Purple KRAS mutation inhibitor and application thereof

Abstract

The invention discloses a generic KRAS mutation inhibitor and application thereof, and belongs to the technical field of biomedicine. The inhibitor disclosed by the invention is a triaromatic ring compound, can selectively inhibit the proliferation of KRAS mutant cells by blocking the function of REG gamma-20S proteasome, and is used for treating KRAS mutant tumors or cancers. The method has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pan-KRAS mutation inhibitor and its application. Background Technology

[0002] The proteasome system is one of the most important protein degradation systems in eukaryotes, primarily responsible for degrading misfolded and other damaged proteins during protein synthesis. It also plays a crucial role in regulating cellular processes such as cell cycle, transcription, cell signaling, cell death, and immune responses. The proteasome complex typically consists of two parts: a 20S core particle and a proteasome activator (19S regulatory particle, 11S activator, and Blm10 / PA200). The 19S proteasome activator binds to the proteasome to form a 19S-20S-19S 26S proteasome complex, degrading substrate proteins in a ubiquitin- and ATP-dependent manner. The 11S proteasome activator binds to the proteasome to form an 11S-20S-11S proteasome complex, degrading substrate proteins in a non-ubiquitin- and ATP-dependent manner.

[0003] REGγ, also known as 11Sγ, PA28γ, PSME3, or Ki, belongs to the 11S proteasome activator family. As a proteasome activator, REGγ binds to the α subunit of the 20S core particle, opening the 20S gating and delivering substrate proteins to the β subunit protease catalytic site of the 20S core particle, leading to substrate protein degradation. The REGγ-20S protein degradation system does not require ubiquitination or ATP, making it a ubiquitin- and ATP-independent protein degradation system. The REGγ-20S protein degradation system regulates a series of physiological and biochemical processes by degrading proteins with important biological functions. REGγ is highly expressed in many human tumor tissues, including malignant tumors such as lung cancer and colon cancer. Most of REGγ's substrate proteins are tumor suppressor proteins, including p53, p21, p19, p16, GSK3β, IκBε, and Lats1. In summary, REGγ is a novel pro-tumorigenic oncogene closely related to tumorigenesis and development.

[0004] KRAS was the first oncogene discovered in the human body. When the KRAS gene mutates, it remains continuously activated, disrupting intracellular signaling, leading to uncontrolled cell proliferation and ultimately cancer. KRAS mutations drive the development of 20% of human malignancies, including the three most common and deadliest cancers: non-small cell lung cancer, pancreatic cancer, and colorectal cancer. KRAS G12C is the most common KRAS mutation in non-small cell lung cancer patients, while KRAS G12D and KRAS G12V are the most common mutations in colorectal and pancreatic cancers.

[0005] KRAS has long been considered a difficult target for drug development. This is mainly due to two reasons: first, the small size and relatively smooth surface of the protein molecule make it difficult to find suitable "pockets" for small molecule drugs to bind to; second, because KRAS has a very strong affinity for the nucleotide GTP, drugs have difficulty competing with GTP, and thus have difficulty binding to the KRAS protein and inhibiting its activity. Advances in structural biology have overturned the notion that KRAS is untreatable. On May 28, 2021, the first inhibitor targeting KRAS G12C, Lumakras (Sotorasib, AM510), was approved by the FDA for the treatment of locally advanced or metastatic non-small cell lung cancer with KRAS G12C mutations; on December 12, 2022, the second KRAS G12C inhibitor, KRAZATI (Adagrasib, MRTX849), was approved by the FDA for the treatment of patients with metastatic non-small cell lung cancer. However, downstream or parallel bypass activation, KRAS secondary mutations, histological transformation (adenocarcinoma to squamous cell carcinoma, EMT), and changes in the tumor microenvironment leading to drug resistance limit the clinical efficacy of KRAS G12C inhibitors. Furthermore, while inhibitors targeting other KRAS mutation types have been studied, no clinical therapeutic drugs have yet been developed. To address these challenges, developing drugs targeting pan-KRAS-mutant cancers may be a promising breakthrough in clinical treatment.

[0006] Therefore, the challenges of developing specific inhibitors targeting different KRAS mutation sites and the difficulties of treatment resistance caused by KRAS drug resistance mutations necessitate the development of new drugs targeting pan-KRAS mutations. Summary of the Invention

[0007] To address the shortcomings and problems of existing technologies, based on the high expression of REGγ in KRAS-mutant tumors and the selective sensitivity of KRAS-mutant tumors to REGγ inhibition, this invention proposes a novel, safe, and highly effective class of triaromatic ring compounds as pan-KRAS mutation inhibitors. These compounds inhibit REGγ-20S proteasome function for the treatment of KRAS-mutant tumors or cancers. The main points of this invention are the high expression of REGγ in KRAS-mutant tumors (compared to KRAS wild-type tumors, REGγ shows a higher expression level in KRAS-mutant tumors; REGγ accelerates lung cancer progression and metastasis in KRAS G12D mice), and the selective sensitivity of KRAS-mutant tumors to REGγ inhibition. This suggests that REGγ plays a crucial role in KRAS-mutant cancers, and that the KRAS / NRF2 / REGγ signaling axis directly upregulates REGγ expression in the KRAS mutation context. This invention further proposes the highly efficient antitumor activity of small molecule triaromatic ring compounds that specifically target and block REGγ-20S proteasome function against KRAS-mutant tumors, and the application of such compounds or pharmaceutical compositions containing such compounds in the treatment of various KRAS-mutant tumors, especially in the treatment of KRAS-mutant lung cancer and KRAS-mutant colorectal cancer.

[0008] This invention proposes that REGγ is highly expressed in KRAS-MUT (KRAS mutant) colorectal and lung cancer tissues, and that REGγ is highly expressed in KRAS-MUT colorectal and lung cancer cells.

[0009] In one specific implementation, it was shown that the protein expression level of REGγ in three KRAS-MUT (G12C, G12D, G12V) lung cancer tissues was higher than that in six KRAS-WT (KRAS wild-type) lung cancer tissues.

