A drug for cancer treatment and a method for discovering its active ingredient.

Abstract

The objective is to provide a novel agent for cancer treatment that can suppress the CDKAL1-dependent translation mechanism specific to cancer cells and / or cancer stem cells. [Solution] The above problems are solved by providing an agent for treating cancer containing as an active ingredient a component that inhibits the phosphorylation of the 43rd threonine of CDKAL1; a method for searching for drug targets for treating cancer, comprising the step of selecting a candidate protein that phosphorylates the 43rd threonine of CDKAL1 as a drug target for treating cancer; and a method for searching for candidate compounds for the active ingredient of an agent for treating cancer, comprising the step of selecting a test compound that inhibits the phosphorylation of the 43rd threonine of CDKAL1 as a candidate compound for the active ingredient of an agent for treating cancer.

Description

[Technical Field]

[0001] This disclosure relates to agents for cancer treatment and methods for searching for their active ingredients. [Background technology]

[0002] Traditionally, cytotoxic anticancer drugs that target cancer cells with active cell cycles have been widely used as anticancer agents. However, cytotoxic anticancer drugs have a strong effect on normal cells, and therefore, the occurrence of side effects is often a clinical problem.

[0003] On the other hand, in recent years, molecularly targeted drugs that act on specific molecules that determine the progression and activity of cancer have been widely used. For example, antibody drugs that target molecules localized on the cell membranes of cancer cells and cells that make up the tumor microenvironment are extremely popular. However, molecularly targeted drugs have a problem in that the rationale for applying them suddenly disappears for cancer types or patients in which the target molecule is poorly expressed.

[0004] These problems stem from a lack of evidence for targeting cancer cells, or from the fact that the methods are too selective and specific, thus lacking universality.

[0005] In contrast, if we can find molecular mechanisms that are specific to cancer cells but on which cancer cells are broadly dependent, we can expect to solve these problems. However, no anticancer drugs that can target such cancer cell-specific molecular mechanisms have yet been launched. This is thought to be due to the fact that the properties of cancer cells are not yet fully understood. If we can find molecular mechanisms on which cancer cells are broadly dependent and elucidate those molecular mechanisms, it is expected that they could become new drug targets for anticancer strategies.

[0006] Previous transcriptome analyses of various cancer types have revealed that cancer cells have a unique transcription phase (the phase from nuclear DNA to mRNA transcription). Now, thanks to recent technological advancements, it is gradually becoming clear that cancer cells also have a unique translation phase (the phase from mRNA to protein translation).

[0007] In particular, our research has revealed that in rhabdomyosarcoma, transcription factors that govern the cancer stem cell characteristics of cancer stem cells (e.g., SALL2 and YAP) are regulated not at the transcriptional level, but at the translational level. Furthermore, it has been discovered that a CDKAL1-dependent translational regulatory mechanism, originally studied as a factor involved in the pathogenesis of type 2 diabetes, plays a crucial role in the translational regulation of these transcription factors (e.g., Patent Document 1, Non-Patent Document 1). Importantly, the CDKAL1-dependent translational regulatory mechanism plays a vital role not only in rhabdomyosarcoma, but also in various other cancers such as malignant melanoma, malignant brain tumors, liver cancer, prostate cancer, and gastric cancer. In other words, by targeting the CDKAL1-dependent translational regulatory mechanism, which is specific to cancer cells but on which a relatively large number of cancer cells depend, it may be possible to provide innovative anticancer drugs unlike anything seen before.

[0008] According to the findings of the present inventors, the N-terminus of CDKAL1 is thought to be necessary for the efficient translation of transcription factors that govern cancer stem cell characteristics. However, many aspects of the mechanism of CDKAL1 activation remain unclear, and drug discovery targeting CDKAL1-dependent translational regulatory mechanisms has not been sufficiently achieved. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] International Publication No. 2002 / 124342 [Patent Document 2] Special Publication No. 2025-511404

Non-Patent Literature

[0010]

Non-Patent Literature 1

Summary of the Invention

Problems to be Solved by the Invention

[0011] The present invention has been made to solve the above problems of the prior art, and in one aspect, an object is to provide a new agent for cancer treatment that can suppress the CDKAL1-dependent translation mechanism specific to cancer cells and / or cancer stem cells.

Means for Solving the Problems

[0012] In the process of intensively researching and striving to solve the above problems, the inventors of the present invention focused on the post-translational modification of CDKAL1 and repeated many trials and errors. As a result, they found that the phosphorylation of the 43rd threonine from the N-terminus of CDKAL1, more specifically the N-terminus of CDKAL1, plays an important role in the formation of the translation initiation factor complex involved in CDKAL1. And as a result of further intensive research efforts, they succeeded in identifying the responsible kinase (phosphorylating enzyme) for phosphorylating the 43rd threonine from the N-terminus of CDKAL1 in cancer stem cells. Furthermore, they found that by suppressing the phosphorylation of the 43rd threonine from the N-terminus of CDKAL1, the CDKAL1-dependent translation mechanism is significantly suppressed and a cancer treatment effect is achieved.

[0013] That is, in one aspect, the present invention solves the above problems by providing an agent for treating cancer containing, as an active ingredient, a component that suppresses the phosphorylation of CDKAL1.

[0014] In one preferred embodiment, the phosphorylation of CDKAL1 may be the phosphorylation of the 43rd threonine of CDKAL1. The 43rd threonine of CDKAL1 may be the 43rd threonine from the N-terminus of CDKAL1, and more specifically, it may be the threonine corresponding to the 43rd threonine from the N-terminus in the amino acid sequence of wild-type CDKAL1. The amino acid sequence of wild-type human CDKAL1 is the amino acid sequence shown in Sequence ID No. 1 of the sequence listing. That is, the threonine corresponding to the 43rd threonine from the N-terminus in the amino acid sequence of wild-type CDKAL1 may be the threonine corresponding to the 43rd threonine from the N-terminus in the amino acid sequence shown in Sequence ID No. 1. According to our findings, CDKAL1 in which the 43rd threonine from the N-terminus is not phosphorylated (unphosphorylated CDKAL1) cannot form a translation initiation factor complex. In other words, a cancer treatment agent containing an active ingredient that inhibits the phosphorylation of CDKAL1, particularly the phosphorylation of the 43rd threonine from the N-terminus of CDKAL1, can suppress the expression of transcription factors that govern cancer stem cell characteristics at the translational level by inhibiting the formation of the translation initiation factor complex that involves CDKAL1, thereby exerting a therapeutic effect on cancer.

[0015] As shown in the experimental examples described later, the inventors have diligently conducted research and found that osimertinib suppresses the phosphorylation of the 43rd threonine from the N-terminus of CDKAL1, thereby suppressing the formation of CDKAL1-dependent translation initiation factor complexes in rhabdomyosarcoma cells. In other words, in one preferred embodiment, osimertinib may be the component that suppresses the phosphorylation of the 43rd threonine of CDKAL1. According to the findings of the inventors, osimertinib can inhibit the interaction between MYLK4 and CDKAL1, thereby suppressing the phosphorylation of the 43rd threonine of CDKAL1 by MYLK4.

[0016] Furthermore, in another aspect, the present invention solves the above problem by providing a method for searching for drug targets for treating cancer, which includes the step of selecting a candidate protein that phosphorylates the 43rd threonine of CDKAL1 as a drug target for treating cancer. Proteins that phosphorylate the 43rd threonine of CDKAL1, that is, the responsible kinases (phosphorylation enzymes) that phosphorylate the 43rd threonine of CDKAL1, are thought to activate CDKAL1-dependent translational regulatory mechanisms and play an important role in maintaining cancer stem cell properties. In other words, candidate proteins that phosphorylate the 43rd threonine of CDKAL1 are promising as drug targets for treating cancer, and by using such candidate proteins as drug targets, it may be possible to provide a new anticancer drug that can induce the loss of cancer stem cell properties through the suppression of CDKAL1-dependent translational regulatory mechanisms and exert a therapeutic effect on cancer.

[0017] Furthermore, in another aspect, the present invention solves the above problem by providing a method for searching for candidate compounds for active ingredients of an agent for treating cancer, which includes the step of selecting a test compound that inhibits the phosphorylation of the 43rd threonine of CDKAL1 by a kinase (phosphorylation enzyme) as a candidate compound for an active ingredient of an agent for treating cancer. As described above, CDKAL1 is activated by phosphorylation, more preferably by phosphorylation of the 43rd threonine from the N-terminus, and forms a translation initiation factor complex. That is, according to the search method in one aspect of the present invention, which selects a test compound that inhibits the phosphorylation of CDKAL1, more preferably by phosphorylation of the 43rd threonine from the N-terminus of CDKAL1, as a candidate compound for an active ingredient of an agent for treating cancer, a new candidate compound for an active ingredient of an anticancer agent that exhibits a therapeutic effect on cancer through a novel anticancer mechanism that causes loss of cancer stem cell characteristics by inhibiting the CDKAL1-dependent translation control mechanism may be provided.

[0018] Furthermore, the kinase used in the above-mentioned search method relating to one aspect of the present invention may be, for example, a kinase obtained using the drug target search method relating to another aspect of the present invention. By combining the two, drug discovery targeting CDKAL1-dependent translational control mechanisms can be accelerated. [Effects of the Invention]

[0019] According to one aspect of the present invention, a method for searching for candidate compounds for treating cancer, and / or a method for searching for drug targets for treating cancer, a novel agent for cancer treatment may be provided that can suppress the CDKAL1-dependent translation mechanism specific to cancer cells and / or cancer stem cells, thereby exhibiting a therapeutic effect on cancer. [Brief explanation of the drawing]

[0020] [Figure 1] (A) This figure shows the results of evaluating the expression levels of each protein in normal skeletal muscle cells (HSMM) and rhabdomyosarcoma cells (RD, RMS-YM, ICH-ERMS-1, RH30) by analyzing cell extracts of each cell type using Western blotting. The numbers below the gel image indicate the relative expression level, with the expression level in normal skeletal muscle cells (HSMM) set to 1.00. (B) This figure shows the results of evaluating the expression levels of each protein in RD cells co-transfected with siRNA against MYLK2 and siRNA against MYLK4 by analyzing cell extracts of each cell type using Western blotting and Phos-tag Western blotting. [Figure 2](A) This figure shows the results of evaluating the sphere formation ability (self-renewal ability) in a scaffold-independent environment after transfection of rhabdomyosarcoma cells, RD cells and RH30 cells, with siRNA against MYLK2 or MLYK4. (B) This figure shows the results of evaluating the expression level of SALL2 mRNA by qPCR and the expression levels of each protein by Western blotting in RD cells transfected with siRNA against MYLK2 or MLYK4. SALL2 is a transcription factor whose translation is initiated by a translation initiation factor complex involving CDKAL1, and CD133 is a cancer stem cell marker. (C) This figure shows the results of evaluating the antitumor effect of administering siRNA against MLYK4 in a xenograft model mouse using RD cells. [Figure 3](A) This figure shows the results of evaluating FLAG-tagged fusion proteins (FLAG-tagged MYLK2 and FLAG-tagged MYLK4) expressed in Expi293 cells, recovered, and purified, using SDS-PAGE and Oriole staining. As indicated by the triangular symbols in the figure, bands were detected at positions corresponding to the molecular weight of MYLK2 or MYLK4. (B) This figure shows the procedure for the in vitro kinase assay. (C) This figure compares wild-type CDKAL1 (indicated as "CDKAL1 WT"), a CDKAL1 mutant in which the 43rd threonine is replaced with alanine (indicated as "CDKAL1 T43A"), and a CDKAL1 mutant in which the N-terminus containing the UPF domain is cleaved (indicated as "CDKAL1 ΔN"). This figure also shows the results of evaluating the degree of phosphorylation of each CDKAL1 after performing the in vitro kinase assay using the procedure in (B) using Phos-tag Western blotting. (D) This figure shows the results of a screening experiment using an in vitro kinase assay in which FLAG-tagged MLYK4 was used instead of Myc-tagged CDKAL1 in the procedure of (B). The degree of phosphorylation of MYLK4 when kinase assays were performed in the presence of alkaline phosphatase (dephosphorylation enzyme BAP) or test compounds (ML-7, ML-9) was evaluated by (bottom) Western blotting and (top) Phos-tag Western blotting. (E) This figure shows the results of evaluating the formation of translation initiation factor complexes involving CDKAL1 in RD cells treated with ML-7 and ML-9 by pull-down with m7GTP beads and Western blotting. [Figure 4] This figure shows the results of screening a total of 2203 FDA-approved drugs using a firefly luciferase reporter plasmid incorporating the 5'-UTR sequence of SALL2. [Figure 5](A) This figure shows the procedure for an in vitro kinase assay using CDKAL1 and MYLK4. As shown in the figure, in this experiment, λ protein phosphatase was used to prepare a dephosphorylated control sample. (B) This is a Western blotting image showing the results of the in vitro kinase assay shown in (A). The numbers in the figure represent the ratio of the signal intensity of the band corresponding to CDKAL1pT43 (CDKAL1 with phosphorylated threonine at position 43) to the signal intensity of the band corresponding to CDKAL1, with the ratio when DMSO was applied set to 1.00. (C) This figure shows the results of co-immunoprecipitation of MYLK4 and CDKAL1 from cell lysates of RD cells transfected with Myc-tagged CDKAL1 and FLAG-tagged MYLK4, in or without the presence of osimertinib. The values ​​in the figure represent the ratio of the signal intensity of the band corresponding to Myc-tagged CDKAL1 (Myc-tag) to the signal intensity of the band corresponding to FLAG-tagged MYLK4 (Flag-tag), with the ratio when DMSO is applied set to 1.00. [Figure 6] (A) This figure shows the results of evaluating SALL2 mRNA expression levels by RT-qPCR (top) and SALL2 protein expression levels by Western blotting (bottom) in RD cells treated with EGF, or EGF and osimertinib. The RT-qPCR results are expressed as mean ± standard deviation (n=3). In the figure, "ns" represents p≧0.05 (vs. EGF "-" · Osimertinib "-"). (B) This figure shows the results of evaluating the self-renewal capacity of RD cells treated with osimertinib based on the number of spheres formed. The results are expressed as mean ± standard deviation (n=4). In the figure, "ns" represents p≧0.05 (vs. DMSO) and "***" represents p<0.001 (vs. DMSO). (C) This figure shows the results of evaluating the formation of CDKAL1-related translation initiation factor complexes in RD cells treated with osimertinib using a pull-down assay with m7GTP beads and Western blotting. [Figure 7](A,B) Figures showing the results of evaluating the IC50 of actinomycin D and vincristine in RD cells and AD-RD cells by cell viability assay. (C) Figure showing the results of evaluating the self-renewal capacity of RD cells and AD-RD cells based on the number of spheres formed. Results are expressed as mean ± standard deviation (n=4). In the figure, "****" represents p<0.0001 (vs.RD). (D) Figures showing the results of evaluating the protein-level expression levels of MYLK4, pT43 (CDKAL1 with phosphorylated threonine at position 43), SALL2, CD133, and CDKAL1 in RD cells and AD-RD cells by Western blotting. (E) Figure showing the results of evaluating the mRNA-level expression level of SALL2 in RD cells and AD-RD cells by qRT-PCR. [Figure 8](A) This figure shows the results of evaluating the self-renewal capacity of AD-RD cells treated with osimertinib based on the number of spheres formed. The results are expressed as mean ± standard deviation (n=4), and in the figure, "****" represents p<0.0001 (vs. DMSO). (B) (Upper panel) This figure shows the results of evaluating the tumorigenic capacity of AD-RD cells treated with actinomycin D and osimertinib based on the number of colonies formed. The results are expressed as mean ± standard deviation (n=3). In the figure, "*" represents p<0.05 (vs. DMSO), and "**" represents p<0.01 (vs. DMSO). (Lower panel) This figure shows the results of evaluating the phosphorylation status of CDKAL1 in AD-RD cells treated with actinomycin D and osimertinib by Western blotting. The numerical values ​​in the figure represent the ratio of the signal intensity of the band corresponding to pT43 (CDKAL1 with phosphorylated threonine at position 43) to the signal intensity of the band corresponding to CDKAL1, with the ratio when DMSO is applied set to 1.00. (C) This figure shows the results of evaluating the formation of the CDKAL1-related translation initiation factor complex in AD-RD cells treated with osimertinib using a pull-down assay with m7GTP beads and Western blotting. (D) This figure shows the results of evaluating the IC50 of actinomycin D in AD-RD cells in the presence and absence of osimertinib using a cell viability assay. (E) This figure shows the results of evaluating the expression of SALL2, CD133, and pT43 (CDKAL1 with phosphorylated threonine at position 43) in AD-RD cells treated with osimertinib using immunostaining. [Modes for carrying out the invention]

[0021] The present invention will be described in more detail below.

