KR102507403B1 - Composition for preventing or treating Acute myelogenous leukemia comprising HSPA1L, inducer thereof or activator thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment of acute myeloid leukemia containing a HSPA1L (Heat shock 70 kDa protein 1L) expression promoter or activator as an active ingredient and a method for screening the same, and more particularly, HSPA1L-overexpressed osteoblast cells are used as a control group Alternatively, as compared to cells in which HSPA1L was knocked out, improved cell proliferation and differentiation effects were shown, and it was confirmed that cell cycle control of osteoblasts was possible through regulation of HSPA1L expression, the HSPA1L expression promoter or activator was acute myeloid It can be provided as a leukemia treatment agent and an osteoblast cell cycle regulator.

Description

A composition for preventing or treating acute myelogenous leukemia comprising HSPA1L, an expression promoter thereof or an activator thereof as an active ingredient

It relates to a pharmaceutical composition for treatment of acute myeloid leukemia containing HSPA1L (Heat shock 70 kDa protein 1L), an expression promoter or activator thereof as an active ingredient, and a screening method thereof.

Leukemia is divided into myelogenous and lymphocytic according to origin, again largely i) Acute myelogenous leukemia (AML), ii) Chronic myelogenous leukemia (CML), iii) Acute lymphocytic leukemia (Acute lymphocytic leukemia) Leukemia, ALL) iv) Chronic lymphocytic leukemia (CLL), which is divided into four types. Such leukemia is a blood tumor in which white blood cells abnormally proliferate in blood cells. That is, immature leukocytes exist in large quantities in the blood, and other organelles including normal hemocytes exist in a much smaller number compared to the abnormal proliferation of leukocytes. As a result, not only basic functions of blood such as oxygen transport and nutrient supply, but also phenomena similar to autoimmune diseases occur, destroying normal tissues.

In double lymphocytic leukemia (ALL), lymphocyte leukocytes change into malignant cells, proliferate in the bone marrow, spread to the peripheral blood, and invade the liver, spleen, lymphatic system, cerebrum, cerebellum, and spinal cord. Lymphoblastic leukemia is usually defined as lymphoblasts occupying more than 20% of the bone marrow or peripheral blood. If an abnormality of chromosome 9:22 is found in this leukemia, it is called Philadelphia chromosome-positive acute lymphocytic leukemia. This leukemia is a typical blood cancer that can be cured and shows much better results than acute myeloid leukemia. However, adults have much worse treatment results than children, and Philadelphia chromosome positivity is rare in children, whereas it appears in about 1/4 of adults.

Lymphocytic leukemia is a rare disease with an incidence rate of 200 to 300 per year in adults. Looking at the treatment results so far, about 30 to 40% of patients can be cured through continuous chemotherapy, but about 50% Patients who develop leukemia relapse during treatment or show refractoriness that does not respond to existing treatment, and in this case, the expected 5-year survival rate is less than 10% of fatal leukemia. In the case of children, it occurs most often in children aged 2 to 5 years, and the complete cure rate reaches 85 to 90%. Representative treatments for lymphocytic leukemia include Besponsa (Pfizer) and Blinatmoab (Blincyto, Amgen).

On the other hand, acute myelogenous leukemia (AML) is a leukemia caused by abnormal proliferation of myeloid leukocytes, and is defined as a case in which immature leukocytes in the bone marrow or peripheral blood are 20% or more according to the WHO diagnostic criteria. Acute myeloid leukemia is a blood cancer in which abnormal cells that interfere with the production of normal white blood cells are produced and accumulated in the red bone marrow. It is most common in adults. Symptoms of acute myelogenous leukemia appear when the bone marrow is filled with leukemia cells and the number of normal blood cells (red blood cells, platelets, and normal white blood cells) decreases rapidly. The main symptoms are fatigue, rapid breathing, easy bruising or bleeding, and infections occur frequently. There are several subtypes of acute myeloid leukemia, and treatment and prognosis vary depending on the subtype, and the 5-year survival rate is 15-70%, and the recurrence rate varies from 33-78%. In the early stage of acute myeloid leukemia, remission induction therapy is performed through chemotherapy, and then additional chemotherapy or hematopoietic stem cell transplantation is performed depending on the patient.

