JP2017146297A - Chronic myelogenous leukemia therapeutic agent and therapeutic agent screening method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To resolve a mechanism that converts normal hematopoietic processing in a chromatic myelogenous leukemia (CML) initial stage into leukemic processing, and to discover a target substance that may serve as a novel chromatic myelogenous leukemia therapeutic agent or bone transplantation therapeutic agent.SOLUTION: In a mechanism in which a model animal having a symptom and disease state in a chromatic myelogenous leukemia (CML) initial stage is used to convert hematopoietic processing into leukemic processing, it is discovered that, under a CML bone marrow environment, a basophil develops a CCL3, and further the CCL3 facilitates predominant proliferation of a LIC with respect to a normal hematopoietic stem cell, which in turn develops the chromatic myelogenous leukemia. Further, it is verified that a function of the basophil is directly or indirectly hindered such as reduction in the basophil and the like, and thereby the developing of the chromatic myelogenous leukemia or bone transplantation can be also suppressed. Accordingly, a novel screening method of a therapeutic agent or preventive agent of the chromatic myelogenous leukemia or bone transplantation with the basophil as a target substance, and the novel therapeutic agent or preventive agent of the chromatic myelogenous leukemia or bone transplantation are completed.SELECTED DRAWING: None

Description

  The present invention relates to a therapeutic agent for chronic myelogenous leukemia and a method for screening the therapeutic agent, and more specifically, in a non-human chronic myelogenous leukemia model animal in which symptoms of an early stage of chronic myelogenous leukemia are induced. The present invention relates to a screening method that targets basophils involved in the above.

(Chronic myeloid leukemia)
Chronic myeloid leukemia (hereinafter sometimes referred to as “CML”) is a myeloproliferative tumor resulting from malignant transformation of hematopoietic stem cells (hereinafter also referred to as “HSC”). The frequency of chronic myelogenous leukemia in Japan is relatively rare (1-2 in 100,000), accounting for about 20% of all leukemia in adults.
The cause of the onset of chronic myelogenous leukemia has not been clearly clarified, but it is divided into three stages: a chronic phase, a transition phase, and an acute phase (acute transformation phase) (Non-patent Document 1). The chronic phase is asymptomatic, and as the disease progresses, the number of white blood cells and platelets in the blood increases, and then gradually becomes anemic. Furthermore, as the white blood cell count increases, symptoms such as general malaise and abdominal fullness appear. More than 90% of CML cases are associated with the presence of the Philadelphia chromosome. This chromosome arises from a reciprocal translocation between chromosomes 9 and 22 and forms a BCR-ABL fusion gene (Non-Patent Document 2). This gene is a pathogen of CML (Non-patent Document 3), and the presence of leukemic stem cells {LEUKEMIA initiating cells (LICs)} expressing BCR-ABL fusion gene product (activated tyrosine kinase) in bone marrow is It is deeply involved in the start of the chronic phase (Non-patent Document 4).

The breakpoint cluster region of the BCR gene encodes a BCR-ABL fusion protein. The BCR-ABL fusion protein exhibits constitutive tyrosine kinase activity that can convert hematopoietic stem cells (HSC) to LIC. LIC can cause pathological pathology of heterogeneous leukemia cells one after another (Non-Patent Documents 3 and 4). The site essential for hematopoiesis is in a limited area of the bone marrow (BM). During the CML initiation process, a small number of BCR-ABL-expressing LIC coexist with a large number of normal hematopoietic cells. Although the exact mechanism is unknown, LIC then overwhelms normal HSCs and becomes dominant throughout the hematopoietic system, converting normal hematopoietic processes into leukemic processes.
Furthermore, CML patients frequently show basophilia in the bone marrow and peripheral blood (PB), which becomes more apparent in the acute phase (Non-Patent Documents 5 and 6). However, little is known about the pathogenic role of basophil-like leukemia cells in the progression of chronic myeloid leukemia.

CCL3 (Chemokine (C-motif) ligand 3) is also known as MIP-1α (Macrophage information protein-1α) and belongs to the CC chemokine family. It has been reported that abnormal expression of CCL3 occurs in the bone marrow by in vivo BCR-ABL expression induction (Non-patent Document 7). Furthermore, it has been reported that CCL3-mediated signals can control in vitro proliferation of normal HSC and leukemia stem cells in different ways, depending on the kinase activity of the Abl protein (Non-patent Documents 8 and 9). ). These reports suggest that CCL3 is potentially involved in the interaction between normal HSCs and leukemic stem cells in the initial process of CML development (Non-Patent Document 7).
However, the exact role of CCL3 was unclear because there was no appropriate experimental model animal.

We have previously shown that CML-like disease can develop after direct injection of a small number of LICs into non-irradiated mice. In this model, we show that leukemia cell-derived CCL3 expression preferentially acts on normal HSPC (hematopoietic stem / progenitor cells) and that this action can ultimately lead to dominant proliferation of LIC in the bone marrow. It was.
However, the cellular source of CCL3 and the mechanism underlying the action have not been clarified (Non-patent Document 10, Patent Document 1).

Patent publication 2014-140336

Lahaye, T., et al. Response and resistance in 300 patients with BCR-ABL-positiveleukemias treated with imatinib in a singlecenter: a 4.5-year follow-up. Cancer 103, 1659-1669 (2005). Bartram, C.R., et al. Translocation of c-ab1oncogene correlates with the presence of a Philadelphia chromosome in chronicmyelocytic leukaemia.Nature 306,277-280 (1983). Sawyers, C.L.Chronic myeloid leukemia.NEngl J Med 340, 1330-1340 (1999). Koschmieder, S., et al. Inducible chronicphase of myeloid leukemia with expansion of hematopoietic stem cells in atransgenic model of BCR-ABL leukemogenesis. Blood 105, 324-334 (2005). Cerny-Reiterer S, Ghanim V, Hoermann G, etal.Identification of basophils as a major source of hepatocyte growth factorin chronic myeloid leukemia: a novel mechanism of BCR-ABL1-independent diseaseprogression. Neoplasia. 2012; 14 (7): 572- 584. Theologides A. Unfavorable signs in patientswith chronic myelocytic leukemia. Ann Intern Med. 1972; 76 (1): 95-99. Zhang, B., et al. Altered microenvironmentalregulation of leukemic and normal stem cells in chronic myelogenous leukemia. Cancer Cell 21, 577-592. Eaves, C.J., Cashman, J.D., Wolpe, S.D. & Eaves, A.C.Unresponsiveness of primitive chronic myeloid leukemia cells tomacrophage inflammatory protein 1 alpha, an inhibitor of primitive normalhematopoietic cells.Proc Natl Acad Sci USA 90, 12015-12019 (1993). Wark, G., et al. Abl protein kinase abrogatesthe response of multipotent haemopoietic cells to the growth inhibitormacrophage inflammatory protein-1 alpha.Oncogene 16, 1319-1324 (1998). Baba T, Naka K, Morishita S, Komatsu N, Hirao A, Mukaida N. MIP-1alpha / CCL3-mediated maintenance of leukemia-initiating cells in theinitiation process of chronic myeloid leukemia. J ExpMed. 2013; 210 (12): 2661 -2673.

  The present inventors have elucidated the mechanism of converting a normal hematopoietic process in the early stage of CML into a leukemic process, and have sought to find a target substance that can be a new therapeutic agent for chronic myeloid leukemia.

In order to solve the above-mentioned problems, the present inventors have used a model animal having symptoms and pathologies in the early stage of CML to convert a normal hematopoietic process into a leukemic process. It was found that basophils express CCL3 and that CCL3 promotes the dominant growth of LIC against normal hematopoietic stem cells, thereby causing chronic myeloid leukemia. Furthermore, it was also confirmed that the onset of chronic myeloid leukemia can be suppressed by directly or indirectly inhibiting the function of basophils, such as reducing basophils.
Thereby, a screening method for a novel therapeutic agent or preventive agent for chronic myeloid leukemia using basophils as a target substance and a novel therapeutic agent or preventive agent for chronic myeloid leukemia were completed.

