CN113234832A - Human EGFR gene missense mutation molecular marker and application thereof in prediction of drug resistance of targeted inhibitor - Google Patents

Abstract

The invention belongs to the fields of biomedicine and gene detection, and particularly relates to a human EGFR gene missense mutation molecular marker and application thereof in predicting drug resistance of a targeted inhibitor. The molecular markers provided by the invention comprise 242 EGFR missense mutants related to the drug resistance of erlotinib, gefitinib and erlotinib and 15 EGFR missense mutants related to the drug resistance of afatinib and oxitinib, and the mutants can be used for predicting the drug resistance of non-small cell lung cancer patients to the targeted inhibitor treatment. The invention constructs a mutant library of EGFR gene tyrosine kinase functional region by using a synthetic biology method, and the EGFR mutant is highly enriched in drug screening; can be clinically used as a potential molecular marker for predicting drug resistance of lung cancer patients treated by targeted inhibitors.

Description

Human EGFR gene missense mutation molecular marker and application thereof in prediction of drug resistance of targeted inhibitor

Technical Field

The invention belongs to the fields of biomedicine and gene detection, and particularly relates to a human EGFR gene missense mutation molecular marker and application thereof in predicting drug resistance of a targeted inhibitor.

Background

EGFR is a high-frequency variation gene in lung cancer tissues, and EGFR variation interpretation is very important for early screening, accurate medication and prognosis monitoring of lung cancer. The world cancer statistics in 2020 show that the incidence and mortality of lung cancer worldwide ranks in the first two. Wherein, the EGFR variation rate in east Asia, female and non-smoking lung cancer patients is as high as 50%. The EGFR protein is localized on the cell membrane and is an important loop in the cell responsible for transmitting growth and proliferation signals. EGFR proteins have a tyrosine kinase domain in the intracellular membrane segment, and the mutation of this domain often leads to abnormal activation of downstream signaling pathways, which in turn leads to cancer. The tyrosine kinase segment of the EGFR gene (especially the No. 18-21 exon segment) is a hot spot segment in the pathogenic mutation set, and the segment is also the key area of the current gene detection. Targeted inhibitors against EGFR have been approved for clinical treatment of first-line non-small cell lung cancer patients in a variety of ways (e.g., afatinib, erlotinib, gefitinib, erlotinib, and oxitinib). The kit has very important significance for accurate detection and interpretation of EGFR variation, accurate medication and prognosis monitoring of lung cancer patients.

A large number of rare EGFR variations are now discovered that lack functional annotation for targeted drug sensitivity. Statistics on EGFR mutation records in the COSMIC (Catalogue of viral Mutations in cancer) database show that, excluding secondary Mutations such as T790M and C797S, L858R (approximately 41%) and exon-19 deletion Mutations (approximately 47%) together account for 88% of the total number of records, whereas rare EGFR Mutations account for approximately 12% of the total number of records. Rare mutations of EGFR consist mainly of missense mutations (proportion greater than 63%) and exon-20 insertional variations (-17%). Despite the low incidence of rare variation of single EGFR, this has led to the clinical discovery of more and more rare mutations of EGFR, given the enormous lung cancer disease base; all rare variations of EGFR were pooled and their numbers were large enough to make us non-negligible. In addition, patients often develop EGFR secondary mutations (often rare missense mutations) as EGFR-targeted therapies continue. However, these numerous rare variations of EGFR are sensitive to targeted drugs and lack functional studies. Currently, the concomitant diagnostic products developed by gene detection companies aiming at EGFR in the market mainly cover few high-frequency EGFR variation, including L858R, G719X, L861X, S768I/V, E709X, T790M, C797S and exon 19 deletion. With the exception of exon 19deletion, the EGFR high-frequency missense mutant species typically covered totaled less than 20. Whereas 3572 missense mutants theoretically exist in the mutation hotspot interval (No. 18-21 exon segments) of EGFR, most of the rare variant drug sensitivities are in need of functional annotation.

Disclosure of Invention

Because the number of EGFR missense mutation mutants with targeted drug sensitivity functional annotation is rare at present, a great number of EGFR missense mutation mutants lack functional annotation on the sensitivity of targeted drugs. Based on the above, the invention provides a systematic EGFR mutant drug sensitivity screening result and discloses an EGFR missense mutant molecular marker with potential targeted drug resistance.

In order to achieve the purpose, the invention adopts the technical scheme that:

a human EGFR gene drug-resistant variation molecular marker comprises 242 mutation sites with strong tolerance to a reversible combined targeted inhibitor and 15 mutation sites with strong tolerance to a non-reversible combined targeted inhibitor.

Preferably, the reversibly binding targeted inhibitor comprises erlotinib, gefitinib, and erlotinib; the non-reversible binding targeted inhibitor comprises afatinib and oxitinib.

