Use of compounds that inhibit nuclear translocation of hibch in the manufacture of a medicament for reversing resistance to pi3ka inhibitors in breast cancer

Abstract

The application discloses application of a compound for inhibiting HIBCH nuclear translocation in preparation of a medicine for reversing resistance of a breast cancer PI3K alpha inhibitor, and relates to the technical field of biological medicines.It is found by the application that valine metabolism is disordered in a breast cancer cell and an animal model resistant to a PI3K alpha inhibitor, and the valine metabolism disorder is caused by translocation of a key enzyme HIBCH of a valine degradation pathway originally positioned in mitochondria to a cell nucleus.Targeting HIBCH and combining the PI3K alpha inhibitor have a synergistic lethal effect on breast cancer, can significantly enhance sensitivity of tumor cells to the PI3K alpha inhibitor, and the treatment effect is not affected by whether PIK3CA itself is mutated or not.The treatment strategy of targeting HIBCH and combining the PI3K alpha inhibitor not only enhances the treatment effect of the PI3K alpha inhibitor, but also expands an adaptability population of the PI3K alpha inhibitor in breast cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of compounds that inhibit HIBCH nuclear translocation in the preparation of drugs that reverse resistance to PI3Kα inhibitors in breast cancer. Background Technology

[0002] The mutation rate of PIK3CA in breast cancer exceeds 30%, making it one of the most significant driver genes in the disease. Overactivity of the PI3K / AKT / mTOR signaling pathway, resulting from PIK3CA mutations or abnormally high expression of its encoded PI3K protein, drives tumor metabolic reprogramming, particularly the Warburg effect, and participates in the regulation of malignant progression such as drug resistance, recurrence, and metastasis. While significant progress has been made in the molecular mechanisms by which PI3Kα regulates breast cancer metabolism and in preclinical research, the development of PI3Kα inhibitors was once considered a major milestone in breast cancer treatment. However, in subsequent large-scale clinical studies, only the SOLAR-II trial demonstrated that PI3Kα inhibitors significantly improved progression-free survival in patients with advanced breast cancer harboring PIK3CA mutations, while the benefit was limited in patients without PIK3CA mutations, and treatment side effects were significant. Therefore, improving the therapeutic efficacy of PI3Kα inhibitors, expanding their application scope, and simultaneously overcoming their side effects are the main challenges facing the clinical application of PI3Kα inhibitors.

[0003] A review of clinical studies on the failure of PI3Kα inhibitors in breast cancer treatment revealed that PI3K reactivation, abnormal activation of parallel pathways, and the tumor microenvironment can all lead to tumor insensitivity to PI3Kα inhibitors. PI3K reactivation is mainly due to acquired amplification of PIK3CA mutation sites and significant upregulation of PI3Kα activity by activating mutations in PIK3CB, leading to abnormal activation of the AKT signaling pathway, resulting in insensitivity to PI3Kα inhibitors and accelerating tumor cell proliferation and metastasis. Studies on parallel pathway activation mainly focus on the RAS-RAF-MEK-ERK signaling pathway and the PI3K-AKT alternative pathway. Mutations and amplification of HRAS in the RAS family can significantly reduce the sensitivity of tumor cells to PI3Kα inhibitors. Simultaneously, the activation of the RAS protein it encodes and its downstream signaling pathways can enhance the invasiveness of breast cancer, promote malignant transformation, and is one of the key determinants of metastasis and poor survival in breast cancer patients. Meanwhile, suppressive immune cells originating from the tumor microenvironment can activate the NF-κB signaling pathway in tumor cells by secreting cytokines, leading to tumor cell insensitivity to PI3Kα inhibitors and promoting tumor progression.

[0004] In summary, this invention reveals that the molecular mechanisms leading to PI3Kα inhibitor insensitivity are often also the molecular mechanisms driving malignant progression in breast cancer. Therefore, elucidating the mechanisms of PI3Kα inhibitor insensitivity not only helps to maximize the therapeutic effect of PI3Kα inhibitors on breast cancer, but also helps to elucidate the driving mechanisms of breast cancer recurrence and metastasis, and explore new targets and strategies for breast cancer treatment.

[0005] PI3K primarily promotes breast cancer development by driving tumor glucose metabolism. Blocking the PI3K signaling pathway with inhibitors can impair tumor cells' access to nutrients, effectively "starving" them. In tumor cells insensitive to PI3Kα inhibitors, inhibition of PI3K allows for metabolic reprogramming to maintain an appropriate metabolic state and survival. This metabolic alternative pathway may be a key mechanism behind PI3Kα inhibitor resistance. Recent studies have shown that active glutamine production in mitochondria leads to reprogramming of the tricarboxylic acid cycle, which, as a metabolic alternative, participates in the regulation of PI3Kα inhibitor resistance. However, research on reversing PI3Kα inhibitor resistance by targeting branched-chain amino acid metabolism remains rare. Summary of the Invention

[0006] The purpose of this invention is to provide the application of compounds that inhibit HIBCH nuclear translocation in the preparation of drugs to reverse PI3Kα inhibitor resistance in breast cancer, thereby addressing the problems existing in the prior art. This invention has found that targeting HIBCH in combination with PI3Kα inhibitors has a synergistic lethal effect on breast cancer, significantly enhancing sensitivity to PI3Kα inhibitors. This treatment strategy of targeting HIBCH in combination with PI3Kα inhibitors not only enhances the therapeutic effect of PI3Kα inhibitors but also expands the target population for PI3Kα inhibitors in breast cancer treatment.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides the use of compounds that inhibit HIBCH nuclear translocation in the preparation of drugs that reverse resistance to PI3Kα inhibitors in breast cancer.

[0009] Furthermore, the compound is C7(C 31 H 30 N4O6), its structural formula is as follows:

[0010]

[0011] This invention also provides a drug for reversing resistance to PI3Kα inhibitors in breast cancer, the active ingredient of which includes a compound that inhibits HIBCH nuclear translocation.

[0012] Furthermore, the compound is C7(C 31 H 30 N4O6), its structural formula is as follows:

[0013]

[0014] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0015] The present invention also provides a composition for treating breast cancer, comprising a PI3Kα inhibitor and a compound that inhibits HIBCH nuclear translocation.

[0016] Furthermore, the compound is C7(C 31 H 30 N4O6), its structural formula is as follows:

[0017]

[0018] The present invention also provides the use of the above-described composition in the preparation of a medicament for treating breast cancer.

[0019] The present invention also provides a medicament for treating breast cancer, wherein the active ingredient comprises the above-described composition.

[0020] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0021] The present invention discloses the following technical effects:

[0022] This invention reveals that valine metabolism is disordered in PI3Kα inhibitor-resistant breast cancer cells and animals. This disorder is attributed to the nuclear translocation of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH), a key enzyme in the valine degradation pathway originally located in mitochondria. Further drug screening identified compound C7, which effectively inhibits HIBCH nuclear translocation. SPR and cell function assays confirmed that C7 has a strong affinity for HIBCH, reducing its binding to c-Myc by inhibiting HIBCH nuclear translocation, thereby downregulating LAT1 expression and thus producing a synergistic lethal effect with BYL719 on PI3Kα-resistant breast cancer cells. Organ models also demonstrated that C7 and BYL719 have a synergistic lethal effect in PI3Kα inhibitor-insensitive breast cancer, regardless of the presence of PIK3CA mutations. Simultaneously, an animal model of xenograft tumors derived from tissues of breast cancer patients resistant to PI3Kα inhibitors and without PIK3CA mutations was constructed. Treatment experiments revealed that compared to BYL719 monotherapy or C7 monotherapy, the combination of BYL719 and C7 exerted a synergistic lethal effect on tumors, thereby reversing the original resistance to PI3Kα inhibitors and significantly inhibiting tumor growth. This demonstrates that targeting HIBCH in combination with PI3Kα inhibitors has a synergistic lethal effect on breast cancer, significantly enhancing sensitivity to PI3Kα inhibitors, and the treatment efficacy is unaffected by the presence or absence of PIK3CA mutations. The treatment strategy of targeting HIBCH in combination with PI3Kα inhibitors not only enhances the therapeutic effect of PI3Kα inhibitors but also expands the target population for PI3Kα inhibitors in breast cancer treatment. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1The diagram shows the results of an experiment on HIBCH-mediated valine metabolic remodeling and its role in resistance to PI3Kα inhibitors in breast cancer. A is a CRISPR-Cas9 Screen diagram; B is the tumor growth curve of nude mice after fat pad tumor formation, under BYL719 and placebo treatments, respectively; C is the CRISPR-Cas9... Screen shows the negative selection gene sequencing; D shows the functional enrichment analysis of negative selection genes; E shows the cell viability statistics of the MDA-MB-231 resistant cell line; F shows the cell viability statistics of the MCF7 resistant cell line; G shows the results of mass spectrometry analysis of small molecule metabolites extracted from the MDA-MB-231 parent and the resistant cell line, representing the changes in each metabolic process after resistance in the logarithmic form of the fold difference; H shows the results of mass spectrometry analysis of small molecule metabolites extracted from the MCF7 parent and the resistant cell line, representing the changes in each metabolic process after resistance in the logarithmic form of the fold difference; I shows the results of the experiment on the effect of branched-chain amino acid deprivation on the sensitivity of 231R to BYL719; J shows the effect of branched-chain amino acid deprivation on M... The results of the sensitivity assay for CF7R cells to BYL719 are shown in the figure. K represents the effect of HIBCH on the proliferation of the breast cancer 231 cell line; L represents the HIBCH knockdown efficiency of the MCF7 cell line; M represents the sensitivity test results of 231R cells to PI3Kα inhibitors under HIBCH knockdown conditions; N represents the sensitivity test results of MCF7R cells to PI3Kα inhibitors under HIBCH knockdown conditions; O represents the sensitivity test results of 231R cells to PI3Kα inhibitors after HIBCH knockdown correction with additional branched-chain amino acids; P represents the sensitivity test results of MCF-7R cells to PI3Kα inhibitors after HIBCH knockdown correction with additional branched-chain amino acids.

