Pharmaceutical composition for treating leukemia, and method for screening active ingredient thereof

Abstract

The present disclosure provides a leukemia therapeutic agent and a method for screening an active ingredient thereof. The leukemia therapeutic agent according to the present disclosure contains an IMPDH1 inhibitor as an active ingredient. The method for screening the active ingredient of the leukemia therapeutic agent comprises a step for selecting, from among a group of compounds to be tested, a compound that exhibits, with respect to leukemia cells, (I) IMPDH inhibitory effect and / or (II) at least one effect selected from the group consisting of (1) to (5): (1) IRBC induction effect, (2) IRBCC formation effect, (3) TP53 protein stabilization effect, (4) TIGAR induction effect, and (5) MYC transcriptional suppression effect.

Description

Pharmaceutical composition for treating leukemia and method for screening its active ingredient

[0001] The present disclosure relates to a pharmaceutical composition for treating leukemia, for example, a pharmaceutical composition for treating acute myeloid leukemia, and to a method for screening an active ingredient of a pharmaceutical composition for treating leukemia.

[0002] Leukemia is a cancerous disease of the bone marrow and blood. Leukemia can be broadly classified into four types: acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia. Of these, acute and chronic myeloid leukemia are hematological malignancies characterized by clonal proliferation of immature myeloblasts. The former, aggressive acute myeloid leukemia with rapid progression, is called "AML," while the latter, less aggressive chronic myeloid leukemia with slow progression, is called "CML." Hereinafter, acute myeloid leukemia may also be referred to as "AML" in this specification.

[0003] AML originates from self-renewing leukemic stem cells (LSCs), which drive AML proliferation in vivo. Therefore, eradicating LSCs is crucial for achieving a cure. In clinical practice, venetoclax, a B-cell lymphoma-2 (BCL-2) inhibitor, has revolutionized the treatment of AML. While BCL-2 is primarily associated with the regulation of apoptosis, recent studies have demonstrated that venetoclax plays a critical role in regulating mitochondrial function, particularly oxidative phosphorylation (OXPHOS). BCL-2 family proteins contribute to maintaining mitochondrial membrane integrity, which is essential for optimal OXPHOS efficiency. Meanwhile, LSCs are particularly dependent on OXPHOS for energy production. Venetoclax may act by disrupting BCL-2-mediated pathways, impairing the OXPHOS-dependent metabolic framework of LSCs.

[0004] The dramatic change in AML treatment brought about by the introduction of venetoclax, which targets OXPHOS and apoptosis, is consistent with recent basic research that has revealed the importance of metabolic processes, including OXPHOS, enhanced branched-chain amino acid metabolic activity, fatty acid metabolism, and increased glutamine dependency, as effective new therapeutic targets for AML. Importantly, these metabolic specificities are adapted to meet the high energy requirements characteristic of AML cells.

[0005] Among the anabolic processes in human cells, ribosome biogenesis is the most energy-consuming. Ribosome biogenesis is a complex and highly regulated intracellular process responsible for generating ribosomes, the cellular machinery essential for protein synthesis. This process begins in the nucleolus and is divided into several key steps, including ribosomal RNA (rRNA) synthesis and processing, ribosomal protein synthesis, transport to the cytoplasm, and ribosomal subunit assembly. Ribosome biogenesis is an energy-intensive process that is closely coupled with the overall metabolic and growth state of the cell, making it a critical mechanism for maintaining cellular homeostasis.

[0006] The impaired ribosome biogenesis checkpoint (IRBC) functions as a critical surveillance system to ensure the fidelity and efficiency of ribosome biogenesis. This checkpoint is activated in response to stresses or disruptions in ribosome biogenesis, such as a lack of ribosome assembly factors or impaired nucleotide or energy supply. Upon activation, the IRBC diverts newly synthesized 60S ribosomal protein precursor complexes containing L5 (RPL5) and L11 (RPL11) and 5S rRNA from assembling into 60S ribosomes. Instead, they bind to the E3 ubiquitin-protein ligase MDM2, forming the IRBC complex (IRBCC). The IRBCC inhibits MDM2-mediated ubiquitination and subsequent degradation of TP53, thereby activating the TP53 pathway. Once activated, TP53 induces cell cycle arrest or apoptosis. The former allows for intracellular correction of ribosome biogenesis defects, while the latter prevents the persistence of dysfunctional ribosomes by removing irreparable defects from the cell.

[0007] Considering the crucial role of IRBCs in maintaining cellular homeostasis through activation of the TP53 pathway, it is speculated that IRBCs function as an important barrier against carcinogenesis. Furthermore, solid tumors exhibit increased ribosome biogenesis, a necessary adaptation to meet metabolic and protein synthesis demands. Therefore, targeting this increased ribosome biogenesis and subsequently inducing activation of IRBCs may be a viable therapeutic strategy for these cancers.

[0008] However, the significance of ribosome biogenesis in human AML has not yet been fully elucidated.

[0009] Kikushige, Y. et al. Human acute leukemia uses branched-chain amino acid catabolism to maintain stemness through regulating PRC2 function. Blood Adv 7, 3592-3603, doi:10.1182 / bloodadvances.2022008242 (2023).Hedstrom L (2009) IMP dehydrogenase: structure, mechanism, and inhibition.Chem Rev 109: 2903 - 2928.Naffouje R, Grover P, Yu HY, Sendilnathan A, Wolfe K, Majd N, Smith EP, Takeuchi K, Senda T, Kofuji S et al (2019) Anti-tumor potential of IMPdehydrogenase inhibitors: A century-long story. Cancer 11: 1346.Xiaoxiao Liu et al., IMPDH inhibition activates TLR-VCAM1 pathway and suppresses the development of MLL-fusion leukemia: EMBO Molecular Medicine (2023) 15: e15631. Maiko Sezaki et al., Repurposing immunosuppressants for antileukemia therapy : EMBO Molecular Medicine (2023) 15: e17042.

[0010] An objective of the present disclosure is to provide a pharmaceutical composition for treating leukemia, for example, a pharmaceutical composition for treating acute myeloid leukemia, and a method for screening an active ingredient of the pharmaceutical composition for treating leukemia.

[0011] The present inventors have conducted extensive research to solve the above-mentioned problems and have found that in human patient AML cells, maintenance of ribosome biogenesis is highly dependent on the de novo guanine nucleotide biosynthetic pathway. They have also confirmed that inhibiting this pathway with an inosine monophosphate dehydrogenase 1 (IMPDH1) inhibitor leads to IRBC induction and IRBCC formation in AML cells, resulting in increased TP53 protein expression and TP53 protein stabilization, as well as increased TIGAR expression, while simultaneously suppressing MYC transcription in AML cells.

[0012] In particular, by using an IMPDH1 inhibitor to inhibit the de novo guanine nucleotide biosynthesis pathway, IC of venetoclax, a conventional AML treatment, was improved. 50 "This study demonstrated that venetoclax significantly reduced leukemia-related blood pressure (LEP) levels, reaching levels between 10 and 1,000 times lower than the initial values. Resistance to venetoclax has become a problem in clinical settings in recent years. Combination therapy with an IMPDH1 inhibitor and venetoclax may be able to overcome this issue."

[0013] The present disclosure was completed through further research based on the above findings, and includes the following embodiments.

[0014] (I) Pharmaceutical Compositions for Treating Leukemia (I-1) A pharmaceutical composition for treating leukemia containing an IMPDH1 inhibitor as an active ingredient. (I-2) The pharmaceutical composition for treating leukemia according to (I-1), wherein the IMPDH1 inhibitor is at least one selected from the group consisting of a compound having an inhibitory effect on IMPDH1, an anti-IMPDH1 antibody and an antigen-binding fragment thereof, an antisense oligonucleotide, an shRNA, and an siRNA. (I-3) The pharmaceutical composition for treating leukemia according to (I-1) or (I-2), wherein the leukemia is AML. (I-4) The pharmaceutical composition for treating leukemia according to any of (I-1) to (I-3), wherein the leukemia is resistant to leukemia treatment with a BCL-2 inhibitor. (I-5) The pharmaceutical composition for treating leukemia according to (I-4), wherein the BCL-2 inhibitor is venetoclax. (I-6) The pharmaceutical composition for treating leukemia according to any one of (I-1) to (I-5), which is used in combination with a BCL-2 inhibitor for the treatment of BCL-2 inhibitor-resistant leukemia. (I-7) The pharmaceutical composition for treating leukemia according to (I-6), wherein the BCL-2 inhibitor is venetoclax. (I-8) The pharmaceutical composition for treating leukemia according to any one of (I-1) to (I-7), wherein the IMPDH1 inhibitor has at least one effect on leukemia cells selected from the group consisting of (1) to (5): (1) IRBC induction, (2) IRBCC formation, (3) TP53 protein stabilization, (4) TIGAR induction, and (5) MYC transcription suppression. (I-9) The pharmaceutical composition for treating leukemia according to any one of (I-1) to (I-7), wherein the IMPDH1 inhibitor is an agent used for at least one application selected from the group consisting of (a) to (e) against leukemia cells: (a) an IRBC inducer, (b) an IRBCC formation agent, (c) a TP53 protein stabilizer, (d) a TIGAR inducer, or (e) a MYC transcription suppressor.

[0015] (II) Method for screening active ingredients of pharmaceutical compositions for treating leukemia (II-1) A method for screening active ingredients of pharmaceutical compositions for treating leukemia, comprising the step of selecting, from a group of test compounds, compounds having, on leukemia cells, (I) an IMPDH inhibitory effect, and / or (II) at least one effect selected from the group consisting of the following effects (1) to (5): (1) an IRBC-inducing effect, (2) an IRBCC-forming effect, (3) a TP53 protein stabilizing effect, (4) a TIGAR-inducing effect, or (5) a MYC transcription suppressing effect. (II-2) The screening method according to (II-1), wherein the leukemia is AML. (II-3) The screening method according to (II-1) or (II-2), wherein the leukemia is resistant to leukemia treatment with a BCL-2 inhibitor. (II-4) The screening method according to (II-3), wherein the BCL-2 inhibitor is venetoclax.

[0016] (III) Methods for Treating Leukemia (III-1) A method for treating leukemia in a leukemia patient, comprising administering a therapeutically effective amount of an IMPDH1 inhibitor to the patient. (III-2) The method for treating leukemia according to (III-1), wherein the IMPDH1 inhibitor is at least one selected from the group consisting of compounds having an inhibitory effect on IMPDH1, anti-IMPDH1 antibodies and antigen-binding fragments thereof, antisense oligonucleotides, shRNA, and siRNA. (III-3) The method for treating leukemia according to (III-1) or (III-2), wherein the leukemia is AML. (III-4) The method for treating leukemia according to any one of (III-1) to (III-3), wherein the AML is AML resistant to leukemia treatment with a BCL-2 inhibitor. (III-5) The method for treating leukemia according to (III-4), wherein the BCL-2 inhibitor is venetoclax.

[0017] (IV) Uses of IMPDH1 Inhibitors (IV-1) An IMPDH1 inhibitor used to treat leukemia. (IV-2) The IMPDH1 inhibitor according to (IV-1), wherein the IMPDH1 inhibitor is at least one selected from the group consisting of compounds having an inhibitory effect on IMPDH1, anti-IMPDH1 antibodies and antigen-binding fragments thereof, antisense oligonucleotides, shRNA, and siRNA. (IV-3) The IMPDH1 inhibitor according to (IV-1) or (IV-2), wherein the leukemia is AML. (IV-4) The IMPDH1 inhibitor according to any of (IV-1) to (IV-3), wherein the AML is AML resistant to leukemia treatment with a BCL-2 inhibitor. (IV-5) The IMPDH1 inhibitor according to (IV-4), wherein the BCL-2 inhibitor is venetoclax.

[0018] (V) Use of an IMPDH1 inhibitor for the manufacture of a pharmaceutical composition for the treatment of leukemia. (V-1) Use of an IMPDH1 inhibitor for the manufacture of a pharmaceutical composition for the treatment of leukemia. (V-2) The use according to (V-1), wherein the IMPDH1 inhibitor is at least one selected from the group consisting of compounds having an inhibitory effect on IMPDH1, anti-IMPDH1 antibodies and antigen-binding fragments thereof, antisense oligonucleotides, shRNA, and siRNA. (V-3) The use according to (V-1) or (V-2), wherein the leukemia is AML. (V-4) The use according to any of (V-1) to (V-3), wherein the AML is AML resistant to leukemia treatment with a BCL-2 inhibitor. (V-5) The use according to (V-4), wherein the BCL-2 inhibitor is venetoclax.

[0019] According to the present disclosure, it is possible to provide a therapeutic agent for leukemia, for example, a therapeutic agent for AML. Preferably, it is possible to provide a therapeutic agent effective against AML resistant to venetoclax, a BCL-2 inhibitor. Furthermore, it is possible to provide a method for selecting and obtaining an active ingredient for a therapeutic agent for leukemia. Preferably, it is possible to provide a method for selecting and obtaining an ingredient effective in producing a therapeutic agent for AML resistant to venetoclax.

