Composition comprising nampt inhibitor and KRAS inhibitor and use thereof

Abstract

The present invention belongs to the technical field of biology. More specifically, the present invention provides a composition for tumor treatment. The composition comprises an NAMPT inhibitor and a KRAS inhibitor. The present invention also provides use of the NAMPT inhibitor and the KRAS inhibitor in the preparation of an anti-tumor drug. Preferably, the tumor is non-small cell lung cancer.

Description

Compositions including NAMPT inhibitors and KRAS inhibitors and their uses

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 2024114287621, filed on October 12, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biotechnology, and more specifically, to a composition comprising a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor and a murine sarcoma virus proto-oncogene (KRAS) inhibitor, and its use in the preparation of an antitumor medicament. Background Technology

[0004] The Kirsten rat sarcoma viral oncogene (KRAS) is a crucial driver gene in the development and progression of non-small cell lung cancer (NSCLC), with abnormally activated missense mutations present in approximately 25% of NSCLC patients. Among these, the activating missense mutation, where the glycine residue at position 12 of KRAS is changed to cysteine ​​(G12C), has the highest mutation rate in NSCLC, affecting approximately 40% of NSCLC patients with KRAS mutations. In 2021, the U.S. Food and Drug Administration (FDA) approved sotorasib (AMG510), a covalent inhibitor specifically targeting the KRAS G12C mutant protein, for the treatment of NSCLC patients with KRAS G12C mutations. The development of sotorasib and its successful clinical application have shattered the myth that KRAS is "untreatable," marking a significant milestone. The clinical approval of a KRAS G12C mutation-specific inhibitor has spurred the development of inhibitors targeting other KRAS mutation types. Researchers developed a novel non-covalent inhibitor targeting pan-KRAS, BI-2865, by removing the covalent warhead portion of the KRAS G12C inhibitor BI-0474. BI-2865 inhibits various KRAS activating mutants G12D, G12V, G12C, and G13D, as well as wild-type KRAS proteins, by binding to the His95 residue in the switch II binding pocket, which is only present in KRAS, without affecting other RAS family proteins. Preclinical studies have shown that BI-2865 can inhibit wild-type KRAS amplification and the growth of various KRAS-mutant xenografts. A Phase I clinical trial of BI 3706674, a clinical compound with an optimized BI-2865 scaffold, is underway, potentially offering new hope for patients with advanced solid tumors carrying wild-type KRAS amplification.

[0005] Recent results from the Phase III clinical trial (CodeBreak 200study, NCT04303780) of the KRAS G12C inhibitor sottorazib for the treatment of KRAS G12C-mutant advanced non-small cell lung cancer (NSCLC) showed that sottorazib improved the overall response rate (ORR) compared to the standard second-line treatment, docetaxel (13.2% vs. 28.1%). However, unfortunately, sottorazib did not improve overall survival (OS). More than 50% of patients with KRAS G12C-mutant NSCLC exhibit innate resistance to KRAS G12C inhibitor monotherapy. To date, no treatment regimen has been developed to overcome innate resistance to KRAS G12C inhibitors in NSCLC and has entered the clinical trial stage.

[0006] Nicotinamide adenine dinucleotide (NAD+) is a metabolite and coenzyme involved in various metabolic pathways and cellular biological processes. NAD+ acts as a redox carrier in maintaining cellular energy homeostasis and serves as a substrate for a series of NAD-consuming enzymes, including NAD+ glycoside hydrolases (NADase), Sirtuin, and PARP, thereby regulating various signal transduction processes. Therefore, cells must maintain a constant NAD+ level through close regulation of NAD+ synthesis, degradation, and cycling to perform normal physiological functions. Cellular NAD+ levels are maintained through three independent biosynthetic pathways: the Preiss-Handler pathway, the de novo synthesis pathway, and the salvage synthesis pathway. Most of the NAD+ in cells is not newly generated but is recovered from nicotinamide (NAM), a byproduct of NAD+-consuming enzymes, in the salvage synthesis pathway. NAM is converted to nicotinamide mononucleotide (NMN) by the rate-limiting enzyme NAMPT in the salvage synthesis pathway, and NMN is further converted to NAD+ by nicotinamide mononucleotide adenosine transferases (NMNAT1-3). The salvage pathway, the most important NAD+ synthesis pathway in cells, relies heavily on the expression and activity of its rate-limiting enzyme, NAMPT, for maintaining NAD+ homeostasis. In recent years, small-molecule inhibitors such as FK866, which target NAMPT to block the NAD+ salvage pathway and exert anti-cancer effects, have entered clinical trials. However, due to poor therapeutic efficacy, they have not been successfully implemented in clinical practice.

