WO2024017406A1 - Use of mek/erk signal pathway inhibitor in preparation of drug for treating myeloproliferative neoplasms - Google Patents

Abstract

The invention relates to the technical field of medicines, in particular to a use of an MEK/ERK signal pathway inhibitor in the preparation of a drug for treating myeloproliferative neoplasms. The MEK/ERK signaling pathway inhibitor is used for treating myeloproliferative neoplasms, providing a new treatment approach for patients with myeloproliferative neoplasms, and more choices for clinicians and patients. For patients with myeloproliferative neoplasms who are resistant to ruxolitinib, the MEK/ERK signaling pathway inhibitor can provide continuous oral drug treatment for the patients, without needing bone marrow transplantation. The MEK/ERK signal pathway inhibitor can be chemically synthesized, and the cost is lower than that of a biological preparation. In addition, the inhibitors in the present specification have all passed phase I clinical trial, and have light side reactions and good tolerance of clinical patients.

Description

Application of MEK/ERK signaling pathway inhibitors in the preparation of drugs for the treatment of myeloproliferative tumors

This application requires priority for the Chinese patent application submitted to the China Patent Office on July 19, 2022, with the application number 202210846544.4 and the invention title "Application of MEK/ERK signaling pathway inhibitors in the preparation of drugs for the treatment of myeloproliferative tumors" rights, the entire contents of which are incorporated herein by reference.

Technical field

The present invention relates to the field of medical technology, and in particular to the use of MEK/ERK signaling pathway inhibitors in the preparation of drugs for the treatment of myeloproliferative tumors.

Background technique

Myeloproliferative neoplasms (MPNs) refer to a group of tumor diseases caused by the clonal proliferation of one or more lines of relatively mature bone marrow cells. The clinical manifestations are the proliferation of one or more blood cells, accompanied by enlargement of the liver, spleen or lymph nodes. In 2016, the World Health Organization (WHO) revised the classification of bone marrow tumors, including polycythemia vera (PV), primary myelofibrosis (PMF), and essential thrombocythemia (ET). Included in the category of Philadelphia-negative classic myeloproliferative neoplasms. Myeloproliferative neoplasms are clonal hematopoietic stem cell diseases. The main disease driver gene mutations include JAK2/V617F, CALR, and MPL mutations. Among them, JAK2/V617F mutation is the most common type and can be found in 95% of PV and 50-60% of cases. ET and 55-65% of patients with PMF. The activation of genes leads to the activation of the JAK-STAT pathway, which leads to the occurrence of diseases. There are approximately 200,000 new patients with myeloproliferative neoplasms worldwide every year, placing a heavy burden on the medical and health systems.

Before the advent of Ruxolitinib (RUX), commonly used therapeutic drugs for myeloproliferative tumors included hydroxyurea and polyethylene glycol-recombinant interferon-α2a. Hydroxyurea can only relieve symptoms but cannot inhibit clonal hematopoiesis. Long-term use may increase the risk of myelodysplastic syndrome and acute myeloid leukemia. However, the use of interferon is limited due to its high toxic and side effects. Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved by the FDA as first-line treatment for intermediate and high-risk bone marrow fibrosis. Myelofibrosis (MF), and as a second-line drug for PV patients who are resistant or intolerant to hydroxyurea (Hydroxyurea, HU). The results of phase II and phase III clinical trials suggest that RUX can reduce spleen volume and symptoms in patients with intermediate and high-risk MF and PV compared with optimal therapy. However, there are many problems during the use of ruxolitinib. The results of the COMFORT and RESPONSE clinical trials showed that MF patients treated with ruxolitinib had more severe anemia. What’s more serious is that long-term use of type I JAK inhibitors such as RUX can induce the occurrence of drug resistance. Among MF patients who received treatment for one year, more than 40% of patients developed drug resistance. Several JAK inhibitors have also been found in clinical studies. Cross-resistance between inhibitors. In August 2019, the US FDA approved the new oral JAK2 selective inhibitor Fedratinib For adults with intermediate- to high-risk primary or secondary (post-PV or post-ET) MF, including patients previously treated with ruxolitinib. The FDA also issued a black box warning that Fedratinib may cause encephalopathy, including the risk of Wernicke's encephalopathy. To further evaluate the efficacy and safety of fedratinib, a new multicenter phase IIIb clinical trial (NCT03755518) is ongoing. Current JAK inhibitors are unable to significantly reduce mutant allele burden and therefore have limited therapeutic potential. Bone marrow transplantation is the only cure for myeloproliferative neoplasms, but there are still issues that need to be addressed. The choice of transplantation modality and regimen is uncertain, and the choice of allogeneic or haploidentical transplantation is unclear. Furthermore, when choosing transplantation, transplantation-related mortality and the long-term nature of myeloproliferative neoplasms must be considered. At present, bone marrow transplantation is mainly used to treat high-risk myelofibrosis patients, but the timing of bone marrow transplantation for patients with other types of myeloproliferative neoplasms needs further exploration and research to confirm. Bone marrow transplantation is expensive, and in the current medical environment, this treatment is not an option for most patients.

Although ruxolitinib is a landmark drug in the treatment of myeloproliferative tumors, ruxolitinib currently has a narrow scope of application, and myelosuppression as a common side effect limits its application in the main indication MF. Ruxolitinib cannot reduce the burden of mutated genes, which means that ruxolitinib treatment cannot achieve molecular-level remission of the disease and cannot fundamentally treat myeloproliferative tumors. Especially after the emergence of ruxolitinib resistance, the limited number of therapeutic drugs is a major challenge.

Contents of the invention

In view of this, the purpose of the present invention is to address the problems existing in the current treatment of myeloproliferative neoplasms, and to treat patients with myeloproliferative neoplasms, especially myeloproliferative neoplasms that are resistant to ruxolitinib. People provide an effective, safe and reliable medicine.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

The present invention provides the use of MEK/ERK signaling pathway inhibitors in preparing drugs for treating myeloproliferative tumors.

Preferably, the myeloproliferative tumor is a myeloproliferative tumor that is resistant to chemotherapy drugs.

Preferably, the chemotherapeutic drug is a chemotherapeutic drug for treating myeloproliferative tumors, and the chemotherapeutic drug includes at least one of ruxolitinib and fizotinib.

Preferably, the myeloproliferative tumor is polycythemia vera, essential thrombocythemia or myelofibrosis, and the myelofibrosis is primary myelofibrosis or myelofibrosis secondary to polycythemia vera. and at least one of myelofibrosis secondary to essential thrombocythemia.

Preferably, the MEK/ERK signaling pathway inhibitor is at least one of Trematinib, Cobimetinib, Binimetinib, Selumetinib, Mirdametinib, Refametinib, TIC10, Ulixertinib, PD184352, and Pimasertib.

