Application of arsenic trioxide and p97 inhibitor in preparation of medicine for treating acute myelogenous leukemia

Abstract

The invention relates to application of arsenic trioxide and a p97 inhibitor in preparation of a medicine for treating acute myelogenous leukemia. Research results of the invention reveal the synthetic lethal effect of ATO and p97 inhibitors in ATO low-sensitive AML cells, and these findings lay a foundation for promoting clinical application of ATO.

Description

Technical Field

[0001] The invention belongs to the technical field of acute myeloid leukemia treatment, and particularly relates to application of arsenic trioxide and a p97 inhibitor in preparing a drug for treating acute myeloid leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by infiltration of abnormal and differentiated cells into the bone marrow and blood. Acute promyelocytic leukemia (APL), which accounts for 5-10% of AML, can be treated with a combination of all-trans retinoic acid and arsenic trioxide (ATO). However, ATO alone does not provide any survival benefit to non-APL AML patients and has limited efficacy when used in combination with other drugs. In order to expand the successful application of ATO in APL to the treatment of other types of AML, in-depth exploration of the general mechanism of ATO in APL is necessary. This exploration will pave the way for identifying the best synthetic lethal drug to improve the therapeutic effect of ATO in non-APL AML patients.

[0003] The main mechanism by which ATO induces remission of APL is to promote the degradation of PML-RARα fusion protein. In addition, ATO has the ability to induce the production of reactive oxygen species (ROS), which promotes cell death. However, studies have shown that ROS may not be necessary for ATO to induce cell death. Therefore, whether PML-RARα degradation and ROS upregulation are sufficient to cause APL cell death remains to be explored. In addition, ATO directly targets certain functional proteins containing thiol groups, suggesting another pathway leading to cell apoptosis. The accumulation of damaged proteins in the endoplasmic reticulum (ER) can cause ER stress. In order to control mild stress and maintain cell homeostasis, cells initiate the unfolded protein response (UPR), which includes three pathways: PERK-eIF2α, IRE1α-XBP1, and ATF6α. However, severe ER stress triggers apoptosis, mainly through the activation of the PERK-eIF2α-ATF4-CHOP pathway, which is also part of the integrated stress response (ISR). The ISR involves four serine / threonine kinases: PERK, PKR, HRI, and GCN2. Among them, GCN2 can be activated by free tRNA without amino acids, ribosome collision or ribosome stalling. Arsenic can induce apoptosis in various cells through abnormal endoplasmic reticulum stress or UPR. However, there is limited exploration of ATO-induced ISR. Therefore, it is of great significance to study whether ATO-induced protein damage activates ISR or UPR in APL cells.

[0004] Irreversibly damaged proteins need to be cleared through the protein degradation system, which includes the ER-associated degradation pathway (ERAD) and ribosome-associated quality control (RQC). ERAD substrates are transferred from the endoplasmic reticulum membrane to the cytoplasm with the help of HRD1, a process coupled to the energy generated by the hydrolysis of ATP by the AAA ATPase VCP / p97. RQC is a degradation pathway that targets defective nascent peptides on stalled ribosomes. Key proteins in this pathway include the ubiquitin ligase Listerin and its cofactor NEMF. NEMF recognizes peptidyl transfer RNA molecules and recruits Listerin to ubiquitinate stalled peptides, which are then extracted from 60S ribosomes with the help of VCP / p97. Ultimately, substrates of both pathways are degraded by the proteasome. Considering the key roles of VCP / p97 in ERAD and RQC, it is necessary to explore p97 inhibitors as potential synthetic lethal drugs for ATO treatment of non-APL AML. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide the use of arsenic trioxide and p97 inhibitors in the preparation of drugs for treating acute myeloid leukemia. The present invention further explores the general mechanism of ATO in treating APL, studies whether ATO-induced protein damage activates ISR or UPR in APL cells, and explores the possibility of p97 inhibitors as potential synthetic lethal drugs for ATO in treating non-APL AML.

