Application of FoXO1 inhibitors in the preparation of drugs for the prevention and treatment of lymphoma

Abstract

The present invention provides the use of a FoXO1 inhibitor in the preparation of a drug for the prevention and treatment of lymphoma. The present invention selected AS1842856 for experiments in a NOD SCID mouse lymphoma tumor model. Administration via intraperitoneal injection effectively prevented and treated the occurrence of PEL (Pelvic Encephalopathy). Dissection of the mice revealed no symptoms of the drug affecting any of the mouse's internal organs. When AS1842856 was used to directly inhibit FoXO1 and FoXO3 in latently KSHV-infected lymphoma cells, it was found that the cells' antioxidant capacity was weakened, thereby increasing intracellular ROS levels, leading to KSHV reactivation and ultimately massive PEL cell death. Inducing oncolytic reactivation of endogenous viruses from latently infected tumor cells is a very attractive cancer treatment option, and inhibitors of FoXOs will have promising application prospects in the prevention and treatment of lymphoma.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the use of a FoXO1 inhibitor in the preparation of drugs for the prevention and treatment of lymphoma. Background Art

[0002] Lymphoma is a malignant tumor of immune cells that develops in the lymph nodes and extranodal lymphoid tissue. It is one of the top ten most common malignant tumors in my country. Lymphoma patients often have no obvious symptoms in the early stages, but once discovered, it is often in the advanced stages. The disease progresses rapidly, is prone to metastasis, and is prone to recurrence. Among hematologic malignancies, its incidence is second only to leukemia. In today's society, young people face high social pressures, excessive late nights, and unhealthy lifestyles, all of which increase their risk of developing lymphoma. Malignant lymphomas are divided into Hodgkin's disease and low-grade or high-grade non-Hodgkin's lymphomas. Primary effusion lymphoma (PEL) is a type of non-Hodgkin's lymphoma with clinical manifestations such as ascites, pleural effusions, and pericardial effusions. This type of lymphoma is highly malignant, with a median survival of less than six months. PEL is often associated with infection with Kaposi's sarcoma-associated herpesvirus (KSHV).

[0003] Currently, the main treatments for lymphoma include radiotherapy, chemotherapy, targeted therapy, immunotherapy, and hematopoietic stem cell transplantation. Chemotherapy is the primary treatment for most lymphoma patients, and the common drug rituximab is widely used in this setting. Rituximab is a human-mouse chimeric antibody that targets the CD20 antigen on the surface of B lymphocytes. It contains a mouse protein structure, which, combined with other drug components, can lead to significant adverse reactions. Another clinically used drug for lymphoma is etoposide, a cell cycle-specific antitumor drug that primarily targets DNA topoisomerase II, forming a stable, reversible drug-enzyme-DNA complex that significantly inhibits DNA repair. However, this capsule-based drug has significant side effects, making single-agent therapy ineffective. Other drugs, such as bortezomib, carfilzomib, dexamethasone, and ibrutinib, often require combination therapy, and their tolerability and efficacy remain uncertain.

[0004] The compound AS1842856 is a synthetic, selective small molecule inhibitor of FoxO1. Its CAS number is 836620-48-5, and it is designated as 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid or 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid. Its structural formula is shown in Formula I below. AS1842856 is an intracellular oxodihydroquinoline that inhibits FoxO1 transcriptional activity by directly binding to it (IC50 of 33 nM), while not binding to ser256-phosphorylated FoxO1 or inactive FoxO1. Reporter gene assays in HepG2 cells revealed that AS1842856 inhibited the functions of FoxO family members FoxO1, FoxO3a, and FoxO4 by 70%, 20%, and 3%, respectively. The study also found that the compound AS1842856 binds to dephosphorylated FoxO1 in a dose-dependent manner, causing FoxO1 to translocate from the nucleus to the cytoplasm, resulting in increased p-FoxO1 protein levels in the cytoplasm and decreased levels in the nucleus, thereby inhibiting FoxO1 activity. However, preclinical evidence for the FoxO1 inhibitor AS1842856 in the treatment of tumors has yet to be reported. Summary of the Invention

