A drug for improving the sensitivity of drug-resistant lung cancer patients to EGFR-TKI inhibitors

Abstract

This invention relates to the field of molecular diagnostics, specifically to a drug that improves the sensitivity of drug-resistant lung cancer patients to EGFR-TKI inhibitors, and discovers that overexpression of MZF1... L Cellular sensitivity to EGFR-TKIs can be increased by modulating the localization of EGFR on the cell membrane. This specific molecule offers a novel treatment option for drug-resistant patients in lung cancer therapy.

Description

Technical Field

[0001] This invention relates to the field of molecular diagnostics, specifically to a drug that improves or enhances the sensitivity of drug-resistant lung cancer patients to EGFR-TKI inhibitors. Background Technology

[0002] Lung cancer is a highly prevalent cancer worldwide, ranking first among malignant tumors in my country. Non-small cell lung cancer (NSCLC) is the most common histological type of lung cancer, accounting for about 85% of lung cancer patients (Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA Cancer J Clin 2022, 72(1):7-33). EGFR mutation is an important oncogenic driver in NSCLC, opening the door to biomarker-guided therapy for patients with advanced disease (Cooper AJ, Sequist LV, Lin JJ. Third-generation EGFR and ALK inhibitors: mechanisms of resistance and management. Nature reviews Clinical oncology 2022.). Many EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been developed, including the commonly used first-generation TKI gefitinib and the highly effective third-generation TKI osimertinib. Despite the remarkable efficacy of EGFR-TKIs in treating patients, resistance to them remains a fundamental challenge that has not yet been resolved.

[0003] Currently, there are two main viewpoints regarding the development of acquired resistance to EGFR-TKIs: alteration of the targeted kinase (such as EGFR C797S mutations (Thress KS, Paweletz CP, Felip E, Cho BC, Stetson D, Dougherty B, Lai Z, Markovets A, Vivancos A, Kuang Y, Ercan D, Matthews SE, Cantarini M, Barrett JC, ...). PA, Oxnard GR. Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M. Nature Medicine 2015, 21(6):560-562) and alterations in downstream signaling pathways of the target (such as BRAF fusion, KRAS mutation, NRAS mutation, MAP2K1 mutation in the RAS-MAPK pathway). Schoenfeld AJ, Chan JM, Kubota D, Sato H, Rizvi H, Daneshbod Y, Chang JC, Paik PK, Offin M, Arcila ME, Davare MA, Shinde U, Pe'er D, Rekhtman N, Kris MG, Somwar R, Riely GJ, Ladanyi M, Yu HA. Tumor Analyses Reveal Squamous Transformation and Off-Target Alterations As Early Resistance Mechanisms to First-line Osimertinib in EGFR-Mutant Lung Cancer. Clinical Cancer Research: An Affiliative Journal of the American Association for Cancer Research 2020, 26(11): 2654-2663. However, most of these studies focus on genetic changes in EGFR-related genes. Epigenetic changes are common in EGFR-TKI resistant patients, and regulating these epigenetic changes can enhance the cytotoxic effects of antitumor therapy (Chen Z, Chen Q, Cheng Z, Gu J, Feng W, Lei T, Huang J, Pu J, Chen X, Wang Z. Long non-coding RNA CASC9 promotes gefitinib resistance in NSCLC by epigenetic repression of DUSP1. Cell death & disease 2020, 11(10): 858.).Compared with permanent gene mutations, epigenetic changes that control gene expression play an important role in gradually regulating cell differentiation and malignant transformation after cell resistance to drugs. They can serve as potential sensitive biomarkers and drug targets for drug resistance biomonitoring.

[0004] Zinc finger proteins bind to Zn 2+ Forming stable, self-folding "finger" structures, and closely associated with various pathological processes including cancer invasion and metastasis and resistance to antitumor drugs, they are considered important molecular targets (Jen J, Liu CY, Chen YT, Wu LT, Shieh YC, Lai WW, Wang YC. Oncogenic zinc finger protein ZNF322A promotes stem cell-like properties in lung cancer through transcriptional suppression of c-Myc expression. Cell death and differentiation 2019, 26(7):1283-1298. Lin S, Ruan H, Qin L, Zhao C, Gu M, Wang Z, Liu B, Wang H, Wang J. Acquired resistance to EGFR-TKIs in NSCLC mediatesepigenetic downregulation of MUC17 by facilitating NF-κB activity via UHRF1 / DNMT1 complex. International journal of biological sciences 2023, 19(3):832-851.). Zinc activates EGFR by activating intracellular Src and metalloproteinases, and activated EGFR induces respiratory inflammation by upregulating the expression of inflammatory proteins. Members of the zinc finger protein family play an important role in regulating the expression and activity of EGFR.

[0005] Methyl-sensitive zinc finger protein myeloid zinc finger 1 (MZF1) belongs to the Krüppel-like family of transcription factors and is involved in cell proliferation and differentiation. MZF1 itself has three known splicing variants (MZF1-V1, MZF1-V2, and MZF1-V3), among which MZF1-V1 and MZF1-V2 share the same coding region, encoding the complete isoform 1 (Isoform1, MZF1). LThe C2H2 type zinc domain has functional domains including an acidic domain, a SCAN domain, a TAD domain, and a DNA-binding domain, namely a zinc finger domain composed of 13 zinc fingers. The core sequences recognized by this C2H2 type zinc domain are all rich in G sites, making it a methyl-sensitive transcription factor. MZF1-V3 encodes a truncated isoform 2 (MZF1) lacking the C-terminus of the carboxyl group. S Therefore, it is similar to a truncated version of the DNA-binding domain of MZF1. In our work, we found that the DNA-binding capacity of the zinc finger domains of the two protein isoforms differs, potentially playing an important role in gene transcriptional regulation. This is because the MZF1 zinc finger domains often cluster together to bind divalent cations (such as Zn). 2+ MZF1 is methylation sensitive. Therefore, the expression of different splicing variants of MZF1 has different effects on EGFR phosphorylation activation, which can provide more reliable molecular markers and drug targets for precision diagnosis and personalized treatment. Summary of the Invention

[0006] This invention discovered, during the research, a variant 2 (MZF1) of the transcription factor bone marrow zinc finger gene 1 splicing. L ) and bone marrow zinc finger gene 1 splice variant 3 (MZF1) s Significant differences were observed in sensitive and resistant cells, namely MZF1. L / MZF1 S MZF1 is co-expressed in drug-resistant lung cancer cells. L MZF1 decrease S Upregulation mode; overexpression of MZF1 was also found. L Cellular sensitivity to EGFR-TKIs can be increased by modulating the localization of EGFR on the cell membrane. Based on this, the present invention was completed.