[0006] The invention provides application of arsenic trioxide and p97 inhibitor in preparing medicine for treating acute myeloid leukemia.

[0007] Preferably, the p97 inhibitor comprises NMS-873.

[0008] Preferably, the drug further comprises a pharmaceutically acceptable carrier and / or excipient.

[0009] Preferably, the pharmaceutically acceptable carrier and / or excipient includes at least one of a diluent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, and a disintegrant.

[0010] Preferably, the dosage form of the drug includes at least one of tablets, pills, powders, solutions, suspensions, emulsions, and granules.

[0011] Preferably, the acute myeloid leukemia comprises AML1-ETO or MLL fusion leukemia.

[0012] Beneficial Effects

[0013] The research results of the present invention reveal the synthetic lethal effects of ATO and p97 inhibitors in ATO-low-sensitive AML cells. These findings lay the foundation for promoting the clinical application of ATO. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 AI showed that PML-RARα degradation and ROS upregulation were not sufficient to cause APL cell death.

[0015] Figure 2 AB show upregulated genes in ATO-resistant NB4-AR cells.

[0016] Figure 3 AI showed that the sensitivity of AML cells to ATO was related to protein synthesis.

[0017] Figure 4 AF show the effects of 4EGI-1 and puromycin on ATO-induced apoptosis of NB4 cells.

[0018] Figure 5 AD shows that ATO targets nascent peptides while activating the GCN2-ATF4 pathway.

[0019] Figure 6 AB shows that in APL, ATO cannot activate ATF6α or XBP1s.

[0020] Figure 7 AI showed that ATO caused ribosome arrest and p97 inhibitor increased the sensitivity of APL to ATO.

[0021] Figure 8 AI showed that ATO and NMS873 had synergistic effects on non-APL AML.

[0022] Fig. 9 AE showed that ATO and NMS873 had synergistic effects on MOLM-13 and MV4-11 cells.

[0023] Fig.10 AC shows the effectiveness of ATO and NMS873 in vivo. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0025] Example 1

[0026] First, to evaluate whether PML-RARα degradation is sufficient to trigger APL apoptosis, the NB4 cell line was exposed to different concentrations of ATO. The results showed that after 24 hours of treatment with 1.5 μM ATO, NB4 cells showed obvious apoptosis ( Figure 1 Interestingly, after 12 h of non-lethal 0.2 μM ATO treatment, PML-RARα protein levels were significantly reduced ( Figure 1 B), indicating that PML-RARα degradation is not sufficient to cause APL cell death. In addition to PML-RARα degradation, ROS is another important mechanism leading to APL apoptosis. DCFH-DA was used to detect the level of ROS induced by ATO. NB4 cells were treated with different concentrations of ATO for 4 hours. The results showed that ROS levels increased after ATO treatment, and there was no significant difference between the groups ( Figure 1 CD). In addition, NB4 cells treated with 10 or 20 μM H2O2 showed ROS accumulation similar to that induced by lethal doses of ATO, however, this ROS level failed to produce a corresponding apoptotic rate ( Figure 1 EH). Therefore, the increase in ROS levels induced by ATO is not sufficient to cause cell apoptosis.

[0027] In order to explore other mechanisms of arsenic sensitivity in APL cells, an ATO-resistant NB4 (NB4-AR) cell line was constructed. Figure 2 A). RNA-seq data showed that defensin family proteins were highly expressed in NB4-AR cells, a class of cysteine-rich peptides ( Figure 2 B). Therefore, it is assumed that arsenic acts mainly by targeting the thiol groups of cysteine ​​in proteins. To test this hypothesis, NB4 cells were pretreated with thiol reagents N-acetyl-L-cysteine ​​(NAC) or glutathione (GSH) for 1 hour, and then treated with 2 μM ATO for another 24 hours. The results showed that both reagents inhibited ATO-induced NB4 cell death ( Figure 1 I). These results reveal the importance of cysteine ​​in proteins as a key target for arsenic attack.