[0025] Figure 2The results of the experiment on HIBCH nuclear translocation and branched-chain amino acid metabolic remodeling mediated by LAT1 are shown. A and B represent the expression levels of HIBCH in PI3Kα-resistant 231R and MFC-7R cells, respectively; C and D represent the results of immunofluorescence detection of HIBCH localization in 231R and MFC-7R cells; E and F represent the localization of HIBCH in MDA-MB-231 and MFC-7 cells under normal culture and valine-deprived culture, respectively, using super-resolution confocal microscopy; G represents the results of detecting HIBCH-regulated gene transcription in the cell nucleus under valine-deprived conditions; H represents the top four transcription factors and their domains that bind to HIBCH under valine-deprived conditions; I represents the results of immunoprecipitation detection of the degree of HIBCH binding to c-Myc under valine-deprived conditions; J represents the results of changes in the degree of c-Myc regulation of gene transcription under valine-deprived conditions; K represents the results of comparison between normal culture and... The results show the changes in the metabolic pathways of c-Myc-regulated genes under valine-deprived culture conditions; L represents the expression level of LAT1 after HIBCH knockdown; M represents the expression level of LAT1 under valine-deprived culture conditions; N and O represent the results of detecting changes in cell sensitivity to BYL719 after HIBCH knockdown and simultaneous LAT1 overexpression in 231R and MCF-7R, respectively; P is a heatmap showing changes in intracellular branched-chain amino acid and TCA pathway metabolite content after HIBCH knockdown and simultaneous LAT1 overexpression, detected by targeted metabolomics; Q represents the results of QPCR detection of LAT1 and c-Myc expression levels after c-Myc knockdown; R represents the results of dual-luciferase reporter system detection of changes in LAT1 transcriptional activity after c-Myc knockdown; S and T represent the results of detecting changes in cell sensitivity to BYL719 after c-Myc knockdown and simultaneous LAT1 overexpression in 231R and MCF-7R, respectively.

[0026] Figure 3 The figures show the results of the experiment on the effect of HIBCH intervention on the sensitivity of BYL719 in animal models; where A is a display of MDA-MB-231 xenografts; B is the growth curve of MDA-MB-231 xenografts; C is a statistical chart of tumor weight of MDA-MB-231 xenografts; D is a display of MCF-7 xenografts; E is the growth curve of MCF-7 xenografts; and F is a statistical chart of tumor weight of MCF-7 xenografts.

[0027] Figure 4 The HIBCH protein was purified using Flag antibody after the nucleo-cytoplasmic separation experiment under valine-deprived conditions, and the result was stained with Coomassie Brilliant Blue after electrophoresis.

[0028] Figure 5 This is a graph showing the succinylation modification of the lysine residue at position 55 of HIBCH as detected by mass spectrometry.

[0029] Figure 6 This is a graph showing the succinylation modification of the lysine residue at position 17 of HIBCH as detected by mass spectrometry.

[0030] Figure 7 The figures show the results of experiments investigating the key mechanisms of HIBCH nuclear translocation. A shows the results of nuclear-cytoplasmic separation after valine-deprived culture, detecting the expression level of mutant HIBCH in the nucleus; B shows the results of nuclear-cytoplasmic separation experiments after valine deprivation based on K55 site mutation, detecting the succinylation level of HIBCH in the nucleus after mutation; C shows the results of immunoprecipitation experiments after K55 site mutation, showing the degree of binding between mutant HIBCH and c-Myc; D shows the results of ChIP-PCR detection of changes in the binding ability of HIBCH and c-Myc to LAT1 after K55 site mutation; E shows the results of HIBCH... The results of LAT1 expression level detection after K55 mutation; F shows the results of nuclear-cytoplasmic separation assay to detect HIBCH cell localization and succinylation after CPT1A and KAT2A knockdown; G shows the results of HIBCH and c-Myc binding to the LAT1 promoter region after CPT1A knockdown; H shows the results of qPCR detection of LAT1 expression level after CPT1A knockdown; I shows the results of dual-luciferase reporter system detection of LAT1 transcriptional activity after CPT1A knockdown; J shows the results of cell viability assay to detect the sensitivity of cells to BYL719 after CPT1A knockdown and simultaneous LAT1 overexpression.

[0031] Figure 8 The results of the experiment detecting the synergistic lethal effect of the small molecule inhibitor C7 targeting HIBCH nuclear translocation and the PI3Kα inhibitor are shown in Figure 1. A is a schematic diagram of the screening process for small molecule inhibitors targeting HIBCH nuclear translocation; B is the result of immunofluorescence showing that C7 exhibits dose-dependent inhibition of HIBCH nuclear translocation; C is a molecular docking diagram showing C7 and lysine residue 55 of the HIBCH protein; D is the result of SPR showing the affinity between C7 and HIBCH; E is the effect of C7 combined with BYL719 reducing the IC50 of breast cancer cells. 50 Figure F shows the results of a synergistic lethal effect between C7 and BYL719 in a PIK3CA-mutated human breast cancer organoid model; Figure G shows the results of a PIK3CA-amplified human breast cancer organoid model showing the synergistic lethal effect between C7 and BYL719; Figures I and II show the results of a synergistic lethal effect between C7 and BYL719 using human xenograft tumor models PDX-1359 and PDX-0595, respectively. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] The molecular formula of the small molecule compound C7 in this invention is C 31 H 30 N4O6, structural formula as follows:

[0038]