Trametinib (GSK1120212, JTP-74057, Mekinist) is a highly specific and effective MEK1/2 inhibitor that was approved by the US FDA in 2013. It is suitable for surgically unresectable melanoma or patients carrying BRAF V600E or V600K mutations. Treatment of adult patients with metastatic melanoma. On January 8, 2014, the FDA approved the combination of dabrafenib and trametinib for the treatment of patients with BRAF V600E/K mutant metastatic melanoma. On May 1, 2018, the FDA approved the combined use of dabrafenib/trametinib as adjuvant therapy. It is the first oral chemotherapy regimen to prevent cancer recurrence in node-positive, BRAF-mutated melanoma after surgical resection of BRAF V600E-mutated stage III melanoma, according to results from the Phase 3 COMBI-AD study. The molecular formula of Trametinib is C ₂₆ H ₂₃ FIN ₅ O ₄ , the molecular weight is 615.39, and the structural formula is shown in Formula I.

Cobimetinib (GDC-0973, RG7420) is an effective and highly selective MEK1 inhibitor that was approved by the FDA on November 10, 2015. It is used in combination with vemurafenib to treat migratory melanoma with BRAF V600E or V600K mutations. NCT03695380 is currently recruiting for a Phase I clinical trial for the treatment of ovarian tumors. The molecular formula of Cobimetinib is C ₂₁ H ₂₁ F ₃ IN ₃ O ₂ , the molecular weight is 531.31, and the structural formula is shown in Formula II.

Binimetinib is a reversible inhibitor of mitogen-activated extracellular signal-regulated kinase 1 (MEK1) and cytokine MEK2 activity. MEK protein is an upstream regulator of the extracellular signal-related kinase (ERK) pathway. In vitro, binimetinib inhibits extracellular signal-related kinase (ERK) phosphorylation immunoassays in cells as well as viability and MEK-dependent phosphorylation of BRAF-mutant human melanoma cells. Binimetinib also inhibits ERK phosphorylation and tumor growth in BRAF mutant mouse xenograft models. The FDA approved binimetinib in combination with encorafenib for the treatment of patients with unresectable or metastatic melanoma who have BRAF V600E or V600K mutations. The molecular formula of Binimetinib is C ₁₇ H ₁₅ BrF ₂ N ₄ O ₃ , the molecular weight is 441.23, and the structural formula is shown in Formula III.

Selumetinib, also known as AZD6244 and Y-142886, is a highly efficient, non-ATP competitive inhibitor of MEK1/2 and ERK1/2. Selumetinib also has significant efficacy in multiple tumor models, significantly inhibiting ERK activity, inhibiting tumor growth, and inhibiting lung metastasis. On April 10, 2020, the U.S. FDA announced the approval of Koselugo (Selumetinib) capsules for the treatment of pediatric patients 2 years old and older with neurofibromatosis type I (NF1). This is the first FDA-approved treatment for NF1. The drug is specifically indicated for the treatment of pediatric patients with symptomatic, inoperable plexiform neurofibromas. The molecular formula of Selumetinib is C ₁₇ H ₁₅ BrClFN ₄ O ₃ ,

Mirdametinib is an oral, small molecule inhibitor of MEK1 and MEK2. Mirdametinib The European Commission (EC) and the US FDA have granted Mirdametinib (formerly known as PD-0325901) orphan drug designation for the treatment of neurofibromatosis type 1 (NF1). The molecular formula of Mirdametinib is C ₁₆ H ₁₄ F ₃ IN ₂ O ₄ , the molecular weight is 482.19, and the structural formula is shown in Formula V.

Refametinib (RDEA119) is a potent, non-ATP competitive, highly selective inhibitor of MEK1 and MEK2 with IC50 of 19nM and 47nM respectively. Refametinib is an oral MEK inhibitor with antitumor activity in combination with sorafenib for the treatment of patients with RAS-mutated hepatocellular carcinoma (HCC). CLIN CANCER RES once published an article reporting the efficacy of Refametinib alone and Refametinib combined with Sorafenib in the treatment of patients with unresectable or metastatic HCC with RAS mutations. Refametinib is currently undergoing a Phase II clinical study in advanced biliary tract cancer. The molecular formula of Refametinib is C ₁₉ H ₂₀ F ₃ IN ₂ O ₅ S, the molecular weight is 572.34, and the structural formula is shown in Formula VI.

TIC10 (ONC201) inhibits Akt and ERK activity, induces TNF-related apoptosis-inducing ligand (TRAIL) through FoxO3a, can penetrate the blood-brain barrier, has super stability, and improved pharmacokinetic properties. TIC10 causes tumor cell cell surface Significant and long-lasting expression of TRAIL on the face. In HCT116p53-/- cells, TIC10 also caused TRAIL-mediated apoptosis. Furthermore, TIC10 simultaneously inactivates Akt and ERK, leading to nuclear translocation of Foxo3a and subsequent upregulation of TRAIL. In tumor xenograft mice, TIC10 has TRAIL-dependent antitumor effects, causing tumor-specific cell death through TRAIL-mediated direct and bystander effects. This molecule has been found to be effective against a variety of solid tumors and has already carried out clinical phase 1 and 2 trials. Currently, it is difficult to advance clinical trials due to compound patent disputes. The molecular formula of TIC10 is C ₂₄ H ₂₆ N ₄ O, the molecular weight is 386.49, and the structural formula is shown in Formula VII.

Ulixertinib (BVD-523, VRT752271) is a potent reversible ERK1/ERK2 inhibitor with an IC50 of ERK2<0.3nM and can be taken orally. Ulixertinib has shown promising results in clinical trials in patients with advanced solid tumors. The molecular formula of Ulixertinib is C ₂₁ H ₂₂ Cl ₂ N ₄ O ₂ , the molecular weight is 433.33, and the structural formula is shown as Formula VIII.

PD184352 (CI-1040) is an ATP non-competitive MEK1/2 inhibitor with an IC50 of 17nM in cell assays and is 100 times more selective for MEK1/2 than MEK5. PD184352(CI-1040) selectively induces apoptosis. PD184352 is the first MEK to enter clinical trials Inhibitors have been stopped in phase II clinical trials due to problems such as poor solubility, short half-life, low oral bioavailability, and large individual differences. The molecular formula of PD184352 is C ₁₇ H ₁₄ ClF ₂ IN ₂ O ₂ , the molecular weight is 478.67, and the structural formula is shown in Formula IX.