[0007] Therefore, there is an urgent need in this field for a new treatment regimen for non-small cell lung cancer that utilizes combination therapy to exert synergistic anti-tumor effects of drugs, thereby overcoming the resistance problem of KRAS inhibitors and improving their therapeutic efficacy. Summary of the Invention

[0008] As mentioned above, existing treatments for non-small cell lung cancer, especially those using KRAS inhibitors, still suffer from problems such as drug resistance and limited efficacy. Therefore, there is an urgent need in the field for a new treatment regimen for non-small cell lung cancer that can overcome the drug resistance problem of KRAS inhibitors and improve its therapeutic effect.

[0009] The inventors unexpectedly discovered that the combined use of NAMPT inhibitors and KRAS inhibitors has a significant synergistic anti-tumor effect. Therefore, they conducted an in-depth study of its mechanism and found that the combined treatment of the two drugs can promote GSDMD-mediated pyroptosis by synergistically downregulating intracellular NAD+ levels. Based on the above findings, the inventors completed this invention.

[0010] In view of this, in a first aspect, the present invention provides a composition comprising a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor and a murine sarcoma virus proto-oncogene (KRAS) inhibitor.

[0011] In one embodiment, the KRAS inhibitor is a KRAS G12C inhibitor or a pan-KRAS inhibitor.

[0012] In one embodiment, the NAMPT inhibitor is FK866, GNE617, or STF118804.

[0013] In one embodiment, the concentration range of the NAMPT inhibitor is 3 nM to 10 nM, and the concentration range of the KRAS inhibitor is 0.3 nM to 10 μM.

[0014] In one embodiment, the composition further includes a pharmaceutically acceptable excipient or carrier.

[0015] In a second aspect, the present invention provides the use of the composition of the first aspect of the present invention in the preparation of an antitumor medicament.

[0016] In one implementation, the tumor is non-small cell lung cancer.

[0017] In one embodiment, the non-small cell lung cancer is a non-small cell lung cancer with a KRAS mutation.

[0018] In one implementation, the drug is used in non-small cell lung cancer patients who have shown resistance to KRAS inhibitor monotherapy.

[0019] This invention provides a new treatment strategy for overcoming the innate resistance of non-small cell lung cancer patients to KRAS inhibitors by utilizing the synergistic effect of a composition comprising a NAMPT inhibitor and a KRAS inhibitor. The application of this invention will significantly improve the treatment effect for patients with KRAS-mutant non-small cell lung cancer. Attached Figure Description

[0020] Figure 1 shows the decrease in intracellular NAMPT expression levels in different KRAS G12C NSCLC cell lines after treatment with the KRAS G12C inhibitor (sotoprazib).

[0021] Figure 2 shows the effects of 1 μM sotoprazib, 3 nM FK866, and the combination of the two drugs on NAD+ levels in congenitally resistant NSCLC cell lines (H1792 and HCC44) for 72 hours (n=3).

[0022] Figure 3 shows the effects of KRAS inhibitors (sottorazib, BI-2865) and NAMPT inhibitors (FK8663 nM, GNE6173 nM, STF11880410 nM) and the combination of the two drugs for 72 hours on the survival of congenitally drug-resistant NSCLC cell lines (H1792 and HCC44) (n=6).

[0023] Figure 4 shows the effects of 1 μM sotoprazib, 3 nM FK866, and the combination of the two drugs on the cell morphology of NSCLC cell lines (H1792 and HCC44) for 72 hours (n=3).

[0024] Figure 5 shows the effects of 1 μM sotorasiduline, 3 nM FK866, and the combination of the two drugs on LDH release levels in congenitally resistant NSCLC cell lines (H1792 and HCC44) for 72 hours (n=3).

[0025] Figure 6 shows the effects of 1 μM sotoprazib, 3 nM FK866, and the combination of the two drugs for 72 hours on the cleavage and activation of intracellular GSDMD protein in NSCLC cell lines (H1792 and HCC44).

[0026] Figure 7 shows the effects of 30 mg / kg sotorasidub, 30 mg / kg FK866, and the combination of the two drugs on the growth of subcutaneous xenografts in nude mice of NSCLC cell lines (H1792 and HCC44) (n=8). Detailed Implementation

[0027] The following description is merely illustrative and is not intended to limit the scope of the invention. The scope of protection of the invention is defined by the appended claims. Furthermore, those skilled in the art will understand that modifications can be made to the technical solutions of the invention without departing from its spirit and intent. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter pertains. Before a detailed description of the invention, the following definitions are provided to better understand it.

