LU602982B1 - Application of compound for inhibiting nuclear translocation of HIBCH in preparation of medicament for reversing resistance to PI3Kα inhibitors in breast cancer - Google Patents

Abstract

The present invention discloses an application of a compound for inhibiting nuclear translocation of HIBCH in the preparation of a medicament for reversing resistance to PI3Kα inhibitors in breast cancer, belonging to the field of biomedicine technology. The study of the present invention found that valine metabolism is disordered in breast cancer cells and animal models resistant to PI3Kα inhibitors, and this disorder of valine metabolism is caused by the translocation of HIBCH, a key enzyme in the valine degradation pathway originally located in mitochondria, to the nucleus. Targeting HIBCH and combining with PI3Kα inhibitors have a synergistic lethal effect on breast cancer, which may significantly enhance the sensitivity of tumor cells to PI3Kα inhibitors, and the therapeutic effect is not affected by whether PIK3CA itself is mutated or not. The therapeutic strategy of targeting HIBCH combined with PI3Kα inhibitors enhances the therapeutic effect of PI3Kα inhibitors. (Fig.1)

Description

DESCRIPTION LU602982

APPLICATION OF COMPOUND FOR INHIBITING NUCLEAR TRANSLOCATION OF

HIBCH IN PREPARATION OF MEDICAMENT FOR REVERSING RESISTANCE TO PI3KA

INHIBITORS IN BREAST CANCER

TECHNICAL FIELD

The present invention relates to the field of biomedicine technology, in particular to an application of a compound for inhibiting nuclear translocation of HIBCH in the preparation of a medicament for reversing resistance to PI3Ka inhibitors in breast cancer.

BACKGROUND

The mutation rate of P/K3CA in breast cancer exceeds 30%, making it one of the main driver genes of breast cancer. Abnormal activation of the PI3K/AKT/MTOR signaling pathway caused by PIK3CA mutation or abnormally high expression of its encoded PI3K protein drives tumor metabolic reprogramming, especially the Warburg effect, and is involved in the regulation of malignant progression such as drug resistance, recurrence and metastasis of tumors. With the significant progress in the research on the molecular mechanism of PI3Ka regulating breast cancer metabolism and preclinical studies, the development of PI3Ka inhibitors isonce regarded as an important milestone in the field of breast cancer treatment. However, in a series of subsequent large-scale clinical studies, only the results of the SOLAR-II clinical study confirmed that PI3Ka inhibitors may significantly improve the progression-free survival rate of advanced breast cancer patients with PIK3CA mutations, while patients with breast cancer without

PIK3CA mutations benefit little, and the treatment has significant side effects. Therefore, how to improve the therapeutic effect of PI3Ka inhibitors, expand their scope of use, and overcome their treatment side effects is a major problem faced in the clinical application of PI3Ka inhibitors.

A review of the failed clinical studies of PI3Ka inhibitors in breast cancer treatment shows that PI3K reactivation, abnormal activation of parallel pathways, tumor microenvironment, etc, may lead to insensitivity of tumors to PI3Ka inhibitors. PI3K reactivation is mainly due to acquired amplification of PIK3CA mutation sites and activating mutations of PIK3CB, which significantly upregulate the activity of PI3Ka, lead to abnormal activation of the AKT signaling pathway, show insensitivity to PI3Ka inhibitors, and accelerate the proliferation and metastasis of tumor cells. Research on parallel pathway activation mainly focuses on the RAS-RAF-MEK-

ERK signaling pathway and the PI3K-AKT alternative pathway.

Mutation and amplification of HRAS in the RAS family may significantly reduce the 692982 sensitivity of tumor cells to PI3Ka inhibitors, and activation of its encoded RAS protein and its downstream signaling pathway may enhance the invasiveness of breast cancer, promote malignant transformation of breast cancer, and is one of the key determinants of metastasis and poor survival in breast cancer patients. Inhibitory immune cells derived from the tumor microenvironment may activate the NF-kB signaling pathway in tumor cells by secreting cytokines, leading to insensitivity of tumor cells to PI3Ka inhibitors and promoting tumor progression.

To sum up, the present invention may find that the molecular mechanisms leading to insensitivity to PI3Ka inhibitors are usually also the molecular mechanisms driving the malignant progression of breast cancer. Therefore, clarifying the mechanism of insensitivity to PI3Ka inhibitor treatment is not only helpful to maximize the therapeutic effect of PI3Ka inhibitors on breast cancer, but also helpful to clarify the driving mechanism of breast cancer recurrence and metastasis, and explore new targets and strategies for breast cancer treatment.

PI3K mainly promotes the occurrence and development of breast cancer by driving tumor glucose metabolism. Blocking the PI3K signaling pathway with inhibitors may cause obstacles to the acquisition of nutrients by tumor cells, achieving the goal of "starving" tumor cells. In tumor cells insensitive to PI3Ka inhibitors, when PI3K is inhibited, tumor cells may undergo metabolic reprogramming to maintain an appropriate metabolic state and survival. The emergence of such metabolic alternative pathways may also be an important mechanism of insensitivity to PI3Ka inhibitor treatment. Recent studies have shown that active mitochondrial glutamine production pathway leads to reprogramming of the tricarboxylic acid cycle, which, as a metabolic alternative pathway, is involved in the regulation of resistance to PI3Ka inhibitors.

However, studies on reversing resistance to PI3Ka inhibitors by targeting branched-chain amino acid metabolism are still rarely reported.

SUMMARY

LU602982

The purpose of the present invention is to provide an application of a compound for inhibiting nuclear translocation of HIBCH in the preparation of a medicament for reversing resistance to PI3Ka inhibitors in breast cancer, so as to solve the problems existing in the prior art. The study of the present invention found that targeting HIBCH and combining with PI3Ka inhibitors have a synergistic lethal effect on breast cancer, and may significantly enhance the sensitivity to PI3Ka inhibitors. The therapeutic strategy of targeting HIBCH combined with PI3Ka inhibitors not only enhances the therapeutic effect of PI3Ka inhibitors, but also expands the population eligible for PI3Ka inhibitors in breast cancer treatment.

To achieve the above purpose, the present invention provides the following solutions:

The present invention provides an application of a compound for inhibiting nuclear translocation of HIBCH in the preparation of a medicament for reversing resistance to PI3Ka inhibitors in breast cancer.

Further, the compound is C7 (C:1HzoN4O6), and its structural formula is as follows:

J a 7 % +.

AN NE

FE > N > Aa, { & F À Ag À v ey X S “ay X >

Vp we

À Fa

The present invention also provides a medicament for reversing resistance to PI3Ka inhibitors in breast cancer, the active ingredient of which includes a compound that inhibits nuclear translocation of HIBCH.

Further, the compound is C7 (Ca1H3oN4Oe), and its structural formula is as follows: ® sl) bl Rn wey”

SN Ÿ \ . A { A “ N £2 i” “a X A rn 5 / £3 dem we gy i x

LA

Further, the medicament further includes pharmaceutically acceptable excipients.

The present invention also provides a composition for treating breast cancer, including a

PI3Ka inhibitor and a compound that inhibits nuclear translocation of HIBCH.

Further, the compound is C7 (C31H30N4Os), and its structural formula is as follows: LU602982 a

Al M * ( # vu au

Lu Pp i

AY

The present invention also provides the use of the above composition in the preparation of a medicament for treating breast cancer.

The present invention also provides a medicament for treating breast cancer, the active ingredient of which includes the above composition.

Further, the medicament further includes pharmaceutically acceptable excipients.

The present invention discloses the following technical effects: the study of the present invention found that valine metabolism is disordered in breast cancer cells and animals resistant to PI3Ka inhibitors, and this valine metabolism 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. Further, a compound C7 that can effectively inhibit nuclear translocation of HIBCH isobtained through drug screening. Moreover, SPR and cell function tests confirmed that C7 has a strong affinity with

HIBCH, reduces the degree of interaction with c-Myc by inhibiting the nuclear entry of HIBCH, thereby downregulating the expression level of LAT1, and thus produces a synergistic lethal effect with BYL719 on drug-resistant breast cancer cells. The organ model also confirmed that

C7 and BYL719 have a synergistic lethal effect in breast cancer insensitive to PI3Ka inhibitors, regardless of the presence or absence of P/K3CA mutations. At the same time, a patient- derived xenograft animal model of breast cancer that is resistant to PI3Ka inhibitors and does not carry PIK3CA mutations isconstructed. Therapeutic experiments found that compared with

BYL719 alone and C7 alone, the combination of BYL719 and C7 exerts a synergistic lethal effect on tumors, thereby reversing the original resistance to PI3Ka inhibitors and significantly inhibiting tumor growth. It can be seen that targeting HIBCH and combining with PI3Ka inhibitors have a synergistic lethal effect on breast cancer, may significantly enhance the sensitivity to PI3Ka inhibitors, and the therapeutic effect is not affected by the presence or absence of PIK3CA mutations. The therapeutic strategy of targeting HIBCH combined with

PI3Ka inhibitors not only enhances the therapeutic effect of PI3Ka inhibitors, but also expands the eligible population for PI3Ka inhibitors in breast cancer treatment.