[0020] Results of Test Example 1 (1-1). In the figure, "Sen" and "VEN-sen#12" refer to VEN-sensitive AML cells, and "Res" and "VEN-res#1" refer to VEN-resistant AML cells. This shows that ATP production is highly dependent on glycolysis. Results of Test Example 1 (1-1). VEN-resistant AML cells (VEN-resistant AML) show that the glycolytic ATP production rate is significantly increased by the inhibition of oxidative phosphorylation (OXPHOS) by VEN. This shows that intracellular metabolites are significantly different between VEN-sensitive and VEN-resistant AML cells. Results of Test Example 1 (1-2). This shows that VEN-resistant AML cells (Res) contain higher amounts of glycolysis pathway-related metabolites (3-phosphoglycerate, fructose 1,6-bisphosphate, and lactate) than VEN-sensitive AML cells (Sen). Results of Test Example 1 (1-2). These results show that VEN-resistant AML cells (Res) have significantly higher levels of intermediate metabolites in the pentose phosphate pathway (6-phosphogluconate, glyceraldehyde 3-phosphate) than VEN-sensitive AML cells (Sen). Results from Test Example 1 (1-2). These results show that the intracellular content of intermediate metabolites in the guanine nucleotide biosynthetic pathway (XMP, GMP, GDP) is significantly elevated in VEN-resistant AML cells (Res). Schematic diagram of in vivo metabolic pathways and nucleotide biosynthetic pathways. Results from Test Example 1 (1-3). These results show that expression of genes encoding rate-limiting enzymes in the de novo pathway (inosine monophosphate dehydrogenase: IMPDH1 and IMPDH2) is higher in VEN-resistant AML cells (Res) than in VEN-sensitive AML cells (Sen). Results from Test Example 1 (1-3). These results show that there is no difference between the two AML cell types (Sen and Res) in the expression of genes encoding salvage pathway-related enzymes (HPRT, PNP). Results of Test Example 1 (1-3). The VEN-resistant MOLM-13 cell line (■) (left panel) and the VEN-resistant THP-1 cell line (■) (right panel) showed a significantly higher IC of VEN compared to their parent cell lines (●). 50The values ​​are significantly elevated, indicating significant increases in the expression of IMPDH1 and IMPDH2. Results of Test Example 2 (1). The figures show the percentage of apoptotic cells when AML cell lines (MOLM13, MV4-11, THP-1) were treated with DMSO, VEN, MPA, VEN + MPA, or VEN + MPA + GMP. Inhibition of the de novo pathway enhances VEN toxicity (effect) on AML cell lines, but this enhancing effect is canceled by GMP. Results of Test Example 2 (1). The figures show the percentage of apoptotic cells when AML cell lines (MOLM13, MV4-11, THP-1) were treated with forodesine, VEN, or VEN + forodesine. Inhibition of the salvage pathway does not enhance VEN toxicity. Results of Test Example 2 (2). IC of VEN alone (●) and VEN + MPA (■) against AML cell lines (MOLM13, MV4-11, THP-1, HNT34, KASUMI-1, KASUMI-3, SKM1, HL-60) 50 The results of Test Example 2 (2) show the IC values ​​of VEN alone (●) and VEN + MPA (■) against VEN-resistant MOLM13 cell line and VEN-resistant THP-1 cell line. 50 The results of Test Example 2 (2) show that knockdown of IMPDH1 or IMPDH2 by sh-RNA ("sh" in the figure) reduced the IC value of VEN in MOLM13 cell line and THP-1 cell line. 50 All values ​​are significantly reduced. Results of Test Example 2 (3). VEN-sensitive AML cells (n = 8) and VEN-resistant AML cells (n = 8) were treated with DMSO, MPA alone, VEN alone, and VEN + MPA in combination, and the results are shown below for a comparison of the proportion of apoptotic cells. Results of Test Example 2 (3). IC values ​​for VEN-sensitive AML cells (n = 8) and VEN-resistant AML cells (n = 9) when treated with VEN alone and VEN + MPA in combination. 501 shows the results of comparing values ​​(relative ratio). Results of Test Example 2 (3). Bliss scores are shown for AML cell lines (MOLM13, MV4-11, THP-1, KASUMI-1, KASUMI-3, HL-60) after combined treatment with VEN and MPA. Results of Test Example 2 (3). Bliss scores are shown for AML cells after combined treatment with VEN and MPA. Results of Test Example 3 (1). This shows that combined treatment with VEN and MPA induces the expression of TP53 target genes in MOLM13 and MV4-11 cell lines, compared with treatment with VEN alone. Results of Test Example 3 (1). This shows that MPA does not affect the transcription of the TP53 gene in AML cells. Results of Test Example 3 (1). This shows that MPA significantly increases TP53 protein in a time-dependent manner in VEN-sensitive and VEN-resistant AML cells. Schematic diagram showing that, among downstream molecules induced by TP53, TIGAR plays an important role in suppressing glycolytic activity. Results of Test Example 3(1). This shows that treatment with MPA does not increase the transcriptional activity of TIGAR in TP53 gene-deficient MOLM13 cell lines (MOLM13_TP53KO). This suggests that MPA inhibits glycolysis by inducing TP53 and TIGAR. Results of Test Example 3(1). This shows that administration of GMP, GDP, and GTP to MOLM13 cell lines inhibits the induction of TP53 and TIGAR proteins, but administration of IMP does not. Results of Test Example 3(1). This shows that MPA significantly inhibits ECAR in AML cell lines and AML cells, but does not affect OCR. Results of Test Example 3(1). This shows that MPA significantly inhibits ECAR in AML cell lines and AML cells, but does not affect OCR. Results of Test Example 3(1). MPA inhibits normal human CD34 + This shows that MPA does not affect the glycolytic activity of HSPCs. +This shows that there is no effect on the glycolytic activity of HSPCs. Seven gene sets show identical changes upon addition of VEN and MPA to both the MOLM13 cell line and MV4-11 cells. Six of these (1-5 and 7 from the top) are related to ribosome biogenesis. Diagram of ribosome biogenesis. Results of Test Example 4. In the MOLM13 cell line and THP-1 cell line, MPA significantly suppressed pre-rRNA expression, but the addition of GMP relieved this suppression. Results of Test Example 4. In AML cells, MPA significantly suppressed pre-rRNA expression, but the addition of GMP relieved this suppression. Results of Test Example 4. Pre-rRNA expression levels in VEN-resistant MOLM13 cell line, VEN-resistant THP-1 cell line, and their parent cell lines are shown (left column). Pre-rRNA expression levels in VEN-sensitive AML cells and VEN-resistant AML cells are also shown (right column). Explanation of IRBC and IRBCC in AML. Under steady-state conditions, RPL5 and RPL11 are utilized for normal ribosome biogenesis. AML cells, on the other hand, are sensitive to intracellular guanine nucleotide depletion. Upon depletion, IRBC is activated and recruits MDM2 to RPL5 and RPL11. As a result, TP53 degradation due to MDM2 functional inactivation is suppressed, resulting in stabilization and accumulation of TP53 protein. Figure 1 shows the results of an immunoprecipitation (IP) assay using an anti-MDM2 monoclonal antibody in Test Example 4. In the MOLM13 cell line, RPL5 and RPL11 co-immunoprecipitate with MDM2 after MPA exposure. Figure 2 shows the results of an immunoprecipitation (IP) assay using an anti-MDM2 monoclonal antibody in Test Example 4. In AML cells, RPL5 and RPL11 co-immunoprecipitate with MDM2 after MPA exposure. Figure 3 shows the results of Test Example 5(1). Figure 4 shows the results of Test Example 5(2). This shows the results of global transcriptome analysis using the MOLM13_TP53KO cell line and the MOLM13_CAS9 cell line. This shows the volcano plot results from Test Example 5(2). This is a conceptual diagram showing that guanine nucleotides (GDP, GTP) are required for activation of the RAS / MAPK pathway and play an important role in the transcriptional induction of MYC. This is the result of Test Example 5(2).This shows that MPA treatment inhibits de novo guanine nucleotide biosynthesis, thereby suppressing phosphorylated MEK1 / 2 in AML cell lines (MOLM13, THP1), regardless of the presence or absence of TP53 mutation. Results from Test Example 5(2). This shows that MPA treatment inhibits phosphorylated MEK1 / 2 in AML cells, regardless of the presence or absence of TP53 mutation. This shows the results of Phosflow analysis in Test Example 5(2). Results from Test Example 5(2). This shows that MYC transcriptional activity is significantly reduced in AML cell lines, regardless of the TP53 mutation status. Results from Test Example 5(2). This shows that MYC transcriptional activity is significantly reduced in AML cells, regardless of the TP53 mutation status. Results from Test Example 6. ROS. high AML cells and ROS low The results of comparing the expression levels of IMPDH1 and IMPDH2 in AML cells and AML cells are shown in Table 1. high AML cells and ROS low 1 shows the results of comparing pre-rRNA levels between AML cells and VEN. Results of Test Example 6: This shows that MPA significantly reduced the number of blast colony-forming cells in AML cells, but VEN did not. Results of Test Example 6: This shows that knockdown of IMPDH1 or IMPDH2 via sh-RNA significantly reduced the number of blast colony-forming cells in AML cells. Results of Test Example 6: This shows that MPA significantly suppresses the estimated frequency of blast colony-forming AML cells by limiting dilution assay (in vitro). Results of Test Example 6: This shows that MPA significantly suppresses the estimated frequency of blast colony-forming AML cells by limiting dilution assay (in vitro). An outline of the xenotransplantation experiment performed in Test Example 7(1) is shown. The xenotransplantation experiment in Test Example 7(1) shows that single-agent administration of MMF provides a significant anti-leukemia effect even in VEN-resistant AML cases (UPN2 and UPN4). Furthermore, the combination of VEN and MMF significantly suppressed the growth of AML cells (hCD45) compared to single-agent administration of VEN or MMF. +The results show that the proportion of AML cells) was significantly decreased. Results of the xenograft experiment in Test Example 7(1). The results show that pre-rRNA synthesis was significantly inhibited by monotherapy with MMF. Results of Test Example 7(1). The results show that TP53 in AML cells was stabilized by monotherapy with MMF. Results of the serial transplantation experiment in Test Example 7(2) are shown. Results of evaluating the effect of combination of MMF+VEN on normal human hematopoiesis in Test Example 7(3) are shown. Test results of evaluating the effect of MPA or VEN on blast-colony formation conducted in Test Example 8(1) are shown. Test results of evaluating the effect of shRNA (shIMPDH1-1, shIMPDH1-2) on blast-colony formation conducted in Test Example 8(1) are shown. An overview of the xenograft experiment conducted in Test Example 8(2) is shown. Results of the xenograft experiment in Test Example 8(2) are shown. Treatment with shRNA (shIMPDH1-1, shIMPDH1-2) shows that the proportion of AML cells (hCD45 + AML cells) was significantly decreased. Results of Test Example 9(1) are shown. Results of Test Example 9(2) are shown.

[0021] (1) Definitions Unless otherwise specified, chemical terms, scientific terms, technical terms, and techniques (including experimental methods) used in this specification are generally understood by those skilled in the art and have the same meaning as these.

[0022] In the present specification and drawings, abbreviations may be used for the convenience of explanation, and unless otherwise specified, each abbreviation means the following: AML: acute myeloid leukemia AML cell: AML cell from a human patient AML cell line: AML cell line established from AML cell from a human patient. Unlike human patient AML cells, these immortalized AML cells can be cultured and passaged in vitro in liquid medium. VEN: Venetoclax IMPDH: Inosine monophosphate dehydrogenase IMPDH1: Inosine monophosphate dehydrogenase 1 IMPDH2: Inosine monophosphate dehydrogenase 2 BCL-2: Derived from B-cell / CLL lymphoma 2, it is the second protein described as being involved in chromosomal translocation between chromosomes 14 and 18 in follicular lymphoma IRBC: Impaired ribosome checkpoint IRBCC: Impaired ribosome checkpoint complex TP53: Tumor suppressor gene TP53 protein: TP53 gene product p53 TIGAR: TP-53-induced Glycolysis and Apoptosis Regulator MYC: A gene belonging to the MYC family, including c-Myc, N-Myc, and M-Myc. Preferably, c-Myc is used. shRNA: small hairpin RNA, siRNA: small interfering RNA

[0023] (I) Pharmaceutical Composition for the Treatment of Leukemia This disclosure relates to a pharmaceutical composition for the treatment of leukemia containing an IMPDH1 inhibitor as an active ingredient. IMPDH is the rate-limiting enzyme in the de novo guanine nucleotide synthesis pathway, responsible for the conversion of IMP to XMP (Figure 1G; see Non-Patent Documents 2 and 3). Guanine nucleotides play important roles in diverse signal transduction pathways and biological processes. Guanine nucleotides are biosynthesized in cells through the salvage pathway and the de novo synthesis pathway. The salvage pathway regenerates guanine nucleotides using preassembled nucleobases (guanine, hypoxanthine) or nucleosides (guanosine). The key enzyme in the salvage pathway is HRPT1, which catalyzes the reaction of guanine or hypoxanthine with PRPP to form GMP or IMP, respectively. The salvage GTP biosynthesis pathway is particularly important in the brain, but is insufficient to meet the needs of most cell divisions. Rapidly proliferating cells, such as lymphocytes and cancer cells, rely heavily on the de novo synthesis pathway to maintain their growth. Two isoforms of IMPDH (IMPDH1 and IMPDH2) have been identified. They share 84% amino acid sequence identity and have indistinguishable enzymatic activity. IMPDH1 is generally expressed at low levels except in certain tissues, whereas IMPDH2 is the predominant isoform in most proliferating cells. Elevated IMPDH2 activity has been observed in various cancer cells, making it a promising therapeutic target (Non-Patent Documents 4 and 5). IMPDH inhibitors have been used clinically for decades as safe and effective immune-stimulating agents (Non-Patent Document 3). The first IMPDH inhibitor, mycophenolic acid (MPA), was identified in fungi over 100 years ago. Subsequent studies have revealed its IMPDH inhibitory function and immunosuppressive properties. MPA, along with its orally bioavailable prodrug, mycophenolate mofetil (MMF), have been successfully tested in multiple clinical trials.

[0024] The pharmaceutical composition for treating leukemia of the present disclosure may act specifically on IMPDH1 and selectively inhibit IMPDH1, or may act on both IMPDH1 and IMPDH2 and inhibit both. As described above, since IMPDH2 is expressed in many proliferating cells, an IMPDH1 inhibitor that selectively acts on IMPDH1 is preferred. Note that, as used herein, "selective" includes, but is not limited to, inhibiting only IMPDH1 activity and not IMPDH2 activity at all (100% selectivity), and also includes a stronger inhibitory activity against IMPDH1 than against IMPDH2. For example, this includes cases where the inhibitory activity against IMPDH1 is at least 2-fold, at least 5-fold, or at least 10-fold stronger than the inhibitory activity against IMPDH2.

[0025] In the present disclosure, the term "inhibition" encompasses both the inhibition of IMPDH1 activity itself and the inhibition of IMPDH1 gene expression or IMPDH1 protein production. It also encompasses the complete or partial elimination of IMPDH1 activity by degrading IMPDH1. In other words, the "IMPDH1 inhibitor" covered by the present disclosure is any inhibitor that inhibits IMPDH1 activity, regardless of the cause. Furthermore, "inhibition" encompasses not only the complete elimination of IMPDH1 activity or expression, but also partial elimination (suppression, attenuation, or reduction), i.e., downregulation.

