Fusion gene STRN3-RARA acquired drug resistance S232F locus and application thereof in ATRA

Abstract

The invention relates to the technical field of molecular oncology and drug resistance mechanism research, and discloses an S232F site of acquired drug resistance of a fusion gene STRN3-RARA and application of the S232F site in ATRA. The product is used for detecting the sensitivity of a patient suffering from acute promyelocytic leukemia to all-transretinoic acid (ATRA) treatment or screening whether the patient suffering from acute promyelocytic leukemia is resistant to ATRA treatment, an S232F mutation site is an STRN3-RARA fusion gene coding protein region mutation site, and the nucleotide sequence of the STRN3-RARA fusion gene is shown as SEQ ID NO: 1. The S232F mutation site is located at the 974th nucleotide site of the nucleotide sequence, and the base C is mutated into T. According to the present invention, the S232F mutation site can be adopted as the drug resistance detection marker, the reagent or the kit can be designed according to the STRN3-RARA gene S232F mutation site so as to be used for clinical drug resistance detection, and the gene mutation state of the patient can be monitored so as to guide the personalized drug use.

Description

Technical Field

[0001] This invention relates to the field of molecular oncology and drug resistance mechanism research technology, and in particular to an S232F site of the fusion gene STRN3-RARA that induces acquired drug resistance and its application in ATRA. Background Technology

[0002] Acute promyelocytic leukemia (APL) is a special type of acute myeloid leukemia, characterized by the unrestricted proliferation of a large number of leukemia cells in the bone marrow and other hematopoietic tissues, which then enter the peripheral blood, while the production of normal blood cells is significantly suppressed.

[0003] For variant acute promyelocytic leukemia (APL), current ATRA (all-trans retinoic acid) treatment is generally ineffective, often resulting in poor disease control and significantly impacting patients' quality of life. Furthermore, the limited available treatment strategies pose a significant challenge to both clinicians and patients. In clinical practice, treatment options for variant APL encompass multiple approaches. Particularly in patients involving the variant fusion gene STRN3-RARA, treatment includes repeated use of ATRA combined with arsenic. While this combination therapy can inhibit leukemia cell growth to some extent, relapse still occurs within a short period. These drugs are highly targeted, but they also face issues such as drug resistance in practical applications. The underlying mechanisms of drug resistance and the causes of relapse are currently unclear, and numerous medical researchers are conducting investigations.

[0004] Therefore, developing drug resistance testing for acute promyelocytic leukemia is of great significance for the rapid diagnosis and treatment of acute promyelocytic leukemia. Summary of the Invention

[0005] The purpose of this invention is to overcome the problem that there is no detection method for drug resistance in the treatment of acute promyelocytic leukemia in the prior art. It provides an S232F site of acquired resistance to all-trans retinoic acid (ATRA) in the fusion gene STRN3-RARA and its application in ATRA. It is based on the acquired mutation (S232F) of the fusion gene STRN3-RARA in patients with acute promyelocytic leukemia (APL) leading to resistance to all-trans retinoic acid (ATRA) treatment and its detection and application.

[0006] In a first aspect, the present invention provides an application of the S232F mutation site in the preparation of an ATRA resistance detection product, the product being used to detect the sensitivity of acute promyelocytic leukemia patients to ATRA treatment or to screen whether acute promyelocytic leukemia patients are resistant to ATRA treatment.

[0007] The present invention provides the application of the S232F mutation site in the preparation of ATRA resistance detection products, clarifying that the S232F point mutation in STRN3-RARA is the cause of drug resistance in STRN3-RARA patients. This mutation can be used to detect the sensitivity of acute promyelocytic leukemia patients to radiotherapy with ATRA or to screen for resistance to ATRA treatment in acute promyelocytic leukemia patients. The above technical solution is used for clinical drug resistance detection, for purposes other than disease diagnosis and treatment.

