Application of DUSP6 in curative effect prediction and prognosis evaluation of KRAS G12C inhibitor

Abstract

The invention belongs to the field of medicines, and discloses application of DUSP6 in curative effect prediction and prognosis evaluation of a KRAS G12C inhibitor. The research of the inventor finds that the expression level of DUSP6 is related to the drug sensitivity of the KRAS G12C inhibitor for the first time, and proves that overexpression of DUSP6 can significantly enhance the sensitivity of tumor cells to the KRAS G12C inhibitor in both in-vivo and in-vitro models. On the basis, clinical doctors can be effectively helped to accurately screen out patients with potential benefits for treatment of the KRAS G12C inhibitor from huge tumor patients carrying KRAS G12C mutation, particularly colorectal cancer and pancreatic cancer patient groups.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to the application of DUSP6 in the prediction of efficacy and prognosis evaluation of KRAS G12C inhibitors. Background Art

[0002] KRAS The mutation of the gene (Kirsten rat sarcoma viral oncogene homolog) is one of the most common carcinogenic mutations in digestive system tumors, and the main types of protein mutations caused by it are G12D, G12V, G13D and G12C. Since 2020, the U.S. Food and Drug Administration has gradually approved the KRAS G12C inhibitors sotorasib and adagrasib (Chinese names are sotorasib and adagrasib respectively) for the clinical treatment or clinical trials of KRAS G12C mutant advanced tumors. In August 2024, my country also approved the first domestically produced KRAS G12C inhibitor fluzeretide for the clinical treatment of advanced non-small cell lung cancer. However, a large number of clinical studies have shown that there are significant differences in the efficacy of these drugs in patients with non-small cell lung cancer, colorectal cancer and pancreatic cancer. According to the results of the Codebreak100 and KRYSTAL-1 clinical trials, the objective response rates of sotorasib monotherapy in patients with non-small cell lung cancer, colorectal cancer, and pancreatic cancer were 37.1%, 9.7%, and 21%, respectively, and the objective response rates of adagrasib monotherapy in patients with non-small cell lung cancer and colorectal cancer were 42.9% and 19%, respectively, indicating that there are large individual differences in the response of colorectal cancer and pancreatic cancer patients to KRASG12C inhibitors. Therefore, how to accurately screen patients with potential benefits from KRAS G12C inhibitor treatment from the huge population of colorectal cancer and pancreatic cancer patients is a clinical problem that urgently needs to be solved.

[0003] The dual-specificity protein phosphatase subfamily inactivates members of the mitogen-activated protein (MAP) kinase superfamily (MAPK / ERK, SAPK / JNK, p38) by dephosphorylating phosphoserine / threonine and phosphotyrosine residues, thereby negatively regulating cell proliferation and differentiation. The members of this family have different tissue distributions and subcellular localizations, as well as different extracellular stimulus-induced expression patterns, and have different substrate specificities for various MAP kinases. Among them, dual specificity phosphatase 6 (DUSP6) can specifically dephosphorylate ERK1 / 2 proteins and negatively regulate cell proliferation signals. However, there are currently no reports suggesting that this protein can be used to predict the efficacy of KRAS G12C inhibitor therapy and evaluate prognosis. Summary of the invention

[0004] The object of the present invention is to overcome at least one deficiency of the prior art and provide an application of DUSP6 in the prediction of efficacy and prognosis evaluation of KRAS G12C inhibitors.

[0005] The technical solution adopted by the present invention is: The first aspect of the present invention provides: use of a reagent for quantifying DUSP6 protein or mRNA in a sample in the preparation of a reagent for predicting the efficacy of a KRASG12C inhibitor.

[0006] Specifically, the high expression level of DUSP6 protein or mRNA relative to adjacent normal tissues indicates that the tumor is sensitive to KRASG12C inhibitors and that patients can benefit from targeted therapy with KRAS G12C inhibitors.

[0007] In some application examples, the sample is tumor tissue.

[0008] In some application examples, the primer set for quantifying DUSP6 mRNA in a sample is: Forward primer: 5'- GCGACTGGAACGAGAATACG-3', Reverse primer: 5′-TCGGCTTGGAACTTACTGAAG-3′.

