PHGDH inhibitors and their use in treatment of disseminated tumor cells

Abstract

The invention belongs to the field of biological medicine, and relates to a PHGDH inhibitor and application thereof in treating disseminated tumor cells. The invention discloses application of a PHGDH inhibitor or pharmaceutically acceptable salt, ester, isomer, prodrug, polymorphic substance or solvate thereof in preparation of a medicine for treating an object with and / or suspected to have DTC. Specifically provided are the use of a PHGDH inhibitor in the preparation of a medicament for treating a subject with and / or suspected of having DTC, a product comprising the PHGDH inhibitor, and the use thereof in combination with other treatments. The present invention also relates to a method of treating a subject with and / or suspected of having disseminated tumor cells (DTC) using the PHGDH inhibitors and pharmaceutically acceptable salts, esters, isomers, prodrugs, polymorphs, or solvates thereof. In addition, the invention further discloses application of the PHGDH and the biological effect of the PHGDH as a treatment target of disseminated tumor cells (DTC) in drugs for inhibiting DTC activation and proliferation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and discloses the use of PHGDH and its biological effects as a therapeutic target for disseminated tumor cells (DTCs) in a drug that inhibits DTC activation and proliferation. The present invention also discloses the use of a PHGDH inhibitor or a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph, or solvate thereof in the preparation of a drug for treating a subject having or suspected of having DTC. Background Art

[0002] Most cancer-related deaths occur due to metastasis, the spread of cancer cells from the primary tumor site to distant organs. Metastasis is the cause of most cancer-related deaths and is the final and most lethal manifestation of cancer. The vast majority of cancer patients die from the consequences of their metastatic disease, not their primary tumor.

[0003] Take hepatocellular carcinoma (HCC) as an example. As a malignant tumor with the highest incidence and mortality rates in my country, most patients are diagnosed in the mid-to-late stage. Advanced patients often present with distant metastases, such as to the lungs, bones, or brain, making treatment challenging and the prognosis extremely poor. The metastatic process of HCC primarily involves dissemination, entry into the bloodstream, and distant colonization. First, some tumor cells from the primary tumor intravasate into proximal blood vessels or lymphatics. They then become circulating tumor cells (CTCs) and spread through the bloodstream or lymphatics. Physical, biochemical, and immune stressors significantly reduce the number of CTCs. Upon reaching distant organs, CTCs are retained in the capillary beds of these organs, ultimately extravasating as disseminated tumor cells (DTCs) and migrating to the organ parenchyma, completing tumor cell colonization and metastasis. Similarly, despite improvements in survival rates for early-stage breast cancer patients, distant recurrence remains a major cause of high mortality and is generally considered incurable. Studies have shown that up to 75% of breast cancer patients may harbor micrometastases at diagnosis, and metastatic recurrence often occurs years or even decades after treatment. Previous literature has reviewed the clinical and biological aspects of DTCs and dormancy in breast cancer, pointing out that during clinical latency, DTCs can enter a cell cycle arrest or dormancy state at distant sites and may be shielded from immune detection and treatment (see, for example, Ring A, Spataro M, Wicki A, Aceto N. Clinical and Biological Aspects of Disseminated Tumor Cells and Dormancy in Breast Cancer. Front Cell Dev Biol. 2022; 10: 929893).

[0004] Systemic treatments such as targeted therapy and immunotherapy have been the preferred methods for distant metastasis of advanced cancer (such as liver cancer) in recent years, but the treatment response rate is low, and most patients are insensitive to targeted and immunotherapy. When the primary cancer (such as liver cancer) is detected, DTC has generally colonized in distant organs, making treatment strategies to prevent the spread of tumor cells generally less effective. At present, there are no treatment options for targeted disseminated tumor cells (DTC) at home and abroad.

[0005] Therefore, the goal of the present invention is to provide a treatment regimen that targets disseminated tumor cells (DTCs), thereby improving the prognosis of cancer patients and benefiting more patients at high risk of metastasis by effectively inhibiting the activation and proliferation of DTCs. Summary of the Invention

[0006] One of the technical problems to be solved by the present invention is to provide a targeted treatment for disseminated tumor cells (DTCs) to overcome the shortcomings of existing drugs for the targeted treatment of DTCs. Through extensive experiments and research, the inventors of the present invention have demonstrated that phosphoglycerate dehydrogenase (PHGDH) and its biological effects can serve as therapeutic targets for disseminated tumor cells (DTCs). By inhibiting the expression or activity of PHGDH, the activation and proliferation of disseminated tumor cells (DTCs) can be effectively inhibited, thereby improving the prognosis of cancer patients.

