Application of aromatic hydrocarbon receptor agonist in treatment of acute myelogenous leukemia

Abstract

The invention provides an application of an aromatic hydrocarbon receptor (AHR) agonist in preparation of a medicine for treating AML (acute myeloid leukemia). The invention also provides application of an aromatic hydrocarbon receptor (AHR) agonist combined with all-transretinoic acid (ATRA) in preparation of a medicine for treating AML (acute myeloid leukemia). The anti-leukemia activity of the AHR provides a new treatment strategy for induced differentiation therapy of AML patients, the AHR agonist can be combined with ATRA for application, and the clinical treatment range of ATRA is expanded.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of an aromatic hydrocarbon receptor agonist and an aromatic hydrocarbon receptor agonist combined with ATRA in the treatment of acute myeloid leukemia (AML). Background Art

[0002] AML is a highly heterogeneous and complex malignancy, characterized by uncontrolled proliferation of myeloid progenitor cells and impaired differentiation ability. Despite extensive research and significant progress in elucidating the molecular mechanisms underlying the pathogenesis of AML, chemotherapy and hematopoietic stem cell transplantation have been the standard clinical treatment for AML for decades. However, patients often experience adverse reactions after chemotherapy or hematopoietic stem cell transplantation, such as decreased immune response, primary and secondary drug resistance to chemotherapy drugs, and rejection reactions, resulting in poor remission of the disease. The incidence of AML increases with age, and a large proportion of elderly patients are not suitable for chemotherapy or hematopoietic stem cell transplantation. The lack of effective and less aggressive treatment leads to a very low cure rate of only 5-15% for these elderly patients. AML remains a highly destructive disease clinically.

[0003] Clinically, the induction differentiation strategy has achieved great success in the treatment of patients with acute promyelocytic leukemia (APL). The pathological feature of APL is the t(15;17) chromosomal translocation, which leads to the formation of the PML-RARɑ fusion oncoprotein. All-trans retinoic acid (ATRA) targets PML-RARɑ and induces cell differentiation, enabling leukemic blast cells to return to normal and develop into terminally differentiated neutrophils, indicating that inducing terminal differentiation is an effective method for treating AML. Although ATRA represents a successful differentiation treatment regimen, it is ineffective for AML other than the APL subtype.

[0004] The persistent overexpression of HOXA9 may represent a commonly dysregulated node suitable for therapeutic targeting of a series of AML subtypes. Previous studies have shown that dihydroorotate dehydrogenase (DHODH) is a potential target for AML treatment, and this study demonstrated the feasibility of using a Hoxa9 overexpression model to study the pro-differentiation targets for AML treatment. However, the effectiveness of DHODH has not been confirmed in clinical trials, which may be because cancer cells survive using the pyrimidine salvage pathway. For many targets, the preclinical potential has not been successfully translated, and there is a great need to explore new promising targets to overcome differentiation blockade. Summary of the Invention

[0005] To solve the above technical problems, the first aspect of the present invention provides the use of an aryl hydrocarbon receptor (AHR) agonist in the preparation of a drug for treating hematological malignancies.

[0006] The second aspect of the present invention provides the use of an aryl hydrocarbon receptor (AHR) agonist in combination with all-trans retinoic acid (ATRA) in the preparation of a drug for treating hematological malignancies.

[0007] The third aspect of the present invention provides the use of an aryl hydrocarbon receptor (AHR) agonist in the preparation of a drug for treating hematological malignancies in combination with all-trans retinoic acid (ATRA).

[0008] The fourth aspect of the present invention provides the use of all-trans retinoic acid (ATRA) in the preparation of a drug for treating hematological malignancies in combination with an aryl hydrocarbon receptor (AHR) agonist.

[0009] In the first to fourth aspects of the present invention, the hematological malignancy is AML.

[0010] In the first to fourth aspects of the present invention, the aryl hydrocarbon receptor (AHR) agonist is selected from FICZ, L-Kynurenine, Tapinarof (WBI-1001), ITE, Indole-3-pyruvic acid, VAF347, β-Naphthoflavone.

[0011] In the second to fourth aspects of the present invention, the aryl hydrocarbon receptor (AHR) agonist and all-trans retinoic acid (ATRA) are administered simultaneously or sequentially.

[0012] In the first to fourth aspects of the present invention, the hematological malignancy is AML.

[0013] The fifth aspect of the present invention provides the use of an agonist of the cannabinoid receptor (CNR2), a downstream target of the aryl hydrocarbon receptor (AHR), in the preparation of a drug for treating hematological malignancies.

[0014] In certain embodiments, the hematological malignancy is AML.

[0015] In certain embodiments, the cannabinoid receptor (CNR2) is a common target of the aryl hydrocarbon receptor (AHR) agonist and all-trans retinoic acid (ATRA).

[0016] The sixth aspect of the present invention provides a drug combination comprising an aryl hydrocarbon receptor (AHR) agonist and all-trans retinoic acid (ATRA).

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The present invention uses a transgenic zebrafish model with blocked myeloid differentiation for phenotypic screening, and determines that AHR is the target of the compound with the best activity. In addition, the present invention conducts a whole-genome transcriptome analysis and determines that CNR2 is a common downstream target induced by ATRA and FICZ differentiation. It is demonstrated that the activation of AHR / CNR2 has strong induced differentiation activity on AML cell lines and zebrafish models both in vitro and in vivo.

[0019] The anti-leukemia activity of AHR provides a new treatment strategy for the induced differentiation therapy of AML patients. The AHR agonist has a synergistic effect with ATRA, and the combined application of the two expands the clinical treatment range of ATRA. It is also demonstrated that CNR2 has a new role in overcoming the immature state of leukemic blast cells, and it is pointed out that its activation is a strategy to overcome the blocked differentiation of AML. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Other features, objects, and advantages of the present invention will become more apparent by reading the detailed description of the non-limiting embodiments with reference to the following drawings:

[0021] Figure 1 Showing that compound screening determines that Khellin overcomes myeloid differentiation block by activating the AHR pathway. (A) An experimental procedure for chemical screening was established to monitor compounds that trigger the differentiation of Tg(drl:hoxa9) by rescuing the number of mpx + neutrophils in the CHT region at 3 dpf. Lef was used as a positive control. (B, C) Tg(drl:hoxa9) embryos were treated with Lef or Khellin, and the expression of the neutrophil gene mpx at 3 dpf was determined by whole-mount in situ hybridization. (D) Cell morphology in U937 or THP-1 cells treated with Khellin or Lef, and the arrows indicate differentiated cells. (E) Flow cytometry analysis of the CD11b + or CD14 + cell ratio in U937 or THP-1 cells after treatment with Khellin or Lef. (F) Immunoblotting was used to detect the expression of AHR protein in U937 cells after treatment with Khellin. (G) Immunofluorescence staining was used to detect the nuclear translocation of AHR (white arrows) in U937 cells after treatment with Khellin. (H, I) qPCR was used to measure the mRNA levels of AHR or CYP1A1 in U937 cells after treatment with Khellin.

[0022] Figure 2Show that AHR induces myeloid differentiation in zebrafish. (A, B) Tg(drl:hoxa9) embryos were treated with FICZ, and the expressions of myeloid progenitor cell marker (cmyb), neutrophil marker (mpx), and macrophage marker (mfap4) were detected by whole-mount in situ hybridization at 3 - 5 dpf. (C) Schematic diagram of the construction of the Tg(drl:AHR) transgenic zebrafish line. (D, E) Whole-mount in situ hybridization was used to determine the expressions of neutrophil marker (mpx) and macrophage marker (mfap4) in embryos with co-overexpression of hoxa9 and AHR in blood cells at 3 dpf. (F, G) Whole-mount in situ hybridization was used to determine the expressions of neutrophil marker (mpx) and macrophage marker (mfap4) in embryos with co-overexpression of hoxa9 and AHR in blood cells at 5 dpf.