[0029] In cases where numerical ranges are provided, such as concentration ranges, percentage ranges, or ratio ranges, it should be understood that, unless the context explicitly specifies otherwise, all intermediate values ​​between the upper and lower limits of the range, up to one-tenth of the lower limit unit, and any other values ​​or intermediate values ​​within the range are included in the subject matter. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and such embodiments are also included in the subject matter, limited by any specific excluded limit values ​​within the range. Where the range includes one or two limit values, the range excluding any one or both of those included limit values ​​is also included in the subject matter.

[0030] In the context of this invention, many embodiments use the expressions "comprising," "including," or "basically / mainly composed of." The expressions "comprising," "including," or "basically / mainly composed of" are generally understood as open-ended expressions, indicating that they include not only the elements, components, parts, or method steps specifically listed after the expression, but also other elements, components, parts, or method steps. However, in this document, the expressions "comprising," "including," or "basically / mainly composed of" can also be understood as closed-ended expressions in certain cases, indicating that they only include the elements, components, parts, or method steps specifically listed after the expression, and do not include any other elements, components, parts, or method steps. In this case, the expression is equivalent to the expression "composed of."

[0031] As used herein and in the appended claims, unless the context clearly specifies otherwise, the indefinite articles (“a”, “an”) and definite articles (“the”) in the singular form include the plural referent. Similarly, the terms indefinite article (“a”, “an”), “one or more”, and “at least one” are used interchangeably herein.

[0032] As described in the background section, KRAS is a key driver gene in the development and progression of non-small cell lung cancer (NSCLC). Approximately 40% of NSCLC patients carry the G12C mutation, but over 50% of KRAS G12C-mutant NSCLC patients exhibit innate resistance to KRAS G12C inhibitor monotherapy. Furthermore, compared to research on the mechanisms of adaptive and acquired resistance, our understanding of the mechanisms of innate resistance to KRAS G12C inhibitors in lung cancer patients is very limited, and no treatment regimens to overcome innate resistance to KRAS G12C inhibitors in NSCLC have yet entered the clinical trial stage.

[0033] To explore the mechanism of resistance to KRAS inhibitors in KRAS G12C-mutant NSCLC patients, the inventors performed transcriptome analysis and found that NAMPT levels were significantly higher in KRAS-mutant lung cancer cell lines than in KRAS wild-type cell lines. Furthermore, NAMPT expression was also significantly elevated in KRAS-mutant non-small cell lung cancer in clinical lung cancer samples, suggesting that KRAS-mutant lung cancer cells adapt to their metabolic needs by upregulating NAMPT, and that NAMPT holds promise as a metabolically dependent target for KRAS-mutant non-small cell lung cancer. Based on this, the inventors investigated the effects of NAMPT inhibitor monotherapy and combination therapy with KRAS inhibitors on the proliferation of KRAS-mutant non-small cell lung cancer cells. Experimental results confirmed that the combination therapy of the two drugs synergistically inhibited the growth of non-small cell lung cancer cells.

[0034] The inventors further explored the molecular mechanism by which KRAS inhibitors and NAMPT inhibitors exert synergistic effects. They discovered that KRAS inhibitors can downregulate NAMPT expression, and in combination with NAMPT inhibitors, downregulate NAD+ levels in KRAS G12C mutant lung cancer cells. This promotes GSDMD-mediated pyroptosis, resulting in a synergistic anti-tumor effect, overcoming the inherent resistance of KRAS inhibitors while enhancing the anti-tumor efficacy. Thus, the inventors completed this invention.

[0035] Therefore, in a first aspect, the present invention provides a composition comprising a NAMPT inhibitor and a KRAS inhibitor.

[0036] In this invention, a composition comprising a NAMPT inhibitor and a KRAS inhibitor refers to the combined administration of the NAMPT inhibitor and the KRAS inhibitor; that is, they can be administered together or sequentially, rather than necessarily forming a complex when administered together. In the case of sequential administration, the NAMPT inhibitor and the KRAS inhibitor can be administered in any order, i.e., the NAMPT inhibitor can be administered first, followed by the KRAS inhibitor; or the KRAS inhibitor can be administered first, followed by the NAMPT inhibitor. Therefore, in the compositions of this invention, the NAMPT inhibitor and the KRAS inhibitor can be stored separately in separate containers.