[0008] As a preferred embodiment of the present invention, the product is a reagent or kit. The reagent or kit is a carrier of the S232F mutation site as a drug resistance marker, which can be directly used to detect the sensitivity or resistance of acute promyelocytic leukemia patients to ATRA treatment. The reagent or kit can continuously monitor the mutation status of the patient's genes, promptly detect the occurrence or change of the S232F mutation, and provide real-time basis for clinical adjustment of treatment plans.

[0009] As a more preferred embodiment of the present invention, the reagent is at least one of an antibody, probe, primer pair, and sequencing reagent designed for detecting the S232F mutation site or targeting the S232F mutation site. These reagents enable regular monitoring of the mutation status of a patient's genes, timely detection of the occurrence or changes of the S232F mutation, and help doctors quickly determine the patient's drug resistance. The reagents are used for drug resistance detection and personalized medication guidance, and are not for disease diagnosis or treatment purposes.

[0010] As a preferred embodiment of the present invention, the S232F mutation site is a mutation site in the protein region encoded by the STRN3-RARA fusion gene. The nucleotide sequence of the STRN3-RARA fusion gene is shown in SEQ ID NO: 1. The S232F mutation site is located at the 974th nucleotide position of the nucleotide sequence, and is a mutation of base C to T.

[0011] In a preferred embodiment of the present invention, the S232F mutation site has a base of T, corresponding to an ATRA drug-sensitive phenotype, and the S232F mutation site has a base of C, corresponding to an ATRA drug-resistant phenotype. Through the above technical solution, a direct correspondence between base type and drug response phenotype is established, enabling rapid formulation of treatment plans based on test results. In a preferred embodiment of the present invention, the ATRA drug includes a pharmaceutically acceptable carrier or a drug for treating promyelocytic leukemia.

[0012] As a preferred embodiment of the present invention, the ATRA drug includes a drug whose active ingredient is all-trans retinoic acid.

[0013] In a second aspect, the present invention provides an S232F mutation site associated with ATRA resistance, wherein the S232F mutation site is a mutation site in the protein region encoded by the STRN3-RARA fusion gene, the nucleotide sequence of the STRN3-RARA fusion gene is shown in SEQ ID NO: 1, and the S232F mutation site is located at the 974th nucleotide position of the nucleotide sequence, which is a mutation of base C to T.

[0014] In a third aspect, the present invention provides a product comprising a reagent or kit for detecting whether an S232F mutation site has occurred, the reagent or kit being used to detect the sensitivity of acute promyelocytic leukemia patients to ATRA treatment or to screen whether acute promyelocytic leukemia patients are resistant to ATRA treatment.

[0015] The product claimed in this invention may include a reagent or kit for detecting whether the S232F mutation site has occurred, the reagent or kit being used to detect the sensitivity of acute promyelocytic leukemia patients to ATRA treatment or to screen whether acute promyelocytic leukemia patients are resistant to ATRA treatment.

[0016] In a fourth aspect, the present invention provides a method for detecting drug resistance in acute promyelocytic leukemia, using one of the aforementioned products for detection.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides an application of the S232F mutation site in the preparation of ATRA drug resistance detection products, revealing for the first time the relevant function of the S232F mutation site in the detection of drug resistance in acute promyelocytic leukemia. The S232F mutation site can be used as a drug resistance detection biomarker. Reagents or kits can be designed based on the S232F mutation site of the STRN3-RARA gene for clinical drug resistance detection. At the same time, it can monitor the mutation status of patients' genes and guide personalized medication.

[0018] 2. This invention provides a product comprising reagents or kits for detecting the presence of the S232F mutation site. These reagents or kits are used to detect the sensitivity of acute promyelocytic leukemia (APIL) patients to ATRA treatment or to screen for ATRA resistance in APIL patients. This facilitates rapid and accurate diagnosis of APIL, accurately assesses the patient's condition and drug resistance, and provides crucial evidence for selecting more effective individualized treatment strategies. Based on the different drug resistance characteristics of each patient, the most suitable treatment plan can be developed, thereby maximizing treatment effectiveness, improving patient quality of life and treatment outcomes, and bringing more hope for survival.