[0007] In a first aspect, the present invention provides a biomarker composition for screening drug-resistant lung cancer patients, the composition comprising MZF1. L and MZF1 S Among them, when MZF1 was detected in lung cancer patients L Low expression level, MZF1 s When expression levels are high, and MZF1 s Expression level relative to MZF1 L When the expression level is high, the patient can be identified as a drug-resistant patient.

[0008] Furthermore, the aforementioned drug-resistant lung cancer patients are those resistant to EGFR-TKI inhibitors.

[0009] Furthermore, the EGFR-TKI inhibitors include, but are not limited to, gefitinib, osimertinib, erlotinib, icotinib, and afatinib.

[0010] Furthermore, the MZF1 L The nucleotide sequence is shown in SEQ ID NO.1; MZF1 L The amino acid sequence is shown in SEQ ID NO.2;

[0011] Furthermore, the MZF1 s The nucleotide sequence is shown in SEQ ID NO.3; MZF1 s The amino acid sequence is shown in SEQ ID NO.3.

[0012] Secondly, the present invention provides a kit for detecting drug-resistant lung cancer patients, the kit containing reagents capable of detecting a biomarker composition, wherein, when MZF1 of the lung cancer patient is detected... L Low expression level, MZF1 s When expression levels are high, and MZF1 s Expression level relative to MZF1 L When the expression level is high, the patient can be identified as a drug-resistant patient. The biomarker composition is as described in the first aspect, MZF1. L and MZF1 S .

[0013] Thirdly, the present invention provides the use of a set of biomarker compositions in the preparation of reagents for diagnosing patients with drug-resistant lung cancer, wherein, when MZF1 of the lung cancer patient is detected... L Low expression level, MZF1 s When expression levels are high, and MZF1 s Expression level relative to MZF1 L When the expression level is high, the patient can be identified as a drug-resistant patient. The biomarker composition is as described in the first aspect, MZF1. L and MZF1 S .

[0014] Furthermore, the aforementioned drug-resistant lung cancer patients are those resistant to EGFR-TKI inhibitors.

[0015] Furthermore, the EGFR-TKI inhibitors include, but are not limited to, gefitinib, osimertinib, erlotinib, icotinib, and afatinib.

[0016] Fourthly, the present invention provides a medicament for improving or enhancing the sensitivity of drug-resistant lung cancer patients to EGFR-TKI inhibitors, said medicament containing MZF1. L protein.

[0017] Furthermore, the MZF1 L The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0018] Furthermore, the MZF1 L The protein amino acid sequence also includes 80%-99% homologous sequences, preferably 80%-85%; more preferably 85%-90%; and even more preferably 90%-95%.

[0019] Furthermore, the MZF1 L The protein amino acid sequence also includes mutations, deletions, substitutions, or additions of 1-20 amino acids in its sequence. Further, MZF1 is preferred. L The protein amino acid sequence preferably contains 1-15 mutated, deleted, substituted, or added amino acids; preferably 1-10; preferably 1-5; preferably 5-20; preferably 10-20.

[0020] Furthermore, the mutation, deletion, substitution, or addition of the amino acid includes mutations, deletions, substitutions, or additions occurring simultaneously or at different times among consecutive amino acids.

[0021] Furthermore, the mutation, deletion, substitution, or addition of the amino acid also includes mutations, deletions, substitutions, or additions occurring simultaneously or at different times between discontinuous amino acids.

[0022] Furthermore, the MZF1 L Proteins also include their fusion proteins, conjugates, nucleic acids encoding their fusion proteins or conjugates, and vectors expressing the aforementioned nucleic acid molecules.

[0023] Furthermore, the drug may also contain a pharmaceutically acceptable carrier.

[0024] Furthermore, the drug includes, but is not limited to, injectable formulations, gels, suspensions, emulsions, polymers, nanoparticles, microspheres, rectal capsules, enemas, pastes, decoctions, and implants, and may optionally be controlled-released and / or sustained-released via dosage forms or devices.

[0025] Furthermore, the aforementioned drug-resistant lung cancer patients are those resistant to EGFR-TKI inhibitors.

[0026] Furthermore, the EGFR-TKI inhibitors include, but are not limited to, gefitinib, osimertinib, erlotinib, icotinib, and afatinib.

[0027] Sixthly, the present invention provides an MZF1 L The protein is used in the preparation of drugs that improve or enhance the sensitivity of patients with drug-resistant lung cancer to EGFR-TKI inhibitors.

[0028] Furthermore, the MZF1 L The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0029] Furthermore, the MZF1 L The protein amino acid sequence also includes 80%-99% homologous sequences, preferably 80%-85%; more preferably 85%-90%; and even more preferably 90%-95%.

[0030] Furthermore, the MZF1 L The protein amino acid sequence also includes mutations, deletions, substitutions, or additions of 1-20 amino acids in its sequence. Further, MZF1 is preferred. L The protein amino acid sequence preferably contains 1-15 mutated, deleted, substituted, or added amino acids; preferably 1-10; preferably 1-5; preferably 5-20; preferably 10-20.

[0031] Furthermore, the mutation, deletion, substitution, or addition of the amino acid includes mutations, deletions, substitutions, or additions occurring simultaneously or at different times among consecutive amino acids.

[0032] Furthermore, the mutation, deletion, substitution, or addition of the amino acid also includes mutations, deletions, substitutions, or additions occurring simultaneously or at different times between discontinuous amino acids.

[0033] Furthermore, the MZF1 L Proteins also include their fusion proteins, conjugates, nucleic acids encoding their fusion proteins or conjugates, and vectors expressing the aforementioned nucleic acid molecules.