[0005] The present invention aims to address the above-mentioned deficiencies in the prior art and to provide a use of a FoXO1 inhibitor in the preparation of a drug for the prevention and treatment of lymphoma, with the aim of providing a new pharmaceutical use of the FoXO1 selective inhibitor AS1842856.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first object of the present invention is to provide a use of a FoXO1 inhibitor in the preparation of a drug for the prevention and treatment of lymphoma. The structural formula of the FoXO1 inhibitor compound is shown in Formula I:

[0008] .

[0009] Furthermore, the lymphoma includes primary effusion lymphoma.

[0010] Furthermore, the primary effusion lymphoma is associated with Kaposi's sarcoma-associated herpes virus.

[0011] Furthermore, the compounds directly inhibit FoXO1 and FoXO3 in lymphoma cells latently infected with Kaposi's sarcoma-associated herpesvirus.

[0012] The second object of the present invention is to provide an anti-lymphoma drug, the effective active ingredient of which is the compound shown in Formula I or a pharmaceutically acceptable salt of the compound shown in Formula I.

[0013] Furthermore, the drug can be introduced into the body by injection, oral administration, spraying, penetration, absorption, physical or chemical mediation methods such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue; or can be introduced into the body after being mixed or encapsulated with other substances.

[0014] When necessary, one or more pharmaceutically acceptable carriers may be added to the above-mentioned drugs. The carriers include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0015] The drug can be prepared into various forms such as injection, suspension, powder, tablet, granule, etc. The drugs in the above various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0016] Experiments conducted in the present invention demonstrate that the FoXO1 inhibitor AS1842856 has no significant toxic side effects in mouse experiments. The inventors conducted experiments on the FoXO1 inhibitor AS1842856 in a NOD SCID mouse lymphoma tumor model. The results demonstrated that, in the NOD SCID mouse lymphoma tumor model, intraperitoneal injection of the drug effectively prevented and treated the occurrence of peritoneal leukemia (PEL). Furthermore, no symptoms affecting the mouse organs were observed after dissection of the mice. The applicants used a FoXO inhibitor to directly inhibit FoXO1 and FoXO3 in lymphoma cells latently infected with KSHV. The results revealed that this weakened the cells' antioxidant capacity, thereby increasing intracellular ROS levels, leading to KSHV reactivation and ultimately massive PEL cell death. Furthermore, in a NOD SCID xenograft mouse tumor model, the FoXO1 inhibitor significantly inhibited the initiation and progression of KSHV-associated PEL tumors. The above results indicate that inducing oncolytic reactivation of endogenous viruses from latently infected tumor cells is a very attractive cancer treatment strategy. Moreover, inhibitors of FoXOs are representative and competitive drugs among them, which will have good application prospects in the prevention and treatment of lymphoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figures 1a to 5d This is a detection graph or statistical graph showing that the FoxO1 selective inhibitor AS1842856 can inhibit the proliferation of PEL cells and induce their apoptosis;

[0018] Figures 6a to 9 This is a detection graph or statistical graph of a study on the induction of KSHV lytic replication by the FoxO1 selective inhibitor AS1842856;

[0019] Figures 10a to 12 Detection graph or statistical graph of the study on knockdown of FoxO1 and FoxO3 triggering KSHV lytic replication;

[0020] Figures 13 to 19 This is a detection graph or statistical graph showing that the FoxO1 selective inhibitor AS1842856 can inhibit the occurrence, development and regression of tumors in a PEL xenograft mouse model;

[0021] Figures 20 to 28d These are detection graphs or statistical graphs for a study in which the FoxO1 selective inhibitor AS1842856 inhibits the occurrence, progression, and regression of PEL tumors by inducing KSHV reactivation. DETAILED DESCRIPTION

[0022] To make the purpose, technical solutions and advantages of the present invention clearer, the specific embodiments of the present invention are described in further detail below in conjunction with specific examples and accompanying drawings. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0023] The FoxO1 selective inhibitor AS1842856 used in the present invention was purchased from Adooq Bioscience with the catalog number A15871;

[0024] The cells used in this invention were donated by Shantou University;

[0025] The model mice used in the present invention were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.

