Use of pevonedistat in combination with medroxyprogesterone acetate for the preparation of a medicament for the treatment of endometrial cancer

Abstract

The application provides a use of a combination of pevonedister and medroxyprogesterone acetate in the preparation of a drug for treating endometrial cancer, and belongs to the technical field of biological medicines. AMBRA1 The application finds that pevonedister can enhance the sensitivity of endometrial cancer cells to progestin treatment by inhibiting CRL4 The experimental results show that the combination of pevonedister and medroxyprogesterone acetate can significantly inhibit tumor growth, restore progestin response in progestin-resistant endometrial cancer cells, patient-derived organoids and xenograft tumor models, and has a significant synergistic effect. The application provides a use of AMBRA1 as a biomarker in the preparation of a product for evaluating the sensitivity of endometrial cancer patients to progestin treatment, and high expression of AMBRA1 indicates that the patient has a risk of drug resistance. The application provides an effective combination therapy strategy and diagnostic tool for progestin-resistant or insensitive endometrial cancer, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the application of pevorinistat combined with medroxyprogesterone acetate in the preparation of drugs for treating endometrial cancer. Background Technology

[0002] Endometrial cancer is one of the most common gynecological malignancies worldwide, with its incidence and mortality rates continuing to rise. Of particular concern is the steadily increasing proportion of patients of reproductive age. While standard surgical treatment can achieve good oncological prognoses for early-stage endometrial cancer, hysterectomy affects fertility preservation and is not suitable for young patients who wish to preserve their fertility. Against this backdrop, fertility-preserving treatments for young patients with early-stage endometrial cancer and endometrial dysplasia are receiving increasing attention. Progestin-based therapy is a first-line fertility-preserving regimen for early-stage, low-grade endometrial cancer patients who wish to preserve their fertility, with medroxyprogesterone acetate (MPA) being the most widely used progestin. Although progestin regimens have high initial response rates, primary and acquired resistance remain major challenges.

[0003] Although progestins have been used clinically for decades, their resistance mechanisms remain incompletely understood. The effects of progestins are primarily mediated by binding to the progesterone receptor (PR). PR expression is crucial for maintaining the endocrine response phenotype, and its expression level is also used as a potential predictor of progesterone therapy response in endometrial cancer. Progestins bind to the ligand-binding domain (LBD) of PR (especially the major subtype progesterone receptor B (PRB)), activating downstream signaling pathways and regulating multiple PRB response genes involved in apoptosis, differentiation, and cell cycle arrest. Abnormal PR signaling is also associated with resistance following continuous progesterone exposure, and increasing evidence suggests that restoring PR expression is a key strategy for overcoming MPA resistance. Studies in endometrial cancer cell lines have shown that demethylating agents and histone deacetylase inhibitors can enhance MPA sensitivity by upregulating PR; chlorpromazine can synergistically interact with MPA by increasing PR expression; and the combined use of neural cell adhesion molecules can maintain PR function and enhance MPA sensitivity. However, few of these potential targets have entered clinical trials. Given the complexity of progesterone-mediated molecular alterations in MPA-resistant endometrial cancer, it is urgent to identify key factors regulating PR stability and function and elucidate their molecular mechanisms.

[0004] AMBRA1 (autophagy / Beclin-1 regulator 1) is a conserved, intrinsically disordered adaptor protein in vertebrates, initially identified as a key positive regulator of autophagy. AMBRA1 interacts with Beclin1 to anchor the Beclin1-VPS34 complex to the dynein motor complex and promotes autophagy initiation by regulating TRAF6-mediated ULK1 ubiquitination. It also participates in mitochondrial clearance and apoptosis. Recent studies have found that abnormal AMBRA1 expression can participate in the progression of tumors such as melanoma and breast cancer through a Cullin-dependent degradation pathway. Cullin-RING ligases (CRLs) are multi-subunit E3 ubiquitin ligase complexes that regulate the proteasome degradation of various substrates and participate in the regulation of numerous cellular processes. Due to their crucial role in tumorigenesis, CRLs have become highly promising therapeutic targets. However, the role of specific CRL components in the pathogenesis of endometrial cancer and whether AMBRA1 is involved in the regulation of MPA resistance remain poorly understood. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the use of pevorinistat combined with medroxyprogesterone acetate in the preparation of a drug for treating endometrial cancer.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of pevorinistat combined with medroxyprogesterone acetate in the preparation of a drug for treating endometrial cancer.

[0007] Preferably, the endometrial cancer is an endometrial cancer that is resistant to or insensitive to progesterone.

[0008] More preferably, the pevornidat inhibits CRL4 AMBRA1 The complex enhances the sensitivity of endometrial cancer to progesterone therapy.

[0009] Preferably, the pevorinistat and medroxyprogesterone acetate are administered simultaneously or sequentially.

[0010] The present invention also provides a pharmaceutical composition comprising pevorisstat, medroxyprogesterone acetate, and a pharmaceutically acceptable carrier.

[0011] Preferably, the pharmaceutical composition is an injection, a lyophilized powder injection, or an oral preparation.

[0012] This invention also provides the use of AMBRA1 in the preparation of products for assessing the sensitivity of endometrial cancer patients to progesterone therapy.

[0013] Preferably, high expression of AMBRA1 suggests that endometrial cancer patients are insensitive to progesterone therapy or have a risk of drug resistance.

[0014] More preferably, the product contains a reagent for detecting the expression level of the AMBRA1 protein.

[0015] The present invention also provides the use of pevorin in the preparation of a drug for enhancing the sensitivity of endometrial cancer cells to progesterone.

[0016] Compared with the prior art, the present invention has the following advantages: (1) This invention discloses for the first time that AMBRA1 passes through CRL4 AMBRA1 A novel mechanism by which the ubiquitin ligase complex mediates the K48-linked polyubiquitination degradation of the progesterone receptor (PR) leads to resistance to medroxyprogesterone acetate (MPA) in endometrial cancer. Based on this mechanism, this invention proposes a therapeutic strategy combining pevorinistat (MLN4924) with MPA. Experimental results show that in MPA-resistant endometrial cancer cell lines (such as ISK_Res), MLN4924 can significantly restore PR protein levels. After combined treatment with MPA, cell viability is significantly reduced, colony-forming ability is significantly decreased, and the proportion of apoptosis is significantly increased, confirming that the combination therapy can effectively reverse acquired resistance to MPA.

[0017] (2) This invention further validated the synergistic antitumor effect of combined drug therapy in a patient-derived endometrial cancer organoid (ECO) model. MPA-resistant ECO7 and ECO8 organoids were treated with a combination of MLN4924 and MPA. The results showed a significant synergistic effect, with synergistic scores of 11.44 and 13.95, respectively. The combined group showed a significantly better inhibitory effect on cell viability than either single-drug group. In an in vivo xenograft tumor model, MPA monotherapy had limited inhibitory effect on the growth of drug-resistant tumors, and MLN4924 monotherapy was also not ideal. However, the combination of the two drugs significantly inhibited tumor growth, resulting in a substantial reduction in tumor volume and weight. This effect was closely related to the recovery of PR expression in tumor tissue.

[0018] (3) This invention also confirms the diagnostic value of AMBRA1 as a biomarker. Immunohistochemical analysis of clinical endometrial cancer specimens and patient-derived organoids revealed a significant positive correlation between AMBRA1 expression levels and MPA resistance, and a negative correlation with PR expression. Patients with high AMBRA1 expression were insensitive to progestin therapy or had a higher risk of resistance. Therefore, detecting AMBRA1 expression levels can be used to assess the sensitivity of endometrial cancer patients to progestin therapy and guide clinical drug selection. Attached Figure Description

[0019] Figure 1To establish and validate MPA-resistant endometrial cancer cell lines; Figure a shows the flowchart of establishing an MPA-resistant subline from the MPA-sensitive endometrial cancer cell line ISK, where the MPA concentration was gradually increased by 2.5 μM every 4 weeks for 6 months. After this treatment, the cells acquired MPA resistance (ISK_Res) compared to the corresponding parental cells (ISK_Parental); b shows the dose-response curves of MPA to the resistant cells (ISK_Res) and their corresponding parental cells (ISK_Parental), with cells cultured in different concentrations of MPA for 3 days (4 biological replicates per group); c shows the cell viability of ISK_Res and ISK_Parental cells after 72 hours of treatment with 20 μM MPA using the CCK8 assay (4 biological replicates per group), and statistical analysis was performed using two-way ANOVA combined with Šídák correction; d shows the crystal violet staining method for detecting the viability of ISK_Res and ISK_Parental cells after 20 μM MPA treatment. Cell growth after 10 days of MPA treatment; e shows the quantitative analysis of clone number in d (3 biological replicates per group), and the data were analyzed using an unpaired two-tailed t-test; f shows the mRNA expression level of the indicated gene in ISK_Res and ISK_Parental cells after 24 hours of treatment with 20 μM MPA, detected by qRT-PCR (3 biological replicates per group), and the data were analyzed using an unpaired two-tailed t-test; g shows the protein immunoblotting analysis of PR expression level in ISK_Res and ISK_Parental cells; h shows the qRT-PCR analysis of PRB mRNA expression level in ISK_Res and ISK_Parental cells (3 biological replicates per group), and the data were analyzed using an unpaired two-tailed t-test; all results are expressed as mean ± standard deviation.

