Use of notch inhibitors in combination with jak-stat inhibitors in the treatment of liver cancer

Abstract

The application discloses application of a NOTCH inhibitor combined with a JAK-STAT inhibitor in treating liver cancer. The application is characterized in that different liver cancer cells and liver cancer organoids are treated by separately or jointly administering a NOTCH inhibitor DAPT and a JAK-STAT inhibitor Ruxolitinib. The results show that Ruxolitinib combined with DAPT can obviously inhibit the growth of in-vitro tumor cells and the formation of liver cancer organoids. Specific small-molecule drug treatment and stable-transfected cell strain detection show that combined inhibition of the NOTCH and JAK-STAT pathways can reduce the expression of AFP. In-vivo results prove that Ruxolitinib combined with DAPT has an obvious inhibiting effect on the tumor proliferation capacity of Hep3B. The experimental results of the application show that combined targeting of the JAK-STAT and NOTCH pathways can block the occurrence and development of liver cancer.

Description

Technical Field

[0001] This invention relates to the application of NOTCH inhibitors in combination with JAK-STAT inhibitors in the treatment of liver cancer, and belongs to the field of biomedical technology. Background Technology

[0002] The International Agency for Research on Cancer (IARC) of the World Health Organization released the latest cancer burden data in December 2020, showing that primary liver cancer ranks sixth in incidence and third in mortality among malignant tumors. However, little is currently known about the underlying mechanisms of liver cancer development. (1,2) China accounts for 45.3% of all liver cancer cases and 47.1% of all liver cancer deaths worldwide. Approximately 80% of liver cancer patients are diagnosed at an advanced stage, losing the opportunity for radical surgical resection, and the 5-year survival rate is less than 18%. (3) Sorafenib, a multi-kinase inhibitor, is approved for the treatment of patients with advanced hepatocellular carcinoma, but it only provides limited extension of patient survival. (4) Therefore, identifying key factors regulating liver cancer progression and pinpointing new therapeutic targets for liver cancer is of great significance for exploring new strategies for anti-tumor treatment or chemotherapy sensitization.

[0003] ROS1 or FRK chromosome mutations lead to the activation of janus kinase (JAK) / signal sensor and the uncontrolled and persistent activation of the transcription activator (STAT) pathway. Regulation of the JAK-STAT signaling pathway using interferon (IFN) can promote innate immunity and inhibit malignant tumor growth, suggesting that the JAK-STAT signaling pathway plays a crucial role in tumorigenesis and development. (5,6) Inhibiting the inflammatory signaling JAK / STAT can reduce prostate cancer spectrum plasticity and inhibit malignant progression. (7) Given that NOTCH and its ligands (Jagged (JAG) and Delta) are both transmembrane proteins, NOTCH plays an important role in signal transduction through autocrine or paracrine secretion of secretory factors. Its oncogenic or tumor-suppressive effects depend on the tissue and microenvironment. (8,9) Targeting both the NOTCH and JAK-STAT pathways simultaneously is highly effective in the treatment of peripheral neuropathy and foregut diseases. (10-12) Currently, there are no reports on whether combined targeting of JAK-STAT and NOTCH can significantly regulate the progression of liver cancer.

[0004] References:

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[0007] 3.Xie DY,Ren ZG,Zhou J,Fan J,Gao Q.2019 Chinese clinical guidelinesforthe management of hepatocellular carcinoma:updates andinsights.HepatobiliarySurg Nutr.2020;9(4):452-63.Epub 2020 / 08 / 25.doi:10.21037 / hbsn-20-480.PubMedPMID:32832496;PubMed Central PMCID:PMCPMC7423548.

[0008] 4.Wang C,Jin H,Gao D,Lieftink C,Evers B,Jin G,et al.Phospho-ERK isabiomarker of response to a synthetic lethal drug combination of sorafeniband MEKinhibition in liver cancer.J Hepatol.2018;69(5):1057-65.Epub 2018 / 07 / 22.doi:10.1016 / j.jhep.2018.07.004.PubMed PMID:30030148.

