Application of intracellular lactic acid level improver in preparation of medicine for enhancing sensitivity of BRAF V600E mutant tumor to BRAF inhibitor

Abstract

The invention provides application of an intracellular lactic acid level increasing agent in preparation of a drug for enhancing sensitivity of BRAF V600E mutant tumors to a BRAF inhibitor or in preparation of a drug for treating BRAF V600E mutant tumors resistant to the BRAF inhibitor. According to the invention, the intracellular lactic acid concentration is improved by using an intracellular lactic acid level improver, SUMOylation of BRAFV600E protein is promoted, and the BRAFV600E protein is locked in an open drug sensitive conformation; in-vivo and in-vitro experiments prove that the combination of the MCT4 inhibitor and the BRAF inhibitor (such as vimofenib) has a remarkable synergistic anti-tumor effect, drug resistance can be effectively relieved, and a new treatment strategy is provided for refractory colorectal cancer and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the use of intracellular lactate level enhancers in the preparation of medicaments for enhancing the sensitivity of BRAF V600E mutant tumors to BRAF inhibitors or in the preparation of medicaments for treating BRAF V600E mutant tumors resistant to BRAF inhibitors. Background Technology

[0002] BRAF gene mutations are among the most common oncogenic mutations in human tumors, with mutations at the V600E site accounting for approximately 90% of all BRAF mutations. BRAF (V600E) mutations lead to persistent activation of the MAPK signaling pathway, driving the development and progression of various tumors, such as melanoma, colorectal cancer, and thyroid cancer. BRAF inhibitors targeting this site (such as vemurafenib and dabrafenib) have shown significant efficacy in melanoma, but their efficacy is minimal in BRAF (V600E)-mutant colorectal cancer, often exhibiting primary or acquired resistance.

[0003] Current research on drug resistance mechanisms mainly focuses on feedback activation of signaling pathways (such as rebound activation of the EGFR signaling pathway). Clinically, multi-target combination strategies (such as BRAF inhibitors combined with EGFR antibodies) are commonly employed, but objective response rates remain limited. In recent years, tumor metabolic reprogramming (especially glycolysis and lactate metabolism) has been found to be closely related to drug resistance, but the specific mechanisms remain unclear. Current technologies have not yet revealed the direct molecular link between intracellular lactate levels and the conformation of the BRAF protein itself, as well as its drug-binding ability.

[0004] The BRAF gene encodes a serine / threonine protein kinase, a key regulatory molecule in the MAPK (mitogen-activated protein kinase) signaling pathway. Mutations at the V600 site of the BRAF gene (especially the V600E mutation) lead to persistent activation of this kinase, which in turn drives abnormal activation of downstream signaling pathways. This is closely related to the development and progression of various malignant tumors, including melanoma, colorectal cancer, thyroid cancer, and non-small cell lung cancer.

[0005] Several small molecule inhibitors, such as vemurafenib, dabrafenib, and encorafenib, have been developed clinically to target BRAF V600E mutations. These drugs have shown significant efficacy in the treatment of melanoma with BRAF V600E mutations. However, their clinical application exhibits significant differences across cancer types, particularly in BRAF (V600E)-mutant colorectal cancer (CRC), where the objective response rate for monotherapy is extremely low (less than 5%). Even with combinations of BRAF inhibitors and EGFR inhibitors, the objective response rate remains below 20%, and patients are highly susceptible to acquired resistance.

[0006] Current research on BRAF inhibitor resistance mechanisms primarily focuses on compensatory activation at the signaling pathway level (such as feedback activation of the EGFR signaling pathway and reactivation of the MEK / ERK pathway). While studies have shown that tumor metabolic reprogramming (especially enhanced glycolysis and lactate accumulation) plays a role in tumor resistance—for example, lactate, as a signaling molecule, may affect the activity of the de-SUMOylase SENP1—current techniques have not yet elucidated the relationship between lactate metabolism and BRAF. V600E The direct molecular link between conformational changes in the protein itself and its ability to bind to drug targets is unclear. Specifically, it remains unclear how fluctuations in lactate levels within tumor cells physically affect the structure of the BRAF protein at the molecular level, thereby preventing effective drug binding. Summary of the Invention

[0007] To address the technical problems in the prior art, the present invention provides the use of an intracellular lactate level enhancer in the preparation of a medicament that enhances the sensitivity of BRAF V600E mutant tumors to BRAF inhibitors or in the preparation of a medicament for treating BRAF V600E mutant tumors resistant to BRAF inhibitors.

[0008] The first aspect of the present invention provides the use of an intracellular lactate level enhancer in the preparation of a medicament for enhancing the sensitivity of BRAF V600E mutant tumors to BRAF inhibitors or in the preparation of a medicament for treating BRAF V600E mutant tumors resistant to BRAF inhibitors.

[0009] In one embodiment of the present invention, the intracellular lactate level enhancer is selected from at least one of lactate efflux inhibitors, glycolysis promoters, or hypoxia mimics.

[0010] In one embodiment of the present invention, the glycolysis promoter is glucose or a compound capable of upregulating the glycolysis pathway.

[0011] In one embodiment of the present invention, the hypoxia simulant is cobalt chloride.

[0012] In one embodiment of the present invention, the lactate efflux inhibitor is an MCT4 inhibitor.

[0013] In one embodiment of the present invention, the MCT4 inhibitor is selected from AZD0095, Syrosingapine, or a pharmaceutically acceptable salt, ester, solvate, or prodrug thereof.

[0014] In one embodiment of the present invention, the MCT4 inhibitor is AZD0095.

[0015] In one embodiment of the present invention, the BRAF inhibitor is selected from at least one of vemurafenib, dabrafenib, encofenib, or PLX8394.

[0016] In one embodiment of the present invention, the BRAF V600E mutant tumor is colorectal cancer, melanoma, thyroid cancer, or non-small cell lung cancer.

[0017] A second aspect of the present invention provides a pharmaceutical composition comprising: an intracellular lactate level enhancer, a BRAF V600E inhibitor, and a pharmaceutically acceptable carrier or excipient; wherein the intracellular lactate level enhancer is selected from lactate efflux inhibitors, glycolysis promoters, or hypoxia mimics.

[0018] In one embodiment of the present invention, the intracellular lactate level enhancer is the MCT4 inhibitor AZD0095, and the BRAF V600E inhibitor is vemurafenib.

[0019] A third aspect of the present invention provides a medicine box, comprising: Pharmaceutical formulations containing intracellular lactate level enhancers; Pharmaceutical formulations of BRAF V600E inhibitors; and manual.

[0020] Compared with the prior art, the embodiments of the present invention have the following beneficial effects: 1. The intracellular lactate level enhancer provided by this invention is used in the preparation of drugs that enhance the sensitivity of BRAF V600E mutant tumors to BRAF inhibitors or in the preparation of drugs for treating BRAF V600E mutant tumors resistant to BRAF inhibitors. It proposes a novel regulatory axis of "lactate efflux-SUMOylation modification-protein conformation-drug sensitivity." When BRAF V600E mutant tumor cells are attacked by drugs, they reduce intracellular lactate concentration by accelerating lactate efflux, leading to increased BRAF levels. V600EThe inhibition of SUMOylation modification of the protein (mainly at the K483 site) leads to a deSUMOylation process that causes the CAT and REG domains of the BRAF protein to bind tightly, forming a "closed" self-inhibiting conformation. This hinders the entry of drugs into the binding pocket, resulting in drug resistance. The intracellular lactate level enhancer provided in this invention promotes BRAF activity by inhibiting intracellular lactate levels. V600E SUMOylation locks the drug into an "open" drug-sensitive conformation, solving the drug resistance problem from the source of conformational biology and metabolic regulation.

[0021] 2. This invention verifies that lactate efflux is mainly mediated by monocarboxylic acid transporter 4 (MCT4). Experimental results show that MCT4-specific inhibitors (such as AZD0095) can significantly inhibit lactate efflux, allowing intracellular lactate concentrations to remain high even under BRAF inhibitor treatment, thus effectively reversing drug resistance. In contrast, MCT1-specific inhibitors (such as AZD3965) are ineffective in overcoming BRAF inhibitor resistance. This may be because the BRAF(V600E) mutant cells studied in this invention mainly rely on MCT4 for lactate efflux, and inhibiting MCT1 is insufficient to significantly increase or maintain high intracellular lactate levels (such as...). Figure 4 As shown in B), this also confirms the core concept of the present invention: only by substantially increasing the intracellular lactate level (such as by inhibiting the key transporter MCT4, or by supplementing glucose, or by simulating hypoxia) can drug sensitization be achieved.

[0022] 3. The pharmaceutical composition provided by this invention comprises an intracellular lactate level enhancer, a BRAF V600E inhibitor, and a pharmaceutically acceptable carrier or excipient. The combination of the intracellular lactate level enhancer and the BRAF inhibitor produces a significant synergistic antitumor effect, showing a breakthrough therapeutic prospect, especially for BRAF V600E-mutant colorectal cancer, which is currently almost untreatable. In vitro cell experiments show that the combination of MCT4 inhibitors (such as AZD0095) and BRAF inhibitors (vemurafenib, dabrafenib, etc.) exhibits a significant synergistic killing effect in various BRAF V600E-mutant cell lines, greatly reducing the half-maximal inhibitory concentration (IC50) of the BRAF inhibitor; particularly effective against vemurafenib-acquired resistance cell lines (RKO). VR The MCT4 inhibitor successfully restored the sensitivity of tumor cells to vemurafenib. In vivo animal models showed that in a human colorectal cancer RKO nude mouse xenograft model, the tumor growth inhibition rate (TGI) of the AZD0095 combined treatment group with vemurafenib was significantly higher than that of the single-drug group. Statistical analysis indicated that the actual inhibition rate of the combined treatment (60.73% based on volume and 50.12% based on mass) was significantly higher than the theoretical additive inhibition rate, confirming a strong synergistic effect between the two drugs.