[0026] To prepare AS1842856, weigh an appropriate amount of powder and dissolve it in 6% cyclodextrin-containing saline, sonicate at 50°C, and use immediately after preparation.

[0027] To prepare D-luciferin, weigh an appropriate amount of powder, dissolve it in PBS buffer solution, and store it in a refrigerator at -80 degrees Celsius.

[0028] Example 1

[0029] FoxO1 selective inhibitor AS1842856 inhibits proliferation and induces apoptosis of PEL cells

[0030] (1) Detection of the effect of AS1842856 inhibitor on PEL cell proliferation

[0031] Five types of PEL cells, namely BJAB, DG75, BCBL1, BC3 and BCP1, were seeded into 24-well cell culture plates with 1 mL of culture medium per well and 2×10 cells per well. 5 AS1842856 powder was prepared and dissolved in DMSO (dimethyl sulfoxide), then added to a 24-well plate to a final concentration of 0, 1, 2, and 5 µM. The cells were cultured in a cell culture incubator. At 24, 48, and 72 hours of culture, trypan blue staining was used and the number of live and dead cells was counted on a hemocytometer.

[0032] The results are as follows Figure 1a-Figure 1e As shown in the results, AS1842856 significantly inhibited the proliferation of primary effusion lymphoma cells with increasing treatment concentrations, and when the concentration reached 5 μM, the inhibition of lymphoma cell growth was extremely significant.

[0033] (2) Cell culture

[0034] PEL cells were cultured in RPMI medium containing 10% fetal bovine serum and double-antibody in a cell culture incubator at 37°C with 5% CO2 in a humidified atmosphere.

[0035] (3) Western blotting

[0036] After the PEL cells were treated with the AS1842856 inhibitor for 72 h, the culture medium was centrifuged to obtain the cell pellet, protein samples were prepared, and protein immunoblotting was performed, and the developed images were saved.

[0037] (4) Cell apoptosis detection

[0038] The cells in the 24-well plate used for cell proliferation assay were removed after 72 h, washed twice with PBS, and stained with Annixin V / PI. Cell apoptosis was detected by flow cytometry, and the c-caspase 3 level was detected by Western blot using β-actin as an internal control.

[0039] The results are as follows Figure 2a-2e As shown in the figure, AS1842856 significantly promoted the apoptosis of lymphoma cells with the increase of treatment concentration, and there was a significant difference at 5μM.

[0040] like Figure 3a-3d The figure shows the trend of the inhibition of the proliferation of PEL cells by knocking down FoxO1. Lentivirus carrying NT shRNA or three different FoxO1 shRNAs (1, 2 and 3) was used to inoculate PEL cells at a rate of 1.5×10 5 / mL of infected cells, and the viable cells were counted 24h, 48h, and 72h after infection.

[0041] like Figure 4a-4d Figure 2 shows the trend of apoptosis induced by FoxO1 knockdown in PEL cells. Flow cytometry was used to assess apoptosis in PEL cells 72 hours after transfection with NTshRNA or three FoxO1 shRNAs (1, 2, and 3). All values ​​are mean ± SD from three independent experiments.

[0042] like Figure 5a-5d As shown in the figure, knockdown of FoxO1 increased the expression of c-caspase 3 protein in PEL cells. 5 The cells were infected with NT / ml and three FoxO1 shRNAs (1, 2, and 3) for 72 h, and the levels of FoxO1 and c-caspase 3 proteins were detected by Western blot.