[0019] 3. In this invention, the S232F mutation site was identified in the STRN3-RARA fusion transcript through RNA-seq analysis of bone marrow from ATRA relapse patients. The S232F mutation was introduced into the STRN3-RARA cDNA using site-directed mutagenesis PCR technology, and a stable cell line expressing the mutation was established. The expression of differentiation markers (CD11b) and cell survival rate of wild-type and mutant cells under ATRA treatment were compared, clarifying that the S232F point mutation in STRN3-RARA is the cause of drug resistance in STRN3-RARA patients. Attached Figure Description

[0020] Figure 1 A schematic diagram of the S232F site of acquired drug resistance in STRN3-RARA patients with fusion genes; Figure 2 A schematic diagram of the structure of the fusion gene in a STRN3-RARA patient; Figure 3 A schematic diagram of the overexpression plasmid of the STRN3-RARA S232F mutation (A), and a comparison diagram of the first-generation sequencing results of the mutant plasmid (B). Figure 4 Fluorescence expression of the differentiation marker (CD11b) in response to ATRA treatment in a stable expression vector constructed for ATRA-treated U937 cell lines, wild-type STRN3-RARA, and STRN3-RARA S232F mutant cell models. Figure 5 Cell survival curves of ATRA treatment response in U937 cell lines with stable expression vectors, wild-type STRN3-RARA, and STRN3-RARA S232F mutant cell models. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0022] Example 1: Identification of the S232F mutation site in acquired drug resistance of STRN3-RARA fusion gene in patients. Clinical data: A 50-year-old male was admitted to the hospital due to leukopenia and coagulation dysfunction. 91.5% of the cells in his bone marrow (BM) were hypergranular promyelocytic cells. Based on clinicopathological features including immunophenotype, the patient was diagnosed with acute promyelocytic leukemia (APL). Initially, the patient achieved complete remission with standard ATRA and arsenic trioxide combination therapy, followed by consolidation therapy with anthracyclines and maintenance therapy with ATRA and arsenic trioxide. However, after 10 months, he rapidly relapsed and developed resistance to ATRA and ATO therapy. The patient subsequently received combination therapy with ATRA and venetoclax, but unfortunately, there was no response. This patient was clinically diagnosed with STRN3-RARA fusion gene-positive acute promyelocytic leukemia.

[0023] Informed patients and their families of the research objective and their informed consent were obtained. Bone marrow samples were collected from patients before and after ATRA treatment for relapse. Samples were collected at baseline before standard ATRA treatment and at the relapse stage after treatment. 5-10 mL of bone marrow fluid was obtained under aseptic conditions via bone marrow aspiration, stored in EDTA anticoagulant tubes, and transported via cold chain at 4°C. Within 2 hours of sample arrival at the laboratory, total RNA was extracted using TRIzol reagent according to standard operating procedures, and RNA concentration and integrity were detected using Nanodrop 2000. A sequencing library was then constructed using the Illumina TruSeq StrandedTotal RNA Library Prep Kit. After library quality control, 150 bp paired-end whole transcriptome sequencing was performed on the Illumina NovaSeq 6000 platform, with a sequencing depth ≥6G clean data. Raw data underwent FastQC quality control and was aligned to the human reference genome GRCh38 using STAR software. Gene expression quantification was performed using HTSeq-count, with a focus on analyzing the expression level and potential mutation sites of the STRN3-RARA fusion gene transcript.

[0024] Using a mutation detection protocol, a C→T mutation was found at position 38508647 on chromosome 17, corresponding to amino acid S232F. The mutation was confirmed by IGV to occur in the fusion transcript, with a mutation frequency of approximately 28%.

[0025] In this study, a mutation detection procedure was used to conduct meticulous analysis of the target sample. After a series of complex and precise steps, a C→T mutation was successfully discovered at a specific site, Chr17:38508647. This corresponds to the replacement of the original serine (S) at position 232 of the amino acid sequence with phenylalanine (F), which manifests as the specific amino acid change S232F, and is the S232F mutation site.

