WO2017110830A1 - Method for predicting effect of combination therapy on cancer using l-asparaginase agent and autophagy inhibitor, and cancer therapeutic agent - Google Patents

Abstract

The present invention addresses the problem of finding an index of whether or not a combination therapy using L-asparaginase and an autophagy inhibitor is effective, and providing a cancer therapeutic agent for which the index can be used, and provides: a method for obtaining data on prediction of the effect of a pharmaceutical agent, wherein, when the functional deletion of p53 due to gene mutation is identified in a cancer specimen, the data is determined to be negative in terms of the effect of a combination therapy using L-asparaginase and an autophagy inhibitor on cancer; and a therapeutic agent which is for a cancer showing resistance to sole administration of an L-asparaginase agent, and which is characterized by containing L-asparaginase and an autophagy inhibitor.

Description

Prediction method of effect of combined therapy of L-asparaginase agent and autophagy inhibitor for cancer, and cancer therapeutic agent Related applications

This application claims priority based on Japanese Patent Application No. 2015-250112 filed on Dec. 22, 2015, the entire disclosure of which is incorporated herein by reference.

The present invention relates to an invention relating to a means for predicting the effect of drug therapy on cancer and an invention relating to a cancer therapeutic agent.

In order for cancer cells to grow, various substances are required, and asparagine (Asp), one of the amino acids that constitute proteins, is one of them. Asparagine is produced by asparagine synthase and degraded by L-asparaginase (L-asp).

Some cancers are deficient in asparagine synthase in the cells and are deficient in asparagine. Further, when L-asp is given, the growth of the cancer cells is known to be inhibited. (Non-patent Document 1), an anticancer agent (L-asp agent) using L-asp has been put into practical use for leukemia, malignant lymphoma and the like.

Acute lymphocytic leukemia (ALL) is known as one of the cancers for which L-asp drugs are key drugs. Although treatment results have improved due to advances in treatment means, 20% of childhood ALL and 40% of adult ALL become treatment refractory and still have a poor prognosis (Non-Patent Documents 2 and 3).

Among them, the L-asp agent alone can bring about complete remission in 60% of ALL, and further, by devising the dose and frequency of administration of L-asp, the disease-free survival rate for ALL (Non-patent Document 4).

However, it is also known that a decrease in the sensitivity of ALL cells to L-asp leads to an increased recurrence rate and treatment refractory (Non-patent Document 5).

By the way, autophagy is one of the intracellular degradation systems by lysosomes. Long-lived proteins, intracellular organelles, and protein aggregation are trapped in autophagosomes and then fused and degraded by lysosomes. It is a highly controlled process. In particular, it is an amino acid supply source that enables cells to survive under adverse conditions of various stresses such as amino acid deficiency and oxidative stress. In recent years, it has been reported that L-asp induces autophagy in ovarian cancer cells and ALL (Non-patent Documents 6 and 7). In chronic myelogenous leukemia, autophagy induced by L-asp works favorably for the survival of cancer cells, and the combined use of L-asp and an autophagy inhibitor reduces the cell killing effect of L-asp. It was reported to be enhanced (Non-patent Document 8).

However, even in the above-mentioned combination therapy of L-asp and an autophagy inhibitor, there are cases where the treatment is successful and resistance is exhibited. The present invention reveals the mechanism of action of this combination of L-asp and an autophagy inhibitor in cancer cells, finds an index as to whether or not the combination therapy is successful, and can utilize the index. The present invention has been made with the object of providing a therapeutic agent for cancer.

The present inventors have now found the following (1) to (6) and found that the p53 gene plays a decisive role in the combined therapy of L-asp and an autophagy inhibitor.
(1) L-asp causes autophagy in ALL cells,
(2) L-asp-induced autophagy is important for the maintenance of mitochondrial quality, and the combined use of L-asp and autophagy inhibitors causes accumulation of oxidative stress in ALL cells,
(3) DNA damage caused by excessively accumulated active oxygen (ROS) induces further oxidative stress via the p53 gene, and induces apoptotic cell death of ALL cells,
(4) p53 gene knockdown ALL strains and ALL clinical specimens having a mutated p53 gene have a poor effect of L-asp and autophagy inhibitor combination therapy,
(5) The effect of combined therapy with L-asp and an autophagy inhibitor is restored by introducing a normal p53 gene into an ALL clinical specimen having a mutant p53 gene lacking function,
(6) L-asp-resistant ALL, which shows increased expression of asparagine synthase, also has the effect of combined therapy with L-asp and an autophagy inhibitor.

The above items (1) to (6) are based on the use of a combination of L-asp and an autophagy inhibitor in cancer patients by using as an index the presence or absence of a mutation that causes a loss of function in the p53 gene of the subject. It shows that it is possible to judge the effectiveness of therapy.

That is, the present invention provides a combination of L-asp (L-asparaginase) or a derivative thereof and an autophagy inhibitor in a cancer sample when a loss of p53 function due to a gene mutation is observed in the cancer sample. Provided is a method for acquiring drug effect prediction data (hereinafter also referred to as the data acquisition method of the present invention), which is negative data on the effect of therapy. The data acquisition method of the present invention is a method for the combination therapy of L-asp or a derivative thereof and an autophagy inhibitor in a cancer sample when a mutation resulting in loss of function of the p53 gene is observed in the cancer specimen. It can also be expressed as a method for predicting the effect of a drug that performs a negative prediction of the effect.

In the present invention, “L-asp or L-asparaginase” means L-asparaginase itself. Examples of the “L-asp derivative or L-asparaginase derivative” include polyethylene glycolated asparaginase. “L-asp or a derivative thereof” is an agent having anti-cancer use, and is an L-asp or L-asp derivative itself as an active ingredient, and a substance obtained by applying some formulation means (pharmaceuticals) Composition). Further, “L-asp or a derivative thereof” is hereinafter referred to as “L-asps”.

Further, the “autophagy inhibitor” is a concept including an autophagy-inhibiting ingredient that is an active ingredient thereof and a substance (pharmaceutical composition) obtained by subjecting it to some formulation means.

The cancer samples subject to the data acquisition method of the present invention are basically confirmed to be effective in the administration of L-asps, that is, the administration of L-asps is selected as an option for the cancer treatment. A specimen of cancer that can be done. This choice includes not only the case generally accepted as a course of cancer treatment but also other challenging choices. Specific examples include leukemia, malignant lymphoma, ovarian cancer and the like, but are not limited thereto. As described above, L-asps are used as first-line drugs for acute lymphoblastic leukemia (ALL), and are typical cancers as subjects for performing the data acquisition method of the present invention.

The cancer specimen used in the data acquisition method of the present invention includes cancer cells directly because of the necessity of detecting a mutation that causes a loss of function of the p53 gene of the cancer cells in the specimen. There is a need. Therefore, a blood sample and / or bone marrow sample containing leukemia cells if it is a blood cancer such as leukemia, and if it is a solid cancer, a biopsy sample of the cancer or a body fluid sample containing the cancer, It is mentioned as a cancer sample used in the data acquisition method of the present invention.

Items (1) to (6) above show that the combined administration of L-asps and autophagy inhibitors is effective even for cancers that are resistant to L-asps alone. Show.

Accordingly, the present invention secondly provides a therapeutic agent for cancer having resistance to single administration of L-asps, which comprises L-asps and an autophagy inhibitor (hereinafter referred to as the present invention). (Also referred to as a cancer therapeutic agent).

The autophagy inhibitor is not particularly limited, and examples thereof include chloroquines such as chloroquine (chloroquinoline) and hydroxychloroquine, 3-methyladenine, bafilomycin A, urtomannin and the like, but are not limited thereto. Absent.

Even if the cancer therapeutic agent of the present invention is a pharmaceutical composition containing (a) L-asps and an autophagy inhibitor, (b) the L-asps and the autophagy inhibitor are separately used. It may be a set of drugs included as a constituent drug.

The cancer therapeutic agent of the present invention may further contain an L-asparagine synthase inhibitor (sulfoximine-based inhibitor, Bioorganic® & Medicinal® Chemistry® 20 (2012) 5915-5927, etc.). This L-asparagine synthase inhibitor is contained in a form separate from (b) L-asps and autophagy inhibitors, even though it is contained in (a) a pharmaceutical composition as described above. It may be contained as a constituent drug.