That is, this invention consists of the following.
1. A screening method for a therapeutic or prophylactic agent for chronic myelogenous leukemia or bone metastasis, which comprises selecting a test compound that inhibits the action or expression of basophils or basophil variants.
2. The screening method according to item 1, comprising the following steps:
(1) A step of administering a test compound to the bone marrow of a non-irradiated non-human animal and a chronic myeloid leukemia model animal into which hematopoietic stem cells into which a BCR-ABL fusion gene has been introduced have been transplanted into the medullary cavity. And (2) measuring the expression level of basophils in the bone marrow.
3. 3. The screening method according to item 1 or 2, wherein the chronic myelogenous leukemia is an early stage chronic myelogenous leukemia.
4). 4. The screening method according to any one of items 1 to 3, wherein the non-human animal is an immunodeficient mouse or an immunodeficient rat.
5. 5. The screening method according to any one of items 1 to 4, wherein the hematopoietic stem cells are hematopoietic stem cells derived from the same species as the model animal.
6). 6. The screening method according to any one of items 1 to 5, wherein the medullary cavity is a medullary cavity of a bone marrow of a tibia or a femur.
7). 7. The screening method according to any one of items 1 to 6, wherein autologous normal hematopoietic stem cells and transplanted cells coexist in the medullary cavity.
8). A therapeutic or prophylactic agent for chronic myelogenous leukemia or bone metastasis comprising a compound or extract that inhibits the action or expression of basophils or basophil variants.
9. 9. The therapeutic or prophylactic agent according to item 8, wherein the bone metastasis is bone metastasis due to breast cancer.
10. 10. The therapeutic or prophylactic agent according to 8 or 9 above, wherein the compound is a CCR5 inhibitor.
11. 11. The therapeutic or prophylactic agent according to any one of 8 to 10 above, wherein the compound is maraviroc.
12 11. The therapeutic or prophylactic agent according to any one of 8 to 10 above, wherein the compound or extract is bicribiroc, baicalin flavonoid, Trigonostema xyphophylloids extract (TXE), or Vatica astrotricha extract.

  According to the present invention, a novel finding "basophils express CCL3 in the CML bone marrow environment, and CCL3 promotes dominant proliferation of LIC against normal hematopoietic stem cells, thereby causing chronic myeloid leukemia. Based on the above, a novel screening method for a therapeutic or preventive agent for chronic myeloid leukemia and a novel therapeutic or preventive agent for chronic myeloid leukemia are provided.

