Application of IGF2BP3 as a target in the preparation of therapeutic drugs for fusion genotype leukemia

Abstract

This invention provides the application of IGF2BP3 as a target in the preparation of therapeutic drugs for fusion gene leukemia, specifically in the preparation of mRNA-highly stable fusion gene leukemia. The highly stable mRNA fusion genes include, but are not limited to, PAX5-ETV6 and STK38-PXT1 fusion genes. The drugs include IGF2BP3 gene-specific interfering RNA and IGF2BP3 inhibitors, including but not limited to venetoclax. This invention is the first to propose the use of IGF2BP3-targeted drugs for the treatment of mRNA-highly stable fusion gene leukemia; it not only reveals the role of IGF2BP3 in the oncogenic function of fusion genes but also provides a new therapeutic target and effective drug for this type of fusion gene leukemia, offering the possibility of further improving the clinical efficacy, prognosis, and survival of leukemia patients.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and specifically to the application of IGF2BP3 as a target in the preparation of mRNA-based drugs for the treatment of leukemia with highly stable fusion gene types. Background Technology

[0002] Leukemia is a heterogeneous group of clonal diseases originating from hematopoietic stem cells or early myeloid progenitor cells. Its main characteristic is the malignant clonal proliferation of hematopoietic stem cells or progenitor cells in the bone marrow, leading to the accumulation of a large number of abnormal white blood cells in the body, resulting in a series of clinical symptoms such as anemia, bleeding, and infection. Acute leukemia progresses extremely rapidly; without timely treatment, patients will die within weeks or months. Currently, the main treatment for leukemia is a comprehensive therapy primarily based on chemotherapy. Bone marrow transplantation is the most effective treatment for intermediate and advanced leukemia, but it has drawbacks such as high cost and limited availability of bone marrow donors. Therefore, the treatment of leukemia remains a major challenge and a key research area in the medical field, urgently requiring the discovery of new treatment strategies.

[0003] Fusion genes play a crucial role in the development and progression of leukemia. For example, over 50% of patients with B-cell acute lymphoblastic leukemia (B-ALL) carry fusion genes such as ETV6-RUNX1 and PAX5-ETV6, which directly drive leukemia. Therefore, fusion genes are currently an important indicator for clinical leukemia classification. However, targeted therapies targeting these fusion genes are relatively lacking in clinical practice. Therefore, elucidating the oncogenic mechanisms of fusion genes and developing corresponding targeted therapies will help improve the treatment efficacy and survival rate of leukemia patients.

[0004] N6-methyladenosine (m6A) modification is the most abundant post-transcriptional modification on eukaryotic mRNA. Dysregulation of m6A modification-regulating 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 highly expressed 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 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 hypothesize that different fusion gene types of leukemia may have different responses, which could potentially expand the indications for drugs targeting IGF2BP3 and is well worth further investigation.

[0005] Venetoclax can effectively suppress leukemia by inhibiting BCL-2 and restoring the apoptosis pathway in tumor cells. However, venetoclax is currently only approved by the US FDA for a small number of leukemia patients, including newly diagnosed and relapsed / refractory chronic lymphocytic leukemia (CLL) / small lymphocytic leukemia (SLL), and newly diagnosed acute myeloid leukemia (AML) patients who are unsuitable for or refuse strong induction chemotherapy due to comorbidities or are older than 75 years. Currently, apart from one study reporting the sensitivity of MLL-rearranged ALL leukemia to venetoclax (Blood. 2016 Sep 8; 128(10):1382-95), no other studies have been conducted. Therefore, discovering clinically sensitive populations for other leukemia subtypes, such as leukemia with different fusion gene types, is expected to broaden the clinical applicability of venetoclax. Summary of the Invention

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

[0007] 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. The highly stable mRNA fusion genes include, but are not limited to, fusion genes such as PAX5-ETV6 and STK38-PXT1. Furthermore, the drug includes IGF2BP3 gene-specific interfering RNA and IGF2BP3 inhibitors, including but not limited to venetoclax. The leukemia is a highly stable mRNA fusion genotype leukemia.

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

[0009] This invention analyzed the fusion genes and their expression levels in ALL leukemia using RNA-seq sequencing, discovering that multiple fusion genes exhibited upregulation of mRNA levels after fusion. Further half-life experiments revealed significantly enhanced mRNA stability in fusion genes, including PAX5-ETV6 and STK38-PXT1. Therefore, this invention proposes several highly mRNA-stable fusion genes for ALL leukemia.

