Application of RRx-001 combined with phenatinib in preparation of medicine for treating extracorporeal NK / T cell lymphoma

Abstract

The invention provides an application of RRx-001 combined with Fidarinib in preparation of a medicine for treating extrajunction NK / T cell lymphoma, the influence of RRx-001 on proliferation, cycle and apoptosis of NK / T cells and a molecular mechanism of RRx-001 are researched, and experimental evidence is provided for development of more effective treatment means for ENKTL patients. Meanwhile, the invention explores the effect of the combined use of the RRx-001 and the phenatinib in the ENKTL, and discovers that the combined use of the RRx-001 and the phenatinib has a synergistic effect on the treatment of the ENKTL.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of RRx-001 combined with Fidatinib in the preparation of a drug for treating extranodal NK / T cell lymphoma. Background Art

[0002] Extranodal NK / T-cell lymphoma (nasal type, ENKTL) exhibits high expression of multidrug resistance genes, making ENKTL resistant to conventional drug treatment and highly sensitive to radiotherapy. Conventional chemotherapy regimens such as the CHOP regimen and other anthracycline-containing chemotherapy regimens are ineffective. However, the antitumor activity of L-asparaginase is not affected by multidrug resistance genes. In recent years, new treatment regimens combining chemotherapy with L-asparaginase and radiotherapy have demonstrated robust antitumor activity and improved clinical outcomes for ENKTL patients. However, even with L-asparaginase therapy, the prognosis for patients with advanced disease remains poor, with a 5-year overall survival of approximately 15%-25%. Over 10% of ENKTL patients exhibit primary resistance to L-asparaginase therapy. The prognosis for refractory / relapsed patients is even worse, and available salvage therapies are limited. Therefore, there is a need to explore more effective treatments with less toxicity and higher cure rates.

[0003] RRx-001 (1-bromoacetyl-3,3-dinitroazetidine) is a multi-targeted small molecule drug that specifically binds to glucose-6-phosphate dehydrogenase (G6PD). This compound effectively blocks key biochemical reactions in the pentose phosphate metabolic pathway (PPP), thereby disrupting energy metabolism, biomacromolecule synthesis, and redox homeostasis in malignant cells. RRx-001 can also downregulate c-Myc and CD47 expression, thereby blocking tumor immune escape signaling and enhancing phagocytosis of tumor cells. However, the role and mechanism of RRx-001 in ENKCL remain unknown. Summary of the Invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0005] As one aspect of the present invention, the present invention provides the use of RRx-001 combined with Fidatinib in the preparation of a medicament for treating extranodal NK / T cell lymphoma.

[0006] Among them, RRx-001 can enhance the apoptosis of extranodal NK / T cell lymphoma cells induced by Fidatinib.

[0007] Among them, the molar ratio of RRx-001 to Fidatinib is 1 to 16:1.

[0008] Among them, the molar ratio of RRx-001 to Fidatinib is 3:1.

[0009] Among them, the RRx-001 induces increased ROS levels in extranodal NK / T cell lymphoma cells.

[0010] Among them, the RRx-001 induces endoplasmic reticulum stress response in extranodal NK / T cell lymphoma cells.

[0011] Beneficial Effects of the Invention: This study investigates the effects of RRx-001 on the proliferation, cycle, and apoptosis of NK / T cells, as well as the underlying molecular mechanisms, providing experimental evidence for the development of more effective treatments for ENKTL patients. Furthermore, this study explored the combined effects of RRx-001 and fedratinib in ENKTL and found that the combination of RRx-001 and fedratinib exhibits a synergistic effect in the treatment of ENKTL. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments, wherein:

[0013] Figure 1 This is the experimental result of RRx-001 inhibiting ENKTL cell proliferation.

[0014] Figure 2 This is the experimental result of RRx-001 blocking ENKTL cell cycle.

[0015] Figure 3 This is the experimental result of RRx-001 affecting the expression of ENKTL cell cycle-related genes.

[0016] Figure 4 This is the experimental result of RRx-001 inducing ENKTL cell apoptosis.

[0017] Figure 5 This is the experimental result of RRx-001 inducing ENKTL exogenous cell apoptosis pathway.

[0018] Figure 6 This is the experimental result of RRx-001 inducing ENKTL exogenous cell apoptosis pathway.