[0007] In some aspects herein, PHGDH and its biological effects are provided as therapeutic targets for disseminated tumor cells (DTC) in drugs that inhibit the activation and proliferation of disseminated tumor cells (DTC). In some embodiments, the biological effects include regulation of serine metabolic pathways, regulation of H3K27me3 histone modification, and the like.

[0008] In some aspects herein, there is provided the use of a PHGDH inhibitor or a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph or solvate thereof as an active substance in the preparation of a medicament for treating a subject having and / or suspected of having immune-escape DTC.

[0009] In some aspects herein, provided is the use of a PHGDH inhibitor or a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph or solvate thereof as an active substance in the preparation of a medicament for inhibiting the activation and proliferation of disseminated tumor cells (DTC).

[0010] In some aspects herein, there is provided a use of a PHGDH inhibitor or a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph or solvate thereof as an active substance in the preparation of a medicament for inhibiting the formation of primary tumor metastases.

[0011] In some embodiments, the PHGDH inhibitor herein inhibits the expression of PHGDH or inhibits the activity of PHGDH. In some embodiments, the inhibition of PHGDH expression herein is to inhibit the transcription or translation of the PHGDH gene, including but not limited to causing the PHGDH gene to not be transcribed, or reducing the transcriptional activity of the PHGDH gene, or causing the PHGDH gene to not be translated, or reducing the translation level of the PHGDH gene. In some embodiments, the inhibition of PHGDH activity herein is to weaken the biological effects of PHGDH, including but not limited to inhibiting the synthesis of the serine synthesis pathway and its downstream products, inhibiting the upregulation of H3K27me3 caused by the PHGDH downstream pathway, etc.

[0012] In some embodiments, the PHGDH inhibitor herein is a peptide, a nucleic acid molecule, an aptamer, a small molecule compound or a polysaccharide. In some embodiments, the PHGDH inhibitor herein is a nucleic acid molecule, including but not limited to antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA (siRNA), short hairpin RNA (shRNA), single guide RNA (sgRNA) or microRNA. In some other embodiments, the PHGDH inhibitor herein is a small molecule compound, including but not limited to NCT-503, CBR-5884, disulfiram, galactone A, Zeste enhancer homolog 2 (EZH2) inhibitor or a compound with the molecular formula C20H23F3N4S (CAS No. 1916571-90-8).

[0013] In some preferred embodiments, the PHGDH inhibitor herein is an enhancer of Zeste homolog 2 (EZH2) inhibitor, including but not limited to BIX-01294, chaetocin, CPI-169, CPI-905, CPI-360, CPI-209, CPI-1205, tazestat, EPZ005687, EPZ011989, 3-deazenplanocin A (3-deazenplanocin A), A, DZNep), EI1, GSK503, GSK126, GSK926, GSK343, JQEZ5, MC3629, OR-S0, OR-S1, PF-06821497, PF-06726304 acetate, SAH-EZH2, SHR2554, sinefungin, UNC1999, UNC2399, ZLD1039, EPZ-6438, CPI-169, CPI-1205, GSK-126, GSK343 ​​and GSK503. In a preferred embodiment, the PHGDH inhibitor herein is 3-deazenplanocin A (3-deazenplanocin A, DZNep).

[0014] In some embodiments, the immune escape DTC herein is a disseminated tumor cell (DTC) having a PHGDH level in a sample obtained from a subject that is higher than a reference level. In some embodiments, the PHGDH level herein is a gene expression level of PHGDH (e.g., a nucleic acid expression level or mRNA expression level of PHGDH), a protein expression level, and / or a protein activity level. In some embodiments, the PHGDH level in a sample obtained from a subject herein is a median, mean, or statistically determined standard value of a gene expression level, protein expression level, and / or protein activity level measured in a sample obtained from a subject; and / or the reference level herein is (1) a median, mean, or statistically determined standard value of a gene expression level, protein expression level, and / or protein activity level of PHGDH in a sample obtained from the subject at a prior time point; (2) a median, mean, or statistically determined standard value of a gene expression level, protein expression level, and / or protein activity level of PHGDH in a reference population; or (3) a pre-specified PHGDH level, e.g., a pre-specified immunohistochemistry (IHC) score value.