[0026] IMPDH1 inhibitors of the present disclosure include compounds that inhibit IMPDH1. In the present disclosure, the term "compound" is understood in the broadest sense, and refers, for example, to a substance composed of two or more elements, in which atoms of the elements are bonded together and present in a certain ratio. Therefore, compounds include small molecule chemical compounds, large molecule chemical compounds, naturally occurring compounds, nucleic acids (oligonucleotides), peptides, proteins, antibodies and fragments thereof, and mixtures thereof. Compounds that inhibit IMPDH1 activity include compounds, anti-IMPDH1 antibodies and their antigen-binding fragments, peptides, antisense oligonucleotides, shRNA, and siRNA. These compounds may be any compounds that inhibit IMPDH1 activity, and include not only compounds previously known as IMPDH1 inhibitors, but also compounds that potentially inhibit IMPDH1 activity. These compounds also include compounds that directly degrade IMPDH1 and compounds that induce IMPDH1 degradation. The latter compounds include compounds called PROTACs (Proteolysis Targeting Chimeras), which utilize the ubiquitin-proteasome system to induce the degradation of HMPDH1 in cells.

[0027] The IMPDH1 inhibitory activity of a compound that acts as an IMPDH1 inhibitor can be evaluated using an in vitro assay system. Examples of such in vitro assay systems include a method for measuring changes in IMPDH1 activity in the presence of a target compound. Such changes in IMPDH1 activity can be measured using the reaction product produced by the enzymatic reaction of IMPDH1 with a substrate as an indicator. The following enzyme activity assays are examples of methods for directly measuring IMPDH1 activity to evaluate the inhibitory effect of a target compound on IMPDH1: 1) Color assay: NADH produced by the enzymatic reaction of IMPDH1 is detected as a change in color (e.g., measuring the absorbance of NADH at 340 nm). 2) Fluorescence assay: NADH emits fluorescence. Therefore, NADH produced by the enzymatic reaction of IMPDH1 is detected as a change in fluorescence intensity. 3) A method for directly measuring the amount of reaction product using a mass spectrometer. A method for measuring the amount of XMP produced from the substrate IMP by the enzymatic reaction of IMPDH1 using a mass spectrometer.

[0028] Whether a target compound inhibits IMPDH1 expression can be evaluated, without limitation, by, for example, Western blotting using an anti-IMPDH1 antibody or quantifying IMPDH1 protein by intracellular staining. Regulation of expression at the transcriptional level can be evaluated by quantifying IMPDH1 gene mRNA using quantitative RT-PCR or RNA sequencing.

[0029] As used herein, the term "RNA," or "ribonucleic acid," refers to an organic molecule consisting of a long chain of nucleotides in which the sugar is ribose (or a variant thereof) and the bases are adenine, cytosine, guanine, and uracil. In this disclosure, the terms "siRNA" and "shRNA" refer to a class of double-stranded RNA molecules that function using the concept of RNA interference (RNAi). The term "RNAi" refers to RNA interference, a process in which RNA molecules inhibit gene function. This interference is based on the ability of double-stranded RNA to interfere with or suppress the expression of genes with corresponding base sequences. For example, two types of small ribonucleic acid (RNA) molecules—microRNA (miRNA) and small interfering RNA (siRNA)—are important for RNA interference. RNA is the direct product of genes, and these small RNAs (siRNA, shRNA) can bind to other specific messenger RNA (mRNA) molecules, thereby enhancing or attenuating their activity, for example, by preventing the mRNA from producing proteins.

[0030] In one example, the siRNA sequence is 15-150 base pairs, 60-100 base pairs, 70-120 base pairs, about 60 base pairs, about 65 base pairs, about 70 base pairs, about 75 base pairs, about 80 base pairs, about 85 base pairs, about 90 base pairs, about 95 base pairs, about 100 base pairs, about 105 base pairs, or about 110 base pairs in length. In another example, the siRNA sequence is at least 15 base pairs, at least 20 base pairs, at least 25 base pairs, at least 30 base pairs, at least 35 base pairs, at least 40 base pairs, at least 45 base pairs, or at least 50 base pairs in length.

[0031] In another example, the shRNA sequence comprises a stem that is 15-30 base pairs, 19-29 base pairs, 15-20 base pairs, 20-30 base pairs, about 18 base pairs, about 19 base pairs, about 20 base pairs, about 21 base pairs, about 22 base pairs, about 23 base pairs, about 24 base pairs, about 25 base pairs, about 26 base pairs, about 27 base pairs, about 28 base pairs, about 29 base pairs, or about 30 base pairs in length.

[0032] In yet another example, the disclosed nucleic acid sequence comprises one of the sequences of SEQ ID NO: 16 or SEQ ID NO: 17. For example, the data shown in Figure 2E disclosed herein shows that knocking down IMPDH1 with shRNA reduces the IC50 of VEN, a BCL-2 inhibitor, in AML cell lines (MOLM13 cell line, THP-1 cell line). 50 The data shown in Figure 6E indicate that knockdown of IMPDH1 by shRNA significantly reduces the number of blast colony-forming cells in AML cells derived from human patients. These results suggest that inhibition of IMPDH1 expression is effective in treating leukemia, particularly AML, and may also be effective in treating BCL-2 inhibitor-refractory AML (BCL-2 inhibitor-resistant AML).

[0033] Leukemias covered by the present disclosure include acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia, with AML being preferred. BCL-2 inhibitor-refractory AML (BCL-2 inhibitor-resistant AML) is more preferred. In the present disclosure, the term "disease" includes leukemia, preferably AML. AML also includes relapsed AML and BCL-2 inhibitor-refractory (BCL-2 inhibitor-resistant) AML. In the present disclosure, the term "symptoms" includes clinical symptoms associated with the disease.

[0034] BCL-2 inhibitors are chemical agents (chemotherapeutics) known to be effective in treating leukemia, including venetoclax (VEN), whose chemical name is 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(3-nitro-4-{[(oxan-4-yl)methyl]amino}phenyl)sulfonyl]-2-[(1H-pyrrolo[2,3-b]pyridin-5-yl)oxy]benzamide and is also known as ABT-199 or GDC-0199.

[0035] As used herein, "BCL-2 inhibitor-refractory AML" or "BCL-2 inhibitor-resistant AML" refers to cases of AML in which treatment with a BCL-2 inhibitor at doses typically used in the treatment of AML is unable to prevent or inhibit the growth of AML cancer cells, and BCL-2 inhibitors are not the first choice for treating AML.

[0036] As used herein, "treatment" refers to the eradication or amelioration of a disease or disorder or one or more symptoms associated with the disease or disorder. In certain embodiments, these terms include prevention of a disease or disorder and inhibiting (minimizing) the development or worsening of a disease or disorder.

[0037] In the present disclosure, "effective amount" or "therapeutically effective amount" refers to the amount of an active agent (active ingredient) required to alleviate the symptoms of a disease compared to an untreated subject. The effective amount of an active agent for therapeutically treating a disease may vary depending on factors such as the method of administration, the age, weight, and health condition of the subject, but the appropriate amount and dosage regimen can be determined by the attending physician or specialist (or veterinarian if the subject is not human).

[0038] In the present disclosure, the term "subject" or "patient" includes humans (including children under 15 years of age and adults 15 years of age or older), non-human primates (e.g., cynomolgus monkeys, rhesus monkeys, etc.), commercially relevant mammals (e.g., pigs, dogs, rabbits, mice, rats, sheep, cattle, horses, etc.), and commercially relevant birds (e.g., chickens, ducks, geese, quail, turkeys, etc.). Humans are preferred. Patients targeted by the present disclosure include patients suffering from leukemia and patients with symptoms or conditions caused by leukemia. Leukemia includes AML. These patients also include patients refractory (resistant) to BCL-2 inhibitors. These patients also include BCL-2 inhibitor-refractory (resistant) patients with reduced expression of TP53 protein. This result is based on a study showing that VEN-refractory AML patients have reduced expression of TP53 protein compared to VEN-sensitive AML patients (see Test Example 19, Figure 9B). Furthermore, such patients also include those who are found to have a tendency toward the aforementioned diseases or pathological conditions and who are at risk of further progression of the pathological conditions.

[0039] The administration route (administration method) of the pharmaceutical composition for treating leukemia of the present disclosure (hereinafter also referred to simply as "the pharmaceutical composition") is not particularly limited, and examples include oral administration; and parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration, transmucosal administration, transdermal administration, and rectal administration. Oral administration and intravenous administration are preferred. The pharmaceutical composition may contain, in addition to the IMPDH1 inhibitor, pharmaceutically acceptable carriers and / or additives depending on the administration route (administration method). The form and preparation method of the pharmaceutical composition can be conventional. For example, when the pharmaceutical composition is in an oral dosage form, examples include, but are not limited to, powders, granules, capsules, pills, tablets, chewable tablets, sublingual tablets, pucker tablets, lozenges, suspensions, emulsions, and syrups, and can be appropriately selected from among these. In addition, these formulations can be modified to have sustained release, stabilization, easy disintegration, difficult disintegration, enteric coating, easy absorption, etc. Known drug delivery system (DDS) technologies can be used for each of the above dosage forms. The DDS formulations referred to in this specification are formulations that are optimally formulated in consideration of the route of administration, bioavailability, side effects, etc., such as sustained-release formulations, topical formulations (troches, buccal tablets, sublingual tablets, etc.), controlled-release formulations, enteric-coated formulations, and gastric-soluble formulations.

[0040] In the present disclosure, the pharmaceutical composition is in a form in which an IMPDH1 inhibitor is administered alone or in combination with at least one other anti-tumor agent, either simultaneously or separately. In the present disclosure, the terms "combined," "used in combination," "administered in combination," and "cooperative administration" refer to any form of administration in which two or more different therapeutic compounds are administered such that a first therapeutic compound is still effective in the body while a second compound is administered. This form includes, for example, cases in which two compounds are effective in a patient simultaneously and exhibit a synergistic effect. For example, different therapeutic compounds can be administered simultaneously or sequentially, either as the same compound (formulation) or as separate compounds (formulations). In certain embodiments, different therapeutic compounds can be administered within 1 hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 1 week of each other.

[0041] In the present disclosure, compounds (or formulations) administered in combination with or in concert with an IMPDH1 inhibitor include compounds (formulations) that can reinforce or enhance the therapeutic effect of an IMPDH1 inhibitor on leukemia. Compounds (or formulations) administered in combination with or in concert with an IMPDH1 inhibitor also include leukemia therapeutic agents that, when administered alone, have low therapeutic efficacy against leukemia. Compounds (formulations) administered in combination with an IMPDH1 inhibitor include those commonly used as anticancer drugs. For example, they can be appropriately selected from alkylating agents, antimetabolites, antitumor antibiotics, microvascular inhibitors, hormones or hormone analogs, platinum compounds, topoisomerase inhibitors, cytokines, hormone therapy agents, radioimmunotherapy agents, molecularly targeted drugs (including antibody drugs and ICIs), nonspecific immunostimulants, and other antitumor agents. Although not limited, BCL-2 inhibitors are preferred, and VEN is more preferred.

[0042] As described in the experimental examples below, research by the present inventors has revealed that enhanced ribosome biosynthesis activity due to guanine nucleotide biosynthesis is a novel and specific molecular mechanism involved in the pathogenesis of AML. This study confirmed that only inhibition of the de novo pathway exerts a potent anti-AML effect. In other words, the de novo guanine nucleotide synthesis system is a potent and specific driver of AML. Therefore, inhibiting guanine nucleotide biosynthesis in the de novo pathway, i.e., targeting IMPDH1 or IMPDH1 and IMPDH2, is a promising therapeutic approach for AML.

[0043] IRBCs are also important mechanisms in normal cell structure, maintaining cellular homeostasis by monitoring the integrity of ribosome biogenesis and the dynamics of intracellular metabolic processes. Elimination of cells with defective ribosomes through TP53-dependent cell cycle arrest and IRBC-mediated apoptosis induction could be an important anti-tumor mechanism. Because IRBCs exert potent anti-AML effects, induction of IRBCs represents a promising therapeutic approach for AML. While triggers for IRBC activation are recognized to include conditions such as energy depletion, ribosome misalignment, and nucleotide depletion, this study demonstrated that IRBCs are strongly induced by guanine nucleotide depletion in human AML cells. As mentioned above, guanine nucleotide biosynthesis in AML cells relies on the de novo pathway. Therefore, strategies for inducing IRBCs in AML cells include targeting IMPDH1 or IMPDH1 and IMPDH2.

[0044] Furthermore, as shown in the experimental examples described below, research by the present inventors has confirmed that the induction of IRBCs and the formation of IRBCCs stabilize TP53 protein and suppress MYC transcription in human AML cells, thereby exerting anti-AML effects. This anti-AML effect is enhanced when used in combination with VEN. In AML cells, the induction of IRBCs stabilizes TP53 protein and suppresses MYC transcription, and suppresses OXPHOS-independent energy production through multiple molecular mechanisms, including glycolysis, demonstrating a synergistic effect with VEN.

[0045] Among the downstream molecules induced by TP53, TIGAR plays an important role in suppressing glycolytic activity (Figure 3D). As shown in the experimental example below, guanine nucleotide depletion induces TIGAR via stabilization of TP53, and TIGAR induction is thought to suppress the glycolytic pathway, on which AML cells (especially VEN-resistant AML cells) are highly dependent for energy production. This suggests that TIGAR induction in AML cells, particularly VEN-resistant AML cells, is an effective method for exerting anti-AML effects.

[0046] In light of the above, in the present disclosure, the IMPDH1 inhibitor can have, in addition to an IMPDH1 inhibitory effect on leukemia cells, at least one effect selected from the group consisting of (1) to (5). Preferably, the inhibitor has two or more, three or more, or four or more of these effects, and particularly preferably has five of these effects: (1) IRBC induction, (2) IRBCC formation, (3) TP53 protein stabilization, (4) TIGAR induction, and (5) MYC transcription suppression.

