NOL4L:: ASXL1 fusion gene and application thereof in leukemia

Abstract

The invention discloses a NOL4L:: ASXL1 fusion gene and an application of the NOL4L:: ASXL1 fusion gene in leukemia. The invention relates to application in preparation of leukemia diagnosis and treatment detection reagents and drugs. According to the research, a novel fusion gene NOL4L:: ASXL1 composed of an N-terminal structural domain of NOL4L and a C-terminal structural domain of ASXL1 is identified in a leukemia patient for the first time. It is found for the first time that NOL4L:: ASXL1 has an independent carcinogenic characteristic for driving leukemia, and is significantly related to chemotherapeutic drug resistance phenotypes and poor prognosis of leukemia patients. According to the invention, the NOL4L:: ASXL1 fusion gene and fusion protein are used as a biomarker for leukemia molecular typing diagnosis, an independent predictive factor for prognosis evaluation and a potential target for precise treatment. The discovery provides an important molecular biological basis for establishing a leukemia diagnosis system based on fusion gene and protein detection thereof and developing a targeted therapy strategy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection, and specifically relates to a NOL4L::ASXL1 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 type of malignant clonal disease originating from hematopoietic stem cells, characterized by abnormal proliferation and apoptosis of myeloid or lymphoid cells in a state of differentiation arrest, and extensive infiltration in the bone marrow, peripheral blood and extramedullary tissues.

[0003] Fusion genes generated by chromosomal translocations are key genetic events that directly drive the development of leukemia. Classic fusion genes such as KMT2A::AF4 and BCR::ABL1 have been shown to directly drive the occurrence of leukemia and are closely associated with low response to clinical chemotherapy. They have been included in clinical guidelines for risk stratification. However, in clinical practice, there are still a large number of leukemia patients with low response to chemotherapy who cannot detect fusion proteins known to be clearly associated with leukemia. There may be other driving events that have not yet been discovered. Therefore, unknown fusion proteins may be an important entry point for studying low response to leukemia chemotherapy, which has positive significance for optimizing leukemia molecular typing, improving prognostic stratification systems and targeted treatment strategies. Based on high-throughput sequencing technology, the present invention team discovered a NOL4L::ASXL1 fusion gene and its protein, which can drive leukemia occurrence and drug resistance. This fusion gene is the first to be discovered and has not been reported either domestically or internationally.

[0004] Although high-throughput sequencing technology has demonstrated high sensitivity and multidimensional analytical capabilities in leukemia genomics research, its clinical translation still faces multiple challenges: high testing costs and long experimental cycles have limited its widespread application as a routine diagnostic tool in clinical practice. Common rapid detection methods for fusion genes include fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR). Currently, no detection kits are available for the NOL4L::ASXL1 fusion gene. Therefore, the development of rapid and universally available detection kits based on the newly discovered oncogenic fusion genes will provide more possibilities for the diagnosis and treatment of leukemia. Summary of the Invention

[0005] The present invention aims to provide a NOL4L::ASXL1 fusion gene, which is formed by fusing exon 4 of the NOL4L gene and exons 2-13 of the ASXL1 gene, wherein the DNA sequences thereof are shown in SEQ ID NO.1 and the corresponding encoded amino acid sequences are shown in SEQ ID NO.2.

[0006] The present invention provides specific PCR primers for detecting the NOL4L::ASXL1 fusion gene: upstream primer NOL4L::ASXL1-F: 5'-CCGGTTCCTGATGAGCTGTAC-3' (SEQ ID NO: 3); downstream primer NOL4L::ASXL1-R: 5'-AAGCTCTGTAGCTGTCCCTGG-3' (SEQ ID NO: 4).

[0007] Another object of the present invention is to provide the use of the NOL4L::ASXL1 fusion gene and its protein in the preparation of diagnostic and therapeutic detection reagents for NOL4L::ASXL1 fusion gene-positive leukemias, including detection reagents for leukemia diagnosis and typing, prognosis assessment, and personalized treatment. The detection reagents comprise a PCR primer combination that specifically detects the NOL4L-ASXL1 fusion gene, including but not limited to SEQ ID NO: 3 and SEQ ID NO: 4.