[0009] 5.Bayard Q,Caruso S,Couchy G,Rebouissou S,Bioulac Sage P,Balabaud C,et al.Recurrent chromosomal rearrangements of ROS1,FRK and IL6 activatingJAK / STAT pathway in inflammatory hepatocellular adenomas.Gut.2020;69(9):1667-76.Epub 2020 / 01 / 08.doi:10.1136 / gutjnl-2019-319790.PubMedPMID:31907296.

[0010] 6.Pilati C,Zucman-Rossi J.Mutations leading to constitutiveactivegp130 / JAK1 / STAT3 pathway.Cytokine Growth Factor Rev.2015;26(5):499-506.Epub 2015 / 07 / 21.doi:10.1016 / j.cytogfr.2015.07.010.PubMed PMID:26188635.

[0011] 7.Chan JM,Zaidi S,Love JR,Zhao JL,Setty M,Wadosky KM,Gopalan A,etal.Lineage plasticity in prostate cancer depends on JAK / STAT inflammatorysignaling.Science.2022 Sep 9;377(6611):1180-1191.doi:10.1126 / science.abn0478.Epub 2022 Aug 18.PMID:35981096.

[0012] 8.Lu J,Ye X,Fan F,Xia L,Bhattacharya R,Bellister S,et al.Endothelialcellspromote the colorectal cancer stem cell phenotype through a soluble formof Jagged-1.Cancer Cell.2013;23(2):171-85.Epub 2013 / 02 / 05.doi:10.1016 / j.ccr.2012.12.021.PubMed PMID:23375636;PubMed Central PMCID:PMCPMC3574187.

[0013] 9.Kopan R,Ilagan MX.The canonical Notch signaling pathway:unfoldingtheactivation mechanism.Cell.2009;137(2):216-33.Epub 2009 / 04 / 22.doi:10.1016 / j.cell.2009.03.045.PubMed PMID:19379690;PubMed Central PMCID:PMCPMC2827930.

[0014] 10. Al-Massri KF, Ahmed LA, El-Abhar HS. Pregabalin and lacosamide ameliorate paclitaxel-induced peripheral neuropathy via inhibition of JAK / STAT signaling pathway and Notch-1 receptor. Neurochem Int. 2018;120:164-71. Epub 2018 / 08 / 18. doi:10.1016 / j.neuint.2018.08.007. PubMed PMID: 30118739.

[0015] 11. Josten F, Fuss B, Feix M, Meissner T, Hoch M. Cooperation of JAK / STAT and Notch signaling in the Drosophila foregut. Dev Biol. 2004;267(1):181-9. Epub 2004 / 02 / 21. doi:10.1016 / j.ydbio.2003.11.016. PubMed PMID: 14975725.

[0016] 12. Al-Massri KF, Ahmed LA, El-Abhar HS. Mesenchymal stem cells therapy enhances the efficacy of pregabalin and prevents its motor impairment in paclitaxel-induced neuropathy in rats: Role of Notch1 receptor and JAK / STAT signaling pathway. Behav Brain Res. 2019;360:303-11. Epub 2018 / 12 / 14. doi:10.1016 / j.bbr.2018.12.013. PubMed PMID: 30543902. Summary of the Invention

[0017] The purpose of this invention is to address the technical problem of improving the treatment efficiency of liver cancer. This invention provides the application of NOTCH inhibitors combined with JAK-STAT inhibitors in the treatment of liver cancer. By combining the specific inhibitor DAPT with Ruxolitinib to target and inhibit the NOTCH and JAK-STAT pathways, this invention can significantly inhibit the growth of tumor cells in vivo and in vitro, as well as the formation of liver cancer organoids. This result reveals that NOTCH and JAK-STAT are effective targets for inhibiting the progression of liver cancer, thus providing a new strategy and approach for effective anti-tumor treatment.