Pimasertib (AS-703026, MSC1936369B, SAR 245509) is a highly selective, ATP-noncompetitive, orally available MEK1/2 allosteric inhibitor with an IC50 of 5nM-2μM in MM cell lines. Pimasertib has been studied in more than 10 Phase 1/2 clinical trials in approximately 900 patients with various tumor types. Pimasertib, in combination with DAY101, is indicated for the treatment of patients ≥12 years of age with recurrent, progressive, or refractory solid tumors with MAPK pathway aberrations. The molecular formula of Pimasertib is C ₁₅ H ₁₅ FIN ₃ O ₃ , the molecular weight is 431.20, and the structural formula is shown in Formula X.

Preferably, the treatment includes inhibiting the proliferation of tumor cells and/or promoting the growth of tumor cells. Apoptosis.

The present invention also provides the use of MEK/ERK signaling pathway inhibitors in preparing drugs that inhibit the proliferation of HEL cells and/or promote the apoptosis of HEL cells.

Wherein, the HEL cells include non-drug-resistant HEL cells and/or drug-resistant HEL cells.

Experiments have shown that for non-drug-resistant HEL cells, Trematinib, Cobimetinib, Binimetinib, Selumetinib, Mirdametinib, Refametinib, Ulixertinib, PD184352, and Pimasertib cannot promote apoptosis of HEL cells, and only TIC10 can promote apoptosis. Among them, Trematinib and TIC10 can inhibit the proliferation of HEL cells, Pimasertib has a weak inhibitory effect on HEL cell proliferation, and the other inhibitors have no inhibitory effect.

For drug-resistant HEL cells, Trematinib, Cobimetinib, Binimetinib, Selumetinib, Mirdametinib, Refametinib, TIC10, Ulixertinib, PD184352, and Pimasertib can all inhibit the proliferation of drug-resistant HEL cells. Among them, only Refametinib can promote the apoptosis of drug-resistant HEL cells. Death. This shows that Refametinib has a more significant therapeutic effect on drug-resistant myeloproliferative tumors.

The above results show that MEK/ERK signaling pathway inhibitors have a strong inhibitory effect on the proliferation of drug-resistant HEL cells, and some inhibitors can also promote cell apoptosis. This indicates that MEK/ERK signaling pathway inhibitors have a significant therapeutic effect on drug-resistant myeloproliferative tumors.

Preferably, the medicine also contains other pharmaceutical ingredients for treating myeloproliferative tumors and pharmaceutically acceptable excipients.

Preferably, the pharmaceutical dosage form is an oral preparation or an injection preparation.

It can be seen from the above technical solutions that the present invention provides the use of MEK/ERK signaling pathway inhibitors in the preparation of drugs for the treatment of myeloproliferative tumors. The myeloproliferative tumors are polycythemia vera, essential thrombocythemia, or myelofibrosis and ruxolitinib-resistant myeloproliferative tumors, and the myelofibrosis is primary myelofibrosis, secondary myelofibrosis, or ruxolitinib-resistant myeloproliferative tumors. Myelofibrosis arising from polycythemia vera or secondary to essential thrombocythemia. The technical effects of the present invention are:

The use of MEK/ERK signaling pathway inhibitors in the treatment of myeloproliferative tumors provides a new treatment approach for patients with myeloproliferative tumors and provides more options for clinicians and patients. select. For patients with ruxolitinib-resistant myeloproliferative tumors, MEK/ERK signaling pathway inhibitors can provide patients with continued oral drug therapy and avoid bone marrow transplantation. MEK/ERK signaling pathway inhibitors can be chemically synthesized and the cost is lower than biological agents. Moreover, the MEK/ERK signaling pathway inhibitors under application have all passed Phase I clinical trials, and some have successfully passed Phase II and III clinical trials. They will be used in clinical treatments in the future and have good clinical application prospects.

Description of drawings

Figure 1 shows Example 1: The establishment results of ruxolitinib-resistant myeloproliferative tumor cell model HEL ^RE ;

Figure 2 shows the results of Example 2: MEK/ERK signaling pathway inhibitors treated myeloproliferative tumor cells, and the cell proliferation was detected by the CellTiter-Lumi ^TM luminescence method; a is the result of HEL cell proliferation treated with Trematinib; b is the result of HEL cells treated with Cobimetinib. Cell proliferation results; c is Binimetinib-treated HEL cell proliferation results; d is Selumetinib-treated HEL cell proliferation results; e is Mirdametinib-treated HEL cell proliferation results; f is Refametinib-treated HEL cell proliferation results; g is TIC10-treated HEL. The cell proliferation result chart; h is the HEL cell proliferation result chart treated with Ulixertinib; i is the HEL cell proliferation result chart treated with PD184352; j is the HEL cell proliferation result chart treated with Pimasertib;

Figure 3 shows the results of Example 3: MEK/ERK signaling pathway inhibitors treated myeloproliferative tumor cells, using AnnexinV-PI staining to detect cell apoptosis by flow cytometry; a is the result of Trematinib treatment of HEL cell apoptosis; b is the result of apoptosis of HEL cells treated with Trematinib; Cobimetinib treated HEL cell apoptosis results; c is Binimetinib treated HEL cell apoptosis results; d is Selumetinib treated HEL cell apoptosis results; e is Mirdametinib treated HEL cell apoptosis results; f is Refametinib treated HEL cell apoptosis Result graph; g is the graph of the apoptosis result of HEL cells treated with TIC10; h is the graph of the apoptosis result of HEL cells treated with Ulixertinib; i is the graph of the apoptosis result of HEL cells treated with PD184352; j is the graph of the apoptosis result of HEL cells treated with Pimasertib;

Figure 4 shows Example 4: MEK/ERK signaling pathway inhibitors treated ruxolitinib-resistant myeloproliferative tumor cells, and the results of detecting cell proliferation by CellTiter-Lumi ^TM luminescence assay; a shows the proliferation of HEL ^RE cells treated with Trematinib. Result chart; b is the result chart of HEL ^RE cell proliferation treated with Cobimetinib; c is the result chart of HEL ^RE cell proliferation treated with Binimetinib; d is Selumetinib The result of HEL ^RE cell proliferation after treatment; e is the result of HEL ^RE cell proliferation treated with Mirdametinib; f is the result of HEL ^RE cell proliferation treated with Refametinib; g is the result of HEL ^RE cell proliferation treated with TIC10; h is the result of HEL ^RE cell proliferation treated with Ulixertinib. Figure; i is the result of HEL ^RE cell proliferation treated with PD184352; j is the result of HEL ^RE cell proliferation treated with Pimasertib;

Figure 5 shows Example 5: MEK/ERK signaling pathway inhibitors treated ruxolitinib-resistant myeloproliferative tumor cells, and the results of flow cytometry detection of cell apoptosis after staining with AnnexinV-PI; a is Trematinib treatment of HEL ^RE Cell apoptosis results; b is Cobimetinib-treated HEL ^RE cell apoptosis results; c is Binimetinib-treated HEL ^RE cell apoptosis results; d is Selumetinib-treated HEL ^RE cell apoptosis results; e is Mirdametinib-treated HEL ^RE cell apoptosis f is the result of apoptosis of HEL ^RE cells treated with Refametinib; g is the result of apoptosis of HEL ^RE cells treated with TIC10; h is the result of apoptosis of HEL ^RE cells treated with Ulixertinib; i is the result of apoptosis of HEL ^RE cells treated with PD184352 Figure; j shows the apoptosis results of HEL ^RE cells treated with Pimasertib.