Preferably, the 242 mutation sites with strong tolerance of the reversible binding targeted inhibitor comprise:

L688P、V689G、E690Q、P691S、P691H、L692P、T693P、T693K、P694R、G696E、A698G、N700I、N700Y、Q701E、A702P、R705K、I706T、I706M、L707S、K708E、E709V、E711G、E711V、E711K、E711Q、F712S、F712C、K713I、K713Q、K713R、K714M、K714N、K714Q、I715M、I715N、K716E、V717G、L718M、L718R、S720F、S720C、S720Y、G721D、F723L、F723I、G724D、T725A、Y727C、K728M、G729E、L730V、L730I、L730R、W731R、I732M、I732N、E734A、K737I、V738L、V738D、K739N、P741A、V742A、V742I、A743T、I744M、E746G、E746D、A750G、A750E、S752A、S752F、S752C、P753A、P753S、K754I、A755D、N756S、N756T、N756H、K757M、K757Q、E758V、E758D、I759V、L760V、L760I、L760H、D761G、D761H、D761N、E762K、E762Q、Y764H、Y764D、V765A、V765E、V765M、M766T、M766R、A767P、V769A、V769L、D770H、P772A、P772L、P772T、H773P、H773Q、V774G、V774A、V774M、C775F、C775Y、R776L、R776H、L777P、L777M、L778R、G779S、G779C、G779R、I780M、C781G、T783I、T783N、S784P、S784T、V786L、Q787L、Q787E、L788V、L788I、I789L、I789M、I789N、T790M、Q791L、L792F、M793V、M793I、P794H、G796C、C797W、L798F、Y801H、V802A、E804D、H805P、H805Y、K806T、K806E、N808S、I809V、I809S、I809T、G810V、G810D、S811F、L815H、N816D、W817L、W817C、C818W、C818R、V819A、V819M、Q820L、I821V、I821S、A822E、K823T、K823E、K823R、G824R、N826I、N826H、N826D、Y827D、L828F、E829G、D830E、R831G、R831L、R831P、R831H、R831C、R832G、R832P、R832S、L833V、L833S、H835P、H835Y、R836G、D837Y、L838P、L838M、A839P、A839T、R841T、V843A、L844P、V845G、V845A、V845L、V845E、V845M、Q849K、H850Y、K852E、T854P、D855V、G857V、L858V、L858Q、A859D、K860I、K860Q、L861M、L861Q、G863A、A864P、A864T、E865A、E865K、E866K、K867E、E868G、E868D、E868K、Y869D、H870L、H870D、E872D、E872K、G873A、G873R、G874C、K875T、K875N、K875Q；

the 15 kinds of mutation sites with strong tolerance of the non-reversible binding targeted inhibitor comprise:

T693K、Q701E、I715M、S720C、I744M、C775Y、I789N、C818W、Q820L、N826H、D830E、A839T、K860Q、A864T、E868G。

preferably, the mutation sites are present within the cell alone, in any combination with each other, or in combination with wild-type EGFR or other EGFR mutation sites.

I.e., in combination, there are two ways: in one way, the molecular markers are on the same DNA molecule or mRNA transcript as the other EGFR mutation site(s) (in cis); alternatively, the molecular marker is on a different DNA molecule or mRNA transcript (in trans) than the other EGFR mutation site(s); the variant site can be obtained by single base variation of the corresponding codon in the wild type gene.

When detecting the mutation sites, the adopted methods comprise CRISPR/Cas gene editing technology detection, fluorescence in-situ hybridization detection, gene chip detection, PCR detection, fluorescence quantitative PCR detection, digital PCR detection, Sanger sequencing, high-throughput sequencing detection and the like; the sample selected in the detection may be genomic DNA, DNA derived from tumor tissue, free DNA in peripheral blood, RNA derived from tumor tissue, or free RNA in peripheral blood.

The invention also provides an application of the molecular marker in constructing recombinant genes, mRNA, proteins, cell lines, organoids and animal models.

The invention also provides application of the molecular marker in formulating a target drug use scheme related to EGFR variation, evaluating the progress of cancers related to EGFR variation, predicting the generation of drug-resistant variation and developing new drugs.

The invention also provides a screening method of the molecular marker, which is characterized by comprising the following steps:

s1, constructing an EGFR missense mutant library;

s2, packaging the EGFR mutant library into a lentiviral vector;

s3, infecting a host cell line by using lentivirus, and obtaining a stable transfer cell line through antibiotic screening or fluorescence screening;

s4, adding a targeted inhibitor drug for treatment, and continuously culturing for 2 weeks;

s5, extracting genome DNA from the cells surviving the drug screening, and amplifying a mutant sequence or a tag sequence by PCR;

s6, determining corresponding enrichment coefficients of various mutants before and after drug screening through biological information analysis.

It should be further noted that the mutant library in step S1 is not limited to the missense mutant library, but may be an insertion mutant (insertion) library, a deletion mutant (deletion) library, a complex mutant library, or a mixed mutant library; step S2 is not limited to lentiviral vectors, but may be other types of viral vectors, or exogenous mutants may be introduced into the host cell using non-viral-mediated transformation, transfection or infection; in step S3, the host cell may or may not knock out the endogenous target gene and its mutant gene by a gene editing method in advance; after the targeted inhibitor is added in step S4, the culture time may be determined according to the requirement, and is not limited to two weeks; the mutant enrichment factor calculation method in step S6 is: the ratio of the number of cells corresponding to the mutant after drug screening to the number of cells corresponding to the mutant before screening; a larger enrichment factor indicates a more resistant mutant and vice versa a more sensitive mutant. In addition, the method provided above is not limited to the study of mutants of EGFR gene, and can also be used to study the sensitivity of mutants of other genes to targeted inhibitors, or to study the function of gene mutants using other functional assay methods.