[0023] 4. In view of the current clinical situation where BRAF V600E-mutant colorectal cancer has a low response rate to BRAF inhibitors and is almost untreatable, the pharmaceutical composition provided by this invention, which includes an intracellular lactate level enhancer and a BRAF V600E inhibitor, does not rely on complex immune microenvironment regulation and directly changes the conformation of cancer proteins through metabolic intervention. It has the characteristics of rapid onset of action and strong targeting, and has great clinical translation potential and application prospects. Attached Figure Description

[0024] Figure 1 For BRAF V600E Results of SUMOylation modification and identification of modification sites; among which, Figure 1 In section A, exogenous HA-BRAF was transfected into HEK293T cells. V600E Subsequently, SUMOylation modified bands were detected by immunoprecipitation (IP); among which, Figure 1 In RKO cells with endogenous BRAF knockout, HA-BRAF is mainly introduced as a secondary inhibitor. V600E Subsequently, endogenous SUMOylation modifications were detected; among which, Figure 1 C represents the analysis of wild-type BRAF and mutant BRAF using nickel-NTA pulldown chromatography. V600E and its BRAF K483R The mutants showed that the K483R mutation significantly reduced SUMOylation levels; among them, Figure 1 D in the image represents the mass spectrometry (LC-MS / MS) spectrum, confirming BRAF. V600E The peptide that underwent SUMOylation modification and the specific site was K483; Figure 2 For lactate metabolism of BRAF V600E Results of the regulatory role of protein SUMOylation modification; among which, Figure 2 In Figure A, the result is from immunoprecipitation. Immunoprecipitation verifies that glucose (which promotes aerobic / anaerobic metabolism) enhances BRAF. V600E SUMOylation modification; where, Figure 2 The result in B is an immunoprecipitation assay, which verifies that CoCL2 (a hypoxia inducer) promotes BRAF. V600E SUMOylation modification, Figure 2 The middle C represents the immunoprecipitation results, which show that treatment with BRAF (V600E) inhibitors (vemurafenib, dabrafenib, and encofenib) induced BRAF. V600E The SUMOylation level decreased; among them, Figure 2 The results of immunoprecipitation (D) show that immunoprecipitation validates the inhibition of BRAF by targeted drugs. V600E SUMOylation modification;

[0025] Figure 3 The effects of SUMOylation modification on the conformation (domain interactions) of the BRAF protein were shown; among them, Figure 3 Image A is a schematic diagram illustrating the experimental principle of NanoBRET (nanobioluminescent resonance energy transfer), demonstrating the interaction design between the donor (BRAF CAT domain) and the acceptor (BRAF REG domain); where, Figure 3 Figure B shows the NanoBRET test results, which indicate that in the SUMOylation defect (K483R) or low SUMOylation state, the binding ability of the REG domain and CAT domain of the BRAF protein is significantly enhanced (closed conformation), while normal SUMOylation modification hinders this binding (open conformation). Figure 4 The effects of BRAF inhibitors and lactate efflux inhibitors on intracellular lactate levels were shown; among them, Figure 4 In Figure A, the FiLa probe was used to detect the inhibitory effect of BRAF (V600E)-targeted drug on intracellular lactate in RKO cells; among which, Figure 4 In section B, the FiLa probe was used to detect the inhibitory effects of monocarboxylic acid transporter inhibitors AZD0095 (MCT4 inhibitor), Syrosingapine (MCT1 / 4 inhibitor), and AZD3965 (MCT1 inhibitor) on intracellular lactate efflux in RKO cells; among them, Figure 4 C represents the effect of the FiLa probe on the lactate-promoting effect of the AZD0095 reversal targeted drug on RKO cells; Figure 5 The study demonstrated the effect of SUMOylation deletion on the sensitivity of BRAF (V600E) inhibitors. Among these, Figure 5 In case A, BRAF was carried under vemurafenib treatment. V600E / K483R Survival rate of RKO cells with SUMOylation deficiency Figure 5 B was treated with dabrafenib and carried BRAF. V600E / K483R Survival rate of RKO cells with SUMOylation deficiency Figure 5 C represents the survival rate of RKO cells carrying BRAF V600E / K483R (SUMOylation defect) under encofenib treatment; vemurafenib ( Figure 5 (A) Dabrafenib Figure 5 (B) and Ncofini ( Figure 5 Under C) processing, carrying BRAF V600E / K483R RKO cells (sumoization defective) had significantly higher survival rates than those carrying BRAF. V600E The cells showed that the K483R mutation led to decreased drug sensitivity; Figure 6The effects of different glucose concentrations (metabolic levels) on vemurafenib sensitivity were shown; among them, Figure 6 In section A, RKO BRAF was cultured under glucose-free conditions. V600E With RKO BRAF V600E / K483R Dose-response curves of cells to vemurafenib. Figure 6 Under the condition of 5 mM glucose culture, RKO BRAF-V600E and RKO BRAF V600E / K483R Dose-response curves of cells to vemurafenib. Figure 6 RKO BRAF was cultured under conditions where C was 5 mM glucose. V600E With RKO BRAF V600E / K483R Dose-response curves of cells to vemurafenib; the results showed that under high glucose conditions, cells with normal SUMOylation ability were most sensitive to the drug, while K483R mutant cells remained resistant to the drug. Figure 7 The results showed the in vitro synergistic effect of the MCT4 inhibitor AZD0095 combined with the BRAF (V600E) inhibitor; among which, Figure 7 In Figure A, the survival rate curve of RKO cells treated with a combination of AZD0095 and vemurafenib is shown. Figure 7 Figure B shows the survival rate curve of RKO cells treated with a combination of AZD0095 and dabrafenib. Figure 7 C represents the survival rate curve of RKO cells treated with a combination of AZD0095 and encofenib; the results show that the combination therapy was significantly superior to the single-drug treatment. Figure 8 The study demonstrated the resistance of MCT inhibitors to vemurafenib-acquired resistant cell lines (RKO). VR The reversal effect of ); among which, Figure 8 In the formula, AZD0095, an MCT4 inhibitor, combined with vemurafenib, can significantly kill drug-resistant RKO cells. VR ; Figure 8 In case B, the combination of the MCT1 inhibitor AZD3965 and vemurafenib was ineffective. Figure 8 Syrosingapine, an MCT1 / 4 inhibitor, combined with vemurafenib, can effectively kill drug-resistant RKO cells. VR ; Figure 9 The in vivo efficacy of AZD0095 in combination with vemurafenib for the treatment of RKO xenografts in nude mice was demonstrated; among which, Figure 9 Figure A shows the body weight change curves of nude mice in each group during the drug administration period. Figure 9 Figure B shows the tumor volume growth curves for each group of nude mice. Figure 9 The graph in the middle (C) represents the statistical chart of tumor weight in each group at the experimental endpoint. Figure 9The comparison of tumor inhibition rates between combination therapy and monotherapy, and the analysis of synergistic effects of combination therapy, are presented in section D. Figure 9 E in the image represents a visual photograph of the tumor removed at the experimental endpoint. Detailed Implementation

[0026] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0027] The present invention will now be further described with reference to embodiments, but these embodiments should not be considered as limitations on the invention, but rather as a more detailed description of certain aspects, features, characteristics, and embodiments of the invention. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as plasmid construction as described in Cold Spring Harbor Laboratory's *Antibody Technology Laboratory Manual* or *Molecular Cloning Manual*, or under conditions recommended by the raw material or commercial manufacturer. Reagents whose specific source is not specified are commercially available, conventional reagents.

[0028] Various modifications and variations can be made to the specific embodiments or methods described in this invention without departing from the scientific scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this invention will also be apparent to those skilled in the art. This invention specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” and “contain” used in this disclosure are all open-ended terms, meaning they include but are not limited to.

[0030] The main compounds, pharmaceuticals, and reagents disclosed herein are all commercially available products. AZD0095 (catalog number: E1549); AZD3965 (catalog number: S7339); AZ-33 (catalog number: S0108); vemurafenib (catalog number: S1267); dabrafenib (catalog number: S2807); and encofenib (catalog number: S7108) were all purchased from Selleck, Inc., USA. The NanoBRET® Transcriptional Protein Assays kit (catalog number: N1870) was purchased from Promega Corporation, Inc., USA. The construction and usage of FiLa and FiLa-C probes were performed according to the literature (Aoxue Wang, et al., Nat Protoc. 2024 Feb 2;19(5):1311-1347).

[0031] In this invention, the term "intracellular lactate level enhancer" refers to any substance, compound, or preparation that can increase the intracellular lactate concentration in tumor cells relative to the untreated state through physiological, pharmacological, or genetic means. Mechanisms for increasing intracellular lactate levels include, but are not limited to: (1) inhibiting lactate efflux, for example by inhibiting monocarboxylic acid transporters (especially MCT4) responsible for lactate transport, thereby blocking lactate transport out of the cell; (2) promoting lactate production, for example by promoting glycolysis pathways (e.g., increasing the supply of substrate glucose, or using drugs to upregulate the activity of key glycolytic enzymes), or by simulating a hypoxic environment (e.g., using hypoxia mimics) to enhance anaerobic metabolism and lactate production. The inventors have discovered that, regardless of the pathway, as long as a high level of intracellular lactate can be maintained, SUMOylation modification of the BRAF V600E protein can be promoted, thereby maintaining its drug-sensitive conformation.

[0032] Example 1 BRAF V600E Validation and site identification of protein SUMOylation modification 1. Detection of exogenous BRAF SUMOylation Modification (1) Cell transfection and treatment HEK293T cells were seeded in 6 cm culture dishes at a density of 1.6 × 10⁻⁶. 6 Cells / dish. When the cell confluence reaches about 80%, HA-BRAF, Flag-SUMO1, Myc-Ubc9, and EGB-SENP1 plasmids are co-transfected using liposome transfection. 24 hours after transfection, half the volume of fresh culture medium is added and the cells are cultured for another 24 hours.

[0033] (2) Cell lysis and protein extraction Discard the culture medium and wash the cells twice with PBS. Add 200 μL of 2% SDS denaturing lysis buffer to each dish, ensuring the lysis buffer fully covers the cells. Scrape off the cells and transfer them to microcentrifuge tubes. Boil at 100°C for 10 minutes, then cool to room temperature. Sonicate for 1.5 minutes (40% power, on for 4 seconds, off for 3 seconds), dilute with 200 μL of PBS, centrifuge at 12,000 × g for 15 minutes, and collect the supernatant as the total protein extract. Use a small amount of the supernatant for input and 100 μL for immunoprecipitation.

[0034] (3) Immunoprecipitation (IP) Dilute 100 μL of sample with 900 μL of Pre-RIPA lysis buffer, add 15 μL of agarose beads with pre-coupled flag for immunoprecipitation, and incubate overnight (approximately 16 hours) at 4°C with slow rotation. Centrifuge at 3,000 ×g for 5 minutes at 4°C and discard the supernatant. Wash the beads three times with 1 mL of RIPA lysis buffer, rotating for 20 minutes each time.

[0035] (4) Elution and Western Blot Detection After the final wash, aspirate any remaining liquid. Add 40 μL of 2× SDS-PAGE loading buffer (containing 5% β-mercaptoethanol), boil at 100°C for 10 minutes, centrifuge, and collect the supernatant as the eluted sample. Perform SDS-PAGE electrophoresis on the input sample and the IP eluted sample, transfer to a PVDF membrane, block with 5% skim milk for 1 hour, and incubate with primary antibody overnight at 4°C. The next day, after thorough washing, incubate with HRP-labeled secondary antibody for 1 hour. Develop with chemiluminescent substrate and acquire signals using an imaging system.

[0036] 2. BRAF V600E Identification of endogenous SUMOylation (1) Cell lysis and protein extraction Stable expression of HA-BRAF V600E RKO cells were seeded in 6 cm culture dishes at a density of 2 × 10⁻⁶ cells / mL. 6 Cells / plate. The next day, when cell confluence reached approximately 90%, discard the culture medium and wash the cells twice with PBS. Add 300 μL of 2% SDS denaturing lysis buffer to each plate, ensuring the lysis buffer fully covers the cells. Scrape off the cells and transfer them to microcentrifuge tubes. Boil at 100°C for 10 minutes, then cool to room temperature. Sonicate for 1.5 minutes (40% power, 4 seconds on, 3 seconds off), centrifuge at 12,000 × g for 15 minutes, and collect the supernatant as the total protein extract. Use a small amount of the supernatant for input and 100 μL for immunoprecipitation.

[0037] (2) Immunoprecipitation (IP) Dilute 100 μL of sample with 900 μL of Pre-RIPA lysis buffer, add 5 μL of rabbit anti-SUMO1 antibody, and add an equal volume of normal rabbit IgG as a negative control to another equal volume of lysis buffer. Add 50 μL of protein A / G agarose beads pre-equilibrated with dilution buffer for immunoprecipitation, and incubate overnight (approximately 16 hours) at 4°C with slow rotation. Centrifuge at 3,000 ×g for 5 minutes at 4°C and discard the supernatant. Wash the beads three times with 1 mL of RIPA lysis buffer, rotating for 20 minutes each time.

[0038] 3. Identification of BRAF and BRAF by nickel column affinity precipitation method V600E SUMOylation sites of proteins (1) Cell transfection and treatment HEK293T cells were seeded in 6 cm culture dishes at a density of 1.6 × 10⁻⁶. 6Cells / dish. When the cell confluence reaches about 80%, His-SUMO1, Myc-Ubc9, HA-BRAF (wild type and three mutants), and plasmids are co-transfected using liposome transfection. 24 hours after transfection, half the volume of fresh culture medium is added and the cells are cultured for another 24 hours.

[0039] (2) Cell harvesting and grouping lysis Discard the culture medium and wash the cells once with pre-chilled PBS. Aspirate the PBS, add 1 mL of PBS to each dish, scrape the cells off with a cell scraper, and collect the suspension into a 1.5 mL centrifuge tube. Aliquot each cell suspension into two centrifuge tubes (tube A and tube B). Tube A (approximately 90% cells, for Ni-NTA precipitation): After centrifugation, discard the supernatant and immediately add 1 mL of pre-chilled Ni-NTAWash Buffer 1 (15 mM imidazole), and quickly disperse the cell clumps with a pipette (do not vortex). Tube B (approximately 10% cells, for input control): After centrifugation, discard the supernatant. Add 100 μL of 2% SDS denaturation and lysis buffer.

[0040] (3) Input Sample Preparation Boil the sample in tube B at 100°C for 10 minutes, then cool to room temperature. Sonicate the sample for 1.5 minutes (40% power, 4 seconds on, 3 seconds off), then centrifuge at 12,000×g for 15 minutes. Collect the supernatant as the input sample. Add 25 μL of 5× SDS-PAGE loading buffer (containing 5% β-mercaptoethanol) and heat at 100°C for 10 minutes. Store at -80°C for later use.

[0041] (4) Ni-NTA pulldown Place the sample in tube A in an ice-water bath and sonicate for 3 minutes (40% power, 4s on, 3s off). Centrifuge at 12,000 rpm for 15 minutes at 4°C, and transfer the supernatant to a new tube. Add 20 μL of Ni-NTA Agarose Resin to the supernatant. Incubate overnight at 4°C (approximately 16 hours). Wash the resin sequentially with the following buffers, rotating for 20 minutes (70 rpm) at room temperature each time, centrifuging at 3,000 × g for 5 minutes at 4°C. Carefully aspirate most of the supernatant, retaining approximately 50-80 μL of liquid to prevent the resin from drying out. After the final wash, aspirate as much liquid as possible. Add 40 μL of Ni-NTA pulldown elution buffer and 10 μL of 5 × SDS-PAGE loading buffer (containing 5% β-mercaptoethanol) to the resin, and heat at 100°C for 10 minutes. After centrifugation, collect the supernatant, which is the Ni-NTA precipitate eluted sample.

[0042] a. Ni-NTA pulldown Wash Buffer 1 (15 mM imidazole): Wash once.

[0043] b. Ni-NTA pulldown Wash Buffer 2: Wash once.

[0044] c. Ni-NTA pulldown Wash Buffer 3: Wash once.

[0045] d. Ni-NTA pulldown Wash Buffer 4: Wash once.

[0046] (5) Western Blot detection Input samples and Ni-NTA precipitated eluted samples were subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% skim milk for 1 hour, and incubated overnight at 4°C with primary antibody. The next day, after thorough washing, the membrane was incubated with HRP-labeled secondary antibody for 1 hour. Development was performed using a chemiluminescent substrate, and signals were acquired using an imaging system.

[0047] 4. Identify BRAF SUMO1 modification sites using immunoprecipitation combined with mass spectrometry (LC-MS / MS).

[0048] (1) Cell transfection and treatment HEK293T cells were seeded in 6 cm culture dishes at a density of 1.6 × 10⁻⁶. 6 Cells / plate. When the cell confluence reaches approximately 80%, HA-BRAF is co-transfected using liposome transfection. V600E The Flag-SUMO1 and Myc-Ubc9 plasmids were transfected, and 24 hours later, the medium was replaced with fresh medium containing 100 μM CoCl2 and cultured for another 24 hours.

[0049] (2) Cell lysis and protein extraction Discard the culture medium and wash the cells twice with PBS. Add 500 μL of 2% SDS denaturing lysis buffer to each dish, ensuring the lysis buffer fully covers the cells. Scrape off the cells and transfer them to microcentrifuge tubes. Boil at 100°C for 10 minutes, then cool to room temperature. Sonicate for 1.5 minutes (40% power, on for 4 seconds, off for 3 seconds), dilute with 500 μL of PBS, centrifuge at 12,000×g for 15 minutes, and collect the supernatant as the total protein extract (approximately 1 mL). Use all of this extract for immunoprecipitation.

[0050] (3) Immunoprecipitation (IP) Dilute 1 mL of sample with 9 mL of Pre-RIPA lysis buffer, add 150 μL of flag-coupled agarose beads for immunoprecipitation, and incubate overnight (approximately 16 hours) at 4°C with slow rotation. Centrifuge at 3,000 ×g for 5 minutes at 4°C and discard the supernatant. Wash the beads three times with 5 mL of RIPA lysis buffer, rotating for 20 minutes each time. After the final wash, aspirate any remaining liquid. Transport to the company for mass spectrometry analysis on dry ice.

[0051] The results showed that after overexpressing plasmids HA-BRAF, Flag-SUMO1, Myc-Ubc9, and EGB-SENP1 in HEK293T cells, a significant SUMOylation band of BRAF was detected in the IP-Flag product. Figure 1 (A) Knockout of endogenous BRAF and reintroduction of HA-BRAF in RKO cells. V600E Subsequently, endogenous SUMOylation modification was also detected. Figure 1 (B) The mutant HA-BRAF was detected using a nickel column pulldown method. K483R HA-BRAF K483R / V600E The SUMOification level compared to HA-BRAF WT and HA-BRAF V600E All significantly reduced ( Figure 1 (C) Prove that K483R is a BRAF WT and BRAF V600E The main SUMO1 modification site. Mass spectrometry analysis also confirmed K483 as an important BRAF SUMO1 modification site. Figure 1 (D).

