Application of IGF2BP3 serving as target spot in preparation of fused genotype leukemia treatment medicine

Abstract

The invention provides application of IGF2BP3 serving as a target spot in preparation of a medicine for treating fused genotype leukemia, and particularly relates to application of IGF2BP3 serving as a target spot in preparation of a medicine for treating mRNA (messenger ribonucleic acid) high-stability fused genotype leukemia. The mRNA high-stability fusion gene comprises but is not limited to fusion genes such as PAX5-ETV6, STK38-PXT1 and the like. The medicine comprises an IGF2BP3 gene specific interfering RNA (Ribonucleic Acid) and an IGF2BP3 inhibitor, and the IGF2BP3 inhibitor comprises but is not limited to Vitonetog. The invention provides the application of the medicine taking the IGF2BP3 as the target spot in treating mRNA high-stability fused genotype leukemia for the first time; the invention discloses the effect of the IGF2BP3 in the carcinogenic function of the fusion gene, provides a new therapeutic target and an effective drug for the fusion genotype leukemia, and provides possibility for further improving the clinical curative effect of leukemia patients and improving the prognosis and survival of the leukemia patients.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and relates to the application of IGF2BP3 as a target in the preparation of mRNA highly stable fusion genotype leukemia therapeutic drugs. Background Art

[0002] Leukemia is a group of heterogeneous clonal diseases originating from hematopoietic stem cells or early myeloid progenitor cells. Its main feature is the malignant clonal proliferation of hematopoietic stem cells or progenitor cells in the bone marrow, which leads to the accumulation of a large number of abnormal white blood cells in the body, thereby causing a series of clinical symptoms such as anemia, bleeding, and infection. Acute leukemia develops very rapidly. If not treated in time, the patient will die within a few weeks or months. At present, the treatment for leukemia is mainly a comprehensive therapy based on chemotherapy. Bone marrow transplantation is the most effective treatment for middle and late stage leukemia, but it has the disadvantages of being expensive and having few bone marrow sources. Therefore, the treatment of leukemia is still a major problem and key research direction in the current medical field, and it is urgent to find new treatment strategies for leukemia.

[0003] Fusion genes play a key role in the occurrence and development of leukemia. For example, more than 50% of B-cell acute lymphoblastic leukemia (B-ALL) patients carry fusion genes including ETV6-RUNX1, PAX5-ETV6, etc., which directly drive leukemia. Therefore, fusion genes are an important indicator for the current clinical leukemia classification. However, there is a relative lack of clinical targeted treatment strategies for these fusion genes. Therefore, analyzing the carcinogenic mechanism of fusion genes and developing corresponding targeted treatment strategies will help improve the treatment effect of leukemia and the survival rate of patients.

[0004] N6-methyladenosine (m6A) modification is the most abundant post-transcriptional modification form on eukaryotic mRNA. Dysregulation of m6A modification regulatory enzymes such as insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) plays an important role in tumors. Literature reports that IGF2BP3 is specifically overexpressed in acute myeloid leukemia (AML), and knocking down IGF2BP3 can inhibit the proliferation of AML cells in vitro and in vivo by reducing RCC2 expression (Exp Mol Med. 2022 Feb; 54(2): 194-205); IGF2BP3 is highly expressed in MLL-AF4 fusion genotype leukemia, and knocking out IGF2BP3 can prolong the survival time of MLL-AF4 leukemia mice (Leukemia. 2022 Jan; 36(1): 68-79). Although the role of IGF2BP3 in AML leukemia has been revealed, its role in other leukemia subtypes is unknown, and there are currently no available IGF2BP3 inhibitors and a lack of suitable sensitive molecular markers. We speculate that leukemias with different fusion genotypes may have different responsiveness, which is expected to expand the indications of drugs targeting IGF2BP3, and is worthy of further study.