BRIEF DESCRIPTION OF THE FIGURES LU602982

To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings may be obtained according to these drawings without creative work.

Fig. 1 is a result diagram of experiments on HIBCH-mediated valine metabolism remodeling involved in resistance to PI3Ka inhibitors in breast cancer; where, A is a schematic diagram of

CRISPR-Cas9 Screen; B is a growth curve of tumors after nude mouse fat pad tumor formation under BYL719 and placebo treatment respectively; C is the ranking of negatively selected genes in CRISPR-Cas9 Screen; D is a functional enrichment analysis diagram of negatively selected genes; E is a statistical diagram of cell activity of MDA-MB-231 drug-resistant cell line;

F is a statistical diagram of cell activity of MCF7 drug-resistant cell line; G is a result diagram of mass spectrometry analysis of small molecule metabolites extracted from MDA-MB-231 parent and drug-resistant cell lines, showing changes in metabolic processes after drug resistance in the form of logarithm of fold change; H is a result diagram of mass spectrometry analysis of small molecule metabolites extracted from MCF7 parent and drug-resistant cell lines, showing changes in metabolic processes after drug resistance in the form of logarithm of fold change; is a result diagram of experiments on the effect of branched-chain amino acid deprivation on the sensitivity of 231R to BYL719; J is a result diagram of experiments on the effect of branched- chain amino acid deprivation on the sensitivity of MCF7R to BYL719; K is the effect of HIBCH on the proliferation ability of breast cancer 231 cell line; L is the HIBCH knockdown efficiency in

MCF7 cell line; M is a test result of the sensitivity of 231R cells to PI3Ka inhibitors under HIBCH knockdown; N is a test result of the sensitivity of MCF7R cells to PI3Ka inhibitors under HIBCH knockdown; O is a test result of restoring the sensitivity of 231R cells to PI3Ka inhibitors caused by HIBCH knockdown under the condition of additional branched-chain amino acid addition; P is a test result of restoring the sensitivity of MCF-7R cells to PI3Ka inhibitors caused by HIBCH knockdown under the condition of additional branched-chain amino acid addition;

Fig. 2 is a result diagram of experiments on HIBCH nuclear entry regulating branched-chain, 502982 amino acid metabolism remodeling through LAT1; where, A and B are the expression levels of

HIBCH in PI3Ka-resistant 231R and MFC-7R respectively; C and D are result diagrams of immunofluorescence detection of HIBCH localization in 231R and MCF-7R respectively; E and

F are ultra-high-resolution confocal microscope images showing HIBCH localization in MDA-

MB-231 and MCF-7 cells under normal culture and valine-deprived culture respectively; G is a detection result diagram of HIBCH in the nucleus regulating gene transcription under valine deprivation; H is the top 4 transcription factors bound to HIBCH and their domains under valine deprivation; | is a result diagram of co-immunoprecipitation detection of the binding degree between HIBCH and c-Myc under valine deprivation; J is a result diagram of changes in the degree of c-Myc regulation on gene transcription under valine deprivation; K is a result diagram of changes in metabolic pathways of genes regulated by c-Myc under normal culture and valine- deprived culture; L is the expression level of LAT1 after HIBCH knockdown; M is the expression level of LAT1 under valine-deprived culture; N and O are result diagrams of detecting changes in cell sensitivity to BYL719 after HIBCH knockdown and simultaneous overexpression of LAT1 in 231R and MCF-7R respectively; P is a heat map of changes in intracellular branched-chain amino acid and TCA pathway metabolite contents after HIBCH knockdown and simultaneous overexpression of LAT1 detected by targeted metabolomics; Q is a result diagram of QPCR detection of LAT1 and c-Myc expression levels after c-Myc knockdown; R is a result diagram of dual luciferase reporter system detection of changes in LAT1 transcriptional activity after c-Myc knockdown; S and T are result diagrams of detecting changes in cell sensitivity to BYL719 after c-Myc knockdown and simultaneous overexpression of LAT1 in 231R and MCF-7R respectively;

Fig. 3 is a result diagram of experiments on the effect of HIBCH intervention on BYL719 sensitivity in animal models; where, A is a display diagram of MDA-MB-231 xenografts; B is a growth curve of MDA-MB-231 xenografts; C is a statistical diagram of tumor weight of MDA-MB- 231 xenografts; D is a display diagram of MCF-7 xenografts; E is a growth curve of MCF-7 xenografts; F is a statistical diagram of tumor weight of MCF-7 xenografts;

Fig. 4 is a result diagram of Coomassie brilliant blue staining after electrophoresis of HIBCH protein purified with Flag antibody in nuclear-cytoplasmic separation experiment under valine deprivation;

Fig. 5 is a result diagram of mass spectrometry detection showing succinylation modification of lysine 55 of HIBCH;

Fig. 6 is a result diagram of mass spectrometry detection showing succinylation modification of lysine 17 of HIBCH;

Fig. 7 is a result diagram of experiments on the key mechanism of HIBCH nuclear 502982 translocation; where, À is a result diagram of detecting the nuclear expression level of mutant

HIBCH after nuclear-cytoplasmic separation under valine-deprived culture; B is a result diagram of detecting the succinylation level of nuclear HIBCH after mutation in nuclear-cytoplasmic separation experiment under valine deprivation on the basis of K55 site mutation; C is a result diagram of co-immunoprecipitation experiment showing the binding degree between mutant

HIBCH and c-Myc after K55 site mutation; D is a result diagram of ChIP-PCR detection of changes in the binding ability of HIBCH and c-Myc to LAT1 after K55 site mutation; E is a detection result of LAT1 expression level after HIBCH K55 mutation; F is a result of nuclear- cytoplasmic separation experiment detecting HIBCH cell localization and succinylation after

CPT1A and KAT2A knockdown; G is a result of detecting the binding degree of HIBCH and c-

Myc to the LAT1 promoter region after CPT1A knockdown; H is a QPCR detection result of

LAT1 expression level after CPT1A knockdown; | is a result of dual luciferase reporter system detection of LAT1 transcriptional activity after CPT1A knockdown; J is a result of cell viability experiment detecting cell sensitivity to BYL719 after CPT1A knockdown and simultaneous overexpression of LAT1;

Fig. 8 is a result diagram of experiments on the synergistic lethal effect of small molecule inhibitor C7 targeting HIBCH nuclear translocation and PI3Ka inhibitor; where, A is a schematic diagram of the screening process of small molecule inhibitors targeting HIBCH nuclear translocation; B is a result diagram of immunofluorescence showing that C7 inhibits HIBCH nuclear entry in a dose-dependent manner; C is a molecular docking diagram showing that C7 binds to lysine 55 of HIBCH protein; D is a result diagram of SPR showing the affinity between

C7 and HIBCH; E is a diagram of C7 combined with BYL719 reducing the ICso of breast cancer cells; F is a result diagram of PIK3CA-mutated human breast cancer organoid model showing the synergistic lethal effect of C7 and BYL719; G is a result diagram of P/K3CA-amplified human breast cancer organoid model showing the synergistic lethal effect of C7 and BYL719;

H-I are result diagrams of human-derived xenograft models PDX-1359 and PDX-0595 showing the synergistic lethal effect of C7 and BYL719 respectively.

DESCRIPTION OF THE INVENTION LU602982

The various exemplary embodiments of the present invention will now be described in detail. The detailed description should not be regarded as a limitation of the present invention, but rather as a more detailed description of certain aspects, features and embodiments of the present invention.