[0023] Figure 3 Show that AHR induces myeloid differentiation through ahr2 in the zebrafish model. (A) An experimental procedure for cell sorting was established to collect fluorescently labeled blood cells from Tg(drl:hoxa9), Tg(drl:GFP), Tg(mpx:GFP), or Tg(mpeg:mcherry). (B) qPCR analysis of the expressions of ahr1a, ahr1b, or ahr2 in drl-GFP + cells sorted from Tg(drl:GFP) at 1 or 4 dpf. (C) qPCR analysis of ahr2 expression in drl-GFP + cells sorted at 1, 2, or 4 dpf. (D) qPCR analysis of ahr2 expression in sorted drl-GFP + cells, mpx-GFP + cells, or mpeg1 mcherry + cells at 4 or 5 dpf. (E) qPCR analysis of ahr2 expression in drl-GFP + cells or drl:hoxa9:GFP +Expression of ahr2 in cells. (F) qPCR analysis of ahr2 expression in GFP-negative or GFP-positive cells sorted from Tg(drl:hoxa9) embryos at 1 dpf. (G) Whole-embryo in situ hybridization assay of the expression of the neutrophil marker (mpx) at 3 dpf in Tg(drl:hoxa9) embryos after knockdown of ahr1a, ahr1b, or ahr2 and treatment with FICZ. (I, J) Whole-embryo in situ hybridization assay of the expression of the neutrophil marker (mpx) and macrophage marker (mfap4) at 3 dpf in WT embryos after knockout of ahr1a, ahr1b, or ahr2 or all ahr paralogs. (K, L) Whole-embryo in situ hybridization assay of the expression of the neutrophil marker (mpx) in FICZ-treated WT embryos at 3 dpf.

[0024] Figure 4 Show that FICZ and ATRA have a combined rescue effect on zebrafish. (A, B) Whole-embryo in situ hybridization assay of the expression of the neutrophil marker (mpx) at 3 dpf in embryos treated with 25 - 125 nM FICZ or 100 - 250 nM ATRA. Red arrows show that ATRA-treated embryos exhibit pericardial edema. (C, D) Whole-embryo in situ hybridization assay of the expression of the macrophage marker (mfap4) at 3 dpf and the neutrophil marker (mpx) at 5 dpf in Tg(drl:hoxa9) embryos treated with 25 nM FICZ or 100 nM ATRA. (E) qPCR analysis of ahr2 expression in whole embryos treated with FICZ or ATRA. (F, G) Whole-embryo in situ hybridization assay of the expression of the neutrophil marker (mpx) at 3 dpf in Tg(drl:hoxa9) embryos after knockout of ahr2 and treatment with ATRA. (H, I) Whole-embryo in situ hybridization assay of the expression of the neutrophil marker (mpx) at 3 dpf in Tg(drl:hoxa9) embryos treated with a combination of 25 nM FICZ and 100 nM ATRA.

[0025] Figure 5Shown is that AHR and ATRA induce AML cell differentiation, and ATRA-induced differentiation intersects with the AHR pathway. (A) Relative expression of AHR and RARɑ in U937 cells. (B) Cell morphology of U937 cells treated with FICZ or ATRA. (C) Flow cytometry analysis of the ratio of CD11b+ or CD14+ cells in U937 cells treated with FICZ or ATRA. (D) Overexpression of FLAG-tagged AHR in U937 cells using lentiviral transfection of plasmids. (E) Flow cytometry analysis of the ratio of CD11b+ or CD14+ cells in AHR-overexpressing U937 cells. (F) Expression of AHR and RARɑ in U937 cells treated with FICZ or ATRA. (G) Knockdown of AHR in U937 cells by shRNA transfection and cultured with ATRA or FICZ. Flow cytometry analysis of the ratio of CD11b+ or CD14+ cells. (H) Number of upregulated and downregulated genes (>2-fold change, adj P≤0.05) in cells treated with FICZ or ATRA compared to untreated controls. (I) GO enrichment analysis of differentially expressed genes in FICZ-treated U937 cells. (J) GO enrichment analysis of differentially expressed genes in ATRA-treated U937 cells. (K) K-Venn diagram of overlapping upregulated genes by FICZ and ATRA. (L) Heatmap showing unsupervised hierarchical clustering of genes differentially expressed in response to 6-hour treatment with FICZ or ATRA in U937 cells. Red-blue scale is used to reflect normalized gene expression, with red indicating higher expression and blue indicating lower expression. (M) qPCR analysis of genes significantly upregulated in cells treated with FICZ or ATRA. (N) Immunoblotting detection of CNR2 expression in U937 cells treated with FICZ or ATRA.

[0026] Figure 6It is shown that CNR2 is a common downstream target induced by FICZ or ATRA. (A) Schematic diagram of the putative AHR binding site on the promoter region of RARɑ predicted by JASPAR. (B) Schematic diagram of the putative RARɑ binding site on the promoter region of AHR predicted by JASPAR. (C) Schematic diagram of the putative RARɑ binding site on the promoter region of CNR2 predicted by JASPAR. (D) Schematic diagram of the putative AHR binding site on the promoter region of CNR2 predicted by JASPAR. (E) ChIP-PCR confirmed the binding of AHR to CNR2 in U937 cells. (F) U937 cells were transfected with shRNA to knockdown AHR and cultured with ATRA. The expressions of AHR and CNR2 in U937 cells were detected by qPCR. (G) U937 cells were treated with GW842166X (3 μM), and the ratios of CD11b+ or CD14+ cells were analyzed by flow cytometry. (H) CNR2 was overexpressed in U937 cells using lentiviral transfection of plasmids. Immunoblotting was used to detect the overexpression of CNR2 in U937 cells. (I) Flow cytometry was used to analyze the ratio of CD11b+ cells in CNR2-overexpressing U937 cells. (J) U937 cells were transfected with shRNA-CNR2 to knockdown CNR2, and the protein expression of CNR2 was analyzed by Western blotting. (K) KU937 cells were transfected with shRNA-CNR2 to knockdown CNR2 and cultured with ATRA. Flow cytometry was used to analyze the ratios of CD11b+ or CD14+ positive cells.

[0027] Figure 7 It is shown that CNR2 promotes cell differentiation, and the rescue ability of FICZ and ATRA exerts its function through CNR2. (A) qPCR analysis of cnr2 expression in sorted drl-GFP+ cells, mpx-GFP+ at 2 dpf, and mpeg1-mcherry+ cells at 5 dpf. (B) Whole-mount in situ hybridization was used to determine the expression of cmyb in WT embryos at 5 dpf after knockdown of cnr2 + myeloid progenitor cells, mpx + neutrophils, mfap4 + macrophages, and rag1 + lymphocytes. (C, D) Whole-mount in situ hybridization was used to determine the expression of mpx + neutrophils and mfap4 + macrophages in Tg(drl:hoxa9) embryos at 3 dpf treated with GW842166X. (E) Whole-mount in situ hybridization was used to detect the expression of mpx in Tg(drl:hoxa9) embryos at 3 dpf treated with FICZ after knockdown of cnr2 +Expression of neutrophils. (F) Whole-mount in situ hybridization was used to detect mpx at 3 dpf in Tg(drl:hoxa9) embryos treated with ATRA after knockdown of cnr2. + Expression of neutrophils. (G) Whole-mount in situ hybridization was used to detect mpx at 3 dpf in Tg(drl:hoxa9) embryos treated with FICZ plus ATRA after knockdown of cnr2. + Expression of neutrophils. (H) Schematic diagram of the molecular mechanism and therapeutic targeting of AHR in AML, and the combination of AHR agonists and ATRA enables ATRA to be used for differentiation therapy.

