RUNX1:: QSER1 fusion gene and application thereof in leukemia

Abstract

The invention provides an RUNX1:: QSER1 fusion gene and an application thereof in leukemia, and mainly relates to an application in preparation of RUNX1:: QSER1 fusion gene positive leukemia diagnosis and treatment products. The fusion gene is formed by fusing exons 1-6 of the RUNX1 gene and exons 2-5 of the QSER1 gene. According to the fusion gene, abnormal high expression of the QSER1 gene is caused by a promoter replacement mechanism, and overexpression of RUNX1: QSER1 significantly enhances the proliferation ability and clone formation ability of cells, and promotes leukemia cell proliferation and chemotherapy resistance. The invention provides a RUNX1:: QSER1 fusion gene and fusion protein as markers for leukemia diagnosis and typing, prognosis evaluation and individualized treatment, and as a target for clinical drug treatment, the RUNX1:: QSER1 fusion gene and fusion protein are used as markers for leukemia diagnosis and typing, prognosis evaluation and individualized treatment. New possibility is provided for precise diagnosis and treatment of leukemia, and the kit can be widely applied to clinical prognosis evaluation and individualized treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection, and specifically relates to a RUNX1::QSER1 fusion gene and its application in leukemia, mainly in the preparation of leukemia diagnosis and treatment detection reagents and drugs. Background Art

[0002] Leukemia is a malignant clonal disease of the hematopoietic system, characterized by the obstruction of proliferation and differentiation of abnormal hematopoietic cells in the bone marrow, leading to impaired normal hematopoietic function. Based on the course of the disease and cell type, leukemia can be divided into acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), etc. AML is a highly heterogeneous disease whose pathogenesis involves multiple genetic abnormalities, including chromosomal translocations, gene mutations, and the formation of fusion genes.

[0003] Fusion genes are formed by connecting partial sequences of two different genes through structural variations such as chromosomal translocation, deletion, or inversion. The fusion proteins encoded by fusion genes play an important role in the occurrence and development of leukemia, but the functions of a large number of fusion genes in leukemia are currently unknown, and their clinical significance is still unclear. Among them, the RUNX1 gene, which is highly active in myeloid cells, can form fusion genes with a variety of partner genes, but the prognosis of different fusion genes varies significantly. RUNX1::RUNX1T1 is generally associated with low risk, while RUNX1::MECOM indicates high risk. However, the clinical value of the remaining majority of RUNX1 fusion genes is unknown, which seriously limits the precise diagnosis and treatment of clinical patients.

[0004] Currently, the main methods for detecting fusion genes include fluorescence in situ hybridization (FISH), multiplex nested PCR, and next-generation sequencing (NGS). Among them, NGS technology has the advantages of high throughput and high sensitivity, and can detect unknown fusion genes, but it is relatively expensive and the data analysis is complex. In contrast, RT-PCR or fluorescence quantitative PCR (qPCR) detection methods designed based on known fusion genes are low-cost, simple to operate, and suitable for clinical promotion. Summary of the Invention

[0005] The present invention aims to provide a novel RUNX1::QSER1 fusion gene. The fusion gene is formed by fusing exons 1-6 of the RUNX1 gene with exons 2-5 of the QSER1 gene, the DNA sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of the encoded fusion protein is shown in SEQ ID NO.2.

[0006] The present invention provides specific PCR primers for detecting the RUNX1::QSER1 fusion gene: upstream primer RUNX1::QSER1-F: 5'-ATGGCTTCAGACAGCATATTTG-3' (SEQ ID NO: 3); downstream primer RUNX1::QSER1-R: 5'-CTACAAGAGGTGGTGAGGCT-3' (SEQ ID NO: 4).

[0007] Another object of the present invention is to provide the use of the RUNX1::QSER1 fusion gene and its protein in the preparation of a diagnostic and therapeutic test reagent for RUNX1::QSER1 fusion gene-positive leukemia, including a test reagent for leukemia diagnosis and typing, prognosis assessment, and personalized treatment. The test reagent comprises a PCR primer combination for specific detection of the RUNX1::QSER1 fusion gene, including but not limited to SEQ ID NO: 3 and SEQ ID NO: 4.