[0010] In one specific implementation, REGγ protein expression was shown to be higher in three KRAS-MUT lung cancer cell lines (A549, H441, H460) than in three KRAS-WT lung cancer cell lines (H661, H838, H522), while REGγ expression was lowest in cell lines derived from normal lung tissue (MRC-5, WI38).

[0011] In one specific implementation, it was shown that overexpression of mutant KRAS in KRAS-WT colon cancer cells (HCT8) resulted in increased transcriptional and protein levels of REGγ.

[0012] Furthermore, this invention proposes that KRAS-mutant tumors exhibit selective sensitivity to REGγ inhibition.

[0013] In one specific implementation, REGγ silencing was shown to have a stronger inhibitory effect on the proliferation of KRAS-MUT cancer cells than on the proliferation of KRAS-WT cancer cells.

[0014] In one specific implementation, in vivo experiments showed that REGγ silencing had a stronger inhibitory effect on KRAS-MUT tumor proliferation than on KRAS-WT tumor proliferation.

[0015] This invention also proposes a pan-KRAS mutation inhibitor comprising a triaromatic ring compound or its stereoisomer targeting the REGγ-20S proteasome, a pharmaceutically acceptable salt, a metabolite, a prodrug, or a solvate, the structure of which is shown in Formula 1 below:

[0016]

[0017] The compound described in this invention is C 21 H 17 ClN4O3.

[0018] This invention also proposes that the above-mentioned inhibitors are small molecule triaromatic ring compounds that specifically target and block the function of the REGγ-20S proteasome, and have highly efficient anti-tumor activity against KRAS-mutant tumors or cancers.

[0019] In this invention, the inhibitor is based on the binding pocket of REGγ's C-terminal to the 20S proteasome. Computer dynamic molecular simulation was performed, and computer virtual screening revealed that this type of compound can bind to the pocket between α6 and α7 of the 20S proteasome, blocking the binding of REGγ to the 20S proteasome and inhibiting the function of the 20S proteasome in a REGγ-dependent ATP-activated and ubiquitin-independent manner.

[0020] Specifically, the KRAS-mutated tumors or cancers include, but are not limited to, KRAS-mutated pancreatic cancer, colorectal cancer, lung cancer, cholangiocarcinoma, multiple myeloma, acute myeloid leukemia, diffuse large B-cell lymphoma, esophageal adenocarcinoma, gastric cancer, uterine cancer, cervical cancer, bladder cancer, liver cancer, breast cancer, etc., or metastatic lesions of the above-mentioned tumors or cancers.

[0021] Specifically, the KRAS mutation includes any one or more of the following: KRAS G12C, KRAS G12D, KRAS G12V, KRAS G12S, KRAS G12A, KRAS G12R, KRAS G12F, KRAS G13D, KRAS G13C, KRAS Q61H, KRAS Q61R, KRAS Y96D, and KRASA146T.

[0022] In one specific embodiment, treatment with the representative compound RLY01 (as shown in Formula 1) resulted in a lower half-maximal inhibitory concentration (IC50) in the KRAS-MUT cancer cell line than in the KRAS-WT cancer cell line, indicating that the representative compound RLY01 selectively kills KRAS mutant cancer cells.

[0023] In one specific implementation, the representative compound RLY01 was shown to significantly inhibit the proliferation of KRAS-mutant colorectal cancer (SW620, SW480) and lung cancer cells (A549, H441, Calu1).

[0024] In one specific implementation, the representative compound RLY01 was shown to significantly inhibit the proliferation of KRAS-mutant lung cancer organoids after treatment.

[0025] In one specific embodiment, the representative compound RLY01 was shown to significantly inhibit the proliferation of KRAS-mutant CDX tumors derived from the KRAS-mutant (G12D) HCT15 cell line.

[0026] In one specific implementation, the representative compound RLY01 was shown to significantly inhibit the proliferation of KRAS-mutant (G12V) patient-derived xenografts (PDX) after treatment.

[0027] In one specific embodiment, the representative compound RLY01 is shown to treat LSL-KRAS. G12D Trp53 flox / flox The mouse model of primary lung cancer induction showed a significant inhibitory effect on the development of KRAS-mutant primary lung cancer.

[0028] The present invention also proposes a method for blocking REGγ-20S proteasome function, wherein the method blocks the binding of REGγ to the 20S proteasome by means of an inhibitor as described above binding to the pocket between α6 and α7 of the 20S proteasome.

[0029] The present invention also proposes a drug / drug composition comprising, as described above, an inhibitor, and / or, a pharmaceutically acceptable carrier.

[0030] Specifically, the drug / drug composition may be used alone and / or in combination with drugs such as AMG510.

[0031] Preferably, the pharmaceutically acceptable carrier refers to a carrier that, when properly administered to animals or humans, does not produce adverse, allergic, or other adverse reactions. Pharmaceutically acceptable carriers include, but are not limited to: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0032] Preferably, the drug / drug composition may further contain physiologically compatible excipients, including buffers, diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc.

[0033] Specifically, the drug / drug composition is administered orally, by injection, nasal administration, transdermal administration, or mucosal administration.

[0034] Preferably, the dosage form of the drug / drug composition includes injections, sterile powders for injection, tablets, pills, capsules, lozenges, liniments, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalers, or suppositories, etc. All of the above dosage forms of the drug / drug composition can be prepared according to conventional methods in the pharmaceutical field.

[0035] Specifically, the drug / drug composition includes liquid dosage forms, gaseous dosage forms, solid dosage forms, and semi-solid dosage forms.

[0036] Preferably, the route of administration of the drug / drug composition is parenteral, injection, or oral. Injection preferably includes intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. The drug / drug composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, gaseous, or liquid form, i.e., aqueous, non-aqueous, or suspension, more preferably tablets, capsules, granules, injections, or infusions. More preferably, it is administered intravascularly, subcutaneously, intraperitoneally, or intramuscularly. Preferably, the drug / drug composition can also be administered as an aerosol or coarse spray, i.e., nasal administration; or intrathecal, intramedullary, or intraventricular administration. More preferably, the drug / drug composition can also be administered transdermally, percutaneously, topically, intraenterically, intravaginally, sublingually, or rectally. The drug / drug composition of the present invention can be formulated into various dosage forms as needed, and the physician can determine the beneficial dosage for the patient based on factors such as patient type, age, weight, general disease condition, and administration method. Administration methods may include injection or other treatment methods.