[0037] In this paper, the term "synergistic effect" refers to the situation where the combined measurable physiological effect, particularly the actual therapeutic effect, provided by the administration of two agents is greater than the effect predicted by the sum of the actual therapeutic effects of the individual agents. Specifically, a synergistic effect occurs when the first agent alone provides some measurable effect, the second agent alone provides some measurable effect, and the combined measurable effect of the two agents is greater than the sum of the measurable effects of the two individual agents. More specifically, a synergistic effect occurs when the first agent alone does not provide any measurable effect, the second agent alone provides some measurable effect, and the combined measurable effect of the two agents is greater than the effect provided by the second agent alone. Even more specifically, a synergistic effect occurs when neither the first nor the second agent alone provides any measurable effect, but the two agents together provide a measurable effect.

[0038] The inventors discovered through experiments that, compared to treatment with KRAS inhibitors alone, combinations of different KRAS inhibitors (including KRAS G12C inhibitors and pan-KRAS inhibitors) and NAMPT inhibitors can significantly enhance cellular sensitivity to KRAS inhibitors to varying degrees, synergistically inhibiting the growth of KRAS G12C-mutant lung cancer cells. Therefore, in one embodiment, the KRAS inhibitor is a KRAS G12C inhibitor or a pan-KRAS inhibitor. In one specific embodiment, the KRAS inhibitor is sotorasirb or BI-2865. In one embodiment, the NAMPT inhibitor is FK866, GNE617, or STF118804. In one specific embodiment, the NAMPT inhibitor is FK866.

[0039] In one embodiment, the concentration range of the NAMPT inhibitor is 3 nM to 10 nM, and the concentration range of the KRAS inhibitor is 0.3 nM to 10 μM. In a preferred embodiment, the concentrations of the NAMPT inhibitor and the KRAS inhibitor are 3 nM and 1 μM, respectively.

[0040] In one embodiment, the composition may further comprise a pharmaceutically acceptable excipient or carrier, such as any one or a mixture of two or more of sustained-release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, adsorbent carriers, surfactants, and lubricants. In one specific embodiment, the excipient includes dimethyl sulfoxide (DMSO), polyethylene glycol 300 (PEG300), or Tween 80.

[0041] In a second aspect, the present invention provides the use of the composition of the first aspect of the present invention in the preparation of an antitumor medicament.

[0042] Lung cancer is one of the most common malignant tumors worldwide, classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Non-small cell lung cancer accounts for 80% of all lung cancers and includes squamous cell carcinoma (squamous cell carcinoma), adenocarcinoma, and large cell carcinoma. Compared to small cell carcinoma, NSCLC cancer cells grow and divide more slowly, and metastasize relatively late; approximately 75% of patients are diagnosed at an intermediate or advanced stage, resulting in a very low 5-year survival rate. In one embodiment, the tumor is non-small cell lung cancer.

[0043] As described in the background section, a missense mutation with abnormal activation of KRAS is present in approximately 25% of non-small cell lung cancer (NSCLC) patients. Among these, the activation missense mutation of KRAS 12-glycine to cysteine ​​(G12C) is the most common in NSCLC, and approximately 40% of NSCLC patients with KRAS mutations carry this mutation.

[0044] Therefore, in one embodiment, the non-small cell lung cancer is non-small cell lung cancer with KRAS mutations, such as KRAS G12C, KRAS G12D or KRAS G12V, which can be inhibited by the pan-KRAS inhibitor BI-2865 (Kim, Dongsung et al. “Pan-KRAS inhibitor disables oncogenic signalling and tumour growth.” Nature vol.619,7968(2023):160-166).

[0045] In one specific implementation, the KRAS mutation is KRAS G12C.

[0046] In one implementation, the drug is used in non-small cell lung cancer patients who have shown resistance to KRAS inhibitor monotherapy.

[0047] In a preferred embodiment, the drug is used in non-small cell lung cancer patients who have shown innate resistance to monotherapy with KRAS inhibitors.

[0048] In a third aspect, the present invention provides a method for treating non-small cell lung cancer, the method comprising administering the composition of the first aspect of the present invention to a patient in need.

[0049] In one embodiment, the non-small cell lung cancer is a non-small cell lung cancer with a KRAS mutation, such as KRAS G12C, KRAS G12D, or KRAS G12V.

[0050] In one specific implementation, the KRAS mutation is KRAS G12C.

[0051] In one implementation, the patient exhibits resistance to monotherapy with a KRAS inhibitor.

[0052] In one specific implementation, the drug resistance is innate drug resistance.

[0053] Example

[0054] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are merely illustrative and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0055] 1. Effects of KRAS G12C inhibitors on intracellular NAMPT protein levels in congenitally resistant NSCLC cells

[0056] The inventors selected two NSCLC cell lines, H1792 and HCC44, which are inherently resistant to the KRAS G12C inhibitor, and detected the intracellular NAMPT protein level after sotoprazib treatment.