[0034] Beneficial effects

[0035] This study found that the transcription factor bone marrow zinc finger gene 1 splice variant 2 (MZF1) L ) and bone marrow zinc finger gene 1 splice variant 3 (MZF1) s Significant differences were observed in sensitive and resistant cells, namely MZF1. L / MZF1 S MZF1 is co-expressed in drug-resistant lung cancer cells. L MZF1 decrease S Upregulation mode; overexpression of MZF1 was also found. L EGFR-TKIs can be increased by modulating the localization of EGFR on the cell membrane. Therefore, this specific molecule can be used as a biomarker composition for screening drug-resistant lung cancer patients during lung cancer treatment, providing a new means of precision diagnosis and treatment for drug-resistant lung cancer patients. Attached Figure Description

[0036] Figure 1The effect of gefitinib / osimertinib resistance on MZF1 splicing variants: A. RT-qPCR and Western blot showing mRNA expression of MZF1 splicing variants in resistant cells; B. Schematic diagram of clinical case collection; C. RT-qPCR showing mRNA expression of MZF1 splicing variants in clinical samples; DG. Multicolor immunofluorescence detection of MZF1 splicing variant expression in clinical tissues with inflammation (CP), pre-treatment (BT), gefitinib resistance (GR), and osimertinib resistance (OR), *p<0.05, **p<0.01.

[0037] Figure 2 Effects of MZF1 splice variant overexpression on the function of gefitinib and osimertinib resistant cells; A. RT-qPCR and Western blot were used to detect the expression of MZF1 splice variants after overexpression in resistant cells; B. EGFR-TKI sensitive and resistant lung cancer cell lines were selected, and transfected cell lines with exogenous stable high expression of MZF1 splice variants were established; The effects of MZF1 splice variant overexpression on cell proliferation and invasion were detected by MTT assay (B), colony formation assay (C), and Transwell assay (D). Quantitative data are shown in the upper half of the figure, and representative images are shown in the lower half of the figure. *p<0.05, **p<0.01.

[0038] Figure 3 Effects of MZF1 splice variant overexpression on the function of gefitinib and osimertinib resistant cells; AC. MTT assay, colony formation assay, and Transwell assay show the quantitative data of the effects of gefitinib (1 μM) / osimertinib (0.1 μM) treatment on cell viability (A), colony formation ability (B), and invasion and migration (C) of MZF1 splice variant-overexpressing resistant cells. The upper half of the figure shows the quantitative data, and the lower half shows the representative images; D. Detection of p-EGFR expression in resistant cells treated with gefitinib, osimertinib, and / or transfected with the MZF1 splice variant; E. Protein levels of EGFR subunits in resistant cells after treatment with gefitinib, osimertinib, and / or transfection with the MZF1 splice variant. *p<0.05, **p<0.01. Detailed Implementation

[0039] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0040] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0041] The term "nucleic acid" as used herein refers to any one or more nucleic acid segments present in a polynucleotide, such as DNA or RNA fragments. For the purposes of this invention, the nucleotides encoding the MZF1 splice variant protein contained in the vector may be isolated or may be the MZF1 splice variant gene sequence.

[0042] The term "fusion protein" as used herein refers to a protein that contains amino acids other than those encoding the original or natural full-length protein or its subsequences, contains an amino acid sequence that replaces the amino acid sequence encoding the original or natural full-length protein or its subsequences, contains fewer amino acid sequences than those encoding the original or natural full-length protein or its subsequences, and / or contains an amino acid sequence different from those encoding the original or natural full-length protein or its subsequences.

[0043] The term "mutation" as used herein refers to an alteration in the nucleotide sequence of an organism's, virus's, or extrachromosomal DNA genome, including base substitutions, DNA insertions, DNA deletions, or DNA duplications that cause sequence changes. The mutation described in this invention can be an amino acid mutation in the amino acid sequence of the MZF1 splice variant protein, or a sequence mutation in its MZF1 splice variant gene.

[0044] The term "composition" or "pharmaceutical composition" as used herein may include compositions containing the MZF1 splice variant described herein, as well as, for example, pharmaceutically acceptable carriers, excipients, or diluents, which are administered to individual subjects.

[0045] The term "pharmaceutically acceptable" means a composition suitable for contact with human and animal tissues without excessive toxicity or other complications, within the bounds of reasonable medical judgment, and in proportion to a reasonable benefit / risk ratio. In some respects, the MZF1 splice variant protein, compositions, and vaccines described herein are pharmaceutically acceptable.

[0046] The MZF1 involved in this invention L The nucleotide sequence is ( SEQ ID NO.1 )：

[0047] catccacggc gatcccccgg gccctggcgc taagcccccg gcccctcctg gtgcgcccga gcctcccggc ccctttccgt

[0048] gcagcgagtg ccgcgagagc ttcgcgcggc gcgccgtgct gctggagcac caggcggtac acacgggcga caagtccttt

[0049] ggctgcgtcg agtgcggcga gcgcttcggc cgccgctcag tgctgctgca gcaccggcgc gtgcacagtg gcgagcggcc

[0050] cttcgcctgt gccgagtgcg gccagagctt ccggcagcgc tccaacctga cgcagcaccg gcgcatccac accggggagc

[0051] ggcccttcgc ctgcgccgag tgtggcaagg ccttccgcca gcggcctacg ctcacgcagc atctccgcgt acacacgggc

[0052] gagaaaccct ttgcctgccc cgagtgtggc cagcgcttca gccagcgcct caagctcacg cgtcatcaga ggacacacac

[0053] cggcgaaaag ccctaccact gcggtgagtg cggcctgggc ttcacgcagg tctcgcggct caccgagcac cagcgcatcc

[0054] acacgggcga acggcccttc gcctgccccg agtgcggcca gagctttcgg cagcacgcca acctcaccca gcaccggcgc

[0055] atccacacgg gtgaacggcc ctacgcatgc cctgagtgtg gcaaggcctt ccgccagcgg cccacgctca cgcagcatct

[0056] gcgcacccac cgacgagaga agcccttcgc ctgccaggac tgtggccgcc gcttccacca gagcaccaag ctcattcagcaccagcgcgt ccacagcgcc gagtag.