[0037] The dosage level of the drug / pharmaceutical composition of the present invention can be adjusted according to the amount of composition required to achieve the desired diagnostic or therapeutic outcome. The administration regimen can also be a single injection or multiple injections, or adjustments thereof. The selected dosage level and regimen are subject to reasonable adjustment based on various factors including the activity and stability (i.e., half-life) of the drug / pharmaceutical composition, the formulation, the route of administration, combination with other drugs or treatments, the disease or condition to be detected and / or treated, and the health status and prior medical history of the subject to be treated.

[0038] The therapeutically effective dose of the drug / pharmaceutical composition of the present invention can initially be estimated in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs, and / or primates. Animal models can also be used to determine suitable concentration ranges and routes of administration. These can then be used to determine the useful dose and route of administration in humans. Generally, the determination and adjustment of the effective amount or dose, and the assessment of when and how to make such adjustments, are known to those skilled in the art.

[0039] For further guidance on formulations, dosages, administration regimens, and measurable treatment outcomes, see Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology, etc.

[0040] The present invention also proposes that the REGγ-20S proteasome inhibitor, as a drug or drug composition, can overcome the drug resistance induced by AMG510 treatment, and that combined treatment with AMG510 can more effectively inhibit the growth of AMG510 resistant cells.

[0041] In one specific implementation, it was shown that introducing either of the two secondary mutations (G13D or Y96D) into Calu1 cells (KRAS-G12C) did not significantly differ the IC50 values ​​after treatment with the representative compound RLY01. However, the IC50 value of AMG510 in Calu1 cells carrying the G12C+G13D or G12C+Y96D mutations was approximately 5-6 times higher than that in parental Calu1 cells. This indicates that the representative compound RLY01 overcomes the resistance induced by AMG510 treatment.

[0042] In one specific implementation, the representative compound RLY01 significantly enhanced the anti-growth effect against AMG510-resistant Calu1 cells carrying G12C+G13D or G12C+Y96D mutations, with all combination indices (CI) less than 0.65, indicating a synergistic effect between RLY01 and AMG510.

[0043] The present invention also proposes an application comprising the use of the REGγ-20S proteasome inhibitor as described above, or the preparation method as described above, or the drug / drug composition as described above in a medicament for treating KRAS-mutant tumors or cancers.

[0044] The KRAS-mutated tumors or cancers mentioned include, but are not limited to, KRAS-mutated pancreatic cancer, colorectal cancer, lung cancer, cholangiocarcinoma, multiple myeloma, acute myeloid leukemia, diffuse large B-cell lymphoma, esophageal adenocarcinoma, gastric cancer, uterine cancer, cervical cancer, bladder cancer, liver cancer, breast cancer, etc., or metastatic lesions of the above-mentioned tumors or cancers.

[0045] The KRAS mutations include any one or more of the following: KRAS G12C, KRAS G12D, KRAS G12V, KRAS G12S, KRAS G12A, KRAS G12R, KRAS G12F, KRAS G13D, KRAS G13C, KRAS Q61H, KRAS Q61R, KRAS Y96D, and KRASA146T.

[0046] This invention also provides the application of the REGγ-20S proteasome as a drug target in the preparation of drugs for the prevention / treatment of KRAS-mutant tumors or cancers. The REGγ-20S proteasome degrades substrate proteins in a ubiquitin- and ATP-independent manner. REGγ, as a proteasome activator, binds to the α subunit of the 20S core particle, opening the 20S gating and delivering the substrate protein to the β subunit protease catalytic site of the 20S core particle, thus degrading the substrate protein. REGγ can regulate cell cycle and apoptosis by degrading cyclin-dependent kinase inhibitors such as p21, p16, p19, and p14. The absence of these cyclin inhibitors can induce normal cells to transform into cancer cells, thereby leading to cancer development. Simultaneously, REGγ can also promote the ubiquitination and degradation of p53 by binding to p53 and MDM2. p53 is a relatively well-studied tumor suppressor with biological functions of accelerating apoptosis and inhibiting tumorigenesis. This evidence further confirms the important regulatory role of the REGγ-20S proteasome in cancer development. Studies have demonstrated that KRAS-mutant tumors or cancers enhance REGγ transcription through the downstream transcription factor NRF2, resulting in high REGγ expression in KRAS-mutant tumors. Furthermore, KRAS-mutant tumors or cancers exhibit selective sensitivity to REGγ inhibition. This evidence makes the REGγ-20S proteasome a novel drug target for the treatment of KRAS-mutant tumors.

[0047] In this invention, the KRAS-mutated tumors or cancers include, but are not limited to, KRAS-mutated pancreatic cancer, colorectal cancer, lung cancer, cholangiocarcinoma, multiple myeloma, acute myeloid leukemia, diffuse large B-cell lymphoma, esophageal adenocarcinoma, gastric cancer, uterine cancer, cervical cancer, bladder cancer, liver cancer, breast cancer, etc., or metastatic lesions of the above-mentioned tumors or cancers.

[0048] The KRAS mutations include any one or more of the following: KRAS G12C, KRAS G12D, KRAS G12V, KRAS G12S, KRAS G12A, KRAS G12R, KRAS G12F, KRAS G13D, KRAS G13C, KRAS Q61H, KRAS Q61R, KRAS Y96D, and KRASA146T.

[0049] The present invention also provides a method for assessing the occurrence, development / metastasis of KRAS-mutant tumors or cancers, or a method for predicting or prognostically assessing whether a patient is suitable for treatment targeting the REGγ-20S proteasome as described above, the method comprising: determining the expression of the REGγ-20S proteasome in a tumor or cancer tissue sample from the patient or subject, wherein the expression of the REGγ-20S proteasome is used to assess the occurrence, development / metastasis of the patient's KRAS-mutant tumor or cancer, or to predict or prognostically assess whether the patient is suitable for treatment targeting the REGγ-20S proteasome as described above.