[0028] Figure 8 Showing that transgenic zebrafish express hoxb4 under the control of the drl regulatory element. (A) Schematic diagram of the drl promoter controlling the expression of Tg(drl:hoxb4-2A-GFP). GFP fluorescence was expressed in the CHT region under the control of the drl promoter (white arrow) at 24 hpf. (B) Whole-mount in situ hybridization was used to detect the expression of the hematopoietic stem cell marker runx1 in embryos from 36 hpf to 3 dpf. (C) Whole-mount in situ hybridization was used to detect the expression of the myeloid progenitor cell marker c-myb in embryos from 36 hpf to 4 dpf. (D) Whole-mount in situ hybridization was used to detect the expression of the neutrophil marker mpx in embryos from 1 to 5 dpf. (E) Whole-mount in situ hybridization was used to detect the expression of the macrophage marker mfap4 in embryos from 3 to 5 dpf. (F) Whole-mount in situ hybridization was used to detect the expression of the erythrocyte marker hbbe1 in embryos at 3 dpf. (G) Quantitative results of whole-mount in situ hybridization (B-E) (n = 50 - 200 embryos per group).

[0029] Figure 9Showing chemical screening to find compounds that overcome the differentiation barrier. (A - B) Whole - mount in situ hybridization was used to detect that the expression of mpx in Tg(drl:hoxa9) embryos could be significantly rescued after Lef drug treatment (50 - 200 embryos per group). (C - D) Whole - mount in situ hybridization was used to detect that the expression of mpx in Tg(drl:hoxb4) embryos could be significantly rescued after Lef drug treatment (50 - 200 embryos per group). (E) Representative images of whole - mount in situ hybridization of Tg(drl:hoxa9) embryos treated with compound 06E05. (F - G) Summary of the primary and secondary screening results of 1295 compounds in the Cayman and Prestwick compound libraries for chemical screening. (H - I) Expression of the neutrophil gene mpx was detected by whole - mount in situ hybridization at 3 dpf in Tg(drl:hoxb4) embryos treated with Lef or Khellin. (J) Western blot results of AHR knockdown by AHR shRNA transfection in U937 cells. (K) Flow cytometry analysis of CD11b expression level after AHR was knocked down by AHR shRNA and treated with Khellin for 3 days + expression level.

[0030] Figure 10 Showing that AHR activation can induce zebrafish myeloid cell differentiation. (A) Chemical structure of FICZ. (B) Whole - mount in situ hybridization was used to detect the expression of the erythrocyte marker (hbbe1) and T - lymphocyte marker (rag1) in Tg(drl:hoxa9) embryos at 3 - 5 dpf after FICZ treatment. (C - D) Whole - mount in situ hybridization was used to detect the expression of the neutrophil marker (mpx) in Tg(drl:hoxb4) embryos at 3 dpf after FICZ treatment (n = 50 - 100 embryos per group). (E - F) Whole - mount in situ hybridization was used to detect the expression of the neutrophil marker (mpx) in Tg(drl:hoxb4) embryos at 3 dpf after treatment with FICZ (25 nM and 70 nM) (n = 50 - 100 embryos per group). (G) Tg(drl:hoxa9) embryos were injected with human AHR mRNA (200 ng / μL, ∼1 - 2 nL), and the expression of the neutrophil marker (mpx) in 3 - dpf embryos was detected by whole - mount in situ hybridization. (H) Whole - mount in situ hybridization was used to detect the expression of the myeloid progenitor cell marker (c - myb), neutrophil marker (mpx), and lymphocyte marker (rag) in Tg(drl:AHR) embryos at 3 - 5 dpf. (I - J) Whole - mount in situ hybridization was used to detect the expression of the neutrophil marker (mpx) in Tg(drl:hoxb4;AHR) embryos at 3 dpf (n = 50 - 100 embryos per group).

[0031] Figure 11 It shows that zebrafish Ahr has three subtypes: ahr1a, ahr1b and ahr2. Alignment of the conservation between zebrafish Ahr and human Ahr.

[0032] Figure 12 It shows that FICZ and ATRA have a synergistic effect in overcoming the blocked differentiation. (A - B) Whole - mount in situ hybridization was used to detect the expression of the neutrophil marker (mpx) at 3 dpf after treating Tg(drl:hoxb4) embryos with FICZ (25 nM) or ATRA (100 nM) (n = 50 - 200 embryos per group). (C) Whole - mount in situ hybridization was used to detect the expression of the macrophage marker (mfap4) at 3 dpf after treating Tg(drl:hoxa9) embryos with FICZ (25 nM) combined with ATRA (100 nM). (D - E) Whole - mount in situ hybridization was used to detect the expression of the neutrophil marker (mpx) at 3 dpf after treating Tg(drl:hoxa9) embryos with transient knockdown of ahr1a, ahr1b or ahr2 by Lef treatment (n = 50 - 200 embryos per group). (F) At 24 hpf, Tg(dr1:hoxa9; hsp70:meis1) embryos were heat - shocked, and after treatment with DMSO or FICZ, whole - mount in situ hybridization was used to detect the expression of the myeloid progenitor cell marker (cmyb), neutrophil marker (mpx), and macrophage marker (mfap4) from 1 to 5 dpf. (G) At 24 hpf, Tg(dr1:hoxa9; hsp70:meis1) embryos were heat - shocked, and after treatment with DMSO or ATRA, whole - mount in situ hybridization was used to detect the expression of the myeloid progenitor cell marker (cmyb), neutrophil marker (mpx), and macrophage marker (mfap4) from 1 to 5 dpf.

[0033] Figure 13 It shows the differentiation - promoting effect of FICZ or ATRA. (A) Flow cytometry was used to analyze the expression of the differentiation proteins CD11b or CD14 after treating U937 cells with FICZ or ATRA. (B) Flow cytometry was used to analyze the expression of the differentiation protein CD11b in U937 cells treated with different concentrations of FICZ. (C) Statistical chart of the flow cytometry results in (A). (D - E) Flow cytometry was used to analyze the expression of the differentiation proteins CD11b or CD14 after treating HL - 60 cells with FICZ (100 nM) and ATRA (1 μM) alone or in combination. (F) Heatmap of the transcriptional profiles of single - cell RAR, AHR, and CNR2 gene expressions in 21 AML patients.

[0034] Figure 14Show the pro-differentiation effect of GW842166X. (A) Chemical structure of GW842166X. (B) Detection of the expression of differentiation proteins CD11b or CD14 in U937 cells treated with GW842166X by flow cytometry. (C) Detection of the expression of differentiation protein CD11b in AML cell lines treated with GW842166X by flow cytometry. (D-E) Detection of the expression of myeloid progenitor cell marker c-myb, neutrophil marker mpx, and macrophage marker mfap4 at 5 dpf in WT embryos treated with GW842166X (30 μM) by whole-mount in situ hybridization (n = 50 - 200 embryos per group). (F) Detection of the expression of myeloid progenitor cell marker cmyb, neutrophil marker mpx, and macrophage marker mfap4 at 4 - 5 dpf in WT embryos treated with GW842166X (50 μM) by whole-mount in situ hybridization (n = 50 - 200 embryos per group). (G) RT-qPCR analysis of the expression of cnr2 in Tg(drl:hoxa9) embryos treated with FICZ and ATRA. (H) Detection of the expression of neutrophil marker mpx at 3 dpf in Tg(drl:hoxa9) embryos treated with GW842166X and transiently knocked down of cnr2 by whole-mount in situ hybridization. Detailed implementation manners

[0035] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] Example 1 Compound screening To find compounds that overcome blocked differentiation