[0020] Figure 2The results confirm that AMBRA1 is highly expressed in medroxyprogesterone acetate (MPA)-resistant endometrial cancer and is positively regulated by IKKα; a) is a volcano plot showing the top 50 enriched progesterone receptor (PR) interacting proteins identified in ISK-resistant cells (ISK_Res) and parental cells (ISK_Parental) by label-free quantitative proteomics analysis, with statistical significance (–log10 (P value)) expressed as a gradient; b) shows the Cullin-RING ligase (CRL) family members in MPA-resistant and MPA-sensitive endometrial cancer. Differential expression (data from GSE121367 dataset); c is a Venn diagram showing the intersection of PR-interacting proteins enriched in ISK_Res and ISK_Parental cells (immunoprecipitation-mass spectrometry analysis) and upregulated CRL family members in MPA-resistant and MPA-sensitive cells; d shows the immunohistochemical staining results of AMBRA1 in tumor specimens from MPA-resistant and MPA-sensitive endometrial cancer patients after progesterone therapy (MPA-sensitive samples n=3, MPA-resistant samples n=3), showing representative images, scale bar: left image 200μm, right image 50μm. μm; e is the quantitative analysis of AMBRA1 immunohistochemical staining intensity in d; f is the immunohistochemical staining image of AMBRA1 in tumor specimens of MPA-resistant endometrial cancer patients before and after progesterone treatment (n=3 samples before treatment, n=3 samples after treatment), showing representative images, scale bar: left image 200μm, right image 50μm; Pre-T: before progesterone treatment, After-T: after progesterone treatment, data were analyzed using unpaired two-tailed t-test; g is the quantitative analysis of AMBRA1 immunohistochemical staining intensity in f, data were analyzed using unpaired two-tailed t-test; h Characteristics of organoids (ECOs) derived from 8 patients with endometrial cancer; green represents MPA-sensitive, and dark red represents MPA-resistant; low (light green) and high (red) expression levels of AMBRA1 and PR in biopsy tissues; histological type: endometrioid carcinoma; grade: G1; molecular subtype: nonspecific molecular profile (NSMP); i shows the dose-response curve of organoids to MPA; organoids were cultured in different concentrations of MPA for 6–10 days, and statistical analysis was performed using an unpaired t-test. Data are expressed as mean ± standard deviation; j represents the half-maximal inhibitory concentration (IC50) of the organoid to MPA. 50k represents the quantitative analysis of AMBRA1 and PR immunohistochemical staining intensity in MPA-sensitive and MPA-resistant organoids, and the data were analyzed using an unpaired two-tailed t-test; l represents the quantitative immunohistochemical staining intensity of AMBRA1 and PR in 8 organoid strains; Spearman correlation analysis was used to determine the correlation between AMBRA1, PR and MPA IC50; m represents the protein immunoblotting analysis of AMBRA1 and PR expression levels in MPA-resistant ISK cells and their corresponding parental cells; n represents the endogenous immunoprecipitation experiment of AMBRA1 and IKKα interaction in ISK_Res cells; enrichment was performed using anti-IKKα antibody. Immunoprecipitation products, with immunoglobulin G (IgG) as a negative control; o represents the Western blot analysis of AMBRA1 and IKKα expressed proteins after ISK_Res cells were treated alone with MPA (20 μM, 48 h) or in combination with BAY11-7082 (30 μM, 24 h); p represents the Western blot analysis of proteins expressed by ISK cells after transient transfection with FLAG-IKKα wild-type (FLAG-IKKαWT), FLAG-IKKα kinase inactivation mutant (FLAG-IKKαK44M), or empty vector, followed by cell collection after 36 hours of culture, and analysis using anti-AMBRA1 and anti-FLAG antibodies; q represents... Figure 4 Quantitative analysis of AMBRA1-histidine (His) protein levels based on band intensity in g was performed. Statistical analysis was conducted using two-way ANOVA with Šídák correction. All results are expressed as mean ± standard deviation.

[0021] Figure 3To validate the finding that AMBRA1 was upregulated and negatively correlated with PR in endometrial cancer patients; Figure a shows the immunohistochemical staining results of AMBRA1 in normal endometrial tissue (n=12) and endometrial cancer tissue (n=12), displaying representative images, scale bar: top image 200μm, bottom image 50μm; b shows the quantitative analysis of AMBRA1 immunohistochemical staining intensity in a, and the data were analyzed using an unpaired two-tailed t-test; c shows the expression of AMBRA1 in tumor tissue (T) and paired adjacent normal endometrial tissue (N) of 12 endometrial cancer patients detected by Western blotting; d shows the expression of AMBRA1 in tumor specimens of 30 endometrial cancer patients. Immunohistochemical staining results of MBRA1 and PR are shown in representative images. Scale bar: left image 200μm, right image 50μm; e shows the quantification of the immunohistochemical intensity of AMBRA1 and PR in d, and Spearman correlation analysis was used to determine the correlation between AMBRA1 and PR expression in endometrial cancer; f shows the hematoxylin-eosin (H&E) staining results of the organoids and their primary tissues, with representative images shown. Scale bar: top image 50μm, bottom image 100μm; g shows representative immunohistochemical staining images of AMBRA1 and PR in the organoids shown, with a scale bar of 50μm; all results are expressed as mean ± standard deviation.

[0022] Figure 4The results show that IKKα kinase phosphorylates AMBRA1 at serine 1043 (S1043) to stabilize AMBRA1 protein; Figure a shows the qRT-PCR analysis of AMBRA1 mRNA expression levels in ISK_Res and ISK_Parental cells (3 biological replicates per group), and the data were analyzed using an unpaired two-tailed t-test; Figure b is a table showing the interaction between IKKα and AMBRA1 in ISK_Res cells; Figures c and d show the results of ISK cells after treatment with MPA (20 μM, 48) h) Reverse endogenous immunoprecipitation experiment of AMBRA1 and IKKα interaction after treatment; immunoprecipitation products were enriched with anti-AMBRA1 antibody (c) or anti-IKKα antibody (d), with IgG as a negative control; e) Endogenous immunoprecipitation experiment of AMBRA1 and IKKα interaction in ISK_Res cells; immunoprecipitation products were enriched with anti-AMBRA1 antibody, with IgG as a negative control; f) Immunoblot analysis of proteins expressed by AMBRA1 and IKKα after ISK cells were treated alone with MPA (20 μM, 48 h) or in combination with BAY11-7082 (30 μM, 24 h); g) Immunoblot analysis of AMBRA1-His and FLAG-IKKα expression in ISK cells co-transfected with AMBRA1-His and empty vector or FLAG-IKKα plasmid and treated with CHX (50 μg / mL) for different time periods. - IKKα was used for Western blotting analysis of proteins; h was for ISK cells transiently co-transfected with FLAG-IKKα and AMBRA1WT-His or AMBRA1S1043A-His, and after 36 hours of culture, cells were collected and Western blotting analysis was performed using anti-His and anti-FLAG antibodies; i was for ISK cells co-transfected with AMBRA1WT-His / AMBRA1S1043A-His and FLAG-IKKαWT / FLAG-IKKαK44M plasmids, and after treatment with CHX (50 μg / mL) for different times, Western blotting analysis of proteins AMBRA1-His and FLAG-IKKα was performed; j was for quantitative analysis of AMBRA1-His protein levels based on the band intensity in i, and statistical analysis was performed using two-way ANOVA combined with Šídák correction; all results are expressed as mean ± standard deviation.