[0050] The present invention also provides a method for treating KRAS-mutant tumors or cancers, the method comprising administering an effective amount of the inhibitor described above to a subject in need (e.g., a mammal). The method may also be in vitro or non-therapeutic.

[0051] The beneficial effects of the present invention include: the REGγ-20S proteasome inhibitor provided by the present invention as described above can be used as a pan-KRAS mutation inhibitor as a drug or pharmaceutical composition, and can be used to treat KRAS-mutant tumors or cancers by inhibiting REGγ-20S proteasome function, and has good clinical application and pharmaceutical use.

[0052] The beneficial effects of the present invention also include: the present invention also proposes that the REGγ-20S proteasome inhibitor overcomes the drug resistance induced by AMG510 treatment, and that combined treatment with AMG510 can more effectively inhibit the growth of AMG510 resistant cells. This means that blocking the biological function of REGγ with RLY01 may enhance the sensitivity of cancers that have acquired resistance to KRAS-G12C inhibitors to AMG510 treatment. Attached Figure Description

[0053] To clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required in the description of the embodiments are now briefly introduced. Obviously, the drawings in the following description pertain to some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0054] Figure 1 This is a Western blot result diagram showing the high expression of REGγ in KRAS-mutant tumor tissues, as demonstrated in Example 1 of this invention.

[0055] Figure 2 This is a Western blot result diagram showing the high expression of REGγ in KRAS mutant tumor cells, as demonstrated in Example 1 of this invention.

[0056] Figure 3This is a diagram showing the results of Example 2 of the present invention, demonstrating that the transcriptional and protein levels of REGγ are upregulated with the overexpression of KRAS mutations.

[0057] Figure 4 This is a diagram showing the selective toxicity of REGγ silencing to KRAS mutant cancer cell lines in Example 3 of the present invention.

[0058] Figure 5 This is a graph showing the selective toxicity of REGγ silencing to a KRAS-mutant cancer cell xenograft model in Example 4 of this invention.

[0059] Figure 6 The antiproliferative effect of RLY01 in different KRAS state cell lines in Example 5 of this invention is shown. The antiproliferative activity is expressed as IC50 (half-maximal inhibitory concentration).

[0060] Figure 7 This is a graph showing the results of RLY01 inhibiting the proliferation of KRAS mutant cell lines in Example 6 of the present invention.

[0061] Figure 8 This is a graph showing the results of RLY01 inhibiting the proliferation of KRAS-mutant tumor organoids in Example 7 of the present invention.

[0062] Figure 9 This is a graph showing the results of RLY01 inhibiting the proliferation of KRAS-mutant tumor CDX and PDX models in Example 8 of the present invention.

[0063] Figure 10 In Embodiment Nine of the present invention, RLY01 is used in LSL-KRAS G12D Trp53 flox / flox Micro-CT results of inhibition of primary tumor development in mice.

[0064] Figure 11 This is a graph showing the results of RLY01 overcoming the drug resistance induced by AMG510 treatment in Embodiment 10 of the present invention.

[0065] Figure 12 In Example 10 of this invention, the combined treatment of RLY01 and AMG510 is used to more effectively inhibit the growth of AMG510 resistant cells. Detailed Implementation

[0066] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0067] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0068] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0069] This invention proposes a pan-KRAS mutation inhibitor and its applications, belonging to the field of biomedical technology. The inhibitor described in this invention is a triaromatic ring compound that selectively inhibits the proliferation of KRAS-mutant cells by blocking REGγ-20S proteasome function, and can be used to treat KRAS-mutant tumors or cancers. This invention has broad application prospects.

[0070] Unless otherwise specified, the experimental materials used in the examples are all conventional biochemical reagents.

[0071] Example 1: High REGγ expression in KRAS-mutant tumor tissues and cells

[0072] Experimental materials: Cells A549 (CRM-CCL-185), H441 (CRM-HTB-174), H460 (HTB-177), H661 (HTB-183), H838 (CRL-5844), H522 (CRL-5810), MRC-5 (CCL-171), and WI38 (CCL-75) were all purchased from ATCC; Antibodies: anti-REGγ (Abcam, Cat#ab157157), β-actin (MBL, Cat#M177-3), Alexa 790-conjugated AffiniPure Goat Anti-Mouse IgG(H+L)(Jackson,115-655-146), Alexa 790-AffiniPure GoatAnti-Rabbit IgG (H+L) (Jackson, 111-655-144).

[0073] Method and process:

[0074] 1. Protein cleavage

[0075] 1) Add RIPA to tissue or cell samples. Tissue samples need to be ground at low temperature until there are no obvious particles, and cell samples need to be lysed by sonication.

[0076] 2) Centrifuge at 4℃, 12000 rpm, for 5 min, and transfer the supernatant to a new EP tube;

[0077] 3) After BCA quantification, add 6× loading buffer and boil the sample in a metal bath for 15 min.

[0078] 2. Western Blot (WB) assay

[0079] (1) Making adhesive

[0080] 1) Clean the glue sheet, clamp it on the glue-making frame, add double-distilled water, and check for leaks.

[0081] 2) Prepare the lower separating gel according to the formula, pour it into the gel plate, quickly add 1 mL of anhydrous ethanol blocking solution, and let it stand for 1 hour to gel;

[0082] 3) Discard the anhydrous ethanol, prepare the upper layer of concentrated gel according to the formula, pour it into the gel plate, quickly insert the comb, and let it stand for 1 hour to gel;

[0083] 4) Remove the comb and take the polyacrylamide gel off the gel holder for electrophoresis.