[0028] Therefore, we hypothesized that extensive protein damage plays a key role in ATO-induced cell death. NB4 cells were treated with 1 μM or 1.5 μM ATO for 16 h, and ubiquitinated proteins and aggregates were detected. Treatment with a lethal dose of ATO significantly increased the levels of ubiquitinated and aggregated proteins ( Figure 3 AB), indicating increased protein damage. In contrast, 10 or 20 μM H2O2 treatment failed to induce protein aggregation at a level comparable to ATO ( Figure 3C), which indicates that APL cell apoptosis is related to ATO-induced protein damage, rather than just mediated by ROS. Lethal doses of ATO disrupt protein balance, and protein synthesis is a key factor affecting protein homeostasis. Therefore, the protein synthesis of different AML cells and their sensitivity to ATO were detected, and it was found that cells with higher protein synthesis were more sensitive to ATO ( Figure 3 DF).

[0029] To further validate that protein synthesis is a key target of arsenic-induced cell death, various protein synthesis inhibitors were used to evaluate their effects on ATO toxicity. The effectiveness of these inhibitors in inhibiting protein synthesis in NB4 cells was confirmed ( Figure 3 GH, Figure 4 A). Then, NB4 cells were pretreated with different inhibitors for 1 h, and then treated with 1.5 μM ATO for 24 h. The results showed that cycloheximide (CHX) and 4EGI-1 could inhibit ATO-induced cell apoptosis, while puromycin promoted ATO-induced cell apoptosis ( Figure 3 I, Figure 4 B). It was further confirmed that 0.2 μg / mL puromycin had an inhibitory effect on protein synthesis ( Figure 4 C). However, due to the high apoptosis rate induced by 0.2 μg / mL puromycin, lower concentrations (0.1 or 0.15 μg / mL) were used to treat NB4 cells. Interestingly, these concentrations still enhanced the apoptosis rate of NB4 cells induced by 1 μM or 1.5 μM ATO ( Figure 4 DF). The apparently different effects of puromycin, CHX, and 4EGI-1 in the presence of ATO raise questions. It is hypothesized that puromycin treatment may generate more nascent polypeptides for ATO to target, leading to more protein damage, because puromycin can enter the ribosomal A site and induce the release of peptidyl-puromycin products, and puromycin is also commonly used as a reagent to label nascent polypeptides.

[0030] To investigate whether ATO induces protein damage by targeting nascent peptides, immunofluorescence analysis was performed. Using biotinylated arsenic (Biotin-As) to mimic the effect of ATO, it was found that nascent peptides labeled with puromycin partially colocalized with aggregates and biotin-As, indicating that there is an interaction between arsenic and nascent peptides ( Figure 5 A). In addition, Western Blot results showed that after 4 hours of treatment with 1 μM biotin-As, the puromycin-labeled nascent polypeptides in the cell lysate could be pulled down by streptavidin magnetic beads, confirming the interaction between arsenic and nascent polypeptides ( Figure 5B). Subsequently, the activation of stress pathways in NB4 cells caused by ATO treatment was detected. Previous studies have shown that arsenic can induce endoplasmic reticulum stress or UPR in a variety of cell types. Therefore, the activation of UPR after ATO treatment was investigated. NB4 cells were treated with 0.5 or 2 μM ATO, and Western Blot results showed that the levels of p-eIF2α and ATF4 in NB4 cells increased significantly after 4 hours of lethal dose ATO treatment ( Figure 5 C), while ATF6, XBP1s and Bip were not activated ( Figure 5 C, Figure 6 AB). The increase in ATF4 levels may be due to the activation of PERK or GCN2. To distinguish whether PERK or GCN2 is activated by ATO, NB4 cells were pretreated with PERK inhibitor (PERKi, GSK2656157) or GCN2 inhibitor (GCN2iB) for 1 hour and then treated with 2μM ATO for another 4 hours. The results showed that GCN2iB could inhibit ATO-induced activation of ATF4 and p-eIF2α, while PERKi could not ( Figure 5 D), indicating that ATO-induced protein damage can activate the GCN2-ATF4 pathway.