[0037] In previous studies, the inventors have constructed a transgenic zebrafish line that overexpresses hoxa9 under the control of the zebrafish drl regulatory element, and analyzed the hematopoietic phenotype using whole-embryo in situ hybridization technology (Wang W, Li H, Huang M, et al. Hoxa9 / meis1-transgenic zebrafish develops acute myeloid leukaemia-like disease with rapid onset and high penetrance. Open Biol. 2022;12(10):220172.). The experimental procedure of whole-embryo in situ hybridization is as follows: Embryos are cultured to an appropriate time point and fixed overnight at 4°C with 4% PFA. The next day, remove 4% PFA and wash the embryos twice with freshly prepared PBT (prepared with DEPC water), bleach the embryos, and the bleaching system is prepared as shown in Table 1 below:

[0038] Table 1 Bleaching system

[0039] 10% KOH 1.6 mL <![CDATA[30% H2O2]]> 0.6 mL 10% Tween-20 200 μL DEPC water up to 20 mL

[0040] The bleaching time of the embryos depends on the age of the embryos and is shown in Table 2 below:

[0041] Table 2 Bleaching time

[0042] 2 dpf ~10 min 3 dpf ~30 min 4 dpf ~45 min 5 dpf ~50 min

[0043] Remove the bleaching solution, wash the embryos twice with freshly prepared PBT (prepared with DEPC water), fix at room temperature with 4% PFA for 1 h, wash three times with freshly prepared PBT (prepared with DEPC water), dehydrate with methanol (three times, 5 min each time), and then place at -20°C overnight. Next, wash the embryos for 5 min according to the ratios of PBT:methanol = 3:1, 1:1, and 1:3 respectively, and wash with PBT (prepared with DEPC water) (three times, 5 min each time) to rehydrate the dehydrated embryos. Permeabilize the embryos with proteinase K (PK, 10 μg / mL), and the permeabilization time depends on the age of the embryos and is as follows:

[0044] Table 3 Proteinase K permeabilization time for embryos

[0045] 1 dpf ~6 min 2 dpf ~12 min 3 dpf ~20 min 4 dpf ~30 min 5 dpf ~35 min

[0046] Remove PK, quickly wash with PBT (prepared with DEPC water) to remove residual PK, fix at room temperature with 4% PFA for 20 min, and wash with PBT (prepared with DEPC water) (five times, 5 min each time). Place the embryos in Hybe +Prehybridize in hybridization buffer in an incubator at 65 - 70 °C for 30 min - 3 h (the longer the prehybridization time, the lighter the background). After prehybridization, remove the Hybe + hybridization buffer and use the Hybe + hybridization buffer containing the probe (200 - 300 ng probe / 200 μL Hybe + hybridization buffer) and incubate overnight in an incubator at 65 - 70 °C. After recovering the probe (which can be recycled 3 - 5 times) and storing it at -20 °C, wash the embryos in an incubator at 65 - 70 °C for 15 min respectively according to the ratios of Hybe - :2X SSC = 3:1, 1:1, 1:3. Subsequently, wash the embryos with 2X SSC for 15 min, and wash the embryos with 0.2X SSC twice for 15 min and 30 min respectively. Wash the embryos for 10 min at room temperature respectively according to the ratios of 0.2X SSC:PBT = 3:1, 1:1, 1:3, and wash the embryos with PBT again for 10 min. After blocking at room temperature for 30 min - 3 h with the blocking solution (PBT + 2% sheep serum), remove the blocking solution, add the blocking solution containing Anti-Digoxigenin-AP antibody diluted 1:5000, and incubate overnight at 4 °C. Wash with PBT (six times, 15 min each time), wash with staining buffer for 5 min. The staining buffer is prepared as shown in Table 4 below:

[0047] Table 4 Formulation of staining buffer

[0048] 1 M Tris PH 9.5 100 μL <![CDATA[1M MgCl2]]> 50 μL 5 M NaCl 20 μL Tween-20 5 μL <![CDATA[dd H2O]]> up to 1 mL

[0049] Remove the staining buffer, add the staining buffer staining solution containing 4.5 mg / mL NBT and 3.5 mg / mL BCIP, transfer to a 24-well plate, protect from light, and stain at room temperature on a shaker. After terminating the staining, count and photograph the staining results of the embryos. The statistical method for in situ hybridization staining results is as follows: Transfer the embryos after terminating the staining to a white ceramic plate and observe the staining of each group of embryos under a stereomicroscope. Compared with the control group, observe and count the staining levels of the corresponding stained parts of the embryos, and divide them into "high staining group" and "low staining group". Count the proportion of embryos in the "high staining group" in each group, and use Graph Pad Prism 6 software to statistically analyze the data results. All experimental results are performed in three independent experiments and expressed as mean ± variance, and statistical tests are performed using the t-test.

[0050] Next, a stable phenotype-based screening model was developed using Tg(drl:hoxa9) embryos to screen for compounds that overcome the blockade of myeloid differentiation. The construction method is as follows: Tg(drl:hoxa9) zebrafish embryos were picked under a stereomicroscope at 1 dpf for fluorescence. Well-developed fluorescent embryos, i.e., embryos with high expression of hoxa9, were selected as the model group, drug treatment group, and positive control group, and non-fluorescent embryos, i.e., embryos with normal expression of hoxa9, were used as the control group. The embryos were placed in 24-well plates, with about 15 embryos per well. In the drug treatment group, a set concentration of the compound (diluted with E3) was added. In the model group, an equal volume of DMSO (diluted with E3) as the compound was added. In the positive control group, Lef (2.5 μM, diluted with E3) was added and cultured in a 37°C incubator until the 3 dpf detection time point. The embryos were fixed overnight at 4°C with 4% PFA, and then whole-mount in situ hybridization experiments were performed to evaluate the expression level of neutrophils labeled by mpx. There were a total of 95 compounds in the Cayman Epigenetic Library, with an initial concentration of 10 mM. The primary screening was carried out at a dilution ratio of 1:300 (final concentration of 33.33 μM), i.e., 2 μL of the compound to be screened was added to 600 μL of E3. There were a total of 1200 compounds in the Prestwick Chemical Library, with an initial concentration of 10 mM. The primary screening was carried out at a dilution ratio of 1:500 (final concentration of 20 μM), i.e., 2 μL of the compound to be screened was added to 1 mL of E3. Compounds that significantly increased the expression level of mpx in the primary screening were rescreened in the same batch. The operation of the rescreening experiment was the same as that of the primary screening, and the working concentration of the rescreened compounds was 20 μM.

[0051] In addition, the expression of HOXB4 rapidly decreases as myeloid cells mature, and overexpression of HOXB4 also significantly increases the risk of leukemia. Since the inventors attempted to find broad-spectrum active compounds that can overcome the blocked myeloid differentiation, a transgenic fish line overexpressing hoxb4 under the control of the zebrafish drl regulatory element was also constructed. The construction method is as follows: Dilute the extracted plasmid drl:hoxb4-2A-GFP to 20 ng / μL, and use a microinjector to inject the plasmid into single cells of AB wild-type zebrafish to construct the Tg(drl:hoxb4) transgenic fish line. One night before injection, mate AB wild-type zebrafish. The next morning, collect embryos 15 minutes after removing the partition, and pick healthy embryos that have developed to single cells under a microscope for microinjection. Inject 1-2 nL of the plasmid diluted to 20 ng / μL into single cells of AB wild-type zebrafish embryos. The successfully injected embryos are placed in a clean petri dish and cultured in an incubator at a constant temperature of 28.5°C. On the day after injection, remove unhealthy embryos such as dead and abnormally developed ones, and pick embryos with GFP fluorescence under a fluorescence microscope and raise them to adulthood (sexual maturity at about 3 months) to obtain F0 generation transgenic zebrafish. Pick embryos with GFP fluorescence from the offspring obtained by crossing F0 generation adult zebrafish with AB wild-type zebrafish and raise them to adulthood to obtain F1 generation transgenic zebrafish. Embryos with GFP fluorescence in the offspring produced by self-crossing F1 generation transgenic zebrafish are F2 generation (self-crossing can increase the copy number of the overexpressed gene and the phenotype is more obvious), that is, the construction of a transgenic fish line with stable inheritance is successful and can be used for subsequent experiments.