[0009] In some application examples, the tumor is selected from one of colorectal cancer, pancreatic cancer and lung cancer.

[0010] The second aspect of the present invention provides: use of a reagent for quantifying DUSP6 protein or mRNA in a sample in the preparation of a reagent for evaluating the prognosis of treatment with a KRASG12C inhibitor.

[0011] Specifically, the high expression of DUSP6 protein or mRNA relative to adjacent normal tissues indicates that the tumor is sensitive to KRASG12C inhibitors, and patients can benefit from targeted therapy with KRAS G12C inhibitors. The increased expression of DUSP6 protein or mRNA indicates that the tumor is more sensitive to KRAS G12C inhibitors, and patients can benefit from targeted therapy with KRAS G12C inhibitors.

[0012] In some application examples, the sample is tumor tissue.

[0013] In some application examples, the primer set for quantifying DUSP6 mRNA in a sample is: Forward primer: 5'- GCGACTGGAACGAGAATACG-3', Reverse primer: 5′-TCGGCTTGGAACTTACTGAAG-3′.

[0014] In some application examples, the tumor is selected from one of colorectal cancer, pancreatic cancer and lung cancer.

[0015] The third aspect of the present invention provides: use of an agent for promoting DUSP6 protein expression in the preparation of a drug resistance reversal agent for targeted therapy with KRAS G12C inhibitors of tumors.

[0016] Specifically, the agent for promoting the expression of DUSP6 protein may be a viral vector carrying a DUSP6 overexpression plasmid.

[0017] In some application examples, the tumor is selected from one of colorectal cancer, pancreatic cancer and lung cancer.

[0018] A fourth aspect of the present invention provides: a method for determining the sensitivity of tumor cells to a KRAS G12C inhibitor, comprising: S1) Quantification of DUSP6 protein or mRNA levels in tumor cells; S2) Determine the sensitivity of tumor cells to KRAS G12C inhibitors based on DUSP6 content.

[0019] Specifically, the high expression level of DUSP6 protein or mRNA relative to adjacent normal tissues indicates that the tumor is sensitive to KRASG12C inhibitors and that patients can benefit from targeted therapy with KRAS G12C inhibitors.

[0020] In some examples of the methods, the tumor is selected from one of colorectal cancer, pancreatic cancer, and lung cancer.

[0021] In some examples of the methods, the primer set for quantifying DUSP6 mRNA in a sample is: Forward primer: 5'- GCGACTGGAACGAGAATACG-3', Reverse primer: 5′-TCGGCTTGGAACTTACTGAAG-3′.

[0022] The fifth aspect of the present invention provides: a method for screening a KRAS G12C inhibitor resistance reversal agent, comprising the following steps: S1) Detecting the initial DUSP6 protein and / or mRNA levels in KRAS G12C inhibitor-resistant tumor cells; S2) Treating KRAS G12C inhibitor-resistant tumor cells with the test substance for a certain period of time; S3) After treatment, detecting the content of DUSP6 protein and / or mRNA in KRAS G12C inhibitor-resistant tumor cells; S4) determining whether the test substance has the potential to reverse KRASG12C inhibitor resistance based on the change in the content of DUSP6 protein and / or mRNA.

[0023] Specifically, compared with before treatment, the expression level of DUSP6 protein or mRNA increased, indicating that the tumor is more sensitive to KRAS G12C inhibitors, indicating that the test substance has a certain potential to reverse KRAS G12C inhibitor resistance. Otherwise, it can be considered that it does not have the potential to reverse KRAS G12C inhibitor resistance.

[0024] In some examples of the screening methods, the tumor is selected from one of colorectal cancer, pancreatic cancer and lung cancer.

[0025] In some examples of screening methods, the primer set for quantifying DUSP6 mRNA in a sample is: Forward primer: 5'- GCGACTGGAACGAGAATACG-3', Reverse primer: 5′-TCGGCTTGGAACTTACTGAAG-3′.