[0008] The present invention, through analysis of clinical samples, showed that RUNX1::QSER1-positive patients exhibited higher MRD levels and experienced post-transplant relapse. Furthermore, it was discovered that the RUNX1::QSER1 fusion gene in leukemia leads to abnormally high expression of the QSER1 gene through a "promoter replacement" mechanism, promoting leukemia cell proliferation and chemotherapy resistance.

[0009] Another object of the present invention is to provide the use of the RUNX1::QSER1 fusion gene and its protein in the preparation of a drug for treating RUNX1::QSER1 fusion gene-positive leukemia. The drug includes a drug targeting the RUNX1::QSER1 fusion gene and its protein.

[0010] This study validated the independent leukemia-driving function of RUNX1::QSER1 using a 32D cell transformation model. Experimental results showed that overexpression of RUNX1::QSER1 significantly enhanced cell proliferation and colony formation. Therefore, drugs targeting the RUNX1::QSER1 fusion gene and protein could be used to treat patients with this type of leukemia.

[0011] The present invention has the following beneficial effects: This study identifies and validates the novel leukemia-driving fusion gene, RUNX1::QSER1, for the first time, revealing its pathogenic mechanism, which involves promoter replacement leading to abnormally high QSER1 expression. Importantly, this fusion gene causes leukemia patients to develop resistance to clinical chemotherapy drugs, leading to relapse and refractory leukemia. Detection methods developed for this fusion gene can be widely used in clinical prognosis assessment and personalized treatment, and drugs developed based on this fusion gene and protein can be used in clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a schematic diagram of the structure of the RUNX1::QSER1 fusion gene and the results of Sanger sequencing verification.

[0013] Figure 2 Figure 1 is an analysis of the expression characteristics of the RUNX1::QSER1 fusion gene, including A, a disease progression diagram of a RUNX1::QSER1 fusion-positive patient, and B, a change in FFPM expression of RUNX1::QSER1 in primary and relapse samples.

[0014] Figure 3 This is an analysis of the impact of high expression of QSER1 on prognosis, including A is the expression level of the QSER1 gene in the ZJUpAML cohort, among which RUNX1::QSER1-positive patients have the highest expression, and B is the survival curve of patients with high and low expression of the wild-type QSER1 gene analyzed by TCGA pan-cancer data.

[0015] Figure 4 This is the in vitro functional verification of the RUNX1::QSER1 fusion gene, including A, the Western blot verification result of lentivirus overexpressing RUNX1::QSER1, and B, the proliferation curve of 32D cells overexpressing RUNX1::QSER1.

[0016] Figure 5 This is a validation of the transformation ability of the RUNX1::QSER1 fusion gene, and is the colony formation result of 32D cells overexpressing RUNX1::QSER1 via lentivirus in soft agar. DETAILED DESCRIPTION

[0017] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below with reference to the accompanying drawings and embodiments. It is apparent that the embodiments described are only some of the embodiments of the present invention, rather than all of them. All other implementations obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative effort are intended to fall within the scope of protection of the present invention.

[0018] Example 1: See also Figure 1Using high-throughput sequencing, a RUNX1::QSER1 fusion gene was detected in a relapsed or refractory AML case in the ZJUpAML cohort. The gene was found to be a fusion of exon 6 of the RUNX1 gene and exon 2 of the QSER1 gene. Based on this gene sequence, specific primers for the RUNX1::QSER1 fusion gene were designed: upstream primer RUNX1::QSER1-F: 5'-ATGGCTTCAGACAGCATATTTG-3', and downstream primer RUNX1::QSER1-R: 5'-CTACAAGAGGTGGTGAGGCT-3'. PCR was performed based on this, and the product was verified by Sanger sequencing, revealing that the fragmentation pattern was identical to that found in high-throughput sequencing.