[0010] Further research results from this invention show that the m6A modification level of highly stable mRNA fusion genes is significantly enhanced. Transcriptome analysis revealed that the m6A regulator IGF2BP3 is specifically highly expressed in highly stable mRNA fusion genotype leukemia. Furthermore, silencing IGF2BP3 using RNA interference technology significantly shortens the mRNA degradation half-life of highly stable mRNA fusion genes such as PAX5-ETV6 and STK38-PXT1, and significantly inhibits the proliferation of these fusion genotype leukemia cells. Therefore, this invention not only discloses the application of interfering RNA for the IGF2BP3 gene but also provides a new therapeutic target for highly stable mRNA fusion genotype leukemia.

[0011] Targeting the IGF2BP3 protein, we aim to develop drugs that downregulate its expression or inhibit its activity, with the potential to combat mRNA-stable fusion genotype leukemia. Through drug screening, we discovered that the marketed drug venetoclax can selectively inhibit the proliferation of leukemia cells with high IGF2BP3 expression. Simultaneously, venetoclax specifically inhibits the proliferation of PAX5-ETV6 and STK38-PXT1 mRNA-stable fusion genotype leukemia cells and induces apoptosis. The chemical name of the marketed drug venetoclax 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, with the molecular formula C1. 45 H 50 ClN7O7S.

[0012] Furthermore, the drug also includes pharmaceutically acceptable excipients. These excipients are any formulation or carrier medium capable of delivering an effective dose of the active substance, without interfering with the biological activity of the active substance, and without toxic side effects on the host or subject. The excipients include fillers, wetting agents, binders, disintegrants, or lubricants. The drug can be formulated according to pharmaceutically available methods. The formulation of the above-described drug is either a liquid or a solid dosage form.

[0013] The beneficial effects of this invention are as follows: This invention is the first to propose the use of drugs targeting IGF2BP3 for the treatment of mRNA-stable fusion gene leukemia; and further discovers that the marketed drug Venatoprazole can treat this type of leukemia by inhibiting IGF2BP3. This not only reveals the role of IGF2BP3 in the oncogenic function of fusion genes, but also provides a new therapeutic target and effective drug for this type of fusion gene leukemia, offering possibilities for further improving the clinical efficacy, prognosis, and survival of leukemia patients. Attached Figure Description

[0014] Figure 1 This is a heatmap showing the mRNA expression levels at both ends of the fusion partners of upregulated fusion genes in ALL leukemia, as well as the mRNA half-lives of PAX5-ETV6 and STK38-PXT1 under the action of actinomycin D. The experiments demonstrate the presence of multiple highly stable mRNA fusion genes in ALL leukemia.

[0015] Figure 2 This represents the m6A modification level of the fusion gene, obtained from an RNA methylation immunoprecipitation assay. The experiment showed that the m6A modification level of the highly stable mRNA fusion gene was significantly enhanced.

[0016] Figure 3 The study compared the mRNA expression of multiple m6A modification regulatory enzymes in 22 patients with the PAX5 fusion gene and 4 patients with the STK38-PXT1 fusion gene to the remaining non-fusion patients. The scatter plot showed that IGF2BP3 was specifically upregulated in patients with the PAX5 and STK38-PXT1 fusion genes.

[0017] Figure 4 This study investigated the effect of RNA interference (IGF2BP3) on the stability of fusion gene mRNA. Results showed that silencing IGF2BP3 significantly shortened the mRNA half-life of the PAX5-ETV6 and STK38-PXT1 fusion genes.

[0018] Figure 5 This study investigated the effect of RNA interference IGF2BP3 on the proliferation of leukemia cells with highly stable mRNA fusion genotypes. Results showed that silencing IGF2BP3 selectively inhibited the proliferation of BaF3 cells overexpressing PAX5-ETV6 and STK38-PXT1.

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

[0020] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0021] Example 1

[0022] Based on RNA-seq data from 679 cases of TARGET-ALL and 172 cases of ZJUCH-ALL, we identified a large number of fusion genes. Furthermore, by combining this with the mRNA expression levels at both ends of the fusion mate, we identified upregulated fusion genes, including the classic PAX5-ETV6 and the novel, previously unknown STK38-PXT1. See the Venn diagram for details. Figure 1A). Subsequently, the PAX5(WT), PAX5(N), and PAX5-ETV6 fusion genes, as well as the STK38(WT), STK38(N), and STK38-PXT1 fusion genes, were overexpressed in 293T cells. Twenty-four hours after plasmid transfection, actinomycin D (5 mg / ml) was applied to inhibit mRNA synthesis. Samples were collected at 0, 1, 3, 6, 9, and 12 hours after actinomycin D treatment to extract mRNA. Specific PCR primers were used to detect the corresponding mRNA expression levels, and mRNA degradation half-life curves were plotted. See [link to relevant documentation]. Figure 1 The results showed that the mRNA half-life of the PAX5-ETV6 and STK38-PXT1 fusion genes was significantly longer than that of the wild-type PAX5 and STK38 genes, indicating that these fusion genes have highly stable mRNA characteristics.

