Related application of DNA methyltransferase inhibitor in treatment of VHL-deficient tumors

Abstract

The invention discloses a related application of a DNA methyltransferase inhibitor in treatment of VHL-deficient tumors, and relates to the technical field of biological medicines. The invention finds that the DNA methyltransferase inhibitor is a synthetic lethal drug for VHL-deficient tumors, can significantly inhibit the VHL-deficient tumors, has no obvious toxicity, and provides a new way for individualized treatment of the VHL-deficient tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of DNA methyltransferase inhibitors in the treatment of VHL-deficient tumors. Background Technology

[0002] The Hipper-Lindau (VHL) gene is a crucial tumor suppressor gene. Its encoded pVHL protein, as part of the E3 ubiquitin ligase complex, induces oxygen-dependent degradation of hypoxia-inducible factor (HIF).

[0003] HIF proteins activate the transcription of genes involved in processes such as angiogenesis and glycolysis by forming heterodimeric complexes and binding to hypoxia response elements (HREs) on target genes. Mutations, deletions, or inactivation of VHL activate HIF, leading to cancers with highly vascularized, glucose-dependent phenotypes. VHL also participates in multiple HIF-independent pathways through interactions with various proteins, such as regulating key molecules in extracellular matrix assembly (e.g., fibronectin and integrins), modulating microtubule function of primary cilia, affecting the transcriptional activity of transcription factor SP1, and mediating cellular senescence through interactions with RB and SWI / SNF complexes.

[0004] VHL is frequently inactivated or mutated in various cancers, including renal cell carcinoma, hemangioblastoma, pancreatic neuroendocrine tumors, and pheochromocytoma. In renal cell carcinoma (RCC), the VHL gene mutation frequency exceeds 50%, and the loss of heterozygosity (LOH) incidence is as high as 90%, thus it is considered one of the key biomarkers for RCC. RCC is a difficult-to-treat cancer, highly resistant to chemotherapy and radiotherapy. Traditional systemic immunotherapies, such as interferon and interleukin-2, have been used to treat RCC. However, only a small percentage of patients (approximately 7%) respond to immunotherapy, and the median survival is only 12 months. Anti-angiogenic therapies (such as sunitinib and bevacizumab) and immune checkpoint inhibitors (such as nivolumab) have been approved for the treatment of RCC. These drugs are widely used in targeted therapy for various cancer types, and their anti-tumor efficacy varies significantly among different patients. Furthermore, these drugs are not specifically targeted at RCC.

[0005] Currently, standard treatment for advanced VHL-deficient tumors mainly relies on drugs targeting downstream HIF pathways, such as VEGF / VEGFR inhibitors (e.g., sunitinib, pazopanib) and mTOR inhibitors (e.g., everolimus). While these therapies have improved patient prognosis to some extent, they still have significant limitations, such as the tendency to develop drug resistance, poor efficacy, high toxicity, and lack of curative effect. There is also a lack of therapies that directly target VHL inactivation.

[0006] Therefore, there is an urgent clinical need to develop a novel, highly effective, and low-toxicity treatment strategy based on VHL genotypes to overcome the limitations of existing therapies.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The purpose of this invention is to provide the application of DNA methyltransferase inhibitors in the treatment of VHL-deficient tumors.

[0009] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide the use of DNA methyltransferase inhibitors in the preparation of products that improve or treat VHL-deficient tumors.

[0010] Secondly, embodiments of the present invention provide a composition comprising a VHL inhibitor and the DNA methyltransferase inhibitor described in the foregoing embodiments.

[0011] Thirdly, the embodiments of the present invention provide the use of the compositions of the foregoing embodiments in the preparation of products for treating tumors.

[0012] Fourthly, embodiments of the present invention provide the application of DNA methyltransferases as targets in screening products that improve or treat VHL-deficient tumors.