[0026] In some examples of the screening methods, the tumor is selected from one of colorectal cancer, pancreatic cancer and lung cancer.

[0027] The beneficial effects of the present invention are: The inventors found for the first time that the expression level of DUSP6 is related to the drug sensitivity of KRAS G12C inhibitors, and confirmed in both in vivo and in vitro models that overexpression of DUSP6 can significantly enhance the sensitivity of tumor cells to KRAS G12C inhibitors. Based on this, it can effectively help clinicians accurately screen patients who have the potential to benefit from KRAS G12C inhibitor treatment from a large number of tumor patients, especially colorectal cancer and pancreatic cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the expression of DUSP6 in different cohorts and its relationship with prognosis.

[0029] Figure 2 It is the paired detection result of DUSP6 mRNA expression level in the parent and drug-resistant models.

[0030] Figure 3 It is the paired test result of DUSP6 protein expression level in the parent and drug-resistant model.

[0031] Figure 4 These are the in vitro evaluation results of the effect of DUSP6 overexpression on the susceptibility of sotorasib-resistant cells.

[0032] Figure 5These are the in vivo evaluation results of the effect of DUSP6 overexpression on the susceptibility of sotorasib-resistant cells. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further explained below in conjunction with experiments.

[0034] Example 1: Prognostic effect of DUSP6 in KRAS mutant colorectal cancer and pancreatic cancer The survival data of lung cancer, colorectal cancer and pancreatic cancer patients and the mRNA expression data of DUSP6 were extracted and analyzed from the Cancer Genome Atlas (TCGA) database. Figure 1 As shown: In the overall colorectal cancer patients, the expression level of DUSP6 is not related to the overall survival of the patients ( p =0.8495), while in patients with KRAS mutation, the expression level of DUSP6 was significantly positively correlated with the overall survival of patients ( p =0.0463), that is, the higher the DUSP6 expression, the better the prognosis of the patients; although in the overall pancreatic cancer patients, the DUSP6 expression level was negatively correlated with the overall survival of the patients ( p =0.0079, but no negative correlation was found in the KRAS mutant patient group ( p =0.1784). At the same time, in the cross-cancer joint analysis, we also found that the expression level of DUSP6 in lung cancer tissues was significantly higher than that in colorectal cancer and pancreatic cancer ( p <0.0001). The above results indicate that DUSP6 protein is a protective factor for patients with KRAS mutations, and its expression level is positively correlated with the overall survival of patients with KRAS mutations, suggesting that DUSP6 may have the ability to predict the response of KRAS G12C mutant colorectal cancer and pancreatic cancer to KRAS G12C targeted therapy.