Constant CCL3 expression by restricted progenitor cell types in mouse bone marrow. (A) Whole bone marrow (BM) cells were collected from the tibia of untreated mice. Rat IgG2a was used as the isotype control shown in the figure. The expression of MPO (not shown), CD16 / 32 (not shown), or CD34 (not shown) was confirmed in whole bone marrow cells or lineage ^low CCL3 expressing cells. Representative results from three independent experiments are shown. (B) CCL3 and c-kit expression was confirmed in lineage ^low CD34 ⁺ MPO ⁺ cells (R1 and R2). The percentage of CCL3 ⁺ cells in the c-kit ^high and c-kit ^low regions is shown. Rat IgG2a was used as an isotype control. Representative results from 3 independent experiments are shown. (C) a non-treated mice femurs and whole bone marrow cells taken from the tibia (1 × 10 ⁷⁾ and primary bone marrow chimeric mice femur and whole bone marrow cells taken from the tibia (1 × 10 ^7), respectively First and second bone marrow chimeras were established by intravenous injection into recipient mice irradiated with a semi-lethal dose. Eight weeks after transplantation, the percentage of CCL3 ⁺ c-kit ⁻ cells in lineage ^low CD34 ⁺ MPO ⁺ cells was compared to that in untreated bone marrow cells. Data represent the mean ± standard deviation from 4 independent experiments. * Indicates P <0.05, N.P. S. Indicates no significant difference by Dunnett's test. Basophils as the main producer of CCL3 in the bone marrow. (A) Femur and tibia bone marrow derived HPC were injected into the bone marrow cavity of the right tibia of 2 Gy irradiated CD45.1 ⁺ congenic mice. The expression of c-kit and CCL3 on CD45.2 ⁺ MPO ⁺ lineage ^low cells (R1 and R2) was confirmed. The percentages of CD45.2 ⁺ MPO ⁺ donor-derived bone marrow cells and c-kit ^low CCL3 ⁺ cells are shown in the upper and lower figures, respectively. Representative results from 3 independent experiments are shown. (B) Lineage ^low CD16 / 32 ^high c-kit ^low cells and lineage ^low CD16 / 32 ^high c-kit ^high GMP were isolated from femur and tibia bone marrow using FACSAria Cell Sorter. The resulting cells are stained with Wright-Giemsa staining solution and representative results from three independent experiments are shown with a 10 μm scale bar. The expression level of CCL3 was confirmed using quantitative PCR analysis. Average values ± standard deviation were obtained by three independent experiments. In the Tukey-Kramer test, * indicates P <0.05. (C) CCL3 expression in ^c-kit low CD49b ^{+ FcεR1 +} cells in the bone marrow of the tibia of untreated mice or chimeric mice after transplantation 8 weeks (R1 and R2). Rat IgG2a was used as an isotype control. Representative results from 4 independent experiments are shown. (D) CCL3 expression in CD200R3 ⁺ FcεR1 ⁺ CD34 ⁺ FSC ^high (BaP1), CD34 ⁺ FSC ^low (BaP2) and CD34 ^low FSC ^low cells (Ba) in tibial bone marrow and peripheral blood (PB). Rat IgG2a was used as an isotype control. Representative results from 3 independent experiments are shown. The intermediate fluorescence intensity (MFI) of CCL3 in each population was compared to MFI in Ba of peripheral blood. ** indicates P <0.01, N.I. S. Indicates no significant difference by Dunnett's test. Excessive hematopoietic reconstitution after CCL3 ^{− / −} donor cell bone marrow transplantation. A total of 1 × 10 ⁷ bone marrow cells collected from the femur and tibia of CD45.2 ⁺ wild-type or CCL3 ^{− / −} mice were intravenously injected into semi-lethal dose irradiated CD45.1 ⁺ congenic mice to generate primary bone marrow. A chimera was established. Total bone marrow cells were collected from primary bone marrow chimeric mice 8 weeks after transplantation and subsequently used for the second bone marrow transplant. (A) Number of donor-derived total WBC, CD19 ⁺ B cells and Ly6G ⁺ granule cells excluding TCR-β chain ⁺ T cells in peripheral blood. The number of each cell in the peripheral blood of untreated wild type and CCL3 ^{− / −} mice is indicated by a bar graph. Data represent mean ± standard deviation from 4-6 independent experiments. (B) The numbers of donor-derived CD34 ⁻ KLS ⁺ HSC and CD34 ⁺ KLS ⁺ MPP were measured in the bone marrow of untreated mice and tibia of each bone marrow chimera 8 weeks after transplantation. Data represent the mean ± standard deviation from 4 independent experiments. (C) Ki67 and CD34 expression in KLS ⁺ cells in the bone marrow of tibia of untreated mice and bone marrow chimeric mice 2 weeks after wild type donor cell transplantation. The percentage of Ki67 ^high cells in the CD34 ⁺ and CD34 ⁻ regions is shown. Representative results from 4 independent experiments are shown. (D) Ratio of Ki67 ⁺ cells in KLS ⁺ cells at each time point after bone marrow transplantation. Data represent the mean ± standard deviation from 4 experiments. (A, B and D) * is P <0.05, ** is P <0.01, N.I. S. Indicates no significant difference by Mann-Whitney U test. Inhibition of HSPC proliferation by basophil-derived CCL3. (A) KLS ⁺ cells were isolated from the bone marrow of the femur and tibia of CD45.1 ⁺ congenic mice and labeled with CFSE. And it co-cultured with or without basophils (Ba) derived from CD45.2 ⁺ wild-type mice or CCL3 ^{− / −} mice. Basophil-mediated growth inhibition values were measured on day 4. Basophil-mediated growth inhibition values were calculated using the following formula: Growth inhibition = (MFI (with Ba) -MFI (without Ba)) / (MFI (pre-culture) -MFI (without Ba)) × 100. Representative results and mean ± standard deviation from three independent experiments are shown in the left and right figures, respectively. * Indicates P <0.05 by independent student t test. (B) CD45.2 ⁺ induced basophil deficiency model (MCPT8-DTR) mouse-derived whole bone marrow cells were intravenously injected into semi-lethal dose irradiated CD45.1 ⁺ congenic mice to establish bone marrow chimeras. Active diphtheria toxin (DT) (400 ng / mouse) was injected intravenously at each indicated time point. An equal amount of inactive diphtheria toxin (mutant DT) (Glu52) was injected as a control in the same manner. (C) Number of CD34 ⁻ KLS ⁺ and CD150 ⁺ CD48 ⁻ KLS ⁺ HSC in the bone marrow of the tibia 24 days after bone marrow transplantation. Data represent the mean ± standard deviation from 4 independent experiments. * Indicates P <0.05 by Mann-Whitney U test. (D) Number of donor-derived total WBC, CD19 ⁺ B cells and Ly6G ⁺ granule cells excluding TCR-β chain ⁺ T cells in peripheral blood. Mean ± standard deviation was calculated from 11 DT-treated mice (black circles) or 8 mutant DT (Glu52) -treated mice (white circles). * Is P <0.05, ** is P <0.01, N.P. S. Indicates no significant difference by Mann-Whitney U test. CML Proliferation of CCL3-expressing basophil-like leukemia cells in bone marrow. (A) CD45.1 ⁺ congenic mouse-derived LIC was used in the CML model. The proportion of Ki67 + cells in CD45.2 ⁺ recipient-derived normal KLS ⁺ cells was measured in the bone marrow of the tibia. Data are expressed as mean ± standard deviation from 4 independent experiments. * Indicates P <0.05, N.D. S. Indicates no significant difference by Dunnett's test. (B) Wild type donor-derived LIC was used in the CML model. Basophil phenotypic marker expression was measured on BCR-ABL (GFP) ⁻ CCL3 ⁺ normal cells (R1) or GFP ⁺ CCL3 ⁺ leukemia cells (R2) in the bone marrow of the tibia 2-3 weeks after transplantation . Representative results from 3 independent experiments are shown. (C) Proportion of Ly6G ⁺ or FcεR1 ⁺ cells in normal tibial bone marrow cells or GFP ⁺ myeloid leukemia cells taken from CML mice 3 weeks after transplantation. Data represent the mean ± standard deviation from 4 independent experiments. * Indicates P <0.05, N.D. S. Indicates no significant difference by Mann-Whitney U test. (D) Expression of Ly6G and FcεR1 on GFP ⁺ leukemia cells in tibial bone marrow and peripheral blood 3 weeks after transplantation. Representative results obtained from 4 individual animals are shown along with the percentage of Ly6G ^- FcεR1 ⁺ cells in GFP ⁺ leukemia cells. Effect of basophil-like leukemia cells on the maintenance of LIC in the development of bone marrow and CML. (A) MCPT8-DTR donor-derived LIC was used for the CML model. DT (400 ng / mouse) was injected intravenously at each indicated time point. As a control, an equal volume of mutant DT (Glu52) was injected in the same manner. The number of WBC in peripheral blood (B), spleen (SP) weight (C), and the number of GFP ⁺ KLS ⁺ LIC in the bone marrow of the tibia (D) were measured. Each circle represents an individual mouse (n = 6). ** indicates P <0.01 by Mann-Whitney U test. (E) DT or mutant DT (Glu52) was injected at each indicated time point. The number of WBCs was measured weekly starting 2 weeks after bone marrow transplantation. Non-leukemia survival was measured (n = 12). Mice that died or had a WBC count of 15,000 / μl were diagnosed with leukemia. * Indicates P <0.05 by log rank test. Prevention of the onset of CML by MVC (maraviroc), which is a CCR5 (C-C chemokine receptor type 5) receptor antagonist (CCR5 inhibitor). Wild-type donor-derived LIC was subjected to the CML model. (A) MVC or Vehicle was administered at a dose of 30 mg / kg body weight by oral gavage every other day 1 to 21 days after LIC transplantation. From 2 weeks after bone marrow transplantation, the number of WBCs was measured weekly. Non-leukemia survival was measured (MV = n = 6, control n = 10). Mice that died or had a WBC count of 15,000 / μl were diagnosed with leukemia. * Indicates P <0.05 by log rank test. The number of WBCs in the peripheral blood (B), SP weight (C), and the proportion of GFP ⁺ KLS ⁺ LIC (D) in the bone marrow of the tibia (C and D, 3 weeks) were measured. Each circle represents an individual mouse (MV = n = 6, control n = 5). ** indicates P <0.01, N.P. S. Indicates no significant difference by Mann-Whitney U test. Differentiation of CCL3-expressing cells in vitro. Bone marrow-derived HPCs from the femur and tibia were cultured as described in Example 1. The expression of c-kit and CCL3 was confirmed in lineage ^low CD34 ⁺ MPO ⁺ cells (R1). The percentage of CCL3 ⁺ cells in the c-kit ^high and c-kit ^low regions is shown. Representative results from three independent experiments are shown. Radiation tolerance of mature T cells. Ten million whole bone marrow cells were collected from the femur and tibia of CD45.2 ⁺ wild type mice and injected intravenously into semi-lethal dose irradiated CD45.1 ⁺ congenic mice to establish bone marrow chimeras. (A) TCR-β chain expression on CD45.1 ⁺ recipient-derived cells was measured in peripheral blood 8 weeks after transplantation. CD45.1 ⁺ CD45.2 ^- percentage of recipient-derived cells and TCR-βchain ⁺ T cells, respectively in the middle and right diagrams in. Representative results from 5 independent experiments are shown. (B) CD45.1 and CD45.2 expression on TCR-βchain ⁺ T cells in peripheral blood and TCR-βchain ^medium immature T cells in thymus were measured 8 weeks after transplantation. CD45.1 ⁺ CD45.2 ^- shows the percentage of CD45.2 ⁺ donor derived cells ^- recipient-derived cells and CD45.1. Representative results from 5 independent experiments (peripheral blood) or 3 independent experiments (thymus) are shown. Long-term excessive hematopoietic reconstitution immediately after transplantation of bone marrow cells derived from CCL3 ^{− / −} mice. Ten million whole bone marrow cells were collected from the femur and tibia of CD45.2 ⁺ CCL3 ^{− / −} mice and injected intravenously into semi-lethal dose irradiated CD45.1 ⁺ congenic mice to establish bone marrow chimeras. The number of total donor-derived WBC excluding TCR-βchain ⁺ T cells in peripheral blood (A) or the number of donor-derived CD34 ⁻ KLS ⁺ HSC in the bone marrow of the tibia (B) was measured. For A, the data represent the mean ± standard deviation from 3 independent experiments. For B, each circle represents an individual mouse (n = 4 after 8 weeks, n = 3 for 20 weeks). N. S. Indicates no significant difference by independent student t-test. High hematopoietic reconstitution capacity following transplantation of CCR1 ^{− / −} and CCR5 ^{− / −} donor-derived bone marrow cells. 5 million total bone marrow cells taken from the femur and tibia of CD45.2 ⁺ wild type, CCR1 ^{− / −} , or CCR5 ^{− / −} mice were mixed with the same number of CD45.1 ⁺ competitive bone marrow cells, followed by Thus, a primary bone marrow chimera was established by intravenous injection into half-lethal dose irradiated CD45.1 / CD45.2 atypical mice. Whole bone marrow cells were collected from primary bone marrow chimeras 8 weeks after transplantation and subsequently used for a series of bone marrow transplants in CD45.1 / CD45.2 atypical recipient mice. (A) CD45.2 ⁺ CD45.1 ⁻ Chimerism of total WBC or Ly6G ⁺ granule cells except TCR-β chain ⁺ T cells derived from donors was measured in peripheral blood. Data represent mean ± standard deviation from 5-6 independent experiments. (B) Chimerism of CD45.2 ⁺ CD45.1 ⁻ donor-derived KLS ⁺ cells was measured in the bone marrow of the tibia 8 weeks after transplantation. Data represent mean ± standard deviation from 5-6 independent experiments. (A and B) * is P <0.05, ** is P <0.01, N.P. S. Indicates no significant difference by Mann-Whitney U test. Long-term persistence of high levels of chimerism after bone marrow transplantation of CCR5 ^{− / −} donor-derived cells. A total of 5 × 10 ⁶ bone marrow cells taken from the femur and tibia of CD45.2 ⁺ wild type mice (open circles) or CCR5 ^{− / −} mice (filled circles) were mixed with the same number of CD45.1 ⁺ competitive bone marrow cells. Subsequently, a bone marrow chimera was established by intravenous injection into CD45.1 / CD45.2 atypical mice irradiated with semi-lethal doses. CD45.2 ⁺ CD45.1 except TCR-βchain ⁺ T cells ^- donors total WBC chimerism was measured in peripheral blood. Data represent mean ± standard deviation from 6 independent experiments (up to 20 weeks after transplantation) and 3 independent experiments (after 40 weeks). * After 40 weeks indicates P <0.05 by independent student t-test. Dysfunction of CCR1 ^{− / −} and CCR5 ^{− / −} c-kit ⁺ HPC mobilization to peripheral blood. The percentage of CCR1 ^{− / −} and CCR5 ^{− / −} CD45.2 ⁺ c-kit ⁺ HPC was measured in the peripheral blood of bone marrow chimeric mice (see: FIG. 11). The percentage of CD45.1 ⁺ CD45.2 ⁻ and CD45.1 ⁻ CD45.2 ⁺ cells in Ly6Gc-kit ⁺ cells (R1) is shown. Representative results from 5-6 independent experiments are shown. In vitro basophil differentiation process. Whole bone marrow cells were cultured for 10 days in S-clone medium supplemented with 10% FBS and 10 ng / ml IL-3. Half of the medium was replaced with fresh medium every 3 days. Basophils were further concentrated using CD49b MACS beads (Miltenyi Biotec). CCL3 expression in differentiated basophils was measured. The ratio of differentiated basophils and mast cells before and after concentration is shown. Selective reduction of CCL3-expressing basophils in MCPT8-DTR mice within an individual. (A) DT (400 ng / mouse) was injected intravenously into MCPT8-DTR mice. Number of basophils in peripheral blood (open circles) and bone marrow of the tibia (filled circles). Data represent mean ± standard deviation from 3 independent experiments. (B) DT or mutant DT (Glu52) was injected intravenously into MCPT8-DTR mice. CCL3 and CD200R3 expression was measured in the bone marrow of the tibia 3 days after injection. The percentage of CCL3 ⁺ CD200R3 ⁺ cells is shown. Representative results from three independent experiments are shown. TKI resistance of intramedullary LIC in mouse CML model. Wild type donor-derived LIC was used in the CML model. (A) Dasanitib (10 mg / kg body weight) was administered daily by oral gavage 7-20 days after LIC implantation. The number of WBCs was measured after 2 weeks, 3 weeks and 4 days after stopping dasanitide administration. Each circle represents an individual mouse (n = 5). (B) Maravirok or Vehicle was administered as described in FIG. BCR-ABL (GFP) expression was measured in lineage ^low c-kit ⁺ Sca-1 ⁺ cells (R1 and R2) 3 weeks after LIC transplantation. The percentage of GFP ⁺ cells is shown. Representative results from 5-6 independent experiments are shown. Attenuation of splenomegaly by marabiroc treatment. Visual appearance of recipient SP (spleen) collected from CML mice 3 weeks after LIC transplantation (described in legend to FIG. 7). The scale bar indicates 10 mm. Inhibition of tumor formation in the tibial bone by administration of maraviroc. (A) Outline of experiment. Malabiloc was orally administered 2 days, 4 days, and 6 days after the day of injection (today 0) injection of 4T1.3 cells into the mouse tibia (Day 0), and the tibia was collected after 7 days. (B) Immunostaining using anti-pan-cytokeratin antibody in a tibial paraffin-embedded tissue section. The staining result of the bone marrow cavity of the tibia of a mouse (MVC) and a control mouse (Control) to which marabiroc was administered is shown. The scale bar indicates 200 μm. (C) The calculation result of the ratio (positive rate;%) of anti-pan-cytokeratin antibody positive staining to the entire area of the bone marrow cavity. Data are expressed as mean ± standard deviation from 4 independent experiments. * Indicates p <0.05.