[0043] Example 2

[0044] FoxO1 selective inhibitor AS1842856 induces KSHV lytic replication

[0045] RT-qPCR was used to detect the mRNA levels of KSHV LANA, RTA, PAN RNA, and ORFK8, ORF57, ORF59, and ORF65 in BCP1 cells after treatment with 0, 1, 2, and 5 μM AS1842856 or 0.3 mM sodium butyrate (NaB). The latency-associated nuclear antigen LANA, expressed during latency, is a key protein for KSHV to establish and maintain latency and is present in all known KSHV latently infected cells. RTA is a replication transcription activator and a key protein controlling the latent and lytic states of the virus. During viral lytic replication, most viral genes, such as RTA, PAN RNA, ORFK8, ORF57, and ORF65, are expressed in a cascade manner, leading to the production of infectious virions. After BCP1 cells were treated with 0, 1, 2, and 5 μM AS1842856 for 72 h, the protein levels of LANA and RTA were detected by Western blotting (B) and immunofluorescence (C). qPCR was used to analyze the expression of 0, 1, 2, and 5 μM AS1842856 in BCP1 cells. Relative virus yields in BCP1 cells treated with AS1842856 and 0.3 mM NaB for 96 h.

[0046] The results are as follows Figures 6a-6f As shown, AS1842856 induces the transcription of KSHV lytic genes. After the inhibitor is treated with KSHV-positive PEL cells BCP1, the expression of KSHV lytic genes is promoted.

[0047] like Figure 7The figure shows a western blot of KSHV ORFK8 protein expression induced by AS1842856 in BCP1 cells. Compared with the control group, the inhibitor treatment of BCP1 cells promoted the upregulation of the protein level of the lytic phase gene ORFK8, that is, the inhibitor promoted the increase of its protein level by upregulating the transcription level of the lytic phase gene.

[0048] like Figure 8 Shown is an immunofluorescence detection diagram of KSHV ORFK8 protein expression induced by AS1842856 in BCP1 cells. The immunofluorescence detection results showed that the inhibitor AS1842856 promoted the expression of ORFK8 in cells.

[0049] like Figure 9 The figure shows the data of the relative production of KSHV virus in BCP1 cells increased by AS1842856. When the treatment concentration of AS1842856 was increased to 5 μM, the number of viral copies in BCP1 cells was significantly increased, which proves that the inhibitor promoted the replication and activation of the virus after treating PEL cells.

[0050] Example 3

[0051] Knockdown of FoxO1 and FoxO3 triggers KSHV lytic replication

[0052] RT-qPCR was used to detect the mRNA levels of KSHV LANA, RTA, PAN RNA, and ORFK8, ORF57, ORF59, and ORF65 in BCP1 cells infected with NT shRNA, FoxO1 shRNA, and FoxO3 shRNA for 72 h. BCP1 cells were transduced with NT shRNA, two different FoxO1 shRNAs, or two different FoxO3 shRNAs for 96 h, and the KSHV ORFK8 protein level was analyzed by Western blot using β-actin as a loading control.

[0053] The results are as follows Figure 10a-10g As shown in the figure, knockdown of FoxO1 or FoxO3 induces the transcriptional expression level of KSHV lytic genes. After knocking down FoxO1 or FoxO3, the same results were seen as when the cells were treated with the inhibitor AS1842856, that is, both led to the transcription of lytic genes, such as RTA, PAN RNA, ORF57, ORF59, etc.

[0054] like Figure 11 The figure shows the expression results of KSHV ORFK8 induced by knocking down FoxO1 or FoxO3 in BCP1 cells. Knocking down FoxO1 or FoxO3 genes in BCBP promoted the upregulation of KSHV ORFK8 protein levels, with obvious differences.

[0055] like Figure 12 Shown is an immunofluorescence detection image of ORFK8 protein expression in BCP1 cells induced by knocking down FoxO1 or FoxO3. Consistent with the previous results of treating cells with the inhibitor AS1842856, after knocking down FoxO1 or FoxO3, ORFK8 expression in BCP1 cells was significantly upregulated compared with the control group.