[0008] The present invention conducted a prognostic analysis of a self-constructed cohort of leukemia clinical samples. The results showed that the proportion of patients with intermediate and high risk, as well as the risk of poor prognosis, was significantly increased in patients carrying the NOL4L::ASXL1 fusion gene. Analysis of pediatric tumor samples from public databases also confirmed that the NOL4L::ASXL1 fusion does exist and that patients with this fusion have a significantly worse prognosis compared to patients with other fusions. This demonstrates the clinical value of the NOL4L::ASXL1 fusion gene and its protein as independent markers of poor prognosis in leukemia.

[0009] The present invention further tested the conventional leukemia treatment regimens VDLD (vincristine, daunorubicin, asparaginase, dexamethasone) and DA (daunorubicin, cytarabine) in BaF3-transformed tumor cells. The results showed that NOL4L-ASXL1-driven leukemia cells were resistant to commonly used clinical chemotherapy regimens. This suggests that the NOL4L-ASXL1 fusion gene and its protein can serve as independent prognostic biomarkers for assessing adverse clinical outcomes in leukemia patients.

[0010] Another object of the present invention is to provide the use of the NOL4L::ASXL1 fusion gene and its protein in the preparation of a drug for treating NOL4L::ASXL1 fusion gene-positive leukemia. The drug includes a drug targeting the NOL4L::ASXL1 fusion gene and its protein.

[0011] This study systematically evaluated the independent leukemia-driving function of NOL4L::ASXL1 based on a normal BaF3 cell transformation model. The results demonstrated that overexpression of NOL4L::ASXL1 significantly promoted cell proliferation and clonogenicity, and induced the formation of xenograft tumors in the axilla of nude mice in xenograft experiments. These experimental evidence confirms for the first time that NOL4L::ASXL1 possesses independent oncogenic function, potentially serving as a novel molecular biomarker. Furthermore, the NOL4L::ASXL1 fusion gene and protein may serve as therapeutic targets for patients with this type of fusion leukemia.

[0012] The present invention has the following beneficial effects: This study identifies and validates, for the first time, the novel leukemia-driving fusion gene NOL4L::ASXL1, which contributes to drug resistance in leukemia patients to clinical chemotherapy drugs. The present invention provides markers for leukemia diagnosis, typing, and prognosis prediction using the NOL4L::ASXL1 fusion gene and fusion protein, as well as targets for clinical drug therapy. Furthermore, the present invention designs specific PCR primers and detection kits for this fusion gene, which can be widely used in clinical practice to improve the detection rate and accuracy of the NOL4L::ASXL1 fusion gene. This invention opens the possibility of precise treatment of leukemia and improved prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the structure of the NOL4L::ASXL1 fusion gene and the Sanger sequencing results of the sequencing verification of the NOL4L::ASXL1 fusion protein using primary leukemia samples.

[0014] Figure 2 To investigate the correlation between NOL4L::ASXL1 fusion gene and non-NOL4L::ASXL1 fusion gene and poor prognosis in leukemia patients.

[0015] Figure 3 The proliferation count results of BaF3 cells overexpressing control plasmid and NOL4L::ASXL1 using the lentiviral system.

[0016] Figure 4 Soft agar colony formation results of BaF3 cells overexpressing control plasmid and NOL4L::ASXL1 using the lentiviral system.

[0017] Figure 5 The figure shows the tumor formation of the nude mouse axillary xenograft tumor model using BaF3 cells overexpressing a control plasmid and NOL4L::ASXL1.

[0018] Figure 6The proliferation count results of BaF3 cells overexpressing control plasmid and NOL4L::ASXL1 after being given clinically commonly used chemotherapy regimens. DETAILED DESCRIPTION

[0019] 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.

[0020] Example 1: See also Figure 1 Using high-throughput sequencing technology, a case of refractory and drug-resistant ALL was found in the ZJUALL cohort, in which the NOL4L::ASXL1 fusion gene was detected. It was found that the gene was formed by the fusion of NOL4L gene exon 4 and ASXL1 gene exons 2-13. Its nucleotide sequence is shown in SEQ ID NO: 1 (as shown in Figure 1 A). Based on the gene sequence, specific primers for the NOL4L::ASXL1 fusion gene were designed: upstream primer NOL4L::ASXL1-F: 5'-CCGGTTCCTGATGAGCTGTAC-3', and downstream primer NOL4L::ASXL1-R: 5'-AAGCTCTGTAGCT GTCCCTGG-3'. PCR was performed on this basis and the product was verified by Sanger sequencing. It was found that the fragmentation pattern was exactly the same as that of high-throughput sequencing (e.g. Figure 1 B).