[0026] To ensure the accuracy and reliability of this mutation discovery, the IGV tool was used for detailed observation and analysis of the fusion transcripts, clearly demonstrating the presence of the mutation. Subsequently, a scientifically sound quantitative analysis method was employed to accurately estimate the frequency of this mutation in the samples. After multiple rigorous calculations and verifications, the final frequency of the mutation was determined to be approximately 28%. This mutation frequency indicates a certain degree of significance in the samples, providing crucial and valuable evidence for subsequent in-depth functional studies and the exploration of related biological mechanisms.

[0027] like Figure 1 The diagram shows the S232F mutation site of the fusion gene in patients with STRN3-RARA. As shown above, the S232F mutation site is located at a key site in RARA, which may change the conformation of the fusion protein and its ability to bind to RXR. The mutation site was not detected before treatment, indicating that it is an acquired mutation.

[0028] Example 2: Construction of an overexpression plasmid with the STRN3-RARA S232F mutation STRN3-RARA is an acute promyelocytic leukemia (APL)-associated fusion gene formed by the fusion of the STRN3 and RARA genes. The STRN3-RARA fusion gene is formed by fusing a portion of the STRN3 (stretch protein 3) gene containing the coiled-coil dimerization domain with almost the entire RARA (retinoic acid receptor α) gene.

[0029] Using wild-type STRN3-RARA fusion cDNA as the initial template, the S232F mutation site was introduced via site-directed mutagenesis. The mutant fragment was amplified by polymerase chain reaction (PCR), and the PCR product was then double-digested with restriction endonucleases XhoI and EcoRI. The digested products were purified and ligated into a vector digested with the same enzymes, transformed into DH5α cells, and positive clones were preliminarily identified by antibiotic selection and colony PCR. Positive clones were selected for amplification culture, and plasmids were extracted. The mutation site was verified by Sanger sequencing to ensure sequence accuracy. The verified mutant plasmid was inserted into the pMSCV-IRES-GFP eukaryotic expression vector using subcloning technology, followed by retroviral packaging using calcium phosphate co-precipitation. Viral supernatant was collected and used to infect the human myeloid leukemia U937 cell line. Green fluorescent protein (GFP) positive cells were sorted by flow cytometry to establish a cell line stably expressing the mutant protein.

[0030] The nucleotide sequence of the STRN3-RARA fusion gene is as follows: The nucleotide sequence of the STRN3-RARA S232F mutation is as follows: like Figure 2 , Figure 3 As shown, sequencing results revealed a specific mutation site in the genome, specifically S232F. This mutation resulted in a change in the corresponding encoded amino acid, with the original serine (Ser) being replaced by phenylalanine (Phe). Comprehensive testing revealed no other additional mutation sites in the sample (see [link to sample details]). Figure 3 B).

[0031] Example 3: Response analysis of STRN3-RARA S232F mutant cells to ATRA treatment The specific operating procedure of the experimental method is as follows: (1) Cell differentiation detection: First, stable expression vectors (pMIG), wild-type STRN3-RARA (SR), and STRN3-RARA S232F mutant cell models (Mut-SR) were constructed using the U937 cell line. The stable expression vector constructed using the U937 cell line served as an empty vector control group. Cells in each group were treated with different concentration gradients of ATRA (0 nM, 2 nM, 5 nM, 10 nM, 100 nM, and 1000 nM) and cultured for 72 hours. Finally, the cells were stained with FITC-labeled anti-CD11b antibody by flow cytometry to detect and calculate the proportion of cell differentiation, in order to assess the changes in the degree of differentiation under different treatment conditions.

[0032] (2) Cell survival analysis: After ATRA treatment for the corresponding time, cell viability was detected by relative counting in flow cytometry. By comparing the changes in cell number between the treatment group and the control group, the relative cell survival rate was calculated, thereby assessing the impact of different experimental conditions on cell viability and its potential mechanisms.