[0047] (1) IRBC-inducing effect IRBC is a checkpoint mechanism induced in vivo by a decrease in ribosome biogenesis activity. The presence or absence and degree of IRBC-inducing effect can be evaluated using ribosome biogenesis activity as an indicator. The most standard method for measuring ribosome biogenesis activity is, but is not limited to, the expression level of preribosome RNA. Methods for measuring the expression level of preribosome RNA include, but are not limited to, quantitative PCR and quantitative methods using next-generation sequencers. Nucleolus size can also be used as an indicator of ribosome biogenesis activity. In this case, the presence or absence and degree of IRBC-inducing effect can be evaluated by measuring and evaluating nucleolus size using, for example, an electron microscope.

[0048] (2) IRBCC Formation Activity IRBCC is a molecular complex formed when MDM2 is recruited to RPL5 and RPL11 as a result of decreased ribosome biogenesis activity. Methods for assessing the presence or absence and extent of IRBCC formation include, but are not limited to, coimmunoprecipitation experiments using anti-MDM2 antibodies, anti-RPL5 antibodies, and / or anti-RPL11 antibodies to measure the presence or absence and extent of association between MDM2 and RPL5 and / or RPL11 (IRBCC formation). The presence or absence and extent of association between MDM2 and RPL5 and / or RPL11 (IRBCC formation) can also be assessed by measuring intracellular colocalization using fluorescently labeled antibodies under a fluorescence microscope.

[0049] (3) TP53 Protein Stabilization Effect The presence or absence and degree of TP53 protein stabilization can be measured by, but not limited to, methods assessing the transcriptional activity of the TP53 gene and methods assessing the amount of TP53 protein. Examples of methods for assessing the transcriptional activity of the TP53 gene include, but are not limited to, quantitative RT-PCR and methods assessing the mRNA level of the TP53 gene by RNA sequencing using a next-generation sequencer. Examples of methods for assessing the amount of TP53 protein include Western blotting using an anti-TP53 antibody and intracellular immunostaining using a fluorescently labeled anti-TP53 antibody. Furthermore, TP53 protein stabilization can be interpreted as an increase in TP53 at the protein level without an increase in TP53 transcriptional activity, and therefore can also be evaluated by combining the above-mentioned transcriptional activity assessment with a protein quantification method.

[0050] (4) TIGAR Induction Activity The presence or absence and degree of TIGAR induction activity can be measured by, but not limited to, methods for assessing the transcriptional activity of the TIGAR gene and methods for assessing the amount of TIGAR protein. Examples of methods for assessing the transcriptional activity of the TIGAR gene include, but are not limited to, quantitative RT-PCR and methods for assessing the mRNA level of the TIGAR gene by RNA sequencing using a next-generation sequencer. Examples of methods for assessing the amount of TIGAR (protein) include Western blotting using an anti-TIGAR antibody and intracellular immunostaining using a fluorescently labeled anti-TIGAR antibody.

[0051] (5) MYC Transcriptional Inhibitory Effect The presence or absence and degree of MYC transcriptional inhibitory effect can be measured by, but not limited to, methods for assessing the transcriptional activity of the MYC gene and methods for assessing the amount of MYC protein. Examples of methods for assessing the transcriptional activity of the MYC gene include, but are not limited to, quantitative RT-PCR and methods for assessing the mRNA level of the MYC gene by RNA sequencing using a next-generation sequencer. Examples of methods for assessing the amount of MYC (protein) include Western blotting using an anti-MYC antibody and intracellular immunostaining using a fluorescently labeled anti-MYC antibody.

[0052] In this sense, the IMPDH1 inhibitor of the present disclosure, which has at least one effect selected from the group consisting of (1) to (5) in addition to the IMPDH1 inhibitory effect, can also be said to be a preparation that simultaneously functions as one of the following depending on each effect: (a) an IRBC inducer, (b) an IRBCC formation agent, (c) a TP53 protein stabilizer, (d) a TIGAR inducer, or (e) a MYC transcription suppressor.

[0053] (II) Screening method for active ingredients of pharmaceutical compositions for treating leukemia The present disclosure provides a screening method for active ingredients of pharmaceutical compositions for treating leukemia, comprising the step of selecting, from a group of test compounds, compounds that have, on leukemia cells, (I) an IMPDH inhibitory activity, and / or (II) at least one activity selected from the group consisting of the following (1) to (5): (1) an IRBC-inducing activity, (2) an IRBCC-forming activity, (3) a TP53 protein-stabilizing activity, (4) a TIGAR-inducing activity, or (5) a MYC transcription-suppressing activity.

[0054] The screening method of the present disclosure may be a method including, but not limited to, the following steps 1, 2, and 3: step 1: adding a test compound to leukemia cells, step 2: evaluating the leukemia cells to which the test compound has been added in step 1 for (I) an IMPDH inhibitory activity and / or (II) at least one activity selected from the group consisting of (1) to (5) above, and step 3: selecting, from the group of test compounds, compounds having (I) an IMPDH inhibitory activity and / or (II) at least one activity selected from the group consisting of (1) to (5) above, as candidate substances for the active ingredient of a pharmaceutical composition for treating leukemia.

[0055] The test compound used in the present disclosure is not particularly limited, and examples thereof include chemical libraries, antibody libraries produced by hybridomas, nucleic acid molecules such as siRNA and shRNA, various naturally occurring extracts, and compositions combining these.

[0056] The cells in step 1 may be leukemia cells, and although not particularly limited, are preferably human-derived leukemia cells, more preferably human AML cells. These leukemia cells may be human patient cells collected from a leukemia patient, or may be a leukemia cell line established from human patient cells.

[0057] In step 2, (I) the IMPDH inhibitory effect can be evaluated by measuring IMPDH activity or IMPDH expression levels in leukemia cells to which a test compound has been added to coexist with the test compound (hereinafter referred to as "test cells") and in leukemia cells to which the test compound has not been added (hereinafter referred to as "control cells"), and comparing the results. If the IMPDH activity or IMPDH expression level in the test cells is reduced compared to the control cells, the test compound added to the test cells can be evaluated as having an IMPDH inhibitory effect.

[0058] Known methods can be used to measure IMPDH activity. Examples of such methods include, but are not limited to, the following enzyme activity assays: 1) Color assay: A method in which NADH produced by the enzymatic reaction of IMPDH is detected as a change in color (e.g., measuring the absorbance of NADH at 340 nm). 2) Fluorescence assay: A method in which NADH produced by the enzymatic reaction of IMPDH is detected as a change in fluorescence intensity. 3) Mass spectrometer assay: A method in which the amount of XMP produced from the substrate IMP by the enzymatic reaction of IMPDH is measured using a mass spectrometer.

[0059] The expression level of IMPDH can also be measured by known methods. For example, but not limited to, Western blotting using an anti-IMPDH antibody or quantification of IMPDH protein by intracellular staining can be used. Expression at the transcription level can be evaluated by quantifying the mRNA of the IMPDH gene using quantitative RT-PCR or RNA sequencing.

[0060] In step 2, (II) evaluation of at least one effect selected from the group consisting of (1) to (5) can be carried out by measuring at least one effect selected from the group consisting of (1) to (5) in test cells and control cells, respectively, and comparing the two, as described above. If the test cells have a higher level of at least one effect selected from the group consisting of (1) to (5) in comparison with the control cells, the test compound added to the test cells can be evaluated as having at least one effect selected from the group consisting of (1) to (5). Note that the method for measuring the effects of (1) to (5) is as described above, but is not limited thereto, and known methods can also be used.

[0061] The compounds selected in step 3 can be candidates for the active ingredient of a pharmaceutical composition for treating leukemia, preferably AML, more preferably BCL-2 therapeutic agent-resistant AML.

[0062] Candidate substances for the active ingredients of pharmaceutical compositions selected by the screening method of the present disclosure can be further subjected to clinical trials in non-human animals and humans, and thus can be provided as therapeutic drugs for leukemia, preferably therapeutic drugs for AML, more preferably therapeutic drugs for BCL-2 therapeutic agent-resistant AML.

[0063] The present invention will be described below using experimental examples to aid in understanding the configuration and effects of the present invention. However, the present invention is not limited by these experimental examples. Unless otherwise specified, the following experiments were carried out at room temperature (25±5°C) and atmospheric pressure. Unless otherwise specified, "%" and "parts" in the following descriptions mean "% by mass" and "parts by mass," respectively.

[0064] Materials (1) Cell Lines: MOLM-13, SKM-1, and HNT-34 cell lines were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ). MV-4-11, THP-1, HL-60, Kasumi-1, Kasumi-3, and human embryonic kidney (HEK) 293T cell lines were purchased from the American Type Culture Collection (ATCC). MOLM-13, MV-4-11, THP-1, HL-60, Kasumi-1, Kasumi-3, SKM-1, and HNT-34 cell lines were grown in RPMI-1640 (Fujifilm Wako Pure Chemical Industries). The HEK293T cell line was grown in Dulbecco's modified Eagle's medium (DMEM) (Fujifilm Wako Pure Chemical Industries) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin plus streptomycin (Nacalai Tesque) at 37°C in a humidified incubator under 5% CO .

[0065] (2) Human samples. Bone marrow (BM) and peripheral blood samples were collected from healthy donors (HDs) (controls) and patients with acute myeloid leukemia (AML) at Kyushu University Hospital and the Fukuoka Bone Marrow Transplant Group (FBMTG). Umbilical cord blood (CB) cells were obtained from full-term infants. Informed consent was obtained from all patients and controls in accordance with the 1975 Declaration of Helsinki (revised in 1983). All human subject studies were approved by the Institutional Review Board of Kyushu University Hospital.

[0066] (3) Animals and their care: 4-6 week old female NSG mice (NOD.Cg-Prkdc scid NSG mice (Il2rgtm1Wjl / Sz, Jackson Labs, Strain #005557) were purchased from Jackson Laboratory Japan. NSG mice were housed in microisolator cages in a specific pathogen-free facility at Kyushu University. Animal experiments were conducted in accordance with the facility guidelines approved by the Kyushu University Animal Care Committee. Daylight hours were 7:00–19:00, temperature was 23+1°C, and water and food were available ad libitum. The animal experiment protocol for this study was approved by Kyushu University.

[0067] (4) Drugs used (IMPDH1 inhibitors) MPA: Mycophenolic acid A compound that inhibits the rate-limiting enzyme (IMPDH) in the de novo guanine nucleotide biosynthesis pathway uncompetitively, reversibly, and specifically, thereby depleting GTP and deoxyGTP and suppressing DNA synthesis. MMF: Mycophenolate mofetil (Cellcept, Chugai Pharmaceutical). A prodrug of MPA. It is rapidly hydrolyzed to MPA in the human body and acts as MPA.

[0068] Test Method (1) Establishment of Venetoclax (VEN)-Resistant AML Cell Lines MOLM13 and THP-1 AML cell lines were exposed to gradually increasing concentrations of VEN (starting from 0.1 nM) over a period of 2–3 months. The concentration was gradually increased by 10–50 nM every 3–4 days, maintaining cell viability at 80–90%. The development of VEN resistance was confirmed using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega).

[0069] (2) Metabolomic Analysis: Extraction of intracellular metabolites was performed according to the protocol provided by Human Metabolome Technologies (HMT). Briefly, purified cells were washed twice with 5% mannitol, treated with 800 μL of methanol, and then allowed to stand for 30 seconds to inactivate enzymes. The cell extract was then treated with 550 μL of Milli-Q water containing an internal standard (H3304-1002, HMT) and allowed to stand for another 30 seconds. The resulting extract was centrifuged at 2,300 × g for 5 minutes at 4°C, and 800 μL of the supernatant was centrifuged through a Millipore 5 kDa cutoff filter (UltrafreeMC-PLHCC, HMT) to remove macromolecules (9,100 × g, 4°C, 120 minutes). The filtrate was concentrated by centrifugation and resuspended in 50 μL of Milli-Q water for metabolomic analysis using HMT.

[0070] Metabolomic analysis was performed using a capillary electrophoresis time-of-flight mass spectrometer (CE-TOFMS) for cation analysis and a CE tandem mass spectrometer (CE-MS / MS) for anion analysis using the C-SCOPE package from HMT, as previously described (Non-Patent Document 1). CE-TOFMS analysis was performed using an Agilent CE capillary electrophoresis system (Agilent Technologies) equipped with an Agilent 6210 time-of-flight mass spectrometer. This system was controlled by Agilent G2201AA ChemStation software version B.03.01 (Agilent Technologies) for electrophoresis (CE) and connected to a fused silica capillary (50 μm id × 80 cm total length) using commercially available electrophoresis buffers (H3301-1001 for cation analysis and I3302-1023 for anion analysis, HMT) as electrolytes. The spectrometer scanned from m / z 50 to 1,000. Peaks were extracted using the automated integration software MasterHands (Keio University) and MassHunter Quantitative Analysis B.04.00 (Agilent Technologies), and peak information, such as m / z, peak area, and migration time (MT), was obtained. Signal peaks were annotated based on m / z values ​​in the MT according to the HMT metabolite database. Metabolite concentrations were calculated by normalizing the peak area of ​​each metabolite to the area of ​​the internal standard and using a three-point calibration curve. Detected metabolites were plotted on a metabolic pathway map using VANTED software.

[0071] (3) Cell Viability Analysis. Human-derived AML cell lines were cultured for 48 hours, and AML cells (CD34+ enriched cells) were cultured for 24 hours in RPMI-1640 supplemented with 10% heat-inactivated FBS. AML cells were treated with DMSO (control), MPA (10 μM), VEN (10 nM), or a combination of VEN (10 nM) and MPA (10 μM). For AML cell lines, a guanine nucleotide depletion rescue experiment was performed by adding guanosine 5'-monophosphate disodium salt hydrate (GMP, Sigma, 50 μM) in addition to VEN (10 nM) or MPA (10 μM). Cell viability was assessed using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer's protocol. Apoptotic cells were identified by FACS analysis as "Annexin V" (Figure 1). + " was defined as

[0072] (4) Dose-response curve analysis and IC 50 Cell viability was assessed using the CellTiter-Glo® 2.0 Assay (Promega) according to the manufacturer's protocol. AML cells and AML cell lines were plated at 5 × 10 cells per well in 100 μL of RPMI-1640 medium supplemented with 10% FBS. 4 Cells were seeded in white flat-bottom 96-well plates at a density of 100 μM. These cells and cell lines were treated with various concentrations of VEN for 48 hours (AML cell lines) or 24 hours (AML cells) under +DMSO or +10 μM MPA conditions. Luminescence was measured using an EnSpire 2300 Multi-mode Microplate Reader (PerkinElmer). The IC of VEN was 50 To determine IC values, luminescence data representing cell viability at 0 nM VEN under both +DMSO and +MPA conditions were normalized to 1.0. Dose-response curves were plotted using the latest version of GraphPad Prism 10.2.3, with VEN concentration on the X-axis and cell viability on the Y-axis. A nonlinear regression model with variable slope was fitted to the data, and IC values ​​were calculated as the concentration resulting in a 50% reduction in viability. 50 The value was calculated.

[0073] (5) BLISS score calculation: To evaluate the synergistic effect of drug combinations, the BLISS independence model was adopted. The actual calculation was performed using SynergyFinder (version 2.4.15) and the following formula was used: expected combination effect E ＡＢ was calculated as follows: E ＡＢ = E Ａ + E Ｂ - (E Ａ　 x E Ｂ ) where E Ａ and E Ｂ represents the individual treatment effect. Actual combined effect C ＡＢ The BLISS score was calculated as follows: BLISS score = C ＡＢ - E ＡＢ

[0074] (6) Short Hairpin RNA (shRNA)-Mediated IMPDH Knockdown in AML Cell Lines and Primary AML Cells. The shIMPDH1 and Scramble plasmids were constructed and packaged using VectorBuilder. Each construct contains the human U6 promoter, EGFP, and a puromycin resistance gene (Puro) linked to a T2A site for selection. The shIMPDH2 plasmid was purchased from Sigma-Aldrich. The shIMPDH2 construct contains the human U6 promoter and a T2A site for selection. HEK293T cells were cultured in 10-cm dishes in DMEM supplemented with 10% FBS. When HEK293T cells were 80% confluent, they were transfected in 10 mL of medium containing 6.0 μg psPAX2 (Addgene), 4.1 μg pMD2.G (Addgene), and 10 μg lentiviral vector, along with 60 μg linear polyethyleneimine (Polysciences). The medium was changed 6 hours after transfection. Lentiviral supernatants were collected 48 and 72 hours after transfection and transfected with Lenti-X. TM The solution was concentrated using a Concentrator (TaKaRa Clontech) according to the manufacturer's protocol.

[0075] AML cell lines and primary AML cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin / streptomycin. Each cell was plated in a 12-well plate (1 × 10 cells per well) containing medium supplemented with 8 μg / mL polybrene (Santa Cruz Biotechnology). 6 The cells were seeded onto AML cells and infected by centrifugation at 800 x g for 90 minutes at 32°C. Selection of knockdown cells was initiated 24 hours after transduction for AML cell lines and 48 hours after transduction for primary AML cells. IMPDH1 knockdown cells were selected for EGFP-positive cells. IMPDH2 knockdown cells were selected with puromycin. IMPDH1 and IMPDH2 double knockdown cells were generated by sequential selection. Specifically, IMPDH2 knockdown cells were first infected with the IMPDH1 vector, and then EGFP-positive cells were selected using a Sony SH800S cell sorter (Sony Biotechnology).

[0076] (7) Blast-colony formation assay and limiting dilution assay. AML cell samples were purified by CD34 selection beads (STEMCELL Technologies). + cells (CD34 +AML cells were enriched and resuspended in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 2% FBS. These cells were then mixed with Methocult H4435 Enriched medium (StemCell Technologies) to achieve a final cell concentration of 5,000–10,000 cells / well in a 48-well plate in a total volume of 200 μL per well. The enriched AML cells were treated with specific drugs and their effect on colony formation was assessed. The cell-medium mixture was plated and incubated at 37°C in a humidified atmosphere containing 5% CO2. Colonies were scored using a fluorescence microscope (BZ-X800, Keyence) 5–7 days after plating. For limiting dilution assays, the frequency of colony-forming cells was determined using ELDA software for limiting dilution analysis (Hu and Smyth, 2009) (https: / / bioinf.wehi.edu.au / software / elda / ). Statistical analysis and estimation of stem cell frequency were performed according to the software guidelines.

[0077] (8) Quantitative real-time PCR analysis. Purified leukemia cell lines and CD34 were analyzed using Isogen II reagent (NIPPON GENE) and RNeasy Micro Kit (Qiagen). + Total RNA was extracted from AML cells. First-stranded cDNA was synthesized using ReverTra Ace® qPCR RT Master Mix (Toyobo) according to the manufacturer's specifications. Quantitative real-time PCR was performed on a Stratagene Mx3000P (Agilent Technologies) using THUNDERBIRD Probe qPCR Mix (Toyobo). Reactions were performed in triplicate wells of a 96-well plate. Primers used were those listed in Table 1.

[0078]

[0079] Data were analyzed using MxPro software (version 4.10) (Agilent Technologies). Gene expression of IMPDH1, IMPDH2, HPRT1, and PNP was calculated using the ΔΔCt method. GAPDH was amplified separately on the same plate as an internal control. For each sample, ΔΔCt was calculated by subtracting the Ct value of GAPDH from the Ct value of the target gene. The ΔΔCt value for each sample was calculated using the mean ΔCt value of VEN-sensitive AML samples as the reference. Relative expression levels were calculated using the 2-ΔΔCt method. Expression analysis of MYC and pre-rRNA was performed using the ΔCt method with GAPDH as the reference gene. Gene expression analysis of MYC and its reference gene (GAPDH) was performed using the TagMan Gene expression assay (Thermo Fisher Scientific).

[0080] (9) RNA Sequencing (RNA-seq). Total RNA was extracted using the RNeasy Mini Kit (Qiagen), and its quality was assessed using a NanoDrop spectrophotometer and an Agilent 2100 Bioanalyzer. RNA libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs, E7760S) according to the manufacturer's protocol. 500 ng of total RNA was used to isolate mRNA using oligo-dT beads (New England BioLabs). mRNA was fragmented by incubation at 94°C for 7.5 minutes. First-strand cDNA synthesis was performed using NEBNext First Strand Synthesis Reaction Buffer and random primers, followed by second-strand synthesis using NEBNext Second Strand Synthesis Reaction Buffer and dUTP Mix. The resulting cDNA was purified using SPRIselect Beads (Beckman Coulter). End repair, A-tailing, and adapter ligation were performed using NEBNext Ultra II Ligation Master Mix and Ligation Enhancer. Adapter-ligated DNA was enriched by PCR amplification using NEBNext Q5 Hot Start HiFi PCR Master Mix and NEBNext Multiplex Oligos for Illumina. Libraries were purified using SPRIselect Beads and assessed using an Agilent 2100 Bioanalyzer and Qubit 2.0 Fluorometer (Thermo Fisher Scientific). Library quality was further confirmed using Agilent Technologies High Sensitivity DNA Chips.Quantification was performed using the Roche KAPA Library Quant Kit (Illumina Universal qPCR Mix, KK4824, 500 reactions, 7960140001) and Roche KAPA Library Quant (Illumina) DNA Quantification Standards (KK4903, 80 μL × 6 tubes). Quantified libraries were pooled in equimolar ratios and sequenced on the Illumina NEXTSeq 1000 / 2000 platform according to the manufacturer's instructions to generate 150-bp paired-end reads. Sequencing data quality was assessed using FastQC, and adapter trimming was performed using Trimomatic. Reads were aligned to the human reference genome (GRCh38) using the STAR aligner. Gene expression quantification was performed using featureCounts, and differential expression analysis was performed using DESeq2.

[0081] (10) BRB Sequencing (BRB-seq) BRB-seq was performed for library preparation with the following modifications: Barcoded oligo-dT-based primer (5'-GCCGGTAATACGACTCACTATAGGGAGTTCTACAGTCCGACGATCNNNNNNNNNNCCCCCCCCCTTTTTTTTTTTTTTTTTTTTTTTTV -3' (SEQ ID NO: 13); (10) N = UMI, (9) C = cell barcode) was used for first-strand cDNA synthesis, and the Second Strand Synthesis Module (NEB, #E6111) was used for double-strand cDNA synthesis. Tagmentation was performed using in-house MEDS-B Tn5 transposase, and the tagmented DNA was amplified by 10 cycles of PCR using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, #M0530) and the following primers: 5'-AATGATACGGCGACCACCGAGATCTAC[index]GTTCAGAGTTCTACAGTCCGA-3' (SEQ ID NO: 14) and 5'-CAAGCAGAAGACGGCATACGAGAT[index]GTCTCGTGGCTCGGAGATGT-3' (SEQ ID NO: 15). Sequencing was performed on an Illumina NovaSeq 6000 platform, generating a 15-bp barcode read (Read 1) and an 81-bp insert read (Read 2).

[0082] (11) BRB-seq Data Processing: Read1 (barcode read) was extracted using UMI-tools (1.1.1) with the command "umi_tools extract -I read1.fastq --read2-in=read2.fastq --bc-pattern=NNNNNNCCCCCCC --read2-stdout." Adapter sequences and low-quality sequences were removed using Trim Galore (0.6.7), and reads shorter than 20 bp were discarded. Reads were mapped to the GRCh38 reference using HISAT2 (2.2.1). Read counts for each gene and sample were obtained using the summarized BAM file generated by featureCounts (2.0.1). Differentially expressed genes were extracted using DESeq2 (1.40.2). The thresholds were |log2FC| > 1 and padj < 0.05.

[0083] (12) Gene Expression Analysis of Reactive Oxygen Species-Defined AML Cell Subpopulations AML cells were gently thawed and cultured in RPMI-1640 medium supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO for 24 hours. The cells were harvested, washed twice with phosphate-buffered saline (PBS), and then diluted to 1 × 10 6Cells were resuspended in RPMI-1640 medium at a concentration of 1000 cells / mL. CellROX® Green Reagent (0.5 μM, Thermo Fisher Scientific) was added and incubated at 37°C for 30 minutes in the dark. After staining, cells were washed twice with PBS. Flow cytometry analysis and sorting were performed using a Sony SH800S Cell Sorter (Sony Biotechnology). Single cells were gated based on forward scatter (FSC) and backward scatter (BSC) parameters, and live cells were gated morphologically. ROS-low and ROS-high cell populations were identified, gated, and then sorted based on the median fluorescence intensity (MFI) of CellROX® Green staining. Specifically, the 15% of AML cells with the lowest CellROX® Green signal intensity were defined as ROS-low cells, and the 15% with the highest signal intensity were defined as ROS-high cells. Total RNA was extracted from ROS-low and ROS-high cells sorted from the same AML cell sample using the RNeasy Micro Kit (Qiagen) according to the manufacturer's protocol. First-stranded cDNA was synthesized using ReverTra Ace® qPCR RT Master Mix (Toyobo) according to the manufacturer's specifications. Quantitative real-time PCR was performed on a Stratagene Mx3000P (Agilent Technologies) using THUNDERBIRD Probe qPCR Mix (Toyobo). Reactions were performed in triplicate wells of a 96-well plate. Data were analyzed using MxPro software (version 4.10) (Agilent Technologies). For each AML sample, the expression level of ROS-low cells was evaluated relative to the expression level of ROS-high cells using the ΔCt method, with the expression level of ROS-high cells set at 1.0.

[0084] (13) Mouse xenograft model and in vivo treatment. (A) CD34+ AML treatment model. To evaluate the efficacy of therapeutic drugs in vivo, purified CD34+ AML cells (0.5-1.0 × 10 6) was injected into the tail vein of NSG mice subcutaneously irradiated with 2.2 Gy of radiation. After confirming AML reconstitution in NSG mice by peripheral blood collection, the mice were randomly assigned to a control or treatment group. Treatment mice received VEN (25 mg / kg, orally), MMF (100 mg / kg, orally), VEN + MMF (same amount, orally), or VEN (25 mg / kg, orally) + azacitidine (3 mg / kg, intraperitoneally) once daily for 21 days. Control mice received the same amount of DMSO. After the treatment period, mice were euthanized, and bone marrow (BM) and peripheral blood (PB) were assessed by FACS, and spleen weights were measured. Chimerism was calculated using the following formula: (hCD45+ / [hCD45+ + mouse CD45+]). For flow cytometry analysis, FITC-labeled anti-human CD34 antibody, PE-labeled anti-human CD33 antibody, APC-labeled anti-human CD45 antibody, Brilliant Violet TM A combination of 421-conjugated anti-mouse CD45.1 antibody and PerCP / Cy5.5-conjugated anti-mouse Ter-119 antibody was used. Dead cells were excluded by propidium iodide (PI) staining. Analysis was performed using an Attune NxT flow cytometer (Thermo Fisher Scientific).

[0085] (B) CB-derived HSPCs treatment model. To evaluate the effects of therapeutic agents on normal hematopoiesis in vivo, CB-derived HSPCs (0.5-1.0 × 10 6 ) was injected into the tail vein of NSG mice that had been subcutaneously irradiated (2.2 Gy). After confirming HSPC reconstitution in NSG mice by peripheral blood sampling, the mice were randomly assigned to one of three groups: DMSO, VEN (25 mg / kg, orally), or VEN (same dose, orally) + MMF (100 mg / kg, orally). After the treatment period, the mice were euthanized, and bone marrow was evaluated by FACS. Flow cytometry analysis was performed using the following antibody combinations: FITC-labeled anti-human CD14 or CD34 antibody, PE-labeled anti-human CD33 or CD38 antibody, PE / Cy7-labeled anti-human CD15 or CD56 antibody, APC / Cy7-labeled anti-human CD3 antibody, and Brilliant Violet. TM421 labeled anti-mouse CD45.1 antibody, Brilliant Violet TM 510-labeled anti-human CD11b or CD19 antibody, PerCP / Cy5.5-labeled anti-mouse Ter-119 antibody. The stem cell fraction was defined as hCD45+Lin-CD34+. Analysis was performed using an Attune NxT flow cytometer (Thermo Fisher Scientific).

[0086] (C) Evaluation of the therapeutic effect of IMPDH1 knockdown on AML (reconstitution ability) The therapeutic effect of IMPDH1 knockdown on AML (reconstitution ability) was evaluated in an in vivo study using mice. Specifically, primary AML cells (hCD45 ＋ AML cells were treated with shRNA (sh IMPDH1-1, sh IMPDH1-2) (IMPDH1 knockdown treatment), and knockdown cells were selected as EGFP-positive cells. These cells were injected into the tail vein of NSG mice irradiated with 2.2 Gy of radiation. Six to eight weeks later, the mice were euthanized and bone marrow (BM) was collected. Flow cytometry analysis revealed that AML cells (hCD45) expressing human leukocyte common antigen (CD45) in the bone marrow were identified. ＋ The percentage of AML cells was measured.

[0087] (14) Sequential Transplantation Experiment After the treatment described in (13)(B) above, hCD34+ AML cells were collected from the bone marrow of the first recipient. The same number of hCD34+ AML cells (0.5 x 10 6 The xenografts (1000 x 1000 cells / mouse) were sequentially re-implanted via the tail vein of age-matched NSG female secondary mice that had been sublethally irradiated (2.2 Gy). Eight to 12 weeks after secondary xenografting, the recipient mice were sacrificed and the tumor burden in the bone marrow was analyzed by FACS as previously described.

[0088] (15) Preparation of protein lysates and Western blotting. Cells were harvested, washed with ice-cold phosphate-buffered saline (PBS), and then lysed in RIPA (radioimmunoprecipitation assay) buffer (Nacalai Tesque) containing protease inhibitors and phosphatase inhibitors. Protein concentrations were measured using Micro BCA. TM Protein Assay Kit (Thermo Fisher Scientific) was used for measurement. Lysates were denatured with an equal volume of 4x SDS sample buffer containing 5% (v / v) 2-mercaptoethanol and heated at 95°C for 5 minutes. Proteins were fractionated by SDS-polyacrylamide gel electrophoresis using precast gels (Wako Chemicals), and proteins were transferred to nitrocellulose membranes (Wako Chemicals). Before incubation with primary antibodies, blots were blocked with 5% nonfat dry milk in Tris-buffered saline supplemented with 0.1% Tween-20 for 0.5–1 hour. The membranes were then incubated with primary antibodies overnight at 4°C, washed, and then incubated with secondary antibodies for 1 hour at room temperature. Images were visualized using an iBright CL1500 Imaging System (Thermo Fisher Scientific).

[0089] (16) Immunoprecipitation Assay: To evaluate the binding of MDM2 to RPL5 and RPL11 under ribosome biogenesis inhibition, co-immunoprecipitation was performed using the Dynabeads Co-immunoprecipitation Kit (Invitrogen) according to the manufacturer's instructions. Briefly, MOLM13 cells or CD34-enriched AML cells (CD34+ AML cells) were treated with 10 μM MPA for the indicated times as described in the instructions. Cells were lysed in Extraction Buffer B (1x IP buffer containing 100 mM NaCl, 2 mM MgCl, 1 mM dithiothreitol, and EDTA-free protease and phosphatase inhibitors) and homogenized by gentle mixing and pipetting. Anti-MDM2 antibody (Santa Cruz Biotechnology) was used. TMAntibody-conjugated proteins were prepared by coupling to M-270 Epoxy beads overnight at 37°C. Anti-mouse IgG was used as a control. The lysate was then incubated with the antibody-conjugated beads for 3 hours at room temperature with gentle rotation. After incubation, the beads were washed twice with lysis buffer, and the bound proteins were eluted with elution buffer. The eluted proteins were mixed with 2-mercaptoethanol and boiled at 95°C for 5 minutes. Samples were then analyzed by SDS-PAGE and immunoblotted using anti-MDM2 (Santa Cruz Biotechnology), anti-RPL5, and anti-RPL11 (Cell Signaling Technology) antibodies.

[0090] (17) Seahorse Assay EasySep TM CD34+ AML cells were enriched using the Human CD34 Positive Selection Kit II (STEMCELL Technologies) according to the manufacturer's instructions. Cells were then treated with DMSO or 10 μM MPA for 12 hours in RPMI-1640 medium supplemented with 10% FBS. After treatment, cells were washed and resuspended in assay medium. A total of 2 × 10 cells per well were plated onto XFp cell culture miniplates (Agilent Technologies) precoated with Cell-Tak (Corning). 5 of cells were seeded.

[0091] [Cell Preparation] Cell Mitochondrial Stress Test and Real-Time ATP Assay Test: Cells were resuspended in XF RPMI supplemented with 10 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine, and the pH was adjusted to 7.4. Glycolysis Stress Test: Cells were resuspended in XF RPMI supplemented with 2 mM L-glutamine, and the pH was adjusted to 7.4.

[0092] Assay Method: Cell Mito Stress Test. 20 μL of oligomycin (1.0 μM), FCCP (1.0 μM), and rotenone / antimycin A (0.5 μM) were loaded onto an XFp sensor cartridge. Analysis was performed using the cell mito stress test assay protocol on a Seahorse XFp extracellular flux analyzer (Agilent Technologies). Results were analyzed using Wave software (version 2.6) (Agilent Technologies).

[0093] Real-Time ATP Assay Test: 20 μL of oligomycin (1.0 μM) and rotenone / antimycin A (0.5 μM) were loaded onto the XFp sensor cartridge. Analysis was performed using the Real-Time ATP Rate Assay test on a Seahorse XFp extracellular flux analyzer (Agilent Technologies). Results were analyzed using Wave software (version 2.6) (Agilent Technologies).

[0094] Glycolysis stress test: 20 μL of glucose (10 mM), oligomycin (1.0 μM), and 2-DG (50 mM) were loaded onto the XFp sensor cartridge. Analysis was performed using the glycolysis stress test assay protocol on a Seahorse XFp extracellular flux analyzer (Agilent Technologies). Results were analyzed using Wave software (version 2.6) (Agilent Technologies).

[0095] (18) Evaluation of TP53 protein levels and expression in AML patients. (A) Primary AML cells were collected from VEN-sensitive patients (UPN7, UPN10, UPN6, UPN15) and VEN-resistant patients (UPN4, UPN16, UPN17, UPN13) without TP53 gene mutation (TP53 WT). The expression levels of TP53 protein, IMPDH1, and GAPDH were evaluated by Western blotting using anti-human TP53 antibody, anti-human IMPDH1 antibody, anti-human IMPDH2 antibody, and anti-human GAPDH antibody.

[0096] (B) Primary AML cells from VEN-resistant patients (n=17) with unmutated TP53 (TP53 WT) were analyzed for TP53 protein expression using Western blotting with anti-human TP53 and anti-human GAPDH antibodies, and the GAPDH / TP53 protein ratio was quantified. The pre-rRNA / GAPDH mRNA ratio was also measured using qPCR. The pre-rRNA / GAPDH mRNA ratio is an index for evaluating the relative ribosome biogenesis activity in AML cells.

[0097] (19) Statistical Analysis. All statistical analyses were performed using GraphPad Prism software (versions 10.0–10.2) and R (version 4.3.1, released June 16, 2023). Data were expressed as mean ± standard deviation (SD). To determine statistical significance, various tests were employed depending on the nature of the data and the number of groups compared. When comparing two groups with normally distributed data, unpaired and paired two-tailed t-tests were used. When comparing two groups with non-normal distributions, the Mann-Whitney U test was applied. When comparing three or more groups, one-way analysis of variance (ANOVA) was performed, followed by Tukey's and Dunnett's post-hoc multiple comparison tests. Survival curves were analyzed using the log-rank test.

[0098] Test Results Test Example 11-1: VEN-Resistant AML Cells Depend on Glycolysis for ATP Production Compared to VEN-Sensitive AML Cells One of the key effects of venetoclax (VEN) in the treatment of AML is its inhibitory effect on mitochondrial oxidative phosphorylation (OXPHOS). First, the dependence of energy production on OXPHOS and glycolysis was evaluated in vitro in clinically VEN-sensitive (n = 5) and VEN-resistant (n = 5) AML cells. AML cases that achieved complete remission within four cycles of a VEN-containing regimen (including VEN + azacitidine and VEN + low-dose cytarabine) were defined as "VEN-sensitive," while AML cases with less than 50% reduction in BM leukemia cells after four cycles of a VEN-containing regimen were defined as "VEN-resistant."

[0099] Real-time ATP rate assays revealed that VEN-resistant AML cells were highly dependent on glycolysis for ATP production, at least in vitro (Fig. 1A). Furthermore, inhibition of OXPHOS by VEN significantly increased the rate of glycolytic ATP production in VEN-resistant AML cells (Fig. 1B).

[0100] These results suggest that VEN-resistant AML cells depend on glycolysis for baseline energy production and that glycolytic activity can be enhanced by inhibiting oxidative phosphorylation (OXPHOS).

[0101] 1-2: VEN-resistant AML cells contain a large amount of metabolites related to the guanine nucleotide biosynthesis pathway. To clarify the metabolic characteristics that led to increased glycolysis in VEN-resistant AML cells, AML cells in which 116 metabolites had been comprehensively quantified in a previous study (Non-patent Document 1) were treated with VEN, and the intracellular metabolites were compared between VEN-sensitive and VEN-resistant AML cells.

[0102] After 18 hours of in vitro treatment with VEN (50 nM), cell viability was assessed by annexin / PI assay. AML cells with >50% viability compared with the control (DMSO) were defined as VEN-resistant, and those with <50% viability were defined as VEN-sensitive.

[0103] Figure 1C shows the intracellular metabolites significantly different between VEN-sensitive (n = 16) and VEN-resistant (n = 10) AML cells. Consistent with the previously reported enhanced glycolysis in VEN-resistant AML cells (Figures 1A and 1B), VEN-resistant AML cells contained significantly higher levels of glycolysis-related metabolites (3-phosphoglycerate, fructose 1,6-bisphosphate, and lactate) than VEN-sensitive AML cells (Figure 1D). Furthermore, VEN-resistant AML cells contained significantly higher levels of intermediate metabolites of the pentose phosphate pathway (PPP) (6-phosphogluconate and glyceraldehyde 3-phosphate) than VEN-sensitive AML cells (Figure 1E). The intracellular contents of XMP, GMP, and GDP, intermediate metabolites of the guanine nucleotide biosynthetic pathway downstream of the PPP, were significantly elevated in VEN-resistant AML cells (Figure 1F).

[0104] 1-3: VEN-resistant AML cells show increased gene expression related to the de novo guanine nucleotide biosynthetic pathway. Eight of the metabolites abundant in VEN-resistant AML cells (Fig. 1C) belonged to the glycolytic pathway, PPP, and the downstream guanine nucleotide biosynthetic pathway (Fig. 1G). This suggests that the guanine nucleotide biosynthetic pathway may be involved in enhancing OXPHOS activity, conferring resistance to OXPHOS inhibition to AML. Two pathways, the de novo pathway and the salvage pathway, are involved in guanine nucleotide biosynthesis (Fig. 1G).

[0105] VEN-resistant AML cells showed higher expression of inosine monophosphate dehydrogenase (IMPDH1 and IMPDH2), a gene encoding a rate-limiting enzyme in the de novo pathway, than VEN-sensitive AML cells (Fig. 1H).In contrast, no difference was observed between VEN-resistant and VEN-sensitive AML cells in the expression of genes encoding enzymes related to the salvage pathway (HPRT, PNP) (Fig. 1I).

[0106] Furthermore, MOLM-13 and THP-1 cell lines were cultured for a long period of time under conditions where VEN concentrations were gradually increased from a low concentration of 0.1 nM, and VEN-resistant MOLM-13 and THP-1 cell lines were established. These cell lines showed a significantly higher IC of VEN compared to the parent cell lines. 50 Similar to VEN-resistant AML cells, the expression of IMPDH1 and IMPDH2 was significantly elevated in VEN-resistant MOLM-13 and THP-1 cell lines compared with their parental cell lines (Fig. 1J, right columns).

[0107] Test Example 2: Inhibition of the de-novo guanine nucleotide biosynthetic pathway synergistically enhances the cytotoxic activity of VEN against AML (1) As shown in Test Example 1, VEN-resistant AML cells and various AML cell lines showed increased expression of IMPDH1 and IMPDH2. Therefore, focusing on the de novo pathway, various AML cell lines were treated with VEN and / or MPA, which specifically inhibit IMPDH1 and IMPDH2, and the cell viability of the AML cell lines was evaluated.

[0108] Inhibition of the de novo pathway with MPA significantly enhanced the cytotoxicity of VEN alone in AML cell lines, including MOLM13, MV4-11, and THP-1, in vitro (Fig. 2A). Supplementation with GMP completely abolished this enhancement (Fig. 2A). In contrast, inhibition of the salvage pathway with the PNP inhibitor forodesine did not enhance the efficacy of VEN in vitro (Fig. 2B).

[0109] (2) Using eight AML cell lines (MOLM13, MV4-11, THP-1, HNT34, KASUMI-1, KASUMI-3, SKM1, HL-60), we evaluated the IC of VEN with and without MPA. 50 As a result, the combined administration of VEN and MPA in vitro increased the IC 50The IC value significantly decreased, from 0.18 to 0.0003 relative to VEN monotherapy (Fig. 2C). In the VEN-resistant MOLM13 cell line and VEN-resistant THP1 cell line, the combination of VEN and MPA significantly reduced the IC value compared to VEN alone. 50 The relative values ​​significantly decreased to 0.005 and 0.17, respectively (Fig. 2D). Knocking down IMPDH1 or IMPDH2 with short hairpin RNA (sh-RNA) significantly reduced the IC50 of VEN in MOLM13 and THP-1 cells. 50 All values ​​were significantly reduced (Fig. 2E). The base sequences of the sh-RNAs used are listed in Table 2.

[0110]

[0111] (3) We evaluated the efficacy of VEN and MPA combination therapy against AML cells in vitro. The incidence of apoptosis was significantly increased in cell populations treated with VEN and MPA combination therapy compared with cell populations treated with VEN or MPA alone. This effect was observed in both VEN-sensitive (n = 8) and VEN-resistant AML cells (n = 8) (Fig. 2F). The IC value of VEN with and without MPA was calculated using AML cells. 50 MPA significantly increased the IC value of VEN in VEN-sensitive (n=8) and VEN-resistant (n=9) AML cells. 50 The values ​​were significantly reduced (Fig. 2G).

[0112] Furthermore, we confirmed the synergistic effect of VEN and MPA using the Bliss independence model, a statistical approach used to predict the synergistic effects of combination therapies. The combination of VEN and MPA showed significantly higher Bliss scores against both AML cell lines (Figure 2H) and AML cells (Figure 2I), suggesting a strong synergistic effect. Conversely, the combination of VEN and forodesine resulted in a negative Bliss score, suggesting an antagonistic effect.

[0113] Example 3: Inhibition of de-novo guanine nucleotide biosynthesis pathway induced TP53 and TIGAR expression and suppressed glycolysis in AML. (1) To elucidate the molecular mechanisms underlying MPA's enhanced cytotoxic activity of VEN, we performed RNA-seq in MOLM13 and MV4-11 cell lines. Combination treatment with VEN and MPA induced TP53 target gene expression in both MOLM13 and MV4-11 cell lines compared with VEN monotherapy (Figure 3A). While MPA did not affect TP53 gene transcription in AML cells (Figure 3B), it significantly increased TP53 protein in a time-dependent manner in both VEN-sensitive and VEN-resistant AML cells (Figure 3C). Among the downstream molecules induced by TP53, TIGAR has been shown to play a critical role in suppressing glycolysis (Figure 3D).

[0114] These results suggest that MPA suppresses glycolysis, on which VEN-resistant AML cells are highly dependent for energy production, by inducing TP53 and TIGAR (Figures 1A and 1B). Using the CRISPR / CAS9 system, we deleted the TP53 gene in MOLM13 cell lines (MOLM13_TP53KO). MPA increased TIGAR protein expression in CAS9-transfected MOLM13 cell lines (MOLM13_CAS9), but not in MOLM13_TP53KO cell lines (Figure 3E). Consistent with IMPDH inhibition, administration of GMP, GDP, and GTP inhibited the induction of TP53 and TIGAR protein in MOLM13 cell lines in vitro, whereas administration of IMP did not (Figure 3F) (see Figure 1G).

[0115] (2) We investigated whether MPA-stimulated upregulation of TP53 and TIGAR suppresses glycolysis in AML cells. Glycolytic activity and oxidative phosphorylation (OXPHOS) were assessed by measuring the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively, using a metabolic flux analyzer. MPA significantly suppressed ECAR in AML cell lines and AML cells, regardless of VEN sensitivity, but did not affect OCR (Figures 3G and 3H). Notably, MPA suppressed normal human CD34 cells.+ Glycolytic activity of HSPCs was not affected (Figures 3I and 3J). These results suggest that guanine nucleotide depletion induces TIGAR and specifically suppresses glycolysis in AML in a TP53-dependent manner.

[0116] Experimental Example 4: Guanine nucleotide depletion leads to stabilization of IRBC and TP53 protein in AML. We investigated the molecular mechanism linking guanine nucleotide depletion and increased TP53 protein. Seven gene sets that showed identical changes upon addition of VEN and MPA in both MOLM13 and MV4-11 cell lines were extracted (p<0.05, FDR q-value<0.10). Six of the seven gene sets were related to ribosome biogenesis (Figure 4A). Ribosome biogenesis is an essential mechanism for maintaining cellular homeostasis. Various ribosome proteins are assembled to form ribosome subunits, which play a central role in protein translation (Figure 4B).

[0117] First, we evaluated pre-rRNA expression, a sensitive indicator of ribosome biogenesis activity. Consistent with the RNA-seq results demonstrating inhibition of ribosome biogenesis, MPA significantly suppressed pre-rRNA expression in vitro in cell lines, including MOLM13 and THP-1 (Fig. 4C), as well as in AML cells (Fig. 4D). Addition of GMP to the culture medium reversed the pre-rRNA suppression. VEN-resistant MOLM13 and THP-1 cell lines exhibited higher pre-rRNA levels than their parental cell lines (Fig. 4E, left panel). Furthermore, VEN-resistant AML cells exhibited higher pre-rRNA levels than VEN-sensitive AML cells (Fig. 4E, right panel).

[0118] These findings indicate that intracellular guanine nucleotides are an essential driving force for maintaining ribosome biogenesis activity and that enhanced ribosome biogenesis is likely involved in the acquisition of VEN resistance in AML.

[0119] Recent studies have suggested a novel molecular mechanism that bridges the gap between impaired ribosome biogenesis and TP53 protein stabilization. Under steady-state conditions, RPL5 and RPL11 are utilized for normal ribosome biogenesis. However, when ribosome biogenesis is disrupted for various reasons, the impaired ribosome checkpoint (IRBC) is activated, recruiting MDM2 to RPL5 and RPL11 in the cell to form the impaired ribosome checkpoint complex (IRBCC). As a result, TP53 degradation due to MDM2 inactivation is suppressed, resulting in the stabilization and accumulation of TP53 protein, which induces cell cycle arrest and apoptosis, suppressing the proliferation of cells with impaired ribosome biogenesis. This is thought to be a checkpoint mechanism that contributes to maintaining homeostasis (Figure 4F).

[0120] Based on the above data, we hypothesized that the mechanism linking increased TP53 protein and impaired ribosome biogenesis is that in human AML cells, IRBCs are highly induced in response to intracellular guanine nucleotide depletion, and that the resulting IRBCCs stabilize TP53 protein via MDM2 in AML (Fig. 4F).

[0121] Immunoprecipitation (IP) assays using anti-MDM2 monoclonal antibody revealed that IRBCC components consisting of RPL5 and RPL11 co-immunoprecipitated with MDM2 after MPA exposure in MOLM13 cell lines (Fig. 4G) and AML cells (Fig. 4H). These results suggest that guanine nucleotide depletion induces IRBCC and stabilizes TP53 protein through the formation of IRBCC in AML.

[0122] Test Example 5: IRBC suppresses MAPK and its downstream MYC pathway in a TP53-independent manner. (1) In MOLM13_TP53KO cells, the IC of VEN was significantly higher than in CAS9-transfected MOLM13 cell lines (MOLM13_CAS9). 50The efficacy of MPA in reducing IRBC-mediated AML was significantly reduced, suggesting that the TP53 pathway plays an important role in the anti-AML effect of IRBC (Figures 5A and 5B). However, the calculated Bliss score for the combination of MPA and VEN still showed a synergistic effect in the MOLM13_TP53KO cell line. In cell lines with TP53 heterozygous mutations, particularly THP-1 and KASUMI-1, and the TP53-deficient cell line HL-60, MPA significantly reduced IC 50 IRBC significantly enhanced the cytotoxicity of VEN, as evidenced by a decrease in the TP53-dependent expression level (Figure 2C). These results suggested that IRBC exerts its anti-AML effects through both TP53-dependent and TP53-independent molecular mechanisms.

[0123] (2) To identify the TP53-independent anti-AML effects induced by IRBCs, we performed global transcriptome analysis using the MOLM13_TP53KO and MOLM13_CAS9 cell lines. GSEA revealed that MPA significantly suppressed genes related to MYC target genes in both the MOLM13_TP53KO and MOLM13_CAS9 cell lines (Fig. 5C). Volcano plots revealed that MYC was one of the most suppressed genes, independent of TP53 (Fig. 5D). Guanine nucleotides containing GDP and GTP are required for activation of the RAS / MAPK pathway and play a crucial role in the transcriptional induction of MYC (Fig. 5E). Inhibition of de novo guanine nucleotide biosynthesis by MPA significantly suppressed phosphorylated MEK1 / 2 in AML cell lines (MOLM13, THP1) (Fig. 5F) and AML cells (Fig. 5G), regardless of TP53 mutation. Phosflow analysis revealed that MPA inhibited MEK phosphorylation and GMP counteracted this effect in both MOLM13_CAS9 and MOLM13_TP53KO cell lines (Fig. 5H). As a result, MYC transcriptional activity was significantly reduced in AML cell lines (Fig. 5I) and AML cells (Fig. 5J), regardless of TP53 mutation status.

[0124] Test Example 6: LSCs express IMPDH1 / 2 at high levels and maintain stem cell properties through enhanced ribosome biosynthesis activity. We focused on the function of the de novo guanine nucleotide biosynthesis pathway in LSCs. Self-renewing human LSCs are able to maintain stem cell properties through enhanced IMPDH1 / 2 expression and enhanced ribosome biosynthesis activity. low ROS are concentrated in the fraction. low AML cells produce ROS high The expression of IMPDH1 and IMPDH2 was significantly higher in AML cells than in AML cells (Fig. 6A). low AML cells produce ROS high The amount of pre-rRNA was also higher in AML cells (Figure 6B), suggesting that LSCs play a key role in ribosome biogenesis. MPA significantly reduced the number of blast colony-forming cells in AML cells, whereas VEN did not (Figure 6C). Furthermore, sh-RNA-mediated knockdown of IMPDH1 or IMPDH2 significantly reduced the number of blast colony-forming cells in AML cells (Figure 6D). Limiting dilution assays revealed that MPA significantly suppressed the estimated frequency of blast colony-forming AML cells in vitro (Figures 6E and 6F).

[0125] These results suggest that, at least in vitro, enhanced ribosome biogenesis plays an important role in maintaining the stem cell properties of LSCs.

[0126] Test Example 7: LSCs maintain their stem cell potential in vivo by relying heavily on ribosome biogenesis via de novo guanine nucleotide biosynthesis. (1) We investigated whether induction of IRBCs could be a novel in vivo therapeutic approach for human AML / LSCs. Patient AML cells purified from five independent AML samples were transplanted intravenously into irradiated NSG mice. Figure 7A shows an outline of the xenotransplantation experiment. Four to 12 weeks after xenotransplantation, human CD45 expression levels in the peripheral blood (PB) of recipient mice were significantly elevated. + cells (hCD45 + The amount of human AML cells was assessed to confirm engraftment of human AML cells.

[0127] After confirming engraftment, recipient mice were randomly divided into four or five groups and administered the following: 1) Control group: DMSO was administered orally daily; 2) VEN group: VEN (25 mg / kg) was administered orally daily; 3) MMF group: MMF (100 mg / kg) was administered orally daily; 4) Combination group 1 (VEN + azacitidine): VEN (25 mg / kg) was administered orally daily and azacitidine (2.5 mg / kg) was administered intraperitoneally; 5) Combination group 2 (VEN + MMF): VEN (25 mg / kg) and MMF (100 mg / kg) were administered orally daily.

[0128] Due to the limited number of available samples, NSG mice transplanted with AML cells were randomly assigned to one of four treatment groups. Twenty-one days after administration, recipient mice were euthanized and evaluated. Azacitidine is a drug used to treat acute myeloid leukemia. Its mechanism of action is known to be its incorporation into DNA and RNA, primarily inhibiting protein synthesis and exerting cytocidal effects.

[0129] In all cases tested, the combination of VEN and MMF significantly reduced the number of AML cells (hCD45 + It was confirmed that the proportion of AML cells was significantly reduced (Figure 7B).

[0130] These results were consistent with the synergistic anti-AML effect demonstrated in vitro. Of particular importance was the significant anti-leukemic effect of MMF monotherapy in VEN-resistant AML cases (UPN2 and UPN4) in vivo. Furthermore, the combination of VEN and MMF significantly inhibited the proliferation of AML cells (hCD45 AML) compared with the combination of VEN and azacitidine in all test samples, including VEN / azacytidine-resistant AML cases (UPN2). +We confirmed that the proportion of AML cells (UPN2, UPN6) significantly decreased in patients with MMF. In vivo, MMF monotherapy significantly suppressed pre-rRNA synthesis (Fig. 7C) and stabilized TP53 protein in AML cells (Fig. 7D). These results suggest that MMF monotherapy suppresses guanine nucleotide biosynthesis, resulting in the induction of IRBCs.

[0131] (2) Next, we performed serial transplantation experiments to evaluate the self-renewal capacity of LSCs. + Human AML cells were harvested from first-round AML recipients and cultured at equal numbers (0.4 × 10 6 ~0.5×10 6 In the serial transplantation experiment, AML cells collected from NSG mice treated with MMF alone or VEN+MMF showed higher hCD45 expression than AML cells collected from other groups. + The proportion of AML cells was lower and reconstitution was significantly less (Fig. 7E). Consistent with the inhibitory effect of MPA on the stem cell properties of LSCs shown in in vitro studies, MMF-treated 1 st Residual AML cells harvested from recipient mice showed impaired reconstitution ability in serial transplantation experiments.

[0132] (3) The effect of the combination of MMF and VEN on normal human hematopoiesis was evaluated. Purified CB-derived CD34 + DMSO, VEN, and the combination of VEN and MMF were administered to NSG mice in which normal human hematopoietic stem and progenitor cells had been xenotransplanted to reconstitute normal human hematopoiesis. The results confirmed that MMF did not enhance the cytotoxicity of VEN against normal human hematopoiesis in vivo (Fig. 7F).

[0133] These results suggest that the combination of MMF and VEN exerts a synergistic anti-AML effect in vivo without enhancing cytotoxicity against normal human hematopoietic stem cells.

[0134] Test Example 8: Inhibition of the de novo pathway significantly reduces the AML propagating potential of LSCs. (1) In vitro evaluation. (A) Inhibition of blast colony formation by MPA or VEN. Clinically VEN-resistant AML cells (VEN-resAML_#1) and VEN-resistant primary AML cells from a UPN9 patient were treated with MPA (an IMPDH1 inhibitor, a de novo pathway inhibitor) or VEN, respectively, and a blast colony formation assay was performed to evaluate the effect on colony formation. The results are shown in Figure 8A. These results demonstrate that inhibition of guanine nucleotide synthesis by IMPDH inhibition strongly inhibits the AML propagating potential of LSCs from primary AML cells evaluated in vitro. These results also demonstrate that suppression of IMPDH1 function inhibits the AML propagating potential of LSCs.

[0135] (B) Inhibition of blast-colony formation by IMPDH knockdown using shRNA. VEN-resistant primary AML cells collected from UPN2 and UPN4 patients were treated with various shRNAs (sh IMPDH1-1 (referred to as "sh IMPDH1#1" in Fig. 8B) and sh IMPDH1-2 (referred to as "sh IMPDH1#2" in Fig. 8B)) and subjected to a blast-colony formation assay to evaluate the effect on colony formation. The results are shown in Fig. 8B.

[0136] These results, similar to the results of the IMPDH1 function inhibition experiments using MPA, indicate that IMPDH1 gene silencing also strongly inhibits the AML reconstitution ability of LSCs from primary AML cells assessed in vitro. These results also demonstrate that suppression of IMPDH1 gene expression inhibits the AML reconstitution ability of LSCs.

[0137] (2) Evaluation by in vivo test: In the method described in (13)(C), human-derived AML cells (hCD45) in the bone marrow of mice transplanted with primary AML cells derived from clinically VEN-resistant human AML patients (UPN2, UPN3) treated with shRNA (sh IMPDH1-1, sh IMPDH1-2) (IMPDH1 knockdown treatment) were used. ＋ The results of comparing the proportions of AML cells are shown in Figure 8D.

[0138] These results indicate that suppression of IMPDH1 gene expression suppresses the AML reconstitution capacity of LSCs in vivo, and suggest that IMPDH1 inhibition is important for the AML reconstitution capacity of LSCs in vivo and may be a therapeutic target for AML.

[0139] Test Example 9: Inverse correlation between IMPDH1 and TP53 protein levels in VEN-resistant AML cells. The results of test method (18)(A) are shown in Figure 9A. As can be seen, in AML cells (TP53WT), the expression level of TP53 protein correlates with VEN resistance / sensitivity, and it was confirmed that the expression level of TP53 protein was high in VEN-sensitive AML cells, whereas the expression level of TP53 protein was low in VEN-resistant AML cells.

[0140] The results of test method (18)(B) are shown in Figure 9B. As can be seen from the figure, there was an inverse correlation between the expression level of TP53 protein and the ribosome biogenesis activity in VEN-resistant AML cells.

[0141] These results indicate that there is a negative correlation between ribosome biogenesis activity, which is positively regulated by IMPDH1 expression, and TP53 protein levels. In other words, enhanced ribosome biogenesis activity constantly suppresses the induction of IRBC in AML cells, thereby indicating the existence of a steady-state mechanism for negatively regulating TP53 function independent of gene mutations. These results also indicate that VEN-resistant AML acquires VEN resistance by enhancing IMPDH1 expression, suppressing IRBC activation and thereby allowing MDM2-mediated constitutive degradation of TP53 protein.

[0142] In this study, we demonstrated that enhanced ribosome biogenesis activity through guanine nucleotide biosynthesis is a novel, AML-specific molecular mechanism involved in AML pathogenesis in which IRBC activation is inhibited. Because IRBCs exert potent anti-AML effects through both TP53-dependent and -independent molecular mechanisms, induction of IRBCs by inhibiting guanine nucleotide biosynthesis should be a promising therapeutic approach for AML.

[0143] IRBCs are essential components of normal cellular structure, maintaining cellular homeostasis by monitoring the integrity of ribosome biogenesis and the dynamics of intracellular metabolic processes. Given that abnormal ribosomes are involved in cancer progression, eliminating cells with defective ribosomes through TP53-dependent cell cycle arrest and IRBC-mediated apoptosis induction is an important anti-tumor mechanism. In contrast to the tumor suppressor function of IRBCs, our experimental results demonstrate that induction of IRBCs stabilizes TP53 protein and represses MYC transcription in human AML, exerting potent anti-AML effects, particularly in combination with VEN. While triggers for IRBC activation include energy depletion, abnormal ribosome positioning, and nucleotide depletion, our study demonstrates that guanine nucleotide depletion robustly induces IRBCs in AML cells. Furthermore, AML cells were found to exhibit a significant dependency on the de novo pathway for guanine nucleotide synthesis, providing substantial rationale for targeting IMPDH1 and / or IMPDH2 as a strategy to induce IRBC in AML cells.

[0144] In addition to the use of IRBCs in AML treatment, this study revealed that enhanced ribosome biogenesis is a novel mechanism for efficiently suppressing TP53 function. Enhanced ribosome biogenesis enables AML cells to constitutively degrade TP53 protein via MDM2, promoting metabolic suppression of TP53 function even in the absence of TP53 mutations. Consequently, enhanced ribosome biogenesis may be characterized as a novel, mutation-independent mechanism of TP53 inhibition in AML.

[0145] Furthermore, LSCs showed significant increases in IMPDH1 and IMPDH2, regardless of VEN sensitivity. Inhibition of IMPDH1 and IMPDH2 strongly impaired the stem cell properties of human LSCs in vitro and in vivo, suggesting an important role for ribosome biogenesis via enhanced de novo guanine nucleotide biosynthesis in LSCs.

[0146] The findings of this study have important clinical implications. Our study employed a unique approach: restoring TP53 function using a clinically available IMPDH inhibitor. MMF, an IMPDH inhibitor, is commonly used in clinical settings to prevent graft-versus-host disease (GVHD) in AML patients after allogeneic stem cell transplantation (allo-SCT). Thus, the safety of MMF in AML patients has already been established.

[0147] In addition to stabilizing TP53 protein, induction of IRBCs by IMPDH inhibitors suppresses MYC transcription in a TP53-independent manner in AML. Furthermore, the concentrations of IMPDH inhibitors (MPA) that effectively inhibit guanine nucleotide biosynthesis in vitro closely matched the trough concentrations of MPA after allogeneic SCT in AML patients. These results suggest that the doses of IMPDH inhibitors currently administered in the clinical management of GVHD may also be therapeutically effective in AML.

[0148] These data suggest that the safety and therapeutic efficacy of VEN plus an IMPDH inhibitor combination therapy for AML patients may be predicted. In summary, this study provides novel evidence that ribosome biogenesis, driven by an enhanced de novo guanine biosynthetic pathway, is involved in the pathogenesis of AML. Given the critical role of ribosome biogenesis in regulating the properties of LSCs, targeting de novo guanine nucleotide biosynthesis represents a promising therapeutic approach to eliminate LSCs in AML.

[0149] On the other hand, in AML cells, IRBC activation has been shown to stabilize TP53 protein, suppress MYC, and inhibit OXPHOS-independent energy production through multiple molecular mechanisms, including glycolysis inhibition, thereby exhibiting a synergistic effect with VEN. Furthermore, conditions for IRBC activation are known to include energy depletion, ribosomal abnormalities, and nucleotide depletion. This disclosure has revealed that IRBC activation is strongly induced in AML cells by guanine nucleotide depletion. Generally, AML cells are known to rely on the de novo guanine nucleotide synthesis pathway rather than the salvage pathway. In this study, only inhibition of the de novo pathway demonstrated a potent anti-AML effect. In other words, the de novo guanine nucleotide synthesis system is a potent and specific driver of AML.

[0150] This study also revealed a novel molecular mechanism for TP53 inactivation in AML, which could be considered a new, independent TP53 suppression mechanism. Enhancement of the de novo pathway for guanine nucleotide synthesis allows AML cells to constantly degrade TP53 protein using MDM2, enabling metabolic suppression of TP53 function even in the absence of Tp53 mutations. In other words, enhanced ribosome biogenesis could be considered a novel, mutation-independent TP53 suppression mechanism in AML.

[0151] Furthermore, it has been reported that restoration of TP53 function strongly suppresses the function of CML LSCs, suggesting that TP53 suppression is important for maintaining the stemness of LSCs. In this study, LSCs also overexpressed IMPDH, and IMPDH inhibition strongly suppressed stemness in vitro and in vivo. These data suggest that enhanced ribosome biogenesis driven by de novo guanine nucleotide biosynthesis is an important TP53 suppression mechanism for AML LSCs. Furthermore, we confirmed that IRBC induction not only restored TP53 function but also suppressed MYC. Dual inhibition of TP53 and MYC is known to have a very strong effect on CML-LSCs.

[0152] In addition to the therapeutic application of IRBCs in AML, this study revealed that enhanced ribosome biogenesis is a novel mechanism for efficiently suppressing TP53 function. Enhanced ribosome biogenesis enables AML cells to constitutively degrade TP53 protein via MDM2, promoting metabolic suppression of TP53 function even in the absence of TP53 mutations. Consequently, enhanced ribosome biogenesis may be characterized as a novel, mutation-independent mechanism of TP53 inhibition in AML.

[0153] This study demonstrated the importance of ribosome biogenesis in LSCs. IMPDH1 / 2, the rate-limiting enzymes of de novo guanine nucleotide biosynthesis, was highly expressed in undifferentiated LSCs or ROS-low LSC fractions, regardless of VEN sensitivity. In fact, IMPDH1 / 2 inhibition strongly suppressed LSC activity in vitro and in vivo. This indicates that activation of the LSC-specific de novo guanine nucleotide biosynthetic pathway plays an important role in maintaining LSC activity. Furthermore, this study demonstrated the important role of ribosome biogenesis in LSCs through enhanced de novo guanine nucleotide biosynthesis.

[0154] Furthermore, LSCs enriched for an immature immunophenotype and reduced reactive oxygen species levels exhibited significantly elevated IMPDH1 and IMPDH2, rate-limiting enzymes in the de novo guanine nucleotide biosynthetic pathway, regardless of venetoclax sensitivity. Inhibition of IMPDH1 and IMPDH2 strongly impaired the stem cell properties of human LSCs in vitro and in vivo, suggesting the important role of ribosome biogenesis driven by enhanced de novo guanine nucleotide biosynthesis in LSCs.

[0155] SEQ ID NO: 1 represents the nucleotide sequence of the GAPDH forward primer, SEQ ID NO: 2 represents the GAPDH reverse primer, SEQ ID NO: 3 represents the IMPDH1 forward primer, SEQ ID NO: 4 represents the IMPDH1 reverse primer, SEQ ID NO: 5 represents the IMPDH2 forward primer, SEQ ID NO: 6 represents the IMPDH2 reverse primer, SEQ ID NO: 7 represents the HPRT1 forward primer, SEQ ID NO: 8 represents the HPRT1 reverse primer, SEQ ID NO: 9 represents the PNP forward primer, SEQ ID NO: 10 represents the PNP reverse primer, SEQ ID NO: 11 represents the pre-rRNA forward primer, and SEQ ID NO: 12 represents the pre-rRNA reverse primer. SEQ ID NO: 13 represents the nucleotide sequence of the barcoded oligo-dT-based primer used to prepare the library in BRB-seq. SEQ ID NOs: 14 and 15 represent the nucleotide sequences of the PCR primers used to amplify the tagmented DNA. SEQ ID NOs: 16 to 19 represent the nucleotide sequences of the sh-RNA used in Test Example 2. Specifically, SEQ ID NOs: 16 and 17 show the base sequences of sh-RNAs for IMPDH1-1 and IMPDH1-2, respectively, and SEQ ID NOs: 18 and 19 show the base sequences of sh-RNAs for IMPDH2-1 and IMPDH2-2, respectively.

Claims

1. A pharmaceutical composition for treating leukemia, comprising an IMPDH1 inhibitor as an active ingredient.

2. A pharmaceutical composition for treating leukemia described in claim 1, wherein the IMPDH1 inhibitor is at least one selected from the group consisting of compounds having an inhibitory effect on IMPDH1, anti-IMPDH1 antibodies and their antigen-binding fragments, antisense oligonucleotides, shRNA, and siRNA.

3. The pharmaceutical composition for treating leukemia according to claim 1, wherein the leukemia is acute myeloid leukemia.

4. The pharmaceutical composition for treating leukemia according to claim 1, wherein the leukemia is resistant to leukemia treatment with a BCL-2 inhibitor.

5. The pharmaceutical composition for treating leukemia according to claim 4, wherein the BCL-2 inhibitor is venetoclax.

6. The pharmaceutical composition for treating leukemia according to claim 1, which is used in combination with a BCL-2 inhibitor for the treatment of BCL2 inhibitor-resistant leukemia.

7. The pharmaceutical composition for treating leukemia according to claim 6, wherein the BCL-2 inhibitor is venetoclax.

8. The pharmaceutical composition for treating leukemia according to claim 1, wherein the IMPDH1 inhibitor has at least one effect on leukemia cells selected from the group consisting of (1) to (5): (1) IRBC induction effect, (2) IRBCC formation effect, (3) TP53 protein stabilization effect, (4) TIGAR induction effect, and (5) MYC transcription suppression effect.

9. The pharmaceutical composition for treating leukemia according to claim 1, wherein the IMPDH1 inhibitor is an agent used for at least one application selected from the group consisting of (a) to (e) against leukemia cells: (a) an IRBC inducer, (b) an IRBCC former, (c) a TP53 protein stabilizer, (d) a TIGAR inducer, or (e) a MYC transcription inhibitor.

10. A method for treating acute myeloid leukemia in a patient, comprising the step of administering a therapeutically effective amount of an IMPDH1 inhibitor to said patient.

11. The method for treating acute myeloid leukemia according to claim 10, wherein the patient is resistant to leukemia treatment with a BCL-2 inhibitor.

12. An IMPDH1 inhibitor for use in treating acute myeloid leukemia, preferably acute myeloid leukemia resistant to BCL-2 inhibitor treatment.

13. The IMPDH1 inhibitor described in claim 12, wherein the IMPDH1 inhibitor is at least one selected from the group consisting of compounds having an inhibitory effect on IMPDH1, anti-IMPDH1 antibodies and antigen-binding fragments thereof, antisense oligonucleotides, shRNA, and siRNA.

14. Use of at least one selected from the group consisting of an IMPDH1 inhibitor, preferably a compound having an inhibitory effect on IMPDH1, an anti-IMPDH1 antibody and its antigen-binding fragment, an antisense oligonucleotide, shRNA, and siRNA, for the manufacture of a pharmaceutical composition for treating acute myeloid leukemia.

15. A method for screening active ingredients of a pharmaceutical composition for treating leukemia, comprising the step of selecting, from a group of test compounds, compounds that have, on leukemia cells, (I) an IMPDH inhibitory effect, and / or (II) at least one effect selected from the group consisting of the following (1) to (5): (1) an IRBC-inducing effect, (2) an IRBCC-forming effect, (3) a TP53 protein-stabilizing effect, (4) a TIGAR-inducing effect, or (5) a MYC transcription-suppressing effect.

Images