[0021] Example 2: See also Figure 2 By performing a prognostic analysis on the ZJUALL cohort, the intermediate- and high-risk ratios and the risk of adverse prognosis (e.g., Figure 2 A). By analyzing the original sequencing data of pediatric tumor samples in a public database (TARGET database), the results showed that NOL4L-ASXL1 fusion does exist and that patients with this fusion have a very poor prognosis compared with patients with other fusions (e.g. Figure 2 B).

[0022] Example 3: See also Figure 3, construct pccl control plasmid and NOL4L::ASXL1 overexpression plasmid, overexpress them in 293T tool cells by lipofectamine transfection technology, and detect the expression of HA tag NOL4L::ASXL1 fusion protein by Western blot technology 48 hours later (as shown in Figure 2). Figure 3 A) Using a lentiviral system to overexpress a control plasmid and NOL4L::ASXL1, normal BaF3 cells were infected at an MOI of 10:1. On the third day of infection, fluorescence was observed under a microscope and the infection rate was found to be 100%. 5 The cells were seeded into six-well plates and cell proliferation was counted from D1 to D9. The counting results showed that NOL4L::ASXL1 could significantly promote the proliferation of BaF3 cells (e.g. Figure 3 B).

[0023] Example 4: A lentiviral system was used to overexpress a control plasmid and NOL4L::ASXL1, and normal BaF3 cells were infected at an MOI of 10:1. 250 cells were seeded into a 6-well plate containing a mixture of low-melting-point agarose and culture medium. After 7 days of culture, the number of cell colonies was observed. The BaF3 cell colonies in soft agar were stained with nitroblue tetrazolium (NBT) and photographed to calculate the colony formation rate. The results showed that NOL4L::ASXL1 could significantly promote the colony formation ability of BaF3 cells (such as Figure 4 AB).

[0024] Embodiment 5: See also Figure 5 , a lentiviral system was used to overexpress a control plasmid and NOL4L::ASXL1, and normal BaF3 cells were infected at an MOI of 10:1. The cells were inoculated into the armpits of nude mice at a volume of 2 million cells / 0.2 mL, with 6 mice in each group. On the 70th day after inoculation, a transplanted tumor was observed in the armpit of a nude mouse inoculated with NOL4L::ASXL1 group cells. After 104 days of inoculation, all mice were killed and the tumor formation rate curve was drawn. The results showed that NOL4L::ASXL1 can significantly promote the tumor formation ability of BaF3 cells in nude mice in vivo (such as Figure 5 AB).

[0025] Example 6: The lentiviral system was used to overexpress a control plasmid and NOL4L::ASXL1, and the conventional clinical chemotherapy regimens VDLD and DA were given. Cell proliferation was counted on D3. The results showed that the leukemia cells driven by NOL4L::ASXL1 were resistant to the commonly used clinical anti-tumor chemotherapy drugs (such as Figure 6 ).

[0026] 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. While the above examples utilize PCR technology to verify the presence of the NOL4L::ASXL1 fusion gene, 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 the present invention.

Claims

1. A NOL4L::ASXL1 fusion gene, characterized in that The fusion gene is formed by fusing exon 4 of the NOL4L gene and exons 2-13 of the ASXL1 gene. The DNA sequence of the gene is shown in SEQ ID NO.1, and the corresponding encoded amino acid sequence is shown in SEQ ID NO.

2.

2. Use of the NOL4L::ASXL1 fusion gene and its protein according to claim 1 as a marker in the preparation of a leukemia diagnosis and treatment detection reagent.

3. The use according to claim 2, characterized in that The detection reagent is a PCR primer combination for detecting the NOL4L::ASXL1 fusion gene, the upstream primer sequence of which is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.

4.

4. Use of the NOL4L::ASXL1 fusion gene and protein thereof according to claim 1 in the preparation of a drug for treating leukemia.

5. The use according to claim 4, characterized in that The NOL4L::ASXL1 fusion gene and its protein serve as targets for clinical drug therapy.

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