[0015] In some embodiments, the gene expression level, polypeptide / protein expression level and / or protein activity level of PHGDH is determined by methods known in the art and understood by the skilled person, including but not limited to immunohistochemistry staining ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, flow cytometry, fluorescence activated cell sorting ("FACS"), spatial transcriptomics, spatial proteomics, MassARRAY, proteomics, blood-based quantitative assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization (ISH), fluorescence in situ hybridization (FISH), Southern blot analysis, Northern blot analysis, whole genome sequencing, massively parallel DNA sequencing (e.g., next generation sequencing), polymerase chain reaction (PCR) (including quantitative real-time PCR (qRT-PCR) and other amplification type detection methods, such as branched DNA, SISBA, TMA, etc.), RNA-seq, microarray analysis, gene expression profiling and / or serial analysis of gene expression ("SAGE"), as well as any of a variety of assays that can be performed by protein, gene and / or tissue array analysis.

[0016] In some embodiments, the subject herein is a mammal, such as a human, non-human primate (e.g., gorilla, ape), rodent (e.g., rat, mouse, guinea pig), pet (e.g., cat, dog), livestock (e.g., horse, cow, sheep, pig, rabbit).

[0017] In some embodiments, the subject of the present invention can be selected from: a subject suspected of having primary cancer; a subject suffering from primary cancer but not yet receiving treatment for the primary cancer; a subject suffering from primary cancer and having received treatment for the primary cancer; a subject suffering from primary cancer and having received treatment for the primary cancer, but not yet receiving treatment for cancer metastasis; a subject suffering from primary cancer and having received treatment for the primary cancer and receiving preventive treatment for cancer metastasis; or a subject with one or more of the foregoing conditions; preferably a subject suspected of having metastatic colonization based on the subject's previous medical history and imaging examination results (e.g., CT, MRI, etc.).

[0018] In some embodiments, the sample herein can be a tissue obtained from the subject, such as a fresh sample, a frozen sample, a fixed sample (such as a formalin-fixed sample, a paraffin-embedded sample). In some embodiments, a biopsy tissue suspected of having metastatic colonization based on the subject's medical history and imaging examination results (e.g., CT, MRI, etc.) is preferred.

[0019] In some embodiments, the subject herein suffers from primary cancer.In some embodiments, the subject herein suffers from hepatocellular carcinoma, cholangiocarcinoma, prostate cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, gastric cancer, melanoma or cervical cancer;Preferably hepatocellular carcinoma, breast cancer, colorectal cancer, lung adenocarcinoma.In some embodiments, the subject as described herein suffers from primary cancer and suffers from suspected cancer metastasis caused by primary cancer and / or is in the risk of suspected cancer metastasis caused by primary cancer, for example, lung (such as liver cancer lung metastasis), bone, brain or lymph node (such as liver cancer lymph node metastasis) metastasis caused by primary cancer.

[0020] In some embodiments, the PHGDH inhibitor herein is contained in a pharmaceutical composition or kit, for example, a pharmaceutical composition or kit in a form suitable for administration by a method selected from the group consisting of oral administration, injection (e.g., direct naked DNA or protein injection, liposome-encapsulated DNA, RNA or protein injection), gold-coated gene gun bombardment, replication-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene-encoded protein, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, and transdermal administration. In some embodiments, the pharmaceutical composition or kit herein further comprises other anti-tumor active ingredients or is used in combination with other anti-tumor active ingredients, for example, the other anti-tumor active ingredients are selected from: tumor immunotherapy agents (e.g., PD-1 antibodies, PD-L1 antibodies, adoptive cell therapy agents, antigen-chimeric receptor T cell therapy agents), and chemotherapeutic drugs; for example, the other anti-tumor active ingredients are selected from: ipilimumab and nivolumab.

[0021] In some aspects herein, a product for treating a subject having and / or suspected of having disseminated tumor cells (DTCs) is provided. In some embodiments, the product comprises (A) a therapeutically effective amount of a PHGDH inhibitor; (B) a pharmaceutically or immunologically acceptable carrier or excipient; and (C) optionally, one or more other cancer treatments.

[0022] Those skilled in the art may arbitrarily combine the technical solutions and technical features described herein without departing from the inventive concept and protection scope of the present invention. Other aspects of the present invention will be apparent to those skilled in the art from the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be further described below with reference to the accompanying drawings, wherein these drawings are only for illustrating the embodiments of the present invention and are not intended to limit the scope of the present invention.

[0024] Figure 1 : Definition of tumor microenvironment (TME) units, where a 50 × 50 μm area surrounding tumor cells was defined based on spatial transcriptomic data. 2 The area is defined as a TME unit, and a TME unit is further divided into 25 10 × 10 μm 2 The area is defined as 10 × 10 μm in the center. 2 The region defines the location of the tumor.