Subjects for administration of the cancer therapeutic agent of the present invention are those that are resistant to the administration of L-asps alone and that do not show a mutation that results in loss of function of the p53 gene. Cancers that have been confirmed to be effective, and specific types of cancer are the same as those of the data acquisition method of the present invention, such as leukemia, malignant lymphoma, ovarian cancer, preferably acute lymphocytic leukemia, acute bone marrow Include, but are not limited to, sexual leukemia, chronic myeloid leukemia, NK / T cell lymphoma. In addition, even if a mutation that results in a loss of function of the p53 gene was originally observed, if the cancer was subsequently repaired, the cancer therapeutic agent of the present invention will be administered. . Repair of mutations resulting in loss of function of the p53 gene can be performed by so-called gene therapy.

Whether or not the cancer therapeutic agent of the present invention is administered using the presence or absence of a mutation causing loss of function of the p53 gene as an index can be determined based on the data obtained by the data acquisition method of the present invention. is there. That is, if the data obtained by the data acquisition method of the present invention indicates the presence of a mutation in the p53 gene that results in loss of function, immediate administration of the cancer therapeutic agent of the present invention is not appropriate, Other treatment means may be selected, or a schedule for administering the cancer therapeutic agent of the present invention may be selected after repair of a mutation that results in loss of function of the p53 gene. When the data acquisition method of the present invention is performed before treatment with L-asps, treatment with L-asps can be selected first. On the other hand, if no mutation that results in a loss of function of the p53 gene is observed in the data, the cancer therapeutic agent of the present invention can be administered. When the data acquisition method of the present invention is performed before treatment with L-asps, treatment with L-asps can be selected first.

The subject of administration of the cancer therapeutic agent of the present invention is at least “cancer having resistance to L-asps alone administration”, and the resistant cancer is administered in advance by in vivo administration of L-asps. Can be defined based on negative anti-cancer effect data against the cancer in question or negative anti-cancer effect data obtained by contacting the cancer cells with L-asps outside the body in advance. .

According to the present invention, a means for predicting the effect of combination therapy with an agent of L-asp or a derivative thereof and an autophagy inhibitor is provided, and further a cancer therapeutic agent capable of utilizing the prediction result is provided.

It is drawing which flow-ized one aspect | mode of the detection process of the function deletion mutation of the p53 gene in the data acquisition method of this invention. Induction of autophagy when L-asp and an autophagy inhibitor are used alone or in combination with three types of ALL cell lines, or when both are not acted on by electrophoresis using LC3B-II as an indicator It is the figure which showed the result. Tissues examined for LC3B-II as an indicator of autophagy induction when RE-cells, an ALL cell line, and L-asp and an autophagy inhibitor, Chloroquine (CQ), are used alone or in combination. It is drawing which showed the immuno-staining image. Regarding autophagy induction when RE-cell, which is an ALL cell line, and L-asp and an autophagy inhibitor, Chloroquine (CQ), are used alone or in combination, or both are not allowed to act, an electron microscope It is drawing shown by the image. 2C is a drawing showing the number and area of autophagy vesicles per cell in the electric microscope image of FIG. 2-C. Results of counting the number of damaged mitochondria based on fluorescent dye staining when RE-cell, an ALL cell line, was allowed to act on L-asp and autophagy inhibitor Chloroquine (CQ) alone or in combination It is drawing which showed. The membrane potential of intracellular mitochondria when L-asp and autophagy inhibitor Chloroquine (CQ) are used alone or in combination with REH cells, which are ALL cell lines, is TMRE (tetramethylrhodamine methylester). It is drawing which showed the result examined using this. DCFDA (5- (and-6) -carboxy-2 ′ when L-asp and autophagy inhibitor Chloroquine (CQ) are allowed to act on REH cells, which are ALL cell lines, alone or in combination. , 7'-dichlorofluorescein diacetate, intracellular reactive oxygen species (ROS) (left figure), and mitochondrial ROS evaluation using MitoSox (registered trademark) -Red. Drawing showing the results of examination of the degree of cell death when L-asp and an autophagy inhibitor were used alone or in combination on three types of ALL cell lines by flow cytometry and Western blotting analysis. is there. Suppression of cell death induced by the action of L-asp and autophagy inhibitor Chloroquine (CQ) against REH cells, an ALL cell line, by z-VAD, a pan-apoptosis-related molecular inhibitor It is drawing which showed the result of examining an effect | action. FIG. 6 is a drawing showing the results of examining the viable cell rate of L-asp resistant strain 697-R with respect to an increase in L-asp addition amount. FIG. 6 is a drawing showing the results of examining the influence of L-asp and autophagy inhibitor CQ on the viable cell rate in L-asp resistant strain 697-R. The protein expression level of asparagine synthase (ASNS) in the L-asp resistant strain 697-R is caused when L-asp and the autophagy inhibitor Chloroquine (CQ) are used alone or in combination, or both are not allowed to act. It is drawing which shows the result of having performed western blotting analysis about the case. L-asp and Chloroquine (CQ), an autophagy inhibitor, alone or in combination in “sh-ASNS1” and “sh-ASNS2” in which the ASNS gene of REH cell, an ALL cell line, was knocked down at different sites It is drawing which shows the result of having examined about the case where it is made to act, or the case where neither is made to act. It is a figure which shows the result of the western blotting analysis which examined the mechanism which causes the apoptotic cell death induced by combined use of L-asp and an autophagy inhibitor. FIG. 3 is a drawing showing the results of examining the effect on cell killing effect when acetylcysteine (NAC), an ROS inhibitor, is added to the cell culture system of FIG. It is drawing which shows the result of having performed cell cycle evaluation by the flow cytometry which used Propidium Iodide dyeing | staining. It is a figure which shows the result of having examined the influence by the continuous drug contact of a day unit by counting the number of living cells. It is a figure which shows the result of having carried out the transvenous vein transplantation to the immunodeficient mouse | mouth, and having investigated the progress by fluorescence distribution detection, for REH / Luc2 cell which introduce | transduced the luciferase gene in order to perform the treatment evaluation in vivo. In order to evaluate the treatment in vivo, REH / Luc2 cells introduced with luciferase gene were transplanted via tail vein into immunodeficient mice, and the results were examined based on the photon flux rate of fluorescence. It is a drawing. FIG. 3 is a graph showing the results of whole survival analysis in which the survival rate over time of immunodeficient mice transplanted with REH / Luc2 cells in FIGS. 3-K and 3-L was examined on a daily basis. FIG. 4 shows the results of Western blotting analysis of DNA damage over time when L-asp and autophagy inhibitor Chloroquine (CQ) are used alone or in combination with REH cells, an ALL cell line. It is a drawing. LHasp and autophagy inhibitor Chloroquine (CQ) were allowed to act on REH cells, which are ALL cell lines, alone or in combination, and ROS inhibitor acetylcysteine (NAC) was allowed to act on this. It is drawing which shows the result of having examined the increase / decrease of p53 protein by Western blotting analysis with (gamma) H2AX which is a parameter | index of DNA damage in the case. In the REH cell line (sh-p53) in which the p53 gene was knocked down by sh (short hairpin) RNA, DNA damage caused by combining L-asp and the autophagy inhibitor Chloroquine (CQ) It is drawing which shows the result examined by blotting analysis. It is a figure which shows the result of having investigated accumulation of intracellular ROS in p53 knockdown REH cell line (sh-p53) similarly to FIG. 4-C. As in FIG. 4-C, the suppression of cell death in the p53 knockdown REH cell line (sh-p53) was examined by Annexin V incorporation (left graph) and Western blotting analysis (right electrophoretic diagram). FIG. The effect of combined administration of L-asp and chloroquine (CQ), which is an autophagy inhibitor, in ALL clinical blood samples (total of 12 cases of initial cases and 2 cases of relapses) It is the figure which showed the result examined together with the presence or absence of the mutation which becomes. In FIG. 5-A, the results of autophagy activity evaluation in 3 clinical blood samples of ALL in which the effect of combined administration of L-asp and chloroquine (CQ), an autophagy inhibitor, was shown by Western blotting analysis. It is a drawing. In FIG. 5-A, a clinical blood sample of ALL (case 14) in which p53 was lost in function due to a gene mutation in which the effect of combined administration of L-asp and the autophagy inhibitor Chloroquine (CQ) was not observed. On the other hand, it is the figure which showed the effect at the time of introduce | transducing the normal p53 gene by adenovirus by Western blotting analysis. In FIG. 5-A, a clinical blood sample of ALL (case 14) in which p53 was lost in function due to a gene mutation in which the effect of combined administration of L-asp and the autophagy inhibitor Chloroquine (CQ) was not observed. On the other hand, it is the figure which showed the result of having examined the effect at the time of introduce | transducing the normal p53 gene by adenovirus by the number of living cells. It is the figure which showed the result of having examined the effect of the administration when the REH cell line (sh-p53) which knocked down the p53 gene by sh (short hairpin) RNA was transplanted into the immunodeficient mouse via the tail vein. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a chart showing the mechanism of apoptosis induction of ALL cells by the combined administration of L-asp and an autophagy inhibitor, Chloroquine (CQ).