[0022] <Medications for cancer treatment> In one aspect, the present invention relates to an agent for cancer treatment that contains a component that inhibits the phosphorylation of CDKAL1 as an active ingredient.

[0023] In this disclosure, CDKAL1 means "Cdk5 regulator subunit associated protein 1-like 1". The inventors have found that CDKAL1 plays a role in mediating the binding of eukaryotic translation initiation factors eIF4A and eIF4G to eIF4E in cancer cells, particularly cancer stem cells, and that this action leads to the formation of a translation initiation factor complex. It has also been found that the genes whose translation is initiated by this translation initiation factor complex are deeply involved in maintaining the stem cell nature, tumorigenicity, and / or self-renewal ability of cancer stem cells (Patent Document 1, Non-Patent Document 1). In other words, by suppressing the translation mechanism involving CDKAL1, it may be possible to treat cancer by causing cancer stem cells to lose their stem cell nature, tumorigenicity, and / or self-renewal ability. In this disclosure, human CDKAL1 may be simply referred to as "CDKAL1".

[0024] In one preferred embodiment, the phosphorylation of CDKAL1 inhibited by an agent according to one aspect of the present invention may be the phosphorylation of the 43rd threonine of CDKAL1. The 43rd threonine of CDKAL1 may be the 43rd threonine from the N-terminus in the amino acid sequence of CDKAL1, and more specifically, it may be the threonine corresponding to the 43rd threonine from the N-terminus in the amino acid sequence of wild-type CDKAL1. The amino acid sequence of wild-type CDKAL1 is shown in Sequence ID No. 1 of the sequence listing. That is, the threonine corresponding to the 43rd threonine from the N-terminus in the amino acid sequence of wild-type CDKAL1 may be the threonine corresponding to the 43rd threonine from the N-terminus in the amino acid sequence shown in Sequence ID No. 1 of the sequence listing. Here, "the threonine corresponding to the 43rd threonine from the N-terminus in the amino acid sequence of wild-type CDKAL1" or "the threonine corresponding to the 43rd threonine from the N-terminus in the amino acid sequence shown in Sequence ID No. 1" refers to the threonine that, when the amino acid sequence of CDKAL1 expressed by the target cell is aligned with the amino acid sequence of wild-type CDKAL1 or the amino acid sequence shown in Sequence ID No. 1, is positioned at the same location as the 43rd threonine from the N-terminus in the amino acid sequence of wild-type CDKAL1 or the 43rd threonine in the amino acid sequence shown in Sequence ID No. 1. In other words, "the threonine corresponding to the 43rd threonine from the N-terminus in the amino acid sequence of wild-type CDKAL1" or "the threonine corresponding to the 43rd threonine in the amino acid sequence shown in Sequence ID No. 1" does not necessarily indicate the absolute position of the threonine in the amino acid sequence constituting CDKAL1, but is intended to indicate the relative position relative to the amino acid sequence of wild-type CDKAL1 or the amino acid sequence shown in Sequence ID No. 1.

[0025] For example, if the CDKAL1 expressed by the target cancer cells has an amino acid sequence in which one or more amino acid residues are deleted, inserted, added, or substituted compared to the amino acid sequence of wild-type CDKAL1 or Sequence ID No. 1, the absolute position of "the threonine corresponding to the 43rd threonine from the N-terminus in the amino acid sequence of wild-type CDKAL1" or "the threonine corresponding to the 43rd threonine from the N-terminus in the amino acid sequence of Sequence ID No. 1" may change accordingly. On the other hand, if the CDKAL1 expressed by the target cancer cells has the amino acid sequence of wild-type CDKAL1 or Sequence ID No. 1, then the "threonine corresponding to the 43rd threonine from the N-terminus in the amino acid sequence of wild-type CDKAL1" or "the threonine corresponding to the 43rd threonine from the N-terminus in the amino acid sequence of Sequence ID No. 1" of that CDKAL1 will coincide with the 43rd threonine from the N-terminus of the CDKAL1 expressed by the target cancer cells.

[0026] Amino acid sequence alignment can be performed by a person skilled in the art using appropriate methods. For example, programs such as FASTA and BLAST can be used.

[0027] The component that inhibits the phosphorylation of CDKAL1 contained in an agent for cancer treatment according to one aspect of the present invention may be any component that inhibits the phosphorylation of CDKAL1, preferably the phosphorylation of the above-mentioned specific amino acid residue of CDKAL1. For example, it may be an inhibitor of a kinase that phosphorylates the above-mentioned specific amino acid residue of CDKAL1, a nucleic acid capable of suppressing the expression of a kinase that phosphorylates the above-mentioned specific amino acid residue of CDKAL1, and / or a vector that expresses a nucleic acid capable of suppressing the expression of a kinase that phosphorylates the above-mentioned specific amino acid residue of CDKAL1. The component may be a component that inhibits only the phosphorylation of the above-mentioned specific amino acid residue of CDKAL1, or it may be a component that also inhibits the phosphorylation of amino acid residues other than the above-mentioned specific amino acid residue.

[0028] According to findings made by the present inventors, the kinase responsible for phosphorylating the 43rd threonine from the N-terminus of CDKAL1 may be myosin light chain kinase 2 (MYLK2) and / or myosin light chain kinase 4 (MYLK4). That is, in one preferred embodiment, the component that inhibits the phosphorylation of CDKAL1 contained in the agent according to one aspect of the present invention may be a component that inhibits the phosphorylation of CDKAL1 by MYLK2 and / or MYLK4. By inhibiting the phosphorylation by MYLK2 and / or MYLK4, which are the kinases responsible for phosphorylating the 43rd threonine from the N-terminus of CDKAL1, the phosphorylation of the 43rd threonine from the N-terminus of CDKAL1 can be suppressed.

[0029] In this disclosure, MYLK stands for "myosin light-chain kinase." MYLK is a serine / threonine kinase that primarily phosphorylates the regulatory light chain of myosin, and four isoforms are known: MYLK1, MYLK2, MYLK3, and MYLK4. In this disclosure, human MYLK may be simply referred to as "MYLK."

[0030] The component that inhibits the phosphorylation of CDKAL1 by MYLK2 and / or MYLK4 can be basically any component as long as it can inhibit the phosphorylation of CDKAL1 by MYLK2 and / or MYLK4, but preferably it may be, for example, an inhibitor of MYLK2 and / or MYLK4, a nucleic acid capable of suppressing the expression of MYLK2 and / or MYLK4, or a vector that expresses a nucleic acid capable of suppressing the expression of MYLK2 and / or MYLK4.

[0031] MYLK2 and / or MYLK4 inhibitors are inhibitors of the phosphorylation reaction by MYLK2 and / or MYLK4, and more preferably, inhibitors of the phosphorylation reaction of the specific amino acid residue (i.e., the 43rd threonine from the N-terminus of CDKAL1) by MYLK2 and / or MYLK4. MYLK2 and / or MYLK4 inhibitors can be essentially any substance as long as they inhibit the phosphorylation reaction by MYLK2 and / or MYLK4, and may be, for example, organic compounds, inorganic compounds, natural products, or artificially synthesized substances. Examples of inhibitors for MYLK2 and / or MYLK4 include, but are not limited to, naphthalene sulfonamide derivatives such as ML-7 (chemical name: 1-(5-iodonaphthalene-1-sulphonyl) 1H-hexahydro-1,4-diazepine hydrochloride) and its derivatives, ML-9 (chemical name: 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine) and its derivatives; peptides such as MLCK inhibitor peptide 18 (Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2); apigenin; K252A; K252BlStaurosporine; and others.

[0032] Nucleic acids capable of suppressing the expression of MYLK2 and / or MYLK4 refer to nucleic acids that target the MYLK2 and / or MYLK4 genes and suppress the expression of MYLK2 and / or MYLK4. Here, "suppression" of expression means a decrease in the expression level of mRNA and / or protein compared to a negative control (e.g., untreated cells). There are no particular restrictions on the degree of decrease in mRNA and / or protein expression, but it is preferable that the expression level of mRNA and / or protein compared to a negative control be, for example, 70% or less, 60% or less, or 50% or less.

[0033] Nucleic acids capable of suppressing the expression of MYLK2 and / or MYLK4 may include, but are not limited to, siRNA (small interfering RNA), shRNA (short hairpin RNA), antisense nucleic acids, or sgRNA (single guide RNA) for the MYLK2 or MYLK4 gene.

[0034] siRNA refers to double-stranded RNA that can suppress (knock down) the expression of a target gene through RNA interference. There are no particular restrictions on the number of base pairs that make up double-stranded RNA, but for example, they can be 18-30 base pairs, 20-27 base pairs, or 21-23 base pairs, and typically 21-23 base pairs. When siRNA is introduced into a cell, it forms an RNA-protein complex called the RNA-induced Silencing Complex (RISC) with the Argonaute protein, suppressing the expression of mRNA that has a sequence complementary to the antisense strand, which is one of the double strands that make up the siRNA. In other words, siRNA against the MYLK2 or MLYK4 gene may be double-stranded RNA containing RNA with a base sequence complementary to the mRNA of MYLK2 or MYLK4.

[0035] In this disclosure, "complementary" means that the first base can form a classical Watson-Crick base pair or a non-Watson-Crick base pair with the second base, thereby forming a hydrogen bond. Furthermore, in this disclosure, when two base sequences are said to be "complementary," this includes not only cases where all consecutive bases of the first base sequence are complementary to the same number of consecutive bases of the second base sequence, i.e., can form a hydrogen bond (this case may also be called "perfectly complementary"), but also cases where, for example, 70%, 80%, 90% or more, or 95% or more of the bases of the first base sequence can form a hydrogen bond with the bases of the second base sequence. In other words, in this disclosure, an siRNA for the MYLK2 or MYLK4 gene may contain RNA having a base sequence that is perfectly complementary to a portion of the mRNA that is the transcript of the MYLK2 or MYLK4 gene, or it may contain RNA having a base sequence in which one, two, or more bases have been changed from a perfectly complementary base sequence. In one preferred embodiment, the 3' ends of the sense strand and antisense strand constituting the siRNA may each have one to five, typically two to five, nucleotide or modified nucleotide overhangs. These overhangs may, for example, be two deoxythymidine (dTdT) molecules, but are not limited to these.

[0036] siRNAs targeting MYLK2, MYLK4, or other kinases can be appropriately designed by those skilled in the art based on the DNA sequence of the gene encoding each protein and / or the RNA sequence of its mRNA (e.g., A. Birmingham et al. Nature Protocols, 2007, 2, 2068-2078; E. Fakhr et al. Cancer Gene Therapy, 2016, 23, 73-82), or by using known software (e.g., Yuki Naito et al. Nucleic Acids Research, Volume 32, Issue suppl 2, 1, July 2004, Pages W124-W129; Simone Sciabola et al. PLoSONE 16(1):e0238753; siRNA Wizard™ (InvivoGen)). The DNA sequences of the MYLK2 or MYLK4 genes can be obtained from public databases, such as the National Center for Biotechnology Information (NCBI) database.

[0037] As described above, siRNA targeting the MYKL2 or MYKL4 gene can be appropriately designed by those skilled in the art, but commercially available siRNAs, such as those used in the experimental examples described later, may also be used.

[0038] On the other hand, shRNA is RNA with a hairpin structure. In cells, the hairpin structure of shRNA is cleaved, generating a double-stranded RNA of about 21-23 base pairs. Similar to siRNA, the generated double-stranded RNA suppresses the expression of mRNA having a sequence complementary to the antisense strand of the double-stranded RNA through RNA interference. That is, shRNA for MYLK2 or MYLK4 may be a hairpin-type RNA containing RNA with a base sequence complementary to the mRNA of MYLK2 or MYLK4, respectively. Similar to siRNA, shRNA for MYLK2, MYLK4, or any kinase can be appropriately designed by those skilled in the art.

[0039] Antisense nucleic acids (ASOs) are single-stranded DNA or RNA that, by hybridizing with the mRNA of a target gene, induce translational repression of that mRNA. That is, antisense nucleic acids against MYLK2 or MYLK4 may be single-stranded DNA or single-stranded RNA containing a base sequence complementary to the mRNA of MYLK2 or MYLK4, respectively. Antisense nucleic acids against MYLK2 or MYLK4 can be appropriately designed by those skilled in the art based on the DNA sequence and / or RNA sequence of MYLK2 or MYLK4 (J. HP Chan et al. Clinical and Experimental Pharmacology and Physiology, 2006, Vol.33, pages 533-540), and known software (e.g., Simone Sciabola et al. PLoS ONE 16(1):e0238753) may be used. Furthermore, antisense nucleic acids may be those to which RNA or DNA complementary to their sequence is bound. Such antisense nucleic acids to which complementary RNA or DNA is bound may include, for example, heteroduplex nucleic acids (DNA / RNA) or homoduplex nucleic acids (DNA / DNA) (K. Nishina et al. Nat. Commun. 2015, 6, 7969; Y. Asami et al. Molecular Therapy, 2021, 29, 838-847, etc.).

[0040] Furthermore, sgRNA is a single-stranded RNA having a sequence complementary to the DNA of the target gene. When sgRNA is introduced into cells together with a specific endonuclease, the DNA double-strand having a sequence complementary to the sgRNA is cleaved, and the gene encoded by that DNA can be specifically knocked out. In a preferred embodiment, the endonuclease is a Cas9 nuclease derived from Streptococcus pyogenes, or a variant thereof. sgRNA can be appropriately designed by those skilled in the art based on the DNA sequence of the target gene.

[0041] On the other hand, vectors expressing nucleic acids capable of suppressing the expression of MYLK2 and / or MYLK4 may be, for example, expression vectors expressing the aforementioned siRNA, shRNA, antisense nucleic acids, or sgRNA. Examples of such expression vectors include plasmids, cosmids, phagemids, and viral vectors. Examples of viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and Sendai virus vectors. These expression vectors can be appropriately prepared by those skilled in the art (G. Sui et al., Proc. Natl. Acad. Sci. USA 2002, 99(8), 5515-5520).

[0042] Although MYLK2 and MYLK4 have been described above as examples of kinases that phosphorylate the 43rd threonine of CDKAL1, MYLK2 and / or MYLK4 are not the only kinases that phosphorylate the 43rd threonine of CDKAL1. For example, the kinase that phosphorylates the 43rd threonine of CDKAL1 may be a kinase obtained as a drug target by the discovery method described later.

[0043] In one preferred embodiment, the component that inhibits the phosphorylation of CDKAL1 contained in an agent relating to one aspect of the present invention may be osimertinib. "Osimertinib" is a compound having the chemical structure represented by the following formula, and its chemical name is N-(2-{2-dimethylaminoethyl-methylamino}-4-methoxy-5-{[4-(1-methylindole-3-yl)pyrimidine-2-yl]amino}phenyl)propa-2-enamide. A method for synthesizing osimertinib is described, for example, in Patent Document 2.