Detailed ways

The present invention discloses the application of MEK/ERK signaling pathway inhibitors in the preparation of drugs for the treatment of myeloproliferative tumors. Persons skilled in the art can learn from the contents of this article and appropriately improve the process parameters for implementation. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant persons can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of this invention.

In the present invention, the inhibitory effect of MEK/ERK signaling pathway inhibitors on myeloproliferative tumor cells (drug-resistant and non-drug-resistant) is clarified through a cell line model.

In some embodiments, the present invention establishes ruxolitinib resistance based on two commonly used human myeloproliferative tumor cell lines containing JAK2-V617F mutations, namely HEL cells (Human erythroleukemia cell line, human erythroleukemia cells). Drug Cell Model HEL ^RE . The drug resistance model is constructed using Start adding ruxolitinib at the IC50 concentration of the cells and slowly increase it to a high concentration to keep the cells from being killed. Compare the IC50 to verify whether the model is successfully constructed.

In some embodiments, the present invention uses increasing concentrations of the MEK/ERK signaling pathway to inhibit The myeloproliferative tumor cell lines were treated with the agent, and the cell proliferation was detected by CellTiter-Lumi ^TM luminescence method. The results show that some MEK/ERK signaling pathway inhibitors can successfully inhibit the proliferation of HEL cells.

In some embodiments, the present invention uses increasing concentrations of MEK/ERK signaling pathway inhibitors to treat myeloproliferative tumor cell lines, and uses AnnexinV-PI staining to detect cell apoptosis using flow cytometry. The results show that some MEK/ERK signaling pathway inhibitors can promote the apoptosis of HEL cells.

It can be seen that some MEK/ERK signaling pathway inhibitors can be used to treat myeloproliferative tumor diseases.

Further, in some embodiments, the present invention uses increasing concentrations of MEK/ERK signaling pathway inhibitors to treat ruxolitinib-resistant myeloproliferative tumor cells, and detect cell proliferation through the CellTiter-Lumi ^TM luminescence method. The results showed that inhibitors of the MEK/ERK signaling pathway could inhibit the proliferation of HEL ^RE cells.

In some embodiments, the present invention uses increasing concentrations of MEK/ERK signaling pathway inhibitors to treat ruxolitinib-resistant myeloproliferative tumor cells, and uses AnnexinV-PI staining to detect the apoptosis of the cells by flow cytometry. The results show that some MEK/ERK signaling pathway inhibitors can promote the apoptosis of HEL ^RE cells.

It can be seen that MEK/ERK signaling pathway inhibitors can be used to treat ruxolitinib-resistant myeloproliferative tumor diseases.

Furthermore, the present invention provides the use of some MEK/ERK signaling pathway inhibitors in the preparation of drugs that inhibit the proliferation of HEL cells and promote the apoptosis of HEL cells.

In summary, the present invention provides the use of MEK/ERK signaling pathway inhibitors in the preparation of drugs for treating myeloproliferative tumors.

Further, the myeloproliferative tumors are polycythemia vera, essential thrombocythemia and myelofibrosis (including primary myelofibrosis, myelofibrosis secondary to polycythemia vera and myelofibrosis secondary to primary myelofibrosis). Thrombocytosis, myelofibrosis) and drug-resistant myeloproliferative neoplasms.

In some embodiments, the drug-resistant myeloproliferative neoplasm is a ruxolitinib-resistant myeloproliferative neoplasm.

In some embodiments, the drug-resistant myeloproliferative neoplasm is drug-resistant polycythemia vera, drug-resistant myelofibrosis (including primary myelofibrosis, secondary myelofibrosis Polycythemia myelofibrosis and myelofibrosis secondary to essential thrombocythemia) and drug-resistant essential thrombocythemia.

Among them, the drugs are Trematinib, Cobimetinib, Binimetinib, Selumetinib, Mirdametinib, Refametinib, TIC10, Ulixertinib, PD184352, and Pimasertib.

Furthermore, the medicine also includes pharmaceutically acceptable excipients.

The medicine can be in any dosage form in the current pharmaceutical field, including oral preparations or injection preparations.

Each pharmaceutical dosage form can be prepared by selecting appropriate acceptable excipients according to the actual needs of the dosage form, which is a conventional dosage form preparation technology in this field. Such as making capsules, tablets, injection powders, etc.

All reagents or instruments used in the present invention can be purchased from the market.

The present invention will be further described below in conjunction with the examples:

Example 1. Establishment of two common ruxolitinib-resistant cell models (HEL ^RE ).

1. Materials and methods

1. Cell line

HEL (Human erythroleukemia cell line) and ruxolitinib-resistant HEL cells were cultured in RPMI medium (Gibco) containing 20% heat-inactivated fetal calf serum (Gibco) and 1% penicillin/streptomycin.

The ruxolitinib-resistant HEL model is the HEL ^RE model. The construction method is to start adding ruxolitinib at a concentration lower than the IC50 of the original cells and slowly increase it to a high concentration to maintain the cells from being killed. Our starting concentration is 0.1 μM, and the drug is added when cells proliferate. The drug addition gradient is 1.25 times increments, and the final concentration is 2.0 μM. Stable drug-resistant cells were obtained after 4-6 weeks.

2. Inhibitors

Ruxolitinib and MEK/ERK signaling pathway inhibitors were purchased from Selleck Company, dissolved in DMSO, the concentration of the mother solution was 10mM, and frozen at -80°C. The working solution was diluted to the specified multiple with RPMI medium before processing the cells. Ruxolitinib is specifically ruxolitinib phosphate.

3. In vitro inhibition test

In order to detect the anti-proliferative effect of inhibitors, the above cell lines were cultured at 3000 cells/100 cells per well. μL system culture, add increasing concentrations of ruxolitinib (HEL cell concentration gradient: 0, 0.1, 0.3, 1, 3, 10 μM; or 0, 0.001, 0.003, 0.01, 0.03, 0.1 μM), and DMSO is added until equal. quantity. Set up 4 parallel replicate groups and set up 3 blank wells (wells containing culture medium without cells). After 48 hours, the cell proliferation was detected by CellTiter-Lumi ^TM luminescence method (Beyotime). Multifunctional microplate reader reading, IC50 is calculated by GraphPadprism.

