Application of combination of mTOR inhibitor and EPAS1 inhibitor in preparation of medicine for treating myeloproliferative tumors

Abstract

The invention provides application of combination of an mTOR inhibitor and an EPAS1 inhibitor in preparation of a medicine for treating myeloproliferative tumors, through combined use of the mTOR inhibitor everolimus and the EPAS1 inhibitor PT2385, JAK2V617F mutation-driven metabolic disorder is targeted, metabolic reprogramming is effectively reversed (the lactic acid level is reduced, the alpha-ketoglutaric acid level is increased, and the lactic acid / alpha-KG ratio is corrected), and the treatment effect on myeloproliferative tumors is improved. The JAK inhibitor can be used for treating MPN, synergistically relieving splenomegaly, reversing myelofibrosis and reducing mutation allele load, the curative effect is remarkably superior to that of existing JAK inhibitor single-drug treatment, and an innovative scheme with disease modification potential is provided for MPN.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a combination of an mTOR inhibitor and an EPAS1 inhibitor in preparation of a medicine for treating myeloproliferative neoplasm. BACKGROUND

[0002] Myeloproliferative neoplasm (MPN) is a group of clonal hematopoietic stem cell diseases characterized by the overproduction of one or more mature myeloid cell lines, and JAK2 V617F is the most common driver mutation. Although JAK inhibitors (such as ruxolitinib) can relieve symptoms of MPN patients to a certain extent, they have limited efficacy in disease modification, reversal of fibrosis and reduction of allelic burden, and cannot effectively correct the deep metabolic abnormalities caused by JAK2 V617F .

[0003] Recent studies have found that JAK2 V617F mutations can drive metabolic reprogramming of tumor cells through multiple signaling pathways (such as mTOR), promote glycolysis, and inhibit oxidative phosphorylation, thereby adapting to the biological energy and biosynthesis required for rapid proliferation. Hypoxia-inducible factor 2 alpha (EPAS1 / HIF-2 alpha) is a key transcription factor downstream of mTOR, which plays a core role in regulating glucose metabolism, angiogenesis and cell proliferation. However, no study has clearly revealed whether JAK2 V617F drives metabolic abnormalities in MPN through the “mTOR-EPAS1” axis, and there is no report on the use of mTOR and EPAS1 double-targeted inhibition strategy to reverse the process and treat MPN.

[0004] Therefore, there is an urgent need in the art for a new treatment strategy that can target the core metabolic mechanism of MPN, effectively reverse JAK2 V617F related metabolic abnormalities, and achieve disease modification. SUMMARY

[0005] To solve the above technical problems, the application provides application of a combination of an mTOR inhibitor and an EPAS1 inhibitor in preparation of a medicine for treating myeloproliferative neoplasm, which creatively uses an mTOR inhibitor (everolimus) in combination with an EPAS1 inhibitor (PT2385), achieving synergistic blocking of the key metabolic pathway of MPN, and having stronger efficacy and disease modification potential compared with single-drug treatment or existing JAK inhibitors.

[0006] To achieve the above purpose, the application adopts the following technical solutions. In a first aspect of the application, there is provided application of a combination of an mTOR inhibitor and an EPAS1 inhibitor in preparation of a medicine for treating myeloproliferative neoplasm.

[0007] Furthermore, the myeloproliferative neoplasm is accompanied by JAK2. V617F Mutations include one of the following: polycythemia vera, essential thrombocythemia, or essential myelofibrosis.

[0008] Furthermore, the mTOR inhibitor includes at least one of everolimus, rapamycin, tesiromolimus, or desfomol.

[0009] Furthermore, the EPAS1 inhibitor includes at least one of PT2385, PT2399, or Belzutifan.

[0010] PT2385 is a specific HIF-2α inhibitor, CAS number: 1672665-49-4, and its structural formula is shown below: .

[0011] PT2399 is an effective selective HIF-2α antagonist, CAS number: 1672662-14-4, with the following structural formula: .

[0012] Belzutifan (PT2977) is a potent and selective HIF-2α inhibitor, CAS number: 1672668-24-4, with the following structural formula:

[0013] In a second aspect of the invention, a pharmaceutical composition for treating myeloproliferative neoplasms is provided, comprising a therapeutically effective amount of an mTOR inhibitor and an EPAS1 inhibitor as a combination composition.

[0014] Furthermore, the mTOR inhibitor is everolimus, and the EPAS1 inhibitor is PT2385. Furthermore, the dosage of everolimus is approximately 5 mg / kg / day, and the dosage of PT2385 is approximately 30 mg / kg / day.