[0013] The present invention has the following beneficial effects: This invention discovers that DNA methyltransferase (DNMT) inhibitors are synthetic lethal drugs for VHL-deficient tumors, which can significantly inhibit VHL-deficient tumors without obvious toxicity, providing a new approach for personalized treatment of VHL-deficient tumors. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1This document describes the identification of DNMT inhibitors as synthetic lethal drugs for VHL-deficient tumors. Specifically, (A) a flowchart of the experimental process for screening VHL synthetic lethal drugs from an epigenetic compound library using VHL-equal genotype 786-O cell pairs; (B) stable overexpression of the VHL-HA plasmid in VHL-deficient 786-O cells; and verification of VHL-equal genotype RCC cell pairs by Western blot analysis; (C) identification of VHL-equal genotype RCC cell pairs by selection index (i.e.,...). Cells and cells (ratio), to screen out the top five synthetic lethal drug candidates; Figure 2 To verify the synthetic lethal effect of DNMT inhibitors in VHL-deficient RCC cells; wherein, (A) the chemical structure of the DNMT inhibitors used in this study; (BG) the synthetic lethal effect induced by DNMT inhibitors (decitabine, azacitidine, RX-3117 and SGI-1027) was verified in RCC cell lines with different VHL states. Figure 3 To verify the synthetic lethal effects of VHL and DNMT in other cell types; specifically, the effects of (AB) decitabine on the growth of control and VHL knockdown H1975 cells; and the effects of (CD) decitabine on the growth of control and VHL knockdown PLC / PRF / 5 cells. Figure 4 The study aimed to evaluate the different apoptotic effects of DNMT inhibitors on VHL and other basal RCC cells. Specifically, (A) Annexin V / PI double staining was used to detect the different apoptotic effects of DNMT inhibitors on VHL and other basal RCC cells; (B) Statistical analysis of the apoptosis rate in (A); and (C) Western blot detection of the apoptosis marker protein Cleaved caspase 3 to assess the different apoptotic effects of DNMT inhibitors on VHL and other basal RCC cells. Figure 5 To verify the synthetic lethal effect between VHL and DNMT by silencing DNMT subtypes using siRNA; specifically, (AB) silencing of DNMT1 in 786-O cells containing VHL and other genes and its inhibitory effect on cell growth; (CD) silencing of DNMT3A in 786-O cells containing VHL and other genes and its inhibitory effect on cell growth; and (EF) silencing of DNMT3B in 786-O cells containing VHL and other genes and its inhibitory effect on cell growth. Figure 6 To validate DNMT inhibitors in vivo as synthetic lethal agents for VHL-deficient tumors; (A) Schematic diagram of RCC tumor xenograft construction and decitabine treatment; (BC) ... 786-O cells were subcutaneously inoculated into the right back of mice; decitabine was administered intraperitoneally twice weekly at the indicated dose; (D) Mouse weight was monitored periodically throughout the treatment period to assess systemic toxicity; Figure 7 VHL regulates DNMT1 expression via HIF-2α; among which, (AB) in and mRNA expression levels of VHL, DNMT and TET subtypes in 786-O cells; (C) in and In 786-O cells, Western blot was used to detect the protein expression levels of VHL, HIF-2α, and DNMT subtypes; (DG) the effect of HIF-2α siRNA on the mRNA expression levels of HIF-2α and DNMT subtypes; (HI) the effect of HIF-2α siRNA (H) or the HIF-2α inhibitor belzutifan (I) on the protein expression level of DNMT1; Cyclin D was used as a positive control for the expression of HIF-2α target genes; (J) clinical data analysis of DNMT1 expression levels in renal cell carcinoma tissues and normal tissues; (K) correlation analysis of DNMT1 and EPAS1 (the HIF-2α encoding gene) transcription levels in tumor samples from renal cell carcinoma patients. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0017] The term "VHL-deficient tumor" in this article refers to tumors caused by VHL deficiency. VHL Gene( von Hippel-Lindau A type of tumor caused by loss or abnormality of gene function, its core feature is the overactivation of the hypoxia-inducible factor (HIF) pathway caused by VHL protein dysfunction, which in turn drives tumor cell proliferation, angiogenesis and metabolic reprogramming.

[0018] The term “treatment” in this article includes preventing or alleviating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0019] For cancer, "treatment" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination thereof.

[0020] The inventors of this application have discovered that DNA methyltransferase (DNMT) inhibitors are synthetic lethal agents for VHL-deficient tumors. FDA-approved DNMT inhibitors (decitabine and azacitidine) and other candidate compounds (such as RX-3117 and SGI-1027) can selectively inhibit the growth of VHL-deficient renal cell carcinomas, and this synthetic lethal effect is also effective in other VHL-deficient cancer cell types.

[0021] DNMT inhibitors can be applied to personalized cancer treatment characterized by VHL deficiency or inactivation, and provide a novel treatment strategy for cancer based on synthetic lethality.

[0022] On the one hand, embodiments of the present invention provide the application of DNA methyltransferase inhibitors in the preparation of products that improve or treat VHL-deficient tumors.