[0039] This invention reveals that valine metabolism is disordered in PI3Kα inhibitor-resistant breast cancer cells and animals. This disorder is caused by the translocation of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH), a key enzyme in the valine degradation pathway originally located in mitochondria, to the nucleus. Knockdown of HIBCH significantly increases the sensitivity of drug-resistant breast cancer cells to the PI3Kα inhibitor (BYL719), exhibiting a co-lethal effect, thereby reversing resistance to BYL719 and inhibiting tumor cell growth. Furthermore, this invention successfully constructed organoid models of breast cancer derived from patient tissues that are sensitive to and insensitive to apelixir (BYL719) treatment. It was found that knockdown of HIBCH combined with BYL719 induces co-lethality in the organoids, significantly inhibiting their growth and reversing resistance to BYL719. Mechanistic studies revealed that during the development of resistance to BYL719 in breast cancer cells, the abundance of methylmalonyl-CoA, a key metabolite in the valine metabolic pathway, significantly decreased, releasing the catalytic activity of succinylated transferase CPT1A. This led to succinylation of the lysine residue at position 55 of the HIBCH protein, resulting in HIBCH translocation into the nucleus. Once inside the nucleus, HIBCH interacts with the transcription factor c-Myc, altering c-Myc's transcriptional regulatory preference from regulating key enzymes related to carbohydrate metabolism to regulating amino acid metabolism, particularly the transcriptional activity of proteins related to branched-chain amino acid uptake and transport. Using a drug library containing 1.5 million compounds, molecular docking based on the HIBCH K55 protein structure was performed using a high-energy computer system combined with artificial intelligence, targeting the succinylated domain of the HIBCH protein. This screened 12 drugs (C1-C12) targeting the HIBCH K55 site. Further analysis of the effects of these 12 small molecule drugs on HIBCH nuclear translocation identified small molecule compound 7 (C7:C12). 31 H 30N4O6 effectively inhibits the nuclear translocation of HIBCH. Furthermore, SPR and cell function studies confirmed that C7 has a strong affinity for HIBCH, reducing its binding to c-Myc by inhibiting HIBCH nuclear translocation, thereby downregulating LAT1 expression levels. This results in a synergistic lethal effect with BYL719 on drug-resistant breast cancer cells. Organ models also demonstrated a synergistic lethal effect of C7 and BYL719 in PI3Kα inhibitor-insensitive breast cancer, regardless of the presence of PIK3CA mutations. Simultaneously, an animal model of xenograft tumors derived from tissues of PI3Kα inhibitor-resistant breast cancer patients without PIK3CA mutations was constructed. Treatment experiments showed that compared to BYL719 monotherapy or C7 monotherapy, the combination of BYL719 and C7 exerted a synergistic lethal effect on tumors, reversing the original resistance to PI3Kα inhibitors and significantly inhibiting tumor growth. Therefore, targeting HIBCH in combination with PI3Kα inhibitors has a synergistic lethal effect on breast cancer, significantly enhancing sensitivity to PI3Kα inhibitors, and the treatment efficacy is unaffected by whether PIK3CA itself is mutated. This HIBCH-targeting combined with PI3Kα inhibitor treatment strategy not only enhances the therapeutic effect of PI3Kα inhibitors but also expands the target population for PI3Kα inhibitors in breast cancer treatment. Details are as follows:

[0040] Example 1

[0041] 1. Experimental Methods

[0042] (1) Establishment of PI3Kα inhibitor-resistant cell lines

[0043] This invention uses MDA-MB-231 and MCF7 cell lines. After digestion, the cells are seeded into plates. Cells in the logarithmic growth phase are treated with 5 μM and 2 μM concentrations of the PI3Kα inhibitor BYL719, respectively. The dosage is changed based on cell condition. The cells are continuously passaged and treated until they no longer die from the inhibitor. The results are then analyzed using IC50 assay. 50 After testing, each sample was named and stored in liquid nitrogen.

[0044] (2) MTS detection experiment

[0045] Cells are cultured in different culture dishes according to the experimental objectives, followed by transfection and drug administration. Cells are resuspended according to cell passage methods, and an appropriate amount of cell suspension is aspirated for cell counting. Depending on the experimental objectives, the cell suspension is diluted to 500-2000 cells / 200μL and evenly seeded into 96-well plates. PBS is added to the outer edge of the plate to prevent experimental errors caused by evaporation of the surrounding culture medium. Each group should have at least three replicates as required by the experimental requirements. When performing IC50 assays on cells using a specific drug... 50In the experiment, multiple concentration gradients of a certain drug were set up between 0-100 μM. After the cells adhered, different concentrations of the drug were added to the corresponding wells of the plate. Based on different cell characteristics, the IC50 was measured at 24-96 h. 50 When performing proliferation experiments, cells were monitored at time points from day 0 to day 5. Before testing, MTS working solution was prepared fresh, and the number of wells was calculated. Using a ratio of 100 μL of culture medium / well and 20 μL of MTS / well, the culture medium in each well was quickly aspirated, and 120 μL of the aforementioned MTS working solution was added. The wells were incubated in the dark for 2-4 hours. The absorbance at 492 nm was measured using a multi-mode microplate reader. Wells surrounded by PBS were designated as control wells. The difference between the absorbance of the test well and the control well was the final absorbance value for each well. Data from each group were expressed as the average of three replicates, and each experiment was performed at least three times independently.

[0046] (3) CRISPR-Cas9 screening

[0047] A metabolism-related library was constructed using CRISPR-Cas9 library technology. This library contains 1677 metabolic genes, each targeted by 10 sgRNAs, each carried by a separate virus. By adjusting the lentiviral vector titer, each cell can be infected with one virus, resulting in the knockout of one target gene. This ensures that each gene can be knocked out 10 times at different sites. By comparing knockouts at different sites, non-specific functional expression caused by the non-specific knockout of other genes by a particular sgRNA can be ruled out. In preliminary experiments, MDA-MB-231 cells were infected with the CRISPR-Cas9 library containing 1677 metabolism-related genes, and stable infected cells were obtained through initial in vitro passage. These stable virus-infected MDA-MB-231 cells were then used to seed xenografts in nude mice. After tumor formation, the PI3Kα inhibitor BYL719 was administered for stress selection. When the PI3Kα inhibitor could no longer control tumor growth, this invention considered that the endpoint of the experiment—the occurrence of drug resistance—had been reached. Tumor tissues from the experimental group and the control group were taken, DNA was extracted and deep sequenced, and then relevant analysis was performed.

[0048] (4) Small molecule metabolite extraction experiment

[0049] Using 6cm dishes, prepare 5 dishes of cells for each group in advance: 4 dishes for metabolite extraction and 1 dish for cell quantification. Prepare 80% mass spectrometry grade methanol solution in advance and dilute the internal standard 13C1Methionine to a concentration of 0.1ppm. Pre-cool the solution to -80℃ and set aside. Prepare 80% mass spectrometry grade acetonitrile solution and pre-cool it to -20℃ and set aside. Pre-cool physiological saline solution to 4℃ and set aside. Change the medium for the cells 2 hours in advance. The procedure begins by discarding the culture medium and washing the dish three times with pre-cooled physiological saline, removing as much residual saline as possible. The culture dish is then placed on liquid nitrogen, and 1 mL of pre-prepared 80% mass spectrometry grade methanol solution is added. The dish is immediately placed in a -80°C freezer for 20 minutes. After removal, the dish is immediately placed on liquid nitrogen again, and cells are scraped off. The cell suspension is transferred to a clean 1.5 mL centrifuge tube, which is placed on ice. 0.5 mL of pre-prepared 80% mass spectrometry grade acetonitrile solution is added to the culture dish, and any remaining cells are scraped off. The cell suspension is transferred to the same 1.5 mL centrifuge tube, and the cells are repeatedly blown and aspirated to disrupt them. The dish is then placed on a centrifuge and rotated at 4°C for 30 minutes. Following this, the dish is centrifuged at 14000g for 10 minutes at 4°C. 1.2 mL of the supernatant is collected, evaporated under vacuum, and the cells are digested, resuspended, and counted in parallel wells for subsequent analysis.

[0050] (5) Cell transfection

[0051] Based on cell adhesion time and their own growth characteristics, cells are seeded into suitable culture dishes 6-18 hours in advance. The culture medium is aspirated, and the cells are gently rinsed with appropriate PBS buffer. The PBS is discarded, and then an appropriate amount of trypsin is added. The dishes are then placed in a cell culture incubator for digestion for an appropriate time. Under a microscope, when the cells become rounded and are about to detach, the trypsin is aspirated, and the culture dishes are gently tapped to detach the cells from the bottom. The cells are resuspended in fresh complete culture medium, and an appropriate amount is aspirated for cell counting. Depending on the experimental purpose, the cells are seeded into the corresponding culture dishes, and the cells are shaken as evenly as possible using the cross-hatching method. The dishes are then placed in a cell culture incubator at 37°C with 5% CO2 for further culture. Transfection can be performed when the cells adhere. For siRNA transfection, using a 6-well plate as an example, the cell density is approximately 20-30%. During transfection, 5 μL of lipofectamine RNAi max is added to 125 μL of opti-MEM medium, and another 5 μL of siRNA / siNC is added to 125 μL of opti-MEM medium. The mixture is incubated at room temperature for 5 minutes, and the liquid from the siRNA tube is transferred to a lipofectamine tube. The mixture is then incubated at room temperature for another 15 minutes. Discard the culture medium from the culture dishes where cells have been inoculated and are evenly distributed. Add the above transfection mixture to each experimental group, and bring the volume of fresh culture medium to 1 mL. Gently shake to mix, then return to the incubator for further culture. Replace the culture medium 6-8 hours after transfection. Continue culturing for 48-72 hours according to the experimental objective, then extract the corresponding RNA and total protein. Verify the transfection efficiency using qPCR and Western blot. If the transfection efficiency meets the requirements, proceed to the next step of the experiment.