[0015] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0016] Furthermore, the pharmaceutically acceptable carrier includes one or more of the following: diluent, isotonic agent, pH adjuster, preservative, thickener, or sustained-release material.

[0017] In a third aspect of the invention, a combination composition of an mTOR inhibitor and an EPAS1 inhibitor is provided for reversing JAK2. V617FUse of mutation-driven metabolic reprogramming, which includes increased lactate levels and / or decreased α-ketoglutarate levels.

[0018] The beneficial effects of the above-described technical solution of the present invention are as follows: 1. This invention provides the application of the combination of mTOR inhibitors and EPAS1 inhibitors in the preparation of drugs for treating myeloproliferative neoplasms. This invention is the first to disclose and verify the "JAK2" inhibitor. V617F The mTOR→EPAS1 signaling axis plays a central driving role in MPN metabolic abnormalities. Single-cell sequencing and Western blotting experiments confirmed the role of JAK2. V617F The discovery that mutations specifically activate the mTOR-EPAS1 pathway provides a novel target theoretical basis for MPN treatment.

[0019] 2. This innovative approach employs a dual-targeting strategy combining an mTOR inhibitor and an EPAS1 inhibitor to synergistically block key MPN metabolic pathways. Compared to traditional JAK inhibitor monotherapy, this strategy demonstrates stronger pathway inhibition and disease-modifying potential.

[0020] 3. Synergistic therapeutic effect (1) Reversal effect of metabolic reprogramming Targeted metabolomics analysis confirmed that combination therapy can significantly correct JAK2. V617F Driven metabolic abnormalities: lactate levels decreased by 60-70%; α-ketoglutarate (α-KG) levels increased by 2-3 times; the lactate / α-KG ratio recovered from the abnormal value of 4.5±0.6 to the normal range of 1.2±0.3.

[0021] (2) Effects on improving cell function CCK-8 assay and flow cytometry showed the following: Cell proliferation inhibition rate: The combination therapy group achieved over 50%, significantly superior to the single-drug group (20-30%). Cell apoptosis promotion rate: The combination therapy increased the apoptosis rate by approximately 3 times. A clear synergistic effect was demonstrated, reflecting a therapeutic efficacy of "1+1>2".

[0022] 4. Significant therapeutic effect: Preclinical studies have shown that this dual-targeted combined therapy can effectively reduce splenomegaly, decrease the load of mutant alleles, reverse myelofibrosis, and significantly correct abnormalities in metabolites (such as the lactate / α-KG ratio) in MPN model mice.

[0023] 5. Broad application prospects: This strategy provides a new treatment option for MPN patients who are resistant to or intolerant of JAK inhibitors, and has important clinical translational value. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of this application, the following detailed description of non-limiting embodiments is provided using accompanying drawings, making their features, objectives, and advantages more apparent: Figure 1 Single-cell sequencing identifies JAK2 V617F Differentially expressed genes and signaling pathways in bone marrow hematopoietic stem cells of mutant mice and wild-type mice. (A) Schematic diagram of bone marrow sample preparation; (B) Identification of different cell populations in bone marrow cells after red blood cell removal based on unsupervised clustering analysis; (C) Specific expressed genes of different cell populations; (D) Wild-type mice and JAK2 mice. V617F Volcano plot of differentially expressed genes in bone marrow hematopoietic stem cells of mutant mice; (E) Wild-type mice and JAK2 V617F (F) Heatmap of differentially expressed genes in bone marrow hematopoietic stem cells of mutant mice, mainly the top 20 differentially expressed genes that are upregulated and downregulated; (F) Signaling pathways for clustering differentially expressed genes by KEGG analysis, mainly related to the mTOR signaling pathway and the hypoxia-induced signaling pathway.

[0025] Figure 2 Identifying JAK2 V617F Differentially expressed metabolites in bone marrow hematopoietic stem cells of mutant mice and bone marrow of wild mice. (A) The corresponding signaling pathways for differentially expressed metabolites from different bone marrow sources were identified and enriched, mainly related to lactate metabolism and ketoglutarate metabolism; (B) The contents of lactate and α-KG in bone marrow supernatant of different groups were detected by targeted metabolomics.