[0018] To address the aforementioned problems, the present invention provides the application of NOTCH inhibitors in combination with JAK-STAT inhibitors in the preparation of drugs for treating liver cancer.

[0019] Preferably, the NOTCH inhibitor is DAPT and the JAK-STAT inhibitor is Ruxolitinib.

[0020] Preferably, the dosage form of the drug includes injections, tablets, powders, suspensions, capsules, pills, or syrups.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This invention firstly demonstrates that by combining the specific inhibitor DAPT with Ruxolitinib to inhibit NOTCH and JAK-STAT, the in vivo and in vitro growth of liver cancer cells and the formation of liver cancer organoids can be significantly suppressed. This result reveals an effective target for inhibiting liver cancer progression, thus providing a new strategy and approach for effective anti-tumor treatment. Attached Figure Description

[0023] Figure 1 Combined inhibition of the NOTCH and JAK-STAT pathways inhibited the formation of hepatocellular carcinoma clones. The formation of hepatocellular carcinoma clones under treatment with NOTCH inhibitor (10 μM DAPT) and JAK-STAT inhibitor (10 μM uxolitinib) respectively / in combination is shown in the figure after crystal violet staining after 7 days of culture.

[0024] Figure 2 Combined inhibition of the NOTCH and JAK-STAT pathways reduced organoid formation in hepatocellular carcinoma; the figure shows the changes in the number and size of organoids formed in hepatocellular carcinoma after different treatment days with NOTCH inhibitor (10 μM DAPT) and JAK-STAT inhibitor (10 μM Ruxolitinib), respectively or in combination.

[0025] Figure 3A: Combined inhibition of the NOTCH and JAK-STAT pathways reduced AFP expression in liver cancer cells; A: Bio-intelligent sensors detected AFP secretion in liver cancer cells under different treatment conditions; Liver cancer cells were treated with 1.5 × 10⁻⁶... 5 Each well was seeded into a 6-well plate and treated with NOTCH inhibitor (10 μM DAPT) and JAK-STAT inhibitor (10 μM Ruxolitinib), NOTCH activator JAG1 (10 μg / ml), and JAK-STAT activator IL6 (10 μg / ml) for 3 days. The culture supernatant was then collected for AFP secretion level detection. C-HepG2 (HepG2 cells treated with conditioned medium of hepatocyte-derived hepatic progenitor cells) served as a control. B: Protein level detection of AFP expression in NOTCH knockout, STAT3 knockout, and combined knockout stable cell lines.

[0026] Figure 4 A: Bright field plots showing changes in Hep3B tumors in vivo and in vitro after combined targeting of the NOTCH and JAK-STAT pathways. Specifically, the bright field plots of tumors in vivo and in vitro in nude mice inoculated with Hep3B for 14 days were compared between control, DAPT group, Ruxolitinib group, and combination treatment group, with n=4. B: Changes in tumor growth rate after drug treatment, specifically including changes in tumor proliferation rate in Saline group, DAPT group, Ruxolitinib group, and combination treatment group. The results showed that the combination treatment group had a statistically significant difference compared to the single drug group. n=4; P<0.05 indicates statistical significance, *P<0.05; **P<0.01. Detailed Implementation

[0027] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0029] Example

[0030] (I) Experimental Methods:

[0031] 1. Plate cloning experiment

[0032] Liver cancer cells were treated with 2×10 3Each well was seeded into a 12-well plate, and the liver cancer cell cloning ability was observed after treatment with NOTCH inhibitor (10 μM MDAPT) and JAK-STAT inhibitor (10 μM Ruxolitinib) for 7 days, respectively or in combination.

[0033] 2. Hepatocellular carcinoma organoid formation experiment

[0034] Three liver cancer samples (Case 1–3) from Renji Hospital affiliated with Shanghai Jiao Tong University School of Medicine were selected. They were treated with NOTCH inhibitor (10 μM DAPT) and JAK-STAT inhibitor (10 μM Ruxolitinib) respectively or in combination. After culturing for 7 days, organoid formation was recorded by photograph.