[0025] Figure 2A : Pseudo-temporal analysis was performed on all tumor cells. The Monocle2 R package was used to determine the pseudo-temporal differentiation trajectories and transcriptome dynamics of all tumor cells collected at different sampling time points. Based on the pseudo-temporal analysis results automatically generated by the R package, the tumor cells were divided into 1 to 4 stages along the pseudo-temporal axis, namely Phase 1-4.

[0026] Figure 2B : For the unsupervised clustering of TME units of all samples, single-cell dimensionality reduction and clustering methods were used to divide TME units into 1 to 4 clusters along the pseudo-time axis, namely Cluster 1 (Cluster 1) to Cluster 4 (Cluster 4).

[0027] Figure 3 : The relationship between the changes in tumor cell gene expression along the pseudo-temporal axis and the cell populations in the TME unit on the timeline found that tumor cell staging and TME unit clustering were synchronized on the timeline, suggesting that tumor cells and the immune microenvironment co-evolve during the process of liver cancer metastasis and colonization.

[0028] Figure 4: The percentage of tumor cells and the number of stage 3 tumor cells at each time point (T1 (0.25 hours), T2 (1 hour), T3 (4 hours), T4 (12 hours), T5 (24 hours), T6 (2 days), T7 (8 days), T8 (21 days) and T9 (35 days)). Figure 4A The proportion of stage 3 tumor cells to all tumor cells and the proportion of all tumor cells to all cells at each time point are shown; the left Y-axis represents the proportion of tumor cells with stage 3 tumor cell characteristics detected in the mouse lung tissue obtained at each sampling time point to the total number of tumor cells at that sampling time point; the right Y-axis represents the proportion of all tumor cells in the mouse lung tissue obtained at each sampling time point to the total number of cells. Figure 4BThe number of stage 3 tumor cells at each time point is shown; wherein the Y-axis represents the absolute value of the number of tumor cells with stage 3 tumor cell characteristics detected in the mouse lung tissue obtained at each sampling time point.

[0029] Figure 5 : Differential expression gene analysis of stage 3 tumor cells and other tumor cells, wherein the x-axis is log2 (fold change) and the y-axis is -log10 (adjusted p-value). This differential expression gene analysis of many genes showed that PHGDH was most significantly upregulated in stage 3 tumor cells (t-test). PHGDH expression overlapped more with stage 3 tumor cells, so stage 3 tumor cells were defined as PHGDH+ tumor cells.

[0030] Figure 6 Pathway analysis of PHGDH+ tumor cells, with the x-axis representing -log10 (adjusted p-value) and the y-axis representing pathways related to cell proliferation and immune response. This pathway analysis revealed that PHGDH+ tumor cells were significantly downregulated in pathways related to cell proliferation and immune response (t-test; NS indicates no significant difference, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001).

[0031] Figure 7 : Immune cells in the tumor TME. The results showed that immune cell infiltration in the TME surrounding PHGDH+ tumor cells was significantly reduced (t-test).

[0032] Figure 8: Histochemical score (H score) of PHGDH and CD45+ immune cell infiltration in lymph node metastases of breast cancer (BRCA), colorectal cancer (CRC), lung adenocarcinoma (LUAD), and liver cancer (HCC); PHGDH+ tumor cells were significantly enriched in micrometastases of breast cancer (BRCA, n=3), colorectal cancer (CRC, n=4), lung adenocarcinoma (LUAD, n=4), and liver cancer (HCC, n=6) compared with macrometastases (t-test; NS indicates no significant difference, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001) ( Figure 8A ), and the infiltration of CD45+ immune cells was significantly reduced (t test; NS represents no significant difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001) ( Figure 8B ).

[0033] Figure 9: Multicolor immunofluorescence analysis of mouse lung tissue at T5 (sgPHGDH represents PHGDH knockout cells; sgCtrl represents control). The results show that PHGDH knockout tumor cells are more infiltrated by immune cells and are closer to the immune cells than the control group.

[0034] Figure 10 Figure : The burden of lung metastases (Hepa1-6 hepatoma cells, H22 hepatoma cells, MC38 colorectal cancer cells, and B16-F10 melanoma cells) in mice at the T8 time point (sgPHGDH represents PHGDH knockout cells; sgCtrl represents control; C57BL6 / J and BALB / C represent immunocompetent mice, and Nod / scid and BALB / C nude represent immunodeficient mice; t-test, NS represents no significant difference, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001). The results showed that in immunocompetent mice, PHGDH knockout tumor cells formed significantly fewer lung metastases than the control group, while in immunodeficient mice, there was no significant difference in the number of lung metastases between PHGDH knockout tumor cells and the control group.