[0052] (6) Viral infection

[0053] Cells in good growth phase during the logarithmic growth phase are seeded at a density of 10-20% in 6-well plates. The cells are gently shaken using a cross-hatching method to ensure even distribution. The plates are then placed in a cell culture incubator for further culture. Once adherent, the cells are ready for viral infection. All procedures during viral infection are performed in a dedicated biosafety cabinet. A preliminary experiment is conducted to determine the MOI (Mean Interval) of the cells. For the formal experiment, the virus is diluted with complete culture medium based on the MOI value from the preliminary experiment. Polybrene transfection aid is added to the virus dilution solution according to the manufacturer's instructions. The original cell culture medium is discarded, and the cells are gently washed with PBS. The diluted virus working solution is then added, shaken until well mixed, and the cells are placed in the cell culture incubator for further culture.

[0054] (7) Data Processing

[0055] All results were statistically analyzed and plotted using GraphPad Prism 10 software. All continuous variables were expressed as mean ± standard deviation, and categorical variables were expressed as percentages. For parametric variables, differences between two groups were analyzed using a two-tailed Student's t-test; differences between more than two groups were analyzed using one-way ANOVA. Nonparametric variables were compared using the chi-square test (χ²).2 (Test). P < 0.05 indicates a statistically significant difference.

[0056] 2. Experimental Results

[0057] This invention constructed a CRISPR-Cas9 library containing 1677 metabolism-related genes, and infected MDA-MB-231 cells to obtain cells stably carrying the library. MDA-MB-231 cells carrying the metabolic library were then seeded into the fat pads of balbc / nu nude mice, and transplanted until the tumor reached 5 mm. 3 Tumors of different sizes were administered the PI3Kα inhibitor BYL719 (trade name: Apelisib) and placebo (sterile saline) by gavage (once every two days) to obtain PI3Kα inhibitor-resistant and placebo-resistant xenografts. Figure 1 (A and B). Next-generation DNA deep sequencing was performed on the xenografts in both groups. The sgRNAs enriched by negative selection were compared with the genome, and genes showing significant changes in negative selection were scored and ranked. The gene encoding 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) was significantly enriched in the negative selection of the PI3Kα inhibitor-resistant group. Figure 1 HIBCH is a nuclear-encoded protein, mostly located in mitochondria, that primarily catalyzes the catabolism of valine, breaking down the valine metabolite 3-hydroxyisobutyryl-CoA (HIBC) into 3-hydroxyisobutyrate, ultimately generating succinyl-CoA, which enters the tricarboxylic acid cycle (TCA cycle). Metabolic pathway enrichment analysis of differentially enriched sgRNAs revealed significantly more active branched-chain amino acid metabolism and TCA cycling in xenografts compared to the placebo group in the PI3Kα inhibitor-resistant group. Figure 1 (D).

[0058] MDA-MB-231 and MCF7 cell lines were treated with BYL719 (2 μM) using a low-dose maintenance method. After 6 months of screening, the IC50 values ​​of the two cell lines to BYL719 were [not specified]. 50 Both are significantly improved. The cell lines that obtained BYL719 resistance in this invention are designated as 231R and MCF7R, respectively. Figure 1 (E and F in the middle). For example Figure 1 As shown in Figure E, a drug-resistant strain was established in the MDA-MB-231 cell line using a low-dose maintenance method with BYL719 (5 μmol / L). IC50 50 The concentration increased from 15.68 μmol / L in the parental strain to 40.8 μmol / L; Figure 1As shown in Figure F, a drug-resistant strain was established in the MCF7 cell line using a low-dose maintenance method with BYL719 (2 μmol / L). The IC50 value was [not specified]. 50 The concentration of the drug increased from 6.722 μmol / L in the parental line to 30.95 μmol / L. Small molecule metabolites were extracted from MDA-MB-231, MCF7, and the drug-resistant cell line for mass spectrometry analysis. The changes in various metabolic processes after drug resistance were represented in logarithmic form of the fold change. Targeted metabolomics analysis showed that, in both the triple-negative breast cancer cell line MDA-MB-231 and the Luminal breast cancer cell line MCF7, significant changes were observed in the abundance of key metabolites in intracellular branched-chain amino acid metabolism and the tricarboxylic acid cycle after BYL719 resistance. Figure 1 (G and H). Furthermore, in 231R and MCF-7R cells, deprivation of branched-chain amino acids leucine, isoleucine, and valine, and deprivation of three branched-chain amino acids, respectively, cell viability assays showed that deprivation of the three branched-chain amino acids had the best effect in reversing BYL719 resistance, followed by deprivation of valine, but deprivation of isoleucine had no significant effect. Figure 1 (I and J).

[0059] HIBCH was knocked down using small interfering RNA. Figure 1 K and L) were found to significantly increase sensitivity to PI3Kα inhibitors in drug-resistant cell lines 231R and MCF7R after knocking down HIBCH. Figure 1 (M and N). Furthermore, supplementing branched-chain amino acids on the basis of HIBCH knockdown revealed that valine supplementation could counteract the increased sensitivity to PI3Kα inhibitors caused by HIBCH knockdown. Figure 1 (O and P).

[0060] Example 2

[0061] 1. Experimental Methods

[0062] (1) Cell transfection

[0063] The method is the same as in Example 1.

[0064] (2) Total RNA extraction and RT-qPCR detection

[0065] Based on cell adhesion time and their own growth characteristics, cells were seeded into suitable culture dishes 6-18 hours in advance. The culture medium in the dish was aspirated, and the cells were gently rinsed with appropriate PBS buffer. The PBS was discarded, and then an appropriate amount of trypsin was added. Total RNA was extracted from the cells. Enzyme-free consumables were used throughout the process, and the culture medium in the dish was completely discarded. Taking a 6-well plate as an example, 1 mL of Trizol reagent was added to the dish in a fume hood, and the cells were incubated at room temperature for 5 minutes to lyse. After thorough pipetting, the cells were transferred to an enzyme-free 1.5 mL EP tube. 200 μL of chloroform was added to the tube, the cap was tightened, and the tube was vigorously shaken. The tube was then incubated for 5 minutes. The centrifuge was pre-cooled to 4°C, and centrifuged at 12000g for 15 minutes. 400 μL of the clear, colorless supernatant was carefully aspirated into a new enzyme-free 1.5 mL EP tube, an equal volume of isopropanol was added, and the mixture was inverted and incubated at room temperature for 10 minutes. Centrifuge at 12000g for 10 min at 4℃. The white precipitate is RNA. Carefully discard the supernatant. Add an appropriate amount of pre-chilled 75% ethanol to each tube. Invert the centrifuge tube to allow the precipitate to separate from the tube wall. Centrifuge at 12000g for 5 min at 4℃. Discard as much supernatant as possible. After the RNA precipitate has completely dried, add an appropriate amount of DEPC water and gently vortex to dissolve the precipitate. Use Nanodrop2000 to detect the concentration and purity of the extracted total RNA. After passing the test, store at 80℃ for later use. Reverse transcription reaction: All reagents and operations in this step are performed on ice. Taking 1 μg of total RNA as an example, determine the required RNA volume based on the RNA concentration. Add DEPC water to make up the volume to 12 μL, then add 4 μL of 4×gDNAwiper Mix. Mix thoroughly and centrifuge at 42℃ for 2 min to remove genomic DNA. After removing the tube, add 4 μL of 5×HiScriptIII qRT Super Mix, mix thoroughly, centrifuge at 37℃ for 15 min, and then at 85℃ for 5 s. The cDNA product obtained in this process can be used for the next qPCR reaction or stored at -20℃ for later use. Real-time quantitative PCR detection (qPCR): All reagents and operations in this step are performed on ice. Prepare the qPCR reaction system according to the manufacturer's instructions. Each reaction system consists of 5 μL of 2×Cham QSYBR qPCR Green Master Mix, 0.4 μL each of the forward and reverse primers for the target gene (10 μM concentration), 1 μL of the cDNA obtained in the above process, and DEPC water to a final volume of 10 μL. After adding the samples according to the experimental requirements, carefully cover with a clear membrane. Brief centrifugation will ensure the reaction system is at the bottom of the plate. Set the qPCR reaction program according to the conditions in Table 1. After the reaction, adjust the reaction time according to the CT value of each well. -ΔΔCT The relative expression level of the corresponding gene can be calculated.