The achievement of functional annotation of mutants is a prerequisite for the development of various gene testing applications. Lung cancer is a cancer species with high worldwide incidence and mortality, and has a large number of new cases each year. Non-small cell lung cancer is the major category of lung cancer, with approximately 50% of patients of asian african small cell lung cancer carrying EGFR mutations. For these patients carrying EGFR-activating variants, treatment with EGFR-targeted inhibitors may be used; however, prior to treatment, a preliminary genetic test is required to determine whether and what mutations a patient carries with EGFR. Currently, EGFR missense mutants that can give medication recommendations by gene detection are mainly: L858R, G719X, L861X, S768I/V, E709X and T790M. Due to the lack of functional annotation, no medication guidance suggestions have been given for a large number of rare missense variant mutants of EGFR.

The invention uses EGFR gene missense mutant library (3572, refer to the invention patent with the application publication number of CN 112725331A) constructed by a synthetic biological method, and finds 242 mutants to have drug resistance to reversibly combined targeted inhibitors (erlotinib, gefitinib and erlotinib) and 15 mutants to have drug resistance to irreversibly combined targeted inhibitors (afatinib and erlotinib) through systematic drug sensitivity screening. These mutants can be used as molecular markers to predict the development of clinical drug resistance.

Compared with the prior art, the invention has the technical advantages that:

(1) the method carries out systematic screening on 3572 mutants in an EGFR gene mutation hotspot region, quantitatively evaluates the sensitivity of the mutants to 5 common clinical targeted inhibitors, determines the sensitivity coefficient of each mutant to the 5 tested targeted inhibitors, and greatly increases the quantity proportion of EGFR missense mutants with actual drug sensitivity function annotations to be more than 98%;

(2) according to the invention, 242 mutants with high tolerance to erlotinib, gefitinib and erlotinib and 15 mutants with high tolerance to afatinib and oxitinib are found through systematic screening, and the mutants are potential drug-resistant molecular markers;

(3) the potential drug-resistant molecular marker found by the invention can be used for guiding clinical medication and monitoring the occurrence of clinical drug-resistant events, and the mutant screening method provided by the invention can be used for evaluating the sensitivity of EGFR mutants to other targeted drugs, such as evaluating the drug sensitivity to dacomitinib, and further evaluating the sensitivity of other mutants with important clinical value to the targeted drugs, thereby greatly expanding the application range and accuracy of the concomitant diagnosis.

Description of the drawings:

FIG. 1 is a schematic flow chart of a drug sensitivity screening experiment;

fig. 2 is a schematic structural diagram of expression vectors for EGFR mutant and Cas 9;

FIG. 3 is a schematic view of a flow chart of biological information analysis of sequencing data;

FIG. 4 is a graph of the results of a sensitivity screen using erlotinib on EGFR mutant libraries;

FIG. 5 is a graph of the results of a sensitivity screen using gefitinib for EGFR mutant libraries;

FIG. 6 is a graph showing the results of a sensitivity screen using icotinib on EGFR mutant libraries;

FIG. 7 is a graph of the results of a sensitivity screen using Afatinib on EGFR mutant libraries;

FIG. 8 is a graph showing the results of a sensitivity screen using oxitinib on EGFR mutant libraries;

FIG. 9 is a graph of the intersection results of potential drug-resistant variants of erlotinib, gefitinib and erlotinib;

figure 10 is a graph showing the intersection results of potential drug-resistant variations of afatinib and axitinib.

Detailed Description

The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. The method and the device are operated according to the conventional technical method and the content of the instrument instruction, wherein the specific technology or condition is not indicated in the embodiment; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example a method for screening drug-resistant variant molecular markers

The screening method of the molecular marker comprises the following processes (figure 1):

s1 and EGFR mutant library construction

For the method of constructing the EGFR missense mutant library, see the patent application with publication No. CN112725331A (a method for constructing high throughput mutant library). In order to knock out the endogenous EGFR of a cell, two sgRNA expression modules targeting EGFR, which have been previously detected, need to be cloned into a mutant library vector. The method comprises the following steps:

endogenous EGFR knockdown: on-line software (http:// chopchopchop. cbu. uib. no /) was used to design 2 sgrnas targeting the EGFR gene. The relevant target sequences are as follows:

target 1: GTTCACATCCATCTGGTACGTGG(TGG is PAM sequence)

sgRNA1-oligo-F:CACCGTTCACATCCATCTGGTACG

sgRNA1-oligo-R:AAAACGTACCAGATGGATGTGAAC

Target 2: GTGGAGATCGCCACTGATGGAGG(AGG is a PAM sequence)

sgRNA2-oligo-F:CACCGTGGAGATCGCCACTGATGG

sgRNA2-oligo-R:AAAACCATCAGTGGCGATCTCCAC

The sgRNA is constructed by adopting a pLenti-CRISPR v2 vector of a Zhang Feng laboratory, a BsmBI restriction endonuclease is used for linearizing the vector, and the annealed and renatured target Oligo primer is connected into the vector through a T4-DNA ligase to obtain the required sgRNA. Related cloning methods, reference may be made to the cloning methods provided by Zhang Feng laboratories (Genome-scale CRISPR-Cas9 knock out screening in human cells Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014). Science,343,83-7.DOI: 10.1126/science.1247005).