Examples of acute myelogenous leukemia treatment include Gleevac (Greevec, Novartis), Decitabine (Ansen, Korea), and daurismo (pifzer). In addition, there is hematopoietic stem cell transplantation known as bone marrow transplantation. Hematopoietic stem cells or hematopoietic stem cells are cells that can potentially differentiate into major constituents of blood. Hematopoietic stem cells can differentiate into cells produced in bone marrow (monocytes, macrophages, neutrophils, basophils, red blood cells, platelets, etc.) and lymphatic system (T cells, B cells, NK cells). Unlike the importance of 1/10,000 of cells in bone marrow tissue, these cells account for a relatively small percentage, but because they have the ability to replicate themselves, they can always produce blood components in time. Accordingly, when an abnormality occurs in a cell differentiation process such as leukemia or when an abnormality occurs due to a decrease in the number of hematopoietic stem cells such as aplastic anemia, these diseases can be fundamentally treated by transplanting hematopoietic stem cells.

However, after hematopoietic stem cell transplantation, the decline in immune function lasts for a long time, and repeated bacterial, viral, or fungal infections may occur. Since there are differences depending on the presence or absence of the disease, the degree of recovery from infection also differs. In the case of allotransplantation, graft-versus-host disease can occur, which together with recurrence is an important factor influencing the success of transplantation.

Accordingly, studies have been conducted to minimize the side effects of hematopoietic stem cell transplantation, but they are still incomplete.

Korean Patent Publication No. 10-2018-0127029 (published on November 28, 2018)

The present invention is a method for minimizing the side effects of hematopoietic stem cell transplantation, which is a treatment method for acute myeloid leukemia, and a pharmaceutical composition for preventing or treating acute myeloid leukemia containing HSPA1L, an expression promoter or activator thereof as an active ingredient, and a screening method thereof want to provide

The present invention provides a pharmaceutical composition for preventing or treating acute myeloid leukemia containing HSPA1L, an expression promoter or an activator thereof as an active ingredient.

The present invention provides a composition for regulating hematopoietic stem cell cell cycle containing HSPA1L, an expression promoter or an activator thereof as an active ingredient.

In addition, the present invention comprises the steps of processing a candidate substance to cells isolated from the subject; Checking the expression or activation level of HSPA1L in the cells treated with the candidate substance; and comparing the HSPA1L expression or activation level with a control group.

According to the present invention, osteoblasts in which HSPA1L was overexpressed showed improved cell proliferation and differentiation effects compared to control cells or cells in which HSPA1L was knocked out, and it was confirmed that cell cycle control of osteoblasts was possible through regulation of HSPA1L expression Accordingly, the HSPA1L and its expression promoter or activator may be provided as a therapeutic agent for acute myelogenous leukemia and an osteoblast cell cycle regulator.