It should be understood that the terms used in the present invention are only for describing specific embodiments and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. These smaller ranges may have independent upper and lower limits, which may be included or excluded from the range, depending on whether the upper and lower limits are specifically excluded.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although the present invention only describes preferred methods and materials, similar or equivalent methods and materials may be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

Without departing from the scope or spirit of the present invention, various modifications and changes may be made to the specific embodiments of the present invention described in this specification, which will be obvious to those skilled in the art. Other embodiments obtained from the description of the present invention will be obvious to technicians. The description and examples of the present invention are only exemplary.

As used herein, "comprising", "including", "having", "containing" and the like are open terms, meaning including but not limited to.

The molecular formula of the small molecule compound C7 in the present invention is

C31H30N4Os, and its structural formula is as follows: : ®

Va C4

The study of the present invention found that valine metabolism is disordered in breast, 602982 cancer cells and animals resistant to PI3Ka inhibitors, and this valine metabolism 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 PI3Ka inhibitor (BYL719), resulting in a synergistic lethal phenomenon, thereby reversing resistance to

BYL719 and inhibiting tumor cell growth. Moreover, the present invention successfully constructed patient-derived breast cancer organoid models sensitive and insensitive to alpelisib (trade name of BYL719) treatment, and found that HIBCH knockdown combined with BYL719 may cause a synergistic lethal phenomenon in organoids, significantly inhibit organoid growth, and reverse resistance to BYL719. Mechanism studies found 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 decreases, releasing the catalytic activity of succinyltransferase CPT1A, leading to succinylation modification of lysine 55 of

HIBCH protein and translocation of HIBCH to the nucleus. HIBCH entering the nucleus interacts with transcription factor c-Myc, changing the transcriptional regulation preference of c-Myc from regulating the transcriptional activity of key enzymes related to glucose metabolism to regulating the transcriptional activity of proteins related to amino acid metabolism, especially 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 isperformed using a high- performance computer system combined with artificial intelligence for the succinylated domain of HIBCH protein, and 12 drugs (C1-C12) targeting the HIBCH K55 site are screened. Further detection of the effect of 12 small molecule drugs on HIBCH nuclear translocation determined that small molecule compound No.7 (C7: Cz1HsoN406) can effectively inhibit nuclear translocation of HIBCH. Moreover, SPR and cell function tests confirmed that C7 has a strong affinity with HIBCH, reduces the degree of interaction with c-Myc by inhibiting the nuclear entry of HIBCH, thereby downregulating the expression level of LAT1, and thus produces a synergistic lethal effect with BYL719 on drug-resistant breast cancer cells. The organ model also confirmed that C7 and BYL719 have a synergistic lethal effect in breast cancer insensitive to PI3Ka inhibitors, regardless of the presence or absence of P/K3CA mutations. At the same time, a patient-derived xenograft animal model of breast cancer that is resistant to PI3Ka inhibitors and does not carry PIK3CA mutations isconstructed. Therapeutic experiments found that compared with BYL719 alone and C7 alone, the combination of BYL719 and C7 exerts a synergistic lethal effect on tumors, thereby reversing the original resistance to PI3Ka inhibitors and significantly inhibiting tumor growth. It can be seen that targeting HIBCH and combining with PI3Ka inhibitors have a synergistic lethal effect on breast cancer, may significantly enhance the sensitivity to PI3Ka inhibitors, and the therapeutic effect is not affected by the presence or absence of PIK3CA mutations.

The therapeutic strategy of targeting HIBCH combined with PI3Ka inhibitors not only 02982 enhances the therapeutic effect of PI3Ka inhibitors, but also expands the eligible population for

PI3Ka inhibitors in breast cancer treatment. The details are as follows:

Embodiment 1 1. Experimental Methods (1) Establishment of PI3Ka inhibitor-resistant cell lines

In the present invention, MDA-MB-231 cell line and MCF7 cell line are used. After digestion and plating, cells in the logarithmic growth phase are given 5 uM and 2 uM PI3Ka inhibitor

BYL719 respectively. During the period, whether to change the drug isdetermined according to the cell status. Continuous passage and drug addition culture are performed until the cells no longer died due to the addition of the inhibitor. After ICso detection, they are named and stored in liquid nitrogen respectively. (2) MTS detection experiment

Cells are cultured in different culture vessels according to the experimental purpose, and transfection and drug addition are performed on this basis. Resuspend cells according to the cell passage method, absorb an appropriate amount of cell suspension for cell counting, dilute the cell suspension to 500-2000 cells/200 uL according to the experimental purpose, and evenly inoculate into 96-well plates. PBS isadded to the peripheral wells to prevent experimental errors caused by evaporation of peripheral medium. According to the experimental requirements, at least 3 replicate wells are set for each group. When performing ICso experiments of a certain drug on cells, set multiple concentration gradients of the drug between 0-100 pM. After the cells adhere to the wall, add different concentrations of drugs to the corresponding wells. According to different cell characteristics, detect ICs for 24-96 h. For proliferation experiments, detect cells at 0-5 days. Prepare MTS working solution before detection (prepared before use), calculate the number of wells, quickly aspirate and discard the medium in the wells to be detected one by one at a ratio of 100 pL medium/well and 20 uL MTS/well, add 120 pL of the above MTS working solution, and incubate in the incubator in the dark for 2-4 h. A multi-functional microplate reader isused to detect the absorbance value of the wells to be tested at a wavelength of 492 nm.

Wells with PBS around are set as control wells. The difference between the absorbance value of the wells to be tested and that of the control wells isthe final absorbance value of each well.

Each group of data isexpressed as the average of 3 replicate wells, and each group of experiments isrepeated at least 3 times independently. (3) CRISPR-Cas9 screening

A metabolism-related library isconstructed by CRISPR-Cas9 library technology. The metabolism library covers 1677 metabolism-related genes, and each metabolism gene is targeted by 10 sgRNAs, which are carried by 10 viruses respectively. By adjusting the titer of the lentiviral vector, each cell may be infected with one virus and knock out one target gene.

This ensures that each gene may be knocked out 10 times at different sites, and through 02982 comparison of knockouts at different sites, non-specific functional performance caused by non- specific knockout of other genes by a certain sgRNA is excluded. In the preliminary experiment,

MDA-MB-231 isinfected with a CRISPR-Cas9 library containing 1677 metabolism-related genes, and stably infected cells are obtained through preliminary in vitro passage. Further, these MDA-

MB-231 cells stably infected with the virus are used to establish nude mouse xenografts. After the xenografts formed tumors, PI3Ka inhibitor BYL719 isgiven for pressure screening. When the

PI3Ka inhibitor could not control tumor growth, the present invention considered that the experimental endpoint—resistance—isreached. Tumor tissues from the experimental group and the control group are taken, DNA isextracted for deep sequencing, and relevant analysis isperformed. (4) Small molecule metabolite extraction experiment

Use 6 cm dishes, prepare 5 dishes of cells in advance for each group, 4 dishes for metabolite extraction, 1 dish for cell quantification, prepare 80% mass spectrometry-grade methanol solution in advance, dilute to 0.1 ppm internal standard 13C1Methionine, pre-cool at - 80°C for standby, prepare 80% mass spectrometry-grade acetonitrile solution, pre-cool at -20°C for standby, physiological saline pre-cool at 4°C for standby, and change the medium for cells 2 hours in advance. During operation, first aspirate and discard the medium, wash 3 times with pre-cooled physiological saline, try to aspirate the residual physiological saline, place the culture dish on liquid nitrogen, add 1mL of pre-prepared 80% mass spectrometry-grade methanol solution to the culture dish, immediately put it into a -80°C refrigerator for 20 minutes, take it out and immediately place the culture dish on liquid nitrogen, scrape off the cells, suck the cell suspension into a clean 1.5 mL centrifuge tube, place the centrifuge tube on ice, add 0.5mL of prepared 80% mass spectrometry-grade acetonitrile solution to the culture dish, try to scrape the remaining cells, suck the cell suspension into the 1.5 mL centrifuge tube just now, repeatedly blow the cells to break them, then place them on a rotating instrument at 4°C for 30 min, then centrifuge at 14000g for 10 min at 4°C, take 1.2 mL of supernatant, vacuum dry, and digest and resuspend the parallel cell quantification wells for counting for subsequent analysis.