[0052] The hematopoietic phenotype in Tg(drl:hoxb4) embryos was analyzed using whole-mount in situ hybridization technology. The experimental results showed that overexpression of hoxb4 was also sufficient to block myeloid differentiation and exhibited stronger leukemia-inducing ability than overexpression of hoxa9 ( Figure 8 A-G). In the study, Tg(drl:hoxb4) was used to further verify the differentiation-promoting effect of active compounds. Since two models of blocked differentiation were used, it is more likely to find broad-spectrum active compounds that can overcome blocked differentiation.

[0053] Compared with untreated embryos, Lef treatment (the dihydroorotate dehydrogenase inhibitor Leflunomide) significantly rescued the mpx + neutrophil expression level at 3 dpf in Tg(drl:hoxa9) or Tg(drl:hoxb4) embryos ( Figure 9 A-D), supporting that Lef has the ability to overcome blocked myeloid differentiation in zebrafish and can be used as a positive control. An experimental model for chemical screening was established by rescuing the mpx + neutrophil expression level at 3 dpf to screen for compounds that can overcome myeloid differentiation in Tg(drl:hoxa9)Figure 1 A and Figure 9 E). 1295 small molecules in the Cayman and Prestwick compound libraries were evaluated ( Figure 9 F). After primary screening and secondary screening, 4 compounds (8.7% of the total) showed good and reproducible differentiation-promoting effects in Tg(drl:hoxa9) embryos ( Figure 9 G). Among them, Khellin was the best at rescuing the mpx + neutrophil expression level in Tg(drl:hoxa9) and Tg(drl:hoxb4) embryos and was selected as the lead compound ( Figure 1 B-C and Figure 9 H-I).

[0054] Next, the anti-leukemia activity of Khellin in AML cell lines was studied, and the cell morphology of U937 and THP-1 cells treated with Khellin for 3 days was analyzed. Reniform nuclei and a decreased nuclear / cytoplasmic ratio were observed in Khellin-treated U937 and THP-1 cells ( Figure 1 D), indicating that Khellin triggered the cell differentiation of U937 cells and THP-1 cells. Treatment with Khellin led to enhanced expression of CD11b or CD14, indicating its ability to promote cell differentiation ( Figure 1 E). According to previous studies, Khellin is an agonist of AHR. Western blotting and immunofluorescence staining with an AHR antibody showed that AHR translocated into the nucleus in Khellin-treated U937 cells, indicating AHR activation ( Figure 1 F-G). qPCR analysis detected a significant upregulation of AHR and CYP1A1 (a typical downstream protein of AHR signaling) in Khellin-treated U937 cells, indicating that Khellin activates the AHR pathway ( Figure 1 H-I).

[0055] To study the biological function of AHR, the inventors knocked down AHR in U937 cells by three independent shRNAs. The shRNA sequences were designed as shown in Table 5 below:

[0056] Table 5 shRNA sequences of AHR

[0057]

[0058] Three pairs of shRNAs were annealed, the reaction system was prepared, and it was placed in a 100 °C water bath. The heating function was immediately turned off, and it was allowed to cool naturally to room temperature (>10 h):

[0059] Table 6 Annealing reaction system

[0060] Forward oligo 1 μL Reverse oligo 1 μL 10× NEB buffer 2 5 μL <![CDATA[ddH2O water]]> 43 μL

[0061] The pLKO.1TRC cloning vector was digested with AgeI and EcoRI. After digestion, the linear plasmid was recovered by gel extraction. After measuring the concentration with Nanodrop, it was ligated with three pairs of annealed shRNAs at room temperature for 30 min. After ligation, it was transformed using competent cells DH5α, and the base sequence was confirmed by Sanger sequencing. The reaction system was prepared as shown below:

[0062] Table 7 Sanger sequencing reaction system

[0063] Annealed oligo 2 μL Digested pLKO.1TRC 20 ng 10× Quick ligase buffer 1 μL Quick DNAligase 1 μL <![CDATA[ddH2O water]]> up to 10 μL

[0064] The obtained plasmid was transfected into U937 cells using the lentiviral transfection method (with HEK-293T cells as the vector), and the knockdown effect was evaluated by detecting the AHR protein using Western blot. The successful knockdown of AHR was confirmed by Western blot, and the specific sequence shRNA-AHR-2 was used in subsequent experimental measurements to significantly inhibit the expression of AHR ( Figure 9 J). The inventors observed that, compared with the control group, the knockdown of AHR inhibited the differentiation efficacy of Khellin ( Figure 9 K), indicating that Khellin induces differentiation through AHR activation. In summary, the screened compound Khellin exhibits the ability to significantly overcome the blocked differentiation in vitro and in vivo, and Khellin achieves the overcoming of blocked differentiation by activating the AHR pathway.

[0065] Example 2 AHR induces myeloid differentiation in zebrafish model

[0066] The low bioavailability of Khellin limits its potential as an in vivo tool compound to study the anti-AML activity of AHR. Therefore, the inventors used a well-validated endogenous high-affinity AHR ligand FICZ to evaluate the pro-differentiation effect of AHR in Tg(drl:hoxa9) embryos ( Figure 10 A).

[0067] The hematopoietic phenotypes of FICZ-treated Tg(drl:hoxa9) embryos were analyzed using whole-mount in situ hybridization. After FICZ treatment, the expression levels of myeloid progenitors (cmyb), neutrophils (mpx), and macrophages (mfap4) at 3 and 5 dpf were significantly rescued, indicating that FICZ rescued the myeloid developmental arrest ( Figure 2 A-B). The expression of hbbe1 at 3 dpf in Tg(drl:hoxa9) embryos was similar in the drug-treated group and the control group ( Figure 10B), indicating that FICZ treatment does not affect the development of red blood cells. In addition, after FICZ treatment at 5 dpf, the expression of the lymphoid marker rag1 was slightly restored in Tg(drl:hoxa9) embryos ( Figure 10 B). In addition, in the differentiation-blocked model Tg(drl:hoxb4), FICZ treatment also significantly rescued the expression of the neutrophil marker mpx at 3 dpf ( Figure 10 C-D). Due to the stronger leukemogenic ability of hoxb4 than hoxa9, FICZ required a higher concentration (from 25 nM to 70 nM) to show an effective rescue effect in Tg(drl:hoxb4) embryos ( Figure 10 E-F). To further investigate the pro-differentiation effect of AHR, human AHR mRNA (200 ng / μL) was injected into Tg(drl:hoxa9) embryos to exogenously supplement AHR, and WISH was performed. Compared with the embryos without AHR supplementation, the Tg(drl:hoxa9) embryos supplemented with AHR significantly rescued the expression of the neutrophil marker mpx, indicating that AHR overcame the differentiation block ( Figure 10 G).

[0068] Next, transgenic zebrafish overexpressing AHR were constructed under the control of the zebrafish drl regulatory element to evaluate the role of AHR in hematopoietic development. AHR was cloned into a plasmid containing an integration marker, and γ-crystallin drives the expression of VenusGFP in the lens. The plasmid ɑ-crystallin-VenusGFP-drl-AHR was injected into single cells of wild-type embryos to generate stable Tg(drl:AHR). At 3 dpf, GFP expression was observed in the eyes of transgenic embryos ( Figure 2 C). The hematopoietic phenotype of Tg(drl:AHR) was analyzed using WISH. Embryos overexpressing AHR inhibited the development of myeloid progenitors (cmyb) at 3-5 dpf ( Figure 10 H). In addition, Tg(drl:AHR) increased the expression of mature neutrophils (mpx) at 3-5 dpf and did not affect lymphoid development, as shown by the similar expression of rag1 between WT and transgenic embryos at 5 dpf ( Figure 10 H). The above experimental results indicate that AHR plays a key role in hematopoietic development.