[0057] The MZF1 involved in this invention LThe amino acid sequence is (SEQ ID NO.2): mrpavlgspd rappedegpvmvkledseee geaalwdpgp eaarlrfrcf ryeeatgpqe alaqlrelcr qwlrpevrsk eqmlellvleqflgalppeiqarvqgqrpg speeaaalvd glrrepggpr rwvtvqvqgq evlsekmeps sfqplpetepptpepgpktp prtmqesplglqvkeesevt edsdflesgp laatqesvpt llpeeaqrcg tvldqifphsktgpegpswrehpralwhee aggifspgfalqlgsisagp gsvsphlhvp wdlgmaglsgqiqspsreggfahalllpsd lrseqdptde dpcrgvgpal ittrwrsprgrsrgrpstgggvvrggrcdvcgkvfsqrsn llrhqkihtg erpfvcsecg rsfsrsshll rhqlthteerpfvcgdcgqgfvrsarleehrrvhtgeqpf rcaecgqsfr qrsnllqhqr ihgdppgpgakppappgapeppgpfpcsec resfarravl lehqavhtgdksfgcvecge rfgrrsvllqhrrvhsgerpfacaecgqsf rqrsnltqhr rihtgerpfa caecgkafrqrptltqhlrvhtgekpfacpecgqrfsqrl kltrhqrtht gekpyhcgec glgftqvsrl tehqrihtgerpfacpecgq sfrqhanltq hrrihtgerpyacpecgkafrqrptltqhl rthrrekpfacqdcgrrfhqstkliqhqrv hsae。

[0058] MZF1 involved in the present invention sThe nucleotide sequence is (SEQ ID NO.3): atgaggcct gcggtgctgggctccccaga ccgagcaccc ccagaagatg aggggcctgt catggtgaag ctagaggact ctgaggaggagggtgaggctgccttatggg acccaggccc tgaagctgca cgcctgcgtt tccggtgctt ccgctatgaggaggccacag ggccccaagaggccctggcc cagctccgag agctgtgtcg ccagtggctg cgtccagaggtacgctccaa ggagcagatg ctggagctgttggtgctgga gcagttcctg ggcgcactgc cccctgagatccaggcccgt gtgcaggggc agcggccagg cagccccgaggaggctgctg ccctagtaga tgggctgcgccgggagccgg gcggaccccg gagatgggtc acagtccagg tgcagggccaggaggtccta tcagagaagatggagccctc cagtttccag cccctacctg aaactgagcc tccaactcca gagcctgggcccaagacacctcctaggact atgcaggaat caccactggg cctgcaggtg aaagaggagt cagaggttacagaggactcagatttcctgg agtctgggcc tctagctgcc acccaggagt ctgtacccac cctcctgcctgaggaggccc agagatgtgggaccgtgctg gaccagatct ttccccacag caagactggg cctgagggtccctcatggag ggagcacccc agggccctgtggcatgagga agctgggggc atcttctccc caggggccggagccggggcc gccccagcac tgggggcggg gtggttaggggcggccgttg cgatgtatgt ggcaaggtgttcagccaacg cagcaacctg ctga。

[0059] MZF1 involved in the present inventions The amino acid sequence is (SEQ ID NO.4): mrpavlgspd rappedegpvmvkledseee geaalwdpgp eaarlrfrcf ryeeatgpqe alaqlrelcr qwlrpevrsk eqmlellvleqflgalppeiqarvqgqrpg speeaaalvd glrrepggpr rwvtvqvqgq evlsekmeps sfqplpetepptpepgpktp prtmqesplglqvkeesevt edsdflesgp laatqesvpt llpeeaqrcg tvldqifphsktgpegpswr ehpralwhee aggifspgagagaapalgag wlgaavamyv arcsanaatc.

[0060] Example 1: Detection of the relationship between the expression of MZF1 splice variant and EGFR-TKI resistance in lung cancer sensitive and drug-resistant cells. 1.1 Materials

[0061] The non-small cell lung cancer cells used in the tests, HCC827 and PC9, were both purchased from the National Biomedical Experimental Cell Resource Bank and cultured and passaged under normal conditions in the applicant's laboratory.

[0062] Bronchoalveolar lavage fluid samples tested: 57 cases, including 10 patients with pneumonia, 22 patients with lung cancer before treatment, 13 patients resistant to gefitinib, and 12 patients resistant to osimertinib.

[0063] Paraffin-embedded tissues tested: 180 tissues, including 61 patients with pneumonia, 47 patients with pre-treatment lung cancer, 38 patients resistant to gefitinib, and 33 patients resistant to osimertinib.

[0064] 1.2 Methods

[0065] 1.2.1 Cell RNA Extraction and Reverse Transcription Process

[0066] (1) The cell RNA extraction process is as follows: select cells in good growth condition, and extract RNA at a rate of 1 mL / 10000 mL / mL. 6 Add Trizol reagent (Invitrogen, USA, catalog number: 15596026) to each cell; the tissue RNA extraction procedure is as follows: remove the frozen tissue from the liquid nitrogen tank, cut approximately 200 mg, and grind the tissue sample in a cold mortar. Add 1 mL of Trizol reagent to every 100 mg of tissue sample.

[0067] (2) After 10 min at room temperature, the mixture was fully lysed and extracted with chloroform. 0.2 mL of chloroform was added to each 1 mL of Trizol. The mixture was shaken vigorously for 15 s, left to stand at room temperature for 5 min, and then centrifuged at 12000 g and 4 °C for 15 min.

[0068] (3) Transfer the colorless upper layer of the separated liquid to a new centrifuge tube, precipitate it with pre-cooled isopropanol (add 0.5 mL of isopropanol per 1 mL of Trizol), and place on ice for 20 min. Centrifuge at 12000 g, 4 °C for 10 min, and discard the supernatant.

[0069] (4) Wash the precipitate with pre-cooled 75% ethanol, add 1 mL of 75% ethanol to 1 mL of Trizol, centrifuge at 7500 g, 4 °C for 5 min, and discard the supernatant. After drying, dissolve in an appropriate amount of DEPC-H2O, confirm by 0.8% agarose gel electrophoresis, determine the RNA concentration by NanoDrop, and store at -80 °C.

[0070] (5) Take 1.0 μg of RNA and reverse transcribe it into cDNA. Use the TransScript II First-Strand cDNA Synthesis SuperMix kit (catalog number: AH301-02) produced by Beijing TransGen Biotech Co., Ltd.: Add 1 μL of Anchored Oligo(dT)20, 10 μL of 2×TS Reaction Mix and 1 μL of RT / RI Enzyme Mix to DEPC-H2O to a final volume of 20 μL. Reaction conditions: 42℃ for 30 min, 85℃ for 5 min. The cDNA obtained by reverse transcription is then stored at -20℃.