Assay for sgRNA gene editing potency: a549 cells were infected after packaging the CRISPR/Cas9 vector as a lentivirus. The viral packaging and infection process is as follows: 500 ten thousand HEK293 cells were seeded in 10cm dishes and the sgRNA vector and lentiviral packaging vector (psPAX and pMD2G) plasmid combinations were co-transfected into HEK293 cells using EZ Transs cationic Polymer transfection reagent from Leymi Bio. After 24 hours, the medium was changed and timing was started, and the virus was collected every 24 hours for 2 times. The harvested virus was filtered using a 0.45 μm filter. The packaged virus was added to a549 cell culture medium in an amount of 0.25 of multiple infection index (MOI), and the culture medium was replaced with fresh medium after 3 days. Subsequent screening with puromycin allowed gene editing potency assays to be performed 7 days after screening.

A549 cells and control cells subjected to EGFR gene editing were stained with Anti-EGFR-PE (352904) flow antibody from BioLegend, and then analyzed by flow cytometry to examine EGFR knock-out efficiency. Subsequently, sgRNA-1 was incorporated into the mutant library vector by a first round of subcloning (double-restriction ligation, at Kpn-I and EcoR-I). The sgRNA-2 was then ligated into the mutant library vector (FIG. 2) in a second round of subcloning (single-restriction ligation, EcoR-I at the cleavage site).

S2 packaging mutant library into lentivirus

And (3) slow virus packaging: 500 ten thousand HEK293 cells were seeded in 10cm dishes and the mutant library vectors and lentiviral packaging vector (psPAX and pMD2G) plasmids were seeded using EZ Trans cationic polymer transfection reagent (Liji organism) at a molar ratio of 1: 1: 1 combination was co-transfected into HEK293 cells. After 24 hours the medium was changed and the timer was started. Thereafter, the virus was collected every 24 hours, filtered through a 0.45 μm filter, and frozen at-80 ℃ for storage in a refrigerator.

And (3) virus titer determination: 100 million PC9 cells were plated on 6-cm plates, 6 gradients of virus were added, and the screening was performed 72 hours later by adding Blasticidin (Blasticidin S). After one week, the cells were counted and virus titers were calculated as they grew steadily.

S3 infection of PC9-Cas9 cell line with lentivirus

Constructing a stable PC9-Cas9 transgenic cell line, namely unloading the sgRNA expression module by using a KpnI/EcoRI double-enzyme cutting method on the basis of a pLenti-CRISPR v2 vector of a Zhang Feng laboratory, wherein the enzyme cutting reaction system is as follows:

enzyme cutting conditions are as follows: 37 ℃ for 3 hours. Subsequently, the vector was blunted using Klenow enzyme (NEB) and self-ligated, i.e., Cas9 expression vector, using T4-DNA ligase (NEB) (fig. 2).

The constructed Cas9 expression vector has puromycin resistance screening gene, is packaged into lentivirus (the virus packaging method is the same as above), and then infects PC9 cells. Selection was performed using puromycin and the Cas9 stably infected cell line was obtained for four weeks of serial selection.

Lentivirus infection of PC9-Cas9 stable transgenic cell line using mutant library: evening of the first day 1500 ten thousand PC9-Cas9 cells were seeded each in 6 15cm dishes. The next morning, mutant library viruses were added according to infection coefficients (Multiplicity of infection, MOI <0.3), and the medium was changed 24 hours after virus infection. After continuous culture for 3 days, puromycin and blasticidin were added for screening. Continuous screening with antibiotics for 2 weeks was performed to ensure: (1) endogenous EGFR is knocked out; (2) the exogenous EGFR mutant library is stably integrated into the genome of the PC9-Cas9 cell and normally expresses the EGFR mutant. By the above-described procedures, replacement of the endogenous EGFR gene (WT and exon 19deletion) with an exogenous EGFR mutant in PC9-Cas9 cells was achieved.

S4, adding targeted inhibitor medicine for treatment

After the endogenous EGFR is completely knocked out by CRISPR/Cas9 gene editing and the exogenous EGFR mutant is fully expressed, drug sensitivity screening can be carried out.

It should be noted that whether or not an endogenous target gene (e.g., EGFR herein) is knocked out depends on the purpose of the experiment. In addition, protein products of some genes in the genome function or are regulated in the form of homodimers or homomultimers. In this case, when two or more different types of mutants of the gene are present in the same cell, there may be a complex interaction between the different mutants, and functional annotation of the mutants requires consideration of the complex interaction relationship. For example, the EGFR gene, which is explained in the present invention, forms homodimers on the cell membrane, thereby performing a signal transduction function. When there is only one EGFR protein class (wild-type WT or single type mutant) in the cell, the EGFR protein dimer formed has only a unique combinatorial morphology; when two or more mutant types are present in the cell, different EGFR mutants will now form a variety of different EGFR protein dimer combinations. The drug sensitivity of the EGFR mutant is evaluated in the case that the EGFR protein (wild-type WT and exon 19deletion mutant) endogenous to PC9 cells is knocked out, and only one exogenously introduced EGFR mutant theoretically exists in the cells. In this case, it is appropriate to assess the tolerance of EGFR mutants to drug treatment, i.e. to find possible types of drug-resistant mutants by drug screening experiments.