(Subject of the present invention)
The screening method for a therapeutic or prophylactic agent for chronic myeloid leukemia of the present invention (hereinafter sometimes abbreviated as “the screening method of the present invention”) is based on the following examples: Expresses CCL3, and further CCL3 promotes the dominant growth of LIC against normal hematopoietic stem cells, thereby finding that chronic myelogenous leukemia develops.
In addition, a CCR5 inhibitor, Malavirok, has confirmed that it dramatically reduced LIC in the bone marrow of chronic myeloid leukemia model animals and prevented the onset of CML.
Bone marrow is rich in blood vessels leading to tissues and organs due to hematopoiesis. Then, cancer (lung cancer, breast cancer, prostate cancer, etc.) cells of each tissue / organ move and settle in the bone marrow through the blood vessels, and bone metastasis occurs. The screening method of the present invention selects a compound that directly or indirectly inhibits the function of basophils. The compound not only becomes a therapeutic or preventive agent for chronic myeloid leukemia by suppressing the dominant growth of LIC in the bone marrow, but also suppresses the dominant growth in the bone marrow of cancer cells that have migrated to the bone marrow. It can also be a therapeutic or preventive agent for metastasis. Therefore, by using the screening method of the present invention to identify a substance that inhibits the action or expression of basophils or basophil variants, a therapeutic agent for chronic myeloid leukemia having a new mechanism of action and / or In addition to prophylactic agents for chronic myelogenous leukemia, therapeutic agents or prophylactic agents for bone metastases can be developed.

(Basophil)
The “basophil” used in the screening method of the present invention is not limited to those derived from chronic myelogenous leukemia model animals, but includes those produced in culture in vitro.
In addition, the “basophil modification” has a function equivalent to that of basophils, and the structure of basophils is compared with that of amino acid sequences 1 to 50, 1 to 30, 1 to 10, Alternatively, it means a structure in which 1 to 3 amino acids are substituted, deleted, added or inserted.
In addition, the “basophil modification gene” of the present invention means a gene sequence in which the protein encoded by the gene has a function equivalent to that of basophils.

  In general, means for introducing mutations such as substitution, deletion, addition, or insertion into an amino acid sequence are known per se, for example, using the technique of Ulmer (Science, 219, 666-671 (1983)). be able to. From the viewpoint of not changing the basic properties (physical properties, functions, immunological activity, etc.) of proteins in introducing such mutations, for example, homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilicity) Mutual substitution between amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) is readily envisioned. Furthermore, these usable proteins can be modified to such an extent that the structural amino group or carboxyl group or the like is not significantly changed in function, for example, by amidation modification.

(Inhibits the action of basophils)
The term “inhibiting the action of basophils” in the present invention means that the function of basophils (in particular, the function of producing a physiologically active substance such as CCL3) is lost or reduced directly or indirectly. Note that “inhibiting the action of a modified basophil” has the same meaning as described above.

(Inhibits basophil expression)
“Inhibiting the expression of basophils” in the present invention means inhibiting any stage in which a basophil gene is translated into a basophil protein. “Inhibiting the expression of a modified basophil” has the same meaning as described above.

(Screening method of the present invention)
The screening method of the present invention is exemplified below. However, there is no particular limitation as long as a test compound that inhibits the action or expression of basophils or basophil variants can be selected.
The “selection of the test compound” in the present invention is to confirm whether the test compound can be used as a therapeutic or preventive agent for chronic myelogenous leukemia, and further as a therapeutic or prophylactic agent for bone metastasis. That is, the test compound of the therapeutic or preventive agent for chronic myeloid leukemia obtained by the screening method of the present invention (or the test compound of the therapeutic agent or prophylactic agent for bone metastasis) has been confirmed to be practical in animal experiments and the like. can do.

In the present invention, a screening method including the following steps can be exemplified.
(1) A step of administering a test compound to the bone marrow of a non-irradiated non-human animal and a chronic myeloid leukemia model animal into which hematopoietic stem cells into which a BCR-ABL fusion gene has been introduced have been transplanted into the medullary cavity. .
(2) A step of measuring the expression level of basophils in the bone marrow.

In addition, although the following method is illustrated as a method of measuring the expression level of a basophil gene or a basophil variant gene, or a basophil or basophil variant, it is not particularly limited. The expression level means, for example, an increase or decrease compared to the control.
1) RT-PCR method 2) Immunoblotting method 3) SAGE
4) Immunoprecipitation method using anti-basophil antibody 5) Pull-down method 6) ELISA
7) Western blot 8) Hybridization 9) Flow cytometry 10) Specific gravity centrifugation 11) Stained specimen of cell suspension 12) Stained specimen of pathological tissue More specifically, when using flow cytometry, Basophils are separated from the cells by basophil selection markers CD200R3 and FcεR1. Then, the ratio of basophils to all cells in the bone marrow is measured.

(Test compound)
Any substance can be used as the “test compound” used in the present invention. The type of the test compound is not particularly limited, and may be a known therapeutic agent (particularly a CCR5 inhibitor), an individual small molecule synthetic compound (particularly siRNA), a compound present in a natural product extract, or a synthetic peptide.
Alternatively, the test compound may be a compound library, a phage display library, or a combinatorial library. The test compound is preferably a low molecular compound, and a compound library of low molecular compounds is preferable. The construction of a compound library is known to those skilled in the art, and a commercially available compound library can also be used.

(CML model animal)
In the “CML model animal” used for screening of the present invention, hematopoietic stem cells into which a BCR-ABL fusion gene has been introduced are transplanted into the medullary cavity of non-radiation non-human animals.
The method for producing a CML model animal of the present invention comprises a step of introducing a BCR-ABL fusion gene into a hematopoietic stem cell, and a bone marrow of a non-human animal that has not been irradiated with the obtained BCR-ABL fusion gene-introduced hematopoietic stem cell Implanting into the cavity.