[0023] In some embodiments, the DNA methyltransferase inhibitor includes inhibitors of any one or more subtypes of DNMT1, DNMT2, and DNMT3.

[0024] In some embodiments, the inhibition mode of the DNA methyltransferase inhibitor includes: inhibiting DNA methyltransferase (DNMT) activity and / or reducing DNA methyltransferase expression levels.

[0025] In some embodiments, the DNA methyltransferase inhibitor may be of any one or more of the following types: nucleoside analog inhibitors, non-nucleoside analog inhibitors, siRNA (small interfering RNA), shRNA (short hairpin RNA), or ASO (antisense oligonucleotide) that inhibit DNA methyltransferase.

[0026] Nucleoside analogue inhibitors (DNA incorporation type) are compounds whose structures are similar to the raw materials for DNA synthesis (nucleosides). They can be taken up by cells and incorporated into the DNA chain, where they irreversibly bind to DNMT, "capturing" and inhibiting enzyme activity, leading to DNMT inactivation. Representative compounds include azacitidine (5-azacytidine), decitabine (5-aza-2'-deoxycytidine), and RX-3117. Azacitidine reduces DNA methylation levels by incorporating into DNA and RNA, inhibiting DNMT and RNA polymerase. Decitabine specifically acts on DNA, covalently binding to DNMT after incorporation, preventing it from participating in methylation.

[0027] Non-nucleoside analogue inhibitors (non-incorporated) are compounds that do not incorporate into DNA. Instead, they inhibit the catalytic function of DNMT by directly binding to its active or allosteric sites, either competitively or non-competitively. They have minimal impact on DNA synthesis and are relatively safe. Representative compounds include RG108, SGI-1027, and zebularine.

[0028] siRNA or shRNA can specifically target DNMT mRNA (such as at least one of DNMT1, DNMT2, DNMT3A, and DNMT3B), inducing mRNA degradation or translational repression, thus reducing protein synthesis. Synthetically synthesized ASO binds complementary to DNMT mRNA, preventing its translation or promoting its degradation, thereby reducing protein expression.

[0029] In some embodiments, the DNA methyltransferase inhibitor includes any one or more of decitabine, azacitidine, RX-3117, SGI-1027, RG108, thioguanine, zabralin, SGI-110, romilutrile, and procainamide.

[0030] In some embodiments, the VHL-deficient tumor includes any one or more of the following: renal cell carcinoma, hemangioblastoma, pancreatic neuroendocrine tumor, and pheochromocytoma.

[0031] In some embodiments, the renal cell carcinoma includes clear cell renal carcinoma (ccRCC).

[0032] In some embodiments, the treatment includes adjunctive therapy.

[0033] In some embodiments, the product includes either a drug or a food.

[0034] On the other hand, embodiments of the present invention provide a composition comprising a VHL inhibitor and the DNA methyltransferase inhibitor described in any of the foregoing embodiments.

[0035] On the other hand, embodiments of the present invention provide the use of the compositions as described in any of the foregoing embodiments in the preparation of products for treating tumors.

[0036] In some embodiments, the VHL inhibitor comprises: inhibiting VHL A reagent for determining the expression level of a gene or its protein.

[0037] In some embodiments, suppress VHL Gene knockout or silencing methods include gene removal. VHL Genes. Gene knockout methods include gene editing technologies (such as CRISPR / Cas9, TALEN, ZFN, etc.), while silencing techniques include RNA interference (RNAi, such as siRNA, shRNA), antisense oligonucleotides (ASO), etc.

[0038] In some embodiments, the tumor includes non-small cell lung cancer and / or hepatocellular carcinoma.

[0039] Furthermore, embodiments of the present invention also provide the application of DNA methyltransferases as targets in screening products for improving or treating VHL-deficient tumors.

[0040] In some embodiments, the screening step includes: using DNA methyltransferase inhibitors as candidate drugs to screen for products that can treat or improve VHL-deficient tumors.

[0041] In some embodiments, the VHL-deficient tumor is the same as described in any of the foregoing embodiments, and will not be repeated here.

[0042] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0043] Example 1 This embodiment constructs genotypes such as VHL (except for...). VHL Apart from the fact that their genetic background is completely identical, the RCC cell pair, namely the VHL mutant ( ) and wild type VHL Overexpression ( Using 786-O cell pairs as a model, a synthetic lethal drug screening was conducted against an epigenetic drug library containing 128 compounds. Figure 1 (A in the middle).