[0044] [ka]

[0045] Osimertinib may also be supplied in the form of a pharmaceutically acceptable salt, for example, as a mesylate. The mesylate of osimertinib is sold under the product name "Tagrisso TM It is marketed as "[product name]".

[0046] As shown in the experimental examples described later, osimertinib inhibits the phosphorylation of the 43rd threonine from the N-terminus of CDKAL1 by MYLK4 in rhabdomyosarcoma by inhibiting the binding of MYLK4 to CDKAL1, thereby significantly reducing the cancer stem cell properties of rhabdomyosarcoma cells. In other words, osimertinib can be particularly suitably used as an active ingredient in a drug for the treatment of rhabdomyosarcoma. Osimertinib is a third-generation EGFR tyrosine kinase inhibitor (TKI) and is approved for the treatment of EGFR gene mutation-positive non-small cell lung cancer (NSCLC) (e.g., Patent Document 2). However, to the best of our knowledge, it is not known that osimertinib inhibits the phosphorylation of the 43rd threonine from the N-terminus of CDKAL1 by MYLK4 by inhibiting the binding of MYLK4 to CDKAL1, and is therefore useful in the treatment of rhabdomyosarcoma. This disclosure provides a new use for osimertinib in cancers other than non-small cell lung cancer that are EGFR gene mutation-positive.

[0047] Furthermore, according to findings made by the present inventors, in cancer stem cells, preferably rhabdomyosarcoma, treated with osimertinib, the translation mechanism involving CDKAL1 is suppressed, and the self-renewal and / or tumorigenicity of cancer stem cells is reduced, attenuating their cancer stem cell characteristics, and they may also show high sensitivity to other anticancer agents. That is, in one preferred embodiment, an agent according to one aspect of the present invention, containing osimertinib as an active ingredient, can be used to improve drug resistance in cancer, preferably rhabdomyosarcoma. Drug resistance may be, for example, resistance to actinomycin D or vincristine. It may also be resistance to appropriate RNA polymerase inhibitors, topoisomerase inhibitors, or microtubule inhibitors used for the treatment of cancer.

[0048] In one preferred embodiment, osimertinib may be used in combination with other anticancer agents. There are no particular restrictions on the anticancer agents used in combination with osimertinib, but for example, when used for rhabdomyosarcoma, it may be one or more selected from vincristine, actinomycin D, cyclophosphamide, ifosfamide, etoposide, doxorubicin, topotecan, irinotecan, and melphalan, preferably one or more selected from vincristine, actinomycin D, and cyclophosphamide, more preferably actinomycin D.

[0049] Rhabdomyosarcoma is a rare and refractory sarcoma that occurs in children and young adults, in which cells differentiating into rhabdomyosarcoma become malignant. Research into its molecular mechanisms and the development of treatments are still insufficient. Therefore, the current treatment for rhabdomyosarcoma involves relatively classic anticancer drugs with significant side effects. In contrast, osimertinib, which significantly reduces the cancer stem cell properties of rhabdomyosarcoma cells and can increase their sensitivity to other anticancer drugs, may allow for a reduction in the amount of anticancer drugs used, thereby reducing concerns about side effects and enabling more effective treatment of rhabdomyosarcoma.

[0050] An agent relating to one aspect of the present invention is primarily an agent for treating cancer and can be used for treating cancer. In this disclosure, "treatment" of cancer may include not only complete cure of cancer, but also reduction of cancer size, delay of cancer growth or progression, and / or alleviation or improvement of cancer symptoms. Furthermore, according to the findings of the inventors, suppressing the translation mechanism involving CDKAL1 can suppress cancer stem cell properties and / or drug resistance. That is, in one embodiment, an agent relating to one aspect of the present invention can be suitably used to suppress cancer stem cell properties and / or drug resistance. As mentioned above, in one embodiment, an agent relating to one aspect of the present invention may be used in combination with other anticancer agents.

[0051] The cancer to which the agent according to one aspect of the present invention can be applied is basically any cancer, as long as the translation mechanism involving CDKAL1 is activated in the cancer cells and / or cancer stem cells that constitute the cancer. However, in one preferred embodiment, it is preferable that the cancer is one to which CDKAL1 is overexpressed. Here, "overexpression" of CDKAL1 means that the amount of CDKAL1 expressed at the protein level is increased compared to normal cells. There are no particular restrictions on the degree of increase in expression, but it is preferable that it is, for example, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, or 5.0 times or more compared to normal cells. It is preferable that the normal cells used as the basis for comparison are normal cells of the same type as the cancer cells.

[0052] The expression level of a protein in a cell can be appropriately evaluated by those skilled in the art. For example, as shown in the experimental examples described later, it may be quantified using Western blotting. When using Western blotting, the signal intensity of the band corresponding to the target protein is measured in cancer cells and normal cells, and the signal intensity in normal cells is compared with that of cancer cells. In Western blotting, the signal intensity of each band reflects the concentration of the protein corresponding to that band.

[0053] Furthermore, in one preferred embodiment, the cancer to which the agent for treating cancer according to one aspect of the present invention may be applied is preferably a cancer in which MYLK2 and / or MYLK4 are overexpressed. There are no particular limitations on the degree of increase in expression, but it is preferable that it is, for example, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, or 5.0 times or more compared to normal cells.

[0054] There are no particular limitations on the type of "cancer" to which the agent relating to one aspect of the present invention can be used. It may be any of the following: hematopoietic malignancies such as leukemia, lymphoma, and myeloma; malignancies arising from epithelial cells such as lung cancer, breast cancer, gastric cancer, and colorectal cancer (carcinoma); and malignancies arising from non-epithelial cells such as osteosarcoma, chondrosarcoma, rhabdomyosarcoma, and leiomyosarcoma (sarcoma). In one preferred embodiment, it is preferable that the agent be a sarcoma or an epithelial malignancy, more preferably a malignant brain tumor such as rhabdomyosarcoma or glioma, and even more preferably rhabdomyosarcoma. The rhabdomyosarcoma may be of the fetal type, alveolar type, pleomorphic type, or spindle-shaped cell (sclerosing type). Experimental examples described later show that CDKAL1, MYLK2, and MYLK4 are overexpressed in rhabdomyosarcoma.

[0055] In one preferred embodiment, an agent relating to one aspect of the present invention may be provided in the form of a pharmaceutical composition comprising, depending on its use, water, buffering agents (e.g., phosphate buffering agents, borate buffering agents, citrate buffering agents, tartaric acid buffering agents, amino acids, etc.), preservatives (e.g., quaternary ammonium salts such as benzalkonium chloride, parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, benzyl alcohol, sorbic acid and its salts, thimerosal, parabens, etc.), chelating agents (e.g., sodium edetate, citric acid, etc.), antioxidants (e.g., sodium bisulfite, sodium sulfite, sodium pyrosulfite, etc.), and / or one or more other pharmaceutically acceptable components commonly found in pharmaceuticals. Needless to say, such pharmaceutical composition may also be a pharmaceutical composition for the treatment of cancer.

[0056] There are no particular restrictions on the route of administration of the agent relating to one aspect of the present invention, and an appropriate route of administration may be selected depending on the cancer to which it is applied. The route of administration may be oral or parenteral, and examples include, but are not limited to, oral administration, sublingual administration, intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, and local administration.

[0057] Furthermore, there are no particular restrictions on the dosage form of the agent relating to one aspect of the present invention, and an appropriate dosage form may be selected depending on the type of cancer to which it is applied and the route of administration. Examples of dosage forms suitable for oral administration include, but are not limited to, tablets, capsules, powders, granules, and syrups. Examples of dosage forms suitable for parenteral administration include, but are not limited to, injectables such as solution-type injectables, suspension-type injectables, and injectables prepared at the time of use; and poultices such as tapes and poultices.

[0058] <Methods for discovering drug targets to treat cancer> In another aspect, the present invention relates to a method for discovering drug targets for treating cancer. The method for discovering drug targets for treating cancer according to one aspect of the present invention may include the step of selecting a candidate protein that phosphorylates the 43rd threonine of CDKAL1 as a drug target for treating cancer. That is, the method for discovering drug targets for treating cancer according to one aspect of the present invention is characterized by selecting a protein that phosphorylates the 43rd threonine of CDKAL1 as a drug target, and the other steps are not particularly limited.

[0059] According to the findings of the present inventors, phosphorylation of CDKAL1, more specifically, phosphorylation of the 43rd threonine from the N-terminus of CDKAL1, plays an important role in the formation of a translation initiation factor complex that regulates transcription factors involved in maintaining stem cell characteristics at the translational level in cancer stem cells. In other words, proteins responsible for such CDKAL1 phosphorylation in cancer stem cells can be important drug targets. The method for searching for drug targets for treating cancer, according to one aspect of the present invention, is based on the above findings discovered by the present inventors.

[0060] Candidate proteins can be basically any protein, but are preferably kinases. Candidate kinases can be basically any protein, but are, for example, protein kinases of the CAMK group, STE group, AGC group, or TKL group, and are more preferably protein kinases of the same CAMK group as MYLK2 and MYLK4. Kinases that can act on a given substrate can be predicted by known methods based on phosphate proteomics (e.g., Jared L. Johnson et al. Nature 2003 Jan 11; 613, 759-766; Tomer M. Yaron-Barir et al. Nature 2024 May 8; 629, 1174-1181, etc.). That is, in one preferred embodiment, the search method according to one aspect of the present invention may include a step of preparing a library of candidate proteins based on kinase prediction based on phosphate proteomics, for example.

[0061] "Drug target" means a target for drug discovery, and unless otherwise specified, it more specifically means a protein that is a target for drug discovery. A drug target for cancer treatment means a target for drug discovery for treating cancer, and unless otherwise specified, it means a protein that is a target for drug discovery for treating cancer. Furthermore, since the exploration method according to one aspect of the present invention selects a protein responsible for the phosphorylation of CDKAL1, or more specifically, the phosphorylation of the 43rd threonine from the N-terminus of CDKAL1, as a drug target, it can also be said that the phosphorylation reaction of CDKAL1 by the said protein, or more specifically, the phosphorylation reaction of the 43rd threonine from the N-terminus of CDKAL1, is the drug target.

[0062] In one preferred embodiment, the above-mentioned search method relating to one aspect of the present invention is: A process to suppress the expression of candidate proteins in cancer cells, If the degree of CDKAL1 phosphorylation in cancer cells with suppressed expression of the candidate protein decreases compared to when the expression of the candidate protein is not suppressed, the candidate protein is selected as a drug target for treating cancer. This may be a method characterized by the following:

[0063] Methods for suppressing the expression of any protein in cancer cells are well known and can be carried out as appropriate by those skilled in the art. There are no particular limitations on the specific method, but for example, a substance that suppresses the expression of the target protein may be brought into contact with the cancer cells. The substance that suppresses the expression of the target protein may be, but is not limited to, nucleic acids that can suppress the expression of the protein, such as siRNA, shRNA, antisense nucleic acids, and sgRNA that target the protein.

[0064] Nucleic acids capable of suppressing the expression of candidate proteins may be introduced into cancer cells in combination with appropriate methods. There are no particular restrictions on the specific methods used, and an appropriate method should be selected depending on the type, characteristics, and environment of the cancer cells to be introduced. Examples of such methods include physical introduction methods such as electroporation, sonoporation, and microinjection; chemical introduction methods such as calcium phosphate, lipofection using liposomes, and transfection methods using appropriate delivery means such as cationic lipids, lipidoids, cationic polymers, membrane-permeable peptides, antibodies, antibody fragments, proteins, nanoparticles, microparticles, emulsions, polymer micelles, and dendrimers.

[0065] After introducing nucleic acids capable of suppressing the expression of candidate proteins by an appropriate method, the expression of the candidate proteins is suppressed by incubation for a predetermined time. There are no particular restrictions on the predetermined time, but for example, it may be 12 to 120 hours, preferably 24 to 96 hours, and more preferably 48 to 96 hours. The suppression of protein expression can be confirmed by an appropriate method, such as Western blotting. If necessary, instead of directly quantifying the amount of protein expression, the suppression of protein expression may be evaluated by quantifying the mRNA of the target protein by qPCR or the like.

[0066] In the above-described search method relating to one aspect of the present invention, a candidate protein can be selected as a drug target for treating cancer if, when the expression of the candidate protein is suppressed, the degree of CDKAL1 phosphorylation in cancer cells where the expression of the candidate protein is suppressed decreases compared to when the expression of the candidate protein is not suppressed. "Degree of phosphorylation" means the amount of phosphorylated CDKAL1, more specifically, the amount of CDKAL1 with threonine 43 phosphorylated. A decrease in the degree of CDKAL1 phosphorylation when the expression of the candidate protein is suppressed compared to when the expression of the candidate protein is not suppressed means that the presence of the candidate protein is involved in CDKAL1 phosphorylation, and that the candidate protein can be an important drug target.

[0067] The degree of CDKAL1 phosphorylation in cancer cells can be evaluated by those skilled in the art using appropriate methods. There are no particular restrictions on the specific method, but for example, a cell extract of cancer cells can be prepared according to a conventional method, and the amount of phosphorylated CDKAL1 contained in the cell extract can be measured by Western blotting, ELISA, mass spectrometry, pull-down assay, immunoprecipitation, etc.

[0068] This is merely one example, but as shown in the experimental example described later, cell extracts may be separated and processed by SDS-PAGE, transferred to a membrane, and then proteins detected. Here, SDS-PAGE is a Phos-tag TM SDS-PAGE may also utilize phosphate group scavenging molecules such as (1,3-bis[bis(pyridin-2-yl methyl)amino]propan-2-olato dizinc(II) complex) or its derivatives. Phos-tag TM SDS-PAGE, which utilizes phosphate group scavenging molecules, allows for clearer separation of phosphorylated and non-phosphorylated proteins based on differences in electrophoretic mobility. Proteins transferred to the membrane can be detected using antibodies that bind to target proteins, such as anti-CDKAL1 antibodies, anti-phosphorylated CDKAL1 antibodies, and anti-phosphorylated threonine antibodies, according to conventional methods, such as luminescence or colorimetric detection. However, the detection methods for proteins transferred to the membrane are not limited to these. For example, Phos-tag TM A quantifiable label can be attached to the phosphorylated protein transferred to the membrane via a phosphate group scavenging molecule, and the phosphorylated protein transferred to the membrane may be detected. Methods for detecting phosphorylated proteins using phosphate group scavenging molecules are described, for example, in Kinoshita, Eiji et al., Biochemistry, Vol. 85, No. 6, pp. 447-455, 2013; Emiko Kinoshita-Kikuta et al. Methods Mol. Biol. 2016, 1355, 17-29; Eiji Kinoshita et al. Molecular & Cellular Proteomics, 2006, Vol. 5, Issue 4, P749-757; Kazuki Inamori et al. Analytical Chemistry 2005, 77, 13, 3979-3985.

[0069] In one preferred embodiment, the degree of phosphorylation of wild-type CDKAL1 and a mutant CDKAL1 in which the 43rd threonine is replaced with another amino acid residue may be compared. This allows for easy evaluation of the degree of phosphorylation of the 43rd threonine in CDKAL1.

[0070] Furthermore, in one preferred embodiment, the above-mentioned search method relating to one aspect of the present invention is The process includes contacting the candidate protein with CDKAL1, This method may involve selecting the candidate protein as a drug target for treating cancer if the degree of phosphorylation of CDKAL1 increases when it is in contact with the candidate protein compared to when it is not in contact with the candidate protein.