[0005] Venetoclax can effectively inhibit leukemia by restoring the apoptotic pathway of tumor cells by inhibiting BCL-2. However, venetoclax is currently only approved by the US FDA for a small number of leukemia patients, including newly diagnosed and relapsed chronic lymphocytic leukemia (CLL) / small lymphocytic leukemia (SLL), as well as newly diagnosed acute myeloid leukemia (AML) patients who are not suitable for or refuse to receive strong induction chemotherapy due to comorbidities or are aged >75 years. Currently, except for one literature report that MLL rearranged ALL leukemia is sensitive to venetoclax (Blood. 2016 Sep 8; 128 (10): 1382-95), there are no other studies. Therefore, the discovery of clinically sensitive populations for other subtypes of leukemia, such as leukemias with different fusion genotypes, is expected to broaden the clinical application scope of venetoclax. Summary of the invention

[0006] The purpose of the present invention is to provide an application of IGF2BP3 as a target in the preparation of a fusion genotype leukemia therapeutic drug, an application of IGF2BP3 as a target in the preparation of a mRNA highly stable fusion genotype leukemia therapeutic drug, and to provide a leukemia therapeutic drug targeting IGF2BP3 to improve patient efficacy, prognosis and survival.

[0007] The gene nucleotide sequence of the IGF2BP3 target is shown in SEQ ID NO: 1, and the amino acid coding sequence is shown in SEQ ID NO: 2. The mRNA highly stable fusion gene includes but is not limited to PAX5-ETV6, STK38-PXT1 and other fusion genes. Further, the drug includes IGF2BP3 gene-specific interfering RNA, and IGF2BP3 inhibitors, including but not limited to venetoclax. The leukemia is a mRNA highly stable fusion genotype leukemia.

[0008] The nucleotide sequence of the IGF2BP3 gene-specific interfering RNA is shown in SEQ ID NO:3.

[0009] The present invention analyzes the fusion genes and their expression levels of ALL leukemia by RNA-seq sequencing, and finds that multiple fusion genes have an upregulated mRNA level after fusion; further combined with half-life experiments, it is found that the mRNA stability of fusion genes including PAX5-ETV6 and STK38-PXT1 is significantly enhanced. Therefore, the present invention proposes multiple fusion genes with high mRNA stability in ALL leukemia.

[0010] Further, the results of the present invention show that the m6A modification level of the mRNA highly stable fusion gene is significantly enhanced. Combined with transcriptome analysis, it was found that the m6A regulator IGF2BP3 is specifically highly expressed in mRNA highly stable fusion genotype leukemia; and the use of RNA interference technology to silence IGF2BP3 can significantly shorten the mRNA degradation half-life of mRNA highly stable fusion genes such as PAX5-ETV6 and STK38-PXT1, and significantly inhibit the proliferation of such fusion genotype leukemia cells. Therefore, the present invention not only discloses the application of interfering RNA of the IGF2BP3 gene, but also provides a new therapeutic target for mRNA highly stable fusion genotype leukemia.

[0011] Targeting IGF2BP3 protein, developing corresponding drug preparations that downregulate the expression of the protein or inhibit the activity of the protein is expected to achieve the purpose of resisting mRNA high-stable fusion genotype leukemia. Through drug screening, we found that the marketed drug venetoclax can selectively inhibit the proliferation of leukemia cells with high expression of IGF2BP3. At the same time, venetoclax can specifically inhibit the proliferation of leukemia cells with high mRNA stable fusion genotypes such as PAX5-ETV6 and STK38-PXT1, and induce their apoptosis. Among them, the chemical name of the marketed drug venetoclax is 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexene-1-yl]methyl}piperazine-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide, with a molecular formula of C 45 H 50 ClN7O7S.

[0012] Furthermore, the drug also includes pharmaceutically acceptable excipients. The excipients are any preparations or carrier media that can deliver an effective dose of active substances, do not interfere with the biological activity of the active substances, and have no toxic side effects on the host or the subject. The excipients include fillers, wetting agents, binders, disintegrants or lubricants. The drug can be prepared into a preparation according to the preparation preparation method recorded in pharmacy. The preparation form of the above-mentioned drug is a liquid preparation or a solid preparation.

[0013] The present invention has the beneficial effects: The present invention proposes for the first time that drugs targeting IGF2BP3 have the use of treating mRNA highly stable fusion genotype leukemia; and further discovers that the marketed drug Venatog can treat this type of leukemia by inhibiting IGF2BP3. It not only reveals the role of IGF2BP3 in the carcinogenic function of fusion genes, but also provides new therapeutic targets and effective drugs for this type of fusion genotype leukemia, providing the possibility of further improving the clinical efficacy of leukemia patients and improving their prognosis and survival. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a heat map of the mRNA expression levels at both ends of the fusion partner of the fusion gene with upregulated mRNA expression in ALL leukemia, and the mRNA half-life of PAX5-ETV6 and STK38-PXT1 under the action of actinomycin D. The experiment showed that there are multiple fusion genes with high mRNA stability in ALL leukemia.