(5) Cell transfection LU602982

According to the cell adherence time and their own growth characteristics, cells are inoculated into appropriate culture vessels 6-18 hours in advance, aspirate and discard the medium in the dish, add appropriate PBS buffer to gently rinse, discard PBS, add appropriate trypsin, put into the cell incubator to digest for an appropriate time, observe under the microscope that the cells become round and are about to fall off, aspirate and discard the trypsin, gently tap the culture dish to make the cells detach from the bottom of the dish, add fresh complete medium to resuspend the cells, aspirate an appropriate amount for cell counting, inoculate into the corresponding culture dish according to the experimental purpose, shake the cells as evenly as possible by cross method, put into a 5% CO», 37°C cell incubator for continuous culture, and transfection may be performed when the cells adhere to the wall. For siRNA transfection, take 6-well plate as an example, the cell density is about 20-30%. During transfection, add 5 pL lipofectamine RNAi max to 125 uL opti-MEM medium, add another 5uL siRNA/siNC to 125 uL opti-MEM medium, let stand at room temperature for 5min, aspirate the si tube liquid into the lipofectamine tube, and continue to stand at room temperature for 15min.

Aspirate and discard the medium in the culture dish where the cells have been inoculated and evenly distributed, add the above transfection mixture according to the experimental group, make up the volume of fresh medium to 1 mL, gently shake and mix, and put back into the incubator for continuous culture. Replace the medium 6-8 h after transfection, continue to culture for 48-72 h according to the experimental purpose, then extract the corresponding RNA and total protein, and perform the next experiment after verifying the transfection efficiency by gPCR and Western blot. (6) Virus infection

Take cells in logarithmic growth phase with good growth status, inoculate them into 6-well plates at a density of 10-20%, shake the cells evenly by cross method to distribute them as evenly as possible in the dish, place them in the cell incubator for continuous culture, and virus infection may be performed after adherence. During virus infection, all operations are performed in a dedicated biosafety cabinet. First, a pre-experiment isperformed to determine the MOI value of the cells. In the formal experiment, the virus isdiluted with complete medium according to the MOI value of the pre-experiment, and polybrene transfection reagent isadded to the virus dilution according to the instructions. Aspirate and discard the original cell medium, gently wash with PBS, add the diluted virus working solution, shake to mix, and put into the cell incubator for continuous culture.

(7) Data processing LU602982

GraphPad Prism10 software isused for all result statistics and graphing. All continuous variables are expressed as mean+standard deviation, and categorical variables are expressed as percentages. For parametric variables, the two-tailed student's t-test isused for differences between two groups, and one-way analysis of variance (ANOVA) isused for differences between more than two groups. The chi-square test (x? test) isused for comparison of non- parametric variables. P<0.05 isconsidered statistically significant. 2. Experimental Results

The present 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 metabolism library are inoculated into the fat pad of balbc/nu nude mice. When the xenografts grew to 5mm°, PI3Ka inhibitor BYL719 (trade name: Apelisib) and placebo (sterile normal saline) are given by gavage (once every 2 days) to obtain xenografts with resistance to PI3Ka inhibitor treatment and placebo treatment (Figs. 1A and B). Second- generation DNA deep sequencing isperformed on the xenografts of the two groups, and the sgRNAs enriched by negative screening are compared with the genome for analysis to obtain the significantly changed genes in negative screening for scoring and ranking. Among them, the gene HIBCH encoding 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) issignificantly enriched in the negative screening of the PI3Ka inhibitor treatment resistance group (Fig. 1C). HIBCH is a nuclear-encoded protein mostly located in mitochondria, mainly catalyzing the decomposition of valine metabolite 3-hydroxyisobutyryl-CoA (HIBC) into 3-hydroxyisobutyric acid in valine catabolism, and finally generating succinyl-CoA (Succinyl-CoA), which enters the tricarboxylic acid cycle (TCA cycle). Enrichment analysis of metabolic pathways for differentially enriched sgRNAs found that branched-chain amino acid metabolism and tricarboxylic acid cycle are significantly active in xenografts of the PI3Ka inhibitor treatment resistance group compared with the placebo group (Fig. 1D).

By means of low-dose maintenance, MDA-MB-231 and MCF7 cell lines are treated with

BYL719 (2 UM) under pressure. After 6 months of screening, the ICs of both cell lines to

BYL719 issignificantly increased. The present invention named the cell lines with BYL719 resistance as 231R and MCF7R respectively (Figs. 1E and F). As shown in Fig. 1E, a drug- resistant strain isestablished by maintaining MDA-MB-231 cell line with low dose of BYL719 (5 pmol/L), and the ICs increased from 15.68 pmol/L of the parent strain to 40.8 umol/L; as shown in Fig. 1F, a drug-resistant strain isestablished by maintaining MCF7 cell line with low dose of

BYL719 (2 pmol/L), and the ICso increased from 6.722 umol/L of the parent strain to 30.95 pmol/L. Small molecule metabolites are extracted from MDA-MB-231, MCF7 parent and drug- resistant cell lines respectively for mass spectrometry analysis, and changes in metabolic processes after drug resistance are reflected in the form of logarithm of fold change.

Targeted metabolomics detection results showed that whether it istriple-negative breast, 602982 cancer cell line MDA-MB-231 or Luminal breast cancer cell line MCF7, the abundance of key metabolites in metabolic pathways such as branched-chain amino acid metabolism and tricarboxylic acid cycle in cells changed significantly after developing BYL719 resistance (Figs. 1G and H). Further, leucine, isoleucine, valine and the three branched-chain amino acids are deprived in 231R and MCF-7R cells respectively. Cell viability test showed that deprivation of the three branched-chain amino acids had the best effect on reversing BYL719 resistance, followed by valine deprivation, but isoleucine deprivation had no obvious effect (Figs. 11 and J).

Small interfering RNA isused to knock down HIBCH (Figs. 1K and L), and it isfound that after knocking down HIBCH in drug-resistant cell lines 231R and MCF7R, the sensitivity to

PI3Ka inhibitors issignificantly increased (Figs. 1M and N). On the basis of HIBCH knockdown, branched-chain amino acids are supplemented, and it isfound that valine supplementation could offset the increase in sensitivity to PI3Ka inhibitors caused by HIBCH knockdown (Figs. 10 and

P).

Embodiment 2 1. Experimental Methods (1) Cell transfection

The method is the same as that in Embodiment 1. (2) Total RNA extraction and RT-qPCR detection

According to the cell attachment time and self-growth characteristics, the cells are inoculated into a suitable culture vessel 6-18 hours in advance, the culture medium in the vessel is sucked off, and the PBS buffer is added for gentle washing, and PBS is discarded, and then a proper amount of pancreatin is added, and the total RNA of the cells is finely extracted: using enzyme-free consumables in the whole process, and completely discard the culture medium in the dish. Take a 6-well plate as an example, add 1 mL of trizol reagent to the dish in the fume hood, let it stand for 5 minutes at room temperature, transfer it to an enzyme-free 1.5 mL EP tube, add 200 pL chloroform to the tube, cover the tube tightly and shake it violently, then let it stand for 5 minutes, precool the centrifuge to 4°C in advance, and centrifuge at 12,000 g for 15 minutes. Carefully suck 400 pL of clear and colorless liquid from the upper layer into a new enzyme-free 1.5 mL EP tube, add the same volume of isopropanol, mix it upside down and let it stand at room temperature for 10min. Centrifuge at 4°C for 10 min at 12000 g, and the white precipitate is RNA. Carefully discard the supernatant, add a proper amount of precooled 75% ethanol to each tube, turn the centrifuge tube upside down, and centrifuge at 4°C for 5 min at 12000 g. Discard all supernatant as much as possible, and after RNA precipitation is completely dried, add appropriate amount of DEPC water to gently shake and dissolve the precipitation.

Using Nanodrop2000 to detect the concentration and purity of the extracted total RNA, and storing it at 80°C after passing the test. Reverse transcription reaction: all reagents in this step are placed on the ice, and all operations are performed on the ice.

