downstream ISGs (CXCL9, IFIT3). Figure 5 IJ). ChIP-qPCR experiments showed that MORC2 overexpression enhanced the levels of H3K9me3 at LTR22C2 and MER74C sites, and SETDB1 knockdown eliminated this effect. Figure 5K). CUT&RUN experiments confirmed that MORC2 and SETDB1 jointly occupy a large number of TE sites across the genome. The above results confirm that MORC2 and SETDB1 synergistically establish H3K9me3 modification at TE sites, leading to TE silencing and inhibiting interferon response.

[0050] Example 8: SETDB1 enhances the protein stability of MORC2 by methylating the K234 and K643 sites. mIHC staining showed that SETDB1 was significantly highly expressed in TNBC tissues and was significantly positively correlated with MORC2 expression levels. Figure 6 AD. CBCGA and CPTAC proteomic datasets confirmed a significant positive correlation between SETDB1 and MORC2 protein levels in TNBC. Figure 6 EF). SETDB1 knockdown significantly reduced MORC2 protein expression levels, but did not affect its mRNA levels ( ). Figure 6 G). CHX tracking experiments showed that SETDB1 knockdown significantly shortened the half-life of MORC2 protein (G). Figure 6 H).

[0051] SETDB1 knockdown reduces MORC2 methylation levels, while wild-type SETDB1 overexpression enhances MORC2 methylation, whereas catalytically inactive mutants (H1224K or C1226A) have no such effect. Figure 6 IJ). Mass spectrometry analysis identified K234 and K643 as the major SETDB1 methylation sites of MORC2, and the methylation level of the double mutant (2KR) was significantly reduced. Figure 6 KL). The SETDB1 inhibitor sclerotinib A (MMA) reduces MORC2 protein expression in a dose-dependent manner. Figure 6 M). CHX tracking experiments showed that the half-life of the 2KR mutant protein was significantly shorter than that of the wild-type MORC2 (M). Figure 6 N). Reintroducing wild-type MORC2 (instead of the 2KR mutant) into MORC2-KO cells inhibited TE activation and ISG induction. These results confirm that SETDB1 enhances the protein stability of MORC2 by methylating the K234 and K643 sites, forming a MORC2 / SETDB1 positive feedback regulatory loop.

[0052] Example 9: ASO-MORC2 combined with anti-PD-1 therapy synergistically inhibits TNBC tumor growth. Three Morc2a-specific ASOs (ASO-Morc2a) were evaluated in 4T1 cells: ASO#1 (sequence shown in SEQ ID NO:3), ASO#2 (sequence shown in SEQ ID NO:4), and ASO#3 (sequence shown in SEQ ID NO:5). All three significantly reduced Morc2a expression at a concentration of 50 nM, with ASO#3 showing the strongest effect and being selected for subsequent animal experiments. Clonogenesis assays confirmed that ASO-mediated Morc2a knockdown did not affect the in vitro growth of 4T1 cells.

[0053] In 4T1 tumor-bearing BALB / c mice, ASO-Morc2a (5 nmol, intratumoral injection, 3 weeks) combined with anti-PD-1 antibody (10 mg / kg, intraperitoneal injection) significantly inhibited tumor growth. Figure 7 AC). ASO-mediated Morc2a silencing was well tolerated, with no significant decrease in mouse body weight. Morc2a knockdown in tumor tissue was confirmed, with significant upregulation of ISGs (Cxcl9, Ifit3). Flow cytometry analysis showed that the combination therapy promoted M1 polarization of tumor macrophages (AC). Figure 7 DF significantly increased CD8+ T cell infiltration and the proportion of IFN-γ+ and GzmB+ cytotoxic T cell subsets. Figure 7 (GI), indicating that combination therapy can broadly reshape the tumor immune microenvironment towards a pro-inflammatory anti-tumor state.

[0054] Example 10: MORC2 expression was negatively correlated with CD8+ T cell infiltration and activation in clinical TNBC samples. mIHC analysis was performed on tissue microarrays containing 143 clinical TNBC samples to assess the relationship between MORC2 expression and T cell infiltration and activation. Results showed that CD8+ T cell infiltration was significantly increased in tumors with low MORC2 expression. Figure 7 JK), GZMB and IFN-γ production significantly increased ( Figure 7 LO), and both were significantly negatively correlated with MORC2 expression levels ( Figure 7 The above clinical data validate the clinical value of MORC2 as a predictive biomarker for the efficacy of TNBC immunotherapy and support the treatment strategy of targeting MORC2 to improve TNBC immunotherapy outcomes.

[0055] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention should be covered within the scope of protection of the present invention.

Claims

1. The application of MORC2 as a target in the preparation of drugs for treating triple-negative breast cancer, characterized in that, The nucleotide sequence of MORC2 is shown in SEQ ID NO:

1.

2. The application as described in claim 1, characterized in that, The expression of the MORC2 gene or protein is suppressed by inhibitors and CRISPR-Cas9 gene knockout.

3. The application as described in claim 2, characterized in that, The inhibitors include antisense oligonucleotides, shRNA, siRNA, MORC2 antibodies, and small molecule inhibitors.

4. The application as described in claim 3, characterized in that, The inhibitor is an antisense oligonucleotide.

5. The application as described in claim 4, characterized in that, The antisense oligonucleotides are designed with a 5-10-5 gapmer and modified with a phosphorus thioyl backbone, and target Morc2a mRNA cleavage and degradation through an RNase H-dependent mechanism.

6. Application of MORC2 gene or protein expression inhibitors combined with immune checkpoint blockers in the preparation of drugs for the treatment of triple-negative breast cancer.

7. The application as described in claim 6, characterized in that, The immune checkpoint blockers include anti-PD-1 antibody, anti-PD-L1 antibody, and anti-CTLA-4 antibody.

8. Application of the MORC2 / SETDB1 epigenetic regulatory axis as a target in the preparation of immunosensitizing drugs for triple-negative breast cancer.

9. Application of MORC2 as a biomarker in the preparation of reagents for the diagnosis of triple-negative breast cancer, prediction of immunotherapy efficacy, and patient stratification.

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