[0023] Example 2

[0024] 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. Forty-eight hours after plasmid transfection, cell samples were collected to extract mRNA. The expression levels of 10% of the mRNA samples were detected using specific PCR primers and recorded as the Input value. The remaining 90% of the mRNA samples were enriched with an m6A antibody, identifying mRNAs with m6A hypermethylation modification. The enriched mRNAs were then directly quantified using qPCR, and their expression levels were recorded as MeRIP values. The ratio of MeRIP values ​​to Input values ​​yielded the m6A modification ratio of the genes. See also... Figure 2 The m6A modification levels of the AB, PAX5-ETV6 and STK38-PXT1 fusion genes were upregulated compared to the wild-type PAX5 and STK38 genes, respectively.

[0025] Example 3

[0026] RNA-seq data were used to analyze the differences in mRNA expression levels of various m6A modification regulatory enzymes (Writers, Eraser, and Readers) among 22 patients carrying the PAX5 fusion gene, 4 patients carrying the STK38-PXT1 fusion gene, and the remaining patients who did not carry either the PAX5 or STK38-PXT1 fusion gene. See [link to relevant documentation]. Figure 3 AB, only IGF2BP3 was specifically upregulated in patients with the PAX5 fusion gene and the STK38-PXT1 fusion gene.

[0027] Example 4

[0028] Three days after infecting 293T cell lines with shIGF2BP3 virus, PAX5(WT), PAX5(N), and PAX5-ETV6, as well as STK38(WT), STK38(N), and STK38-PXT1, were overexpressed in 293T cells via plasmid transfection. Twenty-four hours later, actinomycin D was applied to inhibit mRNA synthesis. Samples were collected at 0, 0.5, 1, 3, 6, and 12 hours after actinomycin D treatment to extract mRNA. Specific PCR primers were used to detect the expression levels of the corresponding mRNAs, and mRNA degradation half-life curves were plotted. See [link to relevant documentation]. Figure 4 AB, silencing IGF2BP3 significantly reduced the mRNA stability of PAX5-ETV6 and STK38-PXT1 fusion genes, while having little effect on the mRNA stability of wild-type PX5 and STK38 genes, indicating that IGF2BP3 has a selective regulatory effect on highly stable mRNA fusion genes.

[0029] Example 5

[0030] IGF2BP3-interfering shRNA virus was used to infect 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 viral infection, cells were seeded into six-well plates at a rate of 100,000 cells / well, and cell counts were performed on day 5. Samples were also collected three days after silencing, and silencing efficiency was assessed using qRT-PCR. Results are shown below. Figure 5 In BaF3 cells overexpressing A and 5C, PAX5(WT), PAX5(N), and PAX5-ETV6, as well as PMSCV, STK38(WT), STK38(N), and STK38-PXT1, IGF2BP3 was significantly silenced. See also Figure 5 B and 5D, silencing IGF2BP3 can relatively selectively inhibit the proliferation of PAX5-ETV6 and STK38-PXT1 fusion leukemia cells.

[0031] Example 6

[0032] Database analysis was performed on the mean sensitivity scores (Z-scores) of 345 antitumor compounds to 14 ALL cell lines with high IGF2BP3 expression and 10 ALL cell lines with low IGF2BP3 expression. See also... Figure 6A. 180 compounds (52.2%) were more sensitive in ALL cell lines expressing high levels of IGF2BP3, while 165 compounds (47.8%) were more sensitive in ALL cell lines expressing low levels of IGF2BP3. Venetoclax showed extremely high sensitivity in cells expressing high levels of IGF2BP3. We then investigated the selective sensitivity of BaF3 cells overexpressing PAX5-ETV6 and STK38-PXT1 to antitumor drugs. See [link to relevant documentation] Figure 6 B. Venetoclax showed the highest selective inhibition rate against both fusion gene leukemias. Finally, we investigated the apoptosis-inducing effect of venetoclax (2 μM) on BaF3 cells overexpressing the PAX5-ETV6 and STK38-PXT1 fusion genes using PI-Annexin V double staining combined with flow cytometry. Results are shown below. Figure 6 CD, Venetoclax has a stronger apoptosis-inducing effect on leukemia cells with highly stable mRNA fusion genotypes.

Claims

1. Application of venetoclax in the preparation of therapeutic drugs for leukemia carrying PAX5-ETV6 or STK38-PXT1 fusion genotypes.

2. The application according to claim 1, characterized in that, The drug is formulated in either a liquid or solid form.

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