For the total RNA obtained in the above step, take the total RNA of 1 ug as an example 502982 and obtain the required RNA volume according to the RNA concentration, make up the volume to 12 pL with DEPC water, then add 4 pL of 4x GDNAWIPER MIX, thoroughly mix and centrifuge, remove the genomic DNA at 42°C for 42°C 2 min, and then add 4 pL of 5x Hiscriptii

QRT Supermix into the tube after taking it out, and thoroughly mix and centrifuge at 37°C for 15 min and 85°C for 5 s. The cDNA product obtained in this process can be used for the next gPCR reaction or stored at -20°C for later use. Real-time fluorescence quantitative PCR detection (QPCR): in this step, all reagents are placed on the ice, and all operations are carried out on the ice. QCPR reaction systems are prepared according to the instruction system. Each reaction system consists of 5 uL 2x Chamgsybr qPCR Green Master Mix, 0.4 uL of upstream and downstream primers of the target amplification gene with a concentration of 10 uM, 1 uL of cDNA obtained in the above process, and 10 uL of DEPC water replenishment volume. After adding the sample according to the experimental requirements, cover it with transparent film carefully, and the reaction system will be located at the bottom of the added sample plate after short centrifugation. QPCR reaction program isset according to the conditions in Table 1. After the reaction, the relative expression of the corresponding gene iscalculated according to the CT value of each hole by 244°T method.

Table 1 qPCR reaction program ewe | tee | mer

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Lt 88 | es 4

LU EL

(3) Western Blot

Extraction of total cellular protein and protein quantification: Prepare RIPA Iysis buffer containing protease inhibitors and phosphatase inhibitors in advance, place on ice for standby, take pre-treated cells, aspirate and discard the medium, add appropriate pre-cooled PBS to wash 3 times, aspirate PBS completely, place on ice, add appropriate amount of lysis buffer according to the amount of cells, shake gently to make the lysis buffer cover the culture dish, lyse on ice for 10 min, gently scrape the cell lysis suspension from the bottom of the dish with a cell scraper, transfer to a clean 1.5 mL EP tube, centrifuge at 12000 g for 20 min at 4°C, carefully aspirate an appropriate amount of supernatant into a new EP tube, which may be stored at -20°C or continue protein quantification.

Protein quantification: Prepare solution A and solution B of BCA kit at a ratio of 50:1, shake 0980 and mix well, dilute protein standards in gradients, mix 10 uL protein sample with 190 uL BCA reagent, incubate at 37°C for 30min, and detect the absorbance value at 562 nm in a multi- functional microplate reader. Draw a standard curve according to the absorbance detection value of the protein standard, calculate the absorbance value of the sample to be detected and calculate the protein sample concentration. Set a unified target loading amount, calculate the required protein volume according to the protein concentration, add loading buffer in proportion, shake gently to mix, centrifuge briefly, heat in a 95-100°C constant temperature water bath for 5 min to denature the protein, centrifuge briefly, and continue loading or store in a -20°C refrigerator.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) experiment:

Prepare separation gel of corresponding concentration according to the experimental purpose: add each gel preparation reagent component into a clean 50 mL centrifuge tube according to the kit instructions, shake fully to mix, pour into a clean gel preparation plate, add an appropriate amount of absolute ethanol to seal the gel, let stand at room temperature for about min (time depends on temperature) until a clear gel line is visible, indicating that the separation gel has solidified, carefully discard the upper absolute ethanol, invert and aspirate the absolute ethanol, prepare an appropriate amount of 5% stacking gel according to the number of gel blocks, pour into the glass plate, immediately insert the required comb, let stand at room temperature for about 30 min (time depends on temperature) until the stacking gel solidifies. The prepared gel may be immediately used for subsequent loading or stored in electrophoresis buffer at 4°C for use within 3 days. During electrophoresis, prepare 1x electrophoresis buffer in advance, pour into the electrophoresis tank and sample tank, pull out the comb, try to clean the residual gel blocks and impurities in the sample wells, add protein samples mixed with loading buffer in turn according to the experimental purpose, and add an appropriate amount of pre-stained protein marker at both ends as a molecular size indicator.

According to the temperature and the number of gels, perform electrophoresis at a constant voltage of 60-80 V until the protein enters the separation gel, adjust the voltage to 100-120 V, and perform electrophoresis at a constant voltage until the protein reaches the bottom of the separation gel.

Protein transfer: Prepare 1x transfer buffer in advance and place on ice for standby, cut a

PVDF membrane of appropriate size (generally 8 cmx6 cm), soak in methanol for activation for standby. After electrophoresis, rinse the device with tap water and remove the gel glass plate.

After removing the glass plate, all assembly processes are completed in a special vessel containing transfer buffer. Assemble the transfer device by sandwich method: cathode-sponge- filter paper-gel-PVDF membrane-filter paper-sponge-anode. Be sure to remove air bubbles between layers, especially between the gel and PVDF membrane.

Clamp the splint and put it into the transfer tank correctly, pour an appropriate amount Of 602982 transfer buffer, place the transfer tank in an ice-water mixture, and transfer at a constant current of 260 mA for 60-300 min according to the molecular weight of the target protein.

Protein blocking and antibody incubation: After the transfer, remove the PVDF membrane, mark the front and back sides, quickly put it into an incubation box containing blocking solution (TBST buffer containing 5% BSA or skimmed milk powder), and incubate on a shaker at room temperature for 1 h with slow shaking. The primary antibody isdiluted with 5% BSA at a certain ratio, the PVDF membrane iscut according to the corresponding position of the pre-stained protein marker, the blocking solution on the membrane isblotted with filter paper, the membrane isput into the corresponding primary antibody working solution, and incubated at 4°C overnight.

The next day, take out the PVDF membrane and put it into an appropriate amount of 1xTBST buffer, wash the membrane 3 times with fast shaking at room temperature, 10 min each time.

Take out the membrane, blot the excess TBST with filter paper, place it in the secondary antibody working solution (diluted with TBST buffer according to the ratio), incubate on a shaker at room temperature for 1.5 h with slow shaking, take out the PVDF membrane and put it into an appropriate amount of 1xTBST buffer, wash the membrane 3 times with fast shaking at room temperature, 10 min each time. After washing, perform subsequent development, imaging and recording. Protein development: The luminescent substrate A:B in the ECL chemiluminescence kit ismixed in equal volume to prepare a luminescent reaction solution, which isprepared before use. An appropriate amount of luminescent reaction solution isevenly dropped onto the PVDF membrane strip, and exposure imaging isperformed with a chemiluminescence imager or darkroom film and recorded. (4) Immunofluorescence experiment

Cell preparation: During subculture, cells are inoculated into confocal culture dishes. After the cells adhered to the wall, they are washed twice with PBS, fixed with an appropriate amount of 4% paraformaldehyde for 15min, and washed with PBS for 3x5 min. Permeabilization: Add 0.1% TritonX-100 for permeabilization at room temperature for 5-15 min. After permeabilization, wash with PBS for 3x5 min, add goat serum for blocking at room temperature, generally for 30min. Primary antibody binding: Dilute the primary antibody with 5% BSA at 1:100, add into the culture dish, and incubate at 4°C overnight. The next day, rinse with PBST 3 times, 5min each time. Secondary antibody binding: Dilute the secondary antibody with PBST at 1:100, incubate at room temperature in the dark for 1 h, rinse with PBST 3 times, 5min each time, blot the excess liquid, add mounting medium containing DAPI, take photos on the machine, and store at -20°C in the dark.

(5) Nuclear-cytoplasmic separation LU602982

Use Biyuntian nuclear protein and cytoplasmic protein extraction kit. Prepare solution:

Dissolve the three reagents in the kit at room temperature, immediately place on ice after dissolution, and mix well. Take an appropriate amount of cytoplasmic protein extraction reagent

A for standby, and add PMSF to make the final concentration of PMSF 1 mM within a few minutes before use. Take an appropriate amount of nuclear protein extraction reagent for standby, and add PMSF to make the final concentration of PMSF 1mM within a few minutes before use. Cell preparation: Wash once with PBS, scrape off the cells with a cell scraper, collect the cells by centrifugation, pay attention to aspirate the supernatant completely, and leave the cell pellet for standby. Try to avoid digesting cells with trypsin to prevent trypsin from degrading the target protein to be extracted. Add 100 pL of cytoplasmic protein extraction reagent A with PMSF to every 20 uL of cell pellet, vortex at maximum speed for 5 seconds to completely suspend and disperse the cell pellet. Ice bath for 10-15 minutes. Add 10 pL of cytoplasmic protein extraction reagent B, vortex at maximum speed for 5 seconds, and ice bath for 1 minute. Vortex at maximum speed for 5 seconds, centrifuge at 12,000-16,000 g for 5 minutes at 4°C, and immediately aspirate the supernatant into a pre-cooled EP tube, which is the extracted cytoplasmic protein. For the pellet, completely aspirate the residual supernatant, add 50 pL of nuclear protein extraction reagent with PMSF, vortex at maximum speed for 15-30 seconds to completely suspend and disperse the cell pellet. Then put back into ice bath, vortex at high speed for 15-30 seconds every 1-2 minutes, for a total of 30 minutes. Centrifuge at 12,000-16,000 g for 10 minutes at 4°C. Immediately aspirate the supernatant into a pre-cooled

EP tube, which is the extracted nuclear protein.