[0069] Next, two transgenic fish lines were crossed to establish Tg(drl:hoxa9; AHR) with co-overexpression of hoxa9 and AHR in blood cells to further evaluate whether the anti-leukemic activity of AHR is hematopoietic cell-autonomous. WT, Tg(drl:hoxa9), and Tg(drl:hoxa9; AHR) embryos were subjected to WISH (whole-mount in situ hybridization) at 3 or 5 dpf. Tg(drl:hoxa9; AHR) embryos showed significantly increased expression of mature neutrophils (mpx) and macrophages (mfap4) inhibited by hoxa9 at 3 and 5 dpf, indicating that endogenous AHR activation could rescue the blocked differentiation and the anti-leukemic activity of AHR is hematopoietic cell-autonomous ( Figure 2 D-G). Since AHR is a ligand-activated nuclear transcription factor, Tg(drl:hoxa9; AHR) embryos were further treated with FICZ to enhance AHR activation. After FICZ treatment, the numbers of mpx + neutrophils and mfap4 + macrophages increased compared with those of untreated Tg(drl:hoxa9; AHR) embryos ( Figure 2 D-E), indicating that AHR activation induced myeloid differentiation in the zebrafish model. In addition, the pro-differentiation effect of AHR also worked in Tg(drl:hoxb4) embryos ( Figure 10 I-J). Taken together, AHR induced strong myeloid differentiation in the zebrafish model and the effect of AHR was hematopoietic cell-autonomous.

[0070] Example 3 AHR Induces Myeloid Differentiation via ahr2 in the Zebrafish Model

[0071] Zebrafish have three Ahr subtypes: ahr1a, ahr1b, and ahr2 ( Figure 11 ). To analyze which subtype contributed to differentiation, fluorescently labeled blood cells were sorted by fluorescence-activated cell sorting (FACS) from Tg(drl:hoxa9), Tg(drl:GFP), Tg(mpx:GFP), or Tg(mpeg:mcherry) ( Figure 3 A). Tg(drl:hoxa9), Tg(drl:GFP), Tg(mpx:GFP), Tg(mpeg1:mcherry) zebrafish embryos at different stages were picked for fluorescence under a stereomicroscope with fluorescence, and well-developed fluorescent embryos were selected for cell sorting. AB wild-type zebrafish embryos were used as the control group. hoxa9-GFP was sorted from 1-dpf Tg(drl:hoxa9) embryos (dechorionated with pronase at 1 dpf, 80 - 100 embryos per group) +Cells; Sorting drl-GFP from Tg(drl:GFP) embryos at 1, 2, and 4 dpf + Cells; Sorting mpx-GFP from Tg(mpx:GFP) embryos at 4 and 5 dpf + Cells; Sorting mpeg-mcherry from Tg(mpeg1:mcherry) embryos at 4 dpf + Cells. The embryos were anesthetized with 0.02% Tricaine and washed three times with sterile E3 solution. After removing the liquid as much as possible, the embryos were chopped with a blade on ice, resuspended in 200 - 400 μL of PBS containing 1% FBS, added with Liberase at a final concentration of 38 μg / mL, and digested at 37°C for 20 min (inverted and mixed every 5 min). Then 10% FBS was added to terminate the digestion, and the mixture was filtered through a 40 μM filter. The blood cells were collected by centrifugation at 5,000 rpm, 4°C for 15 min, the supernatant was removed, the cell pellet was resuspended in FACS buffer (PBS + 1% FBS), filtered through a 40 μM filter, and then sorted on the machine. According to different fluorescent labels, the cells were sorted. After sorting, the cells were collected by centrifugation at 5,000 rpm, 4°C for 10 min, and the total RNA was extracted by lysing the cells with Trizol for subsequent experiments.

[0072] According to the fact that the drl element is active during the early development of all lineages from the anterior and posterior hematopoietic populations, the drl:GFP sorted from Tg(drl:GFP) embryos + cells cover all hematopoietic cells. At 1 or 4 dpf, the expression of ahr2 in hematopoietic cells is higher than that of ahr1a or ahr1b ( Figure 3 B), and compared with 1 - 2 dpf, the expression of ahr2 increases at 4 dpf ( Figure 3 C), supporting its potential function in hematopoiesis. The primer sequences used are shown in Table 8 below:

[0073] Table 8 Primers for qPCR detection

[0074]

[0075]

[0076] Similarly, the expression of ahr2 in blood cells sorted from Tg(mpx:GFP) at 5 dpf is higher than that in cells sorted from Tg(drl:GFP), Tg(mpx:GFP), or Tg(mpeg:mcherry) at 4 dpf, which also indicates that the participation of ahr2 is required for the development of blood cells into more mature cells ( Figure 3 D). Compared with the drl:GFP sorted from Tg(drl:GFP) embryos +Compared with cells, the expression of ahr2 in drl:hoxa9 cells sorted from Tg(drl:hoxa9) embryos was significantly reduced, indicating that overexpression of hoxa9 inhibited ahr2 expression ( + E). Consistent with these, a reduction in ahr2 expression was observed in GFP-positive cells sorted from Tg(drl:hoxa9) embryos at 1 dpf compared with sorted GFP-negative cells ( Figure 3 F), supporting the requirement of ahr2 for the development of blood cells into more mature cells. Therefore, it is speculated that AHR activation in myeloid cells overcomes the differentiation block in the action of Tg(drl:hoxa9) through ahr2 activation. Figure 3

[0077] To test this hypothesis, morpholinos were applied to study the roles of ahr1a, ahr1b, and ahr2 in myeloid differentiation. Knockdown of ahr2 in Tg(drl:hoxa9) inhibited the rescue effect of FICZ ( Figure 3 G-H), indicating that AHR overcomes the myeloid differentiation block in Tg(drl:hoxa9) embryos through ahr2. Next, morpholinos were microinjected to knockout ahr1a, ahr1b, ahr2, or all subtype homologs in wild-type embryos, and WISH was performed. After knockout of ahr1a, ahr1b, and ahr2 respectively, the similar expression of mpx Figure 3 + neutrophils and mfap4 + macrophages indicated that the three subtypes interacted and were complementary ( Figure 3 I-J). Only simultaneous knockout of all subtype homologs could inhibit the expression of mpx and mfap4 ( Figure 3 I-J), indicating that Ahr itself is involved in hematopoietic development. Similarly, increasing the concentration of FICZ (100 nM) in WT to highly activate AHR and performing WISH, the upregulation of mature neutrophils (mpx) also indicated that AHR plays a crucial role in hematopoietic development ( Figure 3 K-L). The results were consistent with the hematopoietic phenotype of Tg(drl:AHR), indicating that AHR is involved in hematopoietic development. Taken together, Ahr plays a crucial role in hematopoietic development, and Ahr induces myeloid differentiation in zebrafish through ahr2.

[0078] Example 4 Combined rescue effect of FICZ and ATRA in zebrafish model

[0079] Given the complexity of leukemia and the multiple factors contributing to its development, single-agent therapy is sometimes ineffective, and thus, combination therapy is very common. First, Tg(drl:hoxa9) embryos were treated with FICZ or ATRA, and the hematopoietic phenotype was analyzed using WISH to compare the pro-differentiation effects and safety of FICZ and ATRA. Increasing the working concentration of FICZ to 5-fold (125 nM) still overcame the differentiation block without toxicity, while when the working concentration of ATRA was increased to 2-fold (250 nM), zebrafish showed pericardial edema and developmental retardation( Figure 4 A-B), indicating that FICZ has a wider therapeutic range and higher safety. FICZ or ATRA rescued the expression of mfap4 + macrophages in Tg(drl:hoxa9), and FICZ also rescued the expression of mpx + neutrophils in Tg(drl:hoxb4), while ATRA did not show a rescue effect in Tg(drl:hoxb4)( Figure 4 C-D and 12A-B), indicating that the rescue effect of FICZ is more effective than that of ATRA. The duration of drug treatment was extended from 2 days to 5 days, and ATRA did not show a rescue effect on Tg(drl:hoxa9) embryos at 5 dpf, probably due to long-term drug toxicity( Figure 4 C-D). However, FICZ still showed a rescue effect after continuous treatment for 5 days, indicating that FICZ has no cumulative toxicity( Figure 4 C-D).