[0015] Figure 2 It is the m6A modification level of the fusion gene obtained by RNA methylation immunoprecipitation experiment. The experiment showed that the m6A modification level of the mRNA high-stable fusion gene was significantly enhanced.

[0016] Figure 3 The mRNA expression changes of various m6A modification regulatory enzymes in 22 PAX5 fusion gene patients and 4 STK38-PXT1 fusion patients compared with the remaining non-fusion patients. The scatter plot shows that IGF2BP3 is specifically upregulated in PAX5 fusion gene and STK38-PXT1 fusion gene patients.

[0017] Figure 4 The results showed that silencing IGF2BP3 could significantly shorten the mRNA half-life of PAX5-ETV6 and STK38-PXT1 fusion genes.

[0018] Figure 5 The effect of RNA interference IGF2BP3 on the proliferation of leukemia cells with high mRNA stability fusion genotype. The results showed that silencing IGF2BP3 could selectively inhibit the proliferation of BaF3 cells with high expression of PAX5-ETV6 and STK38-PXT1.

[0019] Figure 6 This is the drug sensitivity result of ALL leukemia cells with high expression of IGF2BP3. The results showed that venetoclax was more sensitive to leukemia cells with high expression of IGF2BP3, and could selectively inhibit the proliferation of STK38-PXT1 and PAX5-ETV6 fusion genotype leukemia and induce apoptosis. DETAILED DESCRIPTION

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the examples where specific conditions are not specified are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0021] Example 1 Based on the RNA-seq data of 679 TARGET-ALL and 172 ZJUCH-ALL, we analyzed and found a large number of fusion genes, and on this basis, combined with the mRNA expression levels of both ends of the fusion partner, we found mRNA upregulated fusion genes, including the classic PAX5-ETV6 and the new unknown STK38-PXT1. For details, please refer to the Venn diagram ( Figure 1A). Subsequently, PAX5(WT), PAX5(N) and PAX5-ETV6 fusion genes, as well as STK38(WT), STK38(N) and STK38-PXT1 fusion genes were overexpressed in 293T cells. Actinomycin D (5 mg / ml) was used to inhibit mRNA synthesis 24 hours after plasmid transfection. Samples were collected and mRNA was extracted 0, 1, 3, 6, 9 and 12 hours after actinomycin treatment. Specific PCR primers were used to detect the corresponding mRNA expression levels and the mRNA degradation half-life curve was drawn. See Figure 1 BC, the results showed that the mRNA half-life of PAX5-ETV6 and STK38-PXT1 fusion genes was significantly longer than that of wild-type PAX5 and STK38 genes, indicating that these fusion genes have high mRNA stability characteristics.

[0022] Example 2 PAX5(WT), PAX5(N) and PAX5-ETV6 fusion genes, as well as STK38(WT), STK38(N) and STK38-PXT1 fusion genes were overexpressed in 293T cells. Cell samples were collected 48 hours after plasmid transfection to extract mRNA. 10% of the mRNA samples were taken to detect the corresponding mRNA expression level using specific PCR primers and recorded as the Input value. The remaining 90% of the mRNA samples were enriched with m6A antibodies to enrich for mRNA with m6A high methylation modification, and qPCR was used to directly perform quantitative analysis and detection on the enriched mRNA. The expression level of the enriched mRNA was the MeRIP value. The ratio of m6A modification of the gene was obtained by using MeRIP to compare with Input. See Figure 2 The m6A modification levels of AB, PAX5-ETV6 and STK38-PXT1 fusion genes were upregulated compared with the wild-type PAX5 and STK38 genes, respectively.

[0023] Example 3 Combined with RNA-seq data, the mRNA expression levels of various m6A modification regulatory enzymes (Writers, Erasers, and Readers) were analyzed in 22 patients carrying PAX5 fusion gene, 4 patients carrying STK38-PXT1 fusion gene, and the remaining patients who did not carry PAX5 fusion gene or STK38-PXT1 fusion gene. Figure 3 AB, only IGF2BP3 was specifically upregulated in samples of patients with PAX5 fusion gene and STK38-PXT1 fusion gene.