[0071] In the above-described search method relating to one aspect of the present invention, a candidate protein can be selected as a drug target for treating cancer if, when the candidate protein is brought into contact with CDKAL1, the degree of phosphorylation of CDKAL1 in contact with the candidate protein increases compared to when the candidate protein is not brought into contact with CDKAL1. As mentioned above, "degree of phosphorylation" means the amount of phosphorylated CDKAL1, more preferably the amount of CDKAL1 with threonine at position 43 phosphorylated. An increase in the degree of phosphorylation of CDKAL1 when brought into contact with the candidate protein compared to when the candidate protein is not brought into contact with CDKAL1 means that the presence of the candidate protein is involved in the phosphorylation of CDKAL1, indicating that the candidate protein can be an important drug target.

[0072] There are no particular restrictions on the specific method of contacting the candidate protein with CDKAL1, but for example, a solution containing the candidate protein and CDKAL1 can be prepared and allowed to stand for a predetermined time. There are no particular restrictions on the composition of the solution containing the candidate protein and CDKAL1, but it is preferable to include at least a phosphate donor such as ATP (adenosine triphosphate). The candidate protein transfers the phosphate group of the phosphate donor to a predetermined amino acid residue of CDKAL1.

[0073] In one preferred embodiment, CDKAL1 is preferably dephosphorylated before contact with the candidate protein. Here, "dephosphorylation" means removing the phosphate group attached to CDKAL1. Dephosphorylation of CDKAL1 can be carried out, for example, by contacting CDKAL1 with a dephosphorylating enzyme. Examples of dephosphorylating enzymes that can be used include, but are not limited to, bacterial alkaline phosphatases such as alkaline phosphatase (BAP) derived from E. coli, alkaline phosphatase (CIAP) derived from calf small intestine, and alkaline phosphatase (SAP) derived from shrimp.

[0074] Furthermore, in one preferred embodiment, the exploration method according to one aspect of the present invention may use CDKAL1 to which a first substance is bound and a support to which a second substance capable of binding to the first substance is bound. By using CDKAL1 to which a first substance is bound and a support to which a second substance capable of binding to the first substance is bound, the separation and purification of CDKAL1 become easier.

[0075] The first substance can be basically any substance that can bind to the second substance, and there are no particular restrictions on its type, but it is preferable that it can be expressed by fusing with a protein (CDKAL1). An example of such a first substance is FLAG. TMThis includes, but is not limited to, peptide tags such as tags, Myc tags, HA tags, and His tags. Furthermore, the methods for preparing tag fusion proteins are well known, and those skilled in the art can prepare tag fusion proteins for appropriate proteins.

[0076] Similarly, the second substance can be basically anything that can combine with the first substance, and there are no particular restrictions on its type. To give an example, for instance, the first substance could be FLAG TM In the case of peptide tags such as tags, Myc tags, and HA tags, corresponding antibodies, i.e., anti-FLAG tag antibodies, anti-Myc tag antibodies, and anti-HA tag antibodies, may be used as the second substance. Furthermore, for example, if the first substance is a His tag, a chelate ligand coordinated with a metal ion such as a nickel ion may be used as the second substance. The binding of these antibodies to the corresponding tags is reversible, and the proteins bound via antibody-tag binding can be eluted and separated according to conventional methods.

[0077] The support to which the second substance is bound can be basically any kind, but it is preferably a solid support. The solid support may be, for example, a plate such as an ELISA plate or fine particles such as metal particles, but it is preferably fine particles, and more preferably magnetic beads. When the solid support is fine particles, it can support a relatively large amount of protein, and the protein bound to the support via the binding of the first substance and the second substance can be easily separated and washed, for example by centrifugation. In particular, when the fine particles that are the solid support are magnetic beads, the protein bound to the support via the binding of the first substance and the second substance can be easily separated and washed, for example by adsorbing the support using a magnet such as a commercially available magnetic stand.

[0078] When CDKAL1 bound to a first substance and a support bound to a second substance capable of binding to the first substance are used, the exploration method according to one aspect of the present invention preferably includes a step of contacting the CDKAL1 bound to the first substance and the support bound to the second substance capable of binding to the first substance. This step is a step of binding the CDKAL1 bound to the first substance and the support bound to the second substance capable of binding to the first substance, and after binding the CDKAL1 bound to the first substance and the support bound to the second substance capable of binding to the first substance by this step, the CDKAL1 may be dephosphorylated and / or brought into contact with a kinase. In addition, when expressing CDKAL1 bound to the first substance in a predetermined cell and separating and purifying the CDKAL1 bound to the first substance from the cell extract, the CDKAL1 bound to the first substance and the support bound to the second substance capable of binding to the first substance may be brought into contact.

[0079] Furthermore, when a CDKAL1 to which a first substance is bound and a support to which a second substance capable of binding to the first substance is bound are used, the exploration method according to one aspect of the present invention may further include a step of releasing the CDKAL1 to which the first substance is bound and the support to which the second substance capable of binding to the first substance is bound. This step is a step of releasing the CDKAL1 to which the first substance is bound from the support, and the degree of phosphorylation of the released CDKAL1 can be analyzed.

[0080] There are no particular limitations on the specific method for releasing CDKAL1 to which the first substance is bound and the support to which the second substance capable of binding to the first substance is bound. Those skilled in the art can adopt an appropriate method depending on the combination of the first and second substances. As an example, if the first substance is the peptide tag described above, it can be incubated in a predetermined buffer such as a buffer containing a surfactant or a chelating agent, or incubated in a buffer containing a predetermined enzyme that degrades the peptide tag.

[0081] The degree of phosphorylation of CDKAL1 can be determined by a person skilled in the art using appropriate methods. Since the specific methods are as described above, a detailed explanation is omitted here.

[0082] <Method for discovering candidate compounds for active ingredients in cancer treatment agents> Furthermore, in another aspect of the present invention, the present invention relates to a method for searching for candidate compounds for active ingredients of an agent for treating cancer. The method for searching for candidate compounds for active ingredients of an agent for treating cancer according to another aspect of the present invention may include the step of selecting a test compound that inhibits the phosphorylation of threonine at position 43 of CDKAL1 from among test compounds as a candidate compound for the active ingredient of an agent for treating cancer. That is, the method for searching for candidate compounds for active ingredients of an agent for treating cancer according to one aspect of the present invention is characterized by selecting a test compound that inhibits the phosphorylation of threonine at position 43 of CDKAL1 as a candidate compound for the active ingredient of an agent for treating cancer, and other steps are not particularly limited.

[0083] A "candidate compound" for the active ingredient of a drug used to treat cancer may, for example, be a compound that is included as an active ingredient in a drug used to treat cancer, and / or a hit compound, seed compound, or lead compound of said compound, which may serve as a basis for developing the active ingredient of a drug used to treat cancer.

[0084] Candidate compounds or test compounds can be virtually any type of compound. For example, they may be organic or inorganic compounds, natural products or synthesized substances. Examples of organic compounds include, but are not limited to, amino acids, peptides, proteins, antibodies, antibody fragments, nucleotides, oligonucleotides, nucleic acids, sugars, oligosaccharides, polysaccharides, and lipids. There are no particular restrictions on the molecular weight of organic compounds; they may be low-molecular-weight compounds up to about 500, medium-molecular-weight compounds between 500 and 1000, or high-molecular-weight compounds.

[0085] As described above, the phosphorylation of CDKAL1, more specifically the phosphorylation of the 43rd threonine from the N-terminus of CDKAL1, plays a crucial role in the formation of translation initiation factor complexes that regulate transcription factors involved in maintaining stem cell characteristics at the translational level in cancer stem cells. In other words, compounds that suppress such CDKAL1 phosphorylation in cancer stem cells may be useful as active ingredients, hit compounds, seed compounds, or lead compounds for cancer treatment.

[0086] In one preferred embodiment, the above-mentioned search method relating to one aspect of the present invention is: The process involves contacting the kinase with CDKAL1 in the presence of the test compound, The process includes a step of evaluating the degree of phosphorylation of CDKAL1 after contact with the kinase, This method is characterized by selecting the test compound as a candidate compound for an active ingredient of a drug for treating cancer when the degree of phosphorylation of CDKAL1 in contact with the kinase decreases compared to when the test compound is not present.

[0087] There are no particular restrictions on the specific method of contacting the kinase and CDKAL1 in the presence of the test compound, but for example, a solution containing the kinase and CDKAL1 can be prepared and allowed to stand for a predetermined time. There are no particular restrictions on the composition of the solution containing the kinase and CDKAL1, but it is preferable that it contains at least a phosphate donor component such as ATP (adenosine triphosphate). The kinase transfers the phosphate group of the phosphate donor to a predetermined amino acid residue of CDKAL1.

[0088] The kinase (phosphorylation enzyme) used in this process can be basically any kinase that phosphorylates the 43rd threonine from the N-terminus of CDKAL1. Such kinases may be, for example, MYLK2, MYLK4, or the CAMK group protein kinases, STE group protein kinases, AGC group protein kinases, or TKL group protein kinases mentioned above, but MYLK2 and / or MYLK4 are more preferred. Note that one kinase or two or more kinases may be used in this process.

[0089] Furthermore, in one preferred embodiment, CDKAL1 is preferably dephosphorylated before contact with the kinase. The definition and method of "dephosphorylation" have already been described.

[0090] Furthermore, in one preferred embodiment, the exploration method according to one aspect of the present invention may use CDKAL1 to which a first substance is bound and a support to which a second substance capable of binding to the first substance is bound. When CDKAL1 to which a first substance is bound and a support to which a second substance capable of binding to the first substance is bound is used, the exploration method according to one aspect of the present invention preferably includes a step of bringing the CDKAL1 to which the first substance is bound and the support to which the second substance capable of binding to the first substance is bound into contact. As already described in the description of the drug target exploration method according to another aspect of the present invention, if the CDKAL1 to which the first substance is bound and the support to which the second substance capable of binding to the first substance is bound are then dephosphorylated or brought into contact with the kinase, the removal and exchange of buffers and reaction solutions becomes easy, which is very convenient. The first substance, the second substance, the support, etc., are as already described in the description of the drug target exploration method according to another aspect of the present invention.

[0091] Furthermore, in this case, the exploration method according to one aspect of the present invention may further include a step of releasing CDKAL1 to which the first substance is bound and a support to which a second substance capable of binding to the first substance is bound. This step is a step of releasing CDKAL1 to which the first substance is bound from the support, and the degree of phosphorylation can be analyzed using the CDKAL1 released by this step. The specific method for releasing CDKAL1 to which the first substance is bound and a support to which a second substance capable of binding to the first substance is bound, as well as the method for analyzing the degree of phosphorylation, have already been described in the description of the drug target exploration method according to another aspect of the present invention, so a detailed explanation is omitted here.

[0092] Furthermore, in one aspect, the present invention, A method for searching for candidate compounds for active ingredients of drugs used to treat cancer, A step of incubating MYLK2 and / or MYLK4 in the presence of the test compound, A step of evaluating the degree of phosphorylation of MYLK2 and / or MYLK4, A step of selecting the test substance as a candidate compound for treating cancer when the degree of phosphorylation of MYLK2 and / or MYLK4 is reduced compared to when the test compound is not present, This also provides a method that includes this.

[0093] According to the findings of the present inventors, MYLK2 and MYLK4, which are kinases responsible for phosphorylating the 43rd threonine of CDKAL1, have the property of autophosphorylation. By utilizing this property of MYLK2 and MYLK4, more specifically, by using the above-mentioned search method that utilizes MYLK2 and / or MYLK4 as both kinases and phosphorylated proteins, it becomes possible to more efficiently screen for components that suppress the phosphorylation of the 43rd threonine of CDKAL1. Note that the above-mentioned search method is the same as the candidate compound search method described above, except that MYLK2 and / or MYLK4 are used as kinases and MYLK2 and / or MYLK4 are used instead of CDKAL1 as the phosphorylated protein, so a detailed explanation of each step will be omitted.

[0094] The present invention will be described in more detail below based on experimental examples. These experimental examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention in any way.

[0095] <Cells and methods for culturing them> Unless otherwise specified, the cells used in the following experimental examples and their culture methods are as follows: Primary cultured human normal skeletal muscle cells, skeletal muscle myoblasts (HSMMs), were purchased from Lonza (catalog number: CC-2580). HSMM cells were cultured in a special medium (Lonza, SkGM). TM -2 Skeletal Muscle Cell Growth Medium-2 BulletKit TM Using ), the cultures were incubated on a 10 cm culture dish at 37°C, 5% CO2, and in a humid environment according to standard procedures. On the one hand, RD, an ICH-ERMS-1 human rhabdomyosarcoma cell line, RMS-YM, and RH30 were purchased from the JCRB cell bank, the RIKEN BRC, and the ATCC (American Type Culture Collection), respectively. These human rhabdomyosarcoma cells were each cultured on a 10-cm culture dish in a DMEM medium (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1× penicillin / streptomycin / L-glutamine (FUJIFILM Wako Pure Chemical Corporation; catalog number 161-23201) (hereinafter sometimes referred to as "normal medium" in the present disclosure) according to a conventional method at 37°C, 5% CO2, and a humid environment.

[0096] <Experiment 1: Evaluation of Protein Expression Levels in Rhabdomyosarcoma Cells> Each of normal skeletal muscle cells (HSMM) and rhabdomyosarcoma cells (RD, RMS-YM, ICH-ERMS-1, RH30) was lysed with a cell lysis buffer (20 mM Tris-HCl (pH = 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% Triton X-100, cOmplete TM protease inhibitor cocktail (Roche; catalog number 4693116001), PhosSTOP TMCells were dissolved using (Roche; catalog number 4906845001) and a cell extract was prepared according to a standard procedure. To the obtained cell extract, 1 / 3 volume of 4×SDS-PAGE sample buffer (250 mM Tris-HCl (pH=6.8), 8% SDS, 40% glycerol, 0.02% bromophenol blue, 20% 2-mercaptoethanol) was added (i.e., the ratio of cell extract to 4×SDS-PAGE sample buffer was 3:1), and the mixture was heated at 95°C for 5 minutes. The resulting sample was developed by SDS-PAGE according to a standard procedure and then transferred to a PVDF membrane. This membrane was blocked with TBST buffer containing 0.5% bovine serum albumin (BSA) (20 mM Tris-HCl (pH=7.4), 150 mM NaCl, 0.1% Tween20), and then treated with TBST buffer containing 0.5% BSA with the primary antibody at a 1 / 2000 dilution at 4°C for 16 hours. After washing this membrane three times with TBST buffer for 5 minutes each, it was treated with TBST buffer containing 0.5% BSA with the secondary antibody at a 1 / 10000 dilution at room temperature for 1 hour. After washing this membrane three times with TBST buffer for 5 minutes each, it was treated with Clarity Max Western ECL substrate (distributed by Bio-Rad Laboratories) and ChemiDoc TM Each protein was detected using the Touch imaging system (sold by Bio-Rad Laboratories). Comparison of the expression levels of each protein was performed by importing images of the membrane into image analysis software (ImageJ) and analyzing the signal intensity of the band corresponding to each protein. The primary antibodies used for staining each protein in the above procedure are shown in the table below. For secondary antibodies, anti-Mouse IgG antibody, HRP-linked (Sigma-Aldrich; catalog number A9044) and anti-Rabbit IgG antibody, HRP-linked (Cell Signaling Technology; catalog number 7074) were used.