[0019] Figure 7 This is the flow cytometry result of the experiment to detect the increase of ROS level in NK / T lymphoma cells induced by RRx-001.

[0020] Figure 8This figure shows the experimental results of the effect of RRx-001 on the endoplasmic reticulum stress pathway in YT and YTS cells.

[0021] Figure 9 This is the experimental result of western blot detection of the effect of RRx-001 on the JAK / STAT signaling pathway.

[0022] Figure 10 This is the experimental result showing the synergistic effect between RRx-001 and Fedratinib.

[0023] Figure 11 This is the experimental result of RRx-001 promoting Fedratinib-induced apoptosis of ENKTL cells. DETAILED DESCRIPTION

[0024] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0025] Example 1:

[0026] Effects of different concentrations of RRx-001 on ENKTL cell proliferation: ENKTL cells (YT and YTS) were observed to be in good condition and in the logarithmic growth phase. 4 The number of cells were seeded in 96-well plates, and the drug RRx-001 was added to the final concentrations of 0, 0.25, 0.5, 1, 2, 4, and 8 μM. After 24 hours, the cell state was observed and CCK-8 reagent was added. The absorbance was then measured using a microplate reader. The results showed that with the increase of RRx-001 drug concentration, the proliferation rate of both cell lines gradually decreased. The differences in cell proliferation between the drug-treated groups and the control group were statistically significant ( Figure 1 ). Figure 1 The results of the RRx-001 inhibition experiment on ENKTL cell proliferation were shown in Figure 1. YT cells (A) and YTS cells (B) were treated with different concentrations of RRx-001 for 24 hours, and cell proliferation was detected by CCK-8 assay.

[0027] Example 2:

[0028] Effect of RRx-001 on ENKTL cell cycle distribution: To explore the effect of RRx-001 on ENKTL cell cycle, the present invention seeded an appropriate amount of ENKTL cells in a six-well plate and added RRx-001 at final drug concentrations of 0, 3, and 6 μM, respectively. After 24 hours of action, the distribution of ENKTL cell cycle was detected by Pl single staining and flow cytometry. The results showed that after ENKTL cells were treated with different concentrations of RRx-001, the number of G1 phase cells increased with increasing drug concentration compared with the control group, and the differences in cell cycle between each drug-treated group and the control group were statistically significant, indicating that RRx-001 induced cell cycle arrest in the G1 phase ( Figure 2 ). Figure 2 This is the experimental result of RRx-001 blocking ENKTL cell cycle. Figure 2 (A) YT and YTS cells were treated with different concentrations of RRx-001 for 24 h, and the cell cycle was detected by PI flow cytometry. (B) The percentage of cells in different cell cycles (three independent experiments, ＊ P<0.05, ＊＊ P < 0.01, ＊＊＊ P<0.001).

[0029] Example 3:

[0030] Effect of RRx-001 on the expression of ENKTL cell cycle-related genes: NK / T lymphoma cells YT and YTS in the exponential proliferation phase were taken, and after inoculating YT and YTS into 6-well plates, RRx-001 was added at final concentrations of 0, 3, and 6 μM; the cells were collected after incubation in a cell culture incubator for 24 hours, and the protein samples were quantitatively prepared by BCA and then subjected to Western Blot; mRNA was extracted and reverse transcribed to obtain cDNA, which was then quantitatively analyzed by qPCR. The effect of RRx-001 on the expression of cell cycle-related proteins and genes in NK / T lymphoma cell lines was detected by Western Blot and RT-qPCR. The results showed that after the action of RRx-001, the expression levels of p21 and p27 proteins in NK / T lymphoma cell lines were enhanced, and the expression of CyclinD1 was reduced. The results of qPCR were consistent with those of Western Blot ( Figure 3 ), indicating that polydatin regulates the ENKTL cell cycle by affecting the expression of p21, p27 and CyclinD1. Figure 3 The results of the experiment on the effect of RRx-001 on the expression of cell cycle-related genes in ENKTL cells were shown in Figure 2. YT and YTS cells were treated with different concentrations of RRx-001 for 24 h, and the expression of cell cycle-related molecules was detected by western blot (A) and qPCR (B).