[0044] All drugs were aliquoted into 384-well plates and screened using eight concentration gradients to obtain the efficacy of each drug in different cell lines. Value. (For) Drugs with selective cell-inhibiting effects are considered synthetic lethal candidates. Western blot analysis was used to detect the protein levels of VHL overexpression (VHL-HA plasmid) and its substrate HIF-2α to validate this. VHL Isogenotype 786-O RCC cell pairs ( Figure 1 (B in the middle).

[0045] According to the selectivity index (i.e.) cells Value and cells The ratio of values) determines the synthetic lethal candidate drug ( Figure 1 (C in the middle).

[0046] Among the top five candidate drugs screened, the DNA methyltransferase (DNMT) inhibitor decitabine was identified as the second most effective drug after AURKA inhibitors (Alisertib and Hesperadin).

[0047] Example 2: Verification of the synthetic lethal effect of DNMT inhibitors on VHL-deficient RCC This embodiment uses four different DNMT inhibitors ( Figure 2 The study, conducted in RCC cells with four different VHL states (A), included 769-P (VHL mutant), Caki-1 (VHL wild-type), and... and 786-O cells. Results showed that the FDA-approved DNMT inhibitors decitabine and azacitidine selectively inhibited VHL-deficient RCC cells. Figure 2 In addition, the DNMT inhibitors RX-3117 and SGI-1027, which are currently under investigation, have also shown selective inhibitory effects on VHL-deficient RCC cells. Figure 2 (FG in the middle).

[0048] Structurally, decitabine, azacitidine, and RX-3117 are all nucleoside analogs based on cytosine, while SGI-1027 belongs to the non-nucleoside DNMT inhibitor class. Figure 2 (A in the figure) indicates that DNMT inhibitors of various structural types can induce synthetic lethal effects in VHL-deficient RCCs.

[0049] Example 3: Investigating whether the synthetic lethal effect between VHL-deficient type and DNMT inhibition applies to cancer types other than RCC. In this embodiment, VHL expression was silenced by siRNA in the non-small cell lung cancer (NSCLC) cell line H1975 and the hepatocellular carcinoma (HCC) cell line PLC / PRF / 5, and changes in their sensitivity to decitabine were evaluated.

[0050] The results showed that decitabine also had a selective inhibitory effect on NSCLC (H1975) and HCC (PLC / PRF / 5) cells with downregulated VHL expression. Figure 3 (AD in the middle). These findings suggest that the synthetic lethal effect between VHL and DNMT inhibition is conserved across multiple cancer cell types.

[0051] Example 4: Verification of the synthetic lethal effect of DNMT inhibitors in VHL-deficient RCC This embodiment examined the differences in the apoptosis response of VHL and other genotype RCC cells to decitabine-induced apoptosis.

[0052] Annexin V and propidium iodide (PI) staining results showed that decitabine in The degree of apoptosis induced in 786-O cells was significantly higher than that in other cells. 786-O cells ( Figure 4 (AB in the middle).

[0053] Western blot analysis showed that, The level of cleaved-caspase-3 was significantly increased in 786-O cells, further supporting the conclusion that it selectively induces apoptosis. Figure 4 (C in the middle).

[0054] These results indicate that DNMT inhibitors induce synthetic lethality in VHL-deficient RCC cells by promoting apoptosis.

[0055] Example 5 confirms whether the synthetic lethal effect produced by the DNMT inhibitor in VHL-deficient RCC is indeed caused by the DNMT inhibition itself, rather than an off-target effect.

[0056] In this embodiment, three DNMT subtypes (DNMT1, DNMT3A, and DNMT3B) were silenced using siRNA, and their synthetic lethal effects were evaluated in VHL syngeneic RCC cell pairs. The results showed that silencing any DNMT subtype could induce synthetic lethality in VHL-deficient RCC cells to some extent. Figure 5 (AF in the middle).

[0057] Furthermore, the DNMT inhibitor used in this embodiment is known to inhibit all DNMT subtypes simultaneously. The siRNA experimental results suggest that the synthetic lethal effect of the DNMT inhibitor may be due to the inhibition of all DNMT subtypes.

[0058] Example 6: RCC Tumor Xenograft Experiment For carrying Nude mice with 786-O RCC tumor xenografts were treated with decitabine at doses of 1 and 2 mg / kg, respectively, and the results showed that tumor growth was significantly inhibited. Figure 6 (AB in the middle).

[0059] Decitabine treatment at any dose level did not cause significant changes in mouse body weight, indicating that DNMT inhibitors significantly inhibited the growth of VHL-deficient RCC tumors without significant toxicity. Figure 6 (C in the middle).