[0097] [Table 1]

[0098] The results obtained are shown in Figure 1A. As shown in Figure 1A, MYLK2, MYLK4, and CDKAL1 expression was more strongly induced in rhabdomyosarcoma cells (RD, RMS-YM, ICH-ERMS-1, RH30) compared to normal skeletal muscle cells (HSMM). When the expression levels of each protein in various cell types were expressed as relative values ​​with the expression level in HSMM cells set to 1.00, the expression levels of MYLK2, MYLK4, and CDKAL1 in RD cells were 5.06, 5.67, and 6.00, respectively; in RMS-YM cells, the expression levels of MYLK2, MYLK4, and CDKAL1 were 9.40, 8.75, and 8.20, respectively; in ICH-ERMS-1 cells, the expression levels of MYLK2, MYLK4, and CDKAL1 were 20.07, 7.84, and 6.65, respectively; and in RH30 cells, the expression levels of MYLK2, MYLK4, and CDKAL1 were 18.09, 8.01, and 6.21, respectively. Thus, in various rhabdomyosarcoma cell types, an increase of more than five times in the expression levels of MYLK2, MYLK4, and CDKAL1 was observed compared to normal cells. Furthermore, as a result, it was observed that various rhabdomyosarcoma cells showed higher expression levels of SALL2 protein, a core stem cell characteristic, compared to normal skeletal muscle cells.

[0099] <Experiment 2: Evaluation of the effect of knockdown of MYLK2 and MYLK4 on the phosphorylation of CDKAL1> For the rhabdomyosarcoma cell line RD, either a negative control siRNA (siControl) or a combination of siRNA against MYLK2 (siMLYK2#1~siMLYK2#3) and siRNA against MYLK4 (siMLYK4#1~siMLYK4#3) was transfected using the transfection reagent Lipofectamine. TM RNAi Max and Opti-MEM TMTransfection was performed using culture medium. 72 hours after transfection, cell lysis buffer (20 mM Tris-HCl (pH=7.5), 150 mM NaCl, 0.1% Triton X-100, cOmplete) was used. TM Protease inhibitor cocktail (Roche), PhosSTOP TM Cell extracts were prepared according to a standard procedure using (Roche) (SDS-PAGE). To the obtained cell extracts, 1 / 3 volume of 4×SDS-PAGE sample buffer (250 mM Tris-HCl (pH=6.8), 8% SDS, 40% glycerol, 0.02% BPB, 20% 2-mercaptoethanol) was added (i.e., the ratio of cell extract to 4×SDS-PAGE sample buffer was 3:1), and the mixture was heated at 95°C for 5 minutes. The siRNAs used in the above procedure are as follows.

[0100] [Table 2]

[0101] MYLK2, MYLK4, CDKAL1, and GAPDH in the obtained samples were detected by Western blotting according to standard procedures. Specifically, first, the samples were developed by SDS-PAGE according to standard procedures and then transferred to a PVDF membrane. The membrane was blocked with 0.5% BSA-containing TBST buffer (20mM Tris-HCl (pH=7.4), 150mM NaCl, 0.1% Tween20), and then treated with 0.5% BSA-containing TBST buffer containing the primary antibody at a 1 / 2000 dilution at 4°C for 16 hours. After washing the membrane three times with TBST buffer for 5 minutes each, it was treated with 0.5% BSA-containing TBST buffer containing the secondary antibody at a 1 / 10000 dilution at room temperature for 1 hour. After washing the membrane three times with TBST buffer for 5 minutes each, it was treated with Clarity Max Western ECL substrate (distributed by Bio-Rad Laboratories) and ChemiDoc TMEach protein was detected using the Touch imaging system (sold by Bio-Rad Laboratories). The anti-MYLK2 antibody, anti-MLYK4 antibody, anti-CDKAL1 antibody, and anti-GAPDH antibody used for detecting MLYK2, MLYK4, CDKAL1, and GAPDH in this experiment were the same as in Experiment 1.

[0102] Furthermore, CDKAL1 in the obtained samples was phos-tag'd according to a standard method. TM It was also detected by Western blotting. Specifically, first, Phos-tag TM An acrylamide gel containing (distributed by Fujifilm Wako Pure Chemical Industries, Ltd.) was prepared according to the product's protocol, and proteins were separated and developed using SDS-PAGE with the prepared acrylamide gel. The procedure after development was the same as in the Western blotting method described above.

[0103] The results obtained are shown in Figure 1B. The upper part of Figure 1B shows the results of analysis by Western blotting, and the lower part of Figure 1B shows the results of analysis by Phos-tag Western blotting. As shown in the Western blotting analysis results in the upper part of Figure 1B, knockdown of MYLK2 and MYLK4 was observed in RD cells co-transfection with siRNA against MYLK2 and siRNA against MYLK4. Furthermore, as shown in the Phos-tag Western blotting analysis results in the lower part of Figure 1B, it was confirmed that the band corresponding to phosphorylated CDKAL1 disappeared in RD cells in which MYLK2 and MYLK4 were knocked down. This result indicates that CDKAL1 is phosphorylated by MYLK2 and MYLK4 in RD cells, which are rhabdomyosarcoma cells.

[0104] <Experiment 3: Evaluation of antitumor effects by knockdown of MYLK2 / MYLK4> <Experiment 3-1: Effect on self-renewal ability (sphere formation ability)> Rhabdomyosarcoma cell lines RD and RH30 were transfected with negative control siRNA (siControl), siRNA against MYLK2 (siMLYK2#1~siMLYK2#3), or siRNA against MYLK4 (siMLYK4#1~siRNA4#3) using the transfection reagent Lipofectamine. TM RNAi Max and Opti-MEM TM Each cell was transfected using a culture medium according to a standard procedure. 24 hours after transfection, each cell was detached by trypsin treatment according to a standard procedure, and then transfected in 1.5 mL of NB medium (Neurobasal). TM Culture medium (Thermo Fisher), 1×B-27 TM Cell suspensions were prepared by containing 1,000 cells in each of the following: Thermo Fisher supplements, 1x N-2 supplements (Thermo Fisher), 20 ng / mL human EGF (Human Epidermal Growth Factor) (Fujifilm Wako Pure Chemical Corporation), 20 ng / mL human bFGF (Human basic Fibroblast Growth Factor) (Fujifilm Wako Pure Chemical Corporation), 10 μg / mL heparin sodium (Fujifilm Wako Pure Chemical Corporation), and 1x penicillin / streptomycin / L-glutamine (Fujifilm Wako Pure Chemical Corporation). This cell suspension was then placed in a 24-well cell culture plate (Costar TM 1.5 mL of siRNA was seeded per well in 24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates (Corning), with 4 wells used for each condition. Following standard procedures, the cells were incubated in a culture environment for one week, after which the number of spheres (cell aggregates with a diameter greater than 100 μm) in each well was counted. The siRNA used in this experiment was the same as that used in Experiment 2 (shown in Table 2).

[0105] The results obtained are shown in Figure 2A. As shown in Figure 2A, when RD cells and RH30 cells were transfected with siRNA against MYLK2 or siRNA against MLYK4, and after 24 hours of incubation, their sphere formation ability (i.e., self-renewal ability) in a scaffold-independent culture environment was analyzed, the sphere formation ability (self-renewal ability) of RD cells and RH30 cells was significantly reduced by either siRNA against MYLK2 or siRNA against MLYK4, compared to when the cells were transfected with a negative control siRNA (siControl). This indicates that MYLK2 and MYLK4 are essential for maintaining the self-renewal ability of rhabdomyosarcoma, and that knockdown of these genes impairs the self-renewal ability of rhabdomyosarcoma.

[0106] <Experiment 3-2: Effects on translation mechanisms involving CDKAL1> Transfection of rhabdomyosarcoma cell line RD with negative control siRNA (siControl), siRNA against MYLK2 (siMLYK2#1~siMLYK2#3), or siRNA against MYLK4 (siMLYK4#1~siMLYK4#3) using the transfection reagent Lipofectamine TM RNAi Max and Opti-MEM TM Transfection was performed using culture medium according to a standard procedure. 72 hours after transfection, cell lysis was performed using cell lysis buffer (20 mM Tris-HCl (pH=7.5), 150 mM NaCl, 0.1% Triton X-100, cOmplete). TM Protease inhibitor cocktail (Roche), PhosSTOP TM Cell extracts were prepared according to a standard method using Roche (SDS-PAGE). To the obtained cell extracts, 1 / 3 volume of 4×SDS-PAGE sample buffer (250 mM Tris-HCl (pH=6.8), 8% SDS, 40% glycerol, 0.02% bromophenol blue, 20% 2-mercaptoethanol) was added (i.e., the ratio of cell extract to 4×SDS-PAGE sample buffer was 3:1), and the mixture was heated at 95°C for 5 minutes.

[0107] MYLK2, MYLK4, CDKAL1, and GAPDH in the obtained samples were detected by Western blotting according to standard procedures. After developing the samples on SDS-PAGE, they were transferred to a PVDF membrane. The membrane was blocked with 0.5% BSA-containing TBST buffer (20 mM Tris-HCl (pH=7.4), 150 mM NaCl, 0.1% Tween20), and then treated with 0.5% BSA-containing TBST buffer containing the primary antibody at a 1 / 2000 dilution at 4°C for 16 hours. After washing this membrane three times with TBST buffer for 5 minutes each, it was treated with 0.5% BSA-containing TBST buffer containing the secondary antibody at a 1 / 10000 dilution at room temperature for 1 hour. After washing this membrane three times with TBST buffer for 5 minutes each, it was treated with Clarity Max Western ECL substrate (distributed by Bio-Rad) and ChemiDoc. TM Each protein was detected using the Touch imaging system (distributed by Bio-Rad). The primary antibodies used for detection are shown in the table below. For secondary antibodies, anti-mouse IgG antibody, HRP-linked (Sigma-Aldrich; catalog number A9044) and anti-rabbit IgG antibody, HRP-linked (Cell Signaling Technology; catalog number 7074) were used.

[0108] [Table 3]

[0109] In addition, TRIzol TM RNA was recovered from each cell lysate using Reagent (Thermo Fisher). cDNA was prepared from 2000 ng of total RNA using PrimeScript RT Master Mix (distributed by Takara Bio Inc.), and primers for 18S ribosomal RNA and SALL2, and Luna TMSALL2 mRNA expression levels were analyzed using Universal qPCR Master Mix (New England Biolabs) and Rotor-Gene Q (QIAGEN). 18S ribosomal RNA was used as an internal standard for standardization during the analysis. The primers used for qPCR are shown in the table below.

[0110] [Table 4]

[0111] The results obtained are shown in Figure 2B. As shown in Figure 2B, when RD cells were transfected with siRNA against MLYK2 or siRNA against MLYK4, and proteins and RNA were collected after 72 hours, the expression levels of each protein were evaluated. Compared to when cells were transfected with a negative control siRNA (siControl), transfection with either siRNA against MYLK2 or siRNA against MLYK4 reduced the expression level of CD133, a stem cell marker for rhabdomyosarcoma. Importantly, although knockdown of MYLK2 or MLYK4 did not change the mRNA expression level of SALL2, a major factor in cancer stem cell characteristics, the protein expression level of SALL2 decreased significantly. This result indicates that MYLK2 or MLYK4 is required for the translation, not transcription, of SALL2 mRNA, and that knockdown of MYLK2 or MLYK4 suppresses SALL2 expression at the translational level.

[0112] <Experiment 3-3: Antitumor effect> Six-week-old male nude mice (BALB / c-nu / nu) were inoculated with 1,000,000 RD cells per injection site near the left lumbar region. The average tumor volume was 100 mm². 3 At that point, the negative control siRNA (siControl) or siRNA against MYLK4 (siMLYK4#1~siMLYK4#2) is used in vivofectamine.TM 3.0 Reagent was administered intratumorally according to the protocol provided with the product. After intratumoral administration of siRNA approximately every 5 days, the average tumor volume of mice administered with negative control siRNA (siControl) was 800 mm². 3 At that point, all the mice were euthanized, the tumors were removed, and their size was evaluated.

[0113] The results obtained are shown in Figure 2C. As shown in Figure 2C, the tumor size remained smaller in tumors treated with siRNA against MLYK4 compared to tumors treated with negative control siRNA (siControl). This result indicates that administration of siRNA against MLYK4 exerts a significant antitumor effect against rhabdomyosarcoma.

[0114] <Experiment 4: Screening method for candidate compounds to treat cancer> <Experiment 4-1: Preparation of FLAG-tagged proteins> In Expi293 cells (Thermo Fisher), a plasmid expressing FLAG-tagged MYLK2 or MYLK4 was introduced using ExpiFectamine. TM Transfection was performed using the 293 Transfection Kit (Thermo Fisher) according to the protocol provided with the product. Cells were harvested one week after transfection and disrupted with TBST buffer (20mM Tris-HCl (pH=7.4), 150mM NaCl, 0.1% Tween20). Anti-FLAG TMUsing an antibody affinity gel (Sigma Aldrich, catalog number: A2220), FLAG-tagged MYLK2 or MYLK4 was recovered and purified from the lysates. A portion of the solution containing the purified recombinant protein was mixed with 1 / 3 volume of 4×SDS-PAGE sample buffer (250 mM Tris-HCl (pH=6.8), 8% SDS, 40% glycerol, 0.02% bromophenol blue, 20% 2-mercaptoethanol) (i.e., solution:4×SDS-PAGE sample buffer = 3:1) and heated at 95°C for 5 minutes. The resulting sample was developed using SDS-PAGE, and the polyacrylamide gel was stained with Oriole fluorescent gel stain (Bio-Rad Laboratories; catalog number: 1610496) at room temperature for 90 minutes, followed by ChemiDoc TM Observations were made using the Touch Imaging System (Bio-Rad Laboratories).

[0115] The results obtained are shown in Figure 3A. As shown in Figure 3A, Expi293 cells were transfected with plasmids expressing FLAG-tagged MYLK2 or MYLK4. After one week, the cells were collected and purified using FLAG antibody beads, then developed and separated by SDS-PAGE and detected by Oriole staining. Bands were detected at positions corresponding to the molecular weights of MYLK2 and MYLK4. This result indicates that FLAG-tagged MYLK2 and FLAG-tagged MYLK4 were generated.

[0116] <Experiment 4-2: Construction of a Screening System> Except for using plasmids expressing FLAG-tagged MYLK2 or MYLK4, a plasmid expressing Myc-tagged wild-type CDKAL1 was used instead, magnetic beads immobilized with anti-Myc antibody (included in the c-Myc-tagged Protein Magnetic Purification Kit (Medical & Biological Laboratories Co., Ltd.; catalog number 3340)) were used instead of magnetic beads immobilized with anti-FLAG antibody, and RD cells were used instead of Expi293 cells. Myc-tagged CDKAL1 was expressed in RD cells using the same procedure as above, and Myc-tagged CDKAL1 was recovered and purified from the disrupted RD cells. In addition, Myc-tagged fusion proteins were prepared using the same procedure for two mutant CDKAL1 variants: one in which the 43rd threonine of wild-type CDKAL1 was replaced with alanine, and another in which the N-terminus of wild-type CDKAL1 was cleaved. These Myc-tagged fusion proteins were all purified using the c-Myc-tagged Protein Magnetic Purification Kit (Medical & Biological Laboratories, Inc.; catalog number 3340).

[0117] The solution containing Myc-tagged CDKAL1 and magnetic beads immobilized with anti-Myc antibody, obtained by the above procedure, was resuspended in 40 μL of 1× kinase buffer (Cell Signaling Technology; catalog number 9820). Alkaline phosphatase (BAP; Takara Bio Inc.; catalog number: 2120B) derived from E. coli C75 was added to a final concentration of 0.4 units / 40 μL, and the solution was allowed to stand at 25°C for 30 minutes to dephosphorylate the CDKAL1. The magnetic beads were adsorbed using a magnetic stand, and the magnetic beads were washed three times with kinase buffer (Cell Signaling Technology; catalog number 9820). Kinase buffer, kinase buffer containing 200 μM ATP, and kinase buffer containing 200 μM ATP and 300 ng of MYLK2 or MYLK4 were added, and the solution was allowed to stand at 37°C for 1 hour. Subsequently, the magnetic beads were washed three times with kinase buffer while adsorbing them using a magnetic stand. Then, 1 / 3 volume of 4× SDS-PAGE sample buffer (250 mM Tris-HCl (pH=6.8), 8% SDS, 40% glycerol, 0.02% bromophenol blue, 20% 2-mercaptoethanol) was added, and the mixture was heated at 95°C for 5 minutes. The supernatant was collected as a sample for SDS-PAGE while adsorbing the magnetic beads using a magnetic stand.