[0066] Table 1. qPCR reaction procedure

[0067]

[0068] (3) Western Blot

[0069] Extraction and quantification of total cellular protein: Prepare RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors in advance and keep it on ice. Take the pre-treated cells, discard the culture medium, add an appropriate amount of pre-chilled PBS and wash three times. Aspirate the PBS completely and keep the cells on ice. Add an appropriate amount of lysis buffer as needed according to the cell volume, gently shake to cover the culture dish with the lysis buffer, and lyse on ice for 10 min. Gently scrape off the cell lysis suspension from the bottom of the dish with a cell scraper and transfer it to a clean 1.5 mL EP tube. Centrifuge at 12000g for 20 min at 4℃. Carefully aspirate an appropriate amount of supernatant to a new EP tube. It can be frozen to -20℃ or used for further protein quantification. Protein quantification: Prepare solutions A and B of the BCA kit at a ratio of 50:1, vortex to mix, and serially dilute the protein standard. Mix 10 μL of protein sample with 190 μL of BCA reagent, incubate at 37℃ for 30 min, and detect the absorbance at 562 nm using a multi-mode microplate reader. A standard curve is plotted based on the absorbance values ​​of the protein standards. The absorbance of the sample to be tested and the protein concentration are then calculated. A uniform target loading volume is set, and the required protein volume is calculated based on the protein concentration. Loading buffer is added proportionally, gently vortexed to mix, and briefly centrifuged. The mixture is then heated in a 95-100℃ water bath for 5 minutes to denature the protein, followed by brief centrifugation. The mixture is then loaded again or stored at -20℃ for later use.

[0070] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): Prepare separating gels of appropriate concentrations according to the experimental objectives. Following the kit instructions, add each gel preparation reagent component to a clean 50mL centrifuge tube, vortex to mix thoroughly, pour into a clean gel preparation plate, add an appropriate amount of anhydrous ethanol for sealing, and let stand at room temperature for approximately 30 minutes (time depends on ambient temperature). A clear gel line will be visible, indicating the separating gel has solidified. Carefully discard the supernatant anhydrous ethanol, invert the plate and remove all anhydrous ethanol. Prepare an appropriate amount of 5% stacking gel according to the number of gel preparation blocks, pour into a glass plate, immediately insert the required comb, and let stand at room temperature for approximately 30 minutes (time depends on ambient temperature) until the stacking gel solidifies. The prepared gel can be used immediately for subsequent sample loading or stored in electrophoresis buffer at 4°C for up to 3 days before use. During electrophoresis, prepare 1× electrophoresis buffer in advance, pour it into the electrophoresis tank and sample loading well, remove the comb, and clean as much residual gel clumps and impurities as possible from the sample loading wells. Add the protein samples mixed with loading buffer in sequence according to the experimental purpose, and add an appropriate amount of pre-stained protein markers at both ends as molecular size indicators. Depending on the temperature and the amount of gel, perform electrophoresis at a constant voltage of 60-80V. When the protein enters the separating gel, adjust the voltage to 100-120V and continue electrophoresis at a constant voltage until the protein reaches the bottom of the separating gel.

[0071] Protein transfer: Prepare 1× electrotransfer buffer in advance and keep it on ice. Cut a suitable size 0.22μm PVDF membrane (generally 8cm×6cm) and soak it in methanol to activate it. After electrophoresis, rinse the apparatus with tap water and remove the gel glass plate. After removing the glass plate, complete all assembly processes in a special container containing electrotransfer buffer. Assemble the electrotransfer apparatus using the sandwich method: cathode-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-anode. During assembly, be sure to remove air bubbles between each layer, especially between the gel and the PVDF membrane. After clamping the plates, correctly place it in the electrotransfer tank, pour in an appropriate amount of electrotransfer buffer, and place the electrotransfer tank in an ice-water mixture. Electrotransfer at a constant current of 260mA for 60-300min, depending on the molecular weight of the target protein.

[0072] Protein blocking and antibody incubation: After electroporation, remove the PVDF membrane, label both sides, and quickly place it in an incubator containing blocking buffer (TBST buffer containing 5% BSA or skim milk powder) and incubate at room temperature with gentle shaking for 1 hour. Dilute the primary antibody with 5% BSA at a specific ratio, cut the PVDF membrane according to the corresponding position of the pre-stained protein marker, blot dry the membrane with filter paper, and place the membrane in the corresponding primary antibody working solution, incubating overnight at 4°C. The next day, remove the PVDF membrane and place it in an appropriate amount of 1×TBST buffer, washing the membrane three times with rapid shaking at room temperature for 10 minutes each time. Remove the membrane, blot off excess TBST with filter paper, place it in secondary antibody working solution (diluted with TBST buffer according to the specified ratio), and incubate at room temperature with gentle shaking for 1.5 hours. Remove the PVDF membrane and place it in an appropriate amount of 1×TBST buffer, washing the membrane three times with rapid shaking at room temperature for 10 minutes each time. After washing, proceed with subsequent imaging and recording. Protein development: Prepare the luminescent reaction solution by mixing equal volumes of luminescent substrates A and B in the ECL chemiluminescence kit. Use immediately after preparation. Take an appropriate amount of the luminescent reaction solution and add it evenly to the PVDF membrane strip. Expose and image the membrane using a chemiluminescence imager or darkroom film and record the results.

[0073] (4) Immunofluorescence assay

[0074] Cell preparation: During passage culture, seed cells into confocal culture dishes. After cell attachment, wash twice with PBS, fix with 4% paraformaldehyde for 15 min, and wash with PBS 3×5 min. Permeabilization: Add 0.1% Triton X-100 and permeabilize at room temperature for 5-15 min. After permeabilization, wash with PBS 3×5 min, and block with goat serum at room temperature for approximately 30 min. Primary antibody binding: Dilute the primary antibody with 5% BSA at a 1:100 ratio and add to the culture dish. Incubate overnight at 4°C. The next day, wash 3 times with PBST for 5 min each time. Secondary antibody binding: Dilute the secondary antibody with PBST at a 1:100 ratio and incubate at room temperature in the dark for 1 h. Wash 3 times with PBST for 5 min each time. After aspirating excess liquid, add mounting medium containing DAPI, photograph, and then store at -20°C in the dark.

[0075] (5) Nuclear-plasma separation

[0076] Use the Beyotime Cell Nuclear and Cytoplasmic Protein Extraction Kit. Solution Preparation: Dissolve the three reagents in the kit at room temperature, then immediately place on ice and mix well. Take an appropriate amount of cytoplasmic protein extraction reagent A and add PMSF a few minutes before use to bring the final PMSF concentration to 1 mM. Take an appropriate amount of nuclear protein extraction reagent and add PMSF a few minutes before use to bring the final PMSF concentration to 1 mM. Cell Preparation: Wash once with PBS, scrape cells with a cell scraper, centrifuge to collect cells, and carefully aspirate the supernatant, reserving the cell pellet for later use. Avoid using trypsin to digest cells, as this may degrade the target proteins to be extracted. Add 100 μL of cytoplasmic protein extraction reagent A with added PMSF to every 20 μL of cell pellet, and centrifuge at the highest speed (Vortex) for 5 seconds to completely suspend and disperse the cell pellet. Incubate on ice for 10-15 minutes. Add 10 μL of cytoplasmic protein extraction reagent B, centrifuge at the highest speed (Vortex) for 5 seconds, and incubate on ice for 1 minute. Centrifuge at the highest speed (Vortex) for 5 seconds at 4°C, 12,000-16,000g for 5 minutes. Immediately transfer the supernatant to a pre-chilled EP tube; this is the extracted cytoplasmic protein. For the precipitate, completely aspirate any remaining supernatant, add 50 μL of nuclear protein extraction reagent supplemented with PMSF, and centrifuge at the highest speed (Vortex) for 15-30 seconds to completely suspend and disperse the cell pellet. Then return to an ice bath and centrifuge at the highest speed (Vortex) for 15-30 seconds every 1-2 minutes, for a total of 30 minutes. Centrifuge at 4°C, 12,000-16,000g for 10 minutes. Immediately transfer the supernatant to a pre-chilled EP tube; this is the extracted nuclear protein.