[0071] 1.2.2 qPCR detection

[0072] (1) qPCR primer sequences:

[0073] Primer pair 1 (SEQ ID No:1): MZF1 L The mRNA public region primer (NM_198055.2, size 339bp) has the following primers: upstream primer: 5'-GGGGGCATCTTCTCCCCA-3'; downstream primer: 5'-CACCTTGCCACATACATCGC-3'.

[0074] Primer pair 2 (SEQ ID No:2): MZF1 S mRNA primer (NM_001267033.2, size 116bp) upstream primer: 5'-CCGTGCTGGACCAGATCTTT-3'; downstream primer: 5'-GGCCCCTGGGGAGAAGA-3'.

[0075] The internal reference primer used for homogenization is a primer with Beta-Aactin as the internal reference.

[0076] Upstream primer: 5'-TTAGTTGCGTTACACCCTTTC-3';

[0077] Downstream primer: 5'-ACCTTCACCGTTCCAGTTT-3'.

[0078] (2) Reaction system for qPCR amplification

[0079] As shown in Table 1.

[0080] Table 1. Reaction system for qPCR amplification

[0081]

[0082] Note: 2×SYBR-Green: Zymo Research, USA, product number E2004.

[0083] (3) The PCR reaction conditions are as follows:

[0084] The fluorescence was collected after 40 cycles of 50℃ for 2 min, 95℃ for 10 min, 95℃ for 15 s, and 60℃ for 1 min. A melting curve was then constructed using the same cycle. The experimental results were analyzed using 2... -ΔΔCt The analytical data and melting curves ensure the specificity of the product.

[0085] 1.2.3 Western blotting analysis

[0086] (1) Sample preparation

[0087] The lung cancer cells to be tested were seeded in culture dishes with a diameter of 10 cm. When the cells grew to about 60-80%, 3-5 culture dishes were collected from each group.

[0088] (2) Collect and wash the samples

[0089] Wash cells 1-2 times with room temperature PBS, then treat with 1% formalin at 37°C for 10 min. Wash cells twice with ice-cold PBS, then transfer cells to 1 ml of ice-cold PBS using a cell scraper, centrifuge at 3,000 rpm for 2 min at 4°C, and discard the supernatant. Resuspend the cell pellet in 400 μl of lysis buffer and incubate on ice for 10 min.

[0090] (3) Western blotting

[0091] After quantification, calculate the sample loading volume at 30 μg / well and add the sample to 5×SDS gel loading buffer (Genstar, China, catalog number: E153-05). Heat at 100℃ for 10 min, add samples sequentially, and perform SDS-PAGE electrophoresis at 120V. After electrophoresis, transfer the membrane to a wet electroporator. After transfer, remove the PVDF membrane (Millipore, USA, catalog number: IPVH00010). Block with 5% skim milk for 1 h, then incubate with primary antibody: MZF1. L Antibody (Wuhan Daian Biotechnology Co., Ltd., Antigen Immunization: RPFACAECGQSFRQRSNLTQHRRIHTGERPFACAECGKAFRQRPTLTQHLRVHTGEKPFACPECGQRFSQRLKLTRHQRTHTGEKPYHCGECGLGFTQVSRLTEHQRIHTGERPFACPECGQSFRQHANLTQHRRIHTGERPYACPECGKAFRQRPTLTQHLRTHRREKPFACQDCGRRFHQSTKLIQHQRVHSAE), MZF1 S Antibody (Wuhan Daian Biotechnology Co., Ltd., antigen immunization: LGAGWLGAAVAMYVARC) was incubated overnight at 4°C. Secondary antibody labeled with anti-rabbit horseradish peroxidase (Abcam, UK, catalog number: ab6721) was added and incubated for 1 hour. A colorimetric reaction was then performed using an ECL chemiluminescence kit (ThemoFisher, USA, catalog number: 32132), and development was performed using a Smart Gel Image Analysis System.

[0092] 1.2.4 Multicolor immunohistochemical analysis

[0093] Multicolor immunohistochemical analysis of tissue arrays was performed using the Opal 7-color fluorescence immunohistochemistry kit (Akoya Biosciences, USA, catalog number: NEL797001KT). 4μm tissue sections were baked at 70°C for 1 hour. Dewaxing was performed twice with xylene, every 20 minutes. Hydration was carried out sequentially with gradient ethanol (100%, 95%, 80%) for 5 minutes each time. The tissue sections were then placed in citric acid retrieval solution and subjected to high-temperature autoclaving. After cooling to room temperature, they were washed three times with 1×PBS for 5 minutes each time. Incubation with 3% hydrogen peroxide solution at room temperature for 15 minutes, followed by rinsing with double-distilled water and washing three times with 1×PBS for 5 minutes each time. Blocking with 5% skim milk powder for 1 hour, followed by discarding the milk and washing three times with 1×PBS for 5 minutes each time. Primary antibodies were used for incubation: CK antibody (Suzhou Baidao Medical Technology Co., Ltd., catalog number: PA125), MZF1. LAntibody (Wuhan Daian Biotechnology Co., Ltd., Antigen Immunization: RPFACAECGQSFRQRSNLTQHRRIHTGERPFACAECGKAFRQRPTLTQHLRVHTGEKPFACPECGQRFSQRLKLTRHQRTHTGEKPYHCGECGLGFTQVSRLTEHQRIHTGERPFACPECGQSFRQHANLTQHRRIHTGERPYACPECGKAFRQRPTLTQHLRTHRREKPFACQDCGRRFHQSTKLIQHQRVHSAE), MZF1 S Antibody (Wuhan Daian Biotechnology Co., Ltd., antigen immunization: LGAGWLGAAVAMYVARC), β-actin antibody (Sigma-Aldrich, USA, catalog number: A45551). Incubated overnight at 4℃. The next day, incubated at room temperature for half an hour, washed three times with 1×PBS for 5 min each time. Secondary antibody was added, incubated at room temperature for 30 min, washed three times with 1×PBS for 5 min each time. Multicolor fluorescence images were acquired using the TissueFAXS Spectra S system (tissuegnostic, USA), and protein expression was quantified using an automated quantitative analysis system (tissuegnostic, USA, software version: StrataQuest 7.0.1.165).