[0118] Sample analysis was performed by Phos-tag Western blotting. Specifically, first, Phos-tag TMAn acrylamide gel containing (distributed by Fujifilm Wako Pure Chemical Industries, Ltd.) was prepared according to the product's protocol. Proteins were separated and developed using SDS-PAGE on the prepared acrylamide gel and then transferred to a PVDF membrane. This membrane was blocked with 0.5% BSA-containing TBST buffer (20mM Tris-HCl (pH=7.4), 150mM NaCl, 0.1% Tween20), and then treated with 0.5% BSA-containing TBST buffer containing the primary antibody at a 1 / 2000 dilution at 4°C for 16 hours. After washing the membrane three times with TBST buffer for 5 minutes each, it was treated with 0.5% BSA-containing TBST buffer containing the secondary antibody at a 1 / 10000 dilution at room temperature for 1 hour. After washing the membrane three times with TBST buffer for 5 minutes each, it was treated with Clarity Max Western ECL substrate (distributed by Bio-Rad Laboratories) and ChemiDoc TM Each protein was detected using the Touch imaging system (distributed by Bio-Rad Laboratories). In the above procedure, the primary antibody used against Myc-tag was an anti-myc-tag antibody (Medical & Biological Laboratories, Inc., catalog number: M192-3). The primary antibodies for the other proteins were the same as those previously described.

[0119] The results obtained are shown in Figure 3C. As shown in Figure 3C, wild-type CDKAL1 (labeled "CDKAL1WT" in Figure 3C) showed an upward band shift in the presence of MYLK2 and MYLK4, respectively. This indicates that wild-type CDKAL1 is phosphorylated in the presence of MYLK2 and MYLK4. On the other hand, no upward band shift was observed in the mutant CDKAL1 in which the 43rd threonine was substituted with alanine (labeled "CDKAL1T43A" in Figure 3C) or the mutant CDKAL1 with the N-terminus cleaved (labeled "CDKAL1ΔN" in Figure 3C), neither in the presence of MYLK2 nor MYLK4. This result indicates that MYLK2 and MYLK4 phosphorylate the 43rd threonine of CDKAL1.

[0120] <Experiment 4-3: Screening Experiment> In Experiment 4-1, a solution containing FLAG-tagged MLYK4 and magnetic beads immobilized with anti-FLAG antibody was added. 0.4 units of alkaline phosphatase (BAP; Takara Bio Inc.; catalog number: 2120B) derived from E. coli C75 was added, and the solution was allowed to stand at 25°C for 30 minutes to dephosphorylate the MLYK4. The magnetic beads were adsorbed using a magnetic stand, washed three times with kinase buffer (Cell Signaling Technology; catalog number 9802), and kinase buffer containing ATP and 25 μM of the test sample (ML-7 or ML-9) was added. The solution was then allowed to stand at 37°C for 1 hour. Subsequently, the magnetic beads were washed three times with kinase buffer while adsorbing them using a magnetic stand. Then, 1 / 3 volume of 4× SDS-PAGE sample buffer (250 mM Tris-HCl (pH=6.8), 8% SDS, 40% glycerol, 0.02% bromophenol blue, 20% 2-mercaptoethanol) was added, and the mixture was heated at 95°C for 5 minutes. The supernatant was collected as a sample for SDS-PAGE while adsorbing the magnetic beads using a magnetic stand.

[0121] As controls, we prepared the following samples: one in which dephosphorylation with BAP was not performed and the kinase reaction was carried out in a kinase buffer that did not contain ATP (Lane 1 in Figure 3D); one in which dephosphorylation with BAP was performed and the kinase reaction was carried out in a kinase buffer that contained BAP in addition to ATP (Lane 2 in Figure 3D); and one in which dephosphorylation with BAP was performed and the kinase reaction was carried out in a kinase buffer that contained ATP (Lane 3 in Figure 3D). For each sample, the presence or absence of dephosphorylation and the presence or absence of ATP and the test sample in the kinase buffer are shown in the table below.

[0122] [Table 5]

[0123] The obtained samples were analyzed by Western blotting and Phos-tag Western blotting. The procedures for Western blotting and Phos-tag Western blotting were as previously described. For detection, an anti-FLAG tag antibody (distributed by Fujifilm Wako Pure Chemical Industries, Ltd.; catalog number 012-22384) was used as the primary antibody.

[0124] The results obtained are shown in Figure 3D. As shown in Figure 3D, MYLK-4 was phosphorylated (autophosphorylated) in the absence of ML-7 and ML-9 (lane 3), whereas in the presence of ML-7 and ML-9, the phosphorylation of MYLK4 was suppressed (lanes 4 and 5). This result indicates that it is possible to search for components that suppress the phosphorylation of MYLK4 by following the procedure shown in Figure 3B.

[0125] <Experiment 4-4: Effects of MYLK2 or MLYK4 inhibitors on the formation of translation initiation factor complexes> RD cells treated with 15 μM ML-7, 15 μM ML-9, or DMSO (negative control) for 30 minutes were disrupted and purified with TBST buffer. Immobilized γ-aminophenyl-m7GTP beads (Jena Bioscience) were added and the mixture was reacted at 4°C for 16 hours. After washing the beads with TBST buffer, an equal volume of 2× SDS sample buffer was added, and the mixture was heated at 95°C for 5 minutes. The resulting sample was developed by SDS-PAGE and then transferred to a PVDF membrane. The membrane was blocked with 0.5% BSA-containing TBST buffer (20 mM Tris-HCl (pH=7.4), 150 mM NaCl, 0.1% Tween20), and then treated with primary antibody at a 1 / 2000 dilution in 0.5% BSA-containing TBST buffer at 4°C for 16 hours. The membrane was washed three times with TBST buffer for 5 minutes each, and then treated at room temperature for 1 hour in 0.5% BSA-containing TBST buffer containing a 1 / 10000 dilution of secondary antibody. After washing the membrane three times with TBST buffer for 5 minutes each, it was treated with Clarity Max Western ECL substrate (distributed by Bio-Rad Laboratories) and ChemiDoc TM The respective proteins were detected using the Touch imaging system (sold by Bio-Rad Laboratories). The antibodies used in the above procedure are as follows:

[0126] [Table 6]

[0127] The results obtained are shown in Figure 3E. As shown in Figure 3E, in samples obtained from RD cells treated with negative control DMSO, bands corresponding not only to eIF4E bound to m7GTP beads, but also to eIF4A, eIF4G, and CDKAL1 were observed, confirming the formation of a translation initiation factor complex formed by the binding of these components. In contrast, in samples obtained from RD cells treated with ML-7 and ML-9, bands corresponding to eIF4A, eIF4G, and CDKAL1 were not observed. This result indicates that the formation of CDKAL1-dependent translation initiation factor complexes is suppressed in RD cells treated with ML-7 and ML-9. Furthermore, this suggests that substances that suppress phosphorylation by MYLK2 or MLYK4 obtained by the above-described screening system have the property of suppressing the phosphorylation of CDKAL1 and inhibiting the formation of CDKAL1-dependent translation initiation factor complexes.

[0128] <Experiment 5. Screening of substances that inhibit translation mechanisms involving CDKAL1> Using the screening method described in International Publication No. 2022 / 124342 (Patent Document 1), we screened for substances that inhibit the translation mechanism involving CDKAL1.

[0129] Specifically, to evaluate the translation mechanism by CDKAL1, we constructed a plasmid (hereinafter referred to as the "SALL2-5'-UTR-firefly luciferase reporter plasmid") that produces mRNA in cells containing the nucleotide sequence encoding the reporter protein, firefly luciferase, and the nucleotide sequence of the 5'-UTR of SALL2 variant 6 mRNA at its 5'-UTR. Using this SALL2-5'-UTR-firefly luciferase reporter plasmid and a plasmid for numerical standardization encoding sea urchin luciferase (sea urchin luciferase reporter plasmid) (Promega, catalog number: E6921), we evaluated a total of 2203 FDA-approved drug groups. In detail, RD cells cultured to 80% confluence on a 10 cm culture dish according to standard procedures were transfected with a mixture of SALL2-5'-UTR-firefly luciferase reporter plasmid and sea urchin luciferase reporter plasmid in a ratio of 9 μg:6 μg using TransIT-LT1 Reagent (Takara Bio Inc., catalog number: MIR2300) according to the protocol provided with the product. 24 hours after transfection, the cells were detached by trypsin treatment according to standard procedures and seeded in a 96-well plate with 5,000 cells per well. 24 hours after seeding, the medium was replaced with standard medium containing 10 μM of the compound to be screened, and incubated for another 24 hours at 37°C in a humid environment with 5% CO2. The samples were then collected and tested for Dual-Luciferase TM The luminescence intensity of firefly luciferase and sea urchin luciferase was measured using a Reporter Assay System (Promega; catalog number: E1980) and a luminometer (MicroLumat Plus LB 96V; Berthold).

[0130] The results obtained are shown in Figure 4. As shown in Figure 4, the screening results showed that ozanimod, perhexiline, osimertinib, and lomitapide significantly reduced the luminescence intensity of firefly luciferase compared to that of sea urchin luciferase. Since the mRNA produced by the SALL2-5'-UTR-firefly luciferase reporter plasmid is mainly translated by a translation mechanism involving CDKAL1, suppression of the CDKAL1-involved translation mechanism reduces the ratio of firefly luciferase reporter activity to sea urchin luciferase reporter activity (Normalized score = [firefly luciferase luminescence intensity / sea urchin luciferase luminescence intensity]). In other words, the above results suggest that the four compounds ozanimod, perhexylline, osimertinib, and lomitapide are compounds that suppress the translation mechanism involving CDKAL1.

[0131] <Experiment 6. Functional evaluation of screened compounds> <Experiment 6.1. Evaluation by In vitro Kinase Assay> The effects of the four compounds screened in Experiment 5 (ozanimod, perhexylline, osimertinib, and lomitapide) on CDKAL1 phosphorylation were investigated. In this experiment, gefitinib and erlotinib, which are EGFR tyrosine kinase inhibitors similar to osimertinib, were used as control groups. As shown in Figure 4, gefitinib and erlotinib had almost no effect on the ratio of firefly luciferase reporter activity to sea urchin luciferase reporter activity in the screening experiment in Experiment 5.

[0132] A conceptual diagram of the experimental procedure is shown in Figure 5A. Specifically, Myc-tagged CDKAL1 was adsorbed onto magnetic beads included with the c-Myc-tagged Protein Magnetic Purification Kit (Medical & Biological Laboratories, Inc.; catalog number: 3340), and these magnetic beads were left to stand for 30 minutes in 1× kinase buffer (Cell Signaling Technology; catalog number 9802) containing 1 μM ozanimod, perhexylline, osimertinib, lomitapide, gefitinib, erlotinib, or DMSO as a negative control. The 1× kinase buffer containing each compound at a concentration of 1 μM, or containing DMSO as a negative control, was prepared by adding either a DMSO solution of each compound (10 mM in DMSO) or the same amount of DMSO to the 1× kinase buffer. Magnetic beads adsorbed with Myc-tagged CDKAL1 were washed three times with 1× kinase buffer. Then, 40 μL of 1× kinase buffer containing 200 μM ATP and 300 ng of FLAG-tagged MYLK4 was added, and the mixture was allowed to stand at 37°C for 60 minutes. 20 μL of 3× SDS-PAGE sample buffer was added to this reaction mixture, and the mixture was allowed to stand at 95°C for 5 minutes. The supernatant was collected as a sample while adsorbing the magnetic beads using a magnetic stand, and subjected to Western blotting. The Western blotting procedure was as described above. The antibodies used for Western blotting are as follows.

[0133] [Table 7]

[0134] The results obtained are shown in Figure 5B. As shown in Figure 5B, when CDKAL1 was reacted with ozanimod, perhexylline, lomitapide, gefitinib, and erlotinib, and then reacted with MYLK4, the ratio of CDKAL1 with phosphorylated threonine at position 43 (Thr43) (labeled "CDKAL1 pT43" in Figure 5B) remained unchanged compared to when reacted with the negative control DMSO. In contrast, when CDKAL1 was reacted with osimertinib, and then reacted with MYLK4, the ratio of CDKAL1 with phosphorylated Thr43 decreased to 0.63 times compared to when reacted with the negative control DMSO. This result indicates that osimertinib inhibited the phosphorylation of Thr43 of CDKAL1 by MYLK4.

[0135] <Experiment 6.2. Inhibitory effect of CDKAL1 phosphorylation in RD cells> To investigate in more detail the mechanism by which osimertinib suppresses Thr43 phosphorylation of CDKAL1 by MYLK4, the effect of osimertinib on the interaction between MYLK4 and CDKAL1 was evaluated by an immunoprecipitation assay using cytolysates from the rhabdomyosarcoma cell line RD.

[0136] Specifically, RD cells were transfected with a plasmid expressing Myc-tagged CDKAL1 and a plasmid expressing FLAG-tagged MYLK4 using TransIT-LT1 Reagent (Takara Bio Inc., catalog number: MIR2300) according to the protocol provided with the product. 72 hours after transfection, the cells were harvested, lysed with TBST buffer (20mM Tris-HCl (pH 7.4), 150mM NaCl, 0.1% Tween20), and the cell extract was collected. To the collected cell extract, osimertinib DMSO solution (10mM in DMSO) or the same amount of DMSO as a negative control was added to achieve a final osimertinib concentration of 10μM, and the mixture was allowed to stand at room temperature for 1 hour. Anti-FLAG TMFLAG-tagged MYLK4 was immunoprecipitated using an antibody affinity gel (Merck; catalog number: A2220) and washed three times with cell lysis buffer. 100 μL of 1× SDS-PAGE sample buffer diluted with cell lysis buffer was added to the washed beads, and the mixture was allowed to stand at 95°C for 5 minutes. The supernatant after centrifugation was collected as a sample and subjected to Western blotting. The Western blotting procedure was as described above. The antibody used in the experiment was the same as in Experiment 6.1.

[0137] The results obtained are shown in Figure 5C. As shown in Figure 5C, CDKAL1 was immunoprecipitated along with FLAG-tagged MYLK4 from the cell extract of RD cells treated with DMSO, the negative control. This result indicates the interaction between MYLK4 and CDKAL1 in the cell extract of RD cells treated with DMSO, the negative control. That is, it is thought that MYLK4 binds to CDKAL1 and phosphorylates Thr43 of CDKAL1. In contrast, the amount of CDKAL1 immunoprecipitated along with MYLK4 from the cell extract of RD cells treated with osimertinib was reduced to 0.55 times compared to the amount of CDKAL1 immunoprecipitated along with MYLK4 from the cell extract of RD cells treated with DMSO, the negative control. This result indicates that osimertinib has an inhibitory effect on the binding of MYLK4 and CDKAL1. Osimertinib is thought to inhibit the phosphorylation of Thr43 of CDKAL1 by MYLK4 by inhibiting the binding of MYLK4 to CDKAL1.

[0138] <Experiment 7. Effects of osimertinib on RD cells> The effects of osimertinib on SALL2 expression levels in RD cells were investigated at the mRNA and protein levels.