[0084] (2) SDS-PAGE

[0085] 1) Prepare 1L of 1×Running Buffer solution using dd H2O, install the polyacrylamide gel on the gel running tank, and pour in 1×Running Buffer solution;

[0086] 2) Add the protein samples sequentially to the loading wells, add 1 μL of protein marker to both sides of the protein sample, and fill in the gaps with 1× loading buffer;

[0087] 3) Connect to the electrophoresis apparatus and perform electrophoresis at a constant voltage of 80V;

[0088] 4) After the protein markers separate, increase the voltage to 120V and continue electrophoresis until the bromophenol blue indicator reaches the bottom of the gel tank, then stop the gel running.

[0089] (3) Transfer membrane

[0090] 1) Prepare filter paper and NC membrane, and prepare 1L of 1×Transfer Buffer solution using dd H2O;

[0091] 2) Remove the protein glue from the running tank and cut it;

[0092] 3) Add the following to the transfer clamp in sequence: sponge, filter paper, protein gel, NC membrane, filter paper, sponge;

[0093] 4) Install the transfer clamp in the transfer tank, place the ice box inside, and pour in 1× Transfer Buffer solution;

[0094] 5) Connect the electrophoresis apparatus and transfer the membrane at a constant current of 200mA in an ice water bath. The transfer time should be slightly longer than the molecular weight of the target protein.

[0095] (4) Closed

[0096] 1) Prepare the blocking solution: 7% skim milk, with PBS buffer solution as the solvent;

[0097] 2) Remove the NC membrane from the transfer tank and transfer clamp, add PBS buffer solution, and wash on a shaker for 3 minutes. Repeat the washing 3 times.

[0098] 3) Discard the PBS buffer solution, add blocking solution, place on a shaker, and block at room temperature for 1 hour.

[0099] (5) Primary antibody incubation

[0100] 1) Prepare antibody dilution solution: 3% BSA, solvent is sterile PBS solution, filter using a 0.45μM filter and syringe;

[0101] 2) Dilute the primary antibody with antibody dilution buffer;

[0102] 3) Discard the blocking solution, add PBS buffer, and wash on a shaker for 3 minutes. Repeat the washing 3 times.

[0103] 4) Cut the membrane according to the molecular weight of the target protein and the protein marker, retaining the NC membrane containing the target protein;

[0104] 5) Place the NC membrane in a dark box and add the corresponding diluted primary antibody;

[0105] 6) Place on a 4℃ shaker and incubate overnight.

[0106] (6) Secondary antibody incubation

[0107] 1) Dilute the secondary antibody with antibody dilution buffer;

[0108] 2) Recover the primary antibody, add PBST buffer, wash on a shaker for 3 min, and repeat the washing 3 times;

[0109] 3) Discard the PBST buffer solution and add the corresponding secondary antibody;

[0110] 4) Incubate on a shaker at 4°C for 1 hour;

[0111] 5) Recover the secondary antibody, add PBST buffer, wash on a shaker for 3 minutes, and repeat the washing 3 times;

[0112] 6) Discard the PBST buffer solution and add PBS buffer solution;

[0113] 7) Use a membrane scanning instrument to scan and analyze the membrane.

[0114] Experimental results:

[0115] like Figure 1 As shown, the protein expression level of REGγ in the three KRAS-MUT (G12C, G12D, G12V) lung cancer tissues was higher than that in the six KRAS-WT lung cancer tissues.

[0116] like Figure 2 As shown, the REGγ protein expression in the three KRAS-MUT lung cancer cell lines (A549, H441, H460) was higher than that in the three KRAS-WT lung cancer cell lines (H661, H838, H522), while the REGγ protein expression in the cell lines derived from normal lung tissue (MRC-5, WI38) was the lowest.

[0117] Example 2: Transcriptional and protein levels of REGγ were upregulated with overexpression of KRAS mutants.

[0118] Experimental materials: HCT8 (CCL-244) cells were purchased from ATCC. HCT8-KRASG12C / G12D / G12V / G12S / G13D stable cells were obtained by lentiviral infection; Antibody: anti-KRAS (Abcam, Cat#ab275876).

[0119] Method and process:

[0120] 1. HCT8 cells were plated in a six-well plate with a density of approximately 30%.

[0121] 2. After the cells adhere to the wall, prepare two tubes of 100 μl free culture medium for each well, add 2 μg of the target plasmid and 4 μl of EZ transfection reagent to each well, and then mix them together and incubate for 15-20 min.

[0122] 3. Change the medium in advance for the cells. Add 200 μl of the mixture of target plasmid and transfection reagent to each well with 800 μl of free medium.

[0123] 4. Replace with complete culture medium after 6-8 hours;

[0124] 5. Samples were collected after 48 hours, and REGγ expression at the transcriptional and protein levels was detected by qPCR and WB, respectively.

[0125] Experimental results:

[0126] like Figure 3As shown, in KRAS-WT colon cancer cells (HCT8), overexpression of mutant KRAS leads to an increase in both the transcriptional and protein levels of REGγ.

[0127] Example 3: REGγ silencing exhibits selective toxicity to KRAS-mutant cancer cell lines.

[0128] Experimental materials: CCK-8 assay kit (Yeasen, Cat#40203ES80)

[0129] Method and process:

[0130] 1. Construct stable HCT8-KRASWT and HCT8-KRASG13D cells, and then construct their respective shNC and shREGγ stable cells from the two stable cell lines;

[0131] 2. Four cell lines were seeded into 15 wells of a 96-well plate, with 1500 cells per well;

[0132] 3. After the cells adhere to the plate, discard the original culture medium in the 96-well plate, add the prepared CCK-8 solution, place it in a 37℃ cell incubator and let it react for 1 hour. Then take it out and use a microplate reader to measure the absorbance value at 450 nM.

[0133] 4. Measure the absorbance every 24 hours, specifically at 0h, 24h, 48h, 72h, and 96h.

[0134] 5. Use Graphpad software to fit the cell proliferation curve.

[0135] Experimental results:

[0136] like Figure 4 As shown, with HCT8-KRAS WT Compared to other cells, silencing REGγ in HCT8-KRAS cells G13D It has a more pronounced inhibitory effect in cells.

[0137] Example 4: REGγ silencing exhibits selective toxicity in a KRAS-mutant cancer cell xenograft model.