[0035] Figure 11 : The ability of cell lines with knockdown or overexpression of PHGDH to recruit CD3+ T cells and NK cells isolated from tumor tissues in vitro (shPHGDH represents PHGDH knockdown cells; shCtrl represents control; PHGDH represents OE = represents PHGDH-overexpressing cells; t-test, NS represents no significant difference, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001). The results showed that PHGDH-knockdown tumor cells had a stronger ability to recruit T cells and NK cells than the control group, while PHGDH-overexpressing tumor cells had a weaker ability to recruit T cells and NK cells than the control group.

[0036] Figure 12 : The expression levels of chemokines along the pseudo-time axis, among which the chemokine expression of PHGDH+ tumor cells (stage 3 tumor cells) was significantly lower than that of tumor cells in other stages.

[0037] Figure 13 : The activity of related transcription factors in tumor cells, among which the activity of related transcription factors in PHGDH+ tumor cells (stage 3 tumor cells) was significantly lower than that in tumor cells in other stages.

[0038] Figure 14: PCR and ELISA test results of mouse hepatocellular carcinoma cells (Hepa1-6) and human hepatocellular carcinoma cells (Huh7). The results showed that tumor cells with PHGDH knockdown or knockout had significantly higher mRNA expression levels of Ccl20, Cxcl2, and Cxcl10 than the control group, and secreted significantly more CCL2, CXCL2, and CXCL10 than the control group; while tumor cells with PHGDH overexpression had significantly lower mRNA expression levels of Ccl20, Cxcl2, and Cxcl10 than the control group, and secreted significantly less CCL2, CXCL2, and CXCL10 than the control group, suggesting that PHGDH is a key factor in inhibiting the expression of chemokines in tumor cells.

[0039] Figure 15 :Mass spectrometry analysis of serine metabolites such as serine, methionine, and SAM in PHGDH knockout mouse cells or PHGDH knockdown human cells. The results showed that serine metabolites such as serine, methionine, and SAM were significantly decreased, while serine metabolites were significantly increased in cells overexpressing PHGDH (sgPHGDH represents PHGDH knockout cells; sgCtrl represents the control relative to PHGDH knockout cells; shPHGDH represents PHGDH knockdown cells; shCtrl represents the control relative to PHGDH knockdown cells; PHGDH OE represents PHGDH-overexpressing cells; t test, NS represents no significant difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001).

[0040] Figure 16 : Multicolor immunofluorescence and digital pathology analysis of lung tissues of C57BL6 / J mice and lymph node micrometastasis samples of HCC patients at T5 time point showed that PHGDH+ disseminated tumor cells (DTC) co-expressed H3K27me3 significantly more than PHGDH- disseminated tumor cells (DTC).

[0041] Figure 17 Single-cell epigenetic and chromatin accessibility analysis (scCPA-Tag) showed that H3K27me3 modification levels of chemokines and inflammatory transcription factors such as RELA, IRF1, NFKB2, and STAT1 were significantly lower in PHGDH-knockout tumor cells compared to the control group.

[0042] Figure 18: Under the action of EZH2 inhibitor, mouse liver cancer cells (Hepa1-6) ( Figure 18A ) and human hepatocellular carcinoma cells (Huh7) ( Figure 18B) PCR and ELISA test results. The results showed that after the EZH2 inhibitor DZNep was added to the control tumor cells, the mRNA expression levels of Ccl20, Cxcl2, and Cxcl10 were higher than those in the control group, and the secretion of CCL2, CXCL2, and CXCL10 was greater than that in the control group. However, after DZNep was added to the PHGDH-overexpressing tumor cells, the mRNA expression levels of Ccl20, Cxcl2, and Cxcl10 were higher than those in the PHGDH-overexpressing tumor cells, and the secretion of CCL2, CXCL2, and CXCL10 was greater than that in the PHGDH-overexpressing tumor cells.

[0043] Figure 19: In vivo experiments of EZH2 inhibitors in immunocompetent and immunodeficient mice (sgPHGDH represents PHGDH knockout cells; sgCtrl represents control; C57BL6 / J and BALB / C represent immunocompetent mice, and Nod / scid and BALB / C nude represent immunodeficient mice; t-test, NS represents no significant difference, * represents p<0.05, ** represents p<0.01, *** represents p<0.001). Figure 19A The results showed that after collecting mouse lung tissue at T5 time point and performing multicolor immunofluorescence, the number of immune cells around tumor cells in the EZH2 inhibitor DZNep treatment group was significantly higher than that in the control group. Figure 19B The results showed that after collecting mouse lung tissues at T9 time point and performing enzyme-labeled immunohistochemistry, the number of CD45+, CD3, CD8, CD11c, CD68 and NK1.1 positive cells infiltrating in the lung metastases in the EZH2 inhibitor DZNep treatment group was significantly higher than that in the control group.