[0080] Next, the inventors investigated whether the expression of AHR affects the pro-differentiation ability of ATRA. The expression of ahr2 in embryos treated with FICZ or ATRA was analyzed using qPCR. The results showed that the expression of ahr2 increased significantly after treatment with FICZ or ATRA compared with WT and untreated, demonstrating that the participation of ahr2 is required for FICZ or ATRA to overcome the differentiation block( Figure 4 E). To investigate the crosstalk between the differentiation pathway induced by ATRA and AHR, morpholinos were used to knock out ahr1a, ahr1b, or ahr2 in Tg(drl:hoxa9) embryos. Knockdown of ahr2 weakened the rescue effect of ATRA, indicating that the differentiation pathway induced by ATRA functions partially through AHR( Figure 4(F - G). The inventors believe that AHR plays an important role in the pro - differentiation effects of ATRA and FICZ, which may be a common downstream target. It has been reported that co - administration of FICZ with ATRA increases the type and intensity of the dynamic changes induced by ATRA. Therefore, the combined effects of FICZ and ATRA in Tg(drl:hoxa9) embryos were studied to evaluate potential combined effects. Compared with treatment with FICZ or ATRA alone, Tg(drl:hoxa9) embryos co - treated with FICZ and ATRA did exhibit a highly differentiated phenotype, as mpx + neutrophils and mfap4 + macrophages were upregulated to a greater extent ( Figure 4 H - I and Figure 12 C).

[0081] Interestingly, previous studies have reported that Lef is not only an inhibitor of DHODH but also an AHR agonist, and enhances AHR nuclear translocation and activation. However, knocking out ahr1a, ahr1b, or ahr2 separately had no effect on the pro - differentiation effect of Lef ( Figure 12 D - E). The inventors speculate that the activation of AHR may not play a key role in the pro - differentiation function of Lef, or that the differentiation pathway induced by Lef acts partially through AHR. In addition, in an AML model Tg(drl:hoxa9; hsp70:meis1) previously constructed in the laboratory, which has a rapid onset and high penetrance (Wang W, Li H, Huang M, et al. Hoxa9 / meis1 - transgenic zebrafish develops acute myeloid leukaemia - like disease with rapid onset and high penetrance. Open Biol. 2022;12(10):220172), FICZ treatment also significantly rescued the expression of cmyb + bone marrow progenitors, mpx + neutrophils and mfap4 + macrophages at 5 dpf ( Figure 12 F), supporting the ability of AHR to promote myeloid differentiation in the zebrafish AML model. Due to the toxicity problem of long - term drug action, the effectiveness of ATRA in Tg(drl:hoxa9; hsp70:meis1) embryos has not been confirmed ( Figure 12 G). In summary, FICZ and ATRA have a combined rescue effect, and the high efficiency of the combination of FICZ and ATRA enables ATRA to be used for the differentiation treatment of non - APL AML.

[0082] Example 5 Interaction between the ATRA-induced differentiation pathway and AHR

[0083] Next, the differentiation-promoting effects of FICZ and ATRA in AML cells were investigated. In U937 cells, AHR (the target of FICZ) was slightly higher than RARɑ (the target of ATRA), indicating that U937 might be more sensitive to FICZ ( Figure 5 A). Changes in cell morphology and upregulation of differentiation markers (CD11b or CD14) in U937 cells treated with FICZ or ATRA indicated that FICZ and ATRA triggered cell differentiation ( Figure 5 B-C and Figure 13 A). In U937 cells, FICZ tended to enhance CD14 + cells, and ATRA tended to enhance CD11b + cells. While increasing the concentration of FICZ, CD11b + cells also increased ( Figure 13 B). FICZ also induced the differentiation of various AML cell lines ( Figure 13 C), especially in HL60 cells. The combination of FICZ and ATRA showed a synergistic differentiation-promoting effect ( Figure 13 D-E). To further investigate the differentiation-promoting effect of AHR, plasmids were transfected into U937 cells to overexpress AHR. The successful overexpression of FLAG-tagged AHR in U937 cells was confirmed at the protein level by Western blotting ( Figure 5 D). Analysis of the upregulation of CD14 or CD11b-positive cells in AHR-overexpressing U937 cells by flow cytometry showed that overexpression of AHR in U937 induced cell differentiation ( Figure 5 E).

[0084] In addition, U937 cells were transfected with AHR-shRNA-2 to knockdown AHR, thus further investigating the role of AHR in ATRA-induced differentiation. U937 cells were treated with ATRA after knockdown of AHR and analyzed by flow cytometry. The reduction of CD11b or CD14-positive cells characterized that the knockdown of AHR significantly inhibited the differentiation-promoting effects of ATRA or FICZ, indicating that ATRA or FICZ induced differentiation through AHR ( Figure 5 F).

[0085] Next, to clarify how ATRA or FICZ induces differentiation, early genome-wide transcriptional changes 6 hours after ATRA or FICZ treatment were examined by RNA-Seq. By analyzing the expression of the AHR signal in ATRA-treated cells, it was found that the AHR mRNA levels were detectable and unchanged between control and ATRA-treated cells. Nevertheless, CYP1A1 (a typical downstream effector of AHR signaling and an indicator of pathway activity, Fc = 4.62) was significantly upregulated in ATRA-treated cells, suggesting the possibility of differential AHR pathway signaling between control and ATRA-treated cells. In FICZ-treated cells, the inventors found 164 genes upregulated and 217 genes downregulated. In ATRA-treated cells, 2,132 genes were upregulated and 2,383 genes were downregulated (FDR < 0.05 and 2-fold change, Figure 5 G).

[0086] GO enrichment analysis showed that retinoic acid metabolic processes were enriched in FICZ-treated cells, indicating that the activation of AHR plays an important role in the ATRA metabolic process ( Figure 5 H). GO enrichment analysis also showed that several signaling pathways, including myeloid cell differentiation, leukocyte differentiation, cell fate commitment, and B cell differentiation pathways, were enriched in FICZ-treated cells, indicating that AHR activation promotes cell differentiation ( Figure 5 H). The MAPK cascade pathway, ERK1 / 2 cascade pathway, Notch signaling, and EGFR pathway were also significantly activated in FICZ-treated cells, and these pathways are known to be crucial in ATRA-induced differentiation and were also enriched in the GO analysis of ATRA ( Figure 5 H-I).

[0087] When the two groups of upregulated genes were combined, it was found that there were 8 overlapping genes among the genes activated by FICZ or ATRA ( Figure 5 J-K). qPCR was performed to further confirm the significant upregulation of these genes. Among these 8 genes, the inventors identified cannabinoid receptor 2 (CNR2) as a candidate target gene ( Figure 5 L). This observation was confirmed by Western blotting, showing a significant upregulation of CNR2 in U937 cells treated with FICZ or ATRA ( Figure 5 M). When exploring the differential gene expression that might contribute to the differentiation of leukemia stem cells, the published single-cell transcriptional profiles of 21 AML patient samples were examined ( Figure 13 D). Interestingly, in the TISCH dataset, the inventors observed elevated transcription of RARɑ and AHR in Mono / Macro cells, indicating the potential role of RAR and AHR in terminal differentiation ( Figure 13 F).