[0094] 1.3 Results and Analysis

[0095] First, the expression level of MZF1 in EGFR-TKI-sensitive cell lines (HCC827) and drug-resistant cell lines (HCC827 / GR and HCC827 / OR) was detected. The results showed that MZF1 expression was higher in the sensitive cell line HCC827. L As the main expressed splicing variant, MZF1 in drug-resistant cell lines S The main splicing variant ( Figure 1 A).

[0096] MZF1 was further detected in bronchoalveolar lavage fluid samples collected from patients with inflammation (CP, n=10), pre-treatment (BT, n=22), gefitinib resistance (GR, n=13), and osimertinib resistance (OR, n=12). L and MZF1 S The expression ( Figure 1 BD), the results showed that MZF1 in the pre-treatment group L The expression level was higher in the gefitinib and osimertinib resistant groups than in the osimertinib resistant groups (p<0.01). Figure 1 C); MZF1 in the pre-treatment group S The expression was lower in the gefitinib and osimertinib resistant groups than in the gefitinib and osimertinib resistant groups (p<0.01). Figure 1 C).

[0097] Further analysis of the clinical characteristics and MZF1 of 47 tumor samples (22 before treatment, 13 gefitinib resistant, and 12 osimertinib resistant) L With MZF1 S Analyze the expression ( Figure 1 D). MZF1 L MZF1 expression is associated with tumor invasion depth, lymph node metastasis, distant metastasis, and clinical stage. S The expression of [something] is related to lymph node metastasis, distant metastasis, and clinical stage. Figure 1 D, see Table 2 for details).

[0098] Table 2 Relationship between MZF1 expression and clinical characteristics

[0099]

[0100]

[0101] To further validate the correlation between the expression of MZF1 splice variants and clinical phenotypes, multiplex immunohistochemistry (mIHC) was performed on a cohort of 180 tissue samples using antibodies against various MZF1 protein isoforms and cytokeratin (CK), including inflammation (CP, n=61), pre-treatment (BT, n=47), gefitinib resistance (GR, n=38), and osimertinib resistance (OR, n=33).

[0102] Experimental results showed that MZF1 was present in both the CP and BT groups. L The splice variant was expressed at a higher rate than MZF1. S MZF1 in patients in the GR and OR groups S The splice variant was expressed at a higher rate than MZF1. L ( Figure 1 E and F). Quantitative fluorescence analysis showed that MZF1 was higher in the tumor tissue region compared to the adjacent normal tissue region. L Reduced expression, MZF1 S Increased expression of MZF1 in tumor tissue regions of the drug-resistant group. L+ MZF1 S- The expression was significantly reduced compared to the sensitive group. Figure 1 G).

[0103] These results indicate that different splicing variants of MZF1 are differentially expressed in EGFR-TKI resistant cell lines and tissues, meaning that MZF1 is more sensitive in the sensitive group. L MZF1 is the predominantly expressed splicing variant in the drug-resistant group. SThe dominant splicing variant was *p<0.05, **p<0.01.

[0104] Example 2: Detection of the biological function of MZF1 splicing variants in lung cancer sensitive and drug-resistant cells

[0105] The non-small cell lung cancer cells used in the tests were HCC827 and PC9. Both cells were purchased from the National Biomedical Experimental Cell Resource Bank and cultured and passaged under normal conditions in the applicant's laboratory.

[0106] 2.1 MZF1 gene expression in HCC827 and PC9 cells

[0107] Universal empty vector plasmids were purchased from GeneCopoeia, USA, catalog number: EX-NEG-M02; MZF1 L Plasmid, purchased from GeneCopoeia, catalog number: EX-T3148-M02-5; MZF1 S The plasmid, NM_001267033.2, was synthesized by Shanghai Jikai Gene.

[0108] Stable transfection was performed using HCC827 and PC9 cell lines, which were sensitive and resistant to non-small cell lung cancer, respectively. Cells were seeded in 6-well cell culture plates. When the cells reached 60% confluence, 200 μL of DMEM was added to both the 20 nM expression plasmid and the empty vector plasmid, respectively. After mixing, the mixture was incubated at room temperature for 5 min. Then, 200 μL of DMEM was added to each of the following solutions, along with 6 μL of Lipofectamine 2000, and the mixture was incubated at room temperature for 5 min. 200 μL of Lipofectamine 2000 (Thermo Fisher Scientific, catalog number: 11668019) dilution buffer was added to 200 μL of the expression plasmid, and the mixture was incubated at room temperature for 20 min. Finally, 400 μL of the mixture was added to cell culture plates and cultured at 37°C for 8 h without serum. The culture was then replaced with 10% FBSDMEM and incubated at 37°C for 24 h. Detection was performed by RT-qPCR and Western blot.

[0109] 2.2 Cell Biology Functional Experiments

[0110] (1) MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) experiment

[0111] Cell lines constructed in step one, exhibiting good growth and in logarithmic growth phase, containing empty vectors and overexpression of the MZF1 gene from sensitive and resistant HCC827 and PC9 cells, were digested and counted. The cell suspension concentration was adjusted, and 100 μL was added to each well of a 96-well cell culture plate, with 2000 cells per well and 8 replicates per well. Observation was performed for 4 consecutive days. Daily, 10 μL of LTT (Sigma-Aldrich, M2128) was added to each well to a final concentration of 5 μg / mL, and the cells were co-cultured for 4 hours, after which the culture medium was discarded. Four days later, 150 μL of dimethyl sulfoxide (DMSO, Sigma-Aldrich, D2650) was added to each well, and the cells were gently shaken in the dark for 10 minutes. The absorbance of each well was then measured at OD 490 nm using an ELISA reader, and the data were analyzed.

[0112] (2) Cell clone formation experiment

[0113] Cell lines in logarithmic growth phase containing empty vectors and those overexpressing the sensitive and resistant MZF1 gene from HCC827 and PC9 were digested and counted, and seeded into 6-well cell culture plates at 100 cells per well, with the culture medium changed every three days. Once the cell clones reached a visible size, the culture medium was aspirated, and the cells were washed twice with 1×PBS. The cells were fixed with 4% paraformaldehyde for 30 min, washed twice with 1×PBS, stained with 0.5% crystal violet for 30 min, rinsed, air-dried, photographed, and the number of clones was counted.