[0057] Main materials: Human non-small cell lung cancer cells H1792 and HCC44 (purchased from ATCC, routinely cultured at 37℃ and 5% CO2), complete culture medium (RPMI-1640 (Gibco) containing 10% fetal bovine serum (ExCell Bio) and penicillin-streptomycin bispecific antibody (Beyotime), PBS (Solarbio), sotorazib (Beyotime Pharmaceuticals), GAPDH antibody (Santa-Cruz), NAMPT antibody (CST), secondary antibody (Raikon Biotech), skim milk powder (Sangon Biotech), PVDF membrane (Millipore), RIPA lysis buffer (Beyotime), PMSF (Beyotime), BCA protein quantification kit (Beyotime), 10% PAGE gel rapid preparation kit (Yamei), 5× loading buffer (Beyotime), pre-stained protein markers (Genestar), BeyoECL Star ultrasensitive ECL chemiluminescence kit (Beyotime).

[0058] The procedure is as follows: H1792 and HCC44 cells were treated with 1 μM sotorasidib for 0 hours, 4 hours, and 72 hours, respectively. The culture medium was discarded, and the cells were washed twice with PBS. 100 μl of RIPA lysis buffer containing PMSF was added to each well, and the cells were collected into 1.5 ml EP tubes using a scraper and lysed on ice for 20 minutes. Subsequently, the cells were centrifuged at 12000 rpm for 15 minutes at 4°C, and the supernatant was retained for BCA protein concentration determination. Each group of proteins was diluted to the same concentration with lysis buffer, and 1 / 4 volume of 5× loading buffer was added. The cells were then incubated at 99°C for 10 minutes. A 10% gel was used, and the electrophoresis conditions were 80V for 20 minutes and 120V for 60 minutes. Transfer was performed using wet transfer at 250 mA for 90 minutes. After transfer, the cells were blocked with 5% skim milk (TBST) at room temperature for 1 hour, and incubated with primary antibody at 4°C overnight. After incubation, the membrane was washed with TBST 3×10 minutes, and incubated with secondary antibody at room temperature for 1 hour. After incubation, wash the membrane with TBST for 3 × 10 minutes and develop it using a chemiluminescence analyzer.

[0059] The results showed that the intracellular NAMPT protein level was significantly reduced after treatment with sotorasib (Figure 1), indicating that the KRAS G12C inhibitor has an inhibitory effect on the NAD+ anabolic pathway in cells.

[0060] 2. Combination therapy of NAMPT inhibitors and KRAS G12C inhibitors synergistically downregulates intracellular NAD+ levels in KRAS G12C-mutant lung cancer cells.

[0061] The above results suggest that KRAS inhibitors can block the NAD+ synthesis pathway by downregulating NAMPT protein levels, leading to a decrease in NAD+ levels in KRAS G12C mutant lung cancer cells. Previous studies have found that tumor cells with low NAMPT expression have lower NAD+ levels, and after treatment with NAMPT inhibitors, NAD+ levels further decrease to below the threshold for maintaining cell survival, thus making them more sensitive to NAMPT inhibitors. The inventors hypothesize that downregulating NAMPT expression by KRAS inhibitors can, in combination with NAMPT inhibitors, downregulate NAD+ levels in KRAS G12C mutant lung cancer cells, thereby exerting a synergistic anti-tumor effect. To verify this hypothesis, the inventors measured the intracellular NAD+ levels in cells after the combined use of sotorasirb and FK866.

[0062] Main materials: NAD+ / NADH assay kit (WST-8 method) (Beyotime), human non-small cell lung cancer cells H1792 and HCC44 (purchased from ATCC, routinely cultured at 37℃ and 5% CO2), complete culture medium (RPMI-1640 (Gibco) containing 10% fetal bovine serum (ExCell Bio) and penicillin-streptomycin antibiotic (Beyotime), 0.25% EDTA-containing trypsin (Beyotime), 96-well plates (Biofill), 6-well plates (Biofill), PBS (Solarbio), FK866 (Taoshu Biotechnology), sotorazib (Bide Pharmaceuticals).