[0031] Example 4:

[0032] Effect of RRx-001 on ENKTL cell apoptosis: ENKTL cells (YT and YTS) were observed to be in good condition and in the logarithmic growth phase under the microscope. They were seeded in 6-well plates and RRx-001 was added to the final concentrations of 0, 3, and 6 μM. After 24 hours, the cells were collected and flow cytometry was performed to detect cell apoptosis. The results showed that with the increase of RRx-001 drug concentration, the cell apoptosis rate gradually increased, and the differences between each drug-added group and the control group were statistically significant ( Figure 4 A and Figure 4 B). Protein analysis also revealed enhanced Caspase3 cleavage and activation, and increased PARP hydrolysis, further demonstrating that RRx-001 induces apoptosis in ENKTL cells. Figure 4 Figure 2 shows the results of the RRx-001-induced apoptosis experiment on ENKTL cells. YT and YTS cells were treated with different concentrations of RRx-001 for 24 hours, and cell apoptosis was detected by flow cytometry (A). The percentage of apoptotic cells (three independent experiments, *P<0.05, ＊＊ P < 0.01, ＊＊＊ P<0.001)(B).

[0033] Example 5:

[0034] RRx-001 induces activation of the extrinsic apoptosis pathway: To investigate the RRx-001-induced apoptosis signaling pathway, we used RT-qPCR to detect the expression of genes involved in the extrinsic apoptosis pathway. The results showed that under the action of RRx-001, the expression of death receptors DR5 and DR4 gradually increased with increasing drug concentration, as did the expression of Caspase 8. This suggests that RRx-001 can activate the extrinsic apoptosis pathway in ENKTL cells, thereby promoting apoptosis of ENKTL cells. Figure 5 ). Figure 5 Figure 2 shows the experimental results of RRx-001-induced ENKTL exogenous cell apoptosis pathway; YT and YTS cells were treated with different concentrations of RRx-001 for 24 h, and the expression of DR4 (A), DR5 (B), and Caspase 8 (C) genes was detected by RT-qPCR (three independent experiments, *P<0.05, ＊＊ P < 0.01, ＊＊＊ P<0.001).

[0035] Example 6:

[0036] RRx-001 induces activation of the intrinsic apoptosis pathway: To clarify whether RRx-001 activates the intrinsic apoptosis pathway during the process of inducing apoptosis in ENKTL cells, we detected the expression of endogenous apoptosis proteins under the action of RRx-001. The results showed that with the increase of the concentration of the drug RRx-001, the expression of pro-apoptotic proteins Bid, Bax, and Cytc increased, and the expression of anti-apoptotic proteins Bcl-2, Mcl-1, and XIAP decreased, indicating that RRx-001 activates the intrinsic apoptosis pathway of ENKTL cells, thereby promoting the apoptosis of ENKTL cells ( Figure 6 ). Figure 6 Figure 2 shows the experimental results of RRx-001-induced ENKTL exogenous cell apoptosis pathway; YT and YTS cells were treated with different concentrations of RRx-001 for 24 h, and the expression of Mcl-1 gene was detected by RT-qPCR (three independent experiments, *P<0.05, ＊＊ P < 0.01, ＊＊＊ P<0.001). Western blot was used to detect the expression of XIAP, Mcl-1, and Bax proteins.

[0037] Example 7:

[0038] RRx-001 induces increased ROS levels in ENKTL cells: Oxidative stress can directly induce DNA damage and mitochondrial dysfunction in tumor cells, leading to cell apoptosis or necrosis. Therefore, we detected the changes in ROS in NK / T lymphoma cells after treatment with RRx-001. Take the NK / T lymphoma cell lines YT and YTS, load the probe according to the kit instructions, and wash off the probe after incubation for 2 hours. Then add different concentrations of RRx-001, incubate for 12 hours, and use flow cytometry to detect. The results showed that as the concentration of RRx-001 increased, the ROS level increased ( Figure 7 ). Figure 7 This is the flow cytometry result of the experiment to detect the increase of ROS level in NK / T lymphoma cells induced by RRx-001.

[0039] Example 8:

[0040] RRx-001 induces ER stress in ENKTL cells: RT-qPCR revealed that the expression levels of ER stress-related genes lRE1, BIP, ATF4, ATF3, and CHOP increased with increasing drug concentration. Furthermore, we used Western blot to examine the effects of RRx-001 on ER stress-related proteins in YT and YTS cells. The results showed that RRx-001 increased the expression of Bip, ATF3, and CHOP proteins with increasing drug concentration, indicating that RRx-001 induces ER stress. Figure 8Figure 2 shows the experimental results of the effect of RRx-001 on the endoplasmic reticulum stress pathway in YT and YTS cells. YT and YTS cells were treated with different concentrations of RRx-001 for 24 hours, and the expression of IRE1 (A), BIP (B), ATF4 (C), ATF3 (D), and CHOP (E) genes was detected by RT-qPCR (three independent experiments, *P<0.05, ＊＊ P < 0.01, ＊＊＊ P<0.001), and protein expression was detected by western blot (F).