[0114] (3) Transfer Experiment

[0115] The cell lines constructed in Step 1, containing empty vectors and overexpressing the MZF1 gene from sensitive and resistant HCC827 and PC9 cells, were digested and counted. The cells were then added to the upper chamber of an 8.0 μm pore size Transwell chamber (Corning Laboratories, USA, catalog number: CLS3422). 200 μL of serum-free cell suspension was added to each upper chamber, and 2 × 10⁶ cells were added to each well. 4 Cells were cultured in a cell culture incubator at 37°C for 24 hours. Cells were then carefully washed several times with 1×PBS. Cells were fixed with 4% paraformaldehyde for 30 minutes and washed three times with 1×PBS. Cells were stained with 0.5% crystal violet for 30 minutes and rinsed until the wash solution was colorless. Cells were carefully removed from the upper chamber with a cotton swab and the number of migrating cells was counted under a microscope (100×).

[0116] 2.3 Statistical Analysis

[0117] All experiments were repeated at least three times. Results were presented using two-tailed t-tests and are expressed as mean ± standard deviation. *p<0.05 indicates a statistically significant difference, and **p<0.01 indicates an extremely statistically significant difference.

[0118] 2.4 Experimental Results

[0119] A functional screening model was established to clarify the molecular basis of the role of the MZF1 splicing variant in acquired resistance to EGFR-TKIs in non-small cell lung cancer. MZF1 was stably overexpressed by plasmids in EGFR-TKI-sensitive and resistant lung cancer cell lines, respectively. L and MZF1 S ( Figure 2 A).

[0120] In HCC827 and PC9 EGFR-TKIs-sensitive and drug-resistant lung cancer cells that exogenously overexpress the MZF1 splice variant, the cell biological functions are significantly different. Figure 2 BD); Cellular overexpression of MZF1 L Subsequently, proliferation, colony formation, and migration were significantly reduced compared to the control (p<0.01), while overexpression of MZF1... S The proliferation of tumor cells, the number of clones formed, and the number of migrating cells were all significantly increased (p<0.01).

[0121] Example 3: Effects of the MZF1 splice variant on cell function and acquired resistance to EGFR-TKIs

[0122] The non-small cell lung cancer cells used in the tests, HCC827 and PC9, were both purchased from the National Biomedical Experimental Cell Resource Bank and cultured and passaged under normal conditions in the applicant's laboratory.

[0123] 3.1 MZF1 gene expression in HCC827 and PC9 cells

[0124] Universal empty vector plasmids were purchased from GeneCopoeia, USA, catalog number: EX-NEG-M02; MZF1 L Plasmids, purchased from GeneCopoeia, catalog number: EX-T3148-M02-5; MZF1 S The plasmid, NM_001267033.2, was synthesized by Shanghai Jikai Gene.

[0125] Stable transfection was performed using HCC827 and PC9 cell lines, which were sensitive and resistant to non-small cell lung cancer. Cells were seeded in 6-well cell culture plates. When the cells reached 60% confluence, 200 μL of DMEM was added to both the 20 nM expression plasmid and the empty vector plasmid, respectively. After mixing, the mixture was incubated at room temperature for 5 min. Then, 200 μL of DMEM was added to 6 μL of Lipofectamine 2000, and the mixture was incubated at room temperature for 5 min. 200 μL of Lipofectamine 2000 (Thermo Fisher Scientific, catalog number: 11668019) dilution was added to 200 μL of the expression plasmid, and the mixture was incubated at room temperature for 20 min. 400 μL of the mixture was added to cell culture plates and cultured at 37°C for 8 h without serum. The culture was then replaced with 10% FBSDMEM and incubated at 37°C for 24 h. Gefitinib (1 μM) / Osimertinib (0.1 μM) was then added, and the results were analyzed by RT-qPCR and Western blot.

[0126] 3.2 Cell Biology Functional Experiments

[0127] (1) MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) experiment

[0128] Cell lines constructed in step one, exhibiting good growth and in the logarithmic growth phase, containing empty vectors and overexpression of the sensitive and resistant MZF1 gene from HCC827 and PC9, were digested and counted. The cell suspension concentration was adjusted, and 100 μL was added to each well of a 96-well cell culture plate, with 2000 cells per well and 8 replicates per well. Observation was performed for 4 consecutive days. Daily, gefitinib (1 μM) / osimertinib (0.1 μM) and 10 μL LTT (Sigma-Aldrich, M2128) were added to each well to a final concentration of 5 μg / mL. After co-culturing for 4 hours, the culture medium was discarded. Four days later, 150 μL of dimethyl sulfoxide (DMSO, Sigma-Aldrich, D2650) was added to each well. After incubation in the dark with gentle shaking for 10 min, the absorbance of each well was measured at OD 490 nm using an ELISA reader. The obtained data were analyzed.

[0129] (2) Cell clone formation experiment

[0130] Cell lines in logarithmic growth phase containing empty vectors and those overexpressing the sensitive and resistant MZF1 gene from HCC827 and PC9 were digested and counted, and seeded into 6-well cell culture plates at 100 cells per well. Gefitinib (1 μM) / osimertinib (0.1 μM) was added, and the culture medium was changed every three days. After the cell clones reached a visible size, the culture medium was aspirated, and the cells were washed twice with 1×PBS. The cells were fixed with 4% paraformaldehyde for 30 min, washed twice with 1×PBS, stained with 0.5% crystal violet for 30 min, rinsed, air-dried, photographed, and the number of clones was counted.

[0131] (3) Transfer Experiment

[0132] The cell lines constructed in Step 1, containing empty vectors and overexpressing the sensitive and resistant MZF1 gene in HCC827 and PC9, were digested and counted. The cells were then added to the upper chamber of an 8.0 μm Transwell chamber (Corning Laboratories, USA, catalog number: CLS3422). 200 μL of serum-free cell suspension was added to each upper chamber, and 2 × 10⁶ cells were added to each well. 4 Cells were cultured in a cell culture chamber with 600 μL of complete culture medium in the lower chamber. Gefitinib (1 μM) / Osimertinib (0.1 μM) was added, and the cells were cultured in a cell culture incubator at 37°C for 24 h. The cells were then carefully washed several times with 1×PBS. The cells were fixed with 4% paraformaldehyde for 30 min and washed three times with 1×PBS. The cells were stained with 0.5% crystal violet for 30 min and rinsed until the wash solution was colorless. The cells in the upper chamber were carefully removed with a cotton swab and the number of migrating cells was counted under a microscope (100×).