Calculation formula for cell proliferation rate: cell proliferation rate = (Luminescence value of drug-added group - average Luminescence value of blank wells) / (Luminescence value of DMSO control group - average Luminescence value of blank wells) × 100%.

The method to evaluate whether the model is successfully constructed is to compare the IC50 of drug-resistant cells and original cells. The ratio is the drug resistance index. If the ratio is greater than 3, the construction is successful.

2. Result analysis

In Figure 1, the IC50 of HEL cells is 0.801 μM, the IC50 of HEL ^RE cells is 12.27 μM, and its drug resistance index is 15.3, indicating the successful construction of the drug resistance model HEL ^RE . The proliferation rate results in the figure are shown as the mean ± standard deviation. The comparison of the proliferation rates of the two groups in the figure is using t test (***p<0.001), and the IC50 is shown as the mean.

Example 2. Some MEK/ERK signaling pathway inhibitors can inhibit the proliferation of myeloproliferative tumor cells.

In order to detect the effect of MEK/ERK signaling pathway inhibitors on the proliferation of drug-resistant myeloproliferative tumor cells, the method was to use increasing concentrations of ruxolitinib and MEK/ERK signaling pathway inhibitors to treat the HEL original cell line, and then use CellTiter- The Lumi ^TM luminescence method was used to detect cell proliferation. The method was the same as in Example 1. The results are shown in Figure 2.

Figure 2 reflects that in HEL cells, the drug concentrations (average proliferation rate % ± standard deviation) in the ruxolitinib-treated group are: 0 μM (100 ± 1.47) (not shown in the figure), 0.1 μM (81.0 ± 1.35), 0.3 μM (54.9±4.35), 1μM (42.7±4.05), 3μM (34.7±1.14), 10μM (29.4±0.497); Figure 2a reflects that in HEL cells, the drug concentration (average proliferation rate) of the Trematinib treatment group is: 0μM ( 100±9.58) (not shown in the figure), 0.0001μM (93.91±2.37), 0.0003μM (98.17±3.44), 0.01μM (84.82±6.67), 0.03μM (83.96±9.48), 0.1μM (85.99±6.9) . This suggests that Trematinib can inhibit the proliferation of HEL cells. Weaker than ruxolitinib. Figure 2b reflects that in HEL cells, the drug concentrations (average proliferation rate) of the Cobimetinib treatment group are: 0 μM (100±6.6) (not shown in the figure), 0.001 μM (98.53±4.64), 0.003 μM (105.51±7.14), 0.01 μM (99.78±5.17), 0.03μM (99.66±1.93), 0.1μM (96.59±8.57). This suggests that Cobimetinib cannot inhibit the proliferation of HEL cells. Figure 2c reflects that the drug concentrations (average proliferation rate) of the Binimetinib treatment group are: 0μM (100±2.65) (not shown in the figure), 0.1μM (111±13.9), 0.3μM (105±17.2), 1μM (115±22.9) , 3μM (107±14.4), 10μM (92.4±2.44). This suggests that Binimetinib cannot inhibit the proliferation of HEL cells. Figure 2d reflects that in HEL cells, the drug concentrations (average proliferation rate) of the Selumetinib treatment group are: 0 μM (100±1.62) (not shown in the figure), 0.1 μM (107±17.6), 0.3 μM (117±16.3), 1μM (110±26.3), 3μM (103±14.5), 10μM (88.8±2.69). This suggests that Selumetinib cannot inhibit the proliferation of HEL cells. Figure 2e reflects that in HEL cells, the drug concentrations (average proliferation rate) of the Mirdametinib treatment group are: 0 μM (100±1.73) (not shown in the figure), 0.1 μM (107±17.6), 0.3 μM (117±16.3), 1μM (110±26.3), 3μM (103±14.5), 10μM (88.8±2.69). This suggests that Mirdametinib cannot inhibit the proliferation of HEL cells. Figure 2f reflects that in HEL cells, the drug concentrations (average proliferation rate) of the Refametinib treatment group are: 0μM (100±2.63) (not shown in the figure), 0.1μM (96.0±7.44), 0.3μM (99.8±4.36), 1μM (93.4±4.31), 3μM (89.1±7.23), 10μM (84.6±8.89). This suggests that Refametinib cannot inhibit the proliferation of HEL cells. Figure 2g reflects that in HEL cells, the drug concentrations (average proliferation rate) of the TIC10 treatment group are: 0μM (100±4.31) (not shown in the figure), 0.1μM (113±15.0), 0.3μM (116±14.9), 1μM (80.3±3.43), 3μM (19.6±2.64), 10μM (11.1±7.15). This suggests that TIC10 can inhibit the proliferation of HEL cells. This effect increases as the drug concentration increases, and its inhibitory effect strengthens as the concentration increases. Figure 2h reflects that in HEL cells, the drug concentrations (average proliferation rate) of the Ulixertinib treatment group are: 0 μM (100±2.58) (not shown in the figure), 0.1 μM (98.9±8.68), 0.3 μM (109±5.36), 1 μM (96.1±5.13), 3μM (100±4.61), 10μM (95.2±5.83). This suggests that Ulixertinib cannot inhibit the proliferation of HEL cells. Figure 2i reflects that in HEL cells, the drug concentrations (average proliferation rate) of the PD184352 treatment group are: 0μM (100±4.46) (not shown in the figure), 0.1μM (105±16.7), 0.3μM (118±21.2), 1μM (103±22.7), 3μM (99.0±20.3), 10μM (82.3 ±6.50). This suggests that PD184352 cannot inhibit the proliferation of HEL cells. Figure 2j reflects that in HEL cells, the drug concentrations (average proliferation rate) of the Pimasertib treatment group are: 0μM (100±3.07) (not shown in the figure), 0.1μM (89.0±1.42), 0.3μM (95.0±7.62), 1μM (86.9±1.71), 3μM (82.8±2.13), 10μM (77.3±2.52). This suggests that Pimasertib has a weak inhibitory effect on HEL cell proliferation, and its effect is weaker than ruxolitinib. The comparison of proliferation rates between the two groups in the figure uses t test (*p<0.05, **p<0.01, ***p<0.001).

