Use of spermidine in the preparation of a medicament for treating leukemia

Abstract

The present application relates to the application of spermidine in the preparation of drugs for treating leukemia, which is specific to the loss-of-function mutation of Runx1 in acute myeloid leukemia (AML) patients, and induces apoptosis of AML cells by spermidine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of spermidine in the preparation of a drug for treating leukemia. BACKGROUND

[0002] Runx1 is the alpha subunit of the core transcription factor, which forms a heterodimer with CBFβ to regulate the transcription of target genes; and the binding of Runx1 to DNA is dependent on CBFβ. As one of the important transcription factors for maintaining the function of hematopoietic stem cells, 20% of patients with acute myeloid leukemia (AML) have abnormal genes of Runx1 or CBFβ, including chromosomal translocation and mutation. Unlike AML patients with chromosomal translocation, patients carrying Runx1 loss-of-function mutations have a poor prognosis, with a 5-year survival rate of less than 20%. In 2016, WHO classified these AML patients carrying Runx1 loss-of-function mutations into a separate category. At present, chemotherapy is still the main treatment for these patients, and the poor prognosis of patients is caused by Runx1 mutations.

[0003] A large number of studies have shown that protein translation plays a key role in the fate determination of cells. After cells are stimulated by stress (including chemotherapy), selective translation of some proteins promotes cell apoptosis or overcomes environmental stress. The applicant found that Runx1 can regulate protein translation by regulating the transcription of ribosome-related genes and can participate in protein translation by interacting with proteins. Further analysis found that Runx1 controls the stability and activity of the key factor eIF5A of protein translation, thereby regulating cell protein translation; after Runx1 loss-of-function mutation, the activity and abundance of eIF5A decrease. Eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved translation factor in cells that is essential for maintaining cell activity. In addition to promoting the synthesis of the first peptide bond in the initiation stage of protein translation, eIF5A can also promote the extension and termination of protein translation; and mediates autophagy, proliferation, metabolism and apoptosis. And inhibiting the expression or activity of eIF5A produces a phenotype in target cells that is basically the same as that after Runx1 knockout or mutation. This suggests that eIF5A is an important target of Runx1. The activity of eIF5A is controlled by the hydroxylation of lysine of the protein.

[0004] AML patients with Runx1 mutations have a poor prognosis, with a low 5-year survival rate, so there is an urgent need for new and effective treatment methods. SUMMARY

[0005] The application aims to provide an application of spermidine in preparation of a drug for treating leukemia, which is specific to Runx1 loss-of-function mutation in patients with acute myeloid leukemia (AML) and induces apoptosis of AML cells by using spermidine.

[0006] The technical scheme of the application is:

[0007] The application of spermidine in preparation of a drug for treating leukemia.

[0008] The leukemia is acute myeloid leukemia.

[0009] The acute myeloid leukemia is caused by Runx1 loss-of-function mutation.

[0010] The treatment is performed by injection, and the dose is 20 mg / kg.

[0011] Firstly, in previous studies, we found that Runx1 regulates protein translation by controlling ribosome biogenesis, but the activation of mTOR (promoting ribosome biogenesis) cannot completely restore the protein translation rate in Runx1 knockout hematopoietic stem and progenitor cells. This indicates that Runx1 has other ways to regulate protein translation. Then in 293T cells, we observed that overexpression of mutant Runx1 can cause down-regulation of eIF5A. Subsequently, we verified that Runx1 mutation causes down-regulation of eIF5A protein in K562 and Runx1 knockout hematopoietic stem cells.

[0012] We know that Runx1 knockout leads to insensitivity of mouse HSCs to various stresses. Inhibition of hydroxylation lysine modification (i.e. activity) of eIF5A in K562 cells by GC7 increases the resistance of cells to AraC. This suggests that the chemotherapy resistance caused by Runx1 mutation may be related to the down-regulation of eIF5A. We found that a large number of cells carrying mutant Runx1 cells undergo apoptosis when spermidine is added to the culture medium of leukemia cells K562 cells carrying Runx1 mutation to inactivate eIF5A, and this apoptosis phenomenon can be reversed by GC7, which indicates that the apoptosis of Runx1 mutant cells caused by spermidine is dependent on eIF5A. Among them, about 60% of Runx1 mutant cells undergo apoptosis after 24 hours of treatment, and more than 90% of cells undergo apoptosis after 48 hours of treatment. However, there is no obvious effect on leukemia cells expressing wild-type Runx1. This indicates that spermidine can specifically kill Runx1 mutant cells.

[0013] Finally, we continuously intraperitoneally injected spermidine (20 mg / kg) into Runx1 knockout mice and control mice for 7 days, and analyzed the stem cells (Lin - cKit + Sca1 +Apoptosis of LSK cells. As shown, spermidine caused a significant increase in Annexin V positive cells in Runxl knock-out LSK cells (did not cause massive apoptosis, possibly due to drug pharmacokinetics).

[0014] Applicants found that spermidine, a substrate for the post-translational modification of eIF5A, promotes eIF5A activation. The results show that spermidine effectively promotes apoptosis in hematopoietic stem cells (HSCs) from Runxl mutant mice and human leukemia cells. In addition, spermidine is widely distributed in the body, and thus spermidine is an effective drug for treating AML carrying Runxl mutations.