[0023] Figure 5The results show that AMBRA1 overexpression promotes MPA resistance in endometrial cancer cells. Figure a shows the overexpression efficiency of AMBRA1 in ISK cells as verified by Western blotting; b shows the overexpression efficiency of AMBRA1 in ISK cells detected by qRT-PCR (3 biological replicates per group), and the data were analyzed using an unpaired two-tailed t-test; c shows the cell viability of ISK cells after 72 hours of treatment with 20 μM MPA (4 biological replicates per group) after CCK8 assay to detect whether AMBRA1 was overexpressed, and the statistical analysis used two-way ANOVA with Šídák correction; d shows the cell growth of ISK cells after 10 days of treatment with 20 μM MPA after crystal violet staining to detect whether AMBRA1 was overexpressed; e shows the quantitative analysis of clone number in d (3 biological replicates per group), and the data were analyzed using an unpaired two-tailed t-test; f shows the results of empty vector transfection and AMBRA1 overexpressing cells after 72 hours of treatment with 30 μM MPA or DMSO, followed by Annexin... V and 7-AAD staining were used to detect apoptosis by flow cytometry; g shows the statistical proportion of apoptotic cells in f (3 biological replicates per group), and the data were analyzed using an unpaired two-tailed t-test; h is a schematic diagram of the xenograft tumor growth model established by subcutaneous inoculation of ISK cells constructed in a; i shows the growth of xenograft tumors after MPA treatment, whether ISK cells overexpress AMBRA1; j shows the dynamic monitoring results of xenograft tumor volume, and the statistical analysis was performed using two-way ANOVA with Šídák correction; k shows the final tumor weight statistics of xenograft tumor volume (n=5), and the data were analyzed using an unpaired two-tailed t-test; all results are expressed as mean ± standard deviation.

[0024] Figure 6The results show the effects of AMBRA1 ke2 on the sensitivity of endometrial cancer cells to MPA. Figures a and b show the overexpression efficiency of AMBRA1 in ECO1 organoids derived from endometrial cancer patients, verified by Western blotting (a) and quantitative real-time PCR (qRT-PCR) (b) (3 biological replicates per group). Data in b were analyzed using an unpaired two-tailed t-test. Figure c shows representative immunohistochemical staining images of AMBRA1 in ECO1 cells, with a scale bar of 50 μm. Figure d shows the quantitative analysis of immunohistochemical intensity in c (3 biological replicates per group), with data analyzed using an unpaired two-tailed t-test. Figure e shows whether ECO1 cells overexpress AMBRA1, after 20 μM... Cell viability was assessed using the ATPlite assay (3 biological replicates per group) after 6 days of treatment with MPA or dimethyl sulfoxide (DMSO). Statistical analysis was performed using two-way ANOVA with Šídák correction. f shows the Western blotting verification of AMBRA1 knockout efficiency in ISK_Res cells. g shows the CCK8 assay of cell viability after 96 hours of treatment with 20 μM MPA (4 biological replicates per group), with statistical analysis performed using two-way ANOVA with Šídák correction. h shows the crystal violet staining assay of cell growth after 10 days of treatment with 20 μM MPA, with or without AMBRA1 knockout. i shows the quantitative analysis of clone number in h (3 biological replicates per group). Statistical analysis was performed using one-way ANOVA with Šídák correction. j shows the control group (CTL) and AMBRA1 knockout cells after 72 hours of treatment with 30 μM MPA or DMSO, followed by annexin V (Annexin V) staining. V) and 7-aminoactinomycin D (7-AAD) staining were used to detect cell apoptosis by flow cytometry; k represents the statistical proportion of apoptotic cells in j (3 biological replicates per group), and the statistical analysis was performed using one-way ANOVA with Šídák correction; l is a schematic diagram of the xenograft tumor growth model established by subcutaneous inoculation of ISK_Res cells constructed in f; m represents the xenograft tumor growth after MPA treatment with or without AMBRA1 knockout in ISK_Res cells; n represents the dynamic monitoring results of xenograft tumor volume, and the statistical analysis was performed using two-way ANOVA with Šídák correction; o represents the final tumor weight statistics of xenograft tumor volume (o), and the statistical analysis of o was performed using one-way ANOVA with Šídák correction; all results are expressed as mean ± standard deviation.

[0025] Figure 7The results show the inhibition of MPA-induced PRB response gene expression in endometrial cancer cells by AMBRA1. Figures a and b show whether AMBRA1 was overexpressed in ISK cells. After treatment with rapamycin (200 nM, 12 h) (a) or CQ (20 μM, 6 h) (b), autophagic flux was analyzed by detecting LC3B transformation (LC3B-I to LC3B-II during autophagy formation) and the indicated protein levels using Western blotting. Figure c shows ISK cells transfected with AMBRA1 and RANK... L-luciferase (RANKL-luc) and Renilla-luciferase (Renilla-luc) expression plasmids were treated with MPA (20 μM, 24 h), and the relative luciferase activity was measured (3 biological replicates per group). Data were analyzed using an unpaired two-tailed t-test. d represents whether ISK cells overexpress AMBRA1; after MPA (20 μM, 24 h) treatment, the expression of the indicated protein was detected by Western blotting. e represents whether ISK cells overexpress AMBRA1; after MPA (20 μM, 24 h) treatment, the expression of the indicated protein was detected by Western blotting. After 24 hours of MPA treatment, the mRNA expression level of the indicated gene was detected by qRT-PCR (3 biological replicates per group), and the data were analyzed using an unpaired two-tailed t-test. f represents the mRNA expression level of the indicated gene after 24 hours of 20 μM MPA treatment in ECO1 cells with or without AMBRA1 overexpression (3 biological replicates per group), and the data were analyzed using an unpaired two-tailed t-test. g represents the qRT-PCR analysis of PRB mRNA expression level in ISK cells, ECO1 cells with or without AMBRA1 overexpression, and ISK_Res cells with or without AMBRA1 knockout (3 biological replicates per group). The data in g (left and middle) were analyzed using an unpaired two-tailed t-test, and the statistical analysis in g (right) was performed using one-way ANOVA with Šídák correction. All results are expressed as mean ± standard deviation.

[0026] Figure 8The results show the interaction between AMBRA1 and PR; figures a and b show the exogenous immunoprecipitation experiments of the interaction between FLAG-PR and HA-AMBRA1 in HEK293T cells; FLAG-PR and HA-AMBRA1 were overexpressed in HEK293T cells, and the immunoprecipitation products were enriched with anti-FLAG antibody (a) or anti-HA antibody (b); figures c and d show the reverse endogenous immunoprecipitation experiments of the interaction between AMBRA1 and PR in ISK and ISK_Res cells; after treatment with MG132 (20 μM, 6 h), the immunoprecipitation products were enriched with anti-PR antibody (c) or anti-AMBRA1 antibody (d), with IgG as a negative control; figure e shows the structural diagrams of wild-type (WT) and truncated mutant PR; figures f and g show the HEK293T cells. T cells co-expressing FLAG-PR wild-type or truncated mutant and HA-AMBRA1 were cultured for 36 hours and then collected. Immunoprecipitation was performed using FLAG magnetic beads (f) or HA magnetic beads (g), followed by Western blotting analysis. h shows the structural diagram of AMBRA1 wild-type and its different truncated mutants. i shows HEK293T cells co-expressing HA-AMBRA1 wild-type or the truncated mutant shown and FLAG-PR. After 36 hours of culture, the cells were collected and immunoprecipitated using FLAG magnetic beads, followed by Western blotting analysis. j shows HEK293T cells co-expressing HA-AMBRA1 wild-type or the truncated mutant shown and FLAG-PR. After 36 hours of culture, the cells were collected and immunoprecipitated using HA magnetic beads, followed by Western blotting analysis.