1. Data acquisition method of the present invention The basic data of the data acquisition method of the present invention is detection data of a mutation that results in loss of function of the p53 gene, and detection of a mutation that results in loss of function of the p53 gene in a cancer sample Is the premise.

The p53 gene is evolutionarily conserved and is found in species other than humans. The human p53 gene is present on the short arm of chromosome 17 (17p13.1). The p53 gene is known as a tumor suppressor gene, and is known to play a role in regulating cell growth such as maintenance of intracellular homeostasis, metabolic regulation, regulation of intracellular ROS production, and induction of apoptosis. It has been. A mutation resulting in loss of function of p53 is one in which the p53 gene in both alleles on the chromosome is inactivated. The following four types are listed as basic types of this loss-of-function mutation.
(1) A type in which one allele has a loss-of-function mutation of the p53 gene, and the other allele has a loss of heterozygosity (LOH) of the p53 gene existing region of the short arm of chromosome 17 (hereinafter referred to as the first type) Also called). The first type of loss-of-function mutations that occur in cancer cells are those in which loss-of-function missense mutations (such as R175H and R248Q), nonsense mutations, and frameshift mutations have occurred prior to the LOH of the p53 gene.
(2) The above-mentioned loss-of-function mutation occurs in the p53 gene of one allele, and the loss-of-function mutation also occurs in the p53 gene of the other allele, and a homo mutation or a compound hetero mutation Type (hereinafter also referred to as the second type).
(3) Deletion mutation of chromosome 17 short arm or deletion mutation of chromosome 17 (hereinafter also referred to as type 3)
(4) Only the loss-of-function mutation of the p53 gene is observed in one allele, and no mutation is observed in the p53 gene of the other allele. As a subunit, a mutant p53 subunit is randomly combined to exert function inhibition, thereby reducing the overall cancer suppression effect (apoptosis inducing action) efficiency (hereinafter also referred to as a fourth type).

Of these four types of mutations that result in loss of function of the p53 gene, the first type is the most frequent. The mutation that results in loss of function of the first type of p53 gene is in a cancer cell in which the mutation of the p53 gene has advanced to a high degree. Therefore, by detecting only LOH, the loss of function of other alleles that exist together is detected. Detection can include detection of a mutation. Of course, for example, LOH detection and direct sequence analysis can be performed together.

Examples of the detection of the first type of LOH include a genomic instability test (microsatellite instability test) using a microsatellite marker, the FISH method, the method described in JP-A-2000-93185, the Southern blot method, and the like. .

In the microsatellite instability test, the “genomic instability” in the gene of the target cancer cell is expressed as a repetitive sequence of microsatellite ((A) n, (CA) n, etc. in the genome from a single base to several base units). The first type of LOH can be detected using a specific one of the markers (from several to several tens of repetitions and 10 ⁴ to 10 ⁵ repetitions) (Vita Vol .15 NO.3 1998 / 7.8.9).

FISH method [fluorescence in situ hybridization (FISH: Fluorescence in situ hybridization): Yasui, K., Imoto, I., Fukuda, Y., Pimkhaokham, A., Yang, ZQ, Naruto, T., Shimada, Y. , Nakamura, Y., And Inazawa, J: Identification of target genes within an amplicon at 14q12-q13 in esophageal squamous cell carcinoma. Genes Chromosomes Cancer, 32, 112-118, 2001] In addition, the first type of LOH can be detected. In this case, the LOH of the p53 gene can be detected by preparing a FISH probe of the p53 gene region, performing hybridization with a chromosome in a cancer specimen, and observing the change in the number of signals.

The method described in Japanese Patent Application Laid-Open No. 2000-93185 identifies loss of heterozygosity in a gene polymorphism marker in the Na / K-ATPase beta2 subunit gene that is present in the downstream region of the p53 gene of the cancer sample gene. This method can also detect the LOH of the p53 gene.

When Southern blotting is used, genomic DNA obtained from a cancer specimen is digested with a restriction enzyme, gel-electrophoresed, immobilized on a nitrocellulose membrane, and hybridized with labeled p53 gene region DNA. By performing detection, the presence of LOH of the p53 gene in the specimen is detected. Presence of L53 of p53 gene in the specimen due to the presence of a different band in addition to the small quantity of detection obtained from the cancer specimen compared to the detection quantity (band density) obtained from the normal specimen. Can be detected.

The second type of loss-of-function p53 gene mutation can be detected, for example, by direct sequence analysis. More specifically, it can be detected by the Sanger method or the so-called next-generation sequencing method. The p53 gene mutation detected in Examples described later is this second type. However, the second type is less frequent than the first type described above.

The Sanger method consists of i) annealing an oligonucleotide complementary to a single-stranded DNA of the p53 gene, synthesizing a complementary strand with a DNA polymerase using this as a primer, and ii) using four deoxynucleotide triphosphates and dideoxy as substrates. Nucleotide triphosphate is added to synthesize a complementary strand. Iii) Further, a primer or dNTP labeled with a chemiluminescent substance is added to label the synthesized DNA and determine the base sequence. By comparing this sequence with a reference sequence, it is possible to detect a mutation that results in loss of function of the second type of p53 gene.

In the next generation sequencing method, the base sequences of tens of millions of randomly cut DNA fragments can be simultaneously determined. Deep sequencing of the gene region of p53 can be performed with high efficiency by pooled sequencing of PCR products. As a result, a mutation that causes a loss of function of the second type p53 gene can be detected even at a low frequency.

A deletion mutation of the third type of chromosome 17 short arm or a deletion mutation of chromosome 17 can be detected by the above FISH method or chromosome examination. Chromosome testing is typically chromosomal testing such as G-separation. Is an abbreviation for Giemsa, and is used for Giemsa staining after trypsin treatment. Thereby, the presence or absence of the deletion of the short arm of chromosome 17 where the p53 gene is located, or the deletion of the chromosome 17 itself (monosomy) can be confirmed.

In addition to the above-mentioned Sanger sequencing method, the p53 protein mutation lacking function due to the tertiary structure of the subunits of the fourth type of p53 protein has a half-life extended in the cell (particularly in the nucleus). The immunohistochemical staining of the p53 protein utilizing the accumulation in (1), and the detection of autoantibodies using an ELISA kit against the p53 protein having a mutation can be performed.

As described above, it is possible to obtain data from cancer specimens regarding mutations that result in loss of function of the p53 gene. By combining the above detection methods, it is possible to detect the first type, the second type, the third type, the fourth type, and the genetic mutation that results in the loss of function of all p53 genes. It is also possible to perform individual detection for each type. In this case, it is preferable to prioritize the first type with high frequency.

FIG. 1 is a flow chart showing an embodiment of a process for detecting a loss-of-function mutation of the p53 gene in the data acquisition method of the present invention.

Box A1 indicates the start of the detection process when the cancer specimen is a “solid cancer tissue specimen”. Tissue specimens include visceral tissue, bone tissue, muscle tissue, skin tissue, nerve tissue, and the like. Box A2 indicates the start of the detection process when the cancer sample is a “solid cancer liquid sample”. The liquid specimen is pleural effusion, ascites, urine residue and the like. Box A3 shows the start of the detection process when the cancer specimen is “leukemia or malignant lymphoma”.

The first stage of the detection process of the solid cancer tissue specimen (A1) is “LOH analysis by microsatellite marker” (box B1). The first stage of the detection process of the solid cancer liquid specimen (A2) is “LOH analysis by microsatellite marker” (B1) or “FISH analysis” (box B2). In addition, the first stage of the detection process of the leukemia or malignant lymphoma cancer specimen (A3) is “LOH analysis by microsatellite marker” (B1), “FISH analysis” (B2), or “chromosome test”.

If the result of LOH analysis (B1) using a microsatellite marker is “LOH positive” (R1 / 1), detection is performed as “positive for loss of function mutation” (J1 / 1) regardless of the type of cancer specimen. Is called. If the result of the LOH analysis (B1) is “normal” (R1 / 0), a “direct sequence analysis” (box C1), which is a second-stage detection process, is further performed. For the direct sequence analysis, the Sanger method or the NGS method is used.

If the result of “FISH analysis” (B2) or “chromosome test” (B3) is “normal” (R2 / 0), the “direct sequence analysis” ( C1) is further performed. As a result of “FISH analysis” (B2) or “chromosome test” (B3), “deletion of short arm of chromosome 17 (17p−) or deletion of chromosome 17 itself (−17)” (R2 / 0) ), The detection is performed as “positive for loss of mutation” (J1 / 2) regardless of the type of the first cancer specimen.