Example 3. Some MEK/ERK signaling pathway inhibitors can promote apoptosis of myeloproliferative tumor cells

1. Materials and methods

1. Cell lines and inhibitors are the same as in Example 1.

2. Cell apoptosis detection

In order to detect the pro-apoptotic effect of the inhibitor, the HEL original cell line was treated with ruxolitinib and MEK/ERK signaling pathway inhibitors for 24 hours (concentration: 0, 0.1, 0.3, 1, 3, 10 μM), and supplemented with DMSO to equal amounts. Three parallel replicate groups were set up, and cell apoptosis was detected by flow cytometry after AnnexinV and PI staining.

Apoptosis rate calculation formula: Apoptosis rate = early apoptotic cell ratio (Annexin V ⁺ /PI ^- ) + late apoptotic cell and necrotic cell ratio (AnnexinV ⁺ /PI ⁺ ).

2. Result analysis

In Figure 3, the drug concentrations (average apoptosis rate % ± standard deviation) of Trematinib, Cobimetinib, TIC10, Ulixertinib, PD184352, and Pimasertib in the ruxolitinib-treated group of HEL cells in the control group are: 0 μM (7.26 ± 0.360), 0.1 μM (7.39 ±0.0600), 0.3μM (7.40±0.820), 1μM (8.58±0.490), 3μM (7.12±0.180), 10μM (6.04±0.700); Binimetinib, Selumetinib, Mirdametinib, Refametinib control HEL cell ruxolitinib treatment group The drug concentrations (average apoptosis rate %±standard deviation) are: 0μM (2.30±0.720), 0.1μM (2.30±0.210), 0.3μM (1.90±0.360), 1μM (2.4±0.50), 3μM (2.00±0.480) , 10μM (2.30±0.720). Figure 3a reflects that in HEL cells, the drug concentrations (average apoptosis rate) in the Trematinib treatment group are: 0 μM (6.94±0.690) (not shown in the figure), 0.1 μM (7.09±1.35), 0.3 μM (6.32±0.780), 1μM (6.11±1.19), 3μM (6.83±1.83), 10μM (6.41±0.240). This suggests that Trematinib cannot promote apoptosis of HEL cells. Figure 3b reflects that in HEL cells, the drug concentrations (average apoptosis rate) of the Cobimetinib treatment group are: 0 μM (5.09±0.060) (not shown in the figure), 0.1 μM (5.35±0.500), 0.3 μM (6.53±0.370), 1μM (6.31±0.670), 3μM (6.61±1.490), 10μM (5.64±0.470). This suggests that Cobimetinib cannot promote apoptosis of HEL cells. In Figure 3c, the drug concentrations (apoptosis rate) in the Binimetinib treatment group are 0 μM (1.20±0.320) (not shown in the figure), 0.1 μM (1.21±0.110), 0.3 μM (1.55±0.310), 1 μM (1.73±0.06), 3μM (1.39±0.29), 10μM (2.19±0.07), which suggests that Binimetinib cannot promote the apoptosis of myeloproliferative tumor cells. In Figure 3d, the drug concentrations (average apoptosis rate) in the Selumetinib-treated group of HEL cells are: 0 μM (1.91 ± 0.51) (not shown in the figure), 0.1 μM (2.24 ± 0.0580), 0.3 μM (2.14 ± 0.748), 1 μM ( 2.45±0.473), 3μM (1.91±0.225), 10μM (2.40±0.260), which suggests that Selumetinib cannot promote the apoptosis of myeloproliferative tumor cells. Figure 3e reflects that in HEL cells, the drug concentrations (average apoptosis rate) of Mirdametinib treatment group are: 0 μM (1.46±0.146) (not shown in the figure), 0.1 μM (1.99±0.367), 0.3 μM (2.23±0.432), 1μM (2.45±0.473), 3μM (1.91±0.225), 10μM (2.40±0.260). This suggests that Mirdametinib cannot promote apoptosis of HEL cells. Figure 3f reflects that in HEL cells, the drug concentrations (average apoptosis rate) of the Refametinib treatment group are: 0 μM (2.08±0.467) (not shown in the figure), 0.1 μM (2.78±1.02), 0.3 μM (2.21±0.369), 1μM (2.06±0.457), 3μM (2.39±0.713), 10μM (1.79±0.745). This suggests that Refametinib cannot promote apoptosis of HEL cells. Figure 3g reflects that in HEL cells, the drug concentrations (average apoptosis rate) of the TIC10 treatment group are: 0 μM (3.69±0.192) (not shown in the figure), 0.1 μM (3.19±0.516), 0.3 μM (5.63±2.50), 1μM (4.39±0.2880), 3μM (5.16±0.266), 10μM (9.43±0.445). This suggests that TIC10 can promote the apoptosis of HEL cells, and its effect increases with increasing concentration. As reflected in Figure 3h, in HEL cells, the drug concentrations (average apoptosis rate) of the Ulixertinib treatment group are: 0 μM (6.00±0.717) (not shown in the figure), 0.1 μM (5.56±1.22), 0.3 μM (6.22±1.86), 1μM (5.72±0.619), 3μM (5.99±1.73), 10μM (6.12±0.903). This suggests that Ulixertinib cannot promote apoptosis of HEL cells. Figure 3i reflects that in HEL cells, the drug concentration (average apoptosis rate) of the PD184352 treatment group was: 0 μM (3.86±0.198) (not shown in the figure), 0.1 μM (4.65± 0.959), 0.3μM (4.90±0.494), 1μM (4.72±0.161), 3μM (4.19±0.709), 10μM (4.77±0.902). This suggests that PD184352 cannot promote apoptosis of HEL cells. Figure 3j reflects that in HEL cells, the drug concentrations (average apoptosis rate) of the Pimasertib treatment group are: 0 μM (3.98±0.115) (not shown in the figure), 0.1 μM (4.61±0.535), 0.3 μM (5.74±1.68), 1μM (1.37±0.290), 3μM (6.37±0.272), 10μM (4.89±0.182). This suggests that Pimasertib cannot promote apoptosis of HEL cells. The comparison of apoptosis rates between the two groups in the figure uses t test (*p<0.05, **p<0.01, ***p<0.001).

Example 4. MEK/ERK signaling pathway inhibitors can inhibit the proliferation of drug-resistant myeloproliferative tumor cells.

In order to detect the effect of MEK/ERK signaling pathway inhibitors on the proliferation of drug-resistant myeloproliferative tumor cells, the method was to use increasing concentrations of ruxolitinib and MEK/ERK signaling pathway inhibitors to treat HEL ^RE through CellTiter-Lumi ^TM Cell proliferation was detected by luminescence. The method is the same as in Example 1, and the results are shown in Figure 4.