[0088] Example 6 CNR2 is a common downstream target of FICZ- or ATRA-induced differentiation

[0089] JASPAR CORE database analysis (https: / / jaspar.genereg.net) showed that there were four AHR binding sites in the promoter region of RARɑ (consensus binding sequences ‘TGCCTG, CGAGTG, CCCGTG’, relative profile score > 80%) from a collection of experimentally defined transcription factor binding sites in published eukaryotes ( Figure 6 A) There was one RARɑ binding site in the promoter region of AHR (consensus binding sequence “AGGTGAAGTTTAAGCTCA”, relative profile score > 80%) ( Figure 6 B), indicating that AHR and RARɑ may activate each other. In addition, there was no RARɑ binding site in the promoter region of CNR2 ( Figure 6 C), but there were two AHR binding sites (binding sequence “GGCGTG”, relative profile score > 90%) ( Figure 6 D), indicating that AHR may directly activate CNR2, while RARɑ does not directly activate CNR2. The binding of AHR to the CNR2 promoter region (sites 1 and 2) was further confirmed by ChIP-PCR ( Figure 6 E). In addition, knockdown of AHR significantly inhibited ATRA-induced CNR2 expression, confirming that CNR2 is a downstream target of AHR ( Figure 6 F).

[0090] Next, the anti-leukemic effect of CNR2 activation in AML was investigated using the CNR2 agonist GW842166X ( Figure 14 A). As expected, the proportion of intracellular CD11b- or CD14-positive cells increased, indicating that GW842166X induced cell differentiation of U937 cells ( Figure 6 G and Figure 14 B), and the inventors also measured multiple AML cell lines, and the results showed that GW842166X could induce differentiation of multiple AML cells ( Figure 14 C). To confirm that the differentiation-inducing effect of GW842166X was mediated by CNR2, the inventors first overexpressed CNR2 in U937 cells. The successful overexpression of CNR2 was confirmed by Western blot ( Figure 6 H). According to the inventors' hypothesis, overexpression of CNR2 had the same pro-differentiation effect as GW842166X ( Figure 6I). Then, to further confirm that the effect of GW842166X is mediated by CNR2, the inventors knocked down CNR2 using shRNA in U937 cells. The successful knockdown of CNR2 was confirmed by Western blotting, and the expression of CNR2 was inhibited using the specific sequence shRNA-CNR2-3 for subsequent experiments ( Figure 6 J). Knockdown of CNR2 attenuated the anti-leukemic effects of ATRA and FICZ ( Figure 6 K), confirming that CNR2 is a common downstream target gene for ATRA- and FICZ-induced differentiation.

[0091] In summary, the inventors' findings support that the ATRA-induced differentiation pathway interacts with AHR, CNR2 is a common downstream of ATRA- or FICZ-induced differentiation, AHR directly activates CNR2, and RAR activates CNR2 through AHR.

[0092] Example 7 CNR2 promotes cell differentiation, and the rescue ability of FICZ and ATRA functions through CNR2

[0093] The rescue effect of cnr2 on zebrafish was further described. The expression of cnr2 was analyzed using qPCR technology

[0094] Table 9 Primers for cnr2 amplification

[0095] Forward 5′-CCAGAATAAACCCACGGGACA-3’(SEQ ID NO:15) Reverse 5′-ACCAAACAGGCCATGAAACAA-3’(SEQ ID NO:16)

[0096] in blood cells at different embryonic stages. The expression of cnr2 was higher in blood cells sorted from Tg(mpx:GFP) or Tg(mpeg:mcherry) at 5 dpf than in cells sorted from Tg(drl:GFP) at 2 dpf, indicating that cnr2 is required for the development of blood cells into more mature cells ( Figure 7 A). To characterize the biological function of cnr2 in hematopoietic development, morpholinos were used to knockdown cnr2 in wild-type embryos, and WISH was used to analyze hematopoietic phenotypes. In WT embryos, silencing cnr2 did not affect the development of myeloid progenitors (cmyb), erythrocytes (hbbe1), and lymphocytes (rag1) at 5 dpf, but inhibited the expression of the neutrophil marker mpx and the macrophage marker mfap4 at 5 dpf ( Figure 7 B), indicating that cnr2 itself is involved in hematopoietic development.

[0097] In addition, the expression of cmyb, mpx, mfap4, and rag1 in wild-type embryos at 3 - 5 dpf treated with GW842166X (30 μM or 50 μM) did not change ( Figure 14D - F). Compared with untreated embryos, treatment with GW842166X (30 μM) significantly rescued the expression of mpx + neutrophils and mfap4 + macrophages at 3 dpf in Tg(drl:hoxa9) embryos ( Figure 7 C - D), supporting the ability of GW842166X to promote myeloid differentiation in zebrafish. Meanwhile, after knocking down cnr2 using morpholinos, Tg(drl:hoxa9) embryos were treated with GW842166X, and it was shown that the rescue effect of GW842166X was inhibited, indicating that GW842166X indeed induces differentiation through cnr2 activation ( Figure 14 H).

[0098] Next, it was investigated whether cnr2 affects the rescue ability of FICZ or ATRA for blocked differentiation in zebrafish. Compared with the control, the expression of cnr2 was upregulated in whole embryos treated with FICZ or ATRA, demonstrating that the blocked differentiation effect of FICZ or ATRA requires cnr2 ( Figure 14 G). Consistent with this, knocking down cnr2 weakened the rescue effects of FICZ, ATRA, or FICZ combined with ATRA in Tg(drl:hoxa9) embryos, supporting that cnr2 plays a crucial role in the differentiation pathways induced by FICZ or ATRA ( Figure 6 E - G). In summary, CNR2 promotes myeloid differentiation in AML cells and zebrafish, and CNR2 is a common downstream target gene for ATRA - or FICZ - induced differentiation.

[0099] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above - described exemplary embodiments, and without departing from the spirit or basic features of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non - restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention. Any reference signs in the claims should not be regarded as limiting the claimed claims.

[0100] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Use of an aryl hydrocarbon receptor (AHR) agonist in the preparation of a drug for treating hematological malignancies.

2. Use of an aryl hydrocarbon receptor (AHR) agonist combined with all-trans retinoic acid (ATRA) in the preparation of a drug for treating hematological malignancies.

3. Use of an aryl hydrocarbon receptor (AHR) agonist in the preparation of a drug for treating hematological malignancies in combination with all-trans retinoic acid (ATRA).

4. Use of all-trans retinoic acid (ATRA) in the preparation of a drug for treating hematological malignancies in combination with an aryl hydrocarbon receptor (AHR) agonist.

5. The application according to any one of claims 1-4, characterized in that, The aryl hydrocarbon receptor (AHR) agonist is selected from FICZ, L-Kynurenine, Tapinarof (WBI-1001), ITE, Indole-3-pyruvic acid, VAF347, β-Naphthoflavone.

6. The application according to any one of claims 2-5, characterized in that, The aryl hydrocarbon receptor (AHR) agonist and all-trans retinoic acid (ATRA) are administered simultaneously or sequentially.

7. Use of an agonist of the cannabinoid receptor (CNR2), a downstream target of the aryl hydrocarbon receptor (AHR), in the preparation of a drug for treating hematological malignancies.

8. The application according to any one of claims 1-7, characterized in that, The hematological malignancy is AML.

9. The application according to claim 7 or 8, characterized in that, The cannabinoid receptor (CNR2) is a common target of the aryl hydrocarbon receptor (AHR) agonist and all-trans retinoic acid (ATRA).

10. A pharmaceutical combination, characterized in that, Comprising an aryl hydrocarbon receptor (AHR) agonist and all-trans retinoic acid (ATRA).

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