As a result of the direct sequence analysis (C1), when a known homo- or compound heterozygous loss-of-function mutation (R3 / 1) is observed, the “function-deficient mutation” is used regardless of the type of the first cancer specimen. Detection is performed as "positive" (J1 / 1). If the result of direct sequence analysis (C1) indicates “normal” (R3 / 0), detection is performed as “negative loss-of-function mutation” (J0) regardless of the type of the first cancer sample. Is called. If “unknown missense mutation” (R3 / 2) is detected as a result of the direct sequence analysis (C1), the “immunity”, which is the third stage detection step, is performed regardless of the type of the first cancer specimen. If "histochemical staining" (box D1) is performed and the result is "nuclear light staining" (R4 / 0). Detection is performed as “negative for loss of function mutation” (J0), and in the case of “nuclear dark staining” (R4 / 1), detection is performed as “positive for function deletion mutation” (J1 / 2).

Moreover, as a means for detecting a loss-of-function mutation of the p53 gene, a functional assay (Kato, Ishioka et al., PNAS, vol. 100, no. 13, pp. 8424-8429 (2003), Shimada, Kato, Ishioka et al., Cancer Res. 59, 2781-2786 (1999), Ishioka et al., NATURE GENETICS (Oct; 5 (2): 124-129) (1993)). According to this method, even when a novel mutation in the p53 gene is observed, it is possible to evaluate the loss of function.

The purification of cancer sample nucleic acid performed when detecting the above-mentioned loss-of-function p53 gene mutation may be preferable for reducing background noise, ensuring hybridization performance, efficient labeling, and the like. Many. “Purification” is used synonymously with extraction, separation and fractionation. As a means for this, a method using a cartridge carrying a nucleic acid-adsorbing membrane such as silica or cellulose derivative, ethanol precipitation, isopropanol precipitation, phenol-chloroform extraction, hydrophobic substituents such as ion exchange resin or octadecyl group, etc. A solid-phase extraction cartridge using a bound silica carrier or a resin exhibiting a size exclusion effect, a method by chromatography, and the like can be included. Furthermore, purification by electrophoresis can also be included. Solvent replacement is also a purification in a broad sense. A refinement | purification process can be performed once or several times as needed.

The presence / absence of a mutation that results in a loss of function of the p53 gene in the cancer specimen specified by the above detection step is basic data for the data acquisition method of the present invention. In other words, when a mutation causing a loss of function of the p53 gene is observed in a cancer sample (positive), it is predicted that the cancer sample is resistant to a combination therapy of L-asps and an autophagy inhibitor. It can be determined that it is preferable to exclude the combination therapy from at least the initial cancer treatment schedule. In this case, in addition to selecting a treatment means other than the combination therapy, it is also possible to set a schedule for performing the combination therapy after performing a repair treatment of the p53 gene in advance.

The repair treatment of the p53 gene is so-called gene therapy, and is performed by introducing a normal p53 gene into at least a cancer cell. For example, a normal p53 gene is cancerated using a modified viral vector such as a retrovirus vector such as a mouse leukemia virus vector or lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex type I vector, or an HVJ-liposome. Can be introduced into cells.

As an example, introduction by adenovirus vector will be explained. When an adenoviral vector incorporating the p53 gene is injected into a tumor, it binds to a receptor on the tumor cell, and the adenoviral vector is taken into the tumor cell to express the encoded foreign p53 gene. After that, it is expected that the effect of the combination therapy of L-asps and autophagy inhibitors will be effective.

In the data acquisition method of the present invention, it is predicted that a combination therapy of L-asps and an autophagy inhibitor is effective when a mutation resulting in loss of function of the p53 gene is not observed in a cancer sample (negative). In order to be actively incorporated into the treatment schedule.

2. Cancer therapeutic agent of the present invention The cancer therapeutic agent of the present invention comprises L-asps and an autophagy inhibitor as active ingredients.

(1) L-asps (L-asparaginases)
As described above, L-asp is an enzyme that degrades asparagine (Asn), which is one of the amino acids that make up proteins, and is used to treat cancer that is chronically asparagine-deficient due to a lack of asparagine synthase. It has already been used for acute lymphoblastic leukemia and the like as a cancer therapeutic agent that inhibits the growth of the cancer by administration.

For cancer cells to grow, various substances are required, and asparagine (Asn), one of the amino acids that make up proteins, is one of them. Asparagine is produced by asparagine synthase and degraded by L-asparaginase (L-asp).

Some cancers are deficient in asparagine synthase in the cells and are deficient in asparagine. Further, when L-asp is given, the growth of the cancer cells is known to be inhibited. (Non-patent Document 1), an anticancer agent using L-asp has been put into practical use for leukemia, malignant lymphoma and the like. L-asp itself can be produced by a known method using a microorganism transformed with a gene encoding L-asp (utilized in Escherichia coli and potato black rot) as a host, and commercially available products can be obtained. It is also possible to use it. As described above, L-asp derivatives obtained by chemically modifying L-asp are also included in L-asps as long as the above-described anticancer effects are observed. Examples of the derivative include polyethylene glycolated asparaginase. The L-asps may be in a form that has undergone some kind of formulation treatment, and examples thereof include erythrocyte encapsulated asparaginase.

The dose of L-asps (active ingredients) by the cancer therapeutic agent of the present invention is about 50 to 200 units / kg body weight per adult per day, and can be increased or decreased depending on the effect. The dosing interval can be performed once a day and every other day or every few days.

(2) Autophagy inhibitor As mentioned above, autophagy is one of the intracellular degradation systems by lysosomes, and long-existing proteins, intracellular organelles, and protein aggregation are trapped in the autophagosome. It is a highly controlled process that is subsequently fused and degraded by lysosomes, and is known to enable cell survival, particularly under adverse conditions of various stresses such as oxidative stress. Due to this phenomenon, it is considered that cancer in which intracellular ROS produced by mitochondria damaged by L-asps is retained.

The active ingredient of the autophagy inhibitor is a component having a function of inhibiting this autophagy, and specifically includes chloroquines such as chloroquine (chloroquinoline) and hydroxychloroquine, 3-methyladenine, bafilomycin A However, it is not limited to these. These autophagy-inhibiting components can be produced by known methods, and commercially available products can be used.

The dose (active ingredient) of the autophagy inhibitor by the cancer therapeutic agent of the present invention varies depending on the type of the drug and the administration interval. For example, in the case of chloroquine, it is about 200 to 400 mg per adult per day. Yes, it can be increased or decreased depending on the effect. The dosing interval can be performed once a day and every other day or every few days.

(3) Effect of cancer therapeutic agent By administering the cancer therapeutic agent of the present invention, the target cancer cells are depleted of L-asparagine by the action of L-asps, and at the same time, emergency life support is achieved. Because of this, autophagy is induced. In particular, enhancement of asparagine synthase is observed in “cancer resistant to L-asps alone administration” which is a target cancer cell of the cancer therapeutic agent of the present invention. However, the autophagy inhibitor in the cancer therapeutic agent of the present invention inhibits the above autophagy, and at that time, a large amount of active oxygen accumulates in the cancer cell, and then the cancer is caused by the action of the p53 protein. Cells are induced to apoptosis. By repeating the accumulation of active oxygen in the cancer cells and the cycle of apoptosis, the cancer therapeutic agent of the present invention exhibits a very excellent cancer therapeutic effect. This is an effectiveness irrespective of the presence or absence of the above-mentioned “resistance to L-asps alone administration”. However, for cancer cells in which a mutation resulting in loss of function of the p53 gene is observed, the above-described process leading to apoptosis is impaired, and even if the cancer therapeutic agent of the present invention is administered as it is, cancer treatment is performed. The effect cannot be expected.

(4) Aspect of Cancer Treatment Agent The cancer treatment agent of the present invention comprises (a) a form in which L-asps and an autophagy inhibitor are contained in a single pharmaceutical composition, (b) L- The asps and the autophagy inhibitor are roughly classified into two forms: a set of forms combined as separate pharmaceutical compositions.

The single form (a) has an advantage that only one agent is required for administration, but on the other hand, it has a drawback that it is difficult to finely adjust the drug amount and the administration interval in order to combine drugs having different properties.

Although the set form (b) is a two-drug administration, fine adjustment of the drug amount and the administration interval is easy.