(6) ChIP-sequence and ChIP-PCR LU602982

First, add formaldehyde solution to the culture dish for fixation. According to different medium volumes, add 37% formaldehyde solution to dilute to 1% formaldehyde solution. Fix at room temperature for 10 min, add 0.125 M glycine to terminate cross-linking for 10 min, wash 3 times with pre-cooled PBS, scrape off the cells with PBS solution containing protease inhibitors, centrifuge at 500 g for 10min at 4°C, discard PBS, add SDS lysis buffer to lyse the cells on ice for 10 min, and may be temporarily stored in a -80°C refrigerator. Next, ultrasonically break

DNA, use CovarisS220 to break the cells, add ChIP dilution buffer containing protease inhibitors to make the final volume 0.5 mL, take 50 pL for DNA fragment verification, take 10 UL as 2% input, add Flag and IgG antibodies to the rest and incubate at 4°C. The next day, add magnetic beads to the IP sample, 30 UHL/IP, incubate at room temperature for 1.5 h, gradient washing: low-salt buffer, high-salt buffer, TE buffer once, once, twice respectively, 5 min each, add 200

ML of elution buffer (including the input tube above), put the IP tube into a 65°C water bath for 30min, collect the supernatant into a new centrifuge tube, add 5M NaCl, RNase A, incubate at 37°C for 30 min, add 0.5 M EDTA, Tris-HCL, protein K, 55°C for 1h, then purify the obtained sample with a DNA purification kit for subsequent PCR experiments and ChIP-seq submission.

UL for DNA fragment verification: add 100 pL nuclease-free water to the DNA solution, add 5

M NaCl, RNase A, incubate at 37°C for 30 min, add 0.5 M EDTA, Tris-HCL, protein K, 55°C for 1 h, then purify the obtained sample with a DNA purification kit, separate DNA by gel electrophoresis, and display the distribution of DNA bands under ultraviolet light to verify the

DNA fragmentation effect. (7) Co-IP experiment

Cells are cultured in appropriate culture vessels according to the experimental purpose, and transfection or drug addition iscompleted. Prepare IP lysis buffer in advance: add appropriate protease inhibitors and phosphatase inhibitors as needed, and place on ice for standby. Aspirate and discard the medium, wash 3 times with pre-cooled PBS, try to aspirate residual PBS, add appropriate amount of IP lysis buffer according to the area of the culture dish, shake gently to make the lysis buffer evenly spread on the bottom of the dish, lyse on a shaker at 4°C for 20 min. Gently scrape the cell suspension from the bottom of the dish with a cell scraper, transfer to an EP tube, centrifuge at 12000g for 20 min at 4°C, aspirate the supernatant into a new EP tube, detect the protein concentration, retain an appropriate volume to detect the transfection efficiency, retain 5% mass as input according to the protein concentration, and store at -80°C. The remaining supernatant isequally divided into two parts according to the equal mass principle between groups, and incubated with specific protein antibodies or control

IgG antibodies respectively. Seal the tube mouth with parafilm, rotate and incubate at 4°C overnight. The next day, first wash the magnetic beads with IP lysis buffer, add the washed magnetic beads directly into the tube in equal amount, seal the tube mouth with parafilm, and rotate and incubate at room temperature for 1.5 h.

After incubation, place the EP tube on a magnetic stand, invert up and down to make the 502982 magnetic beads completely adsorbed on the tube wall, and aspirate all the supernatant. Add an appropriate amount of IP lysis buffer to each tube to wash the magnetic beads, rotate and wash at room temperature for 5min, repeat 3 times. Place the EP tube on a magnetic stand, invert up and down to make the magnetic beads completely adsorbed on the tube wall, and aspirate all the supernatant. Add the prepared suspension of IP lysis buffer and loading buffer, mix gently, heat at 95°C with gentle shaking for 10min, centrifuge briefly, place the EP tube on a magnetic stand, wait for the magnetic beads to be completely adsorbed on the tube wall, transfer the protein in the tube to a new EP tube for subsequent detection, or temporarily store the obtained protein in a -80°C refrigerator. (8) Dual luciferase reporter experiment

A target vector with firefly and Renilla dual luciferase reporter gene system isconstructed and transfected into cells. The specific transfection method isas described above. After 48 hours, according to the instructions of Promega's dual luciferase reporter assay kit, the cells are lysed and reacted, and the fluorescence value isdetected with a Biotech multi-functional microplate reader. (9) Construction of stable cell lines

Take cells in logarithmic growth phase with good growth status, inoculate them into 6-well plates at a density of 10-20%, shake the cells evenly by cross method to distribute them as evenly as possible in the dish, place them in the cell incubator for continuous culture, and virus infection may be performed after adherence. During virus infection, all operations are performed in a dedicated biosafety cabinet. First, a pre-experiment isperformed to determine the MOI value of the cells. In the formal experiment, the virus isdiluted with complete medium according to the MOI value of the pre-experiment, and polybrene transfection reagent isadded to the virus dilution according to the instructions. Aspirate and discard the original cell medium, gently wash with PBS, add the diluted virus working solution, shake to mix, and put into the cell incubator for continuous culture. 12-24 hours after virus infection, in a dedicated biosafety cabinet, discard the waste liquid into an independent centrifuge tube, gently wash with PBS, replace with ordinary complete medium and continue culturing for 72 hours. According to the cell status and different resistances carried by the virus, add puromycin or G418 and other antibiotics for screening. After screening, verify the infection efficiency, freeze for standby after success, and then the cells are cultured with medium containing antibiotics. (10) MTS detection experiment

The method is the same as that in Embodiment 1. (11) Small molecule metabolite extraction experiment

The method is the same as that in Embodiment 1.

(12) Animal experiment LU602982

Materials and facilities related to experimental animals: BALB/C-nu/nu SPF female nude mice, 3-4 weeks old when purchased, weighing 16-18 g, purchased from Beijing Weitong Lihua

Experimental Animal Center. Animal experiments are carried out in the barrier environment of

Sun Yat-sen University Experimental Animal Center (Area D, North Campus), and the environmental technical indicators of the experimental room met the requirements of GB14925- 2010. This animal experiment isreviewed and approved by the Sun Yat-sen University

Experimental Animal Management and Use Committee and the Sun Yat-sen University

Experimental Animal Ethics Committee (experiment number: North-D2021-0363QX). A total of 80 immunodeficient female nude mice aged 3-4 weeks are selected and randomly divided into groups, with 8 mice in each group. After passing the quarantine, when the nude mice grew to 4 weeks old, subcutaneous tumor formation experiments are performed. MDA-MB-231 blank control knockdown cells, cells with stable HIBCH knockdown, cells with stable HIBCH knockdown and overexpression of LAT1, MCF-7 blank control overexpression cells, and LAT1 overexpression cells are inoculated into the fat pad of nude mice respectively. The inoculation amount of MDA-MB-231 is2x10° cells/100 uL, and that of MCF-7 is5x10° cells/100 uL. For

MCF-7, 17B-estradiol pills (0.72 mg, 60 d release) are subcutaneously implanted into mice 1 week before cell injection. Observe whether tumors are formed every three days after injection of tumor cells. After tumor formation, measure the longest and shortest diameters of the tumor with vernier calipers every 3 days. When the tumor grows to a diameter of about 6mm, perform gavage treatment with PI3Ka inhibitor BYL719 (100 mg/kg, once every 3 days). Each cell line has blank medication and PI3Ka inhibitor medication groups. After starting medication, the drinking water of nude mice isreplaced with sugar water containing tetracycline, protected from light, and the growth of tumors iscontinuously observed. The longest and shortest diameters of the tumor are measured with vernier calipers every 3 days. The tumor volume iscalculated using the formula: tumor volume = 0.5 x long diameter x short diameter“, and the tumor weight isweighed. Experimental endpoint: After injection of tumor cells, it depends on the tumor growth of different cell lines. The humane endpoint is that the animal is unable to eat, is ill, the short diameter of the tumor exceeds 1.5 cm, or has systemic metastasis that seriously affects survival, and other extremely uncomfortable symptoms. Mice are sacrificed by cervical dislocation.