The CML model animal of the present invention reproduces the symptoms, bone marrow state, and cell state of the early stage of human CML onset, and has a leukemia process similar to spontaneous leukemia, in particular, an early stage of onset. Using this model, the basophil expresses CCL3 in the CML bone marrow environment, as shown in the examples below, in a mechanism that converts normal hematopoietic processes into leukemic processes, It was found that chronic myeloid leukemia develops by promoting the dominant growth of LIC against normal hematopoietic stem cells.
Furthermore, when a test compound that becomes a drug for chronic myeloid leukemia is administered to the CML model animal of the present invention, unlike the conventional drug for chronic myeloid leukemia, a drug that is effective in the early stage of human CML onset is screened. Can do.

(animal)
Mammals are preferred as animals other than humans. Examples include wild type (WT) animals, animals lacking a desired gene, and immunodeficient animals. Preferably, it is an immunodeficient animal. Among mammals, mice or rats are preferable. More preferred are immunodeficient mice or immunodeficient rats. Several types of immunodeficient mice and rats have been developed, but nude mice or nude rats lacking the thymus are most preferably used.

(Non-irradiation)
Conventionally, the immune system of the recipient animal has been destroyed by irradiation. However, in the present invention, a recipient animal that is not irradiated with radiation that destroys the immune system is used. Since the model animal of the present invention is not irradiated, normal hematopoietic stem cells are present inside the bone marrow of the recipient. Furthermore, by transplanting hematopoietic stem cells into which the BCR-ABL fusion gene has been introduced into the bone marrow, normal hematopoietic stem cells and hematopoietic stem cells into which the BCR-ABL fusion gene have been introduced coexist in the medullary cavity, and human CML The condition is similar to that in the bone marrow at the initial stage of onset.

(Medullary transplantation)
Hematopoietic stem cells into which the BCR-ABL fusion gene has been introduced are transplanted into the medullary cavity of animals other than humans. The medullary cavity is the lumen of the bone marrow. In the screening method of the present invention, hematopoietic stem cells into which the BCR-ABL fusion gene has been introduced are directly or indirectly transplanted into the medullary cavity with a syringe or other tool. The location of the bone marrow to be transplanted is not limited and may be anywhere as long as transplantation is possible. The medullary cavity of the bone marrow that is easy to transplant is the medullary cavity of the bone marrow of the tibia or femur. Moreover, you may transplant several times. If it is a mouse | mouth, it will transplant to the mouse | mouth 3-9 weeks old, Preferably it is 4-8 weeks old, More preferably, it is 5-7 weeks old.

(BCR-ABL fusion gene-introduced hematopoietic stem cells)
Hematopoietic stem cells into which the BCR-ABL fusion gene is introduced can be obtained from the bone marrow of an animal different from or the same as the recipient animal. Hematopoietic stem cells can be obtained from the collected bone marrow using known molecular markers for hematopoietic stem cells. For example, cells of c-kit ⁺ lineage ^- Sca-1 ⁺ (KLS ⁺ ) can be selected. The hematopoietic stem cells are preferably derived from an animal of the same species as the model animal. More preferably, the animal is the same species as the model animal and is derived from a wild-type animal.

(BCR-ABL fusion gene)
The BCR-ABL fusion gene used in the present invention is a gene obtained by fusing the BCR gene and the ABL gene resulting from reciprocal translocation of chromosomes 9 and 22. The expressed protein is not particularly limited as long as it is a gene having tyrosine kinase activity and causing CML. Examples thereof include gene sequences described in the literature Proc. Natl. Acad. Sci. USA Vol. 83, pp. 9768-9772, December 1986. Also included are partially deleted, substituted, and added sequences of this sequence. These partially deleted, substituted, and added sequences preferably have 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more homology with the above sequences. .

(Introduction of BCR-ABL fusion gene)
The BCR-ABL fusion gene can be introduced into hematopoietic stem cells by a known method. For example, a BCR-ABL fusion gene can be inserted into a viral vector and introduced into hematopoietic stem cells. As the viral vector, a known vector can be used. Examples thereof include retrovirus vectors and cytomegalovirus vectors. Preferably, MSCV vectors can be used. In addition, a gene encoding a detection marker can be added for analysis after gene introduction. For example, a gene encoding GFP which is a fluorescent substance can be added.

(Number of transplanted cells)
The number of transplanted hematopoietic stem cells into which the BCR-ABL fusion gene has been introduced can be adjusted as appropriate. When the model animal is a mouse, 50 to 10000, preferably 100 to 1000, more preferably 200 to 800, and most preferably 400 to 500 are transplanted per mouse. When the model animal is a rat, the same number of transplanted cells, 2 times the number of transplants, or 3 times the number of transplants per rat can be used.

(When to use)
When the CML model animal of the present invention is used as a model animal in the early stage of CML onset, it is desirable to use it for 1 to 5 weeks after transplantation, preferably 2 to 4 weeks after transplantation.

(Symptoms after transplantation)
The number of transplanted hematopoietic stem cells into which the BCR-ABL fusion gene has been introduced is small as described above, and this small amount of hematopoietic stem cells is derived from the recipient's own large number of normal hematopoietic cells and donors in the bone marrow. Coexist with many normal hematopoietic cells. Two to three weeks after transplantation, CML-like disease presents with marked white blood cell increase and spleen enlargement. Thus, this CML model animal is very useful for elucidating the interaction between normal hematopoietic cells in the bone marrow and leukemia stem cells, particularly in the early stages of CML development.
Furthermore, in this CML model, after transplantation directly into the medullary canal of the right tibia, leukemia stem cells migrated spontaneously and proliferated in the medullary canal of the contralateral left tibia. In addition, extramedullary hematopoiesis was similarly reproduced in the spleen and liver as observed in CML patients. Thus, this CML model animal is also useful for examining leukemia stem cell infiltration from the primary site, which is the transplant site, to other hematopoietic organs (eg, spleen and liver).

(Therapeutic or preventive agent for chronic myelogenous leukemia or bone metastasis)
The following examples confirm that a CCR5 inhibitor (particularly, marabiroc) is effective as an active ingredient of a therapeutic or preventive agent for chronic myeloid leukemia or bone metastasis (particularly, bone metastasis due to breast cancer).
Therefore, the therapeutic agent or preventive agent for chronic myeloid leukemia or bone metastasis of the present invention contains a CCR5 inhibitor, particularly maraviroc, as an active ingredient.
In addition, the therapeutic agent or preventive agent for chronic myeloid leukemia or bone metastasis of the present invention and a known therapeutic agent for chronic myelogenous leukemia (eg, tyrosine kinase inhibitor, particularly dasatinib) or a bone metastasis therapeutic agent, It may be administered to patients with chronic myelogenous leukemia or bone metastasis.

(CCR5 inhibitor)
The CCR5 inhibitor that can be used as an active ingredient of the therapeutic agent or preventive agent for chronic myeloid leukemia or bone metastasis of the present invention is Maraviroc {UK-427857 4,4-Difluoro-N-{(1S) -3- [3- (3-isopropyl-5-methyl-4H-1,2,4-triazol-4-yl) -8-azabicclo [3.2.1] oct-8-yl] -1-phenylpropyl} cyclohexanecarbamide)}, bicribiloc {1-[(4,6-dimethyl-5-pyridineminyl) carbonyl] -4- [4- [2-methoxy-1 (R) -4- (trifluoromethyl) phenyl] ethyl-3 (S) -methyl-1 −piperazinyl] − -Methylpiperidine}, baicalin flavonoids, Trigonostema xyphophylloides extract (TXE), there may be mentioned Vatica astrotricha extract or the like, particularly maraviroc is preferred.

(Marabiroc)
Maraviroc is marketed as a CCR5 inhibitor used in HAART therapy for HIV infection. In general, for adults, 300 mg of oral medicine is orally administered twice a day.

The therapeutic or preventive agent for chronic myeloid leukemia or bone metastasis of the present invention contains an antisense nucleic acid that specifically hybridizes to mRNA of a basophil gene as an active ingredient.
The therapeutic agent or preventive agent for chronic myeloid leukemia or bone metastasis of the present invention contains a ribozyme that specifically cleaves mRNA of a basophil gene as an active ingredient.
The therapeutic agent or preventive agent for chronic myeloid leukemia or bone metastasis of the present invention contains an antibody that inactivates basophils as an active ingredient.

  Depending on the purpose of prevention or treatment, etc., various forms such as powders, granules, tablets, capsules, intestinal solvents, liquids, injections (liquids, suspensions) or forms used for gene therapy are conventionally used. Therefore it can be prepared. The therapeutic or preventive agent for chronic myelogenous leukemia or bone metastasis of the present invention is usually preferably prepared as a pharmaceutical composition using one or more pharmaceutical carriers.