[0031] Studies have shown that ribosome stalling triggers stress response pathways mediated by JNK and GCN2. Therefore, it was hypothesized that ATO could induce ribosome stalling to activate the GCN2-ATF4 pathway. To investigate this, multimer mapping was used to detect translation behavior. The results showed that after 6 hours of treatment with 1.5 μM ATO, 80S ribosomes increased, while multimers decreased ( Figure 7 A), indicating the initial ribosome arrest. In addition, it was verified that the JNK pathway can be activated by a lethal dose of ATO, and the JNK inhibitor JNK-IN-8 can attenuate ATO-induced cell apoptosis ( Figure 7 BC). These findings are consistent with the idea that ribosome stalling activates JNK, and that JNK activation is a pathway by which ATO induces apoptosis in APL cells. In addition, ZAKα is essential for JNK activation induced by ribosome stalling. Using Phos-tag agarose to enrich for phosphorylated proteins, it was found that lethal doses of ATO treatment triggered rapid activation of ZAKα, which continued to be activated during long-term treatment. In contrast, non-lethal doses of ATO activated ZAKα only after long-term treatment, indicating that lethal doses of ATO treatment caused more severe ribosome stalling than non-lethal doses ( Figure 7 D). Notably, in NB4 cells treated with non-lethal doses of ATO, activation of ATF4, but not JNK, was observed ( Figure 5 C, Figure 7B), indicating that non-lethal doses of ATO treatment only induced mild ribosome arrest and failed to activate JNK. In addition, it was confirmed that knockdown of ZAKα could reduce ATO-induced apoptosis ( Figure 7 EF), indicating the importance of ZAKα in ATO-induced cell apoptosis.

[0032] To further investigate whether the damaged proteins caused by ATO are processed by RQC or ERAD, the interaction between arsenic and RQC or ERAD-related proteins in NB4 was detected. The results showed that after 4 hours of treatment with 1 μM biotin-As, NEMF (RQC-related protein) in the cell lysate could be pulled down by streptavidin magnetic beads, but HRD1 (ERAD-related protein) could not be pulled down, indicating that the damaged proteins caused by ATO can be processed by RQC ( Figure 7 G). Previous studies have shown that p97 is an important protein in RQC. Therefore, the interaction between p97 and NEMF in NB4 was explored. NB4 cells were treated with 1μM Biotin-As for 4h, and then 20μM puromycin was added for 30min. The results showed that puromycin-labeled nascent polypeptides and p97 could be pulled down by protein G-linked NEMF antibodies ( Figure 7 H). In addition, compared with the untreated group, more puromycin-labeled nascent polypeptides and p97 were pulled down in the biotin-As treated group, indicating that arsenic caused more nascent polypeptide damage ( Figure 7 H). Based on the role of p97 in degrading damaged proteins, we investigated whether p97 inhibitors would increase the sensitivity of NB4 to ATO. NB4 cells were pretreated with the p97 inhibitor NMS873 for 1 hour, and then the indicated doses of ATO were added. The results showed that NMS873 enhanced the sensitivity of NB4 to ATO ( Figure 7 I).