[0035] Example 2: Expression level of DUSP6 in KRAS G12C mutant colorectal cancer and pancreatic cancer resistance models The parental models are two cell lines, SW837 and MIA PaCa2. The drug-resistant model is the drug-resistant cell line SW837 / KIR (deposited in the China Type Culture Collection located in the Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province, with the accession number CCTCC NO: C2024360, classified and named as: Human colorectal cancer cell SW837 / KIR Homo sapiens, and the date of preservation is October 24, 2024) and MIAPaCa2 / KIR (deposited in the China Type Culture Collection located in the Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province, with the accession number CCTCC NO: C2024358, classified and named as: Human pancreatic cancer cell MIA PaCa2 / KIR Homosapiens, and the date of preservation is October 24, 2024) induced by the SW873 and MIAPaCa2 parental cell lines. Of course, other commercially available drug-resistant cell lines of the same type, or drug-resistant cell lines of the same type constructed by known methods, can also be used. The specific methods are as follows: (1) Paired detection of DUSP6 transcription levels in parental and resistant models of KRAS G12C mutant colorectal cancer and pancreatic cancer: S1) The culture medium of the cells in the logarithmic growth phase cultured in a 6 cm cell culture dish was discarded and washed twice with phosphate buffered saline (PBS), 0.5 ml of Trizol reagent was added to each dish, and the dish was left at room temperature for 2 minutes to allow the lysate to be evenly distributed on the cell surface and lyse the cells, then the cells were blown off with a pipette and the lysate was transferred to an RNase-free 1.5 ml EP tube; S2) After standing at room temperature for another 5 minutes, the collected lysate was placed in a 4°C centrifuge. After centrifugation at 12,000 rpm for 15 minutes, the liquid in the EP tube was observed to be divided into three layers, of which the upper layer was the RNA aqueous phase. The supernatant was pipetted into a new RNase-free 1.5 ml centrifuge tube and an equal volume of isopropanol was added. After careful pipetting and mixing, the tube was allowed to stand at room temperature for 10 minutes. S3) The mixed liquid was placed in a 4°C centrifuge again and centrifuged at 12,000 rpm for 10 minutes. At this time, white RNA precipitate can be observed at the bottom of the EP tube. The liquid was discarded, and the remaining liquid was carefully sucked out with a pipette tip. The lid was opened and aired until the white precipitate became transparent. Then about 50ul of DEPC water was added and dissolved by blowing with a pipette tip and the concentration was measured using a NanoDrop instrument. S4) Total RNA was reverse transcribed into cDNA using Evo M-MLV reverse transcription reagent premix (Acori Biotech, Cat. No. AG11706), and then quantitatively detected using SYBR Green Pro Taq HS premixed qPCR kit (with Rox) (Acori Biotech, Cat. No. AG11718) and ABI fluorescent quantitative PCR instrument to evaluate the mRNA expression level of DUSP6 in each cell (forward primer sequence: 5'- GCGACTGGAACGAGAATACG-3', SEQ ID NO.1; reverse primer sequence: 5'-TCGGCTTGGAACTTACTGAAG-3', SEQ ID NO.2). The use of the above kits and instruments was carried out according to the relevant instructions.

[0036] Verification results are as follows Figure 2 As shown: In the colorectal cancer KRAS G12C inhibitor-resistant model, the mRNA expression level of DUSP6 was downregulated 15.4 times compared with the parental (drug-sensitive) model; in the pancreatic cancer KRAS G12C inhibitor-resistant model, the mRNA expression level of DUSP6 was downregulated 14.7 times compared with the parental (drug-sensitive) model.

[0037] (2) Paired detection of DUSP6 protein levels in parental and resistant models of KRAS G12C mutant colorectal cancer and pancreatic cancer: S1) The cells in the logarithmic growth phase cultured in a 6 cm cell culture dish were discarded from the culture medium and washed twice with PBS, then a protein lysis buffer containing 1× protease inhibitor and phosphatase inhibitor was added, and the cells were lysed on ice for 30 minutes, then centrifuged at 12,000 rpm and 4°C for 10 minutes, and the supernatant was collected and quantified by BCA (Bicinchoninic Acid); S2) Add 5× protein loading buffer according to the sample volume and mix well, incubate at 100℃ for 10 minutes. Polyacrylamide gel electrophoresis (10% electrophoresis gel, electrophoresis conditions are constant voltage 80v, adjusted to 100v after 30 minutes and maintained for 90 minutes) is used for protein separation, and transfer to the membrane after the electrophoresis is completed (transfer conditions are constant current 220mA maintained for 120 minutes); S3) After transfer, place the membrane in 8% skim milk solution and block at room temperature for 1 hour; after blocking, wash the membrane three times with 1×TBST buffer (Tris-buffered saline containing Tween 20), cut the required bands, add DUSP6 antibody (Abcam, Catalog No. ab76310) and the corresponding internal reference antibody vinculin (CST, Catalog No. 13901), and place it in a shaker at 4°C overnight; S4) On the second day, the primary antibody was recovered and the membrane was washed three times with 1× TBST buffer for 10 minutes each time. The secondary antibody was incubated at room temperature for 1 hour, and then the membrane was washed three times with TBST buffer for 10 minutes each time. After washing, the membrane was chemically developed.

[0038] Test results such as Figure 3 As shown: In the colorectal cancer KRAS G12C inhibitor-resistant model, the protein expression level of DUSP6 was significantly downregulated compared with the parental (drug-sensitive) model; consistent phenotypes were also observed in the pancreatic cancer KRAS G12C inhibitor-resistant model.