[0027] Figure 9The results show the ubiquitination and degradation of PR mediated by AMBRA1 at lysine 388. Figure a shows representative immunohistochemical staining images of PR and AMBRA1 in ECO1 cells with and without AMBRA1 overexpression (scale bar: 100 μm). Figure b shows the quantitative analysis of immunohistochemical intensity in a (3 biological replicates per group), with data analyzed using an unpaired two-tailed t-test. Figure c shows the Western blot analysis of PR protein levels after AMBRA1 overexpression in ISK cells. Figure d shows the Western blot analysis of PR protein levels after AMBRA1 knockout in ISK_Res cells. Figure e shows the degradation of PR by MG132 (20 μM, 6 h) or chloroquine (CQ, 40 μM, 1 h) after AMBRA1 overexpression in ISK cells. 2h) treatment, Western blot analysis of PR and HA-AMBRA1 expression proteins; f is the Western blot analysis of PR expression proteins after ISK_Res cells were treated with MG132 (20 μM, 6h) or CQ (40 μM, 12h); g is the Western blot analysis of PR and AMBRA1 proteins after ISK cells overexpressed with AMBRA1 were treated with cycloheximide (CHX, 50 μg / mL) for different time periods; h is the quantitative analysis of PR protein levels based on band intensity (3 biological replicates), and statistical analysis was performed using two-way ANOVA combined with Šídák correction; i is the co-expression of AMBRA1-His, HA-ubiquitin (HA-Ub), and FLAG in HEK293T cells. -PR wild-type or ΔIF mutant, treated with MG132 (20 μM, 6 h), then subjected to FLAG magnetic beads for co-precipitation, followed by Western blotting analysis with the indicated antibody; j represents HEK293T cells co-expressing MYC-AMBRA1, FLAG-PR wild-type, and HA-Ub or ubiquitin mutant (HA-Ub-K48R), treated with MG132 (20 μM, 6 h), then subjected to FLAG magnetic beads for co-precipitation, followed by Western blotting analysis with the indicated antibody; k represents HEK293T cells co-expressing MYC-AMBRA1, HA-Ub, and FLAG-PR wild-type or its K388R mutant, treated with MG132 (20 μM, 6 h), then subjected to FLAG magnetic beads for co-precipitation, followed by Western blotting analysis with the indicated antibody; h) After treatment, immunoprecipitation was performed using FLAG magnetic beads, followed by Western blotting analysis of proteins using the indicated antibodies; l) ISK cells co-expressed HA-AMBRA1 and FLAG-PR wild-type or K388R plasmids, followed by Western blotting analysis of proteins using the indicated antibodies; m) ISK cells co-transfected with HA-AMBRA1 and FLAG-PR wild-type or K388R plasmids, treated with CHX (50 μg / mL) for different times, and Western blotting analysis of proteins in FLAG-PR and HA-AMBRA1 was performed; n) FLAG-PR protein level quantification analysis based on band intensity (3 biological replicates per group), statistical analysis was performed using two-way ANOVA combined with Šídák correction;All results are expressed as mean ± standard deviation.

[0028] Figure 10 The figures show the characteristics of AMBRA1-mediated PR ubiquitination. Figure a shows HEK293T cells transiently transfected with MYC-AMBRA1, FLAG-PR, and HA-Ub wild-type / mutant plasmids for 36 hours, treated with MG132 (20 μM, 6 h), and then immunoprecipitated with FLAG magnetic beads. The polyubiquitination level of PR was detected by Western blotting. Figures b-e show HEK293T cells transiently transfected with MYC-AMBRA1, HA-Ub, and FLAG-PR wild-type or 34 mutant plasmids for 36 hours, treated with MG132 (20 μM, 6 h), and then immunoprecipitated with FLAG magnetic beads. The polyubiquitination level of PR was detected by Western blotting.

[0029] Figure 11The results show the effects of AMBRA1 promoting PR ubiquitination in a CRL4-dependent manner in endometrial cancer cells; Table a in the figure shows the core CRL4 components that interact with AMBRA1 in ISK_Res cells; Figure b shows the endogenous immunoprecipitation experiment of AMBRA1 interacting with CRL4 core members CUL4A and DDB1 in ISK_Res cells, with the immunoprecipitation products enriched using anti-AMBRA1 antibody and IgG used as a negative control; Figure c shows ISK cells transiently transfected with AMBRA1-His wild-type or ΔN-WD40 mutant, followed by immunoprecipitation with anti-His antibody 48 hours after transfection, and then Western blot analysis using the antibodies shown; Figure d shows HEK2. 93T cells co-expressing FLAG-PR, HA-Ub, and AMBRA1-His wild-type or ΔN-WD40 mutant were treated with MG132 (20 μM, 6 h) and then subjected to immunoprecipitation with FLAG magnetic beads, followed by Western blotting analysis using the indicated antibodies. e shows ISK cells transiently transfected with AMBRA1-His wild-type or ΔN-WD40 mutant, followed by Western blotting analysis using the corresponding antibodies 48 hours later to detect protein levels. f shows the immunoassay of PR ubiquitination in HEK293T cells overexpressing AMBRA1 and knocking down CUL4A or DDB1 with small interfering RNA (siRNA). Cells were pre-treated with 20 μM MG132. MG132 treatment for 6 hours; g is the Western blot analysis of PR after ISK cells overexpressing AMBRA1 and knocking down CUL4A or DDB1 with siRNA; h is the Western blot analysis of PR after ISK_Res cells knocking down CUL4A or DDB1 with siRNA; i is the Western blot analysis of PR, ubiquitinated CUL4A, and NAE1 after ISK cells exogenously overexpressing AMBRA1 and being treated with MLN4924 (0.1 μM, 8 h); j is the Western blot detection of expression levels of PR, ubiquitinated CUL4A, and NAE1 after ISK cells stably overexpressing AMBRA1 and being treated with MPA (20 μM, 24 h) alone or in combination with MLN4924 (0.1 μM, 8 h).

[0030] Figure 12Targeting the CRL4 complex can attenuate AMBRA1-mediated MPA resistance in endometrial cancer. Figure a shows the cell viability of ISK cells after 72 hours of treatment with 20 μM MPA (three biological replicates per group), as determined by the CCK8 assay, with or without AMBRA1 overexpression and CUL4A / DDB1 knockdown. Statistical analysis was performed using two-way ANOVA with Šídák correction. Figure b shows the cell viability of ISK cells after AMBRA1 overexpression and siRNA transfection, followed by treatment with DMSO or 30 μM MPA for 72 hours, and then treatment with Annexin. V and 7-AAD staining were used to detect apoptosis by flow cytometry; c shows the statistical proportion of apoptotic cells in b (3 biological replicates per group), and the data were analyzed using one-way ANOVA with Šídák correction; d shows the cell viability of ECO1 cells after overexpression of AMBRA1, treated with MPA (20 μM) and MLN4924 (0.1 μM) alone or in combination for different time periods, and the data were detected using the ATPlite method (3 biological replicates per group), and the statistical analysis was performed using two-way ANOVA with Šídák correction; e shows the detection of cell viability of ISK cells after overexpression of AMBRA1, treated with MPA (20 μM) and MLN4924 (0.1 μM) alone or in combination for different time periods using the CCK8 assay. M) Synergistic effect of combined treatment (4 biological replicates per group), statistical analysis was performed using two-way ANOVA with Šídák correction; f) Crystal violet staining method was used to detect cell growth of ISK cells after 10 days of treatment with MPA (20 μM) and MLN4924 (0.05 μM) alone or in combination; g) Quantitative analysis of clone number in f (3 biological replicates per group), data were analyzed using one-way ANOVA with Šídák correction; h) ISK cells after 72 hours of treatment with MPA (20 μM) alone or in combination with MLN4924 (0.1 μM) after AMBRA1 overexpression, followed by Annexin... V and 7-AAD staining were used to detect apoptosis by flow cytometry; i represents the statistical proportion of apoptotic cells in h (3 biological replicates per group), and the data were analyzed using one-way ANOVA with Šídák correction; j is a schematic diagram of the xenograft tumor growth model established by subcutaneous inoculation of ISK cells overexpressing AMBRA1; k represents the growth of xenograft tumors derived from ISK cells overexpressing AMBRA1 after treatment with MPA and MLN4924 alone or in combination; l represents the dynamic monitoring results of xenograft tumor volume, and the statistical analysis was performed using two-way ANOVA with Šídák correction; m represents the final tumor weight statistics of xenograft tumor volume (n=5), and the data were analyzed using one-way ANOVA with Šídák correction; all results are expressed as mean ± standard deviation.