[0063] The steps are as follows: H1792 and HCC44 cells were digested with trypsin and centrifuged at 1200 rpm for 3 minutes, then counted using a hemocytometer. Cells were then seeded at 5*10^5 cells / well into 6-well plates. After cell adhesion, cells were treated with 1 μM sotoprazib, 3 nM NAMPT inhibitor FK866, and a combination of both drugs for 72 hours. After 72 hours, the cell culture medium was discarded, and the cells were washed twice with PBS. Intracellular NAD+ levels were then measured according to the NAD+ / NADH assay kit instructions, as follows:

[0064] 1. Add 200 μl of NAD+ / NADH extraction buffer to each well, lyse on ice for 10 min, then centrifuge at 12,000 g and 4 °C for 5-10 min, and take the supernatant as the sample to be tested.

[0065] 2. Pipette 50-100 μl of the sample to be tested into a centrifuge tube, heat it in a 60℃ water bath or on a PCR instrument for 30 minutes to decompose NAD+, and then pipette 20 μl of the supernatant diluted with NAD+ / NADH extraction solution into a 96-well plate as the sample to be tested.

[0066] 3. Setting up the NADH standard curve: Dilute the 10mM NADH standard with NAD+ / NADH extraction buffer to an appropriate concentration gradient. For the first test, you can set the concentrations to 0, 0.25, 0.5, 1, 2, 4, 6, 8, and 10 μM. Add 20 μl of the standard to each well of the 96-well plate during the test.

[0067] 4. Preparation of alcohol dehydrogenase working solution: Dilute alcohol dehydrogenase 45 times with reaction buffer. For example, add 2 μl of alcohol dehydrogenase to 88 μl of reaction buffer to obtain 90 μl of alcohol dehydrogenase working solution.

[0068] 5. After adding the alcohol dehydrogenase working solution, mix thoroughly and incubate at 37°C in the dark for 10 minutes. Mix the colorimetric solution appropriately, then add 10 μl of colorimetric solution to each well, mix well, and incubate at 37°C in the dark for 10-20 minutes. Measure the absorbance at 450 nm using a microplate reader. The NAD+ concentration of each sample can be calculated based on the standard curve.

[0069] The results showed that FK866 and sotoprazib alone reduced the NAD+ level in tumor cells to some extent, while the combination of the two drugs could further cause a significant reduction in intracellular NAD+ (Figure 2).

[0070] 3. Combination therapy of NAMPT inhibitors and KRAS inhibitors synergistically inhibits the growth of KRAS G12C mutant lung cancer cells.

[0071] Furthermore, the inventors tested the cell viability of H1792 and HCC44 cells after treatment with KRAS inhibitors (including the KRAS G12C inhibitor sotorasibu and the pan-KRAS inhibitor BI-2865) and NAMPT inhibitors (including FK866, GNE617, and STF118804) alone or in combination.

[0072] Main materials: Human non-small cell lung cancer cells H1792 and HCC44 (purchased from ATCC, routinely cultured at 37℃ and 5% CO2), complete culture medium (RPMI-1640 (Gibco) containing 10% fetal bovine serum (ExCell Bio) and penicillin-streptomycin antibiotic (Beyotime), 0.25% solution containing EDTA trypsin and cck-8 (Beyotime), 96-well plates (Biofill), PBS (Solarbio), BI-2865, FK866, GNE617, STF118804 (Taoshu Biotechnology), and sotorazib (Bide Pharmaceuticals).

[0073] The steps are as follows: H1792 and HCC44 cells were digested with trypsin and centrifuged at 1200 rpm for 3 minutes, and counted using a hemocytometer. The cell suspension was diluted to 30,000 cells / ml and seeded into 96-well plates, 100 μl (3000 cells) per well. 100 μl of PBS was added to the blank wells. After cell adhesion, cells were treated with gradient doses of KRAS inhibitors (sottorazib or BI-2865), NAMPT inhibitors (FK8663 nM or GNE6173 nM or STF11880410 nM), and a combination of both drugs for 72 hours. After 72 hours, the cell culture medium was discarded, and 100 μl of complete culture medium containing 10% CCK-8 solution was added to each well. The wells were then incubated in a CO2 incubator for 1 hour, and the absorbance at 450 nm was measured using a microplate reader. The cell viability of the treatment group was calculated using the following formula: Cell viability = (mean absorbance of the treatment group - absorbance of the blank well) / (mean absorbance of the control group - absorbance of the blank well) * 100%.

[0074] The results showed that, compared with sotoprazib monotherapy, the addition of NAMPT inhibitors significantly increased the sensitivity of H1792 and HCC44 cells to KRAS inhibitors (Figure 3).

[0075] 4. The combined use of NAMPT inhibitors and KRAS G12C inhibitors induces pyroptosis.

[0076] To further explore the mechanism by which sotorazib and FK866 exert synergistic anti-tumor effects, the inventors observed the morphology of cells after drug treatment.