[0138] Experimental materials: HT29 (HTB-38) and HCT116 (CCL-247) cells were purchased from ATCC, and BALB / cnude mice were purchased from the Animal Center of East China Normal University.

[0139] Method and process:

[0140] 1. Construct HT29 and HCT116 stable transgenic shNC and shREGγ cells, respectively:

[0141] 1) Construction of shRNA: The shRNAγ targeting sequence is CAGAAGACTTGGTGGCAAA (SEQ ID NO.1). The primers were annealed at 95℃ for 5 min and then gradually brought to room temperature. The vector pLKO.1 (addgene, 8453#) was digested with BshTⅠ and EcoRⅠ and ligated with the annealed primers. DH5α was transformed and the cells were incubated in LB-free solution for 45 min. The cells were plated and incubated overnight. Afterward, bacteria were picked and sequenced. Once the sequencing was successful, the plasmid was extracted after shaking the bacterial culture for 14-16 h to obtain pLKO.1-shREGγ.

[0142] 2) Virus packaging: 293T cells were plated at a density of 30%, and 5 μg and 10 μg of virus packaging plasmids pMD2.G and psPAX2, respectively, and 10 μg of target plasmid were added. The cells were incubated with transfection reagent EZ (Liji Biotechnology, AC04L071) for 15 min and then co-transfected into 293T cells. The viral supernatant was collected after 48 h.

[0143] 3) After infecting HT29 / HCT116 cells with viral supernatant for 12 hours, the culture medium was replaced with fresh medium. After 48 hours, the cells were screened with puro to obtain stable transfected cells.

[0144] 2. Expand cells according to the number of mice to be tumor-bearing (4×10⁻⁶). 6 / cell), digested with trypsin, centrifuged, washed twice with PBS, and the appropriate volume of PBS (4×10⁻⁶) was added according to the cell quantity. 6 (100 μl) and placed on ice.

[0145] 3. The mice were 6-8 weeks old, uniformly weighed nude mice. The cells were gently mixed by pipetting, and the cell suspension was drawn up with a syringe. The mouse was held, and 100 μl of the cell suspension was injected subcutaneously from the right side of the mouse's back. The syringe was then slowly withdrawn to minimize leakage of the cell suspension.

[0146] 4. Tumors formed after one week, and the tumor volume was measured every 4 days.

[0147] like Figure 5 As shown, the experimental results are: compared with HT29-KRAS WT Compared to mouse tumor-bearing models constructed using HCT116-KRAS cells, silencing REGγ in HCT116-KRAS cells significantly improved tumor growth. G13D The cell-based model significantly inhibited tumor growth.

[0148] Example 5: Antiproliferative activity of RLY01 in different cell lines

[0149] Experimental materials: HCT15 (CCL-225), HCT116 (CCL-247), SW620 (CCL-227), SW480 (CCL-228), Calu1 (HTB-54), LoVo (CCL-229), H358 (CRM-CCL-185), H441 (CRM-HTB-174), Capan-2 (HTB-80), Panc-28, Colo 320 (CCL-220), H226 (CRL-5826), H522 (CRL-5810), H3122 (CRL-5985), HCC4006 (CRL-2871), Colo205 (CCL-222), MRC5 (CCL-171), 1459 (CRL-1459), HLF1 (CCL-153), and HPNE (CRL-4023) were all purchased from ATCC.

[0150] Method and process:

[0151] 1. Cell seeding, 3500 cells per well;

[0152] 2. Dissolve RLY01 in DMSO to prepare a 50mM stock solution. Dilute the 50mM stock solution with full culture medium to different concentrations: DMSO, 0.01μM, 0.03μM, 0.1μM, 0.3μM, 1μM, 3μM, 10μM, 30μM, and 100μM. Ensure that each well of the 96-well plate contains 100μL of drug solution, and set up 3 replicates for each drug concentration treatment group.

[0153] 3. Discard the original culture medium and add culture medium solutions containing different concentrations of drugs to the 96 plate;

[0154] 4. Return to a 37℃ cell incubator and culture for 48 hours;

[0155] 5. Cell IC50 assay:

[0156] 1) Observe the degree of cell death after drug-induced cell killing under an optical microscope;

[0157] 2) Using serum-free culture medium, prepare sufficient CCK-8 solution at a ratio of CCK-8:culture medium = 1:10, ensuring that each well contains 100 μL of solution, and keep the entire process in the dark.

[0158] 3) Discard the original drug-containing culture medium in the 96-well plate, add the prepared CCK-8 solution, and place it in a 37℃ cell incubator for static reaction for 1 hour, avoiding light throughout the process;

[0159] 4) Remove the 96-well plate, place it in a microplate reader, and measure its absorbance at 450 nM. Keep the entire process dark.

[0160] 5) Normalize the obtained absorbance values, use Graphpad software to fit the IC50 value of the drug, and observe whether the value is consistent with the cell death observed under an optical microscope. If they are consistent, the value is the IC50 value of the drug.

[0161] Experimental results:

[0162] like Figure 6 As shown, RLY01 selectively kills KRAS mutant cells, with an IC50 value much lower than that of KRAS wild-type cells and normal cells.

[0163] Example 6: RLY01 inhibits the proliferation of KRAS mutant cell lines

[0164] Experimental materials: Cells A549 (CRM-CCL-185), SW620 (CCL-227), SW480 (CCL-228), Calu1 (HTB-54), and H441 (CRM-HTB-174) were all purchased from ATCC.

[0165] 1. Cell seeding in 12-well plates, 2000 cells per well;

[0166] 2. After the cells have adhered to the wall, discard the original culture medium and add 1 mL of drug-treated culture medium of different concentrations to each well;

[0167] 3. Change the medium every two days until a visible cluster of cell clones forms.