  The dose or intake of the therapeutic or preventive agent for chronic myeloid leukemia or bone metastasis of the present invention is not particularly limited as long as the effect of the present invention can be obtained, and the effectiveness of the contained components The administration form, administration route, type of disease, nature of the subject (weight, age, medical condition, presence or absence of use of other medicines, etc.), judgment of the doctor in charge, etc. are appropriately selected. The therapeutic agent or prophylactic agent for chronic myeloid leukemia or bone metastasis of the present invention can be administered or ingested in 1 to several times a day, and intermittently administered once every several days or weeks. Or you may ingest.

  The present invention will be specifically described by the following examples, but the present invention is not limited thereto. This example was performed in accordance with the Kanazawa University animal experiment regulations and the declaration of Helsinki. Furthermore, it has been approved by the Jintendo University Ethics Committee (registration number IRB # 969).

(Materials and methods)
The materials and methods used in the examples are as follows.

(mouse)
Specific pathogen-free 5-6 week old male BALB / c and athymic BALB / c-nu mice were purchased from Charles River Japan and used as wild type and nude mice, respectively. CD45.1 BALB / c congenic mice and CCL3 ^{− / −} mice were obtained from Jackson Laboratories. CCR1 ^{− / −} and CCR5 ^{− / −} mice, respectively, Philip M.M. Provided by Murphy (National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA) and Dr. Tsunaharu Matsushima (University of Tokyo). These gene-deficient mice were obtained by crossing with 8 generations or more of BALB / C mice. MCPT8-DTR mouse {Transgenic mouse in which a DTR (diphtheria toxin receptor) gene is linked to the mMCP-8 gene promoter / enhancer region, and DTR is expressed only in basophils. As a result, administration of diphtheria toxin selectively removes basophils} was obtained by a known method (see: J Clin Invest. 2010; 120 (8): 2867-2875.). Mice were raised under specific pathogen-free conditions.

(antibody)
The following rat anti-mouse monoclonal antibodies were used.
Anti-CD4 (RM4-5; TONBO Biosciences), anti-CD8 (53-6.7; TONBO Biosciences), anti-CD16 / 32 (2.4G2; BD Biosciences), anti-CD11b (M1 / 70; TONBO Biosciences), anti-CD19 (1D3; TONBO Biosciences), anti-CD34 (RAM34; eBioscience), anti-CD45.1 (A20; TONBO Biosciences), anti-CD45.2 (104; TONBO Biosciences), anti-CD45R / B220 (RA3-6B2; TONBO Biosc, TONBO Biosc) Anti-CD48 (HN48-1; eBioscience), anti-CD49b (DX5; BioLegend), anti-CD117 / c-ki (ACK2; TONBO Biosciences), anti-CD150 / SLAM (TC15-12F12.2; BioLegend), anti-CD200R3 (Ba13; BioLegend), anti-FcεR1 (MAR-1; eBioscience), anti-Ki67 (SolA15; eBioscience), anti -6A / E / Sca-1 (D7; eBioscience), anti-Ly-6G / Gr-1 (RB6-8C5; TONBO Biosciences), anti-Ly6G (1A8; TONBO Biosciences), anti-MIP1-α / CCL3 (39624; R & D Systems), and anti-TER-119 (TER-119; TONBO Biosciences).
The mouse strain Ab cocktail set was purchased from BD Biosciences. Mouse anti-rat MPO monoclonal antibody (2D4) with cross-reactivity to mouse myeloperoxidase (MPO) was purchased from Abcam. Armenian hamster anti-mouse TCR-β chain (H57-597) was purchased from TONBO Biosciences. Isotype matched control IgG and control mouse IgG for individual rat monoclonal antibodies were purchased from BD Biosciences.

{Preparation of hematopoietic progenitor cells (HPC) and c-kit ⁺ lineage ^- Sca-1 ⁺ (KLS ⁺ ) bone marrow cells}
Total bone marrow cells were separated by density gradient centrifugation using Histopaque-1083 reagent (Sigma-Aldrich). Thereafter, lineage markers (CD4, CD8, CD11b, Gr-1, B220 and TER-119) ⁻ c-kit ⁺ cells and lineage marker ⁻ c-kit ⁺ Sca-1 ⁺ cells were respectively converted into FACSAria Cell Sorter (BD Biosciences). ) And used as HPC and KLS ⁺ bone marrow cells.

(KLS ⁺ basophil-mediated suppression of cell proliferation)
CD45.1 ⁺ KLS ⁺ bone marrow cells were labeled with 5 μM carboxyfluorescein diacetate (CFSE, Life Technologies). Subsequently, they were supplemented with 1% bovine serum albumin (BSA), 100 ng / ml stem cell factor (SCF), 100 ng / ml thrombopoietin (TPO), and 10 ng / ml IL-3 (all from PeproTech). In S-clone medium, the cells were cultured for 4 days in the presence or absence of CD45.2 ⁺ wild type or CCL3 ^{− / −} basophils differentiated in vitro (FIG. 14). The intermediate fluorescence intensity (MFI) of CFSE in CD45.1 ⁺ c-kit ^high cells was measured before and after culture.

(Production of bone marrow chimera)
A total of 1 × 10 ⁷ bone marrow cells were injected intravenously into 5.5 Gy X irradiated recipient mice. In some examples, 2 × 10 ⁵ HPC was injected into the bone marrow cavity of the right tibia of 2Gy X irradiated recipient mice.

(CML model production)
KLS ⁺ cells purified from bone marrow of wild-type or MCPT8-DTR mice were retrofitted with MSCV-BCR-ABL-ires-GFP by known methods {Reference: Nature. 2010; 463 (7281): 676-680}. Virus was infected to obtain LIC. A 30 μl volume of LIC (400-500 BCR-ABL ⁺ cells contained in 30,000 KLS ⁺ cells) was subsequently injected into the tibia with 29 G needle-conjugated insulated syringe (Terumo). In some embodiments, as detailed in the figure descriptions, diphtheria toxin (DT, Sigma-Aldrich), mutant DT (Sigma-Aldrich), maraviroc (MVC, GlaxoSmithKline) or dasanitib (dasatinib, -Myers).

(RNA isolation, cDNA synthesis, and quantitative real-time PCR)
Total RNA was extracted from cells using RNeasy Mini kit (QIAGEN) and then reverse transcribed using SuperScript III First-Strand Synthesis (Life Technologies). Quantitative real-time PCR consists of Fast SYBR Green Master Mix (Life Technologies), and GAPDH gene (sense: 5′-GCG GCA CGT CAG ATC CA-3 ′ SEQ ID NO: 1; antisense: 5′-CAT GGC TCT CTGC TCT CTGC -3 ′ SEQ ID NO: 2) and CCL3 gene (sense: 5′-GCT GAC AAG CTC ACC CTC TGT-3 ′ SEQ ID NO: 3; antisense: 5′-GGC AGT GGT GGA GAC CTT CA-3 ′ SEQ ID NO: 4) This was carried out on a Life Technologies ViiA7 Real-time PCR system using the following specific primer set. The relative expression of the CCL3 gene was analyzed by the ΔΔCt method using the Ct value of the GAPDH gene.

(Flow cytometry)
Isolated leukocytes were stained with various combinations of fluorochrome labeled antibodies. For intracellular CCL3 and MPO staining, leukocytes were cultured in serum-free S-Clone SF-03 medium (Sanko Junyaku) supplemented with 0.1% GolgiStop reagent (BD Biosciences) for 4 hours.
Next, intracellular CCL3 and MPO are stained with PE-labeled anti-CCL3 antibody and biotin-labeled anti-MPO antibody, followed by APC and APC-Cy7 labeling using Intracellular Cytokine Staining Starter Kit (BD Biosciences), respectively. Incubated with streptavidin. Ki67 in the nucleus was successively stained with biotin-labeled anti-Ki67 antibody and APC-labeled streptavidin using Foxp3 / Transcribion Factor Buffer Set (eBioscience). The expression of each molecule was determined using FACSCanto II (BD Biosciences) and further analyzed with FlowJo software (Tree Star).

{In situ hybridization (IHC) evaluation on bone marrow biopsy from CML patients}
Bone marrow biopsy samples were obtained from CML patients diagnosed at Juntendo University Hospital prior to treatment. For this example, 4 μm sections were prepared from paraffin embedded tissue and placed on positively charged glass slides. Double staining of mRNA was performed using Quantene View RNA ISHkit (Panomics) according to the instructions attached to the kit. Deparaffinized tissue samples were cultured in a pretreatment solution followed by protease treatment. Human CCL3- and ectonucleotide pyrophosphatase / phosphodiesterase (ENPP) 3-specific probes were designed / synthesized by Panomics / Affymetrix. The probe set and amplification molecule hybridized to each pair of oligonucleotides. After removing unbound probe by washing, the sample was incubated with alkaline phosphatase to degrade fast red and fast blue substrates that form precipitates. The target mRNA image was obtained using DP21 microscopic camera (Olympus).