[0035] 3. Intelligent sensors detect AFP secretion in liver cancer cells under different treatment conditions.

[0036] Liver cancer cells were treated at a dose of 1.5 × 10⁻⁶. 5 Each well was seeded into a 6-well plate and treated with NOTCH inhibitor (10 μM M APT) and JAK-STAT inhibitor (10 μM Ruxolitinib), NOTCH activator JAG1 (10 μg / ml), and JAK-STAT activator IL6 (10 μg / ml) for 3 days. The culture supernatant was then collected for AFP secretion level detection. C-HepG2 (HepG2 cells treated with conditioned medium of hepatocyte-derived hepatic progenitor cells) served as a control.

[0037] 4. Construction of stable cell lines with single knockout of NOTCH, single knockout of STAT3, and combined knockout

[0038] Lentiviral construction was performed by Heyuan Biotechnology (Shanghai) Co., Ltd. The STAT3 (gene ID: 6774) knockdown transcript was constructed as NM_139276.3 using the viral vector pSLenti-U6-ShRNA-CMV-EGFP-F2A-Puro-WPRE. The NOTCH1 (gene ID: 4851) knockdown transcript was constructed as NM_017617.5 using the viral vector pSLenti-U6-ShRNA-CMV-mCherry-F2A-BSR_WPRE.

[0039] 5. Effects of small molecule drugs DAPT and Ruxolitinib on the in vivo tumorigenesis ability of Hep3B.

[0040] Hep3B proliferates in the pairwise phase at a rate of 5 × 10 6Sixteen nude mice were injected with 100 μl of ruxolitinib via the axilla of their right forelimb. Fourteen days later, the mice were randomly divided into four groups of four: a control group, a DAPT group, a ruxolitinib group, and a combination therapy group. Mice were treated with 0.4 mg / ml ruxolitinib, 20 mg / kg DAPT, and the combination therapy group via gavage every three days. Tumor length and short diameter, as well as body weight, were recorded using calipers. The experimental endpoint was reached when the control group mice experienced severe weight loss (20% of their body weight) and emaciation, at which point samples were collected for further analysis.

[0041] (II) Experimental Results:

[0042] After 7 days of treatment with the NOTCH inhibitor DAPT or the JAK-STAT inhibitor Ruxolitinib on different hepatocellular carcinoma cell lines, crystal violet staining experiments revealed that combined inhibition of the NOTCH and JAK-STAT pathways reduced the clonogenic ability of hepatocellular carcinoma cells. Figure 1 As shown. The hepatocellular carcinoma organoid formation experiment further corroborates that simultaneous inhibition of the JAK-STAT and NOTCH pathways suppresses hepatocellular carcinoma cell proliferation. Figure 2 Specific small molecule drug treatment and stable cell line detection showed that combined inhibition of the NOTCH and JAK-STAT pathways reduced AFP expression in the HepG2 tumor cell line. Figure 3 In vivo results demonstrated that 0.4 mg / ml Ruxolitinib combined with 20 mg / kg DAPT significantly inhibited the growth rate of Hep3B tumors, while treatment with Ruxolitinib or DAPT alone did not show a significant difference in tumor growth rate compared to the control group. Figure 4 ).

[0043] The above experimental results suggest that combined targeting of the JAK-STAT and NOTCH pathways can inhibit the development and progression of liver cancer. This invention not only enriches the application of small molecule drugs in the treatment of liver cancer, but also provides new molecular targets for intervening in the development and progression of liver cancer.

[0044] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. Use of a NOTCH inhibitor in combination with a JAK-STAT inhibitor for the manufacture of a medicament for the treatment of hepatocellular carcinoma, characterized in that, The NOTCH inhibitor is DAPT and the JAK-STAT inhibitor is Ruxolitinib.

2. Use according to claim 1, wherein The dosage form of the drug includes injection, tablet, powder, suspension, capsule, pill or syrup.

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