Figure 1 shows the results of the mRNA levels of LT-HSC, ST-HSC, MPP, and Lin using qPCR.
Figure 2 shows the results of sorting Bone Marrow's cKit ⁺ , Lin ^- hematopoietic stem progenitor cells using a FACS Arial device and cell survival of BM cKit ⁺ , Lin ^- cells treated with H ₂ O ₂ This is the result of qPCR analysis confirming cell viability and mRNA.
3 is a FACS analysis result of confirming the cell population of stem cells, progenitor cells, and lineage cells of the bone marrow.
4 is a FACS analysis result of confirming the cell population of LT-HSC and ST-HSC in HSPA1L wild type (Wild type), over expression (Over expression) and knockout (Knock out) mouse bone marrow.
Figure 5 shows the MEP (Megakaryocyte-erythroid progenitor), CMP (common myeloid) of myeloid progenitor cells in the bone marrow of each mouse in which HSPA1L is wild type, over-expressed, and knocked out. progenitor), GMP (granulocyte-macrophage progenitor), CLP (common lymphoid progenitor), LK (cKit ⁺ , Lin ^- (myeloid progenitor cell) group percentages.
6 is BFU-E (Burst forming unit-erythroid), CFU-M (Colony forming unit-macrophage), CFU-G (Colony forming unit-granulocyte), which are hematopoietic stem progenitor cells stimulated with cytokines. ) and CFU-GEMM (Colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte).
Figure 7 shows colonies of myeloid progenitor cells in the bone marrow of each mouse in which HSPA1L is wild type, overexpressed, and knocked out, and their BFU-E (Burst forming unit-erythroid), CFU- This is the result of confirming the number of colonies of M (Colony forming unit-macrophage), CFU-G (Colony forming unit-granulocyte), and CFU-GEMM (Colony forming unit-granulocyte erythrocyte, macrophage, megakaryocyte).
Figure 8 shows the lineage cells (B cell, T cell, Monocyte and Neutrophil) of the bone marrow of each mouse in which HSPA1L is wild type, overexpressed, and knocked out using a FACS Arial device. It is the result of classification and the result expressed as a percentage.
9 shows a method of analyzing and classifying HSC-like cells using a FACS Arial device in an EML (Erythroid myeloid lymphoid cell) cell line, which is a research model of hematopoietic stem cells.
10 shows cell viability after treatment of Sca1 ⁺ ckit ⁺ EML cells with H ₂ O ₂ at each concentration, mRNA level of HSPA1L, and HSPA1L expression level, respectively, by H ₂ O ₂ , qPCR and Western blot (Western blot). This is the result of blot) analysis.
11 is a result of confirming the proliferation ability (feration ability) of EML cell cells in which HSPA1L is overexpressed (OE) or knocked out (KD) Sca1 ⁺ ckit ⁺ .
12 shows the results of FACS analysis and Western blot analysis confirming cell cycling of Sca1 ⁺ ckit ⁺ EML cells in which HSPA1L is overexpressed (OE) or knocked out (KD).
13 is a result of confirming glucose uptake and ATP generation through fluorescence absorbance analysis in Sca1 ⁺ ckit ⁺ EML cells in which HSPA1L is overexpressed (OE) or knocked out (KD).

Hereinafter, the present invention will be described in more detail.

As a result of research efforts on a treatment method for acute myeloid leukemia, the present inventors confirmed that HSPA1L of hematopoietic stem cells regulates cell proliferation and differentiation of hematopoietic stem cells. In addition, it was confirmed that HSPA1L reacts highly in hematopoietic stem cells and progenitor cells in response to ROS such as H ₂ O ₂ , and it was confirmed that the cell cycle was regulated using this, thereby completing the present invention.

The present invention HSPA1L [Heat shock 70 kDa protein 1L; NM_005527.4 (homo), 　NM_013558.2 (mice)], a pharmaceutical composition for preventing or treating acute myeloid leukemia containing an expression promoter or an activator thereof as an active ingredient may be provided.

The expression promoter or activator may be any one selected from the group consisting of compounds, peptides, aptamers, primers, probes, and antibodies that specifically bind to the HSPA1L protein or the gene encoding the same.

The HSPA1L may improve cell proliferation and differentiation by regulating the cell cycle of hematopoietic stem cells.

The HSPA1L, its expression promoter or its activator may be included in an amount of 0.01 to 10 parts by weight based on 100 parts by weight of the total pharmaceutical composition.

According to an embodiment of the present invention, FACS aria and Western blot were performed to confirm the cell cycle of control cells and Sca1 ⁺ ckit ⁺ EML cells overexpressing or knocking out HSPA1L, as shown in FIG. 12, HSPA1L knockout compared to control ( It can be seen that the G1 phase of KD) mice was decreased, whereas the G1 phase was increased in HSPA1L overexpressing mice.

From the above results, it can be used as a hematological tumor treatment agent by regulating the cell cycle and differentiation of hematopoietic stem cells through the regulation of HSPA1L expression, and the substance enhancing the expression of HSPA1L can be provided as a treatment agent for hematologic tumors.

In one embodiment of the present invention, the pharmaceutical composition for preventing or treating acute myeloid leukemia containing HSPA1L, its expression promoter or its activator as an active ingredient is an injection, granule, powder, tablet, pill, or capsule according to a conventional method. Any one formulation selected from the group consisting of an agent, suppository, gel, suspension, emulsion, drop, or solution may be used.

In another embodiment of the present invention, suitable carriers, excipients, disintegrants, sweeteners, blood, commonly used in the preparation of a pharmaceutical composition for preventing or treating acute myeloid leukemia containing HSPA1L, an expression promoter or an activator thereof as an active ingredient It may further include one or more additives selected from the group consisting of replication, swelling agents, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersing agents, surfactants, binders and lubricants.

Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil may be used, and solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules. These solid preparations may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral use, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base material of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

According to one embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner through intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered to the subject as

The preferred dosage of HSPA1L, its expression promoter, or its activator may vary depending on the condition and body weight of the subject, the type and severity of the disease, the type of drug, the route and duration of administration, and can be appropriately selected by those skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or divided into several administrations, and the scope of the present invention is not limited thereby.

In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.

The present invention can provide a composition for regulating hematopoietic stem cell cell cycle containing HSPA1L, an expression promoter or an activator thereof as an active ingredient.

The expression promoter or activator may be any one selected from the group consisting of compounds, peptides, aptamers, primers, probes and antibodies that specifically bind to the HSPA1L protein or the gene encoding it, more preferably H ₂ O ₂ , but is not limited thereto.

According to another embodiment of the present invention, Sca1 ⁺ ckit ⁺ erythrocyte myeloid lymphocytes were treated with H ₂ O ₂ at each concentration, and cell viability was confirmed, and quantitative PCR and Western blot were performed to determine the mRNA of HSPA1L. As a result of confirming the level and the level of HSPA1L expression, as shown in FIG. 10, the mRNA level of HSPA1L increased as the concentration of H ₂ O ₂ increased, and the expression of HSPA1L also increased in Western blot analysis, and as the concentration of H ₂ O ₂ increased, As the expression of HSPA1L increased, it was confirmed that the cell cycle progressed rapidly and cell viability decreased.

From the above results, it was confirmed that when ROS is increased, the G1 phase can be increased due to increased HSPA1L secretion, and when ROS is decreased, proliferation and differentiation can be inhibited through the opposite signaling mechanism due to decreased HSPA1L secretion.

In addition, the present invention comprises the steps of processing a candidate substance to cells isolated from the subject; Checking the expression or activation level of HSPA1L in the cells treated with the candidate substance; and comparing the HSPA1L expression or activation level with that of a control group.

Cells isolated from the subject may be hematopoietic stem cells or Sca1 ⁺ ckit ⁺ erythroid myeloid lymphocytes.

The HSPA1L expression or activity level was determined by reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA), immunohistochemistry, Western blotting, and flow cytometry (FACS). It may be measured by any one selected from the group consisting of

As used herein, the term "prevention" refers to any activity that inhibits or delays the onset of blood tumors by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to all activities in which symptoms caused by blood tumors are improved or advantageously changed by administration of the pharmaceutical composition according to the present invention.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Example 1> Identification of HSPA1L from hematopoietic stem cells (HSC)

In order to confirm the expression of HSPA1L in hematopoietic stem cells, the following experiment was performed.

1. Cell identification

To identify long-term hematopoietic stem cells (LT-HSC), short-term hematopoietic stem cells (ST-HSC), and hematopoietic multipotent progenitor (MPP) cells, bone marrow cells were obtained from mice and FACS aria It was separated using the instrument.

As a result, it was confirmed that each cell population was classified with high purity, as shown in FIG. 3 .

2. Confirmation of HSPA1L expression

To confirm the expression of HSPA1L in hematopoietic stem cells (HSC), HSPA1L mouse wild type (WT), HSPA1L overexpression (OE), and HSPA1L knockout (KO) cells were analyzed using FACS aria. Classified as in 4.

As a result, as shown in FIG. 4, it was confirmed that the bone marrow percentage of all LT-HSC, ST-HSC, MPP, and LSK cells was specifically increased when HSPA1L was overexpressed.

In addition, as shown in FIG. 5, it was confirmed that the percentage of bone marrow in HSPA1L mouse bone marrow myeloid progenitor cells was also increased in the cells in which HSPA1L was overexpressed.

Next, in order to confirm the differentiation potential of hematopoietic stem progenitor cells, colony formation analysis was performed as shown in FIG. 6, and BFU-E (Burst forming unit-erythroid) and CFU-M (Colony forming unit-macrophage), CFU-G (Colony forming unit-granulocyte), and CFU-GEMM (Colony forming unit-granulocyte erythrocyte, macrophage, megakaryocyte) were confirmed.