[0077] Main materials: human non-small cell lung cancer cells H1792 and HCC44 (purchased from ATCC and routinely cultured at 37℃ and 5% CO2), complete culture medium (RPMI-1640 (Gibco) containing 10% fetal bovine serum (ExCell Bio) and penicillin-streptomycin bispecific antibody (Beyotime), sotorazib (Bide Pharmaceuticals), and FK866 (Taoshu Biotechnology).

[0078] The steps are as follows: cells were treated with 1 μM sotoprazib, 3 nM NAMPT inhibitor FK866, and a combination of the two drugs for 72 hours respectively, and then cell morphology was observed under a 10x microscope.

[0079] The results showed that after 72 hours of combined treatment with the two drugs, obvious bubble-like protrusions appeared on the cell surface (Figure 4), indicating that the cells may have undergone pyroptosis.

[0080] 5. The combined use of NAMPT inhibitors and KRAS G12C inhibitors promotes the release of LDH from lung cancer cells.

[0081] When pyroptosis occurs, the Gasdermin family member GSDMD is cleaved by upstream caspase, releasing its N-terminal domain. This domain has the activity to bind cell membrane phospholipids and create pores in the cell membrane, leading to the release of intracellular LDH into the culture medium. Therefore, changes in LDH activity in the culture medium can be used to determine whether pyroptosis has occurred.

[0082] Therefore, the inventors tested the LDH levels released by cells after treatment with the drug alone or in combination.

[0083] Main materials: human non-small cell lung cancer cells H1792 and HCC44 (purchased from ATCC and routinely cultured at 37℃ and 5% CO2), complete culture medium (RPMI-1640 (Gibco) containing 10% fetal bovine serum (ExCell Bio) and penicillin-streptomycin bispecific antibody (Beyotime), LDH detection kit (Kaiji Biotechnology), sotorazib (Bide Pharmaceuticals), and FK866 (Taoshu Biotechnology).

[0084] The steps are as follows: H1792 and HCC44 cells were treated for 72 hours with 1 μM sotoprazib, 3 nM NAMPT inhibitor FK866, and a combination of the two drugs, respectively. Then, the cell culture supernatant was collected for LDH level detection.

[0085] The results showed that after 72 hours of combined treatment with the two drugs, the release of LDH in lung cancer cells was significantly increased, indicating that cell death characterized by cell membrane rupture had occurred (Figure 5).

[0086] 6. The combination of NAMPT inhibitors and KRAS G12C inhibitors promotes the cleavage of GSDMD in lung cancer cells.

[0087] As a crucial pyroptosis executor within cells, the cleavage of GSDMD proteins is an important indicator for detecting pyroptosis. Therefore, the inventors investigated the changes in GSDMD, a key protein in the intracellular pyroptosis pathway, after drug treatment.

[0088] Main materials: Human non-small cell lung cancer cells H1792 and HCC44 (purchased from ATCC, routinely cultured at 37℃ and 5% CO2), complete culture medium (RPMI-1640 (Gibco) containing 10% fetal bovine serum (ExCell Bio) and penicillin-streptomycin bispecific antibody (Beyotime), PBS (Solarbio), sotorazib (Bide Pharmaceuticals), FK866 (Taoshu Biotechnology), GAPDH antibody (Santa-Cruz), GSDMD antibody (CST), secondary antibody (Raikon Biotechnology), skim milk powder (Sangon Biotech), PVDF membrane (Millipore), RIPA lysis buffer (Beyotime), PMSF (Beyotime), BCA protein quantification kit (Beyotime), PAGE gel rapid preparation kit 10% (Yamei) 5x loading buffer (Beyotime), pre-stained protein markers (Genestar), BeyoECL Star ultrasensitive ECL chemiluminescence kit (Beyotime).

[0089] The procedure is as follows: H1792 and HCC44 cells were treated with 1 μM sotorasidib, 3 nM NAMPT inhibitor FK866, and a combination of both drugs for 72 hours, respectively. The culture medium was discarded, and the cells were washed twice with PBS. 100 μl of RIPA lysis buffer containing PMSF was added to each well. Cells were collected into 1.5 ml EP tubes using a scraper and lysed on ice for 20 minutes. Then, the cells were centrifuged at 12000 rpm for 15 minutes at 4°C. The supernatant was retained for BCA protein concentration determination. The protein was prepared to the same concentration using lysis buffer, and 1 / 4 volume of 5x loading buffer was added. The cells were incubated at 99°C for 10 minutes. A 10% gel was used, and electrophoresis was performed at 80V for 20 minutes and 120V for 60 minutes. Transfer was performed using wet transfer at 250 mA for 90 minutes. After transfer, the cells were blocked with 5% skim milk (TBST) at room temperature for 1 hour and incubated overnight with primary antibody at 4°C. After incubation, wash the membrane with TBST for 3 x 10 minutes, and incubate with secondary antibody at room temperature for 1 hour. After incubation, wash the membrane with TBST for 3 x 10 minutes, and develop using a chemiluminescence analyzer.