As described above, the cancer therapeutic agent of the present invention can further contain an L-asparagine synthase inhibitor in addition to L-asps and an autophagy inhibitor. This is by inhibiting the depletion of L-asparagine in the cancer cells by inhibiting L-asparagine synthase enhanced in “cancer resistant to L-asps alone administration”. Based on being able to improve the cancer treatment effect.

The L-asparagine synthase inhibitor can be contained in the cancer therapeutic agent of the present invention in any of the above-mentioned (a) single agent type and (b) set type. In the case of the set type, in addition to the case where the three agents are different compositions, there may be a case where the pharmaceutical composition is a combination of any two agents. In particular, a combination composition of L-asps and an L-asparagine synthase inhibitor is a preferred combination because of its mechanism of action.

Both of the cancer therapeutic agents of the present invention are administered to the human body as a “pharmaceutical composition”. Also in the case of direct administration of the active ingredient of the cancer therapeutic agent, for example, an injection or the like is mixed at the time of use, and this is also included in the pharmaceutical composition.

The pharmaceutical composition used as the cancer therapeutic agent of the present invention is prepared in the form of a pharmaceutical composition by blending an appropriate pharmaceutical carrier together with the above active ingredients. As the preparation carrier, it is possible to select a carrier according to the use form, and excipients or diluents such as a filler, a bulking agent, a binder, a moistening agent, a disintegrant, and a surfactant are used. can do. The form of the composition is not particularly limited as long as it can effectively contain the cancer therapeutic agent of the present invention, and it may be a solid agent such as a tablet, powder, granule, or pill. Usually, it is preferably in the form of an injection such as a solution, suspension or emulsion. In addition, the cancer therapeutic agent of the present invention can be made into a dry product that can be made liquid at the time of use by adding an appropriate carrier. Furthermore, in the pharmaceutical composition of the present invention, drug delivery systems such as nanoparticles, polymer micelles, erythrocytes, stable nucleic acid lipid particles (SNALP), multifunctional envelope nanostructures (MEND) composed of cyclodextrin-containing polymers are provided. By utilizing this, it is possible to further improve the effect of the cancer therapeutic agent of the present invention.

The obtained pharmaceutical composition is administered in an appropriate administration route according to its form, for example, an injectable pharmaceutical composition is administered in a solid form by intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal administration, etc. The pharmaceutical composition is administered orally or enterally. The amount of the therapeutic agent for cancer of the present invention in the pharmaceutical composition is appropriately selected according to the administration method, administration mode, purpose of use, patient symptom, etc. of the composition, but is usually constant according to the present invention. It is preferable that the therapeutic agent is prepared in a composition form containing about 0.1 to 95% by mass and administered in the above-mentioned dosage form.

Hereinafter, embodiments of the present invention will be disclosed.

1. Examination of L-asp-induced autophagy Three types of ALL cell lines with different genetic backgrounds, (a) REH (ETV6-RUNX1 translocation positive) (Venuat, AM, Testu, MJ, and Rosenfeld, C. (1981). Cytogenetic abnormalities in a human null cell leukemia line (REH). Cancer Genet Cytogenet 3, 327-334.), (B) 697 (TCF3-PBX1 translocation positive) (Findley, HW, Jr., Cooper, MD, Kim, TH, Alvarado, C., and Ragab, AH (1982). Two new acute lymphoblastic leukemia cell lines with early B-cell phenotypes. Blood 60, 1305-1309.), (C) TS2 (MEF2D -DAZAP1 translocation positive) (Yoshinari, M., Imaizumi, M., Eguchi, M., Ogasawara, M., Saito, T., Suzuki, H., Koizumi, Y., Cui, Y., Sato, A ., Saisho, T., et al. (1998). Establishment of a novel cell line (TS-2) of pre-B acute lymphoblastic leukemia with at (1; 19) not involving the E2A gene. Cancer Genet Cytogenet 101, 95-102 ), The autophagy activity of L-asp (L-asp leinase (registered trademark) derived from E. coli, Kyowa Hakko Kirin Co., Ltd.) was examined. Chloroquinone (Chloroquine (CQ): Sigma-Aldrich Co., Catalog No. C6628) and bafilomycin (Bafilomycin A (Baf): Sigma-Aldrich Co., Catalog No. B1793 for the assessment of autophagy ) Was used. The medium is 500 ml of RPMI 1640 (Wako Pure Chemical Industries, Catalog No. 189-02025), fetal bovine serum (Wako Pure Chemical Industries, Catalog No. 35-010-CV, added concentration 10%), and the antibiotic Penicillin-Streptomycin (ThermoFisher Scientific). Inc., catalog number 15140-122, addition concentration 0.5%) was used.

One well of a 6-well plate was seeded with 1 × 10 ⁶ cells / 2 ml of the above medium. L-asp 1 U / ml was added, and after 45 hours, CQ 10 μM to Bafilomycin A1 100 nM was further added. After 3 hours, the cells were collected, and then analyzed by Western blotting, fluorescent immunostaining, and electron microscopy.

In Western blotting, polyacrylamide gel electrophoresis was performed using a cell lysate and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking using skim milk, LC3B antibody and β-actin antibody (both Sigma-Aldrich) were reacted as the primary antibody, and then SuperSignal West Dura (ThermoFisher Scientific Inc.) was reacted as the secondary antibody. Thereafter, detection was performed with LAS-3000 (GE Healthcare).

In the analysis by fluorescent immunostaining, the cells were collected, adhered to a slide glass with CytoSpin3 (ThermoFisher Scientific Inc.), fixed with methanol, and then fixed with methanol. Fetal bovine serum 1% and Triton X-100 0.01% phosphate buffered physiological Blocking was performed with saline (PBS). Thereafter, an LC3B antibody (Sigma-Aldrich) and VECTASHIELD with DAPI (Vector Laboratories) were reacted and observed with a fluorescence microscope.

In electron microscope analysis, after fixing with paraformaldehyde, dehydration with ethanol and drying. After embedding in an epoxy resin, slicing with a microtome and pasting on a sample stage, electron staining was performed with uranium acetate and lead citrate, and observation was performed with a transmission electron microscope.

FIG. 2-A shows the autophagy induction of autophagosome when L-asp and an autophagy inhibitor are used alone or in combination with the above three types of ALL cell lines, or when both are not applied. The results of investigation by electrophoresis using LC3B-II indicating the presence as an index are shown. As a result, in all three strains, the expression of LC3B-II was increased when L-asp and CQ or Baf were administered in combination, compared to when CQ or Baf was administered alone. This indicates that L-asp induces autophagy compared to the steady state.

FIG. 2-B shows autophagy induction when L-asp and Chloroquine (CQ) are used alone or in combination on REH cells among the above three types, using LC3B-II as an index. It is a tissue immunostaining image examined under the same conditions as in 2-A. As a result, it was confirmed that the protein expression of LC3B was increased in the combined administration of L-asp and CQ as compared with the single administration of CQ and single administration of L-asp, and autophagy was induced by L-asp. It was shown that

FIG. 2-C is an electron microscopic image of autophagy induction in the system of FIG. 2-B, and FIG. 2-D shows the number and area of autophagy vesicles per cell in the electromicroscopic image. ing. When L-asp and CQ were used together, autophagosomes containing mitochondria were also observed (Fig. 2-C triangle) along with many autolysosomes (Fig. 2-C arrowheads). Significantly more were observed in both the number and area of autophagy vesicles per cell when L-asp and CQ were combined.

FIG. 2-E shows the case where L-asp and the autophagy inhibitor Chloroquine (CQ) are used alone or in combination with the above-described system shown in FIG. 2-B, that is, the ALLH cell line REH cells. 3 shows the results of counting the number of impaired mitochondria based on the color development of MitoTracker (registered trademark) Red, which is a staining fluorescent dye for mitochondria. As shown here, when L-asp and CQ were used in combination, the peak shifted to the right, and impaired mitochondria were remarkably observed.

FIG. 2-F shows the result of examining the membrane potential of intracellular mitochondria using TMRE (tetramethylrhodamine methyl ester) in the system of FIG. 2-E. As shown here, when L-asp and CQ were used in combination, the proportion of mitochondria with decreased membrane potential, that is, cells with impaired mitochondria, was significantly increased.

FIG. 2-G shows an evaluation of intracellular reactive oxygen species (ROS) using DCFDA (left figure) and mitochondrial ROS using MitoSox (registered trademark) -Red in the system shown in FIG. 2-E. Results are shown. As shown here, it was revealed that ROS accumulated significantly when L-asp was combined with CQ.

This FIG. 2A-G promotes the induction of autophagy by L-asp administration to the ALL cell line, and this autophagy is very important in the removal of impaired mitochondria and reactive oxygen species in cancer cells. It became clear that there was.