[0024] Example 4 Three days after the 293T cell line was infected with the shIGF2BP3 virus, PAX5(WT), PAX5(N) and PAX5-ETV6, as well as STK38(WT), STK38(N) and STK38-PXT1 were overexpressed in 293T cells by plasmid transfection. Actinomycin D was used to inhibit mRNA synthesis 24 hours later. Samples were collected at 0, 0.5, 1, 3, 6 and 12 hours after the actinomycin treatment to extract mRNA. Specific PCR primers were used to detect the corresponding mRNA expression levels and the mRNA degradation half-life curve was plotted. Figure 4 AB, Silencing IGF2BP3 can significantly reduce the mRNA stability of PAX5-ETV6 and STK38-PXT1 fusion genes, but has little effect on the mRNA stability of wild-type PX5 and STK38 genes, indicating that IGF2BP3 has a selective regulatory effect on mRNA highly stable fusion genes.

[0025] Example 5 The shRNA virus that interferes with IGF2BP3 was used in BaF3 cells overexpressing PMSCV, PAX5 (WT), PAX5 (N) and PAX5-ETV6, as well as PMSCV, STK38 (WT), STK38 (N) and STK38-PXT1. Two days after virus infection, the cells were seeded into six-well plates at a cell density of 100,000 cells / well, and the cells were counted on the fifth day. On the other hand, samples were collected after 3 days of silencing, and the silencing efficiency was investigated by qRT-PCR. The results are shown in Figure 5 A and 5C, IGF2BP3 was significantly silenced in BaF3 cells overexpressing PAX5(WT), PAX5(N), and PAX5-ETV6, as well as PMSCV, STK38(WT), STK38(N), and STK38-PXT1. Figure 5 B and 5D, Silencing IGF2BP3 can relatively selectively inhibit the proliferation of leukemia cells with PAX5-ETV6 and STK38-PXT1 fusion genotypes.

[0026] Example 6 The database analyzes the average sensitivity score Z score of 14 ALL cell lines with high IGF2BP3 expression and 10 ALL cell lines with low IGF2BP3 expression to 345 anti-tumor compounds. Figure 6 A, 180 compounds (52.2%) were more sensitive to ALL cell lines with high expression of IGF2BP3, and 165 compounds (47.8%) were more sensitive to ALL cell lines with low expression of IGF2BP3, among which venetoclax showed strong sensitivity to cells with high expression of IGF2BP3. We then investigated the selective sensitivity of BaF3 cells overexpressing PAX5-ETV6 and STK38-PXT1 to anti-tumor drugs. Figure 6 B, Venetoclax has the highest selective inhibition rate against the two fusion genotypes of leukemia. Finally, we investigated the apoptosis-inducing effect of venetoclax (2 μM) on BaF3 cells overexpressing PAX5-ETV6 and STK38-PXT1 fusion genes by PI-Annexin V double staining combined with flow cytometry. Figure 6 CD, venetoclax has a stronger apoptosis-inducing effect on leukemia cells with highly stable mRNA fusion genotype.

Claims

1. The use of IGF2BP3 as a target in the preparation of mRNA highly stable fusion genotype leukemia therapeutic drugs, characterized in that: The nucleotide sequence of the IGF2BP3 target gene is shown in SEQ ID NO:1, and the amino acid coding sequence is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that: mRNA highly stable fusion genes include but are not limited to PAX5-ETV6 PAX5 fusion gene and STK38-PXT1 fusion gene.

3. The use according to claim 1, characterized in that: Drugs targeting IGF2BP3 include IGF2BP3 gene-specific interfering RNA or IGF2BP3 inhibitors.

4. The use according to claim 3, characterized in that: The nucleotide sequence of the IGF2BP3 gene-specific interfering RNA is shown in SEQ ID NO:

3.

5. The use according to claim 3, characterized in that: The compound of the IGF2BP3 inhibitor is venetoclax, the chemical name of which is 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide, and the molecular formula is C 45 H 50 ClN7O7S.

6. The use according to claim 1, characterized in that: The drug is in the form of a liquid preparation or a solid preparation.

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