[0056] Example 4

[0057] The FoxO1 selective inhibitor AS1842856 inhibits tumor initiation, progression, and regression in a PEL xenograft mouse model

[0058] The expanded Luc-BCBL1 cell line was inoculated into the peritoneal cavity of NOD SCID mice, and each mouse was injected with 1x10 7 After injection, mice were randomly divided into 14 control mice and 7 inhibitor mice. For the first part of the experiment, the day of injection was designated Day 0. Starting on Day 1, the inhibitor group received two injections daily, one in the morning and one in the afternoon, totaling 15 mg / kg of AS1842856. The inhibitor was dissolved in 6% cyclodextrin-based saline and was prepared immediately. The control group received the same dose of 6% cyclodextrin-based saline, solubilized at 50°C, and then injected. The mice were weighed and their health was observed daily. On Days 7 and 14, intraperitoneal imaging was performed, with each mouse receiving 50 mg / kg of D-luciferin. The progress of peritoneal tumor growth and fluorescence signal intensity were recorded using an in vivo imaging device. After imaging, the inhibitor mice were sacrificed, and ascites, heart, liver, spleen, lung, and kidney were collected by autopsy. Organ images were taken and their appearance and morphology were observed.

[0059] Based on the imaging results on Day 14, the control group from the first part of the experiment was further divided into two groups: a control group and an inhibitor group. The second part of the experiment was conducted to observe whether the inhibitor treatment would regress after tumor formation. Therefore, on Day 16, the applicant injected the inhibitor group with two injections of the prepared inhibitor, using the same preparation method as in the first part of the experiment. The control group received an injection of 6% cyclodextrin-containing saline. Starting on Day 20, the injections were repeated three times daily, morning, noon, and evening. The mice were weighed and recorded daily, and their physical condition was observed for any difficulty eating or mobility issues. In vivo imaging was performed on Days 23 and 27 using the same experimental method as in the first part of the experiment. Tumor fluorescence signal images were captured and analyzed for differences in fluorescence signal. On Day 27, all mice were sacrificed, ascites were collected, and the appearance of each organ was photographed. The organs were then removed and immersed in 10% formalin.

[0060] The results are as follows Figure 13Shown is the experimental timeline of the occurrence and development of PEL, and PEL tumor imaging images in mice treated with the control group and the AS1842856 inhibitor. On the 7th day after inoculation, the abdominal tumor signal of mice in the inhibitor-injected group was significantly reduced compared with the control group, and the area was also only a small part. In some mice, no tumor formation was even observed.

[0061] like Figures 14a-14b Shown are the decreased fluorescence signal intensity of PEL tumors in AS1842856-treated mice at day 7 (14a) and day 14 (14b) after inoculation with BCBL1-Luc cells.

[0062] like Figure 15 As shown, the timeline of PEL regression following treatment with AS inhibitors is shown. Luminescent images of PEL tumors in mice in the control group and AS1842856 inhibitor group were taken on the 16th, 23rd, and 27th days after inoculation. It was found that after the inhibitor was injected into the mice, the PEL tumors in the abdominal cavity were significantly inhibited compared with the control group, and even led to tumor regression.

[0063] like Figure 16 As shown, the PEL luminescence signal intensity changes over time in individual mice treated with control solvent or AS1842856. The fluorescence signal intensity of the abdominal tumor in the mice in the inhibitor injection group was obviously suppressed or even weakened.

[0064] like Figure 17 As shown, a comparison of the total fluorescence signal intensity of PEL tumors in mice in the control group and the AS1842856 inhibitor group on the 14th day after inoculation of BCBL1-Luc cells. The total fluorescence signal intensity of PEL tumors in the inhibitor group was significantly downregulated compared with that in the control group, with significant and statistically significant differences.

[0065] like Figure 18 The figure shows the protein expression of FoxO1, FoxO3 and c-caspase 3 in cells extracted from the ascites of mice treated with AS1842856 or the control group. After taking the ascites from the peritoneal cavity of the control and inhibitor groups, the proteins of the tumor cells were extracted and the protein expression of FoxO1, FoxO3 and c-caspase 3 was detected. It was found that the protein levels of FoxO1 and FoxO3 in the ascites tumors of the inhibitor group mice were downregulated, while the protein level of c-caspase 3 was upregulated. This further verified at the in vivo level that the inhibitor AS1842856 promoted the apoptosis of tumor cells by inhibiting the expression of FoxO1 and FoxO3 in vivo, thereby promoting the apoptosis of tumor cells, inhibiting cell proliferation, and even leading to tumor regression.