[0041] Example 9:

[0042] RRx-001 inhibits the JAK-STAT signaling pathway: Abnormalities in the JAK-STAT signaling pathway are closely related to the occurrence and development of tumors. Activation of the JAK-STAT signaling pathway promotes tumor cell proliferation and inhibits tumor cell apoptosis. To explore the regulatory effect of RRx-001 on the JAK-STAT signaling pathway in NK / T cell lymphoma cell lines, WB was used to detect the expression of proteins related to the JAK-STAT signaling pathway in cell samples that had been intervened with different concentrations of RRx-001 for 24 hours. The results showed that after treatment with RRx-001, the tyrosine phosphorylation levels of JAK2 and STAT3 proteins were reduced, indicating that RRx-001 can inhibit the activation of the JAK-STAT signaling pathway in NK / T lymphoma cell lines ( Figure 9 ). Figure 9 This is the experimental result of western blot detection of the effect of RRx-001 on the JAK / STAT signaling pathway.

[0043] Example 10:

[0044] RRx-001 and Fedratinib have a synergistic effect: Take an appropriate amount of ENKTL cells (YT and YTS) in good growth state and in the logarithmic growth phase, and use 2x10 4 YT and YTS cells were seeded in 96-well plates and treated with 0, 0.5, 1, 2, 4, and 8 μM RRx-001 combined with 0.5 μM Fedratinib. After 24 hours, the cells were observed and CCK-8 reagent was added. The absorbance was then measured using a microplate reader. The results showed that the cell inhibition rate significantly increased with increasing RRx-001 concentration. We used CompuSyn software to calculate the drug synergy index (CI) of RRx-001 combined with Fedratinib and found that the CI values ​​for both YT and YTS cells were <1, indicating that the combination of RRx-001 and Fedratinib has a strong synergistic effect. Figure 10The results of the experiment showing the synergistic effect between RRx-001 and Fedratinib were shown; the CCK8 method was used to detect the effects of RRx-001 combined with Fedratinib on YT and YTS cells (A and C), and the Cl index was calculated (B).

[0045] Example 11:

[0046] RRx-001 enhances Fedratinib-induced apoptosis in ENKTL cells: YT and YTS cells were treated with 6μM RRx-001 and 2μM Fedratinib, either alone or in combination. Cells were harvested 24 hours later and apoptosis was analyzed by flow cytometry. The results showed that the apoptosis rate of ENKTL cells in the single-drug group was significantly higher than that in the control group, while the apoptosis rate in the combined group was even higher than that in the single-drug group. The difference in apoptosis rate between the combined group and the single-drug group and the control group was statistically significant. Figure 11 Figure 2 shows the experimental results of RRx-001 promoting Fedratinib-induced apoptosis of ENKTL cells. YT and YTS cells were treated with RRx-001 combined with Fedratinib for 24 h, and cell apoptosis was detected by Annexin V-APC / 7-AAD flow cytometry (A). The percentage of apoptotic cells (three independent experiments, *P<0.05, ＊＊ P < 0.01, ＊＊＊ P<0.001)(B).

[0047] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Application of RRx-001 combined with Fidatinib in the preparation of drugs for the treatment of extranodal NK / T cell lymphoma.

2. The use according to claim 1, characterized in that: RRx-001 can enhance fidatinib-induced apoptosis of extranodal NK / T cell lymphoma cells.

3. The use according to claim 1 or 2, characterized in that: The molar ratio of RRx-001 to fidartinib is 1 to 16:

1.

4. The use according to claim 1 or 2, characterized in that: The molar ratio of RRx-001 to fidartinib was 3:

1.

5. The use according to claim 1 or 2, characterized in that: The RRx-001 induces elevated ROS levels in extranodal NK / T cell lymphoma cells.

6. The use according to claim 1 or 2, characterized in that: The RRx-001 induces endoplasmic reticulum stress response in extranodal NK / T cell lymphoma cells.

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