[0077] (6) ChIP-sequence and ChIP-PCR

[0078] First, add formaldehyde solution to the culture dish for fixation. Depending on the volume of the culture medium, dilute 37% formaldehyde solution to 1% formaldehyde solution. Fixation conditions are room temperature for 10 min. Add 0.125M glycine to terminate cross-linking for 10 min. Wash three times with pre-cooled PBS. Scrape off cells with PBS solution containing protease inhibitors. Centrifuge at 500g, 4℃ for 10 min. Discard the PBS. Add SDSlysis buffer and lyse the cells on ice for 10 min. The cells can be temporarily stored at -80℃. Next, the DNA was sonicated and the cells were lysed using a Covaris S220. ChIP dilution buffer containing protease inhibitors was added to bring the final volume to 0.5 mL. 50 μL was used for DNA fragment verification, and 10 μL was used as 2% input. The remainder was added with Flag and IgG antibody and incubated at 4°C. The next day, magnetic beads were added to the IP sample at 30 μL / IP and incubated at room temperature for 1.5 h. Gradient washing was performed: low-salt buffer, high-salt buffer, and TE buffer were washed 1, 1, and 2 times, respectively, for 5 min each time. 200 μL of dilution buffer (including the previous input tube) was added, and the IP tube was placed in a 65°C water bath for 30 min. The supernatant was collected into a new centrifuge tube, 5M NaCl and RNase A were added, and the tube was incubated at 37°C for 30 min. 0.5M EDTA, Tris-HCl, and protein K were added, and the tube was incubated at 55°C for 1 h. The sample was then purified using a DNA purification kit and used for subsequent PCR experiments and ChIP-seq analysis. 50 μL was used as a DNA fragment for verification: 100 μL of nuclease-free water was added to the DNA solution, along with 5 M NaCl and RNase A. The mixture was incubated at 37 °C for 30 min, followed by the addition of 0.5 M EDTA, Tris-HCl, and protein K. The mixture was then incubated at 55 °C for 1 h. The sample was then purified using a DNA purification kit. DNA was separated by gel electrophoresis, and the distribution of DNA bands was observed under UV light to verify the DNA fragmentation effect.

[0079] (7) Co-IP experiment

[0080] Select appropriate culture dishes for cell culture according to the experimental objectives, and complete transfection or drug addition treatments. Prepare IP lysis buffer in advance: add appropriate amounts of protease inhibitors and phosphatase inhibitors as needed, and keep on ice for later use. Discard the culture medium, wash three times with pre-cooled PBS, and aspirate as much residual PBS as possible. Add an appropriate amount of IP lysis buffer according to the area of ​​the culture dish, gently shake to evenly spread the lysis buffer to the bottom of the dish, and lyse on a shaker at 4°C for 20 min. Gently scrape off the cell suspension from the bottom of the dish with a cell scraper, transfer to an EP tube, centrifuge at 12000g at 4°C for 20 min, aspirate the supernatant to a new EP tube, detect the protein concentration, and reserve an appropriate volume for detecting transfection efficiency. Reserve 5% of the protein concentration as input and store at -80°C. The remaining supernatant was divided into two equal portions according to the principle of equal mass between groups. Specific protein antibodies or control IgG antibodies were added to each portion for incubation. The tubes were sealed with sealing film and incubated overnight at 4°C with rotation. The next day, the magnetic beads were washed with IP lysis buffer, and an equal volume of the washed magnetic beads was added directly to the tubes. The tubes were sealed with sealing film and incubated at room temperature with rotation for 1.5 hours. After incubation, the EP tubes were placed on a magnetic rack and inverted to ensure complete adsorption of the magnetic beads onto the tube walls. All supernatant was discarded. An appropriate amount of IP lysis buffer was added to each tube to wash the magnetic beads, and the tubes were washed with rotation at room temperature for 5 minutes, repeated three times. The EP tubes were placed on a magnetic rack and inverted to ensure complete adsorption of the magnetic beads onto the tube walls. All supernatant was discarded. The prepared IP lysis buffer and loading buffer suspension was added, gently mixed, and heated at 95°C with gentle shaking for 10 minutes. After brief centrifugation, the EP tubes were placed on a magnetic rack until the magnetic beads were completely adsorbed onto the tube walls. The protein in the tubes was transferred to new EP tubes for subsequent detection, or the obtained protein could be temporarily stored at -80°C.

[0081] (8) Dual-luciferase reporter assay

[0082] A target vector containing the firefly and sea urchin dual luciferase reporter gene system was constructed and transfected into cells. The specific transfection method was as described above. After 48 hours, the cells were lysed and reacted according to the instructions of the Promega dual luciferase reporter assay kit. The fluorescence value was then detected using a Biotech multi-mode microplate reader.

[0083] (9) Construction of stable cell lines

[0084] Cells in good logarithmic growth phase were seeded at a density of 10-20% in 6-well plates. The cells were shaken evenly using a cross-hatching method to ensure uniform distribution. The plates were then placed in a cell culture incubator for further culture. Once adhered, the cells were ready for viral infection. All procedures were performed in a dedicated biosafety cabinet. A preliminary experiment was conducted to determine the MOI (Mean Interval) of the cells. During the formal experiment, the virus was diluted with complete culture medium based on the MOI value from the preliminary experiment. Polybrene transfection aid was added to the virus dilution according to the manufacturer's instructions. The original cell culture medium was discarded, and the cells were gently washed with PBS. The diluted virus working solution was added, and the cells were shaken until well mixed before being placed in the cell culture incubator for further culture. 12-24 hours after viral infection, the waste liquid was discarded into a separate centrifuge tube in a dedicated biosafety cabinet. The cells were gently washed with PBS, and the medium was replaced with ordinary complete culture medium for 72 hours. Depending on the cell condition and the different resistances carried by the virus, antibiotics such as puromycin or G418 were added for selection. After selection, the infection efficiency was verified. Successful cells were cryopreserved for future use. Thereafter, cells were cultured in medium containing antibiotics.

[0085] (10) MTS detection experiment

[0086] The method is the same as in Example 1.

[0087] (11) Small molecule metabolite extraction experiment

[0088] The method is the same as in Example 1.

[0089] (12) Animal experiments

[0090] Laboratory animal materials and facilities: BALB / C-nu / nu SPF-grade female nude mice, purchased at 3-4 weeks of age and weighing 16-18g, were purchased from the Beijing Vital River Laboratory Animal Center. Animal experiments were conducted in a barrier environment at the Laboratory Animal Center of Sun Yat-sen University (North Campus, Area D). The environmental technical indicators of the experimental room met the requirements of GB14925-2010. This animal experiment was reviewed and approved by the Laboratory Animal Management and Use Committee and the Laboratory Animal Ethics Committee of Sun Yat-sen University (Experiment No.: North-D2021-0363QX). Eighty immunodeficient female nude mice aged 3-4 weeks were randomly divided into 10 groups of 8 mice each. After passing quarantine, subcutaneous tumor formation experiments were performed when the mice reached 4 weeks of age. MDA-MB-231 knockdown control cells, stably knocked-down HIBCH cells, and cells overexpressing LAT1 after stably knocked-down HIBCH were injected into the fat pads of nude mice, respectively. MCF-7 overexpressing control cells and LAT1 overexpressing cells were also injected into the fat pads of nude mice. The MDA-MB-231 injection density was 2 × 10⁻⁶ cells / year. 6 The MCF-7 inoculum size was 5 × 10⁶ cells / 100 μL. 6One week prior to cell injection, mice were subcutaneously implanted with 0.72 mg of 17β-estradiol pellets (released over 60 days). Tumor formation was observed every three days after cell injection. Once tumors formed, the longest and shortest diameters of the tumor were measured every three days using calipers. When the tumors reached approximately 6 mm in diameter, the mice were administered the PI3Kα inhibitor BYL719 (100 mg / kg, every three days) via gavage. Each cell line had both a control group and a PI3Kα inhibitor group. After starting treatment, the mice's drinking water was changed to a tetracycline-containing sugar solution, and the mice were protected from light. Tumor growth was continued, with the longest and shortest diameters measured every three days using calipers. The formula was used: Tumor volume = 0.5 × longest diameter × shortest diameter. 2 The tumor volume was calculated, and the tumor weight was measured. The experimental endpoint was determined after tumor cell injection, specifically based on the tumor growth of different cell lines. The humane endpoint was defined as the animal's inability to eat, disease, tumor short diameter exceeding 1.5 cm, systemic metastasis severely impacting survival, or other extreme discomfort. Mice were euthanized using cervical dislocation. Tumors were obtained through dissection, and the mouse carcasses were placed in the Animal Experiment Center of Sun Yat-sen University North Campus for standardized and harmless disposal.