[0060] These results indicate a synthetic lethal relationship between DNMT inhibition and VHL inactivation, suggesting that DNMT inhibitors may be promising candidates for personalized treatment of VHL-deficient tumors.

[0061] Example 7 (1) Investigate the lethal mechanism of VHL and DNMT synthesis and evaluate the role of VHL in regulating DNMT and TET expression. DNMTs are enzymes that mediate DNA CpG methylation, while TETs (ten-eleven translocation dioxygenases) are involved in DNA demethylation. The results of this example show that the mRNA level of DNMT1 is significantly increased in VHL-deficient RCC cells. Figure 7 The expression of AB in VHL cells was not significantly different between wild-type and defective cells, while the expression of other DNMT and TET family members was not significantly different between wild-type and defective cells.

[0062] Western blot analysis further confirmed that the level of DNMT1 protein was also significantly increased in VHL-deficient RCC cells compared with VHL wild-type cells. Figure 7 (C in the middle).

[0063] (2) Investigate whether VHL regulates DNMT1 through the transcription factor HIF-2α. In this embodiment, HIF-2α was knocked down in VHL-deficient RCC cells, and DNMT expression levels were detected. The results showed that silencing HIF-2α significantly reduced DNMT1 mRNA levels (…). Figure 7 DG and protein (in the middle) Figure 7The expression level of H) in HIF-2α. Furthermore, the small molecule inhibitor belzutifan can also downregulate the expression of DNMT1 (H). Figure 7 The I in the figure indicates that VHL-deficient RCC cells upregulate DNMT1 expression via HIF-2α.

[0064] It was also observed that the expression level of DNMT1 in renal cell carcinoma tissue was higher than that in normal tissue. Figure 7 In the J), ​​and in tumor samples from renal cell carcinoma patients, the transcriptional levels of DNMT1 and EPAS1 (the gene encoding HIF-2α) were positively correlated. Figure 7 (K in the middle).

[0065] These findings suggest that VHL deficiency activates HIF-2α, thereby upregulating DNMT1 expression and leading to widespread CpG hypermethylation. This alteration in the DNA methylation profile often involves promoter regions of tumor suppressor genes (TSGs), resulting in their epigenetic silencing. Treatment of VHL-deficient cancer cells with DNMT inhibitors may reverse this silencing, reactivating TSGs and thus inhibiting tumor cell growth through TSG reexpression.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of DNA methyltransferase inhibitors in the preparation of products that improve or treat VHL-deficient tumors.

2. The application according to claim 1, characterized in that, The DNA methyltransferase inhibitors include inhibitors of any one or more subtypes of DNMT1, DNMT2, and DNMT3.

3. The application according to claim 1, characterized in that, The inhibition mechanisms of the DNA methyltransferase inhibitors include: inhibiting DNA methyltransferase activity and / or reducing DNA methyltransferase expression levels.

4. The application according to claim 1, characterized in that, The DNA methyltransferase inhibitors include any one or more of the following: nucleoside analog inhibitors, non-nucleoside analog inhibitors, siRNA, shRNA, or ASO that inhibit DNA methyltransferase.

5. The application according to claim 1, characterized in that, The DNA methyltransferase inhibitors include any one or more of decitabine, azacitidine, RX-3117, SGI-1027, RG108, thioguanine, zabraline, SGI-110, romilutri, and procainamide.

6. The application according to any one of claims 1 to 5, characterized in that, The VHL-deficient tumors include any one or more of the following: renal cell carcinoma, hemangioblastoma, pancreatic neuroendocrine tumor, and pheochromocytoma.

7. The application according to any one of claims 1 to 5, characterized in that, The treatment includes adjunctive therapy; Optionally, the product includes either a pharmaceutical or a food product.

8. A composition, characterized in that, It includes VHL inhibitors and DNA methyltransferase inhibitors as described in any one of claims 1 to 5.

9. The use of the composition of claim 8 in the preparation of a product for treating tumors; Optionally, the VHL inhibitor comprises: inhibition VHL Reagents for assessing gene or protein expression levels; Optionally, the tumor includes non-small cell lung cancer and / or hepatocellular carcinoma.

10. Application of DNA methyltransferases as targets in screening products that improve or treat VHL-deficient tumors; Optionally, the VHL-deficient tumor includes: Any one or more of the following: renal cell carcinoma, hemangioblastoma, pancreatic neuroendocrine tumor, and pheochromocytoma.

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