Tumors are obtained by dissection, and mouse corpses are placed in the Animal Experiment

Center of Sun Yat-sen University North Campus for unified harmless treatment.

(13) Data processing LU602982

GraphPad Prism10 software isused for all result statistics and graphing. All continuous variables are expressed as mean+standard deviation, and categorical variables are expressed as percentages. For parametric variables, the two-tailed student's t-test isused for differences between two groups, and one-way analysis of variance (ANOVA) isused for differences between more than two groups. The chi-square test (x? test) isused for comparison of non- parametric variables. P<0.05 isconsidered statistically significant. 2. Experimental Results

Figs. 2A and B show that the expression level of HIBCH in PI3Ka-resistant 231R and MFC- 7R is unchanged compared with the parent cells. The immunofluorescence detection results in

Figs. 2C and D show that HIBCH is mainly located in the nucleus in 231R and MCF-7R. The results of ultra-high-resolution confocal microscope detection (Figs. 2E and F) show that HIBCH enters the nucleus after valine deprivation. ChlP-sequence detection showed that compared with normal culture conditions, valine deprivation allows HIBCH to enter the nucleus and enhance the regulation of gene transcription (Fig. 2G); among the transcription factors analyzed to bind to HIBCH, c-Myc ranks third (Fig. 2H). Further, co-immunoprecipitation experiments confirmed that the interaction between HIBCH and c-Myc is enhanced under valine deprivation, while the expression levels of HIBCH and c-Myc do not change during this process (Fig. 2I).

At the same time, under valine-deprived culture conditions, ChlP-sequence isused to detect changes in c-Myc transcriptional regulation. The results showed that valine deprivation may enhance the regulation of gene transcription by c-Myc (Fig. 2J), and in the normal culture group, c-Myc regulation of metabolism-related genes mainly focused on metabolic pathways related to glucose metabolism, while in the valine-deprived group, c-Myc regulation of various amino acid metabolism-related genes including branched-chain amino acids isenhanced, and regulation of glucose metabolism-related genes isweakened (Fig. 2K). Among them, the neutral amino acid transporter LAT1 is the most significantly regulated gene by c-Myc transcriptional regulation mode conversion. After HIBCH knockdown, the expression level of LAT1 is significantly decreased (Fig. 2L); under valine-deprived culture, the expression level of LAT1 gradually increases with the extension of deprivation time (Fig. 2M).

Cell viability detection showed that knockdown of HIBCH in 231R and MCF-7R cells may 602982 significantly improve cell sensitivity to BYL719, but overexpression of LAT1 while knocking down HIBCH inhibits cell sensitivity to BYL719, showing resistance to BYL719 (Figs. 2N-O).

Targeted metabolomics detection results found that knockdown of HIBCH leads to a significant decrease in branched-chain amino acid content and inhibition of the tricarboxylic acid cycle; overexpression of LAT1 not only increases the content of branched-chain amino acids, but also enhances the tricarboxylic acid cycle (Fig. 2P). When c-Myc isknocked down in 231R, the expression level of LAT1 issignificantly decreased (Fig. 2Q), and the transcriptional activity of

LAT1 isalso significantly inhibited (Fig. 2R). Cell sensitivity to BYL719 isalso improved, while simultaneous overexpression of LAT1 offset the effect of c-Myc knockdown (Figs. 2Q-R). Cell viability detection showed that knockdown of c-Myc in 231R and MCF-7R cells may significantly improve cell sensitivity to BYL719, but overexpression of LAT1 while knocking down c-Myc inhibits cell sensitivity to BYL719, showing resistance to BYL719 (Figs. 2S-T).

The results of animal experiments (Fig. 3) show that for MDA-MB-231 cells, which are less sensitive to BYL719, the sensitivity to BYL719 is significantly enhanced after stable HIBCH knockdown, and the growth of xenografts is significantly inhibited; in the group with stable

HIBCH knockdown and simultaneous overexpression of LAT1, the xenografts have poor responsiveness to BYL719, and tumor growth is not inhibited. For MCF-7, which has good responsiveness to BYL719, tumor growth is significantly inhibited; after overexpression of LAT1 in MCF-7, the sensitivity of xenografts to BYL719 is significantly decreased. It can be seen that

HIBCH entering the nucleus interacts with c-Myc, prompting c-Myc to change the transcription mode, and enhance the transcriptional activity and expression level of LAT1 to take in more branched-chain amino acids to supplement glucose metabolism.

Embodiment 3 LU602982 1. Experimental Methods (1) Mass spectrometry identification of post-translational modifications

First, the target protein ispurified with Flag antibody, the electrophoresis tank iscleaned in advance, the equipment issoaked in ultrapure water, the protein isseparated with precast gel, the target band isdisplayed after Coomassie brilliant blue staining, the target band iscut with a clean surgical blade, the band iscut into 1 mm?® pieces, decolorizing solution isadded and shaken at room temperature until transparent, 100% acetonitrile isadded and shaken for 5 min to make the gel particles white, acetonitrile isdrained, freeze-dried, 10 mM DTT/50 mM

NH4sHCO3 isadded, shaken and mixed, shaken at 56°C for 1h, 100% acetonitrile isadded and shaken for 5min to make the gel particles white, acetonitrile isdrained, freeze-dried, 60 mM

IAA/50mM NH4HCO; isadded, shaken and mixed in the dark, reacted in the dark for 30 min, 100% acetonitrile isadded and shaken for 5 min to make the gel particles white, acetonitrile isdrained, freeze-dried, 50-80 pL of 50 mM NH4HCO4 isadded, then 1-2 uL of trypsin isadded, and incubated with shaking at 37°C for more than 6 h. Add 0.1% FA to each tube and shake for 5 min, then add 0.1 FA/ACN and shake for 5min, combine the supernatants twice, spin dry at room temperature, then perform sample elution and on-machine operation. (2) Nuclear-cytoplasmic separation

The method is the same as that in Embodiment 2. (3) Co-IP experiment

The method is the same as that in Embodiment 2. (4) ChIP-PCR

The method is the same as that in Embodiment 2. (5) Cell transfection

The method is the same as that in Embodiment 1. (6) Total RNA extraction and RT-qPCR detection

The method is the same as that in Embodiment 2. (7) Western Blot

The method is the same as that in Embodiment 2. (8) Coomassie brilliant blue staining

The protein sample processing method and electrophoresis method are the same as those described in western blot above, except that after electrophoresis, a small part of the gel containing marker protein and a little sample needs to be cut for staining: put the gel into an appropriate amount of Coomassie brilliant blue staining solution to ensure that the staining solution can fully cover the gel. Place on a horizontal shaker or side-swing shaker and shake slowly, stain 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 thick and the temperature is low, the staining time should be appropriately prolonged.

If the gel is thin and the temperature is high, the staining time may be appropriately, 502982 shortened. After staining, pour out the staining solution, which may be recycled and reused at least 2-3 times. Then add an appropriate amount of Coomassie brilliant blue destaining solution to ensure that the destaining solution can fully cover the gel. Place on a horizontal shaker or side-swing shaker and shake rapidly, destain at room temperature for 4-24 hours. Replace the destaining solution 2-4 times during the period until the blue background is basically removed and the protein band staining effect meets the expectation. After destaining, the gel may be soaked in ddH20. (9) Dual luciferase reporter experiment

The method is the same as that in Embodiment 2. (10) MTS detection experiment

The method is the same as that in Embodiment 1. (11) Data processing

GraphPad Prism10 software isused for all result statistics and graphing. All continuous variables are expressed as mean + standard deviation, and categorical variables are expressed as percentages. For parametric variables, the two-tailed student's t-test isused for differences between two groups, and one-way analysis of variance (ANOVA) isused for differences between more than two groups. The chi-square test (x? test) isused for comparison of non- parametric variables. P<0.05 isconsidered statistically significant. 2. Experimental Results

HIBCH protein in cytoplasm and nucleus ispurified with Flag tag respectively in nuclear- cytoplasmic separation experiment. After SDS-Page electrophoresis, Coomassie brilliant blue staining isused, and the results are shown in Fig. 4. The band where HIBCH is located isseparated for mass spectrometry analysis, and it isfound that lysine 17 and lysine 55 of

HIBCH in the nucleus had succinylation modification (Figs. 5 and 6).