Figure 4 reflects that in HEL ^RE cells, the drug concentrations (average proliferation rate % ± standard deviation) of the ruxolitinib treatment group are: 0 μM (100 ± 3.79) (not shown in the figure), 0.1 μM (105 ± 2.08), 0.3μM (105±3.60), 1μM (104±9.46), 3μM (109±4.40), 10μM (96.0±3.69); Figure 4a reflects that in HEL ^RE cells, the drug concentration (average proliferation rate) of the Trematinib treatment group is: 0μM (100±2.27) (not shown in the figure), 0.001μM (99.38±2.75), 0.003μM (94.51±3.41), 0.01μM (67.59±6.26), 0.03μM (51.61±2.5), 0.1μM (40.45± 4.5). This suggests that Trematinib can effectively inhibit the proliferation of HEL ^RE cells. Figure 4b reflects that in HEL ^RE cells, the drug concentrations (average proliferation rate) of the Cobimetinib treatment group are: 0 μM (100±2.22) (not shown in the figure), 0.001 μM (94.82±3.95), 0.003 μM (101.27±4.23), 0.01μM (98.06±2.53), 0.03μM (83.06±2.48), 0.1μM (58.69±3.51). This suggests that Cobimetinib can effectively inhibit the proliferation of HEL ^RE cells. Figure 4c reflects that the drug concentrations (average proliferation rate) of the Binimetinib treatment group are: 0μM (100±4.10) (not shown in the figure), 0.1μM (67.9±3.44), 0.3μM (53.8±1.41), 1μM (50.4±3.27) , 3μM (43.1±2.18), 10μM (37.6±1.24). This suggests that Binimetinib can inhibit the proliferation of HEL ^RE cells, and the inhibition rate increases with increasing drug concentration. Figure 4d reflects the HEL ^RE fine In cells, the drug concentrations (average proliferation rate) of the Selumetinib-treated group were: 0 μM (100±1.45) (not shown in the figure), 0.1 μM (77.1±1.86), 0.3 μM (62.0±3.90), 1 μM (54.9±1.63) , 3μM (48.2±2.63), 10μM (41.0±3.57). This suggests that Selumetinib can inhibit the proliferation of HEL ^RE cells, and the inhibition rate increases with increasing drug concentration. Figure 4e reflects that in HEL ^RE cells, the drug concentrations (average proliferation rate) of the Mirdametinib treatment group are: 0 μM (100 ± 1.70) (not shown in the figure), 0.1 μM (54.8 ± 2.74), 0.3 μM (57.9 ± 4.50) , 1μM (44.2±3.38), 3μM (39.6±0.380), 10μM (32.5±3.73). This suggests that Mirdametinib can inhibit the proliferation of HEL ^RE cells, and the inhibition rate increases with increasing drug concentration. Figure 4f reflects that in HEL ^RE cells, the drug concentrations (average proliferation rate) of the Refametinib treatment group are: 0μM (100±3.85) (not shown in the figure), 0.1μM (66.0±3.12), 0.3μM (61.2±2.05), 1μM (50.6±2.09), 3μM (43.3±1.23), 10μM (36.6±2.83). This suggests that Refametinib can inhibit the proliferation of HEL ^RE cells, and the inhibition rate increases with the increase of drug concentration. Figure 4g reflects that in HEL ^RE cells, the drug concentrations (average proliferation rate) of the TIC10 treatment group are: 0μM (100±6.41) (not shown in the figure), 0.1μM (101±7.19), 0.3μM (105±3.69), 1μM (62.5±5.82), 3μM (31.3±1.45), 10μM (19.6±1.01). This suggests that TIC10 can inhibit the proliferation of HEL ^RE cells, and this effect increases with increasing drug concentration. Figure 4h reflects that in HEL ^RE cells, the drug concentrations (average proliferation rate) of Ulixertinib treatment group are: 0 μM (100±1.65) (not shown in the figure), 0.1 μM (95.6±5.27), 0.3 μM (91.4±3.49), 1μM (58.5±14.1), 3μM (48.4±7.37), 10μM (37.5±6.71). This suggests that Ulixertinib can inhibit the proliferation of HEL ^RE cells, and this effect increases with increasing drug concentration. Figure 4i reflects that in HEL ^RE cells, the drug concentrations (average proliferation rate) of the PD184352 treatment group are: 0μM (100±4.73) (not shown in the figure), 0.1μM (81.8±1.27), 0.3μM (79.6±3.79), 1μM (52.2±1.24), 3μM (42.7±0.720), 10μM (33.9±0.700). This suggests that PD184352 can inhibit the proliferation of HEL ^RE cells, and this effect increases with increasing drug concentration. Figure 4j reflects that in HEL ^RE cells, the drug concentrations (average proliferation rate) of the Pimasertib treatment group are: 0μM (100±3.71) (not shown in the figure), 0.1μM (59.3±1.78), 0.3μM (53.8±0.410), 1μM (42.2±3.46), 3μM (36.5±1.66), 10μM (30.3±2.22). This suggests that Pimasertib can inhibit the proliferation of HEL ^RE cells, and this effect increases with increasing drug concentration. In the figure, the proliferation rate of the two groups was compared using t test (*p<0.05, **p< 0.01,***p<0.001).

Example 5. Some MEK/ERK signaling pathway inhibitors can promote the apoptosis of drug-resistant myeloproliferative tumor cells.

In order to detect the effect of MEK/ERK signaling pathway inhibitors on the survival of drug-resistant myeloproliferative tumor cells, the method was to treat HEL ^RE with increasing concentrations of ruxolitinib and MEK/ERK signaling pathway inhibitors, and stain with AnnexinV and PI. The apoptosis of cells was detected by flow cytometry. The method is the same as in Example 3, and the results are shown in Figure 5.