[0091] (13) Data processing

[0092] All results were statistically analyzed and plotted using GraphPad Prism 10 software. All continuous variables were expressed as mean ± standard deviation, and categorical variables were expressed as percentages. For parametric variables, differences between two groups were analyzed using a two-tailed Student's t-test; differences between more than two groups were analyzed using one-way ANOVA. Nonparametric variables were compared using the chi-square test (χ²). 2 (Test). P < 0.05 indicates a statistically significant difference.

[0093] 2. Experimental Results

[0094] Figure 2 Figures A and B show that the expression levels of HIBCH in PI3Kα-resistant 231R and MFC-7R cells were not altered compared to those in parental cells. Figure 2 Immunofluorescence assays of C and D cells showed that HIBCH in 231R and MCF-7R cells was mainly located within the nucleus. Super-resolution confocal microscopy results (…) Figure 2 (E and F) show that HIBCH enters the nucleus after valine deprivation. ChIP-sequence analysis, compared with normal culture conditions, showed that valine deprivation induced HIBCH nucleus entry and enhanced its regulation of gene transcription. Figure 2 (G); Among the transcription factors that bind to HIBCH, c-Myc ranked third (G); Figure 2(H). Furthermore, immunoprecipitation assays confirmed that the interaction between HIBCH and c-Myc was enhanced under valine-deprived conditions, while the expression levels of HIBCH and c-Myc remained unchanged during this process. Figure 2 Middle I).

[0095] Simultaneously, under valine-deprived culture conditions, ChIP-sequence analysis was used to detect changes in c-Myc transcriptional regulation. The results showed that valine deprivation enhanced c-Myc's regulation of gene transcription. Figure 2 In the normal culture group, c-Myc mainly regulated metabolism-related genes in the glucose metabolism pathway, while in the valine deprivation group, c-Myc enhanced the regulation of genes related to the metabolism of multiple amino acids, including branched-chain amino acids, and weakened the regulation of genes related to glucose metabolism. Figure 2 (K). Among them, the neutral amino acid transporter LAT1 is the gene most significantly regulated by the c-Myc transcriptional regulatory mode shift, and the expression level of LAT1 decreased significantly after knocking down HIBCH. Figure 2 LAT1 expression level gradually increased with prolonged deprivation time under valine-deprived culture conditions. Figure 2 (M).

[0096] Cell viability assays showed that knocking down HIBCH in 231R and MCF-7R cells significantly increased cell sensitivity to BYL719. However, simultaneous knockdown of HIBCH and overexpression of LAT1 suppressed cell sensitivity to BYL719, demonstrating resistance to BYL719. Figure 2 (NO). Targeted metabolomics analysis revealed that knocking down HIBCH significantly decreased the content of branched-chain amino acids and inhibited the tricarboxylic acid cycle; while overexpression of LAT1 not only increased the content of branched-chain amino acids but also enhanced the tricarboxylic acid cycle (NO). Figure 2 (P). When c-Myc was knocked down at 231R, the expression level of LAT1 decreased significantly (P). Figure 2 (Q), and the transcriptional activity of LAT1 was also significantly inhibited (Q). Figure 2 (R). Cell sensitivity to BYL719 was also increased, while LAT1 overexpression counteracted the effect of c-Myc knockdown. Figure 2 (QR). Cell viability assays showed that knocking down c-Myc in 231R and MCF-7R cells significantly increased cell sensitivity to BYL719. However, simultaneous overexpression of LAT1 while knocking down c-Myc inhibited cell sensitivity to BYL719, demonstrating resistance to BYL719. Figure 2 (ST).

[0097] Results of animal experiments ( Figure 3 The results showed that MDA-MB-231 cells inherently had low sensitivity to BYL719. Stable knockdown of HIBCH significantly enhanced sensitivity to BYL719, and tumor growth was markedly inhibited. However, in the group with stable HIBCH knockdown and simultaneous LAT1 overexpression, the tumors showed poor responsiveness to BYL719, and tumor growth was not inhibited. MCF-7 cells inherently showed good responsiveness to BYL719, and tumor growth was significantly inhibited. However, overexpression of LAT1 in MCF-7 cells significantly reduced the sensitivity of the tumors to BYL719. This indicates that nuclear HIBCH interacts with c-Myc, prompting c-Myc to alter its transcriptional pattern, enhancing LAT1 transcriptional activity and expression levels to allow for the uptake of more branched-chain amino acids to supplement glucose metabolism.

[0098] Example 3

[0099] 1. Experimental Methods

[0100] (1) Mass spectrometry identification of post-transcriptional modifications

[0101] First, the target protein was purified using Flag antibody. The electrophoresis tank was cleaned beforehand, and the equipment was soaked in ultrapure water. The proteins were separated using a precast gel. After Coomassie brilliant blue staining, the target band was destained and displayed. The target band was then cut off with a clean scalpel blade, and further cut into 1mm pieces. 3 Small pieces of gel were added to a decolorizing solution and shaken at room temperature until transparent. Then, 100% acetonitrile was added and shaken for 5 minutes to whiten the gel particles. The acetonitrile was blotted dry, and the gel was lyophilized. 10 mM DTT / 50 mM NH4HCO3 was added, and the mixture was shaken to mix. The mixture was incubated at 56°C for 1 hour. 100% acetonitrile was added again and shaken for 5 minutes to whiten the gel particles. The acetonitrile was blotted dry, and the gel was lyophilized. 60 mM IAA / 50 mM NH4HCO3 was added, and the mixture was shaken to mix in the dark. The mixture was incubated in the dark for 30 minutes. 100% acetonitrile was added again and shaken for 5 minutes to whiten the gel particles. The acetonitrile was blotted dry, and the gel was lyophilized. 50-80 μL of 50 mM NH4HCO3 was added, followed by 1-2 μL of trypsin. The mixture was incubated at 37°C with shaking for at least 6 hours. 0.1% FA was added to each tube and shaken for 5 minutes, followed by 0.1% FA / ACN and shaken for 5 minutes. The supernatants were combined and evaporated to dryness at room temperature. Sample elution and instrumentation were then performed.

[0102] (2) Nuclear-plasma separation

[0103] The method is the same as in Example 2.

[0104] (3) Co-IP experiment

[0105] The method is the same as in Example 2.

[0106] (4) ChIP-PCR

[0107] The method is the same as in Example 2.

[0108] (5) Cell transfection

[0109] The method is the same as in Example 1.

[0110] (6) Total RNA extraction and RT-qPCR detection

[0111] The method is the same as in Example 2.

[0112] (7) Western Blot

[0113] The method is the same as in Example 2.

[0114] (8) Coomassie brilliant blue staining

[0115] The protein sample processing and electrophoresis methods are the same as described in the Western blot section above. The difference is that after electrophoresis, a small portion of the gel containing marker protein and a small amount of sample needs to be cut for staining: Place the gel in an appropriate amount of Coomassie Brilliant Blue staining solution, ensuring that the staining solution can fully cover the gel. Place it on a horizontal shaker or a side-swing shaker and slowly shake it at room temperature for 1 hour or longer. The specific staining time depends on the thickness of the gel and the temperature during staining. If the gel is thicker and the temperature is lower, the staining time should be appropriately extended. If the gel is thinner and the temperature is higher, the staining time can be appropriately shortened. After staining, pour out the staining solution, which can be recycled and reused at least 2-3 times. Then add an appropriate amount of Coomassie Brilliant Blue destaining solution, ensuring that the destaining solution can fully cover the gel. Place it on a horizontal shaker or a side-swing shaker and shake it rapidly at room temperature for 4-24 hours. During this period, change the destaining solution 2-4 times until the blue background is basically completely removed and the protein band staining effect reaches the expectation. After destaining, the gel can be soaked in ddH2O.

[0116] (9) Dual-luciferase reporter assay

[0117] The method is the same as in Example 2.

[0118] (10) MTS detection experiment

[0119] The method is the same as in Example 1.

[0120] (11) Data processing

[0121] All results were statistically analyzed and plotted using GraphPad Prism 10 software. All continuous variables were expressed as mean ± standard deviation, and categorical variables were expressed as percentages. For parametric variables, differences between two groups were analyzed using a two-tailed Student's t-test; differences between more than two groups were analyzed using one-way ANOVA. Nonparametric variables were compared using the chi-square test (χ²). 2(Test). P < 0.05 indicates a statistically significant difference.