[0066] like Figure 19The figure shows the quantification of FoxO1, FoxO3, and c-caspase 3 protein expression in cells extracted from the ascites of mice treated with the control group or AS1842856. This figure is a grayscale analysis of the protein expression of FoxO1, FoxO3, and c-caspase 3, presented in the form of a quantitative graph, which more intuitively shows that the inhibitor downregulates the expression of FoxO1 and FoxO3 in mouse peritoneal tumor cells and promotes cell apoptosis.

[0067] Example 5

[0068] FoxO1 selective inhibitor AS1842856 inhibits the occurrence, progression and regression of PEL tumors by inducing KSHV reactivation

[0069] After a certain number of BCBL1-Luc cells are expanded and seeded in a gradient pattern from left to right in a 96-well plate, substrate is added and mixed by pipetting. Then, a live imaging instrument is used to perform cell luminescence imaging on the 96-well plate cells to obtain a luminescence signal intensity image and luminescence intensity value.

[0070] The results are as follows Figure 20 Shown are bioluminescent imaging images of BCBL1-Luc cells with different cell numbers in a 96-well plate, which validates the feasibility of the constructed BCBL1-Luc cells in expressing fluorescent signals in vitro;

[0071] like Figure 21 As shown, the total bioluminescence flux is linearly correlated with the cell number. As the cell number increases, the fluorescence signal intensity is linearly related, which proves the accuracy of the fluorescence signal expressed by the cell.

[0072] like Figure 22 As shown, intraperitoneal injection of 10 7 BCBL1-Luc cells were then injected into NOD / SCID mice, and the control group or AS1842856 was used to treat the mice. The weight gain trend chart showed that the inhibitor treatment did not affect the weight of the mice.

[0073] like Figure 23 Shown are NOD / SCID mice implanted with BCBL1-Luc cells and treated with AS1842856, which showed no lesions in the lungs, heart, spleen, liver, pancreas, or kidneys.

[0074] like Figure 24 Shown is the average body weight of NOD / SCID mice treated with the control group or AS1842856. The results also demonstrate that the inhibitor treatment does not affect the body weight changes of the mice.

[0075] like Figure 25Shown is a graph showing the weight gain trend of mice in the control group or treated with AS1842856. Treatment of the mice with the inhibitor had no effect on the weight changes of the mice.

[0076] like Figure 26 As shown, the subcutaneous solid tumor volume of NOD / SCID mouse control group mice was larger, while the subcutaneous solid tumor volume of AS1842856 mice was smaller.

[0077] like Figure 27 As shown, the spleens of NOD / SCID mice treated with AS1842856 were smaller than those of the control group, and no lesions were found in the lungs, heart, liver, pancreas, and kidneys.

[0078] like Figures 28a-28d The figure shows the expression of KSHV lytic genes at the mRNA level detected by RT-qPCR in cells extracted from the ascites of mice treated with control solvent or AS1842856. After RNA was extracted from the ascites of the inhibitor group and the control group mice, the qPCR detection of KSHV-related lytic genes was verified. Consistent with the previous in vitro experimental results, the inhibitor treatment of the mouse peritoneal tumor promoted the massive transcription and replication of KSHV lytic phase cells.

[0079] In the absence of conflict, the above embodiments and features in the embodiments may be combined with each other.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of the compound represented by formula I in the preparation of a drug for inhibiting primary effusion lymphoma by inducing KSHV replication and reactivation, Formula I.

2. The use according to claim 1, characterized in that The compounds directly inhibit Expression of FoXO1 and FoXO3 in Kaposi's sarcoma-associated herpesvirus-latently infected lymphoma cells.

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