In Figure 5, the drug concentrations (average apoptosis rate % ± standard deviation) in the ruxolitinib-treated group of HEL ^RE cells compared with Trematinib and Cobimetinib are: 0 μM (8.36 ± 0.636), 0.1 μM (12.29 ± 0.642), 0.3 μM ( 13.14±1.241), 1μM (10.82±1.245), 3μM (9.74±0.255), 10μM (10.07±0.804); Binimetinib, Selumetinib, Mirdametinib, Refametinib control HEL ^RE cell ruxolitinib treatment group drug concentration (average apoptosis Rate% ± standard deviation) are: 0μM (0.300±0.0240), 0.1μM (0.290±0.0800), 0.3μM (0.330±0.0430), 1μM (0.290±0.0800), 3μM (0.280±0.0560), 10μM (0.360±0.0260) ). The drug concentrations (average apoptosis rate % ± standard deviation) of TIC10, Ulixertinib, PD184352, and Pimasertib control HEL ^RE cells in the ruxolitinib-treated group were: 0 μM (2.15 ± 1.01), 0.1 μM (2.73 ± 0.748), 0.3 μM ( 2.11±0.989), 1μM (2.73±0.748), 3μM (2.09±0.982), 10μM (1.900±0.751); Figure 5a reflects that in HEL ^RE cells, the drug concentration (average apoptosis rate) of the Trematinib treatment group is: 0μM ( 7.13±1.46) (not shown in the figure), 0.1μM (9.42±2.48), 0.3μM (6.49±1.58), 1μM (9.56±1.39), 3μM (8.01±2.04), 10μM (7.72±0.167). This suggests that Trematinib cannot promote apoptosis in drug-resistant myeloproliferative tumor cells. Figure 5b reflects that in HEL ^RE cells, the drug concentrations (average apoptosis rate) in the Cobimetinib treatment group are: 0 μM (4.87±1.37) (not shown in the figure), 0.1 μM (3.53±1.10), 0.3 μM (1.88±0.160) , 1μM (4.09±1.46), 3μM (2.90±0.344), 10μM (2.36±0.414). This suggests that Cobimetinib cannot promote apoptosis of HEL ^RE cells. In Figure 5c, the drug concentrations (apoptosis rate) of the Binimetinib treatment group are 0 μM (0.250±0.0620) (not shown in the figure), 0.1 μM (0.220±0.0300), 0.3 μM (0.160±0.0410), 1 μM (0.270±0.0360), 3μM (0.240±0.0180), 10μM (0.220 ±0.111), which suggests that binimetinib cannot promote apoptosis of drug-resistant myeloproliferative tumor cells. In Figure 5d, the drug concentrations (average apoptosis rate) in the Selumetinib-treated group of HEL ^RE cells are: 0 μM (0.290±0.110) (not shown in the figure), 0.1 μM (0.230±0.0160), 0.3 μM (0.270±0.0790), 1 μM (0.220±0.0400), 3μM (0.310±0.0290), 10μM (0.320±0.00800), which suggests that Selumetinib cannot promote the apoptosis of drug-resistant myeloproliferative tumor cells. Figure 5e reflects that in HEL ^RE cells, the drug concentrations (average apoptosis rate) of Mirdametinib treatment group are: 0 μM (0.270±0.0250) (not shown in the figure), 0.1 μM (0.220±0.0180), 0.3 μM (0.370±0.0640) , 1μM (0.410±0.0340), 3μM (0.140±0.141), 10μM (0.330±0.0220). This suggests that Mirdametinib cannot promote apoptosis in drug-resistant myeloproliferative tumor cells. Figure 5f reflects that in HEL ^RE cells, the drug concentrations (average apoptosis rate) in the Refametinib treatment group are: 0μM (0.320±0.0670) (not shown in the figure), 0.1μM (0.320±0.0590), 0.3μM (0.330±0.0660) , 1μM (0.330±0.0710), 3μM (0.560±0.0420), 10μM (0.800±0.0810). This suggests that Refametinib can promote apoptosis of drug-resistant myeloproliferative tumor cells, and its effect increases with increasing concentration. Figure 5g reflects that in HEL ^RE cells, the drug concentrations (average apoptosis rate) in the TIC10 treatment group are: 0 μM (2.450±0.169) (not shown in the figure), 0.1 μM (2.35±0.286), 0.3 μM (1.89±0.253) , 1μM (2.03±0.271), 3μM (2.29±0.210), 10μM (2.80±0.684). This suggests that TIC10 cannot promote apoptosis in drug-resistant myeloproliferative tumor cells. As reflected in Figure 5h, in HEL ^RE cells, the drug concentrations (average apoptosis rate) in the Ulixertinib treatment group are: 0 μM (2.62±0.210) (not shown in the figure), 0.1 μM (2.28±0.450), 0.3 μM (2.53±0.382) , 1μM (2.39±0.399), 3μM (3.11±0.891), 10μM (3.29±0.284). This suggests that Ulixertinib cannot promote apoptosis in drug-resistant myeloproliferative tumor cells. Figure 5i reflects that in HEL ^RE cells, the drug concentrations (average apoptosis rate) of the PD184352 treatment group are: 0 μM (1.07±0.532) (not shown in the figure), 0.1 μM (0.650±0.157), 0.3 μM (0.990±0.153) , 1μM (0.870±0.0560), 3μM (0.970±0.0600), 10μM (1.46±0.0250). This suggests that PD184352 cannot promote apoptosis of drug-resistant myeloproliferative tumor cells. Figure 5j reflects that in HEL ^RE cells, the drug concentrations (average apoptosis rate) in the Pimasertib treatment group are: 0 μM (1.55±0.372) (not shown in the figure), 0.1 μM (1.45±0.528), 0.3 μM (1.63±0.284) , 1μM (1.43±0.189), 3μM (1.60±0.217), 10μM (1.77±0.167). This suggests that Pimasertib cannot promote drug resistance Apoptosis of myeloproliferative neoplasms. The comparison of apoptosis rates between the two groups in the figure uses t test (*p<0.05, **p<0.01, ***p<0.001).

The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as It is the protection scope of the present invention.

Claims (10)

1. Application of MEK/ERK signaling pathway inhibitors in the preparation of drugs for the treatment of myeloproliferative tumors.
2. The application according to claim 1, wherein the myeloproliferative tumor is a myeloproliferative tumor that is resistant to chemotherapy drugs.
3. The application according to claim 2, wherein the chemotherapeutic drug is a chemotherapeutic drug for treating myeloproliferative tumors, and the chemotherapeutic drug includes ruxolitinib and/or fizotinib.
4. The application according to claim 1, wherein the myeloproliferative tumor is polycythemia vera, essential thrombocythemia or myelofibrosis, and the myelofibrosis is primary myelofibrosis, secondary myelofibrosis. At least one of myelofibrosis arising from polycythemia vera and myelofibrosis secondary to essential thrombocythemia.
5. The application according to claim 1, wherein the MEK/ERK signaling pathway inhibitor is at least one of Trematinib, Cobimetinib, Binimetinib, Selumetinib, Mirdametinib, Refametinib, TIC10, Ulixertinib, PD184352, and Pimasertib.
6. The application according to claim 1, wherein the treatment includes inhibiting the proliferation of tumor cells and/or promoting the apoptosis of tumor cells.
7. The application of MEK/ERK signaling pathway inhibitors in the preparation of drugs that inhibit the proliferation of HEL cells and/or promote the apoptosis of HEL cells.
8. The application according to claim 7, wherein the HEL cells include non-drug-resistant HEL cells and/or drug-resistant HEL cells.
9. The application according to any one of claims 1 to 8, characterized in that the medicine also contains other pharmaceutical ingredients for treating myeloproliferative tumors and pharmaceutically acceptable excipients.
10. The application according to claim 9, characterized in that the dosage form of the drug is an oral preparation or an injection preparation.