[0039] The above results preliminarily confirmed that in KRAS G12C mutant colorectal cancer and pancreatic cancer, the expression level of DUSP6 is positively correlated with the sensitivity of cells to KRAS G12C inhibitors, suggesting that DUSP6 can be used as a predictor of the efficacy of KRAS G12C inhibitors.

[0040] Example 3: Role and biological function of DUSP6 in the formation of KRAS G12C inhibitor resistance The specific method is as follows: (1) Construction of DUSP6 overexpression plasmid: First, search the website of the National Center for Biotechnology Information (NCBI) DUSP6 Transcript information, common transcripts (NCBI number: NM_001946.4) and proteins (NCBI number: NP_001937.2) were selected as objects, and the coding sequence of DUSP6 protein was obtained by reverse transcription and in vitro polymerase chain reaction (PCR), and then cloned into the lentiviral vector plasmid pLV3-CMV-MCS-3×FLAG-CopGFP-Puro. Finally, after molecular biological operations such as transformation, shaking, and plasmid extraction, the DUSP6 overexpression plasmid pLV3-CMV-DUSP6-3×FLAG-CopGFP-Puro was obtained.

[0041] (2) Lentiviral packaging and construction of DUSP6 protein stable overexpression cell line: 293T cells in the logarithmic growth phase were inoculated in a 10 cm cell culture dish at a density of 60%-80%. DUSP6 overexpression plasmid pLV3-CMV-DUSP6-3×FLAG-CopGFP-Puro 6.63ug, packaging plasmid PsPAX 5.05ug and pMD2.G 3.32ug were co-transfected into 293T cells using lipo3000 transfection reagent; The transfected 293T cells were cultured in a cell culture incubator for 48 hours, and the culture medium was collected and filtered through a 0.45um filter to filter the cells and debris. The filtrate obtained was the lentiviral solution. During this period, the SW837 / KIR and MIA PaCa2 / KIR resistant cells to be infected in the logarithmic growth phase were seeded into a 6 cm cell culture dish one day before infection; During lentiviral infection, discard the original culture medium, add 2 ml of fresh culture medium and 2 ml of filtered virus solution, and add 3.2 ul of polybrene to promote virus infection. After 24 hours, discard the virus solution and replace with fresh culture medium for continued culture. After 48 hours of infection, replace with fresh culture medium and add appropriate concentration (1-2 ug / ml) of puromycin for resistance screening. Cells that still survive after 2-4 days are DUSP6 stable overexpression cell lines, and the DUSP6 expression level is detected by WB.

[0042] (3) In vitro and in vivo validation of the biological role of DUSP6 in the formation of KRAS G12C inhibitor resistance: S1) In the in vitro validation experiment, blank and DUSP6-overexpressing SW837 / KIR and MIA PaCa2 / KIR resistant cells were cultured at 5×10 3 The cells were seeded into a 96-well plate at a density of 100 cells / well and placed in a cell incubator for further culture; S2) After the cells adhered (24 hours after seeding), fresh culture medium was replaced, and blank control group and sotorasib gradient treatment group (treatment concentrations of 100 nM and 1000 nM) were set up and treated with corresponding drugs and placed in a cell incubator for further culture for 72 hours; S3) After the treatment, the cell culture medium in the 96-well plate was aspirated and a mixture of CCK8 (CellCounting Kit-8) detection reagent (Dongaren, catalog number CK04) and cell culture medium was prepared at a ratio of 1:9 (prepared immediately before use). Then, 100ul of the above-prepared mixture was added to each well and the plate was placed back in the cell incubator for incubation for 1-3 hours. After the incubation, the absorbance of each well was detected using an ELISA reader (450nm wavelength) and converted into cell viability. The conversion formula is: Cell survival rate = [(experimental well-blank well) / (control well-blank well)] × 100%.