Drug screening experiments combining reversible and non-reversible inhibitors are described below to further illustrate how to search for potential drug-resistant molecular markers by systemic drug sensitivity screening.

Screening for mutants potentially resistant to reversibly binding targeted inhibitors

Two screening groups are arranged, one group is a targeted inhibitor screening group, and a targeted inhibitor with proper concentration is added for evaluating the influence of the EGFR mutant on cell proliferation in the presence of the targeted inhibitor; the other group was an experimental control group to which an equivalent volume of dimethyl sulfoxide (DMSO) was added for evaluation of the effect of the mutants themselves on cell proliferation without inhibitors. Theoretically, the less affected the proliferation of cells containing the mutant is when an external targeted inhibitor is present, the less sensitive the mutant is to the drug carried by the cell. When the targeted inhibitor is used for drug sensitivity screening, the drug concentration can be set by referring to the blood concentration of a clinical patient, and the drug concentrations of erlotinib, gefitinib and erlotinib in the invention are set to 600 nM.

In the EGFR targeted inhibitor evaluated by the invention, the erlotinib, gefitinib and erlotinib have the same mechanism for inhibiting EGFR mutants, and the drugs are competitively combined with the EGFR mutants by ATP in a reversible mode, so that the function of the EGFR mutants is inhibited.

The operation method of the experimental control group comprises the following steps: 1500 ten thousand cells (cells obtained in step S3) having integrated EGFR mutants were seeded on 3 15cm plates, and 500 ten thousand cells were used as a zero-point untreated sample and left for the subsequent steps. In 3 plates, 200. mu.L of DMSO were added. The cells were continuously cultured for 2 weeks, and at the intermediate stage, 1500 thousands of cells were left after counting for passaging, and at the passage, continuous culture was carried out using a fresh RPMI 1640 medium (containing 10% fetal bovine serum, streptomycin antibiotic, puromycin, blasticidin, GlutaMAX, all purchased from ThermoFisher Co.) containing DMSO in an equivalent ratio. Passages were performed every 48 hours until the end of the experiment. After 2 weeks, cell counts were performed and cells were collected in 500 ten thousand cell aliquots for subsequent experiments.

Example drug sensitivity screening of mutant libraries Using erlotinib

The erlotinib drug screen group operation method comprises the following steps: 1500 ten thousand cells (cells obtained in step S3) having integrated EGFR mutants were seeded on 3 15cm plates, and 500 ten thousand cells were used as a zero-point untreated sample and left for the subsequent steps. 200 μ L of erlotinib dissolved in DMSO was added to each of 3 plates to give a final erlotinib concentration of 600nM in RPMI 1640 medium (containing 10% fetal bovine serum, penicillin antibiotic, puromycin, blasticidin, GlutaMAX, all purchased from ThermoFisher, Inc.; the same below). The culture was continued for 2 weeks, and subcultured every 48 hours until the end of the experiment. The cells are counted during passage, and culture dishes with proper sizes (15cm, 10cm or 6cm) are selected according to the number of the cells to be cultured so as to ensure that the cell culture density is not too high or too low. At the initial stage of screening, cells die massively, retaining all cells per passage. As the screening time continued, the cells proliferated gradually, leaving only 1500 million cells per passage when the total amount after proliferation exceeded 1500 million. After each passage, serial cultures were performed using fresh medium containing 600nM erlotinib. After 2 weeks, cell counts were performed and cells were collected in 500 ten thousand cell aliquots for subsequent experiments.

EXAMPLE two drug sensitivity screening of mutant libraries Using Gefitinib

The gefitinib drug screening group operation method comprises the following steps: 1500 ten thousand cells (cells obtained in step S3) having integrated EGFR mutants were seeded on 3 15cm plates, and 500 ten thousand cells were used as a zero-point untreated sample and left for the subsequent steps. In 3 plates, 200. mu.L of gefitinib dissolved in DMSO was added to give a final concentration of 600nM gefitinib in the medium. The culture was continued for 2 weeks, and subcultured every 48 hours until the end of the experiment. The cells are counted during passage, and culture dishes with proper sizes (15cm, 10cm or 6cm) are selected according to the number of the cells to be cultured so as to ensure that the cell culture density is not too high or too low. At the initial stage of screening, cells die massively, retaining all cells per passage. As the screening time continued, the cells proliferated gradually, leaving only 1500 million cells per passage when the total amount after proliferation exceeded 1500 million. After each passage, serial cultures were performed using fresh medium containing 600nM gefitinib. After 2 weeks, cell counts were performed and cells were collected in 500 ten thousand cell aliquots for subsequent experiments.