[0044] Figure 20 Effects of a PHGDH inhibitor on lung metastatic burden (sgPHGDH represents PHGDH knockout cells; sgCtrl represents control; C57BL6 / J represents immunocompetent mice, and Nod / scid represents immunodeficient mice; t-test, NS represents no significant difference; * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001). In immunocompetent mice injected with control tumor cells, the number of lung metastases was significantly reduced in the EZH2 inhibitor DZNep group compared with the control group. However, in immunocompetent mice injected with PHGDH knockout tumor cells, no significant effect was observed in either group. In immunodeficient mice, no significant differences were observed, suggesting that the EZH2 inhibitor DZNep can suppress lung metastatic burden in immunocompetent mice. DETAILED DESCRIPTION

[0045] Through extensive experiments and research, the inventors discovered that phosphoglycerate dehydrogenase (PHGDH) and its biological effects can serve as therapeutic targets for disseminated tumor cells (DTCs). By inhibiting the expression or activity of PHGDH, the activation and proliferation of DTCs can be effectively inhibited, thereby improving the prognosis of cancer patients. Based on this, the inventors innovatively proposed using PHGDH inhibitors or pharmaceutically acceptable salts, esters, isomers, prodrugs, polymorphs, or solvates thereof as active substances to prepare drugs for treating subjects with and / or suspected of having immune-evading DTCs.

[0046] All numerical ranges provided herein are intended to expressly include all values ​​falling between the endpoints of the ranges and ranges therebetween. Features described herein or in the embodiments may be combined. All features disclosed herein may be used in any combination, and any feature disclosed herein may be replaced by any alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed are intended only to be general examples of equivalent or similar features.

[0047] As used herein, “containing,” “having,” or “including” encompasses “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of”; “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0048] The numerical ranges herein include their endpoints and each specific numerical point and sub-range within the numerical range. For example, 1 to 3 includes endpoints 1 and 3, the specific integer numerical point 2 therein and non-integer numerical points (for example, but not limited to: 1.2, 1.5, 1.8, 2.1, 2.3, 2.4, 2.8, etc.), and sub-ranges thereof (for example, but not limited to: 1 to 2, 2 to 3, 1 to 1.2, 1.5 to 1.8, etc.). Phosphoglycerate dehydrogenase (PHGDH)

[0049] Phosphoglycerate dehydrogenase (PHGDH), also known as phosphoglycerate dehydrogenase, is a key metabolic enzyme that plays a crucial role in the serine biosynthesis pathway as the first, rate-limiting enzyme in the synthesis of glucose-derived serine. PHGDH catalyzes the conversion of 3-phosphoglycerate (3-PG) to 3-phosphohydroxypyruvate (3-PHP), the first and rate-limiting step in serine biosynthesis and crucial for maintaining intracellular homeostasis. Serine, a non-essential amino acid, is crucial for cell proliferation and various biosynthetic processes. It participates in the one-carbon metabolic pathway, promoting the biosynthesis of nucleotides, S-adenosylmethionine (SAM), reduced nicotinamide adenine dinucleotide phosphate (NADPH), and glutathione (GSH) through the transfer of one-carbon units. The PHGDH-catalyzed conversion of 3-phosphoglycerate to 3-phosphohydroxypyruvate uses NAD+ / NADH as a cofactor. PHGDH is currently known to play a role in cancer progression and has been found to be amplified in a significant proportion of primary tumors, including 6% of breast cancers, 70% of estrogen receptor-negative breast tumors, and 40% of melanomas. PHGDH plays a key role in the metabolic reprogramming of cancer cells. Primary cancer cells with high PHGDH expression have increased serine biosynthesis flux, thereby supporting high cancer cell proliferation and tumor growth, where these cancer cells use PHGDH for serine synthesis. PHGDH activity is regulated by multiple signaling pathways, and changes in its expression and activity are closely associated with various biological processes, including tumor development, cell proliferation, and metabolic regulation. In addition, heterogeneous expression of PHGDH in tumor cells is associated with tumor invasiveness and metastatic propensity. Its activity drives cancer proliferation, while the loss of PHGDH-dependent sialic acid synthesis and integrin glycosylation increases metastatic spread. A previous paper on breast cancer reported that low expression of PHGDH in primary tumors often indicates susceptibility to distant metastasis of breast cancer (see Rossi, Matteo et al. "PHGDH heterogeneity potentiates cancer cell dissemination and metastasis." Nature vol. 605, 7911 (2022): 747-753).