2. Examination of cell death induction by combined use of L-asp and autophagy inhibitor One well of a 6-well plate was seeded with 1 × 10 ⁶ REH cells / 2 ml of medium. At the same time, L-asp and CQ were added at the concentrations shown in the figure (L-asp 1 U / ml, CQ 10 μM if not indicated), and cells were collected 48 hours later.

Western blotting was carried out in the same manner as in FIG. 2, and the primary antibody was cPARP antibody, cCASP3 antibody, CASP3 antibody, CHOP antibody was cell Signaling, ASNS was Sigma-Aldrich, and ATF4 antibody was Santa Cruz Biotechnology.

The number of dead cells was analyzed using Annexin V staining with MEBCYTO-Apoptosis Kit (MBL) and using flow cytometer Accuri C6 (Becton, Dickinson and Company).

In the evaluation of the number of dead cells by the pan-apoptotic factor inhibitor zVAD-fmv (Peptide Co., Ltd.), L-asp and CQ were added to the medium 24 hours later, and the number of dead cells was evaluated 24 hours later. As a control, dimethyl sulfoxide (DMSO) which is a solvent of zVAD-fmv was used.

Establishment of a strain resistant to L-asp (697-R) was performed as described previously (Hutson RG, et al. Am J Physiol 1997; 272: c1691-1699). That is, in 697 cells, L-asp was added to the medium and cultured, and the cells that proliferated after acquiring resistance to L-asp were collected. The L-asp to be added started from 0.1 U / ml and gradually increased to 1 U / ml while gradually increasing over 6 months.

Anti-ROS drug NAC (Sigma-Aldrich) was administered 2 mM simultaneously with L-asp and CQ, and the number of dead cells using Annexin V staining was evaluated 48 hours later using a flow cytometer.

For cell cycle analysis, L-asp and CQ were added, and the cells were collected 48 hours later, washed with PBS and fixed at 70% ethanol at -20 ° C. Eight hours later, the plate was washed with PBS, RNase was added, left to stand at 38 ° C. for 30 minutes, stained with productiodide (Thermo Fisher Scientific), and then measured with an Accu C6 flow cytometer.

Expressing sh (short hairpin) -ASNS1 (target sequence: 5'-GCTGTATGTTCAGAAGCTAAA-3 '(sequence number 1)) or sh-ASNS2 (target sequence: 5'-CGTCAAGTCTTTGAACGCCAT-3' (sequence number 2)) for the following ASNS Created a lentivirus. First, the Inverse PCR method was performed according to the protocol using KOD-Plus-Mutageness Kit (TOYOBO CO., LTD.). That is, PCR was performed using circular DNA (plasmid) as a template and the following primers, and the entire plasmid was amplified. Next, the PCR product was cloned into the BamHI-EcoRI region of the lentiviral vector pGreenPuro shRNA Cloning and Expression Lentivector (System Biosciences) to prepare a recombinant vector. Thereafter, the PCR product, which is a linear plasmid, was ligated to cyclize. Next, E. coli was transformed, and the plasmid was extracted and then introduced into HEK293TN cells to prepare sh-ASNS1-expressing lentivirus and sh-ASNS2-expressing lentivirus. About each recombinant lentivirus obtained in this way, the titer was measured using Global UltraRapid Lenticular Titer Kit (System Biosciences). MOI (multiplicity of infection) was determined according to the attached manual, the viral load was adjusted to 5 MOI, and REH cells were transfected.

sh-ASNS1-sense:
5'-GATCCGCTGTATGTTCAGAAGCTAAACTTCCTGTCAGATTTAGCTTCTGAACATACAGCTTTTTG-3 '(SEQ ID NO: 3)
sh-ASNS1-antisense:
5'-AATTCAAAAAGCTGTATGTTCAGAAGCTAAATCTGACAGGAAGTTTAGCTTCTGAACATACAGCG-3 '(SEQ ID NO: 4)
sh-ASNS2-sense:
5'-GATCCCGTCAAGTCTTTGAACGCCATCTTCCTGTCAGAATGGCGTTCAAAGACTTGACGTTTTTG-3 '(SEQ ID NO: 5)
sh-ASNS2-antisense:
5'-AATTCAAAAACGTCAAGTCTTTGAACGCCATTCTGACAGGAAGATGGCGTTCAAAGACTTGACGG-3 '(SEQ ID NO: 6)

In REH / Luc2 cells, E. coli was transformed with a lentiviral vector in which the luciferase gene Luc2 was incorporated, and the recombinant E. coli was amplified to produce a plasmid. This plasmid was introduced into HEK293TN cells to produce Luc2-expressing lentivirus. About each recombinant lentivirus obtained in this way, the titer was measured using Global Ultra Rapid Lenticular Titer Kit (System Biosciences). MOI was determined according to the attached manual, the viral load was adjusted to 5 MOI, and REH cells were transfected to establish REH / Luc2 cells. It was injected from the tail vein of immunocompromised mice (NOD / SCID mice) at a concentration and amount of the REH / Luc2 cells 5 × ¹⁰ 6 / PBS100μl. From 7 days after the injection, L-asp 6000 U / kg mouse body weight and CQ 50 mg / kg mouse body weight were intraperitoneally administered once a day, and the in vivo leukemia growth amount and survival period were analyzed. For measurement of tumor growth in vivo in mice, D-luciferin (Synchem) 150 mg / kg body weight of mouse was intraperitoneally administered, and bioluminescence imaging analysis was performed using Photon Imager (Biospace Lab). Fig. 3-A shows the degree of cell death when L-asp and an autophagy inhibitor are used alone or in combination with the above three ALL cell lines using flow cytometry and Western blotting analysis. Shows the results. The combination group of L-asp and autophagy inhibitor CQ (L-asp + CQ group, both administered for 48 hours) is divided into CQ single agent administration group (CQ group) and L-asp single agent administration group (L-asp group). Compared with that, cell death was significantly induced.

The evaluation of dead cells by flow cytometry was made clear by the ratio of Annexin V staining positive cells. Western blotting analysis also showed increased expression of cCASP3 and cPARP, which are apoptosis-related proteins, in the L-asp + CQ group compared to the L-asp group.

FIG. 3-B is a pan-apoptosis-related molecular inhibitor against cell death caused by the action of L-asp and the autophagy inhibitor Chloroquine (CQ) on REH cells among the above three types. The result of having investigated the inhibitory effect by a certain z-VAD is shown. As shown here, z-VAD inhibited the cell death.

FIG. 3-A and FIG. 3-B show that cell death induced by the combined use of L-asp and an autophagy inhibitor is mediated by apoptosis.

FIG. 3-C shows the results of examining the viable cell rate of the established L-asp resistant strain 697-R with respect to the increase in the amount of L-asp added. It can be confirmed that the resistant strain has a certain resistance to L-asp.

FIG. 3-D shows the results of examining the effect of L-asp and autophagy inhibitor (CQ) in combination on the viable cell rate in the L-asp resistant strain 697-R. As shown here, even in the L-asp resistant strain, the death-cell rate increased in a dose-dependent manner by the combined addition of L-asp and an autophagy inhibitor.

FIG. 3E shows the expression level of asparagine synthase (ASNS) in L-asp resistant strain 697-R when L-asp and an autophagy inhibitor are used alone or in combination, or both. The result of performing Western blotting analysis is shown for the case where it is not used. As shown here, in the resistant strain, the ASNS expression level was increased compared to the 697 strain in any drug administration environment. The results shown in FIG. 3-C show that, despite the increase in the ASNS level, the dead cell rate of the L-asp resistant strain was increased in a dose-dependent manner by the combined addition of L-asp and an autophagy inhibitor. Will rise.

FIG. 3F shows L-asp and Chloroquine (autophagy inhibitor) in “sh-ASNS1” and “sh-ASNS2” in which the ASNS gene of REH cell, which is an ALL cell line, was knocked down at different sites. The result of examining the case where CQ) is allowed to act alone or in combination, or the case where both are not acted on is shown. As shown here, it was found that knocking down the ASNS gene enhances the cell killing effect by the combined addition of L-asp and an autophagy inhibitor.

From the results shown in FIGS. 3-A to 3-F above, it was shown that the combined therapy of L-asp and an autophagy inhibitor is also effective in L-asp resistant ALL cells. Moreover, it was shown that the triple combination of ASNS inhibitor, L-asp and autophagy inhibitor is very useful in ALL treatment.