[0015] Structure of spermidine

[0016] BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to make the objects, technical solutions and advantages of the present application clearer, the present application is described below with the following drawings:

[0018] Figure 1 Runxl mutation causes down-regulation of eIF5A. In the figure, Figure 1 A. In vitro culture of HSCs (LSK Flt3 - After treatment with PTEN inhibitor (PTENi), p-S6 in the cells was shown to increase (A, left), but the protein translation efficiency in Runxl knock-out HSCs was still lower than that in the control group. Figure 1 B. Runxl mutation or knock-out causes down-regulation of eIF5A in 293T, K562 and mouse HSCs.

[0019] Figure 2 Inhibition of eIF5A increases the resistance of cells to chemotherapy drug AraC. Activation of eIF5A by spermidine promotes apoptosis in leukemia cells carrying Runxl mutations. In the figure, Figure 2 A-C. Runxl knock-out causes HSCs to be insensitive to various stresses; Figure 2 A. Radiotherapy (3 Gy); Figure 2 B. Chemotherapy (AraC); Figure 2 C. Endoplasmic reticulum stress (Tu: Tunicamycin, an endoplasmic reticulum stress inducer); Figure 2 D. Inhibition of eIF5A activity has no significant effect on apoptosis, but can resist AraC-induced apoptosis; Figure 2 E. Spermidine induces apoptosis in cells carrying Runxl mutations (R174Q), but GC7 can inhibit spermidine-induced apoptosis to some extent; Figure 2F is spermidine. It has no effect on wild type Runxl K562 cells, but can significantly kill cells carrying Runxl mutations. 24h treatment can cause nearly 60% of cells to undergo apoptosis, and 48h more than 90% of cells to undergo apoptosis. Spe: spermidine, spermidine. GC7: DHS inhibitor, thereby inhibiting the activity of eIF5A.

[0020] Figure 3 Supplementing spermidine in vivo can also increase the apoptosis of Runxl knockout hematopoietic stem and progenitor cells, as shown in the figure, Figure 3 A is a schematic diagram of administration. Mice received intraperitoneal injection of spermidine (20mg / kg) for 7 consecutive days, and on the 8th day, the apoptosis (Annexin V) of HSCs was analyzed by flow cytometry; Figure 3 B is that spermidine can significantly increase the apoptosis of Runxl knockout LSK cells. Specific embodiments

[0021] The preferred embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0022] Example 1

[0023] 1. Bone marrow of 8-week-old C57 / B6J Runxl f / f; VavCre and wild type control mice was isolated, and Lin- cells were enriched according to the Miltenyi mouse hematopoietic stem and progenitor cell separation kit (Miltenyi, cat: 130-110-470), and then stained with lineage (including CD3e (BD, cat: 558214), B220 (BD, cat: 558108), Gr1 (BD, cat: 562709), Mac1 (BD, cat: 562605) and Ter119 (BD, cat: 563998), ckit (Thermo, cat: 47-1171-82), Sca1 (Thermo, cat: 25-5981-82), Flt3 (Thermo, cat: 12-1351-82) antibodies, and finally the Flt3-LSK cells sorted by flow cytometry were cultured (Stemcell, cat: 09600), while adding PTEN inhibitor (Selleck, cat: S8174) or DMSO, after 24 hours, the phosphorylation of S6 and the protein translation efficiency of the cells were analyzed.

[0024] Example 2 Western blot

[0025] For small amount of HSCs cells, the method published in 2018 JOVE (Cai X, et al. 2018) was used. For cell lines, the routine method was used. After cells were lysed with RIPA buffer, 20-50 ug total protein was separated by SDS-PAGE, transferred, blocked, and then detected with eIF5A (Abclonal, cat: A4414), actin (Abclonal, cat: AC043), p-S6 (CST, cat: 2211) antibodies and corresponding secondary antibodies.

[0026] Example 3 Analysis of apoptosis:

[0027] (1) In vitro experiment: K562 cells stably expressing mutant Runxl (R174Q) or empty vector were cultured with RPMI160 + 10 FBS. Cells were treated with GC7 (Selleck, cat: S2961) (100 uM), AraC (Selleck, cat: S1648) (10 uM), GC7 (100 uM) + AraC (10 uM), spermidine (Selleck, cat: S3569) (50 uM), spermidine (50 uM) + GC7 (100 uM) or DMSO for 24 hours, then stained with AnnexinV (Thermo, cat: BMS147FI) and 7-AAD and analyzed by flow cytometry.

[0028] (2) In vivo experiment: Mice were sacrificed after receiving radiation, chemotherapy drugs or Tunicamycin (Selleck, cat: S7894) treatment for 24 hours, and bone marrow cells were isolated, then the bone marrow cells were stained, including (lineage, including CD3e, B220, Gr1, Mac1 and Ter119), Sca1, Flt3, CD34 (Thermo, cat: 17-0349-42), then stained with AnnexinV and 7-AAD, and finally analyzed by flow cytometry for the proportion of early apoptotic cells (AnnexinV + 7AAD - ) in each cell population (Cai X, et al. 2015). In the spermidine-induced apoptosis experiment, mice (Saiye Biotechnology) received intraperitoneal injection of spermidine (20 mg / kg) for 7 consecutive days, and apoptosis was analyzed on the 8th day, with staining including lineage (including CD3e, B220, Gr1, Mac1 and Ter119), Sca1, ckit, AnnexinV and 7-AAD.

Claims

1. The application of spermidine in the preparation of drugs for treating leukemia, wherein the leukemia is acute myeloid leukemia, and the acute myeloid leukemia is caused by Runx1 loss-of-function mutation.

2. The application according to claim 1, characterized in that: The treatment is administered via injection.

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