[0168] 4. Plate staining

[0169] 1) Discard the original culture medium, add 500 μL of PBS buffer solution to each well, let stand and wash for 3 min, and repeat the washing 3 times;

[0170] 2) Discard the PBS buffer solution, add 500 μL of 4% paraformaldehyde to each well, and let it stand for 20 min to fix;

[0171] 3) Discard the 4% paraformaldehyde solution, add 500 μL of PBS buffer to each well, let stand and wash for 3 min, and repeat the washing 3 times;

[0172] 4) Discard the PBS buffer solution, add 500 μL of 0.9% crystal violet solution to each well, and let it stand for 20 min to stain;

[0173] 5) Discard the 0.9% crystal violet solution, add 500 μL of PBS buffer to each well, let stand and wash for 3 min, and repeat the washing 3 times;

[0174] 6) After letting it stand and dry, take photos and make statistics.

[0175] Experimental results:

[0176] like Figure 7 As shown, RLY01 effectively killed KRAS mutant cell lines A549, SW620, SW480, Calu1, and H441 at all tested doses.

[0177] Example 7: RLY01 inhibits the proliferation of organoids from KRAS-mutant tumors.

[0178] 1. Pretreatment:

[0179] 1) Wash the fresh sample (KRAS G12F lung cancer tissue) several times with washing solution to remove the transport preservation solution (collect the transport solution and washing solution);

[0180] 2) Take a portion of the tissue for T2 for pathological testing. Cut the tissue into a paste in a 5ml centrifuge tube. Take a portion of the cut tissue for T1 and freeze it with 1ml of cryopreservation solution.

[0181] 3) Add 4-5 ml of digestion solution (DB) to a 5 ml centrifuge tube to resuspend the tissue fragments, seal the tube with sealing film, and place it on a shaker at 37°C for 30 min (observe whether the digestion solution is cloudy and whether single cells are digested).

[0182] 4) Grind the tissue and filter the cell suspension using the syringe piston handle and filter screen, then centrifuge at 1500 rpm for 5 min;

[0183] 5) Discard the supernatant, add 1 ml of lysinogen lysate to resuspend the cells, place on ice for 1-2 min for lysinogen lysate, and centrifuge at 1500 rpm for 5 min;

[0184] 6) Discard the supernatant, add 1 ml of washing buffer to resuspend the cells, count the cells using AO / PI staining, and centrifuge at 1500 rpm for 5 min.

[0185] 7) Resuspend cells using matrix gel (10 4 Dispense 10 μl of gel into a 48-well plate, incubate at 37°C for 10-15 min, and then add (AEBL) medium.

[0186] 2. Drug sensitivity testing:

[0187] 1) After the 96-well plates were plated, the drugs were added after the organoids grew to 30-100 μm. The control group was DMSO and the experimental group was RLY01.

[0188] 2) After 96 hours, the drug was collected, and the organoid viability was detected by Calcein-AM / PI fluorescence staining (Calcein-AM:PBS = 1:1000, PI:PBS = 1:40) and CTG activity detection, followed by light microscopy and photographing.

[0189] Experimental results: such as Figure 8 As shown, treatment of the KRAS G12F lung cancer organoids with RLY01 at doses of 50 μM and 100 μM significantly inhibited organoid growth.

[0190] Example 8: RLY01's ability to inhibit the proliferation of KRAS-mutant colorectal cancer CDX and PDX models

[0191] Experimental materials: BALB / c nude mice were purchased from the Animal Center of East China Normal University.

[0192] Method and process:

[0193] 1. Construction of a subcutaneous tumor-bearing model;

[0194] 2. Mice were divided into groups, and tumors were allowed to grow to 70mm. 3 (Approximately 2 weeks) Mice were randomly divided into four groups (vehicle, 25 mg / kg, 50 mg / kg, BTZ: 0.2 mg / kg), with 6 mice in each group.

[0195] 3. Mouse administration: Mice were administered RLY01 (25 mg / kg and 50 mg / kg) and BTZ (0.2 mg / kg) via intraperitoneal injection (once daily). The control group was treated with the same volume of solvent. Tumors in mice were measured every 3 days, and the shortest and longest diameters were recorded. Tumor volume was calculated using these measurements (tumor volume = shortest diameter² × longest diameter × 0.5²). After 21 days of continuous administration, the tumor volume in the control group mice reached approximately 1500 mm². 3 At that time, the mouse tumor was removed, photographed, and weighed.

[0196] Experimental results:

[0197] like Figure 9 As shown, HCT15-KRAS G13D The full-length translational region of KRAS G13D was amplified in cells via PCR. The PCR program was designed according to the instructions for 2×Phanta Max Master Mix (Vazyme, P515-01). The sequence was ligated into the vector pLVX-puro (Ubibio, VT1465) using homologous recombination, resulting in pLVX-puro-KRAS. G13D Plasmid transformation and resuscitation, bacterial selection, culture shaking and sequencing; after successful sequencing, pLVX-puro-KRAS... G13D / Y96D The plasmid and viral packaging plasmid were co-transfected into 293T cells to obtain viral supernatant. The viral supernatant was used to infect Calu-1 cells and screened to obtain stable transfected cells. HCT15-KRAS was established by subcutaneous injection into the lateral aspect of 4-5 week old male athymic nude mice. G13DXenograft tumor mouse model. Continuous treatment with RLY01 at all tested doses significantly inhibited tumor growth. KRAS G12V Patient-derived tissue containing mutated colorectal cancer was minced and subcutaneously implanted onto the lateral side of 4-5 week old male athymic nude mice to construct KRAS. G12V A colorectal cancer tissue xenograft model. After 4 weeks of treatment, RLY01 treatment showed significant inhibition of tumor growth.

[0198] Example 9 RLY01 in LSL-KRAS G12D Trp53 flox / flox Inhibition of primary tumor development and progression in mice

[0199] Experimental materials: Loxp-Stop-Loxp KRAS G12D / FVB / 129Trp53 flox / flox Mice (PK mice). 6-8 week old PK mice were intranasally inoculated with 1×10 6 PFU adenovirus Cre (adeno-Cre) activates lung-oncogenic p53 and KRAS. G12D

[0200] Method and process:

[0201] 1. Loxp-Stop-Loxp KRAS was obtained by mating Trp53-Flox (Nanmo Biotechnology, NM-CKO-18005) and Kras-LSL-G12D (Nanmo Biotechnology, NM-KI-190003) mice. G12D / FVB / 129Trp53 flox / flox Mice (PK mice);

[0202] 2. Cre virus (Hanheng, HBAD-1011) was injected into the lungs of PK mice via nasal drops;

[0203] 3. About 3 months later, the lungs of the mice were scanned by micro-CT. The PK mice were divided into two groups according to the tumor development. The degree of tumor development was uniform between the two groups.