[0133] 3.3 Separation experiments of cell membrane, cytoplasm and nuclear proteins

[0134] Cells were washed twice with ice-cold PBS using an animal cell nuclear-cytoplasmic protein isolation kit (Beijing Aoqing Biotechnology Co., Ltd., catalog number: AQ805-50T) and a membrane protein extraction kit (Beijing Aoqing Biotechnology Co., Ltd., catalog number: AQ802-50T). Cytoplasmic proteins were extracted: per 75 cm³... 2 Culture area (approximately 0.5–1 × 10⁻⁶) 7Add 500 μL of pre-chilled solution A to each cell and gently shake on ice for 5 min. Transfer the supernatant to a new centrifuge tube; the supernatant should be clear and free of cells and debris. Scrape cells off with a cell scraper and transfer to a centrifuge tube. Centrifuge at 500 × g, 4 °C for 10 min. Carefully aspirate the supernatant and mix with the previous step. Extract cell membrane proteins: Estimate the precipitate volume, add 500 μL of solution B, resuspend the precipitate by pipetting, and gently shake on ice for 30 min. Centrifuge at 5000 × g, 4 °C for 10 min, and save the supernatant, which contains membrane proteins. The precipitate is used for nucleus extraction. Extract nucleoproteins: Resuspend the precipitate in 250 μL of solution C and homogenize 5 times at medium speed using a homogenizer. Gently shake on ice for 10 min. Centrifuge at 7000 × g, 4 °C for 10 min, and save the supernatant, which contains nucleoproteins.

[0135] 3.4 Western blotting

[0136] After quantification, calculate the sample loading volume at 30 μg / well and add the sample to 5×SDS gel loading buffer (Genstar, China, catalog number: E153-05). Heat at 100℃ for 10 min, add samples sequentially, and perform SDS-PAGE electrophoresis at 120V. After electrophoresis, transfer the membrane to a wet electroporator. After transfer, remove the PVDF membrane (Millipore, USA, catalog number: IPVH00010). Block with 5% skim milk for 1 h, then incubate with primary antibody: MZF1. L Antibody (Wuhan Daian Biotechnology Co., Ltd., Antigen Immunization: RPFACAECGQSFRQRSNLTQHRRIHTGERPFACAECGKAFRQRPTLTQHLRVHTGEKPFACPECGQRFSQRLKLTRHQRTHTGEKPYHCGECGLGFTQVSRLTEHQRIHTGERPFACPECGQSFRQHANLTQHRRIHTGERPYACPECGKAFRQRPTLTQHLRTHRREKPFACQDCGRRFHQSTKLIQHQRVHSAE), MZF1 SAntibodies (Wuhan Daian Biotechnology Co., Ltd., antigen immunization: LGAGWLGAAVAMYVARC), p-EGFR antibody (Abcam, UK, catalog number: ab40815), EGFR antibody (Proteintech, China, catalog number: 66455-1-Ig), β-actin antibody (Sigma-Aldrich, USA, catalog number: A45551). Incubate overnight at 4°C. Incubate with secondary antibody labeled with anti-rabbit horseradish peroxidase (Abcam, UK, catalog number: ab6721) for 1 hour, then perform a colorimetric reaction using an ECL chemiluminescence kit (Themo Fisher, USA, catalog number: 32132), and develop using a Smart Gel Image Analysis System.

[0137] 3.5 Statistical Analysis

[0138] All experiments were repeated at least three times. Results were presented using two-tailed t-tests and are expressed as mean ± standard deviation. *p<0.05 indicates a statistically significant difference, and **p<0.01 indicates an extremely statistically significant difference.

[0139] 3.6 Experimental Results

[0140] In a cell model overexpressing the MZF1 splice variant, gefitinib (1 μM) or osimertinib (0.1 μM) was added to investigate the effect of the MZF1 splice variant on acquired resistance to EGFR-TKIs. MTT assays and clonogenic assays were used to examine whether overexpression of the MZF1 splice variant after adding gefitinib or osimertinib could inhibit the proliferation of drug-resistant cells.

[0141] MTT results showed that, compared with the drug-resistant cell line transfected with the empty vector control, overexpression of MZF1 was significantly better. L It can significantly inhibit the ability of drug-resistant cells to promote proliferation (p<0.01). Figure 3 A). Clonogenesis assay results showed that, compared with the control, overexpression of MZF1... L It can significantly inhibit the ability of drug-resistant cells to promote clonal formation (p<0.01). Figure 3 B). The effect of MZF1 splice variant overexpression on the invasion and migration ability of drug-resistant cells after the addition of gefitinib or osimertinib was examined by Transwell assay. The results indicated that, compared with the control, overexpression of MZF1... L It can significantly inhibit the ability of drug-resistant cells to promote invasion and migration (p<0.01, working basis-) Figure 3 C).

[0142] GR / OR cells overexpressing the MZF1 splicing variant were treated with gefitinib / osimertinib, and the expression of EGFR-related proteins was detected. The results showed that gefitinib / osimertinib did not reduce p-EGFR levels in GR / OR cells. However, overexpression of MZF1... L Drug treatment significantly reduced p-EGFR expression and overexpressed MZF1. S The opposite result will be presented (work basis - Figure 3 D).

[0143] To further investigate the effect of the MZF1 splicing variant on EGFR membrane translocation after EGFR-TKI resistance, the nucleus, cytoplasm, and membrane of resistant cells were isolated. L Overexpression of [a substance] increases mEGFR expression, while decreasing cEGFR and nEGFR expression. Figure 3 E). These results indicate that overexpression of MZF1 L Cellular sensitivity to EGFR-TKIs can be increased by modulating the localization of EGFR on the cell membrane.

Claims

1.MZF1 L The use of the protein MZF1 in the preparation of medicaments to improve or enhance the sensitivity of drug-resistant lung cancer patients to EGFR-TKI inhibitors. L The amino acid sequence of the protein is shown in SEQ ID NO. 2; the EGFR-TKI inhibitors are gefitinib and osimertinib.

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