(Statistical analysis)
Data were statistically analyzed using the methods indicated in the figure legends. p <0.05 was judged to be statistically significant.

(Confirmation of constant CCL3 production by spinal cord progenitor cells)
The present inventors ^{have, BCR-ABL + lineage - c} -kit - immature leukemic cells and to a lesser extent ^{BCR-ABL - lineage - c-} kit - non leukemic cells express CCL3 in the bone marrow of CML onset mice It was reported previously (Reference: Non-patent document 10).
In this example, CCL3 expression was examined in normal mouse bone marrow. CCL3 expression was consistently detected in lineage ^low cells in untreated normal bone marrow (Ref: FIG. 1A). These CCL3-expressing cells show the phenotype of immature granulocyte cells with the expression of MPO, which is a functional marker of bone marrow cells, and CD34 and CD16 / 32, which are hematopoietic progenitor cells, and are cCF receptors -Kit (FIG. 1B) was not expressed. Furthermore, CCL3 was also detected in a similar cell population after bone marrow transplantation (FIG. 1C).

(Confirmation of whether basophils are the main production cells of CCL3)
First, in order to confirm the characteristics of bone marrow progenitor cells expressing CCL3, differentiation from HPC to bone marrow progenitor cells was induced in vitro and in vivo. Under both conditions, HPC differentiated into bone marrow cells and concomitantly generated lineage ^low c-kit ^low CCL3 ⁺ cells (FIGS. 2A and 8). Among bone marrow cells, CCL3 was abundantly and selectively detected in a freshly isolated lineage ^low CD16 / 32 ^high c-kit ^low cell population. The lineage ^low CD16 / 32 ^high c-kit ^low cell population had segmented nuclei and cytoplasmic basophil granules, and was smaller than lineage ^low CD16 / 32 ^high c-kit ^high granule cell macrophage progenitor (GMP) cells (FIG. 2B). ^{Furthermore, c-kit low FcεR1 + CD49b} + basophils, CCL3 was constitutively expressed in untreated mice and bone marrow chimeric bone marrow (Fig. 2C).
These results indicate that basophils can express CCL3. Basophils in bone marrow expressed the basophil selection markers CD200R3 and FcεR1 and could be classified into three subsets: CD34 ^high forward scatter (FSC) ^high progenitor cells, CD34 ^high FSC ^low progenitors Cells and CD34 ^low FSC ^low mature population cell (FIG. 2D). CCL3 expression was evident in basophil progenitors but decreased with basophil maturation (FIG. 2D).
From the above, it was confirmed that basophils in the bone marrow (basophils-lineage cells) constantly express CCL3.

(Confirmation of the role of intramarrow CCL3 as a negative regulator of HSPC transient growth during bone marrow transplantation-induced hematopoietic reconstitution)
No difference in hematopoietic cell population could be observed between untreated wild type and CCL3 ^{− / −} mice. This hematopoietic cell population includes total white blood cells (WBC), B cells, and granule cells (FIG. 3A) in peripheral blood, and HSC and pluripotent progenitor cells (MPP) in bone marrow (FIG. 3B). . Next, bone marrow transplantation was performed to examine the role of CCL3 in hematopoiesis under stress. As a pretreatment for bone marrow transplantation, recipient mice were whole-body irradiated with 5.5 Gy. The dose was sufficient and minimal required to exchange with donor-derived hematopoietic cells to reduce recipient mouse damage. Under these conditions, approximately 10% of CD45.1 ⁺ recipient-derived cells (mostly TCR-βchain ⁺ T cells) remained in the peripheral blood (FIG. 9A). However, CD45.1 ⁺ recipient-derived cells could not be observed in TCR-β chain ^medium immature T cells differentiated in the thymus (FIG. 9B). Thus, under these conditions, the hematopoietic system was largely replaced with donor-derived cells, with the exception of mature T cells in peripheral blood. Therefore, T cell counts were excluded in order to determine hematopoietic reconstitution in subsequent examples using bone marrow chimeras. When donor cells were derived from CCL3 ^{− / −} mice, donor-derived whole cells, B cells and granule cells were excessive in peripheral blood compared to untreated mice and mice transplanted with wild type derived bone marrow cells. (Fig. 3A). Similarly, HSC and MPP reconstituted more efficiently in bone marrow cells transplanted from CCL3 ^{− / −} donors than bone marrow cells transplanted from wild type donors (FIG. 3B). In bone marrow reconstituted with CCL3 ^{− / −} donor cells, an unnatural increase in total WBC in peripheral blood from donors and HSC in bone marrow persisted even 20 weeks after the first bone marrow transplantation (FIG. 10).
Furthermore, higher hematopoietic reconstitution capacity of CCL3 ^{− / −} donor cells compared to wild type donor cells was also observed in the second bone marrow transplant (FIGS. 3A and 3B). Two weeks after wild-type bone marrow cell transplantation, Ki67 expression in donor-derived KLS ⁺ cells was transiently enhanced (FIG. 3C) and then decreased below baseline levels (FIG. 3D). On the other hand, Ki67 constitutive expression persisted in CCL3 ^{− / −} chimeras (FIG. 3D). Several groups, including the inventors, have reported that CCL3 receptors CCR1 and CCR5 are expressed on normal HSPC. Competitive bone marrow transplantation analysis confirmed that removal of CCR1 or CCR5 in donor cells caused a more advanced chimerism of WBC and bone marrow KLS ⁺ cells after successive bone marrow transplants (FIGS. 11A and 11B).
In addition, an increase in the percentage of donor-derived WBC in peripheral blood was evident up to 40 weeks after the first bone marrow transplantation of CCR5 ^{− / −} donor-derived cells (FIG. 12). The inventors have previously confirmed that intramarrow infusion of CCL3 can induce mobilization of c-kit ⁺ HPC from the bone marrow into the peripheral blood (see: Non-Patent Document 10). However, no defect could be confirmed in the recruitment process of either CCR1 ^{− / −} or CCR5 ^{− / −} HPC in the peripheral blood of competitive bone marrow chimeric mice (FIG. 13).
Thus, these results indicate that endogenously produced CCL3 can primarily control the proliferation of HSPCs in the bone marrow, but not their mobilization.

(Role of basophil-derived CCL3 as a negative regulator of HSPC proliferation)
Regarding the CCL3 expression ability of basophils in bone marrow, the effect of basophil-derived CCL3 on HSPC was confirmed. CD49b ⁺ c-kit ^low FcεR1 ⁺ basophils were generated in vitro (FIG. 14) and used for co-culture with HSPC. KLS ⁺ HSPC proliferation was markedly reduced in the presence of wild type basophils but not in the presence of CCL3 ^{− / −} mouse basophils (FIG. 4A). Administration of DT to MCPT8-DTR mice selectively reduced the number of basophils in peripheral blood and bone marrow as well as the number of CCL3-expressing basophils in the bone marrow (FIG. 15). On the other hand, administration of mutant DT (Glu52) did not reduce basophils. In order to investigate the function of basophils in vivo, wild type mice were transplanted with bone marrow cells derived from MCPT8-DTR mice as donor cells for bone marrow transplantation, and DT or mutant DT was administered intravenously after bone marrow transplantation. (FIG. 4B). DT treatment reduced basophils in bone marrow chimeric mice and ultimately increased donor-derived CD34 ⁻ KLS ⁺ and CD150 ⁺ CD48 ⁻ KLS ⁺ HSC in the bone marrow compared to mutant DT treatment (FIG. 4C). Furthermore, DT increased the reconstitution of donor-derived whole cells, B cells and granule cells in peripheral blood compared to mutant DT (FIG. 4D), resulting in bone marrow transplantation of CCL3 ^{− / −} donor-derived cells. Similar (Figure 3).
Therefore, it was confirmed that basophils in bone marrow can negatively control HSPC proliferation in a CCL3-dependent manner.