[0204] 4. One group was administered RLY01-50mg / kg intraperitoneally daily, while the other group was treated with the same volume of solvent.

[0205] 5. Four weeks later, the mice's lungs were scanned again using micro-CT.

[0206] Experimental results: such as Figure 10 As shown, in the control group, the tumor exhibited aggressive growth and rapid spread throughout the lung tissue within one month. RLY01 treatment significantly inhibited tumor growth compared to the control group.

[0207] Example 10: Results of RLY01 overcoming AMG510 treatment-induced drug resistance

[0208] 1. Construct Calu1-KRAS G13D Calu1-KRAS Y96D Stable cell lines: The full-length translational regions of KRAS G13D and KRASY96D were amplified by PCR. The PCR program was designed according to the instructions of 2×PhantaMax Master Mix (Vazyme, P515-01). The sequences were ligated into the vector pLVX-puro (Ubibio, VT1465) using homologous recombination, resulting in pLVX-puro-KRAS. G13D / Y96D Plasmid transformation and resuscitation, bacterial selection, culture shaking and sequencing; after successful sequencing, pLVX-puro-KRAS... G13D / Y96D The plasmid and viral packaging plasmid were co-transfected into 293T cells to obtain viral supernatant. The viral supernatant was used to infect Calu-1 cells and screened to obtain stable transfected cells.

[0209] 2. Detect RLY01 and AMG510 in Calu1 and Calu1-KRAS respectively. G13D Calu1-KRAS Y96D IC50 value.

[0210] Experimental results:

[0211] like Figure 11 As shown, when either of the two secondary mutations (G13D or Y96D) was introduced into Calu1 cells (KRAS-G12C), the IC50 values ​​after treatment with the representative compound RLY01 showed no significant difference. However, the IC50 value of AMG510 in Calu1 cells carrying the G12C+G13D or G12C+Y96D mutations was approximately 5-6 times higher than that in parental Calu1 cells. This indicates that RLY01 overcomes the drug resistance induced by AMG510 treatment.

[0212] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0213] As used in this invention, the terms "comprising" and "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0214] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0215] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A pan-KRAS mutation inhibitor, characterized in that, The inhibitors include triaromatic ring compounds or their stereoisomers that target the REGγ-20S proteasome, pharmaceutically acceptable salts, metabolites, prodrugs, or solvates, wherein the structure of the triaromatic ring compound is shown in Formula 1 below:

2. The inhibitor as described in claim 1, characterized in that, The inhibitor can selectively inhibit the proliferation of KRAS mutant cells by blocking REGγ-20S proteasome function, and can be used to treat KRAS mutant tumors or cancers; and / or, the inhibitor is a small molecule triaromatic ring compound that specifically targets and blocks REGγ-20S proteasome function, and has highly efficient anti-tumor / cancer activity against KRAS mutant tumors or cancers.

3. The inhibitor as described in claim 1, characterized in that, The inhibitor is based on the binding pocket of REGγ to the 20S proteasome. Computer dynamic molecular simulation was performed, and computer virtual screening showed that the compound can bind to the pocket between α6 and α7 of the 20S proteasome, blocking the binding of REGγ to the 20S proteasome and inhibiting the function of the 20S proteasome in a REGγ-dependent, ATP- and ubiquitin-independent manner.

4. The inhibitor as described in claim 2, characterized in that, The KRAS-mutated tumors or cancers include pancreatic cancer, colorectal cancer, lung cancer, cholangiocarcinoma, multiple myeloma, acute myeloid leukemia, diffuse large B-cell lymphoma, esophageal adenocarcinoma, gastric cancer, uterine cancer, cervical cancer, bladder cancer, liver cancer, breast cancer, or metastatic lesions of the above tumors or cancers.

5. The inhibitor according to any one of claims 1-4, characterized in that, The KRAS mutations include any one or more of KRAS G12C, KRAS G12D, KRAS G12V, KRAS G12S, KRAS G12A, KRAS G12R, KRAS G12F, KRAS G13D, KRAS G13C, KRAS Q61H, KRAS Q61R, KRAS Y96D, and KRAS A146T.

6. A method for blocking REGγ-20S proteasome function, characterized in that, The method blocks the binding of REGγ to the 20S proteasome by the inhibitor as described in claim 1 binding to the pocket between α6 and α7 of the 20S proteasome.

7. A drug / drug composition, characterized in that, The drug / drug composition comprises the inhibitor as described in claim 1, and / or further comprises a pharmaceutically acceptable carrier.

8. The pharmaceutical / pharmaceutical composition according to claim 7, characterized in that, The drug / drug composition may be used alone and / or in combination with AMG510.

9. An application characterized in that, The applications include the use of the inhibitor as described in any one of claims 1-5, or the method as described in claim 6, or the drug / drug composition as described in claim 7 or 8 in the preparation of a medicament for treating KRAS-mutant tumors or cancers.

10. The application of a REGγ-20S proteasome as a drug target in the preparation of drugs for the prevention / treatment of KRAS-mutant tumors or cancers, characterized in that, The KRAS mutation includes any one or more of KRAS G12C, KRAS G12D, KRAS G12V, KRAS G12S, KRAS G12A, KRAS G12R, KRAS G12F, KRAS G13D, KRAS G13C, KRAS Q61H, KRAS Q61R, KRAS Y96D, and KRAS A146T; and / or, the KRAS-mutated tumors or cancers include KRAS-mutated lung cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, multiple myeloma, acute myeloid leukemia, diffuse large B-cell lymphoma, esophageal adenocarcinoma, gastric cancer, uterine cancer, cervical cancer, bladder cancer, liver cancer, breast cancer, or metastatic lesions of the above tumors or cancers.

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