As a result of confirming the number of colonies by culturing colonies of cells whose differentiation potential was confirmed as described above, it was confirmed that the number of colonies overexpressing HSPA1L increased as shown in FIG. 7 .

<Example 2> Confirmation of cell number change in myeloid lineage cells according to HSPA1L expression

Populations of each myeloid lineage cell (B cell, T cell, monocyte, neutrophil) in HSPA1L wild type, HSPA1L overexpression and HSPA1L knockout mice were analyzed by FACS Classification was performed using an Arial device.

As a result, as shown in FIG. 8, it was confirmed that the change in the number of cells of the myeloid lineage depending on whether or not HSPA1L was expressed was not significantly affected.

<Example 3> Sca1 ⁺ cKit ⁺ Determination of HSPA1L expression levels in EML cells

In order to make up for the difficult research problem due to the scarcity of hematopoietic stem cells (HSC), hematopoietic stem cell-like cells Sca1 ⁺ ckit ⁺ erythrocyte myeloid lymphocytes (Sca1 ⁺ ckit ⁺ EML cells) were identified using FACS aria as shown in FIG. classified.

The sorted Sca1 ⁺ ckit ⁺ erythroid myeloid lymphocytes were treated with H ₂ O ₂ at concentrations of 0, 10, 20, 50 and 100 μM, and then cell viability was confirmed, and quantitative PCR and Western blotting were performed. HSPA1L mRNA level and HSPA1L expression level were confirmed.

As a result, cell viability decreased as the concentration of H ₂ O ₂ increased, as shown in FIG. 10, the mRNA level of HSPA1L increased as the concentration of H ₂ O ₂ increased, and the expression of HSPA1L also increased in Western blot analysis. I was able to confirm that

<Example 5> Sca1 ⁺ cKit ⁺ Confirmation of cell proliferation and differentiation effects by HSPA1L stimulation in EML cells

The proliferative ability of control cells and Sca1 ⁺ ckit ⁺ EML cells overexpressing or knocking out HSPA1L was confirmed by Western blotting.

As a result, as shown in FIG. 11, it was confirmed that the number and proliferative capacity of cells overexpressing HSPA1L were increased, whereas the number and proliferative capacity of cells knocked out of HSPA1L were decreased.

In addition, by performing FACS aria and Western blotting, the cell cycles of control cells and Sca1 ⁺ ckit ⁺ EML cells in which HSPA1L was overexpressed or knocked out were confirmed.

As a result, as shown in FIG. 12, compared to the control group, the G0 phase decreased and the G1 phase and the S-G2-M phase increased in the cells in which HSPA1L was overexpressed, whereas in the cells knocked out of HSPA1L, the G0 phase increased, the G1 phase and the S-G2 phase increased. It was confirmed that the G2-M phase was reduced.

From the above results, it was confirmed that HSPA1L regulates the cell cycle.

On the other hand, changes in ATP production and glucose uptake in Sca1 ⁺ ckit ⁺ EML cells in which HSPA1L was overexpressed or knocked out and HSPA1L were overexpressed were confirmed by fluorescence absorbance.

As a result, as shown in FIG. 13, it was confirmed that ATP production and glucose uptake were increased compared to the control group in the cells overexpressing HSPA1L. On the other hand, it was confirmed that ATP production and glucose uptake were reduced in cells in which HSPA1L was knocked out.

From the above results, it was confirmed that an increase in HSPA1L expression increases intracellular ATP production and glucose uptake together.

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

delete delete delete delete delete delete treating osteoblasts or Sca1+ ckit+ erythroid myeloid lymphocytes isolated from the subject with a candidate substance;
Checking the expression or activation level of HSPA1L in the cells treated with the candidate substance; and
A method for screening an acute myeloid leukemia treatment comprising determining a candidate having a higher expression or activation level as an acute myeloid leukemia treatment when the HSPA1L expression or activation level is compared with a control group. delete The method according to claim 7, wherein the HSPA1L expression or activity level is determined by reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA), immunohistochemistry, Western blotting and flow cytometry An acute myeloid leukemia therapeutic agent screening method, characterized in that measured by any one selected from the group consisting of analysis method (FACS).

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