Further, combined with the protein database (Uniprot), the potential succinylation sites on

HIBCH protein are mutated one by one (K to A), and overexpression vectors of HIBCH wild type and mutant type are constructed. MDA-MB-231 cells are transfected with the above overexpression vectors respectively. Under valine deprivation, WB detection after nuclear- cytoplasmic separation showed that when the K55 site ismutated, the expression level of

HIBCH in the nucleus issignificantly decreased (Fig. 7A), and the level of HIBCH succinylation isalso significantly decreased (Fig. 7B).

Further, co-immunoprecipitation experiment isperformed on the basis of K55 mutation, and it isfound that the binding between HIBCH and c-Myc issignificantly weakened after K55 mutation (Fig. 7C). At the same time, mutation of HIBCH K55 site led to weakened transcriptional regulation of LAT1 by HIBCH and c-Myc (Figs. 7D-E).

Further, succinyltransferases catalyzing succinylation modification of HIBCH K55 are £02982 screened, and genes with potential succinyltransferase activity are knocked down one by one.

After 72 hours of valine-deprived culture, cells in each group are collected for nuclear- cytoplasmic separation experiment. WB detection of HIBCH localization found that after CPT1A knockdown, the expression level of HIBCH in the nucleus issignificantly decreased, while knockout of other genes did not affect the nuclear localization of HIBCH. At the same time, detection of HIBCH succinylation after CPT1A knockdown also confirmed that CPT1A catalyzes succinylation modification of HIBCH K55 site (Fig. 7F). Moreover, knockout of CPT1A significantly downregulated the binding of HIBCH and c-Myc to the LAT1 promoter region (Fig. 7G), and inhibited the transcriptional activity and expression level of LAT1 (Figs. 7H-I). Cell viability detection showed that knockout of CPT1A may significantly improve cell sensitivity to

BYL719, but simultaneous overexpression of LAT1 may offset the effect caused by CPT1A knockout (Fig. 7J).

Embodiment 4 1. Experimental Methods (1) Small molecule compound-protein molecular docking

The structure of HIBCH protein isdownloaded from the PDB database website (https://www.rcsb.org/), with its substrate binding sites as docking sites, including amino acids 121, 146, 169, 177, etc. The Quickprep module in MOE (version 2022) software isused to optimize the structure of HIBCH protein, hydrogenate the protein, complement the side chain amino acid structure, reset the protonation state of the protein under physiological pH conditions, set the temperature to 300 K, and place the protein in Amber10: EHT force field for optimization.

The Quickprep module of MOE isused to process the structure of small molecules, hydrogenate the small molecules, calculate the minimized energy of small molecules, and generate multi- conformation files of compounds. The small molecules with the best conformation are selected for docking experiments. The receptor file isgenerated by structure optimization, the docking region isdetermined according to the binding site, MOE-DOCK isused for docking, and the docking engine used Placement: TriangleMatcher. 300 docking modes are retained in the first round using London AG scoring function, and then each docking mode isoptimized using

GBVI/ISAG scoring function. Finally, the conformation with the best scoring value for each molecule isretained as the output file. For each compound, the top 5 conformations are retained according to the scoring system, and the lower the value, the better the affinity effect. (2) Immunofluorescence experiment

The method is the same as that in Embodiment 2. (3) MTS detection experiment

The method is the same as that in Embodiment 1.

(4) Organoid model construction LU602982

Fresh breast cancer tumor fragments are gently digested with 0.6 mg/mL dispase II, 1 mg/mL collagenase IV, 50 ug/ML deoxyribonuclease | and 10uM Y-27632 at 37°C for at least 1 hour with shaking. Tumor cells are centrifuged, washed twice with PBS, and cultured in BC organoid medium (KBR-1000, K2 oncology) containing 10% Matrigel (356230, Corning). (5) PDX xenograft model construction

To establish patient-derived xenograft models (PDX), breast cancer samples are collected from patients who underwent tumor resection surgery at Shantou Central Hospital from 2022 to 2024. Six-week-old NOD-SCID female mice are anesthetized with isoflurane. Breast cancer samples are cut into 1mm3 fragments and directly implanted into the mouse mammary fat pad to obtain first-generation PDX. When the first-generation PDX grew to 1cm in diameter, it istaken out, cut into 1 mm? fragments, and directly implanted into the mammary fat pad to obtain second-generation PDX for treatment. The experimental animal facility has been accredited by the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), and all animal experimental protocols used in the present invention have been approved by the Institutional Animal Care and Use Committee (IACUC) of Guangdong

Laboratory Animal Monitoring Institute. (6) Data processing

GraphPad Prism10 software isused for all result statistics and graphing. All continuous variables are expressed as mean+standard deviation, and categorical variables are expressed as percentages. For parametric variables, the two-tailed student's t-test isused for differences between two groups, and one-way analysis of variance (ANOVA) isused for differences between more than two groups. The chi-square test (x? test) isused for comparison of non- parametric variables. P<0.05 isconsidered statistically significant. 2. Experimental Results

Aiming at the active pocket of HIBCH K55, small molecule compound C7 with affinity for the active pocket isscreened from a library containing 1.5 million compounds through computer simulation and molecular docking technology. The screening process of small molecule inhibitors targeting HIBCH nuclear translocation is shown in Fig. 8A.

Immunofluorescence experiments confirmed that C7 may inhibit nuclear translocation of

HIBCH, and nuclear-cytoplasmic separation experiments confirmed that C7 inhibits succinylation modification of HIBCH. Fig. 8B shows that C7 inhibits HIBCH nuclear entry in a dose-dependent manner. Fig. 8C shows that C7 can specifically bind to lysine 55 of HIBCH protein. Further SPR detection confirmed that C7 has good affinity with HIBCH (Fig. 8D).

In MDA-MB-231 cells insensitive to PI3K inhibitor (BYL719), combination with C7 may 602982 reduce the |Cso of tumor cells to BYL719 and significantly improve sensitivity (Fig. 8E). A human breast cancer tissue-derived organoid model isconstructed to verify the synergistic lethal effect of C7 and BYL719. The results showed that regardless of whether PIK3CA mutation iscarried or not, C7 and BYL719 had a strong synergistic lethal effect in breast cancer organoid models insensitive to PI3Ka inhibitors (Figs. 8F-G). A human breast cancer-derived xenograft animal model isconstructed to verify the role of C7 and BYL719 in inhibiting tumor growth. The results showed that in breast cancer PDX models without PIK3CA mutations, whether it istriple- negative breast cancer originally insensitive to BYL719 or ER-positive/HER-2-negative breast cancer with certain sensitivity, C7 and BYL719 showed a good synergistic lethal effect, significantly inhibiting tumor growth (Figs. 8H-1).

The above-described embodiments are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Various modifications and improvements made to the technical solutions of the present invention by those of ordinary skill in the art without departing from the design spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims (10)

CLAIMS LU602982

1. An application of a compound for inhibiting nuclear translocation of HIBCH in the preparation of a medicament for reversing resistance to PI3K inhibitors in breast cancer.

2. The application according to claim 1, characterized in that the compound is C7, and the structural formula is as follows: FF { X A { \ à PEN TS NY es À $ ul i \ { N rg W \ y a

3. A medicament for reversing resistance to PI3K inhibitors in breast cancer, characterized in that the active ingredient comprises a compound for inhibiting nuclear translocation of HIBCH.

4. The medicament according to claim 3, characterized in that the compound is C7, and the structural formula is as follows: FF { X A { \ à PEN TS SoA ad X 4 i \ { N rg W \ y a

5. The medicament according to claim 4, characterized in that the medicament further comprises pharmaceutically acceptable excipients.

6. A composition for treating breast cancer, comprising a PI3Ka inhibitor and a compound that inhibits nuclear translocation of HIBCH.

7. The composition according to claim 6, characterized in that the compound is C7, and he 502982 structural formula is as follows: ; e OR, =] AN X FON A A 4 em 4 x NA ai a Q \ be

8. An application of a composition according to claim 6 or 7 in the preparation of a medicament for treating breast cancer.

9. A medicament for treating breast cancer, characterized in that the active ingredient comprises the composition according to claim 6 or 7.

10. The medicament according to claim 9, characterized in that the medicament further comprises pharmaceutically acceptable excipients.