[0122] 2. Experimental Results

[0123] Nuclear-cytoplasmic separation experiments were performed using Flag tags to purify HIBCH proteins from the cytoplasm and nucleus, respectively. After SDS-Page electrophoresis, Coomassie Brilliant Blue staining was used. Results are shown below. Figure 4 Mass spectrometry analysis of the bands separating the HIBCH protein revealed succinylation modification of lysine residues at positions 17 and 55 of the HIBCH protein in the cell nucleus. Figure 5 and Figure 6 ).

[0124] Furthermore, using the Uniprot protein database, potential succinylation sites on the HIBCH protein were mutated one by one (K to A) to construct wild-type and mutant overexpression vectors for HIBCH. MDA-MB-231 cells were transfected with these overexpression vectors, and under valine-deprived conditions, Western blotting after nuclear-cytoplasmic separation showed that the expression level of HIBCH in the nucleus significantly decreased when the K55 site was mutated. Figure 7 (A), and the level of HIBCH succinylation also decreased significantly ( Figure 7 (B)

[0125] Furthermore, immunoprecipitation experiments were performed based on the K55 mutation, revealing that the binding of HIBCH to c-Myc was significantly weakened after the K55 mutation. Figure 7 (C). Simultaneously, mutation at the HIBCH K55 site led to a weakening of both HIBCH and c-Myc transcriptional regulation of LAT1. Figure 7 middle DE).

[0126] Further screening of succinylated transferases catalyzing HIBCH K55 succinylation was conducted. Potentially active genes with succinylated transferase activity were knocked down one by one. After culturing cells deprived of valine for 72 hours, cells from each group were collected for nuclear-cytoplasmic separation experiments. Western blotting analysis of HIBCH localization revealed that knockdown of CPT1A significantly decreased HIBCH expression in the nucleus, while knockdown of other genes did not affect HIBCH nuclear localization. Simultaneously, detection of HIBCH succinylation after CPT1A knockdown confirmed that CPT1A catalyzes succinylation modification at the HIBCH K55 site. Figure 7 Furthermore, knocking out CPT1A significantly downregulated the binding of HIBCH and c-Myc to the LAT1 promoter region. Figure 7 (G), inhibiting the transcriptional activity and expression level of LAT1 (G), Figure 7(HI). Cell viability assays showed that CPT1A knockout significantly increased cell sensitivity to BYL719, but LAT1 overexpression counteracted the effect of CPT1A knockout. Figure 7 (J).

[0127] Example 4

[0128] 1. Experimental Methods

[0129] (1) Small molecule compound-protein molecule docking

[0130] The structure of the HIBCH protein was downloaded from the PDB database website (https: / / www.rcsb.org / ), using its substrate binding site as the docking site, including amino acids 121, 146, 169, and 177. The structure of the HIBCH protein was optimized using the Quickprep module in MOE (version 2022). This involved hydrogenation of the protein, completion of the side chain amino acid structure, resetting the protonation state of the protein under physiological pH conditions, setting the temperature to 300K, and placing the protein in an Amber10:EHT force field for optimization. Small molecules were also structurally processed using the Quickprep module in MOE, including hydrogenation, calculation of the minimum energy for the small molecule, and generation of multiconformation files for the compound. The small molecule with the best conformation was selected for docking experiments. Receptor files were generated through structural optimization, docking regions were determined based on binding sites, and docking was performed using MOE-DOCK. The docking engine used was Placement:TriangleMatcher. In the first round, the LondonΔG scoring function was used to retain 300 docking patterns. Then, the GBVI / WASΔG scoring function was used to optimize each docking pattern. Finally, the conformation with the best score for each molecule was retained as the output file. For each compound, the top 5 conformations were retained according to the scoring system. The lower the score, the better the affinity.

[0131] (2) Immunofluorescence assay

[0132] The method is the same as in Example 2.

[0133] (3) MTS detection experiment

[0134] The method is the same as in Example 1.

[0135] (4) Construction of organoid models

[0136] Freshly collected breast cancer tumor fragments were digested at 37°C with gentle agitation for at least 1 hour using 0.6 mg / mL type II dispersase, 1 mg / mL type IV collagenase, 50 μg / mL deoxyribonuclease I, and 10 μM Y-27632. The tumor cells were then centrifuged, washed twice with PBS, and cultured in BC organoid medium (KBR-1000, K2 oncology) containing 10% Matrigel (356230, Corning).

[0137] (5) Construction of PDX xenograft model

[0138] To establish a patient-derived xenograft model (PDX), breast cancer samples were collected from patients who underwent tumor resection surgery at Shantou Central Hospital between 2022 and 2024. Six-week-old female NOD-SCID mice were anesthetized with isoflurane. Breast cancer samples were cut into 1 mm sections. 3 Small fragments were directly implanted into the mammary fat pads of mice to obtain the first-generation PDX. When the first-generation PDX grew to a diameter of 1 cm, it was removed and then cut into 1 mm pieces. 3 Fragments were directly implanted into the mammary fat pad to obtain a second-generation PDX for treatment. This laboratory animal facility is accredited by the International Committee for Assessment and Accreditation of Laboratory Animals (AAALAC), and all animal experimental protocols used in this invention have been approved by the Institutional Animal Care and Use Committee (IACUC) of the Guangdong Provincial Laboratory Animal Monitoring Institute.

[0139] (6) Data processing

[0140] All results were statistically analyzed and plotted using GraphPad Prism 10 software. All continuous variables were expressed as mean ± standard deviation, and categorical variables were expressed as percentages. For parametric variables, differences between two groups were analyzed using a two-tailed Student's t-test; differences between more than two groups were analyzed using one-way ANOVA. Nonparametric variables were compared using the chi-square test (χ²). 2 (Test). P < 0.05 indicates a statistically significant difference.

[0141] 2. Experimental Results

[0142] Targeting the active cavity of HIBCH K55, small molecule compound C7 with affinity for the active cavity was screened from a library containing 1.5 million compounds using computer simulation and molecular docking techniques. The screening process for small molecule inhibitors targeting HIBCH nuclear translocation is as follows: Figure 8 As shown in Figure A.

[0143] Immunofluorescence assays confirmed that C7 inhibits nuclear translocation of HIBCH, and nucleoplasmic separation assays confirmed that C7 inhibits succinylation modification of HIBCH. Figure 8The B-cell curve shows that C7 exhibits dose-dependent inhibition of HIBCH nuclear translocation. Figure 8 The C7 assay showed that C7 specifically binds to lysine 55 of the HIBCH protein. Further SPR assays confirmed that C7 has a good affinity for HIBCH. Figure 8 (D).

[0144] In MDA-MB-231 cells that are insensitive to the PI3Kα inhibitor (BYL719), the combination of C7 and other treatments reduced the IC50 of tumor cells against BYL719. 50 Significantly improves sensitivity ( Figure 8 (E). An organoid model derived from human breast cancer tissue was constructed to verify the synergistic lethal effect of C7 and BYL719. Results showed that, regardless of whether the patient carried the PIK3CA mutation, C7 and BYL719 exhibited a strong synergistic lethal effect in a breast cancer organoid model insensitive to PI3Kα inhibitors. Figure 8 (FG). An animal model of human breast cancer xenograft was constructed to verify the roles of C7 and BYL719 in inhibiting tumor growth. Results showed that in a breast cancer PDX model without PIK3CA mutations, both C7 and BYL719 exhibited good synergistic lethal effects, significantly inhibiting tumor growth in both previously insensitive triple-negative breast cancer and somewhat sensitive ER-positive / HER-2-negative breast cancer. Figure 8 (China HI).

[0145] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of compounds that inhibit HIBCH nuclear translocation in the preparation of drugs that reverse resistance to PI3Kα inhibitors in breast cancer treatment, characterized in that, The breast cancer PI3Kα inhibitor is BYL719; The compound is C7, and its structural formula is as follows:

2. A drug for reversing resistance to PI3Kα inhibitors in breast cancer treatment, characterized in that, The active ingredients include compounds that inhibit HIBCH nuclear translocation; The breast cancer PI3Kα inhibitor is BYL719; The compound is C7, and its structural formula is as follows:

3. The drug according to claim 2, characterized in that, The drug also includes pharmaceutically acceptable excipients.

4. A composition for treating breast cancer, characterized in that, Including PI3Kα inhibitors and compounds that inhibit HIBCH nuclear translocation; The PI3Kα inhibitor is BYL719; The compound is C7, and its structural formula is as follows:

5. Use of the composition as described in claim 4 in the preparation of a medicament for treating breast cancer.

6. A drug for treating breast cancer, characterized in that, The active ingredient includes the composition of claim 4.

7. The drug according to claim 6, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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