[0139] Specifically, RD cells were cultured for 1 hour in a DMSO solution of osimertinib (Selleck, catalog number: S7297) (10 mM in DMSO), or in 2.5 μM osimertinib prepared by adding the same amount of DMSO to standard medium, or in standard medium containing DMSO as a negative control. Subsequently, standard medium containing human epidermal growth factor (hEGF) was added to achieve a final hEGF concentration of 40 ng / mL, or the same amount of standard medium containing pure water as a negative control, and the cells were cultured for 24 hours. RNA and proteins were extracted from these cells, and the extracted RNA and proteins were subjected to quantitative real-time PCR and Western blotting, respectively.

[0140] RNA extraction was performed using TRIzol Reagent (Thermo Fisher Scientific; catalog number 15596018) according to the protocol provided with the product. The extracted RNA was treated with DNase I (Takara Bio Inc.; catalog number 2270A), and cDNA (complementary DNA) was prepared based on this. PrimeScript RT Master Mix (Takara Bio Inc.; catalog number RR036A) was used to prepare the cDNA. Quantitative real-time PCR was performed using the obtained cDNA, Luna Universal qPCR Master Mix (New England Biolabs; catalog number M3003E), and primers corresponding to the target genes on a Rotor-Gene Q 2plex HRM system (Qiagen). The expression level of SALL2 mRNA was standardized by the expression level of ribosomal RNA 18S. The sequences of the primers corresponding to each target gene are shown in Table 4.

[0141] On the other hand, the protein extraction procedure was as follows: First, the RD cells processed according to the above procedure were lysed with cell lysis buffer, sonicated, and then centrifuged at 13,500 rpm and 4°C for 15 minutes. The supernatant was collected as the protein extract. To the collected protein extract, 1 / 3 the volume of 4× SDS-PAGE sample buffer was added, and the mixture was allowed to stand at 95°C for 5 minutes. Using the obtained sample, SDS-PAGE and protein detection were performed using the same procedure as in Experiment 3-2. The primary and secondary antibodies used in this experiment were the same as in Experiment 3-2.

[0142] The results obtained are shown in Figure 6A. As shown in Figure 6A, in RD cells treated with osimertinib, the amount of SALL2 mRNA produced did not change compared to when treated with the negative control DMSO, while the amount of SALL2 protein produced decreased significantly compared to when treated with the negative control DMSO, and almost no band corresponding to SALL2 protein was observed in SDS-PAGE. This result indicates that SALL2 expression is suppressed in RD cells by osimertinib, and that this suppression of SALL2 expression occurs at the level of translation from mRNA to protein. Furthermore, the expression of the target gene SALL2 did not change at either the mRNA or protein level with the addition of EGF. This result indicates that SALL2 expression in RD cells is less affected by the classical EGFR pathway. Unlike EGFR mutation-positive lung cancer, which is treated with osimertinib, rhabdomyosarcoma does not have EGFR mutations, and is translated by a translation mechanism involving CDKAL1. It is thought that factors such as SALL2, which governs cancer stem cell characteristics in RD cells, are less affected by the EGFR pathway. In other words, the above results indicate that the mechanism of action of osimertinib in rhabdomyosarcoma is entirely different from the EGFR inhibitory effect observed in EGFR mutation-positive lung cancer.

[0143] <Experiment 7-2. Effects on the self-renewal capacity of RD cells> Next, the effect of osimertinib on the self-renewal capacity of RD cells was evaluated based on sphere formation ability.

[0144] Specifically, RD cells cultured in standard medium according to conventional methods were detached by trypsin treatment. The detached RD cells were resuspended in NB medium to a cell count of 1000 cells / 1.5 mL, and then placed in a 24-well ultra-low adhesion cell culture plate (Costar). TM Four wells were seeded under each condition using 24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates (catalog number CLS3473; Corning), with 1.5 mL of osimertinib per well (i.e., 1000 cells per well). Osimertinib in DMSO solution (10 mM in DMSO) was then added to achieve a final concentration of 1 μM or 2.5 μM. DMSO (0.375 μL) was added as a negative control. The cells were cultured for one week in a humid incubator at 37°C and 5% CO2, and the number of spheres (cell aggregates with a diameter greater than 100 μm) formed was counted.

[0145] The results obtained are shown in Figure 6B. As shown in Figure 6B, when RD cells were treated with osimertinib, the number of RD cell spheres decreased in a concentration-dependent manner. This result indicates that osimertinib reduces the self-renewal capacity of RD cells.

[0146] <Experiment 7-3. Effects of CDKAL1 on the formation of translation initiation factor complexes> Next, the effect of osimertinib on the formation of the CDKAL1-mediated translation initiation factor complex was evaluated using a pull-down assay with m7GTP beads.

[0147] Specifically, the culture medium of RD cells cultured to 80% confluence on a 10 cm culture dish was replaced with osimertinib in DMSO solution (10 mM in DMSO), or 2.5 μM osimertinib prepared by adding the same amount of DMSO to standard medium, or standard medium containing DMSO as a negative control. The cultures were incubated for 24 hours at 37°C in a humid environment with 5% CO2. Subsequently, 1 mL of cell lysis buffer was added, and the cells were lysed using a 23-gauge needle and syringe. The cells were then centrifuged at 13,500 rpm at 4°C for 15 minutes, and the supernatant was collected as a protein extract. 40 μL of m7GTP beads (Jena Bioscience; catalog number AC-155S), washed with cell lysis buffer, were added to each protein extract, and the mixture was allowed to stand at 4°C for 4 hours. The m7GTP beads were washed three times with cell lysis buffer, and 100 μL of 4× SDS-PAGE sample buffer, diluted fourfold with cell lysis buffer, was added. The mixture was then allowed to stand at 95°C for 5 minutes. After centrifugation, the supernatant was collected as a sample and subjected to Western blotting. The Western blotting procedure was as described above, and the antibodies used in the experiment are as follows.

[0148] [Table 8]

[0149] The results obtained are shown in Figure 6C. As shown in Figure 6C, in addition to eIF4E, which shows binding affinity to m7GTP beads, eIF4G, eIF4A, and CDKAL1 were pulled down from the protein extract of RD cells not treated with osimertinib (Osimertinib "-" in Figure 6C), while in the protein extract of RD cells treated with osimertinib, only eIF4E was pulled down, and eIF4G, eIF4A, and CDKAL1 were hardly pulled down at all (Osimertinib "+" in Figure 6C). This result indicates that treatment of RD cells with osimertinib suppresses the formation of the CDKAL1-involved translation initiation factor complex in RD cells.

[0150] <Experiment 8. Establishment and evaluation of drug-resistant RD cell lines> <Experiment 8-1. Drug resistance of AD-RD cells> RD cells resistant to actinomycin D (AD-RD cells) were established by culturing RD cells while gradually increasing the concentration of actinomycin D according to a standard method. AD-RD cells or RD cells detached using trypsin were suspended in basal medium and seeded into 96-well cell culture plates at a density of 5,000 cells per well. The following day, the culture medium was changed to basal medium containing a predetermined concentration of actinomycin D or vincristine. 72 hours after the medium change, cell viability was calculated using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega; catalog number: G3582) according to the protocol provided with the product.

[0151] The results obtained are shown in Figures 7A and 7B. As shown in Figure 7A, the IC50 of actinomycin D against RD cells was 0.47 nM, while the IC50 of actinomycin D against AD-RD cells increased to 3.07 nM. This result indicates that AD-RD cells have acquired drug resistance to actinomycin D. Also, as shown in Figure 7B, the IC50 of vincristine against RD cells was 0.66 nM, while the IC50 of vincristine against AD-RD cells was 2.94 nM. These results indicate that AD-RD cells exhibit drug resistance not only to actinomycin D but also to vincristine, which is consistent with reports that RD cells that acquire resistance to actinomycin D exhibit resistance to multiple anticancer drugs (Melguizo, C. et al. Actinomycin D causes multidrug resistance and differentiation in a human rhabdomyosarcoma cell line. Cell Mol Biol (Noisy-le-grand) 40, 137-145 (1994).).

[0152] <Experiment 8-2. Evaluation of the self-renewal capacity of AD-RD cells> Next, the self-renewal capacity of AD-RD cells was evaluated based on their sphere-forming ability. Specifically, RD cells and AD-RD cells cultured according to a standard method were detached with trypsin, and the detached cells were resuspended in NB medium to a cell concentration of 1,000 cells / 1.5 mL, and then cultured in a 24-well ultra-low adhesion cell culture plate (Costar). TM Four wells were seeded under each condition using 24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates (catalog number CLS3473; Corning), with 1.5 mL of seed per well (i.e., 1,000 cells per well). After culturing for one week in a humid incubator at 37°C and 5% CO2, the number of spheres (cell aggregates with a diameter greater than 100 μm) in each well was counted.

[0153] The results obtained are shown in Figure 7C. As shown in Figure 7C, the number of spheres formed by AD-RD cells was greater than that formed by RD cells. This result indicates that AD-RD cells have a higher self-renewal capacity than their parent RD cell line, i.e., they have higher cancer stem cell characteristics.

[0154] <Experiment 8-3. Activation of the CDKAL1-related translation mechanism in AD-RD cells> We compared the expression levels of various proteins related to the CDKAL1-mediated translation mechanism in AD-RD cells and RD cells.

[0155] Specifically, RD cells and AD-RD cells cultured according to standard methods were lysed using cell lysis buffer, sonicated, and then centrifuged at 13,500 rpm at 4°C for 15 minutes. The supernatant was collected as a protein extract. To the collected protein extract, 1 / 3 volume of 4× SDS-PAGE sample buffer was added, and the mixture was allowed to stand at 95°C for 5 minutes. The resulting reaction mixture was developed using SDS-PAGE and transferred to a PVDF membrane (Bio-Rad Laboratories; catalog number 1620177). The transferred membrane was blocked by immersion in BSA-TBST at room temperature for 1 hour. Subsequently, it was immersed in BSA-TBST containing the primary antibody at 4°C for 16 hours. After washing, the membrane was immersed in TBST for 5 minutes three times, and then immersed in BSA-TBST containing the secondary antibody at room temperature for 1 hour. After treating the membrane with the secondary antibody, it was washed by immersing it in TBST for 5 minutes three times. Then, it was reacted with Clarity Max Western ECL Substrate (Bio-Rad Laboratories), and the reacted membrane was imaged using the ChemiDoc Touch Imaging System (Bio-Rad Laboratories). The antibodies used in the experiment are as follows:

[0156] [Table 9]

[0157] The results obtained are shown in Figure 7D. As shown in Figure 7D, AD-RD cells showed increased expression of CD133, a cancer stem cell marker, and SALL2, which is translated by a translation mechanism involving CDKAL1, compared to RD cells. This result indicates that the translation mechanism involving CDKAL1 is activated in AD-RD cells and that cancer stem cell characteristics are enhanced compared to RD cells. On the other hand, no difference was observed in CDKAL1 expression levels between AD-RD cells and RD cells. In contrast, the expression levels of MYLK4 and the amount of Thr43-phosphorylated CDKAL1 (labeled "pT43" in Figure 7D) were significantly increased in AD-RD cells. This result suggests that phosphorylation of Thr43 of CDKAL1 by MYLK4 plays an important role in activating the CDKAL1-mediated translation mechanism in AD-RD cells.

[0158] <Experiment 8-4. Comparison of SALL2 mRNA expression levels> We compared the mRNA expression levels of SALL2, a protein translated by a CDKAL1-mediated translation mechanism, in AD-RD cells and RD cells.

[0159] Specifically, RNA was extracted from RD cells and AD-RD cells cultured according to a standard method using TRIzol Reagent (Thermo Fisher Scientific; catalog number 15596018) according to the protocol provided with the product. The extracted RNA was treated with DNase I (Takara Bio Inc.; catalog number 2270A), and cDNA (complementary DNA) was prepared based on this. PrimeScript RT Master Mix (Takara Bio Inc.; catalog number RR036A) was used to prepare the cDNA. Quantitative real-time PCR was performed using the obtained cDNA, Luna Universal qPCR Master Mix (New England Biolabs; catalog number M3003E), and primers corresponding to the target gene on a Rotor-Gene Q 2plex HRM system (Qiagen). The expression level of SALL2 mRNA was standardized by the expression level of ribosomal RNA 18S. The sequences of the primers corresponding to each target gene are shown in Table 4.

[0160] The results obtained are shown in Figure 7E. As shown in Figure 7E, there was no difference in the expression level of SALL2 mRNA between AD-RD cells and RD cells. Considering this result together with the result in Figure 7D, it can be seen that SALL2 expression in AD-RD cells is upregulated at the translation level from mRNA to protein. These results indicate activation of the MYLK4-CDKAL1-SALL2 translation mechanism in AD-RD cells. It is thought that the activation of this translation mechanism contributes to the cancer stem cell characteristics of AD-RD cells.

[0161] <Experiment 9. Effects of osimertinib on AD-RD cells> <Experiment 9.1. Effects on the self-renewal ability of AD-RD cells> The effect of osimertinib on the self-renewal capacity of AD-RD cells was evaluated based on the sphere formation ability of AD-RD cells treated with osimertinib. Sphere formation ability is considered an indicator of the self-renewal capacity of cancer stem cells.

[0162] AD-RD cells cultured according to standard methods were detached with trypsin. The detached AD-RD cells were resuspended in NB medium to a cell concentration of 1,000 cells / 1.5 mL and placed in a 24-well ultra-low adhesion cell culture plate (Costar). TM Four wells were seeded under each condition using 24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates (catalog number CLS3473; Corning), with 1.5 mL of osimertinib per well (i.e., 1,000 cells per well). Osimertinib was dissolved in DMSO and added to NB medium to a final concentration of 1 μM. DMSO was used as a negative control. After adding osimertinib or DMSO, the cells were incubated for 1 week in a humid incubator at 37°C and 5% CO2 according to standard procedures. After incubation, the number of spheres (cell aggregates with a diameter greater than 100 μm) in each well was counted.

[0163] The results obtained are shown in Figure 8A. As shown in Figure 8A, the number of spheres formed by AD-RD cells treated with osimertinib was smaller than that of AD-RD cells not treated with osimertinib, approximately half. This result indicates that osimertinib treatment weakens the self-renewal capacity of AD-RD cells.

[0164] <Experiment 9.2. Effects of AD-RD cells on tumorigenicity> The effect of osimertinib on the tumorigenicity of AD-RD cells was evaluated based on the colony-forming ability of AD-RD cells treated with osimertinib. Colony-forming ability is considered an indicator of the tumorigenicity of cancer stem cells.

[0165] AD-RD cells cultured according to standard procedures were detached with trypsin. The detached AD-RD cells were resuspended in standard medium to a cell concentration of 5,000 cells / 3 mL in the suspension, and seeded in 3 wells of a 6-well cell culture plate, with 3 mL per well (i.e., 5,000 cells per well), for each condition. Actinomycin D in DMSO solution (10 mM in DMSO) was added to achieve a final concentration of 1 nM of actinomycin D, and / or osimertinib in DMSO solution (10 mM in DMSO) to achieve a final concentration of 1 μM of osimertinib. DMSO (0.3 μL) was used as a negative control. After the addition of actinomycin D, osimertinib, and / or DMSO, the cells were cultured for 5 days in a humid incubator at 37°C and 5% CO2 according to standard procedures. After culturing, the cells were fixed by removing the culture medium, adding 4% paraformaldehyde, and incubating at room temperature for 20 minutes, according to standard procedures. After fixation, the cells were stained by adding an aqueous solution containing 0.01% crystal violet and incubating at room temperature for 30 minutes, and then washed with a sufficient amount of water according to standard procedures. After drying, the bottom surface of the plate was imaged using a scanner. The obtained images were analyzed using ImageJ software, and the number of formed colonies was counted.