[0026] Figure 3 Western blotting and immunofluorescence were used to identify the phosphorylation of mTOR protein related to metabolic pathways and the expression of HIF2α in corresponding bone marrow hematopoietic stem cells in different treatment groups. (A) (B) (C): First, bone marrow hematopoietic stem cells from different groups were isolated, mainly from wild-type mice and JAK2 mice. V617F Mutant mice and JAK2 mice treated with JAK2 inhibitors V617F Mutant mice were used, and then the expression of phosphorylated STAT3, phosphorylated STAT5, phosphorylated mTOR, phosphorylated 4EBP1, HIF1α, HIF2α, GLUT1 and VEGF in hematopoietic stem cells of different groups was identified by western blotting; (D) Immunofluorescence was used to identify the expression of phosphorylated mTOR and HIF2α in bone marrow hematopoietic stem cells of different groups in vivo.

[0027] Figure 4 Identifying the effects of mTOR and HIF2α inhibitors on JAK2 V617FThe proliferation, apoptosis, and metabolism of mutant hematopoietic stem cells. (A) CCK8 assay to identify the effects of different groups on JAK2. V617F The effects of different groups on the proliferation of mutant hematopoietic stem cells, including Ruxolitinib (a Jak2 inhibitor), Everolimus (an mTOR inhibitor), and PT2385 (an EPAS1 inhibitor); (B) Flow cytometry analysis of the effects of different groups on JAK2. V617F The effect of mutant hematopoietic stem cell apoptosis; (C) Targeted metabolomics analysis of different groups on JAK2 V617F The effects of lactate and α-KG metabolism on mutant hematopoietic stem cells.

[0028] Figure 5 : Validating the effects of different treatment regimens on JAK2 V617F The effect of the mutant mice on spleen weight. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings. The present invention and its embodiments are described below; however, this description is not restrictive, and actual embodiments are not limited thereto. If those skilled in the art, inspired by this description, design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

[0030] Example 1: The mTOR-EPAS1 pathway in JAK2 V617F Activation verification in the MPN model 1. Animal model: JAK2 was selected. V617F Transgenic mice (MPN model) and littermate wild-type (WT) control mice.

[0031] 2. Grouping and processing: Group 1: Wild-type control group (solvent); Group 2: JAK2 V617F Model group (solvent); Group 3: JAK2 V617F +Ruxolitinib treatment group (90 mg / kg / day, positive control) Sample collection: After processing for a certain period of time, mouse bone marrow was collected.

[0032] 3. Detection method: (1) Western Blot: Detect the expression levels of proteins such as p-mTOR, p-4EBP1, EPAS1, VEGF, GLUT1, and p-STAT3 / 5.

[0033] (2) Immunohistochemistry (IHC): Bone marrow sections were stained to observe the expression of p-mTOR (mTOR activity marker) and EPAS1 and their interaction with CD34. + Co-localization of stem cells.

[0034] 4. Results The results are as follows Figure 3 As shown: Western blot results showed that, compared with the WT group, key proteins of the mTOR and EPAS1 pathways were significantly activated in the model group. Ruxolitinib only partially inhibited STAT phosphorylation, but had limited inhibitory effect on the mTOR-EPAS1 pathway.

[0035] Immunohistochemistry (IHC) showed that bone marrow section staining revealed significantly elevated expression of p-mTOR (a marker of mTOR activity) and EPAS1 in the model group, and these expression levels correlated with CD34 expression. + Stem cell co-localization indicates that this pathway is specifically activated in the mutation model.

[0036] Example 2: Single-cell sequencing analysis of metabolic gene profiles 1. Method: Take group 1 (wild type) and group 2 (JAK2) from Example 1. V617F Bone marrow mononuclear cells (BM-MNCs) from model mice were used for single-cell RNA sequencing using the 10×Genomics platform. Data analysis included cell clustering, differentially expressed gene screening, and KEGG pathway enrichment analysis, with a focus on glycolysis-related genes (such as HK2 and LDHA) and HIF target genes (such as VEGFA and SLC2A1).

[0037] 2. Results: Differential gene analysis showed that JAK2 V617F Glycolysis genes (HK2, LDHA) and HIF target genes (VEGFA, SLC2A1) were significantly upregulated in mutant cells. KEGG pathway enrichment analysis confirmed significant enrichment of the mTOR signaling pathway and the hypoxia-inducible factor signaling pathway. Figure 1 and Figure 2 ).

[0038] Example 3: Single-target therapy and dual-target therapy for JAK2 V617F Impact of treatment effect 1. Method This study aims to systematically evaluate the difference in efficacy between single-target and dual-target therapies.