[0050] Currently, there are no reports documenting the expression of PHGDH in disseminated tumor cells (DTCs) or their specific applications. However, through long-term and in-depth research, the inventors of this application have discovered that one of the biological effects of PHGDH expression is to regulate H3K27me3-mediated DTC transcriptional silencing through SAM, inhibiting chemokine expression, reducing immune cell recruitment, and thereby promoting the formation of a local immune desert microenvironment, which is beneficial for the metastasis and colonization of immune-evading DTCs.

[0051] As used herein, the terms "phosphoglycerate dehydrogenase," "phosphoglycerate dehydrogenase," "PHGDH," or "PHGDH molecule" are used interchangeably and have a broad meaning, including one or more detectable PHGDH molecules present in any type of normal and tumor / cancer subject, such as PHGDH gene, PHGDH mRNA, PHGDH cDNA, PHGDH precursor, PHGDH antisense transcript, PHGDH miRNA, PHGDH polypeptide, PHGDH protein, PHGDH protein fragment, or any other active fragment or marker fragment that indicates PHGDH gene expression level, polypeptide / protein expression level, and / or protein activity level. In addition, the term also includes molecules obtained by modifying natural PHGDH molecules for detection purposes, such as derivative molecules obtained by fragmentation, tagging, chemical modification, binding to specific molecules, etc.

[0052] An exemplary (Homo sapiens) genomic DNA sequence of PHGDH can be shown as NCBI NG 009188.1 (SEQ ID NO: 1), its polypeptide sequence can be shown as NP_006614.2 (SEQ ID NO: 3), and its mRNA sequence can be shown as NM_006623.3 (SEQ ID NO: 2). It should be understood that nucleotide substitutions in codons are acceptable when encoding the same amino acid. Similarly, it should be understood that nucleotide substitutions are also acceptable when conservative amino acid substitutions are generated by nucleotide substitutions. In addition, it should be understood that the above-mentioned specific sequences are exemplary sequences, and any molecules having a sequence homology, similarity, identity, etc. of more than 70% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or more, or any value or range of values ​​therebetween) and that can indicate the level of PHGDH in a sample fall within the scope of the terms "phosphoglycerate dehydrogenase", "phosphoglycerate dehydrogenase", "PHGDH" or "PHGDH molecule" described herein.

[0053] As used herein, the term "biological effect of PHGDH" refers to any function exhibited or performed by phosphoglycerate dehydrogenase (also known as phosphoglycerate dehydrogenase or PHGDH) and / or artificially modified phosphoglycerate dehydrogenase when detected or observed in vivo (i.e., the natural physiological environment of the protein / polypeptide) or in vitro (i.e., under laboratory conditions), including but not limited to regulating serine synthesis and / or metabolic pathways, regulating the trimethylation status of histone 3 lysine 27 (H3K27Me3), regulating H3K27me3 histone modification, etc. For example, "biological effect of PHGDH" can be regulating H3K27me3-mediated DTC transcriptional silencing through SAM, inhibiting chemokine expression, reducing immune cell recruitment, and thereby promoting the formation of a local immune desert microenvironment, which is conducive to the metastasis and colonization of immune-escape DTCs. PHGDH inhibitors

[0054] As used herein, the term "PHGDH inhibitor" means any substance that inhibits the expression of PHGDH or inhibits the activity of PHGDH. As used herein, "inhibiting the expression of PHGDH" means inhibiting the transcription or translation of the PHGDH gene, including but not limited to preventing the PHGDH gene from being transcribed, or reducing the transcriptional activity of the PHGDH gene, or preventing the PHGDH gene from being translated, or reducing the translation level of the PHGDH gene. As used herein, "inhibiting the activity of PHGDH" means weakening the biological effects of PHGDH, including but not limited to inhibiting the synthesis of the serine synthesis pathway and its downstream products, inhibiting the upregulation of H3K27me3 caused by the downstream pathway of PHGDH, reducing the level of H3K27me3 in the cell to restore the expression level of the suppressed chemokine, etc.

[0055] In some embodiments, the PHGDH inhibitor may include a peptide, a nucleic acid molecule, an aptamer, a small molecule or a polysaccharide. In some embodiments, the PHGDH inhibitor may be a nucleic acid molecule, for example, an antisense oligonucleotide, a double-stranded RNA (dsRNA), a ribozyme, a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a single guide RNA (sgRNA) or a microRNA. In some embodiments, the PHGDH inhibitor may be a small molecule compound, for example, NCT-503, CBR-5884, disulfiram, galactone A, an enhancer of zeste homolog 2 (EZH2) inhibitor or a C 20 H 23 The compound F3N4S (CAS No. 1916571-90-8) (its structural formula is shown below).

[0056] In some embodiments, the PHGDH inhibitor can be an enhancer of Zeste homolog 2 (EZH2) inhibitor. As used herein, the terms "enhancer of Zeste homolog 2 inhibitor," "EZH2 inhibitor," and "EZH2 methyltransferase inhibitor" are used interchangeably and refer to any EZH2 inhibitor currently known in the art and / or to be identified in the future, and include any chemical entity that, when administered to a subject, causes the subject's biological activity related to EZH2 activity (including any downstream biological effects derived from the binding of EZH2 to its natural ligand in other cases) to be inhibited. Such EZH2 inhibitors include any agent that can block EZH2 methyltransferase or any downstream biological effects of EZH2 methyltransferases associated with the treatment of cancer in a patient. Such inhibitors can act by directly binding to EZH2 and inhibiting its methyltransferase activity. Alternatively, such inhibitors can act by occupying the non-EZH2 domain of the polycomb repressive complex 2 (PRC2), thereby making EZH2 inaccessible to chromatin, and thus preventing or reducing its normal biological activity. Alternatively, such inhibitors may act by regulating the association of PRC2 proteins, or enhancing the ubiquitination and endocytic degradation of EZH2. EZH2 inhibitors include, but are not limited to, low molecular weight inhibitors, antibodies or antibody fragments, antisense constructs, small inhibitory RNAs (i.e., RNA interference via dsRNA; RNAi), and ribozymes.

[0057] In some embodiments, inhibitors of Enhancer of Zeste homolog 2 (EZH2) include but are not limited to BIX-01294, chaetocin, CPI-169, CPI-905, CPI-360, CPI-209, CPI-1205, tazemestat, EPZ005687, EPZ011989, 3-deazenplanocin A, A, DZNep), EI1, GSK503, GSK126, GSK926, GSK343, JQEZ5, MC3629, OR-S0, OR-S1, PF-06821497, PF-06726304 acetate, SAH-EZH2, SHR2554, sinefungin, UNC1999, UNC2399, ZLD1039, EPZ-6438, CPI-169, CPI-1205, GSK-126, GSK343 ​​and GSK503. In one embodiment, 3-deazenplanocin A (DZNep) is used as a PHGDH inhibitor and is used to treat subjects with and / or suspected of having immune-escape DTC. Cancer metastasis and dissemination of tumor cells (DTC)

[0058] As used herein, the terms "sample," "biological sample," or "test sample" are used interchangeably and refer to a sample obtained from a subject and used to detect PHGDH. The sample can be a tissue obtained from the subject, such as fresh tissue, a frozen sample, a fixed sample (e.g., a formalin-fixed sample, a paraffin-embedded sample), etc., preferably fresh tissue, formalin-fixed or paraffin-embedded tissue, and particularly preferably a biopsy tissue suspected of metastasis and colonization based on the subject's medical history and imaging examination results (e.g., CT, MRI, etc.).

[0059] It is known in the art that cancer metastasis involves a series of biological events, primarily dissemination, entry into the bloodstream, and distant colonization. Each of these events is driven by the ability of tumor cells to adopt distinct phenotypic cell states and co-opt surrounding immune and stromal cells within the tumor environment to support their growth and evade the immune system. Specifically, during dissemination, cancer cells from the primary tumor first acquire the ability to penetrate the basement membrane, thereby invading surrounding tissues. These cells then gain access to the bloodstream or lymphatic system through intravasation, i.e., entry into adjacent blood vessels or lymphatic vessels. Once in the bloodstream, cancer cells form circulating tumor cells (CTCs). Although CTCs are severely lost during this stage due to physical, biochemical, and immune factors, some CTCs can survive as single cells or cell microclusters and become encapsidated by platelets, neutrophils, or tumor-derived stromal cells, thereby assisting them in evading immune surveillance and increasing their metastatic potential. During the distant colonization phase, CTCs ultimately exit the blood vessels through extravasation and migrate to the parenchymal tissues of distant organs, forming disseminated tumor cells (DTCs). In distant organs, although DTCs may be cleared by local immune defenses, some cells can still survive and enter a dormant state. Although these surviving DTCs have not yet expanded to form clinically detectable metastases, they can adapt to and escape immune surveillance in the organ-specific microenvironment, ultimately leading to the formation of clinically detectable macroscopic metastases (see, