Application of KRAS inhibitor in combination with glucocorticoid in preparation of medicine for treating cancer

Abstract

The invention provides application of a KRAS inhibitor combined with glucocorticoid in preparation of a medicine for treating cancers, and belongs to the technical field of biological medicines. The invention provides a combined application of a KRAS inhibitor and glucocorticoid in preparation of a medicine for treating cancers. Compared with the single use of the KRAS inhibitor or prednisolone, the combined use of the KRAS inhibitor and prednisolone can more effectively inhibit the proliferation and survival of KRAS G12C mutated NSCLC cells and slow down the growth of tumor volume. In addition, prednisolone can relieve the acquired drug resistance problem of KRASi by blocking generation of TNF and I-type IFN induced by a KRAS inhibitor, and prednisolone and prednisolone have a synergistic anti-tumor effect. The two can be directly combined for medication, can also be prepared into a combined agent for treating cancers, and has a good clinical transformation prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of KRAS inhibitors combined with glucocorticoids in the preparation of drugs for treating cancer. Background Technology

[0002] RAS were the first human oncogenes discovered, including KRAS, NRAS, and HRAS, which play an important role in the development and progression of cancer. More than 30% of cancers are driven by mutations in one of these three RAS genes [1]. Among them, KRAS is the most common mutation among the three RAS genes, and KRAS mutations can be detected in more than 90% of pancreatic cancers, 30% to 40% of lung adenocarcinomas, and colorectal adenocarcinomas [2-5]. RAS mutations give cancer the characteristic of "self-sufficient growth signaling", which is one of the most critical features of abnormal excessive proliferation of malignant tumors [6, 7].

[0003] RAS regulates cell growth, proliferation, differentiation, and survival through its involvement in the classic RTK-RAS-RAF-MAPK signaling pathway. RTK (Receptor Tyrosine Kinase) is a cell surface receptor protein that autophosphorylates upon sensing external growth factors, activating intracellular RAS proteins. This, in turn, cascades the activation of RAF kinase and MAPK, ultimately activating many transcription factors and other proteins, regulating gene expression and cellular function. Tumors often promote their development and progression through overactivation of this pathway caused by high RTK expression or mutations, or mutations in the RAS or RAF genes. However, the shallow GTP pocket of RAS, despite its strong affinity for the substrate GTP, has long hindered the development of small-molecule targeted inhibitors, leading to its long-standing reputation as an "untreatable" drug.

[0004] Encouragingly, in 2021, AMG510 and MRTX849, targeted drugs against KRAS G12C mutations, received FDA approval for the first time for the treatment of non-small cell lung cancer, breaking the long-standing "undruggable" curse of the RAS gene. AMG510, in the form of a molecular glue, locks KRAS G12C in the inactive state of KRAS-GDP, thereby inhibiting the continuous activation of the KRAS mutant-GTP. However, challenges remain. Drug resistance to molecularly targeted drugs is almost unavoidable, and acquired resistance to KRAS-G12C inhibitors is also foreseeable. Clinical practice and research over the past three years have shown that even if initial treatment is effective, acquired resistance after treatment remains a serious problem, affecting subsequent treatment outcomes and prognosis.

[0005] The emergence of secondary resistance means that a subset of cancer cells that were not completely eliminated during the initial exposure to targeted therapy still exists. Therefore, more effective elimination of cancer cells during the initial exposure to targeted therapy may delay or eliminate the emergence of secondary resistance.

[0006] References: [1].Stephen, AndrewG., et al., Dragging Ras Back in the Ring. CancerCell, 2014.25(3): p. 272-281. [2]. Jones, Sn, et al., Core Signaling Pathways in Human Pancreatic Cancers Revealed by Global Genomic Analyses. Science, 2008.321(5897): p. 1801-1806. [3].Ding, L., et al., Somatic mutations affect key pathways in lung adenocarcinoma. Nature, 2008.455(7216): p. 1069-1075. [4]. Raphael, BJ, et al., Integrated Genomic Characterization of Pancreatic Ductal Adenocarcinoma. Cancer Cell, 2017.32(2): p. 185-203.e13. [5].Network., CGA, Comprehensive molecular characterization of human colon and rectal cancer. Nature, 2012.487(7407): p. 330-337. [6]. Hanahan D, WR, The hallmarks of cancer. Cell. ;, 2000 Jan 7.100(1):: p. 57-70. [7].Hanahan, D. and RobertA. Weinberg, Hallmarks of Cancer: The Next Generation. Cell, 2011.144(5): p. 646-674. Summary of the Invention In our previous experimental studies, we discovered that the KRAS inhibitor AMG510 significantly upregulated the expression of innate immune and inflammation-related cytokines such as IFN-I and TNF-α, and that these cytokines mediate acquired resistance to KRAS inhibitors. To overcome the resistance problem of KRAS inhibitors, we further investigated and found that glucocorticoids can effectively inhibit the expression of these cytokines through mechanisms such as blocking NF-κB nuclear translocation and inhibiting the STING / RIG-I-TBK1-IRF3 signaling axis. More importantly, we found that the combination therapy of KRAS inhibitors and glucocorticoids significantly enhanced the therapeutic effect of KRAS inhibitors in cell lines and animal tumor models by inhibiting the acquired reactivation of the JAK-STAT and MAPK (ERK) pathways. Based on these findings, the combination therapy of KRAS inhibitors and glucocorticoids can overcome secondary resistance in NSCLC.

[0007] The purpose of this invention is to provide the use of KRAS inhibitors in combination with glucocorticoids in the preparation of medicaments for treating cancer, aiming to overcome the acquired resistance problem caused by KRAS inhibitor treatment of cancer. A further purpose of this invention is to provide a medicament for treating cancer, including non-small cell lung cancer.

[0008] This invention combines a KRAS inhibitor (KRASi) with glucocorticoids for the treatment of non-small cell lung cancer, effectively inhibiting tumor growth in both short-term and long-term treatment. Glucocorticoids suppress acquired resistance to KRAS inhibitors by blocking KRASi-induced production of type I interferon and TNF, and by inhibiting the adaptive activation of the KRASi-induced bypass RTK signaling pathway.

[0009] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides the use of KRAS inhibitors, glucocorticoids, and / or TNF inhibitors in the preparation of medicaments for treating cancer.

[0010] Secondly, the present invention provides the use of a composition containing a KRAS inhibitor and glucocorticoids and / or TNF inhibitors in the preparation of a medicament for treating cancer. In addition to containing KRAS inhibitors and glucocorticoids and / or TNF inhibitors, the composition may also contain other substances with cancer-treating effects.

[0011] Thirdly, the present invention provides a medicament for treating cancer, comprising a KRAS inhibitor, as well as glucocorticoids and / or TNF inhibitors, and may also contain other substances with cancer-treating effects, and may further contain pharmaceutically acceptable excipients.

[0012] In some implementations, the KRAS inhibitors include one or more of AMG 510, MRTX849, BI-2865, fluzole, and gexole.

[0013] In some embodiments, the glucocorticoids include one or more of beclomethasone, betamethasone, cortisone, dexamethasone, hydrocortisone, and prednisolone.

[0014] In some implementations, the TNF inhibitors include one or more of lenalidomide, thalidomide, prednisone, etanercept, adalimumab, infliximab, golimumab, pecelizumab, and etanercept.

[0015] In some implementations, the cancer is KRAS wild-type or cancer carrying a KRAS G12C activating mutation.

[0016] In some implementations, the cancers include one or more of lung cancer, pancreatic cancer, colorectal cancer, ovarian cancer, bile duct cancer, and appendiceal cancer.

[0017] In some implementations, the cancer is a cancer that is resistant to KRAS inhibition.

[0018] In some implementations, the cancer is non-small cell lung cancer. Further, the non-small cell lung cancer is KRAS-mutated non-small cell lung cancer, and even further, the non-small cell lung cancer is non-small cell lung cancer carrying a KRAS G12C activating mutation.

[0019] In some embodiments, the KRAS inhibitor is AMG 510, MRTX849, or BI-2865; the glucocorticoid is prednisolone; and the TNF inhibitor is etanercept or lenalidomide.

[0020] This invention has the following advantages and beneficial effects: the combination of glucocorticoid prednisolone and KRASi AMG510 significantly reduces the survival rate of KRAS G12C mutant NSCLC cells. Prednisolone significantly inhibits the upregulation of cytokines such as TNF and IFN-I in KRASi-induced NSCLC, thereby improving the problem of acquired drug resistance. The combination of KRAS inhibitors and glucocorticoids for the treatment of non-small cell lung cancer has good prospects for clinical translation. Attached Figure Description

[0021] Figure 1 The structural formulas of KRASi AMG 510 (a) and the glucocorticoid prednisolone (b).

[0022] Figure 2Effects of hormone drugs combined with KRASi on cell survival. In the figure, ad represents the MTT cell proliferation assay results of prednisolone combined with AMG510 in H23, H2122, H1373, and H358, respectively; eh represents the MTT cell proliferation assay results of prednisolone combined with MRTX-849 in H23, H2122, H1373, and H358, respectively; and ik represents the colony formation assay of prednisolone combined with AMG510.

[0023] Figure 3 A living xenograft model demonstrated the highly effective inhibition of tumor growth by the combined use of KRASi and prednisolone. In Figure ac, nude mice were subcutaneously injected with H23 cells. After tumor formation, they were treated with prednisolone and AMG510. Figure de shows the combined treatment with prednisolone and AMG510 in the H2122 xenograft model.

[0024] Figure 4 The living xenograft model demonstrated the long-term inhibitory effect of combined KRASi and prednisolone on tumor growth. Figures ab show the H23 xenograft model treated with long-term combination therapy of prednisolone and AMG510. Figures cd show the acquisition of drug resistance in large tumors established in the H23 xenograft model, treated with AMG510 alone and in combination with prednisolone and AMG510.

[0025] Figure 5 Effects of pan-KRAS inhibitor combined with prednisolone on cell survival. Figure ac shows the MTT assay of pan-KRAS inhibitor BI-2865 combined with prednisolone. Figure de shows the colony formation assay of BI-2865 combined with prednisolone.

[0026] Figure 6 KRASi-induced RTK signaling was inhibited by prednisolone. Figure ab shows the Western blot results of the hormone prednisolone inhibiting KRASi-induced RTK signaling in H23, H2122, H1373, and H358 cells.

[0027] Figure 7 The effects of hormone drugs combined with KRASi on KRAS G12C mutant drug-resistant cell lines. Figure ad shows the MTT cell proliferation and colony formation assays of prednisolone combined with AMG510 in drug-resistant cell lines H358AR and H2122AR. Figure eg shows the qPCR results of TNF, IFN-α, and IFN-β expression levels in H358AR. Figure hj shows the qPCR results of TNF, IFN-α, and IFN-β expression levels in H2122AR.

[0028] Figure 8 KRASi upregulates IFN signaling in NSCLC cell lines and animal models carrying the KRAS G12C activating mutation. Non-small cell lung cancer cell lines were treated with AMG510 for a specified time, followed by RNA extraction and detection of IFN-α and IFN-β mRNA by qRT-PCR. Figure 8 In Figures ad, H23, H2122, H1373, and H358 cells were treated with AMG510 at specified time points, followed by RNA extraction for qRT-PCR detection of IFN-α. Figure eh shows similar experiments performed on H23, H2122, H1373, and H358 cells to detect IFN-β mRNA. Figure ij shows a CDX xenograft model constructed by subcutaneous injection of H23 cells into nude mice. After tumor formation, mice were administered AMG510 at days 0, 1, 2, 4, and 7. Subsequently, the mice were sacrificed, and the tumors were removed for quantitative qRT-PCR analysis of IFN-α and IFN-β.

[0029] Figure 9 Knockdown of the type I interferon receptor IFNAR1 led to enhanced sensitivity of KRAS G12C mutant NSCLC to KRASi. Figures a, b, and clonogenic assays showed that shRNA knockdown of IFNAR1 in H23 and H2122 cells, in combination with AMG510, inhibited tumor cell survival. IFNAR1 silencing was confirmed by Western blot. Figure eh shows that AMG510 induced phosphorylation of STAT1 in non-small cell lung cancer cells. Figure ij shows that IFNAR1 knockdown inhibited STAT1 phosphorylation, resulting in the blocking of AMG510-induced upregulation of p-STAT1 in H23 and H2122 cells. Figure kn shows the combination of IFNAR1 inhibition and AMG510 in a mouse model. IFNAR1 inhibition led to enhanced sensitivity of KRAS G12C mutant NSCLC cells to KRASi. Figure 1 shows that exogenous IFN-α can protect H23 and H2122 cells from AMG510-induced cell death.

[0030] Figure 10KRASi leads to activation of the TBK1 / IRF3 signaling axis in NSCLC with KRAS G12C activating mutations. Figure ad shows cells treated with AMG510 at different time points, followed by cell lysate preparation and Western blot analysis using p-TBK1 and p-IRF3 antibodies. Figure e shows a CDX xenograft model constructed by subcutaneous injection of H2122 cells into nude mice. After tumor formation, mice were administered AMG510 at days 0, 1, 2, 4, and 7. Subsequently, the mice were sacrificed, and the tumors were removed and lysed for Western blot analysis. Figure fm shows that MTT and colony formation assays demonstrate that shRNA knockdown of TBK1 and IRF3 in H23 and H2122 cells, in combination with AMG510, inhibits tumor cell survival. Knockdown of TBK1 and IRF3 was confirmed by Western blot analysis.

[0031] Figure 11 Cells stably silenced with TBK1 or IRF3 are sensitive to KRASi. Figure ah shows stable cell lines shTBK1, shIRF3, or the control shCtrl treated with AMG510 for 72 hours, followed by RNA extraction for qRT-PCR detection of IFN-α and IFN-β. Figure in shows animal experiments using stably silenced TBK1 or IRF3.

[0032] Figure 12 Hormonal drugs combined with KRASi inhibited the expression of type I interferon and downstream signaling pathways in NSCLC cells and animal models. Figure ad shows that AMG510-induced upregulation of IFN-α was blocked by prednisolone. Figure eh shows that AMG510-induced upregulation of IFN-β was blocked by prednisolone. Figure ij shows that a similar experiment was performed in the H2122 CDX model. Figure ko shows that Western blot showed that prednisolone blocked AMG510-induced TBK1-IRF3 signaling axis transduction.

[0033] Figure 13 KRASi upregulates TNF signaling in NSCLC cell lines and animal models carrying the KRAS G12C activating mutation, promoting NSCLC resistance to KRAS inhibition. Figure ac shows NSCLC cell lines treated with AMG510 for a specified time, followed by RNA extraction and TNF quantitative PCR. Figure de shows H2122 cells subcutaneously injected into nude mice. After tumor formation, AMG510 treatment was administered, followed by tumor resection and TNF mRNA quantification by qPCR.

[0034] Figure 14KRASi promotes TNF-dependent NF-κB nuclear translocation. Figure ad shows H23, H212, H1373, and H358 cells treated with AMG510 for a specified time, followed by cell lysate preparation and Western blot analysis using NF-κB antibody. Figure eh shows H23, H212, H1373, and H358 cells exposed to AMG510 for 24 hours, followed by dual-luciferase reporter gene assay. Figure il shows NSCLC cell lines treated with AMG510 for a specified time, followed by cell lysate preparation and Western blot analysis of IκBα and RELB.

[0035] Figure 15 Inhibition of TNF induces the sensitivity of KRAS G12C-mutant NSCLC cells to KRASi. Figures ab and b show the observation of cell survival after knocking down TNFR1 in HCC827 cells and treating them with AMG510. Figures cd and d show a similar experiment performed in H2122 cells. Figure el shows an MTT cell proliferation assay validating the combination of TNF inhibitor and AMG510.

[0036] Figure 16 Hormonal drugs combined with KRASi inhibited the upregulation of TNF and the nuclear translocation of TNF-dependent NF-κB in NSCLC. Figures a, e, and f, show the qRT-PCR results of in vitro and in vivo AMG510-upregulated TNF being blocked by prednisolone. Figure fi, NSCLC cell lines treated with AMG510 and / or prednisolone at specific time points, with NF-κB levels detected by Western blot. Figure jm, Western blot showing that prednisolone blocked IκB degradation and increased RELB levels. Figure nr, the NF-κB activity increased by AMG510 was blocked by prednisolone in a luciferase reporter gene assay.

[0037] Figure 17 Schematic diagram illustrating the inhibition of KRASi-induced type I interferon and TNF-driven adaptive responses in NSCLC by a combination of hormone drugs and KRASi. AMG 510 inhibits the TBK1-IRF3 and NF-κB pathways simultaneously activated by KRAS G12C, inducing the secretion of IFN-I and TNF, promoting cell survival and adaptive tolerance; combined with prednisolone, it can block both axes, reduce IFN-I / TNF levels, disrupt the inflammatory circuit, and restore the sensitivity of NSCLCs to KRASi. Detailed Implementation

[0038] This invention provides the use of a combination of a KRAS inhibitor and a glucocorticoid and / or a TNF inhibitor in the preparation of a medicament for treating cancer, wherein the use of the medicament is preferably to provide a patient suitable for the use of the medicament with an effective dose of the KRAS inhibitor AMG 510 and the glucocorticoid prednisolone.

[0039] To make the problems to be solved, the technical solutions and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

[0040] As used herein, the phrase "KRAS inhibitor" (also known as KRAS TKl, KRASi) or "drug that inhibits KRAS activity" refers to any drug (molecule) that can inhibit the biological activity of the KRAS G12C mutant. These include AMG510, MRTX-849, etc., which, in the form of a molecular gel, lock KRAS G12C in the inactive state of KRAS-GDP, thereby inhibiting the sustained activation of the KRAS mutant-GTP.

[0041] In this invention, the term "treatment" or "manipulation" of a subject includes applying the compounds or pharmaceutical compositions of this invention to the cells or tissues of that subject with the aim of stabilizing, curing, alleviating, reducing, altering, remedying, preventing its deterioration, improving or affecting the disease or condition, or the risk of the disease or condition, or susceptibility to the disease. The term "treatment" refers to any indication of success in treating or improving an injury, pathological condition, or symptom, including but not limited to any objective or subjective parameters such as reduction, relief, degree of relief, etc. This includes slowing the rate of disease progression; stabilizing, reducing symptoms, or making the injury, pathology, or condition more tolerable for the subject; slowing the rate of degeneration; reducing the degree of deterioration at the final point of degeneration; or improving the physical or mental health of the subject. In one embodiment, the term "treatment" may include increasing the life expectancy of the subject.

[0042] In this context, an "effective amount" of a compound or composition used to treat a specific disease (such as cancer) refers to an amount sufficient to improve or alleviate symptoms associated with that disease in some way. This amount may be administered as a single dose or according to a specific dosing regimen to exert its therapeutic effect. This amount may cure the disease, but in some specific embodiments, the administration is intended to improve the symptoms of the disease. In certain embodiments, multiple doses may be required to achieve the desired symptom improvement. A "therapeutic effective amount" or "therapeutic effective dose" may refer to an agent, compound, material, or composition containing at least a compound sufficient to produce a therapeutic effect. An effective amount is the amount of a therapeutic agent necessary to prevent, cure, improve, inhibit, or partially inhibit the symptoms of a disease or disorder.

[0043] In this context, "patient" or "subject of treatment" includes humans and non-human animals, including mammals. Mammals include primates such as humans, chimpanzees, gorillas, and monkeys, as well as domesticated animals.

[0044] In this article, "combination" refers to any association between two or more projects. This association can be spatial or refers to the use of two or more projects for a common purpose.

[0045] The specific cancers treated in this article are primarily those with wild-type KRAS or those carrying KRAS G12C activating mutations. In some instances, the specific cancers being treated include lung cancer, pancreatic cancer, colorectal cancer, ovarian cancer, appendiceal cancer, and bile duct cancer. Currently, the specific cancers being treated are resistant to KRAS G12C inhibition. KRAS G12C-resistant tumors may include non-small cell lung cancer.

[0046] KRAS inhibitors include targeted drugs AMG 510 and MRTX849 against KRAS G12C mutations, and the pan-KRAS inhibitor BI-2865. Glucocorticoids include beclomethasone, betamethasone, cortisone, dexamethasone, hydrocortisone, and prednisolone. A specific implementation involves a combination of the KRAS inhibitor AMG 510 and prednisolone.

[0047] The term "in combination with" refers to the simultaneous administration of KRASi and prednisolone; or the administration of one of the compounds prior to the administration of other inhibitory compounds.

[0048] In this document, "TNF inhibitor" or "agent that inhibits TNF activity" refers to any known agent (molecule / compound) capable of reducing or inactivating the biological activity of tumor necrosis factor (TNF). TNF inhibitors include lenalidomide, thalidomide, prednisone, etanercept, adalimumab, infliximab, golimumab, pecelizumab, and etanercept. In specific implementations, KRASi and TNF inhibitors can be combined, including: AMG510 and lenalidomide; and AMG510 and etanercept.

[0049] It is understood that the appropriate dosage depends on a range of factors typically possessed by a physician, veterinarian, or researcher with the relevant skills. Dosage for small molecules can vary, for example, depending on the identity, size, and condition of the subject or sample being treated, as well as the route of administration, if applicable, and the desired effect the practitioner expects in the subject. Furthermore, it is understood that the appropriate dosage depends on efficacy. These appropriate dosages can be determined using detection methods known in the art. When one or more of these compounds need to be administered to animals (e.g., humans), the determination should be made by a physician or veterinarian.

[0050] The phrase “cancer resistance to KRAS inhibition” as used in this article refers to a well-known mechanism in which cancer or tumor cells are inherently resistant to KRAS inhibition; or to the acquisition of such resistance after initial susceptibility to treatment with a well-known KRAS inhibitor. For example, many tumors with KRAS G12C mutation activation, such as NSCLC, show remarkable initial clinical responses to treatment with KRAS tyrosine kinase inhibitors (TKIs), but it is well known that the development of secondary resistance is inevitable with effective treatment of a particular KRAS inhibitor.

[0051] Statistical analysis was performed, with error bars representing the mean ± SEM of the three independent experiments. All data were analyzed using t-tests with GraphPadPrism 7.0 software to determine significance, where P < 0.05 was considered statistically significant.

[0052] Example 1: Prednisolone combined with KRASi more effectively inhibits the growth of NSCLC in in vitro and in vivo models. (1) The tumor cells and drugs involved KRAS-mutated NSCLC cell lines and drugs: KRAS G12C mutant NSCLC cells: H23, H2122, H1373, H358; AMG 510 resistant KRAS G12C mutant NSCLC cells: H358AR, H2122AR.

[0053] Non-KRAS G12C mutant NSCLC cells: H3122, H441, A549.

[0054] AMG 510 (Sotorasib, S8830, SelleckChem, USA), MRTX-849 (Adagasib, S8884, SelleckChem, USA), BI-2865 (HY-153724, MCE, USA).

[0055] Prednisolone (PRDN, P276607, Aladdin).

[0056] (2) Experimental methods Experimental Groups: a) Control group: Cells were treated with DMSO (Servicebio).

[0057] b) AMG 510 group, MRTX-849 group and BI-2865 group: cells were treated with AMG 510 or MRTX-849 or BI-2865 alone.

[0058] c) Prednisolone group: Cells were treated with prednisolone alone.

[0059] d) KRASi combined with prednisolone group: cells were treated with AMG 510 or MRTX-849 or BI-2865 and prednisolone simultaneously.

[0060] 2.1 MTT cell proliferation assay All experiments were performed using RPMI-1640 medium (Servicebio) containing 10% fetal bovine serum (FBS, Cellmax) and 1% penicillin-streptomycin (BioSharp, USA). NSCLC cells in the logarithmic growth phase were treated with different drugs.

[0061] a) Control group: DMSO was added.

[0062] b) AMG 510 group: AMG510 was added at concentrations of 0.01 μM (H358), 0.1 μM (H1373), or 2 μM (H23, H2122) depending on the different cells.

[0063] c) MRTX-849 group: MRTX-849 at a concentration of 0.1 μM was added.

[0064] d) BI-2865 group: BI-2865 at a concentration of 0.1 μM was added.

[0065] e) Prednisolone group: Prednisolone at a concentration of 10 μM was added.

[0066] f) AMG 510 + prednisolone group: 0.01 μM (H358), 0.1 μM (H1373) or 2 μM (H23, H2122) of AMG 510 and 10 μM of prednisolone.

[0067] g) MRTX-849 + Prednisolone group: MRTX-849 at a concentration of 0.1 μM and prednisolone at a concentration of 10 μM were added.

[0068] h) BI-2865 + prednisolone group: BI-2865 at a concentration of 0.1 μM and prednisolone at a concentration of 10 μM were added.

[0069] Each group of drugs was prepared using fresh complete culture medium, with the same final volume (100 μL) for each well. After culturing for 72 h, MTT reagent (Biofrox) was added, and the absorbance was measured using a microplate reader (450 nm). MTT reagent can rapidly and sensitively detect cell proliferation and cytotoxicity, indirectly reflecting the number of viable cells through OD values.

[0070] 2.2 Cloning Experiment Colony formation assays are experiments used to assess cell viability, evaluating the ability of single cells to proliferate and form clones after drug treatment. Logarithmic growth phase liver cancer cells were uniformly seeded into 6-well plates. After cell adhesion, they were cultured for 3-4 days, followed by treatment with different groups of drugs. Specific group treatments were as follows: a) Control group: DMSO was added.

[0071] b) AMG 510 group: AMG510 was added at a concentration of 0.01 μM (H358) or 2 μM (H23, H2122) depending on the different cells.

[0072] c) Prednisolone group: Prednisolone at a concentration of 10 μM was added.

[0073] e) AMG 510 + prednisolone group: AMG 510 at a concentration of 0.01 μM (H358) or 2 μM (H23, H2122) and prednisolone at a concentration of 10 μM were added.

[0074] Prepare each group of drugs using fresh complete culture medium, with the same final volume (2 mL) for each well. After adding the corresponding drugs, continue culturing for 5-10 days (replacing the culture medium and drugs every 72 hours). After colony formation, fix and stain the cells, and observe the number of cell clones.

[0075] 2.3 Animal Experiments 2 million (2×10 6 H23 and H2122 cells were subcutaneously injected into the flanks of nude mice. Subcutaneous tumors appeared in the mice approximately two weeks later. The mice were randomly assigned to an indicator group. Figure 3 or Figure 4The groups and dosages described herein were administered to mice via intragastric administration of the drugs. The AMG 510 monotherapy group received 5 mg / kg / day or 200 mg / kg / day, and the PRDN monotherapy group received 5 mg / kg / day. For combination therapy, both drugs were administered simultaneously for the specified period. Tumor size was measured every 2–4 days, and tumor volume was calculated using the following formula: Volume = 0.5 × length × width × width. Eight mice were used per group at the time of injection, with a tumor formation rate of 5–8 per group.

[0076] 2.4 Western blot assay Western blot experiments were performed according to standard procedures. p-ERK (Try204) and p-MEK (S217 / 221) antibodies were from Cell Signaling Technology; Actin, ERK, MEK, and KRAS antibodies were from Proteintech gurop.

[0077] Each group of drugs was prepared using fresh complete culture medium, with the same final volume (2 mL) for each well. Logarithmic-phase NSCLC cells were evenly seeded into 6-well plates. After cell adhesion, different groups of drugs were added for treatment. Drugs were added at corresponding time points, and treatments were performed for 2 h, 6 h, 24 h, or 48 h. Total cell protein was extracted, and Western blotting was conducted to detect the expression of the corresponding proteins.

[0078] The specific grouping process is as follows: a) Control group: DMSO was added.

[0079] b) AMG 510 group: AMG510 was added at concentrations of 0.5 μM (H358), 0.5 μM (H1373), or 5 μM (H23, H2122) depending on the different cells.

[0080] c) Prednisolone group: Prednisolone at a concentration of 5 μM was added.

[0081] d) AMG 510 + prednisolone group: AMG 510 at concentrations of 0.5 μM (H358), 0.5 μM (H1373) or 5 μM (H23, H2122) and prednisolone at a concentration of 5 μM were added.

[0082] (3) Experimental results Figure 2 The effect of hormone drugs combined with KRASi on cell survival. MTT ( Figure 2 ah) and clonal formation ( Figure 2The experimental results showed that prednisolone combined with AMG 510 had a synergistic effect on inhibiting KRAS G12C mutation in NSCLC, indicating that prednisolone can enhance the sensitivity of NSCLC cells to KRASi.

[0083] Figure 3 The combined use of KRASi and prednisolone demonstrated highly effective inhibition of tumor growth in a living xenograft model. Immunodeficient nude mice were subcutaneously injected with H23 ( Figure 3 ac) and H2122 ( Figure 3 (de) cells were treated with drugs after tumor formation. Results showed significant tumor growth in the control group. Tumor growth was slightly slowed in groups treated with AMG 510 and prednisolone alone, but not statistically significant. However, the combined use of AMG 510 and prednisolone significantly and effectively inhibited tumor growth in the short term (one month).

[0084] Figure 4 A live xenograft model was used to demonstrate the long-term inhibitory effect of the combined use of KRASi and prednisolone on tumor growth. Immunodeficient nude mice were subcutaneously injected with H23 cells, and after tumor formation, they were treated with the drugs for an extended period. The results showed that the combined use of KRASi and prednisolone had a long-term inhibitory effect on tumor growth. Figure 4 ab). Prednisolone combined with KRASi inhibits acquired resistance in large tumors ( Figure 4 cd).

[0085] Figure 5 The effect of pan-KRAS inhibitors combined with prednisolone on cell survival. MTT ( Figure 5 ac) and clonal formation ( Figure 5 The experimental results showed that prednisolone combined with BI-2865 had a synergistic effect on inhibiting non-KRAS G12C mutant NSCLC, indicating that prednisolone can enhance the sensitivity of cells to BI-2865.

[0086] Figure 6 KRAS-induced RTK signaling was inhibited by prednisolone. Western blot results showed that prednisolone significantly inhibited the reactivation of the KRASi-induced MEK-ERK signaling pathway in NSCLCL cells with four KRAS G12C activating mutations.

[0087] Figure 7 The effect of hormone drugs combined with KRASi in AMG 510-resistant NSCLCL cell lines. Figure 7As can be seen from the results, AMG 510 combined with prednisolone also produced a good inhibitory effect on cancer cell survival in H358 AR and H2122 AR cells. Furthermore, Figure 7 The results showed significant upregulation of TNF and IFN-I in H358 AR and H2122 AR, which further confirms that KRASi may lead to acquired resistance in NSCLC by upregulating TNF and IFN-I.

[0088] Example 2: The regulatory effect of KRASi combined with the hormone drug prednisolone on interferon. (1) The tumor cells and drugs involved KRAS G12C NSCLC cell lines: H23, H2122, H1373 and H358.

[0089] KRAS G12C mutant NSCLC drug-resistant cells: H358AR, H2122AR.

[0090] AMG 510 (Sotorasibu, S8830, SelleckChem, USA).

[0091] Prednisolone (P276607, Aladdin).

[0092] IFN-α 1b and IFN-β (Human, ACROBiosystems, USA).

[0093] (2) Establish stable cell lines for xenotransplantation and cell experiments. Lentiviral viruses, including shIFNAR1, shTBK1, shIRF3 human lentiviral particles and control shCtrl lentiviral particles, were constructed using the vector PKLO.1 in combination with molecular cloning and 293T. Among them, lentiviral vectors (PLKO.1) with the target IFNAR1 shRNA sequence (5'-GCCAAGATTCAGGAAATTATT-3', SEQ ID NO.1), TBK1 shRNA sequence (5'-GCGGCAGAGTTAGGTGAAATT-3', SEQ ID NO.2), and IRF3 shRNA sequence (5'-GATCTGATTACCTTCACGGAA-3', SEQ ID NO.3) were used to knock down IFNAR1, TBK1, and IRF3 through RNA interference. PLKO.1 was used as a control. Cells H23 and H2122 were infected with the corresponding lentiviral particles, and 0.6 μg / mL or 0.6 μg / mL puromycin was added to select stable clones, resulting in stably transfected cells of shIFNAR1, shTBK1, shIRF3, and shCtrl. Silencing of IFNAR1, TBK1, and IRF3 was confirmed by Western blot.

[0094] (3) Experimental methods Experimental Groups: a) Control group: Cells were treated with DMSO.

[0095] b) AMG 510 group: Cells were treated with AMG 510 alone.

[0096] c) Prednisolone group: Cells were treated with prednisolone alone.

[0097] b) AMG 510 combined with prednisolone group: Cells were treated with both AMG 510 and prednisolone.

[0098] d) IFNA group: Cells were treated with IFNA alone.

[0099] e) IFNB group: Cells were treated with IFNB alone.

[0100] f) AMG 510 combined with IFNA group: Cells were treated with both AMG 510 and IFNA.

[0101] g) AMG 510 combined with IFNB group: cells were treated with AMG 510 and IFNB simultaneously.

[0102] h) shCtrl group control group: shCtrl cells were treated with DMSO.

[0103] i) shIFNAR1 control group: shIFNAR1 stable cells were treated with DMSO.

[0104] j) shTBK1 control group: shTBK1 stable cells were treated with DMSO.

[0105] k) shIRF3 control group: shIRF3 stable cells were treated with DMSO.

[0106] l) shCtrl group AMG 510 treatment group: shCtrl stable cells were treated with AMG 510.

[0107] m) shIFNAR1 group AMG 510 treatment group: shIFNAR1 stable cells were treated with AMG 510.

[0108] n) shTBK1 group AMG 510 treatment group: shTBK1 stable cells were treated with AMG 510.

[0109] o) shIRF3 group AMG 510 treatment group: shIRF3 stable cells were treated with AMG 510.

[0110] 3.1 MTT cell proliferation assay.

[0111] All experiments were performed using RPMI-1640 medium (Servicebio) containing 10% fetal bovine serum (FBS, Cellmax) and 1% penicillin-streptomycin (BioSharp, USA). H23 and H2122 cells in the logarithmic growth phase were treated with different drugs.

[0112] a) Control group: DMSO was added.

[0113] b) AMG 510 group: Add AMG 510 at a concentration of 2 μM or 10 μM.

[0114] c) IFNA group: IFNA at a concentration of 5 ng / mL was added.

[0115] d) IFNB group: IFNB at a concentration of 5 ng / mL was added.

[0116] e) AMG 510 combined with IFNA group: AMG 510 at a concentration of 10 μM and IFNA at a concentration of 5 ng / mL were added.

[0117] f) AMG 510 combined with IFNB group: AMG 510 at a concentration of 10 μM and IFNB at a concentration of 5 ng / mL were added.

[0118] g) shCTRL control group: Add DMSO.

[0119] h) shIFNAR1 control group: DMSO was added.

[0120] i) shTBK1 control group: DMSO was added.

[0121] j) shIRF3 control group: DMSO was added.

[0122] k) shCTRL group AMG 510 treatment group: AMG 510 at a concentration of 2 μM was added.

[0123] l) shIFNAR1 group AMG 510 treatment group: AMG 510 at a concentration of 2 μM was added.

[0124] m)shTBK1 group AMG 510 treatment group: AMG 510 at a concentration of 2 μM was added.

[0125] n) shIRF3 group AMG 510 treatment group: AMG 510 at a concentration of 2 μM was added.

[0126] Each group of drugs was prepared using fresh complete culture medium, with the same final volume (100 μL) for each well. After culturing for 72 h, MTT reagent (biofroxx) was added, and the absorbance was measured using a microplate reader (450 nm). MTT reagent can rapidly and sensitively detect cell proliferation and cytotoxicity, indirectly reflecting the number of viable cells through OD values.

[0127] 3.2 Cloning experiment Hepatocellular carcinoma cells in the logarithmic growth phase were evenly seeded into 6-well plates. After cell adhesion, different treatment groups were added. The specific grouping and treatment were as follows: a) shCTRL control group: DMSO was added.

[0128] b) shIFNAR1 control group: DMSO was added.

[0129] c) shTBK1 control group: DMSO was added.

[0130] d) shIRF3 control group: DMSO was added.

[0131] e) shCTRL group AMG 510 treatment group: AMG 510 at a concentration of 2 μM was added.

[0132] f) shIFNAR1 group AMG 510 treatment group: AMG 510 at a concentration of 2 μM was added.

[0133] g) shTBK1 group AMG 510 treatment group: AMG 510 at a concentration of 2 μM was added.

[0134] h) shIRF3 group AMG 510 treatment group: AMG 510 at a concentration of 2 μM was added.

[0135] Continue culturing for 5-10 days (replacing the culture medium and drugs with fresh ones every 72 hours). After colony formation, fix and stain the cells, and observe the number of cell clones.

[0136] 3.3 Animal Experiments 2 million (2×10 6 Stable cell lines derived from these strains (shCtrl, shTBK1, shIRF3, and shIFNAR1) were subcutaneously injected into the flanks of nude mice. Subcutaneous tumors appeared in the mice approximately two weeks later. Mice were randomly assigned to an indicator group. Figure 9 Mice were treated with AMG 510 via intragastric administration, as described in the groupings described above. Treatment lasted 28–36 days. Tumor size was measured every 2–4 days, and tumor volume was calculated using the following formula: Volume = 0.5 × length × width × width. Mice were processed and tumor tissue was obtained after administration. Eight mice were used per group at the time of injection, with a tumor formation rate of 5–8 per group.

[0137] 3.4 Western blot assay Western blot experiments were performed according to standard procedures. p-STAT1 (Tyr701), p-TBK1 (Ser172), and TBK1 antibodies were from Cell Signaling Technology; the p-IRF3 (S386) antibody was from Abcam. IFNAR1, STAT1, IRF3, and KRAS antibodies were from Proteintech Group.

[0138] Each group of drugs was prepared using fresh complete culture medium, with the same final volume (2 mL) for each well. Logarithmic growth phase cells were evenly seeded into 6-well plates. After cell adhesion, different groups of drugs were added for treatment. Drugs were added at corresponding time points, and treatments were performed for 2 h, 6 h, and 24 h. Total cell protein was extracted, and Western blotting was conducted to detect the expression of the corresponding proteins.

[0139] The specific grouping process is as follows: a) shCtrl group AMG 510 treatment group: Add AMG 510 at a concentration of 5 μM.

[0140] b) shIFNAR1 group AMG 510 treatment group: AMG 510 at a concentration of 5 μM was added.

[0141] c) shTBK1 group AMG 510 treatment group: AMG 510 at a concentration of 5 μM was added.

[0142] d) shIRF3 group AMG 510 treatment group: AMG 510 at a concentration of 5 μM was added.

[0143] e) Control group: DMSO was added.

[0144] f) AMG 510 group: Add AMG 510 at a concentration of 5 μM.

[0145] g) Prednisolone group: Prednisolone at a concentration of 5 μM was added.

[0146] h) AMG 510 combined with prednisolone group: Cells were treated with 5 μM AMG 510 and 5 μM prednisolone at the same time.

[0147] 3.5 cDNA Synthesis and Real-Time PCR Each group of drugs was prepared using fresh complete culture medium, with the same final volume (2 mL) for each well. Logarithmic growth phase NSCLC cell lines H23, H2122, H1373, and H358 were evenly seeded in 6-well plates. After cell adhesion, different groups of drugs were added for treatment. Specific grouping and treatment details are as follows: a) Control group: DMSO was added.

[0148] b) AMG 510 group: Add AMG 510 at a concentration of 5 μM.

[0149] c) Prednisolone group: Prednisolone at a concentration of 5 μM was added.

[0150] d) AMG 510 combined with prednisolone group: cells were treated with 5 μM AMG 510 and 5 μM prednisolone at the same time.

[0151] Drugs were added at appropriate time points, and cells were treated for 2 hours, 6 hours, and 24 hours, respectively. Total RNA was then separated from cells using TRIzol reagent (Am-bion). cDNA reverse transcription was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems). PCR primers were synthesized by Qingke Biotechnology. Each PCR was performed in triplicate in 20 μL volumes. Initial denaturation was performed using a SYBR GreenMasterMix system at 95°C for 15 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds in a ViiA7 real-time quantitative PCR system (Applied Biosystems). Three independent experiments were conducted. The values ​​for each gene were normalized to the expression level of Actin mRNA. Primer sequences are as follows.

[0152] IFNA: Forward: 5'-GTGAGGAAATACTTCCAAAGAATCAC (SEQ ID NO.4), Reverse: 5'-TCTCATGATTTCTGCTCTGACAA (SEQ ID NO.5); IFNB: Forward: 5'-AGCTGAAGCAGTTCCAGAAG (SEQ ID NO.6), Reverse: 5'-AGTCTCATTCCAGCCAGTGC (SEQ ID NO.7); Actin: Forward: 5'-CACCATTGGCAATGAGCGGTTC (SEQ ID NO.8), Reverse: 5'-AGGTCTTTGCGGATGTCCACGT (SEQ ID NO. 9).

[0153] (4) Experimental results Figure 8 The results showed that KRASi upregulated NSCLC cell lines carrying the KRAS G12C activation mutation. Figure 8 ah) and animal models ( Figure 8 (ij) IFN signaling. As shown in the figure, KRASi upregulated the immune cytokine IFN-α (ij) in NSCLC cell lines H23, H2122, H1373, and H358. Figure 8 ad) and IFN-β ( Figure 8 The expression of eh) and the upregulation of these cytokines have also been confirmed in animals. Figure 8 ij).

[0154] Figure 9 Knockdown of the type I interferon receptor IFNAR1 resulted in increased sensitivity of KRAS G12C mutant NSCLC to KRASi. KRASi upregulated IFN signaling in NSCLC cell lines and animal models carrying KRAS G12C activating mutations, promoting resistance to KRASi in NSCLC. Figure 9 ad MTT and colony formation assays showed that IFNAR1 knockdown conferred sensitivity of drug-resistant KRAS G12C cell lines to KRASi. Furthermore, Western blot results showed that KRASi led to STAT1 activation in multiple KRAS G12C mutant NSCLC cell lines. Figure 9 eh). Knockdown of IFNAR1 blocks AMG510-induced STAT1 activation, confirming that KRASi-induced type I interferon upregulation is essential for STAT1 activation. Figure 9 Stable knockdown of IFNAR1 led to increased sensitivity of xenografted H2122 tumors to KRASi. Figure 9 kn). The MTT assay showed that exogenous type I interferon could prevent KRASi-induced oncogene-addicted cell death (kn). Figure 9 op).

[0155] Figure 10 The results showed that KRASi leads to activation of the TBK1 / IRF3 signaling axis in NSCLC cells carrying the KRAS G12C activating mutation. The results also indicated that KRASi induces activation of TBK1 and IRF3 in NSCLC cell lines carrying the KRAS G12C activating mutation (…). Figure 10 ad) and animal models ( Figure 10 Rapid and potent activation occurs in e). Cells stably silenced by TBK1 or IRF3 are more sensitive to KRASi ( Figure 10 fm).

[0156] Figure 11 The figure shows that cells stably silenced with TBK1 or IRF3 are sensitive to KRASi. As indicated by the figure, H23 and H2122 cells stably silenced with TBK1 or IRF3 are sensitive to KRASi, and type I interferon is significantly suppressed. Figure 11 Animal experiments were conducted using cells with stable TBK1 or IRF3 silencing, and the results showed that H23 cells stably silenced by TBK1 or IRF3 (ah). Figure 11 ik) and H2122 ( Figure 11Ln) cells were more sensitive to KRASi, which significantly suppressed tumor volume in xenograft models. This indicates that the TBK1-IRF3-IFN signaling pathway mediates acquired resistance to KRASi in NSCLC.

[0157] Figure 12 The study showed that the combination of hormone-based drugs with KRASi inhibited the production of type I interferon in NSCLC cells and animal models. Figure 12 As can be seen from the results, the combination of the hormone drug prednisolone and KRASi significantly inhibited the transcriptional levels of IFN-a and IFN-β in various NSCLC cell lines carrying KRAS G12C activating mutations. Figure 12 The results showed that the blocking effect of prednisolone on IFN-α and IFN-β was also verified at the animal level. Westrn blot results showed the same effect. Figure 12 (ko), the reactivation of TBK1-IRF3 induced by KRASi in vitro and in vivo was also inhibited by prednisolone.

[0158] Example 3: The hormone drug prednisolone inhibits KRASi-induced upregulation of TNF expression in lung cancer cell lines and xenograft tumors. (1) The tumor cells and drugs involved KRAS G12C NSCLC cell lines: H23, H2122, H1373 and H358.

[0159] AMG 510 (Sotorasibu, S8830, SelleckChem, USA).

[0160] Prednisolone (P276607, Aladdin).

[0161] TNF (Human, ACROBiosystems, USA).

[0162] TNF inhibitors lenalidomide (MCE) and etanercept (Enbrel, Pifzer).

[0163] (2) Establishing stable cell lines for xenotransplantation and cell experiments. Lentiviral viruses, including shTNFR1 human lentiviral particles and control shCtrl lentiviral particles, were constructed using molecular cloning and 293T. TNFR1 was knocked down by RNA interference using a lentiviral vector (PLKO.1) with the target TNFR1 shRNA sequence (5'-GCCATGCAGGTTTCTTTCTAA-3', SEQ ID NO.10), with PLKO.1 used as a control. Cells were infected with the corresponding lentiviral particles according to the manufacturer's protocol, and 0.6 μg / mL or 0.8 μg / mL puromycin was added to select stable clones, obtaining shTNFR1 and shCtrl stably transfected cells.

[0164] (3) Experimental methods Experimental Groups: a) Control group: Cells were treated with DMSO.

[0165] b) AMG 510 group: Cells were treated with AMG 510 alone.

[0166] c) Prednisolone group: Cells were treated with prednisolone alone.

[0167] d) TNF group: Cells were treated with TNF alone.

[0168] e) AMG 510 combined with TNF group: cells were treated with AMG 510 and TNF at the same time.

[0169] f) AMG 510 combined with prednisolone group: Cells were treated with both AMG 510 and prednisolone.

[0170] e) shCtrl control group: shCtrl cells were treated with DMSO.

[0171] f) shTNFR1 control group: shTNFR1 stable cells were treated with DMSO.

[0172] g) shCtrl group AMG 510 treatment group: shCtrl stable cells were treated with AMG 510. h) shTNFR1 group AMG 510 treatment group: shTNFR1 stable cells were treated with AMG 510.

[0173] i) Control group: Cells were treated with DMSO.

[0174] j) AMG 510 group: Cells were treated with AMG 510.

[0175] k) Enbrel group: Cells were treated with Enbrel.

[0176] l) Lenalidomide group: Cells were treated with Lenalidomide.

[0177] m) AMG 510 combined with Enbrel group: Cells were treated with both AMG 510 and Enbrel.

[0178] n) AMG 510 combined with Lenalidomide group: cells were treated with both AMG 510 and Lenalidomide.

[0179] 3.1 MTT cell proliferation assay.

[0180] All experiments were performed using RPMI-1640 medium (Servicebio) containing 10% fetal bovine serum (FBS, Cellmax) and 1% penicillin-streptomycin (BioSharp, USA). H23 and H2122 cells in the logarithmic growth phase were treated with different drugs.

[0181] a) Control group: DMSO was added.

[0182] b) AMG 510 group: Add AMG 510 at a concentration of 5 μM or 2 μM.

[0183] c) TNF group: TNF at a concentration of 5 μM was added.

[0184] d) AMG 510 combined with TNF group: AMG 510 at a concentration of 5 μM and IFN-α at a concentration of 10 μM were added.

[0185] e) shCTRL control group: Add DMSO.

[0186] f) shTNFR1 control group: DMSO was added.

[0187] g) shCTRL group AMG 510 treatment group: shCTRL stable cells were treated with AMG 510.

[0188] h) shTNFR1 group AMG 510 treatment group: shTNFR1 stable cells were treated with AMG 510.

[0189] i) Lenalidomide group: Lenalidomide at a concentration of 5 μM was added alone.

[0190] j) Enbrel group: Enbrel was added alone at a concentration of 20 μg / mL.

[0191] k) AMG 510 combined with Enbrel group: AMG 510 and Enbrel at a concentration of 5 μM were added simultaneously.

[0192] l) AMG 510 combined with Lenalidomide group: AMG 510 and Lenalidomide were added at a concentration of 5 μM.

[0193] Each group of drugs was prepared using fresh complete culture medium, with the same final volume (100 μL) for each well. After culturing for 72 h, MTT reagent (biofroxx, USA) was added, and the absorbance was measured using a microplate reader (450 nm). MTT reagent can rapidly and sensitively detect cell proliferation and cytotoxicity, indirectly reflecting the number of viable cells through OD values.

[0194] 3.2 Cloning experiment Hepatocellular carcinoma cells in the logarithmic growth phase were evenly seeded into 6-well plates. After cell adhesion, different treatment groups were added. The specific grouping and treatment were as follows: a) shCtrl control group: Add DMSO.

[0195] b) shTNFR1 control group: DMSO was added.

[0196] c) shCtrl group AMG 510 treatment group: Add AMG 510 at a concentration of 2 μM.

[0197] d) shTNFR1 group AMG 510 treatment group: AMG 510 at a concentration of 2 μM was added.

[0198] Continue culturing for 5-10 days (replacing the culture medium and drugs with fresh ones every 72 hours). After colony formation, fix and stain the cells, and observe the number of cell clones.

[0199] 3.3 Animal Experiments 2 million (2×10 6 The stable cell lines shCtrl and shTNFR1 derived from AMG were subcutaneously injected into the flanks of nude mice. Subcutaneous tumors appeared in the mice approximately two weeks later. Mice were randomly assigned to an indicator group. Mice were treated with 5 mg / kg AMG 510 via gavage. Tumor size was measured every 2–4 days, and tumor volume was calculated using the following formula: Volume = 0.5 × length × width × width. Eight mice were used in each group at the time of injection, with a tumor formation rate of 5–8 per group. Mice were sacrificed 28–30 days after treatment.

[0200] 3.4 Western blot assay Western blot experiments were performed according to standard procedures. The NF-κB (D14E12) antibody was from Cell Signaling Technology. The TNFR1 antibody was from Santa Cruz Biotechnology; the Actin, IκBα, RELB, and LaminB antibodies were from the Proteinch group.

[0201] Each group of drugs was prepared using fresh complete culture medium, with the same final volume (2 mL) for each well. Logarithmic-phase NSCLC cells were evenly seeded into 6-well plates. After cell adhesion, different groups of drugs were added for treatment. Drugs were added at corresponding time points, and treatments were performed for 2 h, 6 h, and 24 h. Total cellular protein and nuclear protein were extracted, and Western blotting was conducted to detect the expression of the corresponding proteins.

[0202] The specific grouping process is as follows: a) Control group: Cells treated with DMSO b) AMG 510 group: AMG 510 at a concentration of 5 μM was added alone.

[0203] c) Prednisolone group: Prednisolone at a concentration of 5 μM was added alone.

[0204] d) AMG 510 combined with prednisolone group: AMG 510 and prednisolone at a concentration of 5 μM were added simultaneously.

[0205] 3.5 cDNA Synthesis and Real-Time PCR Each group of drugs was prepared using fresh complete culture medium, with the same final volume (2 mL) for each well. Logarithmic growth phase NSCLC cell lines H23, H2122, H1373, and H358 were evenly seeded in 6-well plates. After cell adhesion, different groups of drugs were added for treatment. Specific grouping and treatment details are as follows: a) Control group: DMSO was added.

[0206] b) AMG 510 group: Add AMG 510 at a concentration of 5 μM.

[0207] c) Prednisolone group: Prednisolone at a concentration of 5 μM was added.

[0208] d) AMG 510 combined with prednisolone group: cells were treated with 5 μM AMG 510 and 5 μM prednisolone at the same time.

[0209] Drugs were added at appropriate time points, and total RNA was isolated using TRIzol reagent (Am-bion) after treatment for 2h, 6h, and 24h. cDNA reverse transcription was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems). PCR primers were synthesized by Qingke Biotechnology. Each PCR was performed in triplicate in 20μL volumes. Initial denaturation was performed at 95℃ for 15 minutes using a SYBR GreenMasterMix system, followed by 40 cycles of 95℃ for 15 seconds and 60℃ for 60 seconds using a ViiA7 real-time quantitative PCR system (Applied Biosystems). Three independent experiments were conducted. The values ​​for each gene were normalized to the expression level of Actin mRNA. Primer sequences are as follows.

[0210] TNF: Forward: 5'-CCCAGGGACCTCTCTCTAATCA (SEQ ID NO.11), Reverse: 5'-GCTACAGGCTTGTCACTCGG (SEQ ID NO. 12).

[0211] 3.6 Luciferase assay Cells were spread in 48-well plates and transfected with NF-κB-LUC and Renilla-LUC plasmids (Addgene) using Lipofectamine 2000. Twenty-four hours after transfection, cells were treated with the drug (AMG 510, 5 μM) for another 24 hours. A dual luciferase reporter assay system was used according to the manufacturer's instructions. Firefly luciferase activity was measured in a luminometer and normalized to Renilla luciferase activity. Experiments were performed three times, for a total of three independent experiments.

[0212] (4) Experimental results: Figure 13 This indicates that KRASi upregulates TNF signaling in NSCLC cell lines and animal models carrying the KRAS G12C activating mutation, promoting resistance to KRASi in NSCLC. Tumor necrosis factor TNF (TNF) is a key mediator of inflammatory responses, produced by various tissues, and can be induced to express TNF and bind to its corresponding receptors TNFR1 or TNFR2 in response to inflammatory stimuli such as LPS, thereby activating a series of inflammatory signaling networks. It is known that malignant cells and cells in the tumor microenvironment can produce TNF, and experimental evidence from various models has shown that TNF can promote tumor growth. To investigate the mechanism by which KRASi induces resistance in lung cancer cells, cells were treated and TNF mRNA levels were measured. The results showed that NSCLC cell lines (… Figure 13Exposure to AMG 510 led to elevated TNF mRNA levels in four cell lines. Resistance to AMG 510 also induced tumor growth in mice. Figure 13 e) Upregulation of TNF mRNA levels.

[0213] Figure 14 This indicates that KRASi promotes TNF-dependent NF-κB nuclear translocation. Previous studies have shown that TNF is a key activator of NF-κB and may mediate NF-κB-induced drug resistance in tumor cells. This study investigated whether TNF enhancement plays a role in AMG 510-induced NF-κB activation. NF-κB has been reported to be rapidly activated in lung cancer cells expressing EGFR activating mutations. This study confirmed, using Western blot and luciferin reporter gene assays, that AMG 510 activates NF-κB in the KRASG12C mutant cell line. Figure 14 (ah), which also confirms that IκBa degrades ( Figure 14 Since TNF is the main activator of NF-κB, AMG 510 may activate NF-κB by affecting TNF levels.

[0214] Figure 15 This indicates that TNF inhibition induces the sensitivity of KRAS G12C-mutant NSCLC cells to KRASi. In the four lung cancer cell lines and the tested animal models, KRASi induced upregulation of TNF levels. This example further investigates whether the upregulation of TNF is biologically significant. TNFR1 is widely expressed, while TNFR2 expression is limited to immune cells and endothelial cells. The role of TNFR1 knockdown in lung cancer cell lines was first investigated. Figure 15 It is known that TNFR1 knockdown increases the sensitivity of NSCLC cells to KRASi. Enbrel, a fusion protein of TNFR and IgG1, is used clinically as a stable and effective TNF blocker for autoimmune diseases. Enbrel, in combination with AMG 510, also blocks and inhibits the survival of tumor cells. Figure 15 Similarly, this embodiment also used lenalidomide, a drug known to reduce TNF levels. MTT assay results also showed that lenalidomide in combination with AMG 510 effectively inhibited tumor cell survival (eh). Figure 15 This indicates that inhibiting TNF can enhance the sensitivity of KRAS G12C-activating mutant NSCLC cells to KRASi.

[0215] Figure 16This indicates that hormonal drugs can block KRASi-induced TNF upregulation in H23 and H2122 cells. q-RCR results showed that the hormonal drug prednisolone combined with KRASi significantly inhibited TNF transcription levels in various NSCLC cell lines (ad) carrying KRAS G12C activating mutations and xenografted H2122 tumors (e). Similarly, Western blot analysis confirmed that prednisolone inhibited KRASi-induced TNF-dependent NF-κB nuclear translocation, blocked IκBα degradation, and increased the level of RELB, a component of the atypical NF-κB pathway, further alleviating acquired resistance. Prednisolone's inhibition of KRASi-induced NF-κB activation (nr) was determined by the NF-κB reporter gene assay. This indicates that prednisolone inhibits KRASi-induced TNF-dependent NF-κB activation.

[0216] This invention demonstrates that the combined use of KRASi and the hormonal drug prednisolone effectively inhibits the proliferation and survival of KRAS G12C-mutant NSCLC cells, significantly reducing tumor volume. Its efficacy is significantly superior to using AMG510 or MRTX-849 alone, exhibiting a synergistic anti-tumor effect. Furthermore, prednisolone can alleviate acquired resistance to KRASi by blocking the production of TNF and type I IFN induced by KRASi, allowing for direct combination therapy for lung cancer. KRASi AMG510 and MRTX-849 are FDA-approved KRAS G12C targeted therapies, demonstrating reliable safety in relevant clinical trials. Therefore, the findings of this invention are expected to support subsequent Phase II clinical trials of KRASi combined with glucocorticoids, or Phase I-II clinical trials of hormone-drug conjugate KRASi, leading to better clinical benefits for patients carrying the most prevalent mutation gene types in non-small cell lung cancer.

[0217] According to the present invention, MEK-ERK reactivation has been demonstrated as a common adaptive resistance response to KRAS signaling inhibition in tumor cells. Firstly, KRASi induces MEK-ERK reactivation, allowing tumor cells to regain growth signals under sustained stress. The glucocorticoid prednisolone combined with KRASi significantly reduces the survival rate of KRAS G12C-mutant NSCLC and inhibits MEK-ERK reactivation. Furthermore, activation of the TBK1-IRF3 signaling pathway leads to increased type I interferon transcription, and activation of NF-κB leads to increased TNF transcription. Sensitivity to KRASi is enhanced when the adaptive responses mediated by type I interferon and TNF are blocked. Increased type I interferon and TNF mediate intrinsic resistance to KRAS inhibition, while biological or chemical inhibition of type I interferon and TNF signaling makes KRAS G12C-expressing NSCLC cell lines and corresponding CDX models highly sensitive to KRASi. In tumor gene-dependent cells, blocking type I interferon and TNF enhances the efficacy of KRAS inhibitors. Furthermore, KRASi combined with lenalidomide or etanercept is highly effective in inhibiting tumor growth, while KRASi or lenalidomide or etanercept alone are ineffective. According to the present invention, a combined inhibition of KRAS G12C and type I interferon, TNF is also provided as a useful strategy for treating human cancers, such as patients with non-small cell lung cancer (NSCLC).

[0218] This invention also investigated the combined effects of prednisolone and KRASi in the AMG 510-resistant KRAS G12C cell lines H358AR and H2122AR. The combination of lower doses of AMG 510 and prednisolone also showed good inhibitory effects on cell survival in the AMG 510-resistant KRAS G12C cell lines, while KRASi or prednisolone alone were less effective.

[0219] The biological upregulation of type I interferon and TNF following KRAS inhibition may have significant implications for the treatment of lung cancer. Lung cancer is the most common cancer worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of all lung cancers. Most NSCLCs express KRASwt, with a smaller subset expressing KRAS activating mutations. The drug combination of this invention is applicable to most NSCLCs carrying KRAS G12C activating mutations. According to this invention, in tumor subpopulations with KRAS G12C activating mutations, the combination therapy of a KRAS inhibitor and the hormonal drug prednisolone holds promise for more effective elimination of tumor cells in the initial treatment phase, and may also eliminate or delay the development of secondary resistance. Several hormonal drugs have been proven safe and have been used in various rheumatic and immunological diseases, making it feasible to test this therapy in patients. The combined use of prednisolone and a KRAS inhibitor can overcome the resistance of these cells to KRAS inhibitors, suggesting that this strategy may be effective in tumors with secondary resistance. The expression of KRAS G12C mutations is prevalent in other types of cancer in humans, and the combined use of prednisolone and KRAS inhibitors has certain clinical significance.

[0220] The description of the disclosed embodiments is intended to enable any person skilled in the art to implement or use the invention. Various modifications to these embodiments will readily occur to those skilled in the art, and the general principles described herein can be applied to other embodiments without departing from the spirit of the invention. The spirit or scope of the invention should be understood in such a way that the descriptions and illustrations provided herein represent only currently preferred embodiments of the invention and thus only represent the broad subject matter covered by the invention. It should also be understood that the scope of the invention fully encompasses other embodiments that are obvious to those skilled in the art, and therefore the scope of the invention is not limited.

Claims

1. Application of KRAS inhibitors, glucocorticoids, and / or TNF inhibitors in the preparation of drugs for treating cancer.

2. Use of compositions containing KRAS inhibitors, glucocorticoids, and / or TNF inhibitors in the preparation of medicaments for treating cancer.

3. A drug for treating cancer, characterized in that: It includes KRAS inhibitors, as well as glucocorticoids and / or TNF inhibitors.

4. The application according to claim 1 or 2, or the drug according to claim 3, characterized in that: The KRAS inhibitors include one or more of AMG 510, MRTX849, and BI-2865.

5. The application according to claim 1 or 2, or the drug according to claim 3, characterized in that: The glucocorticoids mentioned include one or more of beclomethasone, betamethasone, cortisone, dexamethasone, hydrocortisone, and prednisolone.

6. The application according to claim 1 or 2, or the drug according to claim 3, characterized in that: The TNF inhibitors mentioned include one or more of lenalidomide, thalidomide, prednisone, etanercept, adalimumab, infliximab, golimumab, pecelizumab, and etanercept.

7. The application according to claim 1 or 2, or the drug according to claim 3, characterized in that: The cancer in question is either KRAS wild-type or carries a KRAS G12C activating mutation; Alternatively, the cancers mentioned include one or more of the following: lung cancer, pancreatic cancer, colorectal cancer, ovarian cancer, bile duct cancer, and appendiceal adenocarcinoma; Alternatively, the cancer in question may be a cancer resistant to KRAS inhibition.

8. The application according to claim 1 or 2, or the drug according to claim 3, characterized in that: The cancer in question is non-small cell lung cancer.

9. The application according to claim 1 or 2, or the drug according to claim 3, characterized in that: The cancer in question is KRAS-mutated non-small cell lung cancer.

10. The application according to claim 1 or 2, or the medicament according to claim 3, characterized in that: The cancer in question is non-small cell lung cancer carrying the KRAS G12C activating mutation.

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