[0031] Figure 13The results show the synergistic inhibition of MPA-resistant endometrial cancer cells and organoid growth by MLN4924 and MPA. Figures a and b show organoids treated for 6–10 days, with cell viability detected using the ATPlite method. SynergyFinder software (ZIP model) was used to plot the synergistic effect and calculate the synergistic score. Figures c and d show organoids treated with MPA (20 μM) and MLN4924 (0.1 μM) alone or in combination for 6–10 days, with cell viability detected using the ATPlite method (3 biological replicates per group). Statistical analysis used two-way ANOVA with Šídák correction. Figure e shows ISK_Res cells treated with MPA (20 μM, 24 h) alone or in combination with MLN4924 (0.1 μM, 8 h), showing the PR and ubiquitination C levels. Western blot analysis of UL4A and NAE1 expression levels; f: CCK8 assay for the synergistic effect of MPA (20 μM) and MLN4924 (0.1 μM) combined treatment of ISK_Res cells (4 biological replicates), statistical analysis was performed using two-way ANOVA with Šídák correction; g: Crystal violet staining analysis for ISK_Res cell growth after 10 days of treatment with MPA (20 μM) and MLN4924 (0.05 μM) alone or in combination; h: Quantitative analysis of clone number in g (3 biological replicates), data were analyzed using one-way ANOVA with Šídák correction; i: ISK_Res cells treated with MPA (20 μM) and MLN4924 (0.1 μM) alone or in combination for 72 hours, followed by Annexin... V and 7-AAD staining were used to detect apoptosis by flow cytometry; j represents the statistical proportion of apoptotic cells in i (3 biological replicates), and the data were analyzed using one-way ANOVA with Šídák correction; k is a schematic diagram of the in vivo growth model of xenograft tumors established by subcutaneous seeding of ISK_Res cells; l represents the growth of xenograft tumors derived from ISK_Res cells after treatment with MPA and MLN4924 alone or in combination; m represents the dynamic monitoring results of xenograft tumor volume, and the statistical analysis was performed using two-way ANOVA with Šídák correction; n represents the final tumor weight statistics of xenograft tumor volume (n=5), and the data were analyzed using one-way ANOVA with Šídák correction; all results are expressed as mean ± standard deviation.

[0032] Figure 14 The results show that MLN4924 promotes the expression of PRB response genes in MPA-resistant organoids; Figures a and b show the mRNA expression levels of the indicated genes (3 samples per group) in ECO7 and ECO8 cells after treatment with MPA (20 μM, 24 h) and MLN4924 (0.1 μM, 8 h) alone or in combination, detected by qRT-PCR; one-way ANOVA combined with Šídák correction was used for statistical analysis; all results are expressed as mean ± standard deviation. Detailed Implementation

[0033] This invention provides the use of pevorinistat in combination with medroxyprogesterone acetate in the preparation of a medicament for treating endometrial cancer. The endometrial cancer is preferably progestin-resistant or insensitive endometrial cancer. Experiments of this invention confirm that AMBRA1-mediated PR ubiquitination depends on CRL4. AMBRA1 The complex, and MLN4924, can effectively reverse MPA resistance by restoring PR levels in vitro, in patient-derived organoids, and in xenograft tumor models. Although MLN4924 is not CRL4 AMBRA1 While specific inhibitors exist, the genetic evidence of this invention (including CUL4A and DDB1 knockdown and disruption of the AMBRA1-DDB1 binding domain) strongly suggests that CRL4 is the most likely candidate for a specific inhibitor. AMBRA1 This is its primary target. These findings suggest that combining NEDD8 pathway inhibition with progesterone therapy may broaden the therapeutic window for fertility-preserving treatment in endometrial cancer patients.

[0034] In this invention, the pevorinistat works by inhibiting CRL4. AMBRA1 The complex enhances the sensitivity of endometrial cancer to progesterone therapy. This invention was genetically and pharmacologically validated in MPA-resistant ISK_Res cells. Knockdown of the CRL4 core component CUL4A or DDB1 using small interfering RNA completely blocked progesterone receptor (PR) degradation induced by AMBRA1 overexpression, and MPA treatment reduced cell viability and increased apoptosis. Constructing an AMBRA1 mutant lacking the WD40 domain (ΔN-WD40) resulted in the loss of DDB1 binding ability; this mutant could not promote PR ubiquitination, and PR levels were not reduced. However, treatment with pevorinitol inhibited CUL4A ubiquitination, restoring PR protein levels in a dose-dependent manner. The above evidence suggests that pevorinitol inhibits CRL4... AMBRA1 The complex activity stabilizes PR, thereby reversing progesterone resistance.

[0035] In this invention, pevorisstat and medroxyprogesterone acetate are preferably administered simultaneously or sequentially.

[0036] This invention also provides a pharmaceutical composition comprising pevorinilstat, medroxyprogesterone acetate, and a pharmaceutically acceptable carrier. To meet different routes of administration and clinical needs, this composition can be formulated as an injection, a lyophilized powder for injection, or an oral formulation. All of the above formulations require stability studies and content uniformity testing to ensure the stability of the active ingredient within its shelf life. In clinical use, an appropriate dosage form can be selected according to the patient's condition to achieve combined administration of pevorinilstat and medroxyprogesterone acetate.

[0037] This invention also provides the application of AMBRA1 in the preparation of a product for assessing the sensitivity of endometrial cancer patients to progesterone therapy. The product is preferably an immunohistochemical detection kit. High expression of AMBRA1 indicates that the patient is insensitive to progesterone therapy or at risk of drug resistance; such patients should avoid progesterone therapy alone and instead prioritize combination therapy.

[0038] In this invention, the product preferably contains a reagent for detecting the expression level of the AMBRA1 protein. The reagent may be a monoclonal or polyclonal antibody against AMBRA1.

[0039] This invention also provides the application of pevorin in the preparation of a drug for enhancing the sensitivity of endometrial cancer cells to progesterone. To verify the effect of pevorin in enhancing the sensitivity of endometrial cancer cells to progesterone, this invention uses an MPA-resistant ISK_Res cell model. Results showed that the cell survival rate in the MPA monotherapy group without pevorin pretreatment was higher than that in the combination group pretreated with pevorin. Further Western blot analysis revealed that progesterone receptor (PR) protein expression was restored in drug-resistant cells after pevorin pretreatment. In patient-derived MPA-resistant organoids ECO7, pretreatment with 0.1 μmol / L pevorin followed by MPA treatment resulted in decreased cell viability compared to the MPA monotherapy group. These results indicate that pevorin can enhance the sensitivity of drug-resistant endometrial cancer cells to progesterone and can be used to prepare sensitizing drugs.

[0040] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0042] Example 1: High expression of AMBRA1 is positively correlated with MPA resistance in endometrial cancer. To identify key regulatory factors of progesterone resistance, this embodiment established paired MPA-resistant cell lines by subjecting MPA-sensitive endometrial cancer cells to long-term MPA treatment. Figure 1 (a) Progesterone binds to the ligand-binding domain of the major PRB subtypes, activating downstream targets and inhibiting endometrial cancer cells. This was demonstrated after confirming differences in the sensitivity of ISK parental cells (ISK_Parental) and ISK resistant cells (ISK_Res) to MPA. Figure 1In this embodiment, the progesterone-driven signaling pathway was examined, and it was found that some identified PRB response genes were significantly downregulated in ISK_Res cells. Figure 1 (f). Compared with ISK_Parental cells, the PRB protein level in ISK_Res cells was significantly reduced, while the PRB mRNA level remained essentially unchanged. Figure 1 Zhongg and Figure 1 (h). Based on this, it is speculated that other post-translational regulatory mechanisms may be involved in the downregulation of PR in MPA-resistant cells.

[0043] To identify post-translational regulators of progestin-releasing protease (PR) during progestin therapy, this study employed immunoprecipitation combined with 4D label-free quantitative proteomics analysis to analyze PR-interacting proteins in ISK_Parental and ISK_Res cells. The results showed that a group of interacting proteins specifically binding to PR existed in ISK_Res cells, with AMBRA1 showing an approximately 6.2-fold increase in enrichment. Figure 2 (a). Given the increasing evidence that CRLs are important targets for cancer therapy, this embodiment analyzed the transcriptional profiles of CRLs in published endometrial cancer datasets and found that AMBRA1 was the most differentially expressed gene in MPA-resistant endometrial cancer compared to MPA-sensitive endometrial cancer. Figure 2 (b). Subsequently, by integrating proteomics results and database analysis, this embodiment identified AMBRA1 as a high-confidence candidate target ( Figure 2 (c) AMBRA1, as a substrate recognition component of the CRL4 ubiquitin E3 ligase complex, can regulate the degradation of various proteins. Therefore, this embodiment hypothesizes that AMBRA1 may be involved in the regulation of PR stability and progesterone response in endometrial cancer. To explore its clinical relevance, this embodiment performed AMBRA1 immunohistochemical (IHC) detection on endometrial cancer specimens from individuals receiving progesterone therapy. Quantitative analysis showed that, compared with MPA-sensitive tumors, the staining intensity of AMBRA1 was significantly enhanced in MPA-resistant tumors. Figure 2 d and Figure 2 (e). Staining analysis of MPA-resistant specimens before and after treatment revealed that progesterone treatment significantly upregulated AMBRA1 expression. Figure 2 f and Figure 2 (g).

[0044] This embodiment further evaluated the role of AMBRA1 in endometrial cancer. Immunohistochemical and Western blot results showed that AMBRA1 expression was significantly increased in tumor tissue compared with normal endometrial tissue. Figure 3Furthermore, immunohistochemical analysis of the relationship between AMBRA1 and PR in endometrial cancer revealed a negative correlation between the two. Figure 3 d and Figure 3 The results suggest that AMBRA1 may regulate MPA sensitivity in endometrial cancer. Subsequently, this embodiment constructed eight endometrial cancer organoids (ECOs) using surgically removed endometrial cancer patient specimens. Figure 2 (h), hematoxylin and eosin (H&E) staining confirmed that the cultured organoids reproduced the histological features of the corresponding primary tumor. Figure 3 (f). The expression of AMBRA1 and PR in organoids was detected by immunohistochemistry, and their sensitivity to MPA was evaluated. Figure 3 g, Figure 2 Zhong i and Figure 2 In the study of eight organoids, the results showed that higher levels of endogenous AMBRA1 protein were associated with higher half-maximal inhibitory concentrations (IC50) of MPA, and the two were strongly positively correlated (r=0.8987, p=0.0024). Figure 2 k and Figure 2 In addition, organoids that are sensitive to MPA and resistant to MPA were compared. Figure 2 k and Figure 2 (middle l) and ISK_Parental cells and ISK_Res cells ( Figure 2 The study found that AMBRA1 expression was negatively correlated with PR levels. In summary, these data indicate that AMBRA1 is upregulated in MPA-resistant endometrial cancer and is negatively correlated with PR expression.

[0045] Example 2: IKKα stabilizes AMBRA1 via phosphorylation under MPA treatment. This embodiment examined the expression level of AMBRA1 in ISK_Parental cells and ISK_Res cells, and found that compared with a slight increase in mRNA levels ( Figure 4 In ISK_Res cells, the protein level of AMBRA1 was significantly increased (a). Figure 2 The change in protein stability suggests that the upregulation of AMBRA1 expression during MPA resistance may be the reason. Previous studies have reported that IKKα can stabilize AMBRA1, and immunoprecipitation-mass spectrometry (IP-MS) analysis of AMBRA1-interacting proteins in ISK_Res cells revealed an interaction between IKKα and AMBRA1. Figure 4 (b) Therefore, it is speculated that IKKα is involved in the upregulation of AMBRA1 expression in MPA-resistant cells. Endogenous reverse immunoprecipitation (Co-IP) experiments confirmed that AMBRA1 and IKKα interact in ISK cells, and that the protein levels of both increased after MPA treatment. Figure 4c and Figure 4 (d); Endogenous immunoprecipitation further confirmed the binding of AMBRA1 and IKKα in ISK_Res cells ( Figure 2 n, Figure 4 (e). Subsequently, this example investigated the effects of the IKKα-specific irreversible inhibitors BAY 11-7082 and MPA on AMBRA1 expression. The results were consistent with the above, showing that the pharmacological inhibition of IKKα by BAY 11-7082 was observed in ISK cells ( Figure 4 (f) and ISK_Res cells ( Figure 2 The expression of wild-type IKKα significantly blocked MPA-induced upregulation of AMBRA1. Furthermore, transient overexpression of wild-type IKKα in ISK cells increased AMBRA1 protein abundance, while overexpression of the kinase-inactivated IKKαK44M mutant had no such effect. Figure 2 (p). This example assesses the protein stability of AMBRA1 in ISK cells after treatment with the protein synthesis inhibitor cyclohexylimide (CHX). The results showed that, compared with the control group, IKKα overexpression prolonged the half-life of AMBRA1. Figure 4 g, Figure 2 (q). In summary, these data indicate that activation of IKKα kinase is a necessary condition for the upregulation of AMBRA1 in MPA resistance.

[0046] Previous studies have found that IKKα can phosphorylate AMBRA1 at the Ser1043 site to regulate mitophagy. Therefore, this study investigated whether phosphorylation at this site is involved in the regulation of AMBRA1 stability in endometrial cancer cells. Transient co-transfection of FLAG-IKKα with histidine-tagged wild-type AMBRA1 (AMBRA1WT) or the phosphorylation-deficient AMBRA1 S1043A mutant into ISK cells showed that IKKα overexpression increased the abundance of wild-type AMBRA1, but had no effect on the S1043A mutant. Figure 4 (h). Consistency results showed that the protein half-life of the AMBRA1S1043A mutant was shorter than that of wild-type AMBRA1, while the kinase-inactivated IKKαK44M mutant had no effect on wild-type AMBRA1. Figure 4 Zhong i and Figure 4 (j). These data indicate that IKKα-mediated phosphorylation at Ser1043 of AMBRA1 under MPA treatment can stabilize AMBRA1.

[0047] Example 3: AMBRA1 promotes MPA resistance in endometrial cancer To investigate the role of AMBRA1 in regulating MPA response, this embodiment first constructed an AMBRA1 overexpression model in the MPA-sensitive endometrial cancer cell line ISK. Figure 5 a and Figure 5 (b) The results showed that AMBRA1 overexpression significantly attenuated the growth inhibition induced by MPA, as evidenced by enhanced cell proliferation and colony formation compared to the MPA-treated control group. Figure 5 (ce), while apoptosis is reduced ( Figure 5 f and Figure 5 (g). Subsequently, AMBRA1 was overexpressed in MPA-sensitive organoid ECO1. Figure 6 As expected, AMBRA1 overexpression reduced the MPA-induced inhibition of cell viability in ECO1 cells. Figure 6 (e). This embodiment further constructed ISK cell-derived xenograft models with AMBRA1 overexpression and non-overexpression, and subjected them to MPA treatment ( Figure 5 h), the results showed that, compared with the control group, AMBRA1 overexpression significantly reduced the sensitivity of ISK xenografts to MPA treatment, manifested as tumor growth restriction ( Figure 5 In the middle i), the tumor volume and weight decreased ( Figure 5 j and Figure 5 (middle k).

[0048] To further verify that AMBRA1 is an effective target for overcoming MPA resistance in endometrial cancer, this embodiment utilizes CRISPR-Cas9 technology to construct AMBRA1 knockout (KO) ISK_Res cells (… Figure 6 (f). The results showed that AMBRA1 knockout significantly increased the sensitivity of ISK_Res cells to MPA, as evidenced by decreased cell viability and colony-forming ability after MPA treatment. Figure 6 In the middle gi), compared with the control group, MPA-induced apoptosis was significantly increased ( Figure 6 j and Figure 6 (k). Finally, in this example, AMBRA1 knockout and control ISK_Res cells were subcutaneously seeded into the lateral ventral region of nude mice and treated with MPA (k). Figure 6 The results were consistent with in vitro experiments, showing that AMBRA1 knockout significantly inhibited tumor growth, reduced tumor volume and weight, and improved the in vivo efficacy of MPA in the MPA resistance model. Figure 6 (m). In summary, these results confirm that AMBRA1 can promote MPA resistance in endometrial cancer.

[0049] Example 4: AMBRA1 interacts with PR and attenuates the progesterone signaling pathway Previous studies have reported that AMBRA1 can promote autophagy; however, this study, using the specific autophagy activator rapamycin and the autophagy inhibitor chloroquine (CQ), found that AMBRA1 overexpression did not cause a reproducible change in autophagic flux in ISK cells, regardless of MPA treatment. Figure 7 a and Figure 7 (b) This embodiment investigates the mechanism of AMBRA1-mediated MPA resistance using a luciferase reporter gene assay. RANKL is a direct downstream target of PR and is inhibited by MPA treatment. The results show that, compared with the control group, ISK cells overexpressing AMBRA1 exhibit significantly increased luciferase activity driven by the RANKL promoter after MPA treatment. Figure 7 (c); Consistency results showed that in ISK cells overexpressing AMBRA1, MPA could not effectively inhibit RANKL protein levels ( Figure 7 (d). Real-time quantitative PCR further confirmed that AMBRA1 overexpression significantly reduced MPA-induced upregulation of PRB response genes ( Figure 7 China and Figure 7 (f). In summary, these data indicate that AMBRA1 can significantly attenuate the progesterone signaling pathway in endometrial cancer cells.

[0050] To investigate the mechanism by which increased AMBRA1 expression weakens the progesterone signaling pathway, this example first verified the interaction between AMBRA1 and PR suggested by proteomics analysis. Figure 2 (a) In HEK293T cells ectopically expressing FLAG-PR and HA-AMBRA1, exogenous immunoprecipitation experiments were performed, and the binding of AMBRA1 to PR was discovered and confirmed for the first time. Figure 8 (a) Reverse immunoprecipitation further verified this interaction ( Figure 8 (b); Endogenous immunoprecipitation assays confirmed that endogenous AMBRA1 and PR interact in ISK cells and ISK_Res cells ( Figure 8 c and Figure 8 (d). To further elucidate the structural determinants mediating AMBRA1-PR binding, this embodiment constructed a series of truncated mutants of the two proteins. Immunoprecipitation analysis using the PR deletion mutants revealed that the IF domain of PR is essential for its interaction with AMBRA1. Figure 8 China and Figure 8 (f), reverse immunoprecipitation analysis verified this result ( Figure 8(g). Localization experiments on AMBRA1 revealed that the binding affinity of its N-terminal WD40 domain (amino acids 1-199) and C-terminal region (F2, F3a, F3b; amino acids 533-1298) to PR is comparable to that of the full-length AMBRA1, while the amino acid fragment from 200-532 (F1ΔN-WD40) cannot interact with PR. Figure 8 h and Figure 8 (i), reverse immunoprecipitation confirmed the above results ( Figure 8 (j). In summary, these data suggest that AMBRA1 can interact with PR and attenuate the progesterone signaling pathway in endometrial cancer cells.

[0051] Example 5: AMBRA1 downregulates PR protein levels in endometrial cancer cells via ubiquitination-mediated degradation. AMBRA1 as CRL4 AMBRA1 The substrate recognition subunit of the ubiquitin ligase is hypothesized in this embodiment to bind to PR and degrade PR via the ubiquitin-proteasome pathway, thereby downregulating its protein level. To detect the relationship between AMBRA1 and PR expression, immunohistochemical staining was first performed on ECO1 organoids overexpressing AMBRA1. Quantitative analysis revealed that, compared with control organoids, PR staining was significantly weakened in AMBRA1-overexpressing organoids. Figure 9 a and Figure 9 (b). Consistency results showed that stable overexpression of AMBRA1 significantly reduced the protein level of PR in ISK cells ( Figure 9 (c), while PR protein levels are elevated in ISK_Res cells with AMBRA1 knockout ( Figure 9 (d). Neither AMBRA1 overexpression nor knockout altered the mRNA levels of PR in endometrial cancer cells and organoids. Figure 7 The data (g) suggests that AMBRA1 regulates PR at the posttranscriptional level.

[0052] To clarify whether this regulation depends on the ubiquitin-proteasome system, this study ectopically expressed AMBRA1 in ISK cells and treated them with either the proteasome inhibitor MG132 or the lysosomal inhibitor CQ. The results showed that AMBRA1 overexpression reduced the protein level of endogenous proteasome protein (PR), while MG132 (but not CQ) restored PR expression and significantly reversed the AMBRA1-induced decrease in PR levels. Figure 9 (e); Consistency results showed that MG132 (but not CQ) increased the protein level of PR in ISK_Res cells ( Figure 9 (f). Subsequently, this example assessed the stability of PR by detecting its degradation after CHX treatment, and found that compared with the control group, the half-life of PR in ISK cells overexpressing AMBRA1 was significantly shortened (f). Figure 9Zhongg and Figure 9 (h), suggesting that AMBRA1 promotes PR instability through proteasome degradation.

[0053] This study investigated whether AMBRA1 promotes PR ubiquitination. Histidine-tagged AMBRA1, HA-ubiquitin (HA-Ub), and wild-type (WT) or ΔIF mutant FLAG-PR (with impaired AMBRA1 binding) were co-expressed in HEK293T cells. The results showed that AMBRA1 significantly enhanced ubiquitination of wild-type PR. Figure 9 In contrast, the ΔIF mutant showed only a slight increase in ubiquitination after AMBRA1 expression, consistent with the reduced AMBRA1 binding ability. Figure 9 i. Figure 8 (f, g). To determine the type of ubiquitin linkage involved, in this embodiment, the AMBRA1 expression plasmid was co-transfected with wild-type ubiquitin with the HA tag or ubiquitin mutants with single lysine mutations (K6R, K11R, K27R, K29R, K33R, K48R, K63R). Analysis revealed that AMBRA1 mainly mediates K48-linked polyubiquitination of PR (f, g). Figure 10 a, Figure 9 (j). In summary, these results confirm that AMBRA1 promotes K48-linked polyubiquitination and proteasome degradation of PR in endometrial cancer cells.

[0054] To identify the lysine binding site of PR in AMBRA1-mediated ubiquitination, this study constructed 34 PR mutants, mutating lysine residues individually or in combination to arginine, covering all 41 lysine residues of PR. Ubiquitination of these PR variants mediated by AMBRA1 was then detected in HEK293T cells. In vivo ubiquitination experiments revealed that only the double mutant of PR, K386R+K388R (lysines at positions 386 and 388 mutated to arginine), could not be effectively ubiquitinated in the presence of AMBRA1. Figure 10 The two lysine residues (bd) suggest that they are potential sites for AMBRA1-mediated PR ubiquitination. To further identify the main sites, this example mutated lysine at positions 386 or 388 to arginine and performed in vivo ubiquitination experiments. The results showed that exogenous overexpression of AMBRA1 in HEK293T cells increased ubiquitination in wild-type FLAG-PR, and a similar increase in ubiquitination was observed in the K386R mutant of FLAG-PR, while ubiquitination in the K388R mutant was almost completely inhibited. Figure 10 e, Figure 9(k), confirming that lysine 388 of PR is the main ubiquitination site required for AMBRA1-mediated ubiquitination. Furthermore, HA-AMBRA1 overexpression significantly reduced wild-type PR levels, but had no effect on the K388R mutant. Figure 9 (l); and the K388R mutant of the PR protein has a longer half-life than the wild-type PR ( Figure 9 m and Figure 9 (n).

[0055] In summary, these findings indicate that AMBRA1 mediates K48-linked ubiquitination of PR, with lysine at position 388 being the main ubiquitination site for AMBRA1 to target and regulate the MPA response in endometrial cancer cells.