[0033] Cell differentiation results as follows Figure 4 As shown, after 72 hours of ATRA treatment, the CD11b positive expression rate in wild-type cells significantly increased, reaching approximately 65%. This data clearly indicates that wild-type cells underwent significant differentiation under the influence of the drug, with cell morphology and function gradually shifting towards a specific differentiation direction. For the STRN3-RARAS232F mutant cell model, the CD11b positive rate was extremely low, approximately 1%. To ensure the reliability of the results, the empty vector group served as a control, and the results for the mutant cells were largely consistent with those of the empty vector group. This phenomenon clearly suggests that the S232F mutation severely interferes with the cell differentiation process, preventing cells from responding normally to ATRA stimulation and significantly hindering the differentiation process.

[0034] Cell survival results as follows Figure 5As shown, after 72 hours of ATRA treatment, the survival rate of both the empty vector group and wild-type cells showed a significant decreasing trend with increasing ATRA concentration. This result indicates that the empty vector group and wild-type cells exhibit concentration-dependent sensitivity to ATRA; higher concentrations of ATRA exert significant stress on cell survival, inhibiting cell growth and even leading to cell death. This may be because ATRA acts on certain intracellular signaling pathways, affecting normal cellular metabolism and survival mechanisms. However, the S232F mutant cells exhibited a completely different response from the empty vector group and wild-type cells. The survival rate of S232F mutant cells did not change significantly under different ATRA concentrations. This phenomenon suggests that the S232F mutation may confer ATRA resistance, enabling cells to resist the toxic effects of ATRA to a certain extent and maintain their own survival. This result further confirms that the S232F mutation has an important impact on cell survival behavior, possibly involving a series of complex intracellular signal transduction and gene expression regulation mechanisms.

[0035] The results showed that the S232F mutation weakened ATRA-induced differentiation and conferred drug resistance to cells.

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An application of the S232F mutation site in the preparation of ATRA resistance detection products, characterized in that, The product is used to detect the sensitivity of acute promyelocytic leukemia patients to ATRA treatment or to screen for resistance to ATRA treatment in acute promyelocytic leukemia patients.

2. The application of the S232F mutation site according to claim 1 in the preparation of ATRA resistance detection products, characterized in that, The product is a reagent or kit.

3. The application of the S232F mutation site according to claim 2 in the preparation of ATRA resistance detection products, characterized in that, The reagent is at least one of the following: antibodies, probes, primer pairs, and sequencing reagents designed for detecting the S232F mutation site or targeting the S232F mutation site.

4. The application of the S232F mutation site according to claim 1 in the preparation of ATRA resistance detection products, characterized in that, The S232F mutation site is a mutation site in the protein region encoded by the STRN3-RARA fusion gene. The nucleotide sequence of the STRN3-RARA fusion gene is shown in SEQ ID NO:

1. The S232F mutation site is located at the 974th nucleotide position of the nucleotide sequence, and it is a mutation of the base C to T.

5. The application of the S232F mutation site according to claim 4 in the preparation of ATRA resistance detection products, characterized in that, The S232F mutation site has a base of T, which corresponds to the ATRA drug-sensitive phenotype, and the S232F mutation site has a base of C, which corresponds to the ATRA drug-resistant phenotype.

6. The application of the S232F mutation site according to claim 1 in the preparation of ATRA resistance detection products, characterized in that, ATRA drugs include pharmaceutically acceptable carriers or drugs used to treat promyelocytic leukemia.

7. The application of the S232F mutation site according to claim 6 in the preparation of ATRA resistance detection products, characterized in that, ATRA drugs include those whose active ingredient is all-trans retinoic acid.

8. The S232F mutation site associated with ATRA resistance, characterized in that, The S232F mutation site is a mutation site in the protein region encoded by the STRN3-RARA fusion gene. The nucleotide sequence of the STRN3-RARA fusion gene is shown in SEQ ID NO:

1. The S232F mutation site is located at the 974th nucleotide position of the nucleotide sequence, and it is a mutation of the base C to T.

9. A product characterized in that, The product includes reagents or kits for detecting the presence of the S232F mutation site, which are used to detect the sensitivity of acute promyelocytic leukemia patients to ATRA treatment or to screen for resistance to ATRA treatment in acute promyelocytic leukemia patients.

10. A method for detecting drug resistance in acute promyelocytic leukemia, characterized in that, The product described in claim 9 is used for testing.

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