EXAMPLE III drug sensitivity screening of mutant libraries Using Excotinib

The method for operating the icotinib drug sieve group comprises the following steps: 1500 ten thousand cells (cells obtained in step S3) having integrated EGFR mutants were seeded on 3 15cm plates, and 500 ten thousand cells were used as a zero-point untreated sample and left for the subsequent steps. In 3 plates, 200. mu.L of icotinib dissolved in DMSO was added to give a final concentration of 600nM icotinib in the medium. The culture was continued for 2 weeks, and subcultured every 48 hours until the end of the experiment. The cells are counted during passage, and culture dishes with proper sizes (15cm, 10cm or 6cm) are selected according to the number of the cells to be cultured so as to ensure that the cell culture density is not too high or too low. At the initial stage of screening, cells die massively, retaining all cells per passage. As the screening time continued, the cells proliferated gradually, leaving only 1500 million cells per passage when the total amount after proliferation exceeded 1500 million. After each passage, serial cultures were performed using fresh medium containing 600nM icotinib. After 2 weeks, cell counts were performed and cells were collected in 500 ten thousand cell aliquots for subsequent experiments.

Screening for mutants potentially resistant to non-reversibly binding targeted inhibitors

Two screening groups are arranged, one group is a targeted inhibitor screening group, and a targeted inhibitor with proper concentration is added for evaluating the influence of the EGFR mutant on cell proliferation in the presence of the targeted inhibitor; the other group was an experimental control group to which an equivalent volume of dimethyl sulfoxide (DMSO) was added for evaluation of the effect of the mutants themselves on cell proliferation without inhibitors. Theoretically, the less affected the proliferation of cells containing the mutant is when an external targeted inhibitor is present, the less sensitive the mutant is to the drug carried by the cell. When the targeted inhibitor is used for drug sensitivity screening, the setting of the drug concentration can be set by referring to the blood drug concentration of a clinical patient, the drug concentration of afatinib in the invention is set to be 50nM, and the drug concentration of oxitinib is set to be 200 nM.

In the EGFR targeted inhibitor evaluated by the invention, the mechanism of inhibiting the EGFR mutant by afatinib and axitinib is the same, and the drugs are irreversibly combined with cysteine at 797 of EGFR protein in a covalent combination mode, so that the combination of ATP and the EGFR mutant is hindered, and the purpose of inhibiting the function of EGFR is achieved.

The operation method of the experimental control group comprises the following steps: 1500 ten thousand cells (cells obtained in step S3) having integrated EGFR mutants were seeded on 3 15cm plates, and 500 ten thousand cells were used as a zero-point untreated sample and left for the subsequent steps. In 3 plates, 200. mu.l of DMSO were added. After continuous culture for 2 weeks, 1500 ten thousand cells were counted and passaged at an intermediate stage when the number of cells was excessive, and fresh RPMI 1640 medium (containing 10% fetal bovine serum, streptomycin antibiotic, puromycin, blasticidin, GlutaMAX, all purchased from ThermoFisher) containing DMSO in an equivalent ratio was used for the passage. Passages were performed every 48 hours until the end of the experiment. After 2 weeks, cell counts were performed and cells were collected in 500 ten thousand cell aliquots for subsequent experiments.

Example drug sensitivity screening of mutant libraries Using Afatinib

The operation method of the Afatinib drug screening group comprises the following steps: 1500 ten thousand cells (cells obtained in step S3) having integrated EGFR mutants were seeded on 3 15cm plates, and 500 ten thousand cells were used as a zero-point untreated sample and left for the subsequent steps. 200 μ L of Afatinib dissolved in DMSO was added to each of 3 plates to give a final Afatinib concentration of 50nM in RPMI 1640 medium (containing 10% fetal bovine serum, penicillin antibiotic, puromycin, blasticidin, GlutaMAX, all purchased from ThermoFisher, Inc.; the same below). The culture was continued for 2 weeks, and subcultured every 48 hours until the end of the experiment. The cells are counted during passage, and culture dishes with proper sizes (15cm, 10cm or 6cm) are selected according to the number of the cells to be cultured so as to ensure that the cell culture density is not too high or too low. All cells were retained for each passage during the screening process, and a suitable dish was selected according to the number of cells. After each passage, serial cultures were performed using fresh medium containing 50nM afatinib. After 2 weeks, cell counts were performed and cells were collected in 500 ten thousand cell aliquots for subsequent experiments.

EXAMPLE two drug susceptibility screening of mutant libraries Using Oxecitinib

The operation method of the oxitinib drug sieve group comprises the following steps: 1500 ten thousand cells (cells obtained in step S3) having integrated EGFR mutants were seeded on 3 15cm plates, and 500 ten thousand cells were used as a zero-point untreated sample and left for the subsequent steps. In 3 plates, 200 μ L of each of the oxitinib dissolved in DMSO was added to give a final concentration of 200nM in the culture medium. The culture was continued for 2 weeks, and subcultured every 48 hours until the end of the experiment. The cells are counted during passage, and culture dishes with proper sizes (15cm, 10cm or 6cm) are selected according to the number of the cells to be cultured so as to ensure that the cell culture density is not too high or too low. During the screening process, all cells are retained for passage at each passage, and a proper culture dish is selected according to the number of the cells. After each passage, continuous culture was performed using fresh medium containing 200nM of ocitinib. After 2 weeks, cell counts were performed and cells were collected in 500 ten thousand cell aliquots for subsequent experiments.