[0090] The results showed that the combined treatment with the two drugs could induce the cleavage and activation of intracellular GSDMD, producing a 30KD N-terminal protein with cell membrane binding and membrane pore-forming activity (Figure 6).

[0091] 7. Synergistic antitumor effects of NAMPT inhibitors and KRAS G12C inhibitors in vivo

[0092] Furthermore, the inventors used a nude mouse subcutaneous xenograft model of the KRAS G12C mutant lung cancer cell line to study the antitumor effect of combined treatment with sotoprazib and FK866.

[0093] Main materials: nude mice (Jicui Yaokang), sotorazib (Bide Pharmaceutical), FK866 (Taoshu Biotechnology), human non-small cell lung cancer cells H1792 and HCC44 (purchased from ATCC and routinely cultured at 37℃ and 5% CO2), and complete culture medium (RPMI-1640 (Gibco) containing 10% fetal bovine serum (ExCell Bio) and penicillin-streptomycin bispecific antibody (Beyotime).

[0094] The procedure is as follows: 5 × 10^6 H1792 or HCC44 cells are injected subcutaneously into 5-6 week old nude mice. When the tumor volume reaches 400 mm², the tumor is allowed to grow. 3 Mice were randomly assigned to different groups for drug administration. The mice were divided into a solvent group (5% DMSO, 30% PEG300, 5% Tween 80 (prepared with sterile water)), a sotoprazib group (30 mg / kg), an FK866 group (30 mg / kg), and a combination of the two drugs.

[0095] The results showed that H1792 and HCC44 xenografts exhibited drug resistance to sotoprazib, and FK866 monotherapy did not inhibit the growth of the xenografts. However, the combination of the two drugs significantly inhibited tumor growth (Figure 7).

[0096] The results showed that the combination of sotoprazib and FK866 could effectively reverse the innate drug resistance of KRAS inhibitor-resistant NSCLC cells.

[0097] In summary, the inventors discovered that the combined use of NAMPT inhibitors and KRAS inhibitors exhibits a significant synergistic anti-tumor effect against lung cancer cells inherently resistant to KRAS inhibitors. Mechanistically, the combined use of the two drugs downregulates NAD+ levels in KRAS G12C-mutant lung cancer cells, leading to the cleavage and activation of GSDMD, inducing pyroptosis, and thereby inhibiting tumor growth. The findings of this invention will provide a new therapeutic strategy for overcoming inherent resistance of NSCLC to KRAS G12C inhibitors.

Claims

1. A composition comprising a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor and a murine sarcoma virus proto-oncogene (KRAS) inhibitor.

2. The composition according to claim 1, wherein the KRAS inhibitor is a KRAS G12C inhibitor or a pan-KRAS inhibitor, such as sotorasidub or BI-2865.

3. The composition according to claim 1 or 2, wherein the NAMPT inhibitor is FK866, GNE617 or STF118804, preferably FK866.

4. The composition according to any one of claims 1-3, wherein the concentration range of the NAMPT inhibitor is 3 nM to 10 nM, and the concentration range of the KRAS inhibitor is 0.3 nM to 10 μM, preferably, the concentrations of the NAMPT inhibitor and the KRAS inhibitor are 3 nM and 1 μM, respectively.

5. The composition according to any one of claims 1-4, wherein the composition further comprises a pharmaceutically acceptable excipient or carrier, such as dimethyl sulfoxide (DMSO), polyethylene glycol 300 (PEG300), or Tween 80.

6. Use of the composition according to any one of claims 1-5 in the preparation of an antitumor medicament.

7. The use according to claim 6, wherein the tumor is non-small cell lung cancer.

8. The use according to claim 7, wherein the non-small cell lung cancer is a non-small cell lung cancer with a KRAS mutation such as KRAS G12C, KRAS G12D or KRAS G12V, preferably, the KRAS mutation is KRAS G12C.

9. The use according to claim 7 or 8, wherein the drug is used in patients with non-small cell lung cancer who have shown resistance to monotherapy with KRAS inhibitors, preferably, the resistance is congenital resistance.

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