(Role of basophil-like leukemia cells as a major factor in the development of CML)
We have previously described that "direct injection of BCR-ABL-converted LIC into the bone marrow cavity of non-irradiated mice accurately represents the interaction between normal hematopoietic and leukemic cells early in the onset of CML. And using this model, endogenously produced CCL3 acts on CCR1 or CCR5 expressing non-leukemic HSPCs to promote the maintenance of LIC in the bone marrow. ”(See : Non-patent document 10).
In this example, in this model, Ki67 expression was transiently enhanced in recipient-derived normal KLS ⁺ cells in bone marrow (FIG. 5A), similar to the results in bone marrow transplantation (FIG. 3D). It was confirmed. Considering the ability of basophil-derived CCL3 to suppress HSPC proliferation, this result indicates the involvement of CCL3 in this transient increase in Ki67 expression. In fact, during the CML onset, CCL3 expressed BCR-ABL ^- normal hematopoietic cells (Figure 5B) and basophil-like leukemic cells are similar showing a phenotype ^{CD49b + FcεR1 + CD200R3 + c-} kit low is the CCL3 It was expressed and proliferated in bone marrow but not in peripheral blood (FIGS. 5C and 5D). Consistent with these results, CCL3 expression could be confirmed primarily in ENPP3 ⁺ basophil-like cells in bone marrow biopsies from CML patients. To confirm the role of basophil-like leukemia cells in CML pathophysiology, MCPT8-DTR donor-derived LICs were transplanted into non-irradiated nude mice. Subsequently, the non-irradiated nude mice were treated with DT to selectively reduce basophil-like leukemia cells (FIG. 6A). DT treatment reduced the increase in the number of peripheral blood WBC (FIG. 6B), spleen (SP) weight (FIG. 6C), and bone marrow LIC (FIG. 6D). Furthermore, sustained DT treatment significantly delayed the onset of CML (FIG. 6E).
These results indicated that basophil-like leukemia cells in the bone marrow are the major producers of CCL3 that promote the predominant growth of LIC in CML bone marrow.

(Effects of Maraviroc, a CCR5 inhibitor, as a potent preventive agent for CML)
In a non-irradiated CML model, dasatinib treatment, a daily tyrosine kinase inhibitor (TKI) one week after LIC transplantation, reduced CML-like leukocyte increase (FIG. 16A), but many LICs were found in the bone marrow. (FIG. 16B). Furthermore, cessation of dasatinib treatment rapidly induced the progression of leukocytosis (FIG. 16A).
In order to confirm whether the above results can be used as a therapeutic agent for CML, maraviroc, an anti-HIV drug having a strong CCR5 antagonism, was administered to this CML model. Marabiroc, when administered immediately after LIC infusion, dramatically decreases with the disappearance of LIC in the bone marrow (FIGS. 7D and 16B), as shown by decreased leukocytosis and splenomegaly weight reduction (FIGS. 7B, 7C and 17). CML development was prevented (FIG. 7A).

(Effects of Maraviroc, a CCR5 inhibitor, as a potent preventive agent for bone metastasis)
It was investigated whether bone metastasis was suppressed when malaviroc, a CCR5 inhibitor, was administered. The outline is shown in FIG. 18A.
Mouse breast cancer cell line 4T1.3 cells that cause bone metastasis with high frequency (the present inventors inoculated mice with 4T1 cell line obtained from JCRB Cell Bank, National Institute of Biomedical Innovation, Health and Nutrition) Cells that metastasize to bone were selected and established cell lines) were suspended in HBSS (Hanks' Balanced Salt Solution) at a concentration of 5 × 10 ³ cells. 20 μL of the cell suspension was injected into CD45.2 ⁺ BALB / c mouse tibia.
Two days after injection, 4 days, and 6 days, 30 mg / kg body weight of maraviroc was orally administered. As a control, 0.1 mL of sterilized water per animal was administered on each day instead of Malabiloc.
Seven days after the injection, the tibia was collected and decalcified {Ion-Exchange Decal Unit; E. D Unit (Biocare Medical)} and decalcified overnight at room temperature. After decalcification, paraffin-embedded tissue sections were prepared.
Anti-pan-cytokeratin antibody (BioLegend), which is a tumor marker, is used as the primary antibody, and M. as the secondary antibody. O. The positive rate of tumor cells relative to the entire bone marrow cavity area was calculated by immunostaining using M ImmPress Peroxidase Polymer kit (VECTOR LABORATORIES) and ImmPACT DAB Substrate (VECTOR LABORATORIES). Image acquisition of immunostaining and detection of positive staining were performed using a B2-710 all-in-one fluorescence microscope (Keyence) according to the manufacturer's instructions.
As a result of immunostaining, it was confirmed that when maraviroc was administered, the area that was positively stained was significantly smaller than that of the control (FIG. 18B). As a result of calculating the positive rate with respect to the entire area of the bone marrow cavity, in the control, the positive rate with respect to the entire area of the bone marrow cavity was about 32.34%, whereas when maraviroc was administered, the positive rate with respect to the entire area of the bone marrow cavity was positive. The rate was about 1.91% (FIG. 18C). That is, when maraviroc was administered, tumor formation was suppressed to about 1/17.
From the above, it has been clarified that administration of maraviroc can suppress colonization and tumor formation of cancer cells in the bone marrow and suppress bone metastasis. Therefore, it was confirmed that maraviroc, which is a CCR5 inhibitor, is a potent therapeutic / preventive agent for bone metastasis that suppresses the proliferation of cancer cells in the bone marrow.

(Summary)
From the above Examples 1 to 8, the following points were confirmed.
(1) CCL3 is selectively expressed in basophils. Furthermore, basophils in the bone marrow express CCL3 without inflammatory stimulation. Therefore, CCL3 functions as a homeostatic chemokine in the bone marrow, similar to normal physiological conditions.
(2) Basophils in bone marrow can negatively control HSPC proliferation in a CCL3-dependent manner. That is, a compound that inhibits the action or expression of basophils can be an active ingredient of a therapeutic or prophylactic agent for chronic myeloid leukemia in which the proliferation of HSPC is suppressed.
(3) Basophil-like leukemia cells amplify the expression of CCL3 by the CML model of this example. CCL3 then amplifies LIC in the bone marrow and promotes the onset of CML. More specifically, CCL3 from basophil-like leukemia cells plays an important role in the interaction between normal hematopoiesis and leukemia processes and promotes the dominant growth of LIC against normal hematopoietic stem cells in the CML bone marrow environment .
(4) According to the CML model of this example, Marabiroc dramatically reduced LIC in the bone marrow and prevented the onset of CML. On the other hand, dasatinib, which is a CML standard therapeutic agent, was able to reduce the number of leukemia cells in peripheral blood, but was not able to reduce LIC in the bone marrow of this model. In addition, all mice succumbed to leukemia following discontinuation of dasatinib. That is, marabiroc is more effective against LIC of CML, especially in the early stage of chronic leukemia, compared to dasatinib, which is a CML standard therapeutic agent.
(5) LIC is known to be resistant to tyrosine kinase inhibitors. Therefore, combined administration of a CCR5 inhibitor (eg, maraviroc) and a CML standard therapeutic agent (eg, tyrosine kinase inhibitor, particularly dasatinib) is superior to conventional CML standard treatment.
(6) In this example, marabiroc suppressed tumor formation in the bone marrow and further suppressed bone metastasis due to breast cancer. Administration of a CCR5 inhibitor (eg, maraviroc) is effective in treating and preventing bone metastasis.

  The CML model animal of the present invention can be used for the development of new therapeutic methods and new drugs by using it for the study of chronic myelogenous leukemia. It can also be used for screening for leukemia therapeutic agent candidates.

Claims (12)

A screening method for a therapeutic or prophylactic agent for chronic myelogenous leukemia or bone metastasis, which comprises selecting a test compound that inhibits the action or expression of basophils or basophil variants.
The screening method according to claim 1, comprising the following steps:
(1) A step of administering a test compound to the bone marrow of a non-irradiated non-human animal and a chronic myeloid leukemia model animal into which hematopoietic stem cells into which a BCR-ABL fusion gene has been introduced have been transplanted into the medullary cavity. And (2) measuring the expression level of basophils in the bone marrow.
The screening method according to claim 1 or 2, wherein the chronic myelogenous leukemia is an early stage chronic myelogenous leukemia.
The screening method according to any one of claims 1 to 3, wherein the non-human animal is an immunodeficient mouse or an immunodeficient rat.
The screening method according to any one of claims 1 to 4, wherein the hematopoietic stem cells are hematopoietic stem cells derived from the same species as the model animal.
The screening method according to claim 1, wherein the medullary cavity is a medullary cavity of a bone marrow of a tibia or a femur.
The screening method according to any one of claims 1 to 6, wherein autologous normal hematopoietic stem cells and transplanted cells coexist in the medullary cavity.
A therapeutic or prophylactic agent for chronic myelogenous leukemia or bone metastasis comprising a compound or extract that inhibits the action or expression of basophils or basophil variants.
The therapeutic or prophylactic agent according to claim 8, wherein the bone metastasis is bone metastasis due to breast cancer.
The therapeutic or prophylactic agent according to claim 8 or 9, wherein the compound is a CCR5 inhibitor.
The therapeutic or preventive agent according to any one of claims 8 to 10, wherein the compound is maraviroc.
  The therapeutic agent or prophylactic agent according to any one of claims 8 to 10, wherein the compound or extract is bicribiroc, baicalin flavonoid, Trigonostema xyphophylloids extract (TXE), or Vatica astrotricha extract.