S5, extracting cell genome DNA and extracting tag sequence by PCR amplification

Genomic DNA (gDNA) preparation and tag sequence amplification. Genomic DNA of cells in each group of specimens was extracted using QIAGEN genomic DNA medium extraction kit. Amplifying DNA fragments containing tag sequences (the tag sequences are positioned in the middle of the amplified DNA fragments, each tag sequence corresponds to a unique mutant, and the corresponding relation between the tag sequences and the mutants is determined during library preparation) from a genome by using Q5 high-fidelity DNA polymerase of NEB company, wherein the amplification primer sequences are as follows:

Tag-F：5’-GCTGCCCTCTGGTTATGTGTG-3’

Tag-R：5’-GTAATCCAGAGGTTGATTACCG-3’

the number of PCR reactions is determined according to the total amount of the genome DNA, and all the genome DNA is ensured to be used for amplifying and extracting the Tag sequence so as to ensure the maximum representativeness. Specifically, 100ng of genomic DNA was added per PCR reaction. The PCR reaction system is as follows:

the PCR amplification conditions were as follows: at 95 ℃ for 3 minutes; (98 ℃, 20 seconds; 55 ℃, 30 seconds; 72 ℃, 30 seconds) x 35 cycles; 72 ℃ for 5 minutes; preserving at 10 deg.C.

After the PCR products of the same genome DNA are combined, the PCR products are fully and uniformly mixed. It should be noted here that the DNA of a sample is used as completely as possible for PCR amplification, so that mutant information can be extracted from the cell genome more completely, and assuming that 2000ng of sample DNA and 100ng of sample DNA are added for each PCR reaction, 20 PCR amplification tubes are theoretically required for the sample DNA. The amplified Tag DNA fragment was purified using Tiangen DNA purification kit. Tag-tagged DNA was submitted to commercial high-throughput sequencing companies for sequencing, and PE150(Paired-End 150) high-throughput sequencing was performed using Illumina HiSeq 4000 sequencing platform or other high-throughput sequencing platform.

S6 determination of enriched mutants by bioinformatic analysis

FIG. 3 shows a flow of analysis of sequencing data. The sequencing data analysis flow is as follows:

(1) merging pairs of raw reads (read1 and read2) into a single read;

(2) splitting sequencing data corresponding to each sample into 3 replicates;

(3) converting the tag sequence obtained by sequencing into a corresponding mutant according to the corresponding relation of the mutant-tag sequence determined during library construction of the mutant library;

(4) and (4) calculating the enrichment coefficients of various mutants in each repeated group in each drug screening experiment. The enrichment coefficient calculation method is as follows:

(a) and calculating the corresponding theoretical cell number of each mutant before drug screening. Calculating the proportion of each mutant in all qualified reads according to the sequencing result, and multiplying the proportion by the number of cells at the starting point to obtain the theoretical number of cells n corresponding to each mutant;

(b) and calculating the theoretical cell number corresponding to each mutant after drug screening. Calculating the proportion of each mutant in all qualified reads according to a sequencing result, and multiplying the proportion by the number of cells at the end of drug screening to obtain the theoretical number of cells n' corresponding to each mutant;

(c) calculating an enrichment coefficient: the enrichment factor is n'/n.

The matrix plots in fig. 4-8 show the results of a sensitivity screen for 3572 EGFR variants using erlotinib, gefitinib, erlotinib, afatinib, and oxitinib, respectively. The vertical axis represents the amino acid position (688-875), and the horizontal axis represents the 20 amino acid variation types. The dots in the graph show the enrichment factor (Log2) for each variation (or wild type WT) at each amino acid position in the drug screening experiment. The circles indicate that the enrichment factor for the mutant is less than 0 and the triangles indicate that the enrichment factor for the spot is greater than 0.

Due to the same action mechanism, the drug screening results of erlotinib, gefitinib and erlotinib are respectively sequenced, and mutants with enrichment coefficients (log2) larger than 0 are extracted, namely 841 mutants, 1802 mutants and 2102 mutants. We get the intersection of three mutants with more than 0 in the experiment result of reversible combination TKI drug screening to obtain 813 mutants. Similarly, we ranked the drug screening results of afatinib and axitinib, respectively, and extracted 280 mutants with enrichment coefficients (log2) greater than 0, respectively, 271. We took intersection of two non-reversible combination TKI drug screening test results with the mutant larger than 0 to obtain 51 mutants.

Here, we classified the mutants into two categories: (1) complex missense mutants (Complex Substistion). This type of missense mutant, in the wild type gene sequence corresponding to the codon, needs to undergo 2 or 3 base variations to obtain. (2) Simple missense mutant (Simple disruption). This type of missense mutant needs to undergo 1 base mutation at the codon corresponding to the wild-type gene sequence. Due to the randomness of gene variation, the probability of multiple point mutations in the same codon is very low (about 2.2X 10)^-9/(bases. times.years)), the frequency of occurrence of complex missense mutants was therefore 10 times lower than that of simple missense mutants^-9To 10^-18. In fact, most of the clinically found missense mutants are simple missense mutants, and few are complex missense mutants.

Based on the above rules, further, we divided the mutants enriched in the 813 reversible TKI drug screening experiments into two groups: 571 complex missense mutants and 242 simple missense mutants. The dot plot in fig. 9 shows the enrichment factor (log2) for the 242 simple missense mutants enriched in the reversible TKI drug screening experiment. Similarly, we divided the 51 mutants enriched in the irreversible TKI drug screening experiments into two groups: 36 complex missense mutants and 15 simple missense mutants. The dot plot in fig. 10 shows the enrichment factor (log2) for the 15 simple missense mutants enriched in the irreversible TKI drug screening experiment.