[0033] Next, the synergistic effect of ATO and NMS873 on non-APL AML was investigated. The CCK-8 method was used to detect cell viability after 72 hours of drug treatment, and the Annexin V method was used to detect cell apoptosis. The results showed that NMS873 treatment could increase the sensitivity of Kasumi-1 to ATO. When both ATO and NMS873 were 1 μM, the CI value was 0.36 ( Figure 8 AB), indicating a synergistic effect. Similar results were observed for MOLM-13 and MV4-11 ( Fig. 9 AC). Protein aggregation analysis of Kasumi-1 and MOLM-13 showed that combined treatment with ATO and NMS873 induced more protein aggregation ( Figure 8 C, Fig. 9D), indicating increased production of damaged proteins. Previous research results have shown that lethal doses of ATO can induce severe ribosome arrest and activate JNK in NB4. Therefore, the activation of JNK in different AML cell lines was studied. It was found that after ATO combined with NMS873 treatment, the JNK pathway in Kasumi-1, MOLM-13 and MV4-11 was activated ( Figure 8 D, Fig. 9 E). At the same time, p-eIF2α and ATF4 in Kasumi-1 were activated under combined drug treatment ( Figure 8 D), indicating increased stress, similar to the effects of a lethal dose of ATO on NB4 cells. In addition, ZAKα activation was observed in Kasumi-1 cells under ATO and NMS873 treatment ( Figure 8 E), indicating that the combined treatment induced more ribosome stalling and activated the JNK pathway. To further confirm the combined effect, p97 knockdown was performed in Kasumi-1 and was found to promote ATO-induced apoptosis ( Figure 8 FG). In addition to non-APL AML cell lines, the effects of the drugs on patient AML cells were also tested in vitro. First, it was confirmed that the combined treatment had no significant effect on apoptosis in normal peripheral blood mononuclear cells, although it appeared to induce more apoptosis ( Figure 8 H). Next, it was demonstrated that ATO combined with NMS873 could induce cell death in patients with AML ( Figure 8 I), emphasizing its role in non-APL AML.

[0034] In vitro data showed a synergistic effect between ATO and NMS873. Next, the effects of NMS873 and ATO were investigated in vivo. The Kasumi-1 xenograft mouse tumor model was prepared by injecting luciferase-GFP-Kasumi-1 tumor cells into the tail vein of NSG mice ( Fig.10 A). Tumor growth was monitored by a quantitative imaging system. It was found that single-drug treatment and untreated mice had no significant inhibitory effect on leukemia progression, while combined drug treatment significantly delayed tumor progression ( Fig.10 B). The median survival time of mice treated with control, ATO, NMS873 or combination therapy was 51, 54, 60 and 94 days, respectively ( Fig.10 C). Therefore, in vivo, the combination of ATO and the p97 inhibitor NMS873 has a good inhibitory effect on ATO-low-sensitive AML cells.

[0035] In the present invention, it was determined that PML-RARα degradation and ROS upregulation were not sufficient to cause APL cell death. It was further demonstrated that ATO caused nascent polypeptide damage and ribosome arrest, accompanied by activation of the ISR and ZAKα-JNK pathway mediated by GCN2-ATF4. In addition, RQC can also degrade damaged proteins. Notably, the results revealed the synthetic lethal effects of ATO and p97 inhibitors in ATO-low-sensitive AML cells. These findings lay the foundation for promoting ATO treatment of non-APL AML, especially for the treatment of AML1-ETO and MLL fusion leukemias.

Claims

1. Application of arsenic trioxide and p97 inhibitors in the preparation of drugs for the treatment of acute myeloid leukemia.

2. The use according to claim 1, characterized in that: The p97 inhibitors include NMS-873.

3. The use according to claim 1, characterized in that: The medicine further comprises a pharmaceutically acceptable carrier and / or excipient.

4. The use according to claim 3, characterized in that: The pharmaceutically acceptable carrier and / or excipient includes at least one of a diluent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, and a disintegrant.

5. The use according to claim 1, characterized in that: The dosage form of the drug includes at least one of tablets, pills, powders, solutions, suspensions, emulsions, and granules.

6. The use according to claim 1, characterized in that: The acute myeloid leukemia includes AML1-ETO or MLL fusion leukemia.

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