[0043] Test results such as Figure 4 As shown, compared with blank SW837 / KIR cells, the inhibition rate of SW837 / KIR cells overexpressing DUSP6 increased by 24.03% at most after treatment with the same concentration of sotorasib ( p<0.05); similarly, compared with blank MIA PaCa2 / KIR cells, the inhibition rate of MIA PaCa2 / KIR cells overexpressing DUSP6 increased by 31.14% at most after treatment with the same concentration of sotorasib ( p <0.05).

[0044] In the in vivo validation experiment, we selected 4-6 week old balb / c-nu / nu mice, a total of 24 mice, of which 12 mice were subcutaneously inoculated with DUSP6 overexpressing MIA PaCa2 / KIR cells, and the other 12 mice were subcutaneously inoculated with blank MIA PaCa2 / KIR cells. Each mouse was inoculated with 5 million cells, and then the two groups of mice were randomly divided into 2 groups (control group and sotorasib treatment group). After the tumor cell inoculation, the mice were routinely raised for 2 weeks, and then the long and short diameters of the tumor were recorded every three days and the tumor volume was calculated. The calculation method is: tumor volume = 0.523 × long diameter × short diameter × short diameter. When the mouse tumor volume reaches about 100mm 3 At 1:10 pm, drug treatment was started (sotorasib was administered by oral gavage at 100 mg / kg, once a day). After 10 consecutive days of administration, the mice were sampled and the tumor weights of each group were weighed, and the volume growth curves were plotted. Figure 5 As shown in the results, under the premise of sotorasib treatment, the tumor volume and weight of the DUSP6 overexpression group were significantly lower than those of the blank control group, that is, the upregulation of DUSP6 expression can increase the cell sensitivity to sotorasib. This experiment further confirmed the application value of DUSP6 in the prediction of KRAS G12C inhibitor efficacy and prognosis evaluation.

[0045] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. Application of reagents for quantifying DUSP6 protein or mRNA in samples in the preparation of reagents for predicting the efficacy of KRAS G12C inhibitors.

2. Application of reagents for quantifying DUSP6 protein or mRNA in samples in the preparation of reagents for evaluating the prognosis of KRAS G12C inhibitor treatment.

3. The use according to claim 1 or 2, characterized in that: The sample is tumor tissue.

4. The use according to claim 3, characterized in that: The tumor is selected from one of colorectal cancer, pancreatic cancer and lung cancer.

5. The use according to claim 1 or 2, characterized in that: The primer set for quantifying DUSP6 mRNA in the samples was: Forward primer: 5'- GCGACTGGAACGAGAATACG-3', Reverse primer: 5′-TCGGCTTGGAACTTACTGAAG-3′.

6. Application of reagents that promote DUSP6 protein expression in the preparation of agents for reversing resistance to KRAS G12C inhibitor targeted therapy of tumors.

7. A method for determining the sensitivity of tumor cells to a KRAS G12C inhibitor, comprising: Quantify the amount of DUSP6 protein or mRNA in tumor cells; The sensitivity of tumor cells to KRAS G12C inhibitors was determined based on DUSP6 content.

8. The method according to claim 7, characterized in that The primer set for quantifying DUSP6 mRNA in the samples was: Forward primer: 5'- GCGACTGGAACGAGAATACG-3', Reverse primer: 5′-TCGGCTTGGAACTTACTGAAG-3′.

9. A method for screening a KRAS G12C inhibitor resistance reversal agent, comprising the following steps: Detect the initial DUSP6 protein and / or mRNA levels in KRAS G12C inhibitor-resistant tumor cells; Treat KRAS G12C inhibitor-resistant tumor cells with the test substance for a certain period of time; After treatment, the levels of DUSP6 protein and / or mRNA in KRAS G12C inhibitor-resistant tumor cells were detected; According to the change value of the content of DUSP6 protein and / or mRNA, it is determined whether the test substance has the potential to reverse the resistance to KRAS G12C inhibitor.

10. The screening method according to claim 9, characterized in that The primer set for quantifying DUSP6 mRNA in the samples was: Forward primer: 5'- GCGACTGGAACGAGAATACG-3', Reverse primer: 5′-TCGGCTTGGAACTTACTGAAG-3′.

Images