[0056] Example 6 CRL4 AMBRA1 The complex is essential for AMBRA1-mediated PR ubiquitination. AMBRA1 belongs to the DDB1-CUL4-associated factor (DCAF) family and can specify the substrate of the CRL4 ubiquitin ligase complex. Simultaneously, AMBRA1 also acts as an important cofactor of the RING-E3 ubiquitin ligase TRAF6, regulating the ubiquitination of its substrate. To elucidate the mechanism of AMBRA1-mediated PR ubiquitination, this example identified AMBRA1-interacting proteins in endometrial cancer cells using IP-MS. The results showed that the core components of CRL4, CUL4A and DDB1, bind to AMBRA1, while TRAF6 does not. Figure 11 (a) Based on this, it can be inferred that CRL4 AMBRA1 The ligase activity of the complex is essential for regulating PR protein levels. This example first confirmed the interaction between AMBRA1 and the two core subunits CUL4A and DDB1 of the CRL4 ubiquitin ligase using endogenous immunoprecipitation in ISK_Res cells. Figure 11 (b) Previous studies have reported that the N-terminal region containing the WD40 domain mediates the binding of DDB1 and also affects CRL4. AMBRA1 Assembly of the ubiquitin ligase complex is crucial; therefore, this embodiment constructed an N-terminal deletion AMBRA1 mutant (ΔN-WD40, amino acids 1-179). Subsequently, heterologous wild-type or ΔN-WD40 AMBRA1 was subjected to semi-endogenous immunoprecipitation with endogenous DDB1 in ISK cells. The results showed that, unlike wild-type AMBRA1, the ΔN-WD40 mutant lost its ability to bind to DDB1. Figure 11 (c), and cannot increase PR ubiquitination ( Figure 11 (d). Consistency results showed that ectopic expression of the ΔN-WD40 mutant had no effect on PR protein abundance, while full-length AMBRA1 significantly reduced PR levels in ISK cells. Figure 11(e) suggests that AMBRA1-mediated PR expression regulation depends on CRL4. AMBRA1 ubiquitin ligase complex.

[0057] Furthermore, knocking down endogenous DDB1 or CUL4A using siRNA significantly reduced AMBRA1-driven PR ubiquitination in 293T cells. Figure 11 In ISK cells stably overexpressing AMBRA1, silencing CUL4A or DDB1 significantly blocked the AMBRA1-induced decrease in PR protein levels (f); Figure 11 (g); Consistency results showed that silencing CUL4A or DDB1 in ISK_Res cells significantly increased PR protein abundance (g); Figure 11 (h). MLN4924 is a specific inhibitor of NEDD8 activator, which is essential for CRL activity. Therefore, this example investigated whether pharmacological inhibition of CRL4 regulates AMBRA1-mediated PR expression and MPA response. After confirming that MLN4924 effectively inhibits NAE1 expression and reduces CUL4A ubiquitination, this example found that MLN4924 treatment reversed the AMBRA1-induced decrease in PR protein levels in ISK cells (h). Figure 11 (i), and increased the protein level of PR in ISK cells that stably overexpress AMBRA1 (i). Figure 11 (j). In summary, these findings indicate that AMBRA1 primarily functions as a CRL4 receptor antagonist. AMBRA1 The substrate receptor of the ubiquitin ligase complex is used to downregulate PR.

[0058] Example 7: In vitro experiment on reversing MPA resistance by pevorinistat combined with medroxyprogesterone acetate Because AMBRA1 overexpression can promote CRL4 AMBRA1 PR-dependent degradation confers MPA resistance in endometrial cancer cells; this embodiment hypothesizes that inhibition of CRL4... AMBRA1 The complex can restore MPA sensitivity. Therefore, in this embodiment, siRNAs were used to knock down CUL4A or DDB1 in ISK cells overexpressing AMBRA1, and the knockdown efficiency was confirmed by immunoblotting. Figure 11 The results showed that knockdown of CUL4A and DDB1 significantly reversed MPA resistance induced by AMBRA1 overexpression, as evidenced by decreased cell viability in cell viability assays after MPA treatment. Figure 12 (a) Flow cytometry analysis showed an increased proportion of apoptotic cells. Figure 12 b and Figure 12(c). Consistency results showed that the NEDD8 activator enzyme inhibitor MLN4924 significantly restored the PR protein level in ISK cells that was reduced by AMBRA1 overexpression. Figure 12 (j), suggesting that MLN4924 is a potential therapeutic agent for inhibiting CRL4 activity in MPA-resistant endometrial cancer. Subsequently, this example examined whether MLN4924 could restore the sensitivity of an AMBRA1-overexpressing endometrial cancer model to MPA. It was found that MLN4924 significantly improved AMBRA1-induced MPA resistance in patient-derived organoids (ECO1), manifested as decreased cell viability (j). Figure 12 (d); A similar effect was also observed in ISK cells overexpressing AMBRA1, manifested as decreased cell viability after MLN4924 treatment ( Figure 12 (e) Decreased settlement formation capacity ( Figure 12 f and Figure 12 (g) and increased apoptosis (g) Figure 12 h and Figure 12 (i). Consistent with in vitro results, MLN4924 effectively eliminated AMBRA1-induced MPA resistance in ISK xenografts overexpressing AMBRA1 in vivo. Figure 12 (j), manifested as inhibited tumor growth ( Figure 12 (k), tumor volume and weight decreased ( Figure 12 China and Figure 12 (m). In summary, these results confirm that CRL4 AMBRA1 The complex is a key mediator of MPA resistance in endometrial cancer and targets CRL4. AMBRA1 Complexes may be an effective strategy to overcome MPA resistance.

[0059] Example 8: Synergistic effect of pevorinistat combined with medroxyprogesterone acetate in organoids and in vivo models This study investigated the synergistic antitumor effect of MLN4924 and MPA in an MPA-resistant endometrial cancer model. Patient-derived ECO7 and ECO8 organoids were treated with MPA alone or in combination with MLN4924. The results showed a significant synergistic effect between MPA and MLN4924, with synergistic scores of 11.44 and 13.95, respectively. Figure 13 a and Figure 13 (b) Based on this synergistic effect, this embodiment selected the lowest effective concentration of MLN4924 and MPA for subsequent combined experiments. The results showed that the combination with MLN4924 significantly enhanced the sensitivity of ECO7 and ECO8 organoids to MPA, manifested as a significant decrease in cell viability ( Figure 13 c and Figure 13(d); In addition, MLN4924 can significantly enhance the ability of MPA to induce the expression of PRB response genes ( Figure 14 a and Figure 14 (b). Consistency results showed that a similar combined effect was also observed in MPA-resistant ISK_Res cells, with PR expression recovering after MPA and MLN4924 co-treatment. Figure 13 (e) Cell viability was significantly reduced ( Figure 13 (f) Decreased settlement formation capacity ( Figure 13 (g, h) and increased apoptosis ( Figure 13 Zhong i and Figure 13 (j). This embodiment evaluated the therapeutic effect of the combination regimen in vivo. Figure 13 (k) The results showed that, despite MPA treatment, tumors in the control group continued to proliferate rapidly, while the therapeutic effect of MLN4924 monotherapy was limited. However, the combination of MPA and MLN4924 significantly restored the tumor's sensitivity to MPA, leading to a significant inhibition of tumor growth. Figure 13 (ln). In summary, these results indicate that for CRL4 AMBRA1 Pharmacological inhibition of the complex can enhance the sensitivity of endometrial cancer cells to progesterone therapy.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of pevorinistat combined with medroxyprogesterone acetate in the preparation of drugs for the treatment of endometrial cancer.

2. The application according to claim 1, characterized in that, The endometrial cancer mentioned is endometrial cancer that is resistant to or insensitive to progesterone.

3. The application according to claim 2, characterized in that, The pevorinistat works by inhibiting CRL4. AMBRA1 The complex enhances the sensitivity of endometrial cancer to progesterone therapy.

4. The application according to claim 1, characterized in that, The pevorinistat and medroxyprogesterone acetate were administered simultaneously or sequentially.

5. A pharmaceutical composition, characterized in that, It contains pevorin, medroxyprogesterone acetate, and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition is an injection, a lyophilized powder injection, or an oral preparation.

7. Application of AMBRA1 in the preparation of products for assessing the sensitivity of endometrial cancer patients to progestin therapy.

8. The application according to claim 7, characterized in that, High expression of AMBRA1 suggests that endometrial cancer patients are insensitive to progesterone therapy or have a risk of drug resistance.

9. The application according to claim 8, characterized in that, The product contains reagents for detecting the expression level of AMBRA1 protein.

10. The use of pervorinesta in the preparation of drugs for enhancing the sensitivity of endometrial cancer cells to progesterone.

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