[0019] Example 2: See also Figure 2 The RUNX1::QSER1 fusion-positive patient failed to completely eliminate the proportion of primitive cells in the bone marrow after medication in the initial stage. The proportion of tumor cells was reduced to 0% after combining intensive chemotherapy and bone marrow transplantation. However, relapse occurred 16 months after transplantation (such as Figure 2 A). Further analysis revealed that the FFPM value of this fusion gene in relapse samples was significantly higher than that in primary samples (0.68 vs 3.85) (e.g. Figure 2 B). These results suggest that RUNX1::QSER1 fusion is highly likely to be an important driver of relapse in patients.

[0020] Example 3: Among 149 AML patients in the ZJUpAML cohort, RUNX1::QSER1-positive patients had the highest QSER1 expression level among all patients ( Figure 3 A). Considering that the wild-type RUNX1 gene is strongly activated in myeloid blood cells, we proposed that RUNX1::QSER1 drives the abnormally high expression of QSER1 by utilizing the strong promoter of RUNX1. Further analysis of the TCGA pan-cancer database showed that patients with high expression of wild-type QSER1 had a significantly poor prognosis (e.g. Figure 3 B). Our results reveal that RUNX1::QSER1 leads to poor prognosis in cancer patients by significantly upregulating QSER1 expression.

[0021] Example 4: See also Figure 4 , construct PCCL control plasmid and RUNX1::QSER1 overexpression plasmid, use lentiviral packaging system to package them in 293T tool cells, collect viral supernatant after 48 hours to infect 32D cells. Western blot technology was used to detect the expression of HA tag RUNX1::QSER1 fusion protein (such as Figure 4 A) Normal 32D cells were infected at an MOI of 10:1. On the third day of infection, fluorescence observation under a microscope revealed that the infection rate reached 100%. 150,000 cells were seeded into six-well plates, and cell proliferation was counted from D1 to D9. The count results showed that RUNX1::QSER1 significantly promoted the proliferation rate of 32D cells (e.g., Figure 4 B).

[0022] Embodiment 5: See also Figure 5 , a lentiviral system was used to overexpress a control plasmid and RUNX1::QSER1, and normal 32D cells were infected at an MOI of 10:1. 1000 cells were seeded into a 6-well plate containing a mixture of 0.3% low-melting point agarose and complete medium and cultured in a 37°C, 5% CO2 incubator for 9 days. The 32D cell colonies in soft agar were stained with nitroblue tetrazolium (NBT), photographed, and cell colonies were counted. The results showed that RUNX1::QSER1 could significantly promote the cloning ability of 32D cells (such as Figure 5 ).

[0023] It is worth noting that the above examples are only partial examples of the present invention and should not be construed as limiting the present invention. The above examples utilize PCR technology to verify the presence of the RUNX1::QSER1 fusion gene. On this basis, other methods for detecting fusion genes and fusion proteins, such as fluorescence in situ hybridization, immunohistochemistry, and Western blot, also fall within the scope of protection of the present invention.

Claims

1. A RUNX1::QSER1 fusion gene, characterized in that The fusion gene is formed by fusing exons 1-6 of the RUNX1 gene with exons 2-5 of the QSER1 gene. Its DNA sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded fusion protein is shown in SEQ ID NO.

2.

2. Use of the RUNX1::QSER1 fusion gene and its protein according to claim 1 in the preparation of a diagnostic and therapeutic detection reagent for RUNX1::QSER1 fusion gene-positive leukemia.

3. The use according to claim 2, characterized in that The detection reagent comprises a PCR primer combination for specifically detecting the RUNX1::QSER1 fusion gene, including but not limited to SEQ ID NO: 3 and SEQ ID NO:

4.

4. Use of the RUNX1::QSER1 fusion gene and its protein according to claim 1 in the preparation of a drug for treating RUNX1::QSER1 fusion gene-positive leukemia.

5. The use according to claim 4, characterized in that RUNX1::QSER1 fusion gene and its protein serve as targets for clinical drug therapy.

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