It should be noted that although the above embodiments have been described, once the basic inventive concept is obtained, other variations and modifications can be made to these embodiments by those skilled in the art, so that the above embodiments are only examples of the present invention, and not to limit the scope of the present invention, and all the modifications made by the equivalent structures or equivalent processes in the present specification, or directly or indirectly applied to other related technical fields are included in the scope of the present invention.

Sequence listing

<110> Shenzhen Shenfeng Life technology Limited

<120> human EGFR gene missense mutation molecular marker and application thereof in prediction of drug resistance of targeted inhibitor

<130> 2021.6.30

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> DNA

<213> Target 1(Target 1)

<400> 1

gttcacatcc atctggtacg tgg 23

<210> 2

<211> 24

<212> DNA

<213> sgRNA1-oligo-F

<400> 2

caccgttcac atccatctgg tacg 24

<210> 3

<211> 24

<212> DNA

<213> sgRNA1-oligo-R

<400> 3

aaaacgtacc agatggatgt gaac 24

<210> 4

<211> 23

<212> DNA

<213> Target 2(Target 2)

<400> 4

gtggagatcg ccactgatgg agg 23

<210> 5

<211> 24

<212> DNA

<213> sgRNA2-oligo-F

<400> 5

caccgtggag atcgccactg atgg 24

<210> 6

<211> 24

<212> DNA

<213> sgRNA2-oligo-R

<400> 6

aaaaccatca gtggcgatct ccac 24

<210> 7

<211> 21

<212> DNA

<213> Tag-F

<400> 7

gctgccctct ggttatgtgt g 21

<210> 8

<211> 22

<212> DNA

<213> Tag-R

<400> 8

gtaatccaga ggttgattac cg 22

Claims (7)

1. A human EGFR gene drug-resistant variation molecular marker is characterized by comprising 242 kinds of mutation sites highly enriched in reversible binding targeted inhibitor screening experiments and 15 kinds of mutation sites highly enriched in non-reversible binding targeted inhibitor screening experiments.

2. The molecular marker of claim 1, wherein the reversibly binding targeted inhibitor comprises erlotinib, gefitinib, and erlotinib; the non-reversible binding targeted inhibitor comprises afatinib and oxitinib.

3. The molecular marker of claim 1, wherein the 242 highly enriched mutation sites in the screening assay for reversibly binding targeted inhibitors comprise: l688, V689, E690, P691, L692, T693, P694, G696, A698, N700, Q701, A702, R705, I706, L707, K708, E709, E711, F712, K713, K714, I715, K716, V717, L718, S720, G721, F723, G724, T725, Y727, K728, G729, L730, W731, I732, E734, K737, V738, K739, P741, V780, A742, A746, I744, E762, L762, W731, I732, I769, V779, S77779, S779, P77779, P779, K76779, K769, K779, K769, S779, K779, S779, K779, S779, K779, q787, L788, I789, T790, Q791, L792, M793, P794, G796, C797, L798, Y801, V802, E804, H805, K806, N808, I809, G810, S811, L815, N816, W817, C818, V819, Q820, I821, a822, K823, G824, N826, Y827 872, L872, E829, D830, R831, R832, L833, L826, H872, R835, D838, L847, L868, L845, V868, G868, K868, K868, K868, K868, K868, K868, G845, K868, K868, K868, G845, K868, K, G845, G831, K868, K868, G845, K868, G845, G831, R831, G845, R831, L845, G845, L845, R831, K868, R831, L845, G845, K868, G845, L845, R831, L845, K868, L845, L832, K, L845, K868, L845, R831, L845, K868, L845, L;

the 15 highly enriched mutation sites in the screening experiment of the irreversible binding targeted inhibitor comprise: T693K, Q701E, I715M, S720C, I744M, C775Y, I789N, C818W, Q820L, N826H, D830E, a839T, K860Q, a864T, E868G.

4. The molecular marker of claim 1, wherein the mutation sites are present within the cell alone, in any combination with each other, or in combination with wild-type EGFR or other EGFR mutation sites.

5. Use of the molecular marker of claim 1 for constructing recombinant genes, mRNA, proteins, cell lines, organoids, animal models.

6. Use of the molecular marker of claim 1 for the development of a targeted drug regimen associated with EGFR mutation, for the assessment of cancer progression associated with EGFR mutation, for the prediction of the development of resistance mutations, and for the development of new drugs.

7. A method for screening a molecular marker according to claim 1, comprising the steps of:

s1, constructing an EGFR missense mutant library;

s2, packaging the EGFR mutant library into a lentiviral vector;

s3, infecting a host cell line by using lentivirus, and obtaining a stable transfer cell line through antibiotic screening or fluorescence screening;

s4, adding a targeted inhibitor drug for treatment, and continuously culturing for 2 weeks;

s5, extracting genome DNA from the cells surviving the drug screening, and amplifying a mutant sequence or a tag sequence by PCR;

s6, determining the enrichment coefficients of various mutants before and after drug screening through biological information analysis.

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