[0166] The results obtained are shown in the upper panel of Figure 8B. As shown in the upper panel of Figure 8B, the number of colonies formed in AD-RD cells treated with actinomycin D alone was 80.09% of that of AD-RD cells treated with DMSO (negative control), while the number of colonies formed in AD-RD cells treated with osimertinib and actinomycin D in combination decreased to 42.1% of that of AD-RD cells treated with DMSO (negative control). This result indicates that the tumorigenic potential of AD-RD cells is more efficiently attenuated by combining actinomycin D and osimertinib.

[0167] Furthermore, the phosphorylation status of CDKAL1 in AD-RD cells treated with actinomycin D and / or osimertinib was evaluated by Western blotting. Specifically, the culture medium of RD cells cultured to 80% confluence on a 10 cm culture dish was replaced with a standard medium containing 1 nM actinomycin D and / or 2.5 μM osimertinib, prepared by adding a DMSO solution of actinomycin D (10 mM in DMSO) and / or a DMSO solution of osimertinib (10 mM in DMSO) to standard medium. The culture medium was incubated for 24 hours at 37°C in a humid environment with 5% CO2 after the medium change. On the other hand, as a negative control, the culture medium was replaced with a standard medium to which the same amount of DMSO as the DMSO solution of osimertinib was added, and the culture medium was incubated for 24 hours at 37°C in a humid environment with 5% CO2 after the medium change. Subsequently, 1 mL of cell lysis buffer was added, and the cells were disrupted using a 23-gauge needle and syringe. The cells were then centrifuged at 13,500 rpm at 4°C for 15 minutes, and the supernatant was collected as a protein extract. The collected protein extract was subjected to Western blotting. The antibodies used for Western blotting are as follows:

[0168] [Table 10]

[0169] The results obtained are shown in the lower panel of Figure 8B. As shown in the lower panel of Figure 8B, when AD-RD cells were treated with actinomycin D alone, the proportion of CDKAL1(pT43) with phosphorylated threonine at position 43 was increased compared to when DMSO was treated. Thus, it is thought that the enhancement of Thr43 phosphorylation of CDKAL1 when actinomycin D is treated alone is the main culprit in maintaining and enhancing the cancer stem cell properties of rhabdomyosarcoma. In contrast, when osimertinib was treated together with actinomycin D, the proportion of CDKAL1(pT43) with phosphorylated threonine at position 43 was significantly reduced. Thus, it is thought that osimertinib inhibits the phosphorylation of Thr43 of CDKAL1, which is the main culprit in maintaining and enhancing the cancer stem cell properties of rhabdomyosarcoma, thereby suppressing the cancer stem cell properties of AD-RD cells, and that the combination of actinomycin D and osimertinib exhibits a superior antitumor effect on AD-RD cells.

[0170] <Experiment 9.3. Effects on the formation of translation initiation factor complexes in AD-RD cells> The effect of osimertinib on the formation of translation initiation factor complexes in AD-RD cells was evaluated using a pull-down assay with m7GTP beads.

[0171] Specifically, AD-RD cells cultured to 80% confluence on a 10 cm cell culture dish were mixed with osimertinib dissolved in DMSO to a final concentration of 1 μM, or DMSO as a negative control, and cultured for 24 hours in a humid incubator at 37°C and 5% CO2 according to standard procedures. After removing the culture medium, 1 mL of cell lysis buffer was added, and the cells were lysed using a 23-gauge needle and syringe according to standard procedures. The solution containing the lysed cells was centrifuged at 13,500 rpm at 4°C for 15 minutes, and the supernatant was collected as a protein extract. 40 μL of m7GTP beads (Jena Bioscience; catalog number AC-155S), washed with cell lysis buffer, were added to each protein extract, and the mixture was allowed to stand at 4°C for 4 hours. After standing, the m7GTP beads were washed three times with cell lysis buffer, and 100 μL of 4×SDS-PAGE sample buffer, diluted fourfold with cell lysis buffer, was added. The mixture was then allowed to stand at 95°C for 5 minutes. The solution was then centrifuged, and the supernatant was used as a sample for Western blotting. The Western blotting procedure was as described above. The antibodies used in this experiment are as follows.

[0172] [Table 11]

[0173] The results obtained are shown in Figure 8C. As shown in Figure 8C, from the protein extract of AD-RD cells not treated with osimertinib (Osimertinib "-" in Figure 8C), eIF4E, which binds to m7GTP beads, along with eIF4G, eIF4A, and CDKAL1 were pulled down. In contrast, from the protein extract of AD-RD cells treated with osimertinib (Osimertinib "+" in Figure 8C), only eIF4E, which binds to m7GTP beads, was pulled down, while eIF4G, eIF4A, and CDKAL1 were hardly pulled down at all. This result indicates that treatment with osimertinib suppresses the formation of the CDKAL1-involved translation initiation factor complex in AD-RD cells.

[0174] <Experiment 9.4. Effects on drug resistance in AD-RD cells> Next, the effect of osimertinib treatment on drug resistance in AD-RD cells was investigated in combination with actinomycin D.

[0175] Specifically, 5,000 RD cells or AD-RD cells were seeded in each well of a 96-well cell culture plate, and the following day the culture medium was changed to a standard medium containing actinomycin D at a predetermined concentration. For AD-RD cells, a basic medium containing 1 μM osimertinib in addition to actinomycin D, or a basic medium containing DMSO as a negative control, was prepared, and the cells were changed to these media. After culturing for 72 hours in a humid incubator at 37°C and 5% CO2 according to standard procedures, cell viability was calculated using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega; catalog number G3582) according to the protocol provided with the product.

[0176] The results obtained are shown in Figure 8D. As shown in Figure 7A, the IC50 for AD-RD cells treated with actinomycin D alone was 3.07 nM, whereas, as shown in Figure 8D, when osimertinib was used in combination, the IC50 of actinomycin for AD-RD cells decreased to 1.06 nM. This result indicates that osimertinib reduces the drug tolerance of AD-RD cells to actinomycin D.

[0177] <Experiment 9.5. Immunostaining> The expression levels of stem cell markers and the phosphorylation level of CDKAL1 Thr43 in AD-RD cells treated with osimertinib were evaluated by immunohistochemical staining.

[0178] AD-RD cells coated with collagen (collagen acidic solution I-PC 3 mg / mL; Koken Co., Ltd.) in an 8-well chamber slide (Nunc TM Lab-Tek TM After culturing on a II Chamber Slide System (catalog number 154534PK; Thermo Fisher Scientific), the cells were replaced with standard medium containing 1 μM osimertinib or DMSO as a negative control, and incubated for 48 hours in a humid incubator at 37°C and 5% CO2 according to standard procedures. After incubation, the medium was removed, 4% paraformaldehyde was added, and the cells were fixed by incubation at room temperature for 20 minutes. After removing the paraformaldehyde and washing with PBS, the cells were blocked at room temperature for 1 hour using PBST (Phosphate-buffered saline with tween20) (BSA-PBST: 1% BSA, 150 mM NaCl, 10 mM sodium phosphate (pH 7.5), 0.05% Tween20) containing 1% bovine serum albumin (BSA). Subsequently, the cells were reacted with BSA-PBST containing the primary antibody at 4°C for 16 hours. After washing three times with PBST, the samples were reacted with BSA-PBST containing the secondary antibody at room temperature for 1 hour. After washing three times with PBST, the samples were treated with DAPI-Fluoromount-G. TMThe specimens were mounted using Southern Biotechnology Associates (catalog number 0100-20). After mounting, the specimens were imaged using a confocal laser microscope LSM780 (ZEISS). The antibodies used for staining are as follows:

[0179] [Table 12]

[0180] The results obtained are shown in Figure 8E. As shown in Figure 8E, the expression level of CD133, a cancer stem cell marker, was reduced in AD-RD cells treated with osimertinib. Furthermore, the amount of Thr43-phosphorylated CDKAL1 was attenuated, and the expression level of SALL2, which is translated by a translation mechanism involving CDKAL1, was also reduced. These results indicate that osimertinib suppresses Thr43 phosphorylation of CDKAL1, inhibits the translation mechanism involving CDKAL1, and thereby reduces cancer stem cell characteristics.

[0181] [Explanation of the sequence list] Sequence ID 1: Amino acid sequence of wild-type human CDKAL1 (CDKAL1 WT) MPSASCDTLLDDIEDIVSQEDSKPQDRHFVRKDVVPKVRRRNTQKYLQEEENSPPSDSTIPGIQKIWIRTWGCSHNNSDGEYMAGQLAAYGYKITENASDADLWLLNSCTVKNPAEDHFRNSIKKAQEENKKIVLAGCVPQAQPRQDYLKGLSIIGVQQIDRVVEVVEETIKGHSVRLLGQKKDNGRRLGGARLDLPKIRKNPLIEIISINTGCLNACTYCKTKHARGNLASYPIDELVDRAKQSFQEGVCEIWLTSEDTGAYGRDIGTNLPTLLWKLVEVIPEGAMLRLGMTNPPYILEHLEEMAKILNHPRVYAFLHIPVQSASDSVLMEMKREYCVADFKRVVDFLKEKVPGITIATDIICGFPGETDQDFQETVKLVEEYKFPSLFINQFYPRPGTPAAKMEQVPAQVKKQRTKDLSRVFHSYSPYDHKIGERQQVLVTEESFDSKFYVAHNQFYEQVLVPKNPAFMGKMVEVDIYESGKHFMKGQPVSDAKVYTPSISKPLAKGEVSGLTKDFRNGLGNQLSSGSHTSAASQCDSASSRMVLPMPRLHQDCALRMSVGLALLGLLFAFFVKVYN Sequence ID 2: Amino acid sequence of a mutant (CDKAL1 T43A) in which the 43rd threonine in the amino acid sequence of wild-type human CDKAL1 is replaced with alanine. MPSASCDTLLDDIEDIVSQEDSKPQDRHFVRKDVVPKVRRRNAQKYLQEEENSPPSDSTIPGIQKIWIRTWGCSHNNSDGEYMAGQLAAYGYKITENASDADLWLLNSCTVKNPAEDHFRNSIKKAQEENKKIVLAGCVPQAQPRQDYLKGLSIIGVQQIDRVVEVVEETIKGHSVRLLGQKKDNGRRLGGARLDLPKIRKNPLIEIISINTGCLNACTYCKTKHARGNLASYPIDELVDRAKQSFQEGVCEIWLTSEDTGAYGRDIGTNLPTLLWKLVEVIPEGAMLRLGMTNPPYILEHLEEMAKILNHPRVYAFLHIPVQSASDSVLMEMKREYCVADFKRVVDFLKEKVPGITIATDIICGFPGETDQDFQETVKLVEEYKFPSLFINQFYPRPGTPAAKMEQVPAQVKKQRTKDLSRVFHSYSPYDHKIGERQQVLVTEESFDSKFYVAHNQFYEQVLVPKNPAFMGKMVEVDIYESGKHFMKGQPVSDAKVYTPSISKPLAKGEVSGLTKDFRNGLGNQLSSGSHTSAASQCDSASSRMVLPMPRLHQDCALRMSVGLALLGLLFAFFVKVYN Sequence ID 3: Amino acid sequence of a mutant (CDKAL1 ΔN) in which the N-terminal amino acid sequence of wild-type human CDKAL1 is cleaved. MPLIEIISINTGCLNACTYCKTKHARGNLASYPIDELVDRAKQSFQEGVCEIWLTSEDTGAYGRDIGTNLPTLLWKLVEVIPEGAMLRLGMTNPPYILEHLEEMAKILNHPRVYAFLHIPVQSASDSVLMEMKREYCVADFKRVVDFLKEKVPGITIATDIICGFPGETDQDFQETVKLVEEYKFPSLF INQFYPRPGTPAAKMEQVPAQVKKQRTKDLSRVFHSYSPYDHKIGERQQVLVTEESFDSKFYVAHNQFYEQVLVPKNPAFMGKMVEVDIYESGKHFMKGQPVSDAKVYTPSISKPLAKGEVSGLTKDFRNGLGNQLSSGSHTSAASQCDSASSRMVLPMPRLHQDCALRMSVGLALLGLLFAFFVKVYN [Industrial applicability]

[0182] According to one aspect of the present invention, the agent, the method for searching for candidate compounds for treating cancer, and / or the method for searching for drug targets for treating cancer, the discovery of novel cancer therapeutics that target a translation process specific to cancer cells and / or cancer stem cells, and on which many cancer cells and / or cancer stem cells depend, such as the CDKAL1-dependent translation mechanism, can be facilitated.

Claims

1. A cancer treatment agent containing an active ingredient that inhibits the phosphorylation of threonine at position 43 of CDKAL1.

2. The agent according to claim 1, wherein the aforementioned component is a component that suppresses the phosphorylation by MYLK2 and / or MYLK4.

3. The agent according to claim 2, wherein the component is an inhibitor of MYLK2 and / or MYLK4, a nucleic acid that suppresses the expression of MYLK2 and / or MYLK4, and / or a vector that expresses a nucleic acid that suppresses the expression of MYLK2 and / or MYLK4.

4. The agent according to claim 2, wherein the aforementioned component is osimertinib.

5. An agent according to any one of claims 1 to 4 for suppressing cancer stem cell properties and / or suppressing drug resistance.

6. The agent according to any one of claims 1 to 5, wherein the cancer overexpresses CDKAL1 and / or MYLK2 and / or MYLK4.

7. The agent according to claim 6, wherein the cancer is rhabdomyosarcoma.

8. A method for discovering drug targets to treat cancer, The process of selecting candidate proteins that phosphorylate the 43rd threonine of CDKAL1 as drug targets for cancer treatment. A method that includes this.

9. The process includes suppressing the expression of the candidate protein in cancer cells, If the degree of phosphorylation of threonine 43 of CDKAL1 in cancer cells where the expression of the candidate protein is suppressed is reduced compared to when the expression of the candidate protein is not suppressed, the candidate protein is selected as a drug target for treating cancer. The method according to claim 8.

10. The process includes contacting the candidate protein with CDKAL1, If the degree of phosphorylation of the 43rd threonine of CDKAL1 increases when it is in contact with the candidate protein compared to when it is not in contact with the candidate protein, the candidate protein is selected as a drug target for treating cancer. The method according to claim 8.

11. A method for searching for candidate compounds for active ingredients of drugs used to treat cancer, A process for selecting a test compound that inhibits the phosphorylation of threonine at position 43 of CDKAL1 by kinase as a candidate compound for treating cancer. A method that includes this.

12. A step of contacting the kinase with CDKAL1 in the presence of the test compound, The step includes evaluating the degree of phosphorylation of the 43rd threonine of CDKAL1 after contact with the kinase, If, compared to the case where the test compound is absent, the degree of phosphorylation of the 43rd threonine of CDKAL1 in contact with the kinase decreases in the presence of the test compound, then the test compound is selected as a candidate compound for an active ingredient in a drug for treating cancer. The method according to claim 11.

13. The method according to claim 11 or 12, wherein the kinase is MYLK2 and / or MYLK4.

14. A method for searching for candidate compounds for active ingredients of drugs used to treat cancer, A step of selecting a test compound that inhibits the autophosphorylation of MYLK2 and / or MYLK4 by MYLK2 and / or MYLK4 as a candidate compound for treating cancer. A method that includes this.

15. A step of incubating MYLK2 and / or MYLK4 in the presence of the test compound, The process includes evaluating the degree of phosphorylation of MYLK2 and / or MYLK4, If the degree of phosphorylation of MYLK2 and / or MYLK4 incubated in the presence of the test compound decreases compared to when the test compound is not present, the test compound is selected as a candidate compound for an active ingredient in a drug for treating cancer. The method according to claim 14.

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