[0052] Example 2 Lactic acid metabolism regulation BRAF V600E SUMOylation of proteins.

[0053] 1. Glucose or 2-DG on BRAF V600E The effects of protein SUMOylation modification.

[0054] Cell transfection and processing HEK293T cells were seeded in 6 cm culture dishes at a density of 1.6 × 10⁻⁶. 6 Cells / plate. When the cell confluence reaches approximately 80%, HA-BRAF is co-transfected using liposome transfection. V600E The cells were transfected with Flag-SUMO1 and Myc-Ubc9 plasmids. Twenty-four hours after transfection, the medium was replaced with fresh medium containing pre-prepared 0, 10, and 20 mM glucose, or fresh medium containing 0, 10, and 20 mM 2-DG, respectively. The cells were then returned to the incubator (37°C, 5% CO2) and treated continuously for 24 hours.

[0055] (2) Cell lysis and protein extraction Discard the culture medium and wash the cells twice with PBS. Add 500 μL of 2% SDS denaturing lysis buffer to each dish, ensuring the lysis buffer fully covers the cells. Scrape off the cells and transfer them to microcentrifuge tubes. Boil at 100°C for 10 minutes, then cool to room temperature. Sonicate for 1.5 minutes (40% power, on for 4 seconds, off for 3 seconds), dilute with 500 μL of PBS, centrifuge at 12,000×g for 15 minutes, and collect the supernatant as the total protein extract (approximately 1 mL). Use all of this extract for immunoprecipitation.

[0056] (3) Immunoprecipitation (IP) Dilute 1 mL of sample with 9 mL of Pre-RIPA lysis buffer, add 150 μL of flag-coupled agarose beads for immunoprecipitation, and incubate overnight (approximately 16 hours) at 4°C with slow rotation. Centrifuge at 3,000 ×g for 5 minutes at 4°C and discard the supernatant. Wash the beads three times with 5 mL of RIPA lysis buffer, rotating for 20 minutes each time. After the final wash, aspirate any remaining liquid. Transport to the company for mass spectrometry analysis on dry ice.

[0057] 2. The effect of the hypoxia inducer CoCl2 on BRAF V600E The effects of protein SUMOylation modification.

[0058] Cell transfection and processing HEK293T cells were seeded in 6 cm culture dishes at a density of 1.6 × 10⁻⁶. 6 Cells / plate. When the cell confluence reaches approximately 80%, HA-BRAF is co-transfected using liposome transfection. V600E The cells were transfected with Flag-SUMO1 and Myc-Ubc9 plasmids. Twenty-four hours later, the culture medium was replaced with pre-prepared fresh medium containing 0, 50 μM, and 100 μM CoCl2, respectively. The cells were then returned to the incubator (37°C, 5% CO2) and treated continuously for 24 hours.

[0059] Cell lysis and protein extraction Discard the culture medium and wash the cells twice with PBS. Add 500 μL of 2% SDS denaturing lysis buffer to each dish, ensuring the lysis buffer fully covers the cells. Scrape off the cells and transfer them to microcentrifuge tubes. Boil at 100°C for 10 minutes, then cool to room temperature. Sonicate for 1.5 minutes (40% power, on for 4 seconds, off for 3 seconds), dilute with 500 μL of PBS, centrifuge at 12,000×g for 15 minutes, and collect the supernatant as the total protein extract (approximately 1 mL). Use all of this extract for immunoprecipitation.

[0060] Immunoprecipitation (IP) Dilute 1 mL of sample with 9 mL of Pre-RIPA lysis buffer, add 150 μL of flag-coupled agarose beads for immunoprecipitation, and incubate overnight (approximately 16 hours) at 4°C with slow rotation. Centrifuge at 3,000 ×g for 5 minutes at 4°C and discard the supernatant. Wash the beads three times with 5 mL of RIPA lysis buffer, rotating for 20 minutes each time. After the final wash, aspirate any remaining liquid. Transport to the company for mass spectrometry analysis on dry ice.

[0061] 3. BRAF-targeting inhibitors on BRAF V600E Effects of protein SUMOylation Cell transfection and processing HEK293T cells were seeded in 6 cm culture dishes at a density of 1.6 × 10⁻⁶. 6 Cells / plate. When the cell confluence reaches approximately 80%, HA-BRAF is co-transfected using liposome transfection. V600E The cells were transfected with the Flag-SUMO1 and Myc-Ubc9 plasmids. After 24 hours, half the volume of fresh culture medium was added and the cells were cultured for another 24 hours. Two hours before receiving the samples, the cells were treated with the BRAF inhibitors vemurafenib (10 μM), dabrafenib (1 μM), and encorafenib (1 μM) for 2 hours.

[0062] Cell lysis and protein extraction Discard the culture medium and wash the cells twice with PBS. Add 500 μL of 2% SDS denaturing lysis buffer to each dish, ensuring the lysis buffer fully covers the cells. Scrape off the cells and transfer them to microcentrifuge tubes. Boil at 100°C for 10 minutes, then cool to room temperature. Sonicate for 1.5 minutes (40% power, on for 4 seconds, off for 3 seconds), dilute with 500 μL of PBS, centrifuge at 12,000×g for 15 minutes, and collect the supernatant as the total protein extract (approximately 1 mL). Use all of this extract for immunoprecipitation.

[0063] Immunoprecipitation (IP) Dilute 1 mL of sample with 9 mL of Pre-RIPA lysis buffer, add 150 μL of flag-coupled agarose beads for immunoprecipitation, and incubate overnight (approximately 16 hours) at 4°C with slow rotation. Centrifuge at 3,000 ×g for 5 minutes at 4°C and discard the supernatant. Wash the beads three times with 5 mL of RIPA lysis buffer, rotating for 20 minutes each time. After the final wash, aspirate any remaining liquid. Transport to the company for mass spectrometry analysis on dry ice.

[0064] 4. Effects of lactate efflux inhibitors on BRAF V600E Effects of protein SUMOylation Cell transfection and processing HEK293T cells were seeded in 6 cm culture dishes at a density of 1.6 × 10⁻⁶.6 Cells / plate. When the cell confluence reaches approximately 80%, HA-BRAF is co-transfected using liposome transfection. V600E The cells were transfected with Flag-SUMO1 and Myc-Ubc9 plasmids. Twenty-four hours after transfection, the medium was replaced with pre-prepared fresh culture medium containing AZ-33 (1 μM, 10 μM), MCT4 inhibitor AZD0095 (1 μM, 10 μM), reserpine ethyl acetate (1 μM, 10 μM), or MCT1 inhibitor AZD3965 (1 μM, 10 μM), respectively. The cells were then returned to the incubator (37°C, 5% CO2) and treated continuously for 24 hours.

[0065] Cell lysis and protein extraction Discard the culture medium and wash the cells twice with PBS. Add 500 μL of 2% SDS denaturing lysis buffer to each dish, ensuring the lysis buffer fully covers the cells. Scrape off the cells and transfer them to microcentrifuge tubes. Boil at 100°C for 10 minutes, then cool to room temperature. Sonicate for 1.5 minutes (40% power, on for 4 seconds, off for 3 seconds), dilute with 500 μL of PBS, centrifuge at 12,000×g for 15 minutes, and collect the supernatant as the total protein extract (approximately 1 mL). Use all of this extract for immunoprecipitation.

[0066] Immunoprecipitation (IP) Dilute 1 mL of sample with 9 mL of Pre-RIPA lysis buffer, add 150 μL of flag-coupled agarose beads for immunoprecipitation, and incubate overnight (approximately 16 hours) at 4°C with slow rotation. Centrifuge at 3,000 ×g for 5 minutes at 4°C and discard the supernatant. Wash the beads three times with 5 mL of RIPA lysis buffer, rotating for 20 minutes each time. After the final wash, aspirate any remaining liquid. Transport to the company for mass spectrometry analysis on dry ice.

[0067] The results showed that high glucose or CoCl2 treatment significantly enhanced BRAF. V600E SUMOification ( Figure 2 Treatment with BRAF (V600E)-targeting inhibitors vemurafenib, dabrafenib, and encolafenib can all reduce BRAF levels. V600E SUMOification level ( Figure 2 (C). AZD0095 (MCT4 inhibitor) can increase BRAF. V600E The SUMOylation level was significantly reduced, while AZD3965 (an MCT1 inhibitor) had no significant effect. Figure 2 (Middle D). These results suggest that intracellular lactate accumulation is a promoter of BRAF. V600E SUMOylation of proteins is a key factor, and inhibiting lactate efflux (e.g., using MCT4 inhibitors) is essential for maintaining BRAF. V600E An effective way to modify SUMOylation.

[0068] Example 3 The effect of SUMOylation on the conformation of BRAF protein was verified using bioluminescent resonance energy transfer (BRET) technology.

[0069] NanoBRET™ is a live-cell protein interaction detection system based on bioluminescent resonance energy transfer (BRET). The experimental principle is as follows: Figure 3 As shown in Figure A, its core components include: a. Donor: fused to BRAF using NanoLuc® luciferase. CAT a. Protein: Upon addition of a substrate (such as Nano-Glo® Substrate), it emits blue light (peak at 460nm); b. Acceptor: Using HaloTag® protein, fused to BRAF REG On the protein. Pre-labeled with HaloTag® NanoBRET™ 618 fluorescent ligand, which emits fluorescence at 618 nm; c. Energy transfer conditions: when the protein BRAF CAT With protein BRAF REG When the donor and acceptor interact or come into close proximity (typically ≤10 nm), a nonradiative energy transfer occurs. The blue light energy emitted by the donor is absorbed by the acceptor, exciting the acceptor's fluorescent ligand to emit red light (618 nm).

[0070] (1) Cell transfection and treatment HEK293T cells were seeded in 6 cm culture dishes at a density of 1.6 × 10⁻⁶. 6 Cells / plate. When the cell confluence reaches approximately 80%, co-transfect His-SUMO1, Myc-Ubc9, and NL-BRAF-CAT using liposome transfection. WT NL-BRAF-CAT K483R NL-BRAF-CAT V600E NL-BRAF-CAT V600E / K483R BRAF-REG-HT plasmid was transfected. 24 hours later, HEK293T cells were digested, and a portion was resuspended in assay medium (100 nM 618 ligand group and no-ligand group) and reseeded into white transparent 96-well cell culture plates, approximately 8 × 10⁶ cells per well. 4 One cell per well, 100 μL, with at least 3-4 replicates per group; another portion is reseeded into 6 cm dishes, approximately 2 × 10⁶ cells per dish. 6 One cell, continue culturing for 24 hours.

[0071] (2) Cell lysis, protein extraction and Western Blot detection Cell lysis and protein extraction were performed in 6cm dishes for Western blot analysis. The culture medium was discarded, and cells were washed twice with PBS. 200 μL of 2% SDS denaturing lysis buffer was added to each dish, ensuring the buffer completely covered the cells for lysis. Cells were scraped off and transferred to microcentrifuge tubes. The tubes were boiled at 100°C for 10 minutes and cooled to room temperature. Sonication was performed for 1.5 minutes (40% power, 4 seconds on, 3 seconds off), followed by centrifugation at 12,000×g for 15 minutes. The supernatant was collected as the protein extract. 50 μL of 5× SDS-PAGE loading buffer (containing 5% β-mercaptoethanol) was added, and the tubes were boiled at 100°C for 10 minutes. SDS-PAGE electrophoresis was then performed, and the cells were transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 1 hour and incubated with primary antibody overnight at 4°C. The next day, after thorough washing, the membranes were incubated with HRP-labeled secondary antibody for 1 hour. Chemiluminescent substrate was used for development, and the signal was acquired using an imaging system.

[0072] (3) BRET signal detection was performed on cells in a 96-well plate.

[0073] a. Substrate solution preparation: Prepare a 5X concentration (5X) NanoBRET™ Nano-Glo® substrate solution in phenol red-free Opti-MEM™ I low serum medium, which is a 100-fold dilution of the stock reagent.

[0074] b. Substrate addition and mixing: Add 25 μL of the above substrate solution to the cells in the 96-well plate, and then shake the culture plate for 30 seconds to achieve uniform mixing.

[0075] c. Endpoint detection: Within 10 minutes after substrate addition, use a chemiluminescence analyzer with dual-channel detection to simultaneously measure the 460nm signal (donor fluorescence) and the 618nm signal (acceptor fluorescence).

[0076] Calculate the NanoBRET™ ratio: NanoBRET™ Ratio = Signal / (Signal Ratio) 618nm / Signal 460nm To eliminate background interference, the ratio of the ligand-free control group needs to be subtracted to obtain the corrected NanoBRET™ ratio.

[0077] like Figure 3 As shown in Figure B, SUMOylation (at site K483) significantly weakens the interaction between the REG and CAT domains of the BRAF protein. When this modification is absent, the two domains bind more tightly (enhanced "closed" conformation). Conversely, SUMOylation promotes domain separation, resulting in a more "open" protein conformation. These results indicate that SUMOylation stabilizes the BRAF protein in an "open" conformation by blocking the CAT-REG domain interaction.

[0078] Example 4 Lactate efflux inhibitors can reverse the decrease in intracellular lactate levels caused by BRAF (V600E) inhibitors.

[0079] 1. To verify the effect of BRAF (V600E) inhibitor treatment on lactate concentration in RKO cells. (1) Grouping and processing: RKO-FiLa-C Sensor and RKO-FiLa Sensor cells were divided into four groups each. Control group: DMSO was added.

[0080] Vemurafenib treatment group: Vemurafenib was added to a final concentration of 5 μM.

[0081] Dabrafenib treatment group: Dabrafenib was added to a final concentration of 5 μM.

[0082] Encorafenib treatment group: Encorafenib was added to a final concentration of 1 μM.

[0083] (2) After 24 hours of drug treatment, confocal imaging was performed. The cell fluorescence intensity at excitation wavelengths of 488 and 405 was calibrated using Image J, and the relative ratio was quantitatively calculated.

[0084] 2. Evaluate the effects of different specific monocarboxylic acid transporter inhibitors on intracellular lactate levels under basal conditions. (1) Grouping and processing: The RKO-FiLa Sensor cells were divided into five groups: Control group: DMSO was added.

[0085] AZD3965 treatment group: AZD3965 was added to a final concentration of 0.1 μM and 1 μM.

[0086] AZD0095 treatment group: AZD0095 was added to a final concentration of 0.1 μM and 1 μM.

[0087] Syrosingapine treatment group: Syrosingapine was added at final concentrations of 0.1 μM and 1 μM.

[0088] AZ-33 treatment group: AZ-33 was added to a final concentration of 0.1 μM and 1 μM.

[0089] (2) After 24 hours of drug treatment, confocal imaging was performed. The cell fluorescence intensity at excitation wavelengths of 488 and 405 was calibrated using Image J, and the relative ratio was quantitatively calculated.

[0090] 3. Verify the effect of MCT inhibition on intracellular lactate recovery induced by BRAF (V600E) inhibitors.

[0091] (1) Grouping and processing: The RKO-FiLa Sensor cells were divided into four groups: Control group: DMSO was added. DMSO group: DMSO was added; AZD0095 group: 1 μM AZD0095 was added.

[0092] Vemurafenib treatment group: Vemurafenib was added to a final concentration of 5 μM. DMSO group: DMSO was added. AZD0095 group: Add 1 μM AZD0095.

[0093] Dabrafenib treatment group: Dabrafenib was added to a final concentration of 5 μM. DMSO group: DMSO was added. AZD0095 group: Add 1 μM AZD0095.

[0094] Encorafenib treatment group: Encorafenib was added to a final concentration of 1 μM. DMSO group: DMSO was added; AZD0095 group: 1 μM AZD0095 was added.

[0095] (2) After 24 hours of drug treatment, confocal imaging was performed. The cell fluorescence intensity at excitation wavelengths of 488 and 405 was calibrated using Image J, and the relative ratio was quantitatively calculated.

[0096] The results showed that BRAF (V600E) inhibitors could trigger a decrease in lactate concentration in colorectal cancer cells. Figure 4 Inhibition of MCT4 can completely block the BRAF (V600E) inhibitor-induced decrease in intracellular lactate. Figure 4 (Middle B and C). This demonstrates that lactate efflux inhibitors (especially MCT4 inhibitors) can reverse the lactate decrease induced by BRAF (V600E) inhibitors.

[0097] Example 5 The effect of SUMOylation on the sensitivity of BRAF (V600E) inhibitors.

[0098] Quantitative assessment of BRAF V600E The effect of SUMOylation on the sensitivity of vemurafenib, dabrafenib and encorafenib.

[0099] (1) Cell inoculation RKO BRAF will be in the logarithmic growth phase. V600E and RKO BRAF V600E / K483RCells were digested with trypsin to prepare single-cell suspensions. Cells were counted using an automated cell counter, and the cell density was adjusted with complete culture medium. Cells were seeded at a density of 3000 cells per well in the middle 60 wells of a 96-well cell culture plate (the edge wells were filled with 100 μL PBS to reduce evaporation). At least three replicates were set for each cell line and each drug concentration. The culture plates were incubated overnight (approximately 24 hours) at 37°C in a 5% CO2 incubator to allow for full cell adhesion and entry into the logarithmic growth phase.

[0100] (2) Drug treatment and concentration gradient setting Prepare the following concentration gradients using complete culture medium: 0, 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM stock solutions of vemurafenib, dabrafenib, or encobrafenib (1 mM, 5 mM, 10 mM, 50 mM, and 100 mM). Remove the old culture medium from each well and carefully add 100 μL of fresh complete culture medium containing different concentrations of the drug. Return the cells to the incubator and culture continuously for 72 hours.

[0101] (3) Cell viability assay (CCK-8 assay) After 72 hours of incubation, add 10 μL of CCK-8 solution directly to each well. Gently shake the culture plate to mix the reagents. Return the culture plate to the incubator and continue incubation for 1 hour. Use a microplate reader to measure the absorbance (OD) of each well at a wavelength of 450 nm. 450 nm Meanwhile, a well containing only culture medium and CCK-8 reagent (cell-free) was set up as a blank control, and its OD value was used for calibration.

[0102] (4) Data Analysis The corrected absorbance value is obtained by subtracting the average OD value of the blank control well from the OD values ​​of each experimental well.

[0103] Cell viability was calculated using the average OD value of the solvent control group (0 μM drug) after correction as the 100% viability baseline.

[0104] Cell viability (%) for each drug-treated well = (corrected OD value of that well / average corrected OD value of the solvent control group) × 100%. Plot RKO BRAF values ​​on the x-axis and cell viability (%) on the y-axis. V600E and RKO BRAF V600E / K483R Dose-response curves of cells to vemurafenib, dabrafenib, or encorafenib.

[0105] RKO BRAF V600E / K483R Cells compared to RKO BRAF V600E Cellular responses to three types of BRAFV600E Sensitivity to inhibitors was significantly reduced. Figure 5 (A, B, and C). This indicates that, at any given drug concentration, mutations at the SUMOylation modification site (K483) lead to BRAF. V600E The protein showed significantly reduced sensitivity to BRAF inhibitors, demonstrating that SUMOylation is essential for maintaining BRAF. V600E A key molecular switch for protein sensitivity to targeted drugs.

[0106] Example 6 Effects of SUMOylation on vemurafenib sensitivity under different glucose concentrations.

[0107] 1. BRAF under sugar-free conditions V600E The effect of SUMOylation on vemurafenib sensitivity.

[0108] (1) Synchronization of cell seeding and cell metabolic state RKO BRAF will be in the logarithmic growth phase. V600E and RKO BRAF V600E / K483R Cells were digested with trypsin and resuspended in glucose-free DMEM medium to prepare single-cell suspensions. Cells were counted using an automated cell counter, and cell density was adjusted with glucose-free DMEM medium. Cells were seeded at a density of 5000 cells per well in the middle 60 wells of a 96-well cell culture plate (the edge wells were filled with 100 μL PBS to reduce evaporation). At least three replicates were set for each cell line and each drug concentration. The culture plates were incubated overnight (approximately 24 hours) at 37°C in a 5% CO2 incubator to allow for full cell adhesion and homogenization of their metabolic state.

[0109] (2) Drug treatment and concentration gradient setting Vemurafenib stock solutions (1 mM, 5 mM, 10 mM, and 50 mM) were diluted 1:1000 using glucose-free DMEM to prepare the following concentration gradients: 0, 1 μM, 5 μM, 10 μM, and 50 μM. The old culture medium was removed from each well, and 100 μL of fresh complete culture medium containing different concentrations of the drug was carefully added. The cells were then returned to the incubator and cultured continuously for 72 hours.

[0110] (3) Cell viability assay (CCK-8 assay) After 72 hours of incubation, add 10 μL of CCK-8 solution directly to each well. Gently shake the culture plate to mix the reagents. Return the culture plate to the incubator and continue incubation for 1 hour. Use a microplate reader to measure the absorbance (OD) of each well at a wavelength of 450 nm. 450 nmMeanwhile, a well containing only culture medium and CCK-8 reagent (cell-free) was set up as a blank control, and its OD value was used for calibration.

[0111] (4) Data Analysis The corrected absorbance value is obtained by subtracting the average OD value of the blank control well from the OD values ​​of each experimental well.

[0112] Cell viability was calculated using the average OD value of the solvent control group (0 μM drug) after correction as the 100% viability baseline.

[0113] Cell viability (%) for each drug-treated well = (corrected OD value of that well / average corrected OD value of the solvent control group) × 100%. Plot RKO BRAF values ​​on the x-axis and cell viability (%) on the y-axis. V600E and RKO BRAF V600E / K483R Dose-response curves of cells to vemurafenib, dabrafenib, or encorafenib.

[0114] 2. BRAF under moderate glucose concentration (5 mM) conditions V600E The effect of SUMOylation on vemurafenib sensitivity. Quantitative comparisons were made between SUMOylation-intact RKO BRAF in a strictly controlled 5 mM glucose culture environment. V600E Cells and SUMOylation-deficient RKO BRAF V600E / K483R The study of cellular differences in vemurafenib demonstrates that SUMOylation remains a key factor determining drug efficacy at glucose concentrations close to those in certain physiological or tumor microenvironments.

[0115] (1) Synchronization of cell seeding and cell metabolic state RKO BRAF will be in the logarithmic growth phase. V600E and RKO BRAF V600E / K483R Cells were digested with trypsin and resuspended in 5 mM glucose DMEM medium to prepare single-cell suspensions. Cells were counted using an automated cell counter, and the cell density was adjusted with 5 mM glucose DMEM medium. Cells were seeded at a density of 5000 cells per well in the middle 60 wells of a 96-well cell culture plate (the edge wells were filled with 100 μL PBS to reduce evaporation). At least three replicates were set for each cell line and each drug concentration. The culture plates were incubated overnight (approximately 24 hours) at 37°C in a 5% CO2 incubator to allow for full cell adhesion and homogenization of their metabolic state.

[0116] (2) Drug treatment and concentration gradient setting Vemurafenib stock solutions (1 mM, 5 mM, 10 mM, and 50 mM) were diluted 1:1000 in 5 mM glucose DMEM medium to prepare the following concentration gradients: 0, 1 μM, 5 μM, 10 μM, and 50 μM. The old medium was removed from each well, and 100 μL of fresh complete medium containing different concentrations of the drug was carefully added. The cells were then returned to the incubator and cultured continuously for 72 hours.

[0117] (3) Cell viability assay (CCK-8 assay) After 72 hours of incubation, add 10 μL of CCK-8 solution directly to each well. Gently shake the culture plate to mix the reagents. Return the culture plate to the incubator and continue incubation for 1 hour. Use a microplate reader to measure the absorbance (OD) of each well at a wavelength of 450 nm. 450 nm Meanwhile, a well containing only culture medium and CCK-8 reagent (cell-free) was set up as a blank control, and its OD value was used for calibration.

[0118] (4) Data Analysis The corrected absorbance value is obtained by subtracting the average OD value of the blank control well from the OD values ​​of each experimental well.

[0119] Cell viability was calculated using the average OD value of the solvent control group (0 μM drug) after correction as the 100% viability baseline.

[0120] Cell viability (%) for each drug-treated well = (corrected OD value of that well / average corrected OD value of the solvent control group) × 100%. Plot RKO BRAF values ​​on the x-axis and cell viability (%) on the y-axis. V600E and RKO BRAF V600E / K483R Dose-response curves of cells to vemurafenib, dabrafenib, or encorafenib.

[0121] 3. BRAF under high glucose concentration (10 mM) conditions V600E The effect of SUMOylation on vemurafenib sensitivity. Quantitative comparisons were made between SUMOylation-intact RKO BRAF in a 10 mM glucose culture environment. V600E Cells and SUMOylation-deficient RKO BRAF V600E / K483R Differences in cellular sensitivity to vemurafenib. The aim was to demonstrate that SUMOylation-mediated drug sensitization is maximized under conditions of high metabolic activity, providing phenotypic evidence for the "metabolism-SUMOylation-susceptibility" axis.

[0122] (1) Synchronization of cell seeding and cell metabolic state RKO BRAF will be in the logarithmic growth phase. V600Eand RKO BRAF V600E / K483R Cells were digested with trypsin and resuspended in 10 mM glucose DMEM medium to prepare single-cell suspensions. Cells were counted using an automated cell counter, and the cell density was adjusted with 10 mM glucose DMEM medium. Cells were seeded at a density of 5000 cells per well in the middle 60 wells of a 96-well cell culture plate (the edge wells were filled with 100 μL PBS to reduce evaporation). At least three replicates were set for each cell line and each drug concentration. The culture plates were incubated overnight (approximately 24 hours) at 37°C in a 5% CO2 incubator to allow for full cell adhesion and homogenization of their metabolic state.

[0123] (2) Drug treatment and concentration gradient setting Vemurafenib stock solutions (1 mM, 5 mM, 10 mM, and 50 mM) were diluted 1:1000 in 10 mM glucose DMEM medium to prepare the following concentration gradients: 0, 1 μM, 5 μM, 10 μM, and 50 μM. The old medium was removed from each well, and 100 μL of fresh complete medium containing different concentrations of the drug was carefully added. The cells were then returned to the incubator and cultured continuously for 72 hours.

[0124] (3) Cell viability assay (CCK-8 assay) After 72 hours of incubation, add 10 μL of CCK-8 solution directly to each well. Gently shake the culture plate to mix the reagents. Return the culture plate to the incubator and continue incubation for 1 hour. Use a microplate reader to measure the absorbance (OD) of each well at a wavelength of 450 nm. 450 nm Meanwhile, a well containing only culture medium and CCK-8 reagent (cell-free) was set up as a blank control, and its OD value was used for calibration.

[0125] (4) Data Analysis The corrected absorbance value is obtained by subtracting the average OD value of the blank control well from the OD values ​​of each experimental well.

[0126] Cell viability was calculated using the average OD value of the solvent control group (0 μM drug) after correction as the 100% viability baseline.

[0127] Cell viability (%) for each drug-treated well = (corrected OD value of that well / average corrected OD value of the solvent control group) × 100%. Plot RKO BRAF values ​​on the x-axis and cell viability (%) on the y-axis. V600E and RKO BRAF V600E / K483R Dose-response curves of cells to vemurafenib, dabrafenib, or encorafenib.

[0128] The results are as follows Figure 6As shown, under 10 mM glucose conditions, RKO BRAF V600E The cell curve showed the steepest downward shift to the left, indicating that its sensitivity to vemurafenib reached the highest level in the series of experiments. RKO BRAF... V600E / K483R The similarity in the curves of cells under different glucose concentrations indicates that SUMOylation defects affect BRAF. V600E / K483R No significant improvement was observed in the sensitizing effect on the metabolic environment. The results indicate that the sensitizing effect of SUMOylation on vemurafenib was maximized under 10 mM glucose (metabolic abundance) conditions. Cells with intact SUMOylation function exhibited strong drug sensitivity, while SUMOylation-deficient cells were completely unable to utilize their metabolic advantage and showed refractory resistance. This directly demonstrates that sufficient metabolic flux (via lactate) is the key driving force for amplifying the sensitizing effect of SUMOylation.

[0129] Example 7 In vitro effects of combining MCT4 inhibitor AZD0095 with BRAF (V600E) inhibitor.

[0130] (1) Cell inoculation RKO cells in the logarithmic growth phase were digested with trypsin to prepare a single-cell suspension. Cells were counted using an automated cell counter, and the cell density was adjusted with complete culture medium. Cells were seeded at a density of 3000 cells per well in the middle 60 wells of a 96-well cell culture plate (the edge wells were filled with 100 μL PBS to reduce evaporation). At least three replicates were set for each cell line and each drug concentration. The culture plate was incubated overnight (approximately 24 hours) at 37°C in a 5% CO2 incubator to allow for full cell adhesion and entry into the logarithmic growth phase.

[0131] (2) Drug treatment and concentration gradient setting BRAF (V600E) inhibitor stock solutions (vemurafenib, dabrafenib, and encobrafenib) (10 mM and 50 mM) were diluted 1:1000 using complete culture medium to prepare working concentrations of 10 μM and 50 μM. AZD0095 stock solutions (1 mM, 10 mM, 50 mM, and 100 mM) were then further diluted 1:1000 to prepare the following concentration gradients: 0, 1 μM, 10 μM, 50 μM, and 100 μM. The old culture medium was removed from each well, and 100 μL of fresh complete culture medium containing different drug concentrations was carefully added. The cells were then returned to the incubator and cultured continuously for 72 hours.

[0132] (3) Cell viability assay (CCK-8 assay) After 72 hours of incubation, add 10 μL of CCK-8 solution directly to each well. Gently shake the culture plate to mix the reagents. Return the culture plate to the incubator and continue incubation for 1 hour. Use a microplate reader to measure the absorbance (OD) of each well at a wavelength of 450 nm. 450 nm Meanwhile, a well containing only culture medium and CCK-8 reagent (cell-free) was set up as a blank control, and its OD value was used for calibration.

[0133] (4) Data Analysis The corrected absorbance value is obtained by subtracting the average OD value of the blank control well from the OD values ​​of each experimental well.

[0134] Cell viability was calculated using the average OD value of the solvent control group (0 μM drug) after correction as the 100% viability baseline.

[0135] Cell viability (%) for each drug-treated well = (corrected OD value of that well / average corrected OD value of the solvent control group) × 100%. Plot RKO BRAF values ​​on the x-axis and cell viability (%) on the y-axis. V600E and RKO BRAF V600E / K483R Dose-response curves of cells to vemurafenib, dabrafenib, or encorafenib.

[0136] The results showed that, at the same AZD0095 concentration, the inhibitory efficiency of the combination with a BRAF (V600E) inhibitor against colorectal cancer cells was significantly higher than that of BRAF (V600E) inhibitor monotherapy. Figure 7 (A, B, and C). The MCT4 inhibitor AZD0095 can produce a clear synergistic anti-proliferative effect with BRAF (V600E) targeted drugs, significantly reducing the effective dose of BRAF (V600E) targeted drugs and enhancing their killing efficacy against tumor cells.

[0137] Example 8 Evaluation of the killing effect of MCT4 inhibitors combined with vemurafenib on acquired resistant cell lines.

[0138] 1. Evaluation of the effect of the MCT4 inhibitor AZD0095 in combination with vemurafenib on vemurafenib-acquired resistance RKO cells. VR The destructive effect.

[0139] (1) Cell inoculation RKO, which is in the logarithmic growth phase VRCells were digested with trypsin to prepare a single-cell suspension. Cells were counted using an automated cell counter, and the cell density was adjusted with complete culture medium. Cells were seeded at a density of 3000 cells per well in the middle 60 wells of a 96-well cell culture plate (the edge wells were filled with 100 μL PBS to reduce evaporation). At least three replicates were set for each drug concentration. The culture plates were incubated overnight (approximately 24 hours) at 37°C in a 5% CO2 incubator to allow for full cell adhesion and entry into the logarithmic growth phase.

[0140] (2) Drug treatment and concentration gradient setting Vemurafenib stock solutions (1 mM, 2.5 mM, 5 mM, 10 mM, and 50 mM) were diluted 1:1000 using complete culture medium to prepare the following concentration gradients: 0, 1 μM, 2.5 μM, 5 μM, 10 μM, and 50 μM. AZD0095 stock solutions (1 mM, 10 mM, and 50 mM) were then further diluted 1:1000 to prepare the following concentration gradients: 0, 1 μM, 10 μM, and 50 μM. The old culture medium was removed from each well, and 100 μL of fresh complete culture medium containing different concentrations of the drug was carefully added. The cells were then returned to the incubator and cultured continuously for 72 hours.

[0141] (3) Cell viability assay (CCK-8 assay) After 72 hours of incubation, add 10 μL of CCK-8 solution directly to each well. Gently shake the culture plate to mix the reagents. Return the culture plate to the incubator and continue incubation for 1 hour. Use a microplate reader to measure the absorbance (OD) of each well at a wavelength of 450 nm. 450 nm Meanwhile, a well containing only culture medium and CCK-8 reagent (cell-free) was set up as a blank control, and its OD value was used for calibration.

[0142] (4) Data Analysis The corrected absorbance value is obtained by subtracting the average OD value of the blank control well from the OD values ​​of each experimental well.

[0143] Cell viability was calculated using the average OD value of the solvent control group (0 μM drug) after correction as the 100% viability baseline.

[0144] Cell viability (%) for each drug-treated well = (corrected OD value of that well / average corrected OD value of the solvent control group) × 100%. Plot RKO BRAF values ​​on the x-axis and cell viability (%) on the y-axis. V600E and RKO BRAF V600E / K483R Dose-response curves of cells to vemurafenib, dabrafenib, or encorafenib.

[0145] 2. Evaluation of the effect of the MCT1 selective inhibitor AZD3965 combined with vemurafenib on vemurafenib-resistant RKO cells. VR The destructive effect.

[0146] (1) Cell inoculation RKO, which is in the logarithmic growth phase VR Cells were digested with trypsin to prepare a single-cell suspension. Cells were counted using an automated cell counter, and the cell density was adjusted with complete culture medium. Cells were seeded at a density of 3000 cells per well in the middle 60 wells of a 96-well cell culture plate (the edge wells were filled with 100 μL PBS to reduce evaporation). At least three replicates were set for each drug concentration. The culture plates were incubated overnight (approximately 24 hours) at 37°C in a 5% CO2 incubator to allow for full cell adhesion and entry into the logarithmic growth phase.

[0147] (2) Drug treatment and concentration gradient setting Vemurafenib stock solutions (1 mM, 2.5 mM, 5 mM, 10 mM, and 50 mM) were diluted 1:1000 using complete culture medium to prepare the following concentration gradients: 0, 1 μM, 2.5 μM, 5 μM, 10 μM, and 50 μM. AZD3965 stock solutions (1 mM, 10 mM, and 50 mM) were then further diluted 1:1000 to prepare the following concentration gradients: 0, 1 μM, 10 μM, and 50 μM. The old culture medium was removed from each well, and 100 μL of fresh complete culture medium containing different concentrations of the drug was carefully added. The cells were then returned to the incubator and cultured continuously for 72 hours.

[0148] The same applies below

[0138] to

[0143] .

[0149] 3. Evaluation of the effect of the MCT1 / 4 dual inhibitor yrosingapine combined with vemurafenib on vemurafenib-resistant RKO cells. VR The destructive effect.

[0150] (1) Cell inoculation RKO, which is in the logarithmic growth phase VR Cells were digested with trypsin to prepare a single-cell suspension. Cells were counted using an automated cell counter, and the cell density was adjusted with complete culture medium. Cells were seeded at a density of 3000 cells per well in the middle 60 wells of a 96-well cell culture plate (the edge wells were filled with 100 μL PBS to reduce evaporation). At least three replicates were set for each drug concentration. The culture plates were incubated overnight (approximately 24 hours) at 37°C in a 5% CO2 incubator to allow for full cell adhesion and entry into the logarithmic growth phase.

[0151] (2) Drug treatment and concentration gradient setting Vemurafenib stock solutions (1 mM, 2.5 mM, 5 mM, 10 mM, and 50 mM) were diluted 1:1000 using complete culture medium to prepare the following concentration gradients: 0, 1 μM, 2.5 μM, 5 μM, 10 μM, and 50 μM. Syrosingapine stock solutions (1 mM, 10 mM, and 50 mM) were then further diluted 1:1000 to prepare the following concentration gradients: 0, 1 μM, 10 μM, and 50 μM. The old culture medium was removed from each well, and 100 μL of fresh complete culture medium containing different concentrations of the drug was carefully added. The cells were then returned to the incubator and cultured continuously for 72 hours.

[0152] (3) Cell viability assay (CCK-8 assay) After 72 hours of incubation, add 10 μL of CCK-8 solution directly to each well. Gently shake the culture plate to mix the reagents. Return the culture plate to the incubator and continue incubation for 1 hour. Use a microplate reader to measure the absorbance (OD) of each well at a wavelength of 450 nm. 450 nm Meanwhile, a well containing only culture medium and CCK-8 reagent (cell-free) was set up as a blank control, and its OD value was used for calibration.

[0153] (4) Data Analysis The corrected absorbance value is obtained by subtracting the average OD value of the blank control well from the OD values ​​of each experimental well.

[0154] Cell viability was calculated using the average OD value of the solvent control group (0 μM drug) after correction as the 100% viability baseline.

[0155] Cell viability (%) for each drug-treated well = (corrected OD value of that well / average corrected OD value of the solvent control group) × 100%. Plot RKO BRAF values ​​on the x-axis and cell viability (%) on the y-axis. V600E and RKO BRAF V600E / K483R Dose-response curves of cells to vemurafenib, dabrafenib, or encorafenib.

[0156] In vemurafenib-acquired resistance cells RKO VR In this study, the MCT4 inhibitor AZD0095 effectively alleviated the resistance of colorectal cancer cells to vemurafenib, restoring antitumor activity to previously ineffective doses of vemurafenib. Figure 8 (A) This directly proves that targeting MCT4 is an effective way to overcome acquired resistance to vemurafenib. Compounds that selectively inhibit MCT1, such as AZD3965, are completely ineffective in alleviating RKO. VR Acquired resistance of cells to vemurafenib Figure 8(Middle B). The combination of the MCT1 / 4 dual inhibitor syrosingapine and vemurafenib can also effectively alleviate RKO. VR Cellular drug resistance ( Figure 8 (C). Therefore, MCT4 is a key metabolic target for alleviating acquired resistance to vemurafenib, and drugs that selectively inhibit MCT4, such as AZD0095, can effectively enhance the sensitivity of resistant cells to vemurafenib.

[0157] Example 9 Evaluation of AZD0095 in combination with crizotinib for the treatment of human colorectal cancer RKO nude mouse xenografts.

[0158] Using BALB / c nude mice as test animals, the antitumor effects of vemurafenib and AZD0095 alone or in combination on subcutaneous xenografts of human colorectal cancer cells RKO in nude mice were evaluated and compared.

[0159] (1) Cell preparation RKO cells in the logarithmic growth phase were digested with trypsin to prepare a single-cell suspension, which was then suspended in DPBS buffer. Cells were counted using an automated cell counter, and the cell density was adjusted with DPBS and a proportional amount of matrix gel. Cells were subcutaneously seeded at a rate of 5 × 10⁻⁶ cells per 50 nude mice. 5 Prepare cells (0.1 mL / cell / animal). (2) Animal inoculation and grouping Forty 6-8 week old female nude mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were used to subcutaneously implant RKO cells until the tumors grew to approximately 80-100 mm. 3 (In this example, the duration is 12 days), with an average tumor volume of approximately 80-85 mm. 3 The standard procedure randomly divided the experimental animals into four groups, with eight nude mice in each group receiving oral gavage (PO) twice daily (BID).

[0160] Control group: Oral administration of a solvent (0.5% CMC + 0.1% Tween80) twice daily.

[0161] AZD0095 group: AZD0095 was administered orally by gavage (solvent: 0.5% CMC + 0.1% Tween 80) at a dose of 100 mg / kg twice daily.

[0162] Vemurafenib group: Vemurafenib was administered orally by gavage (solvent: 0.5% CMC + 0.1% Tween 80) at a dose of 75 mg / kg twice daily.

[0163] AZD0095+Vemurafenib group: AZD0095+Vemurafenib (solvent: 0.5% CMC + 0.1% Tween 80) was administered orally via gavage at doses of 100 mg / kg and 75 mg / kg, twice daily.

[0164] Dosage volume: 10 μL / g based on the nude mouse's body weight. If the animal's body weight decreases by more than 15%, discontinue administration; resume administration once the body weight has recovered to the 10% reduction level.

[0165] During the drug administration period, the weight and tumor volume of the nude mice were measured every 2-3 days. The formula for calculating tumor volume is: V = D / 2 × d 2 (V: tumor volume; D: major axis; d: minor axis). Results showed that, while ensuring animal weight changes remained within ethically acceptable limits, the tumor growth curve in the combination therapy group was significantly lower than that in the single-drug group (see attached). Figure 9 A&B) The tumor volume inhibition rate was calculated as follows: T / C(%) = (TT0) / (CC0) × 100%, where T and C are the tumor volumes at the end of the experiment and control group, respectively, and T0 and C0 are the tumor volumes at the beginning of the experiment and control group, respectively. The tumor growth inhibition rate % (TGI%) = 100T / C(%). Tumor volumes for each group are expressed as Mean ± SEM. The statistical method for the difference in tumor volume between the experimental group and the control group was Student t-test (unpaired two-tailed heteroscedasticity), and p < 0.05 was considered statistically significant.

[0166] Table 1. Dosing regimens and efficacy of single or combined drugs against RKO human colorectal cancer cell subcutaneous xenografts in nude mice (based on relative tumor volume) During different drug treatments, the combined effect of combination therapy versus monotherapy was calculated and compared based on changes in relative tumor volume. The theoretical formula for calculating the combined effect is as follows: C(%) = (S1) TGI + S2 TGI - S1 TGI × S2 TGI ) × 100%, where C represents the theoretical additive inhibition rate and S represents the actual single-drug inhibition rate. If C 理论值 >C 实际值 Represents a combined antagonistic effect; if C 理论值 =C 实际值 Represents a combined superposition effect; if C 理论值 <C 实际值This represents a combined synergistic effect. Since the theoretical value is fixed, we used a one-tailed one-sample t-test for statistical analysis. In this embodiment, the theoretical additive inhibition rate C of the relative tumor volume of the combined use of AZD0095 and vemurafenib is... 理论值 = (0.2291+0.3328-0.2291× 0.3328) × 100% =48.56%, C 实际值 = 60.73%, C 理论值 < C 实际值 p = 0.0679 (marginally significant). This indicates that the combined drug therapy in this embodiment (based on volumetric inhibition rate) has a strong potential synergistic effect (as shown in Table 1).

[0167] In this embodiment, 19 days after the animals were given the drug, the experimental mice were euthanized with 45% CO2, the tumors were removed and their mass was weighed, and the drug treatment effects of different treatment regimens were compared and evaluated.

[0168] The tumor quality inhibition rate was calculated using the same method as above (the tumor volume inhibition rate was calculated as follows: T / C(%) = (TT0) / (CC0) × 100%, where T and C are the tumor volumes at the end of the experiment and control group, respectively, and T0 and C0 are the tumor volumes at the beginning of the experiment and control group, respectively. The tumor growth inhibition rate % (TGI%) = 100T / C(%). The tumor volume values ​​of each group are expressed as Mean ± SEM. The statistical method for the difference in tumor volume between each experimental group and the control group was Student t-test (unpaired two-tailed heteroscedasticity). p < 0.05 was considered to be statistically significant, and T0 and C0 were both set to zero.

[0169] Table 2. Dosing regimens and efficacy of single or combined drugs in RKO human colorectal cancer subcutaneous xenografts in nude mice (based on tumor quality) The combined effect of the combined medication is calculated based on tumor mass, using the same method as above (the theoretical combined effect calculation formula is as follows: C(%) = (S1) TGI + S2 TGI - S1 TGI × S2 TGI ) × 100%, where C represents the theoretical additive inhibition rate and S represents the actual single-drug inhibition rate. If C 理论值 >C 实际值 Represents a combined antagonistic effect; if C 理论值 = C 实际值 Represents a combined superposition effect; if C 理论值 <C 实际值This represents a combined synergistic effect. In this embodiment, the theoretical additive inhibition rate Ctheoretical of the combined use of AZD0095 and vemurafenib is (0.1886 + 0.1576 - 0.1886 × 0.1576) × 100% = 31.65%, Cactual = 50.12%, Ctheoretical < Cactual, p = 0.0376. This also demonstrates that the combined use (based on mass inhibition rate) in this embodiment has a significant combined synergistic effect (as shown in Table 2 and Appendix). Figure 9 C&D).

[0170] Animal experimental results show that the lactate efflux inhibitor AZD0095 can significantly enhance the efficacy of the BRAF (V600E) inhibitor vemurafenib in the treatment of colorectal cancer, and has a synergistic effect of the two drugs combined.

[0171] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of application of the present invention. Various modifications, improvements, or substitutions made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the invention should fall within the protection scope claimed by the claims of the present invention.

[0172] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. Use of an intracellular lactate level enhancer in the preparation of a medicament for enhancing the sensitivity of BRAF V600E mutant tumors to BRAF inhibitors or in the preparation of a medicament for treating BRAF V600E mutant tumors resistant to BRAF inhibitors.

2. The use according to claim 1, characterized in that, The intracellular lactate level enhancer is selected from at least one of lactate efflux inhibitors, glycolysis promoters, or hypoxia mimics.

3. The use according to claim 2, characterized in that, The glycolysis promoter is glucose or a compound that can upregulate the glycolysis pathway.

4. The use according to claim 2, characterized in that, The hypoxia simulant is cobalt chloride.

5. The use according to claim 2, characterized in that, The lactate efflux inhibitor is an MCT4 inhibitor.

6. The use according to claim 5, characterized in that, The MCT4 inhibitor is selected from AZD0095, Syrosingapine, or pharmaceutically acceptable salts, esters, solvates, or prodrugs thereof.

7. The use according to claim 6, characterized in that, The MCT4 inhibitor is AZD0095.

8. The use according to claim 1, characterized in that, The BRAF inhibitor is selected from at least one of vemurafenib, dabrafenib, encofenib, or PLX8394.

9. The use according to claim 1, characterized in that, The BRAF V600E mutated tumors are colorectal cancer, melanoma, thyroid cancer, or non-small cell lung cancer.

10. A pharmaceutical composition, characterized in that, include: The intracellular lactate level enhancer, the BRAF V600E inhibitor, and the pharmaceutically acceptable carrier or excipient; wherein the intracellular lactate level enhancer is selected from lactate efflux inhibitors, glycolysis promoters, or hypoxia mimics.

11. The pharmaceutical composition according to claim 10, characterized in that, The intracellular lactate level enhancer is the MCT4 inhibitor AZD0095, and the BRAF V600E inhibitor is vemurafenib.

12. A medicine box, characterized in that, include: Pharmaceutical formulations containing intracellular lactate level enhancers; Pharmaceutical formulations of BRAF V600E inhibitors; as well as manual.

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