The results of Western blotting analysis in FIG. 3-G show the results of examining the mechanism responsible for apoptotic cell death induced by the combined use of L-asp and an autophagy inhibitor for the REH cell line. In L-asp, it has been reported that apoptotic cell death is induced by ATF4-CHOP pathway (Ye, J. et al., The EMBO journal 29, 2082-2096, doi: 10.1038 / emboj.2010.81 ( 2010)), there was no clear difference in the expression of ATF4 and CHOP between the L-asp group and the L-asp + CQ group, suggesting that apoptotic cell death due to other causes was induced in the L-asp + CQ group. In addition, the expression of ASNS downstream of ATF4 was not clearly different between the L-asp group and the L-asp + CQ group.

FIG. 3-H shows the results of examining the effect on cell killing effect when acetylcysteine (NAC), an ROS inhibitor, is added to the cell culture system of FIG. 3-G. As shown here, the addition of NAC attenuated the cell killing effect in the L-asp + CQ group. From this, it became clear that accumulation of ROS in the target cells is an important factor for exerting the combined effect of L-asp and an autophagy inhibitor.

Fig. 3-I shows the evaluation of the cell cycle using flow cytometry with Propidium Iodide staining when L-asp and autophagy inhibitor were used alone or in combination, or when both were not allowed to act. Results are shown. As shown here, the G0 / G1 phase increased in the L-asp alone group, but the G0 / G1 phase decreased and the sub-G1 phase increased in the L-asp + CQ group. Therefore, when the L-asp single agent is added, the cell cycle is stopped at the G0 / G1 phase to escape cell death, but it is difficult to stop the cell cycle by using an anti-autophagy inhibitor in combination with this. It was shown that apoptosis was induced without escape from cell death.

Fig. 3-J shows the effect of continuous drug contact for 9 days by counting the number of living cells. This count was checked with a TC20 automatic cell counter using Trypan blue staining. Each drug was added when the cell culture medium was changed every 72 hours, and the number of viable cells was measured. As a result, a gradual cell increase was observed in the CQ group and the L-asp group, but in the L-asp + CQ group, the ratio of the number of viable cells decreased with time.

Fig. 3-K and Fig. 3-L show the establishment of REH / Luc2 cells introduced with a luciferase gene for evaluation of treatment in vivo, and immunodeficient mice (NOD / SCID mice (Charles River Laboratories Japan)). The result of transcranial vein transplantation was examined and the course was examined. FIG. 3-K shows the results of examining the degree of proliferation based on the fluorescence distribution 7, 16, and 22 days after transplantation of REH / LUC2 cells. FIG. 3-L shows the degree of proliferation of REH / Luc2 cells over time. The photon flux rate was investigated. Both figures show that the transplanted REH / Luc2 cells proliferated rapidly in the mice of the control group and the single administration group, but the proliferation was significantly suppressed in the L-asp + CQ group.

FIG. 3-M shows the results of an overall survival analysis in which the survival rate over time of immunodeficient mice transplanted with the above REH / Luc2 cells was examined on a daily basis. As shown here, a significant prolongation of the survival period was observed in the L-asp + CQ group compared to the control group, CQ group and L-asp group.

Thus, it was shown that the combined therapy of L-asp and anti-autophagy inhibitor in ALL is effective in vitro and in vivo.

3. Relationship between combined use of L-asp and autophagy inhibitor and reactive oxygen species 6 well plates were seeded with 1 × 10 ⁶ REH cells / well 2 ml of medium. L-asp 1 U / ml and CQ 10 μM were added and observed over time or after 48 hours.

Western blotting was performed in the same manner as in FIG. 2, and the primary antibody was purchased from Abcam for γH ₂ AX antibody, and from Santa Cruz Biotechnology for PUMA antibody.

The knock-down of p53 with sh (short hairpin) RNA was performed in the same manner as in FIG. 3 described above, and a lentivirus expressing sh-p53 (target sequence: 5′-GACTCCAGTGGTAATCTAC-3 ′ (SEQ ID NO: 7)) was prepared. A recombinant vector was prepared using the following primers, and Escherichia coli was transformed. After amplification of the recombinant E. coli, a plasmid was generated. This plasmid was introduced into HEK293TN cells to produce Luc2-expressing lentivirus. About each recombinant lentivirus obtained in this way, the titer was measured using Global UltraRapid Lenticular Titer Kit (System Biosciences). The MOI was determined according to the attached manual, and the viral load was adjusted to 5 MOI and transfected into REH cells.

sh-p53-sense:
5'-GATCCGACTCCAGTGGTAATCTACCTTCCTGTCAGAGTAGATTACCACTGGAGTCTTTTTG-3 '(SEQ ID NO: 8)
sh-p53-antisense:
5'-AATTCAAAAAGACTCCAGTGGTAATCTACTCTGACACAGGAAGGTAGATTACCACTGGAGTCG-3 '(SEQ ID NO: 9)

FIG. 4-A shows the results of examination of DNA damage over time by Western blotting analysis when L-asp and an autophagy inhibitor are used alone or in combination with the REH cell line which is an ALL cell line. Is shown. Accumulation of ROS is known to cause DNA damage (Kruiswijk, F., Labuschagne, C. F. & Vousden, Nature reviews. Molecular cell biology 16, 393-405, doi: 10.1038 / nrm4007 (2015) ). As shown here, in the REH cell line, in the L-asp + CQ group, the protein expression of γH2AX, which is an indicator of DNA damage, increased, and at the same time, P53 and PUMA (P53-upregulated modulator-ofapoptosis) also increased.

FIG. 4-B shows the increase / decrease in p53 protein as well as γH2AX, which is an indicator of DNA damage, when acetylcysteine (NAC), a ROS inhibitor, is further acted on in the test system of FIG. 4-A. The result examined by blotting analysis is shown. As shown here, the addition of ROS inhibitors suppressed DNA damage and P53 activity.

From FIG. 4-A and FIG. 4-B, P53-induced apoptosis is caused by DNA damage due to accumulation of excess ROS in the cells by the combined use of L-asp and an autophagy inhibitor in ALL cells. Rukoto has been shown.

FIG. 4-C shows DNA damage when LHasp and CQ are combined in an REH cell line (sh-p53), which is an ALL cell line in which the p53 gene is knocked down by sh (short hairpin) RNA. It is the result examined by Western blotting analysis. As shown here, in sh-p53, not only the PUMA downstream of the p53 gene but also DNA damage was not induced.

FIG. 4-D shows the result of examining intracellular ROC accumulation in sh-p53. As shown here, sh-p53 significantly suppressed the accumulation of intracellular ROS in the L-asp + CQ group.

FIG. 4-E shows the results of examination of inhibition of cell death in sh-p53 by Annexin V uptake (left graph) and Western blotting analysis (right electrophoretic diagram). As shown here, sh-p53 significantly suppressed cell death in the L-asp + CQ group, and p53 expression was also strongly suppressed.

Based on the results shown in FIG. 4-A to FIG. 4-E, the combination therapy of L-asp and anti-autophagy inhibitor showed that (a) ROS accumulation, (b) DNA damage, (c) p53 activity (D) additional ROS accumulation, forming a “ROS-DNA damage-p53 loop” and inducing cell death, and this loop is broken in the p53 knockdown strain, It was shown that cell death was suppressed.

4). Examination using clinical specimens and mouse organisms Clinical specimens used leukemia cells isolated from bone marrow specimens of patients with more than 90% occupied by ALL leukemia cells using density gradient centrifugation with Ficoll. The collection was approved by the parents in writing after approval by the Ethics Committee. For the analysis of the number of viable cells, L-asp 1 U / ml and CQ 10 μM were added, and after 48 hours, the cells were examined using an automatic cell counter TC20 (Bio-Rad) using trypan blue staining.

Western blotting was performed in the same manner as in FIG.