[0039] (1) Animal model and grouping: JAK2 was selected. V617F Transgenic mice (MPN model) were randomly divided into 5 groups: Group 1 was the solvent control group; Group 2 was the everolimus monotherapy group (5 mg / kg / day); Group 3 was the PT2385 monotherapy group (30 mg / kg / day); Group 4 was the combination therapy group of everolimus and PT2385 (dose was the same as the single drug group); Group 5 was the ruxolitinib positive control group (90 mg / kg / day).

[0040] Six mice were administered the drug to each group for four consecutive weeks.

[0041] (2) Detection indicators and methods: Spleen weight assessment: After euthanizing the mice, the spleen was separated and weighed, and the spleen index (spleen weight / body weight) was calculated to assess the degree of splenomegaly.

[0042] Cell proliferation and apoptosis analysis: Bone marrow hematopoietic stem cells were collected, and cell proliferation activity (expressed as OD value) was detected by CCK-8 assay. Annexin V / PI double staining was performed by flow cytometry to quantify the apoptosis rate.

[0043] Metabolite detection: The levels of lactate and α-ketoglutarate (α-KG) in bone marrow supernatant were detected using targeted metabolomics technology (LC-MS), and the lactate / α-KG ratio was calculated.

[0044] Statistical analysis: Data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups, and p < 0.05 was considered statistically significant.

[0045] 2. Results The results are as follows Figure 4 and Figure 5 As shown: The combination therapy group showed the best performance in reducing splenomegaly, with spleen weight significantly lower than that of the monotherapy group and the ruxolitinib group (the spleen index of the combination therapy group decreased by about 60%, while that of the monotherapy group decreased by only 20-30%).

[0046] At the cellular level, the combination therapy significantly inhibited JAK2. V617F The mutations increased the proliferation of hematopoietic stem cells (CCK-8 showed a decrease in OD value of more than 50%) and promoted apoptosis (the apoptosis rate increased by about 3 times).

[0047] Metabolomics analysis showed that the combination therapy was the most effective in correcting metabolic abnormalities: lactate levels decreased to near normal range, α-KG levels increased significantly, and the lactate / α-KG ratio recovered from 4.5±0.6 in the model group to 1.2±0.3 (normal reference value 1.0±0.2).

[0048] Mechanistically, the dual-targeted therapy synergistically inhibited cell proliferation, promoted apoptosis, and significantly improved the cellular glycolytic and oxidative phosphorylation metabolic phenotypes.

[0049] In summary, this invention, through detailed methodological design, demonstrates that the combined use of everolimus and PT2385 produces a synergistic effect both in vivo and in vitro. The combination therapy not only significantly improves the disease phenotype of MPN (such as splenomegaly) but also mechanistically reverses metabolic reprogramming, with effects far exceeding those of monotherapy or existing JAK inhibitors. This provides solid evidence for the clinical translation of dual-targeting strategies.

[0050] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a combination of mTOR inhibitors and EPAS1 inhibitors in the preparation of a medicament for the treatment of myeloproliferative neoplasms.

2. The application according to claim 1, characterized in that, The myeloproliferative neoplasm with JAK2 V617F Mutations include one of the following: polycythemia vera, essential thrombocythemia, or essential myelofibrosis.

3. The application according to claim 1, characterized in that, The mTOR inhibitors include at least one of everolimus, rapamycin, tesiromolimus, or desfomol.

4. The application according to claim 1, characterized in that, The EPAS1 inhibitor includes at least one of PT2385, PT2399, or Belzutifan.

5. A pharmaceutical composition for treating myeloproliferative neoplasms, characterized in that, The combination contains therapeutically effective amounts of an mTOR inhibitor and an EPAS1 inhibitor.

6. The pharmaceutical composition according to claim 5, characterized in that, The mTOR inhibitor is everolimus, and the EPAS1 inhibitor is PT2385.

7. The pharmaceutical composition according to claim 5, characterized in that, The dosage of everolimus is approximately 5 mg / kg / day, and the dosage of PT2385 is approximately 30 mg / kg / day.

8. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutically acceptable carriers include one or more of the following: diluents, isotonic agents, pH adjusters, preservatives, thickeners, or sustained-release materials.

10. A combination of mTOR inhibitors and EPAS1 inhibitors for reversing JAK2 V617F The application of mutation-driven metabolic reprogramming is characterized by, The metabolic reprogramming includes an increase in lactate levels and / or a decrease in α-ketoglutarate levels.

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