The detection of mutations that resulted in loss of function of p53 was carried out by direct sequence analysis by the Sanger method. After the addition of PUREGENE Cell Lysis Solution and Protein Precipitation Solution (both from QIAGEN) to the collected cells, DNA was precipitated with ethanol and dissolved with Tris-EDTA Buffer. The exon part (exon 2-11) was amplified by reverse transcription polymerase chain reaction using the following primers. The amplified product was subjected to direct sequencing (double-strand analysis) using Applied Biosystems 3130xl Genetic Analyzer (Thermo Fisher). Mutation analysis was performed from the obtained analysis waveform chart using software GENETYX (Genetics Co., Ltd.).

exon2-3 sense: 5'-tctcatgctggatccccact-3 '(SEQ ID NO: 10)
exon2-3 antisense: 5'-agtcagaggaccaggtcctc-3 '(SEQ ID NO: 11)
exon4 sense: 5'-tgaggacctggtcctctgac-3 '(SEQ ID NO: 12)
exon4 antisense: 5'-agaggaatcccaaagttcca-3 '(SEQ ID NO: 13)
exon5-6 sense: 5'-tgttcacttgtgccctgact-3 '(SEQ ID NO: 14)
exon5-6 antisense: 5'-ttaacccctcctcccagaga-3 '(SEQ ID NO: 15)
exon7 sense: 5'-cttgccacaggtctccccaa-3 '(SEQ ID NO: 16)
exon7 antisense: 5'-aggggtcagaggcaagcaga-3 '(SEQ ID NO: 17)
exon8-9 sense: 5'-ttgggagtagatggagcct-3 '(SEQ ID NO: 18)
exon8-9 antisense: 5'-agtgttagactggaaacttt-3 '(SEQ ID NO: 19)
exon10 sense: 5'-caattgtaacttgaaccatc-3 '(SEQ ID NO: 20)
exon10 antisense: 5'-ggatgagaatggaatcctat-3 '(SEQ ID NO: 21)
exon11 sense: 5'-agaccctctcactcatgtga-3 '(SEQ ID NO: 22)
exon11 antisense: 5'-tgacgcacacctattgcaag-3 '(SEQ ID NO: 23)

Recombinant adenovirus was used for gene transfer of p53. Recombinant adenovirus was prepared using Adenovirus Expression Vector Kit (Takara). Specifically, a cosmid vector (pAxCAwtit-p53) into which wild-type p53 was inserted was prepared, a recombinant adenovirus genome containing the p53 gene was excised and introduced into HEK293TN cells, and a p53 recombinant adenovirus was prepared. About each recombinant adenovirus obtained in this way, the titer was measured using Global UltraRapid Lenticular Titer Kit (System Biosciences). The MOI was determined according to the attached manual, the amount of virus was adjusted to 5 MOI, and the target cells were transfected.

Knockdown of p53 was performed on REH cells using shRNA (sh-p53) in the same manner as in FIG. The p53 knockdown REH cells were injected from the tail vein of immunodeficient mice (NOD / SCID mice) at a concentration and volume of 100 μl of 5 × 10 ⁶ / PBS. From 7 days after the injection, L-asp 6000 U / kg mouse body weight and CQ 50 mg / kg mouse body weight were intraperitoneally administered once a day, and the survival period was analyzed.

FIG. 5-A shows the effect of combined administration of L-asp and chloroquine (CQ), which is an autophagy inhibitor, on ALL clinical blood samples (total of 14 cases of initial cases and 2 cases of relapses). The results are shown together with the presence or absence of a mutation that causes a loss of function of the gene. Among these 14 specimens, 7 specimens with a sufficient amount of preserved specimens were subjected to p53 analysis by the Sanger method, and a homozygous mutation (R248Q) of the p53 gene was observed in 1 case (case 14). In comparison between the L-asp group and the L-asp + CQ group, the number of viable cells was significantly decreased in the L-asp + CQ group in 13 cases that did not have a mutation that resulted in loss of p53 function. In one case (case 14) having a loss mutation, the combined effect of L-asp and an autophagy inhibitor was not observed.

FIG. 5-B shows the results of autophagy activity evaluation in 3 clinical blood samples of ALL ( cases 4, 5, and 7) in which the effect of combined administration of L-asp and CQ was recognized among the 14 cases described above. Shown by Western blotting analysis. The numbers at the bottom of the figure indicate the relative ratio of LC3B-II / ACTB when the control is 1. In all cases, LC3B-II protein expression was increased when L-asp was combined with CQ. This indicates that autophagy activity is enhanced upon administration of L-asp, which is consistent with the in vitro findings described above.

In FIG. 5-C and FIG. 5-D, the clinical blood sample of ALL (case 14) in which the effect of the combined administration of L-asp and CQ was not observed among the 14 cases described above was normal due to adenovirus. The effect of introducing the p53 gene was shown by Western blotting analysis (FIG. 5-C) and viable cell count (FIG. 5-D). As shown above, when the normal p53 gene was introduced by adenovirus, p53 protein expression was increased in the L-asp + CQ group, and cell death was induced.

FIG. 5-E shows the results of examining the effects of medication when the above-mentioned REH cell line knocked down from the p53 gene (sh-p53) was transplanted into the immunodeficient mouse via the tail vein. Specifically, the luciferase gene was introduced into p53 knockdown REH cells by shRNA, and transvenous vein transplantation into NOD / SCID mice was performed for treatment experiments. As a result, as shown in this figure, in the p53 knockdown strain, sh-p53, the combined use of L-asp and CQ did not extend the survival time.

This example shows that the p53 gene plays an essential role in the combination therapy of L-asp and an anti-autophagy inhibitor and is extremely important in stratifying treatments such as ALL. It was. FIG. 5-F shows a chart showing the mechanism of apoptosis induction of ALL cells by the combined administration of L-asp and the autophagy inhibitor Chloroquine (CQ).

Claims (15)

1. When a mutation resulting in a loss of function of the p53 gene in a cancer sample is observed, negative data on the effect of a combination therapy of an L-asparaginase or a derivative thereof and an autophagy inhibitor in the cancer, A method for obtaining prediction data of drug effects.
2. Mutations resulting in loss of function of the p53 gene are:
   (1) Mutation in which one of the alleles has a loss-of-function mutation of the p53 gene, and the other allele has LOH (loss of heterozygosity) in the region of the short arm of chromosome 17 in which the p53 gene is present (2) P53 gene function-deficient homo mutation or compound hetero mutation in the allele, (3) deletion mutation of chromosome 17 short arm, or deletion mutation of chromosome 17, and (4) one allele Only p53 gene lacking function mutation is observed in p53 gene, and no mutation is observed in p53 gene of the other allele, but mutant p53 subunits are randomly combined as subunits of p53 molecule having a tetrameric structure. The method for obtaining predicted data according to claim 1, wherein the mutation is one or more mutations selected from mutations in which functional inhibition is observed.
3. Detection of LOH (loss of heterozygosity) in which one of the alleles has a loss-of-function mutation in the p53 gene and the other allele has a p53 gene existing region in the short arm of chromosome 17 is detected by detecting LOH. The prediction data acquisition method according to claim 2, wherein the prediction data is acquired.
4. The method for acquiring predicted data according to any one of claims 1 to 3, wherein the cancer type is a cancer in which the effectiveness of administration of an agent of L-asparaginase or a derivative thereof is basically recognized.
5. The method for obtaining prediction data according to any one of claims 1 to 4, wherein the type of cancer is leukemia, malignant lymphoma, or ovarian cancer.
6. A therapeutic agent for cancer having resistance to single administration of an agent of L-asparaginase or a derivative thereof, comprising an agent of L-asparaginase or a derivative thereof and an autophagy inhibitor.
7. Cancer resistance to single administration of L-asparaginase or a derivative thereof can be determined based on negative anti-cancer effect data against the cancer caused by prior administration of L-asparaginase or a derivative thereof in advance. The cancer treatment according to claim 6, characterized in that it is defined based on negative anticancer effect data by contact with an agent of L-asparaginase or a derivative thereof outside the body of the cancer cell. Agent.
8. The cancer therapeutic agent according to claim 6 or 7, wherein the autophagy inhibitor is chloroquine, 3-methyladenine, bafilomycin A, or wortmannin.
9. The cancer therapeutic agent according to claim 8, wherein the chloroquine is chloroquine (chloroquinoline) or hydroxychloroquine.
10. The cancer therapeutic agent according to any one of claims 6 to 9, which is a pharmaceutical composition containing an agent of L-asparaginase or a derivative thereof and an autophagy inhibitor.
11. 10. The therapeutic agent for cancer according to any one of claims 6 to 9, which is a set of drugs containing L-asparaginase or a derivative thereof and an autophagy inhibitor as separate constituent drugs.
12. The cancer therapeutic agent according to any one of claims 6 to 11, further comprising an L-asparagine synthase inhibitor.
13. Cancer types that are resistant to single administration of L-asparaginase or a derivative thereof are cancers that do not show mutations that result in loss of function of the p53 gene, or that subsequently lack of function of the p53 gene. The cancer therapeutic agent according to any one of claims 6 to 12, which is a cancer in which a mutation to be lost is repaired.
14. The type of cancer having resistance to single administration of an agent of L-asparaginase or a derivative thereof is basically a cancer in which the effectiveness of administration of an agent of L-asparaginase or a derivative thereof is recognized. The therapeutic agent for cancer according to 13.
15. 15. The cancer therapeutic agent according to claim 13 or 14, wherein the type of cancer resistant to single administration of L-asparaginase or a derivative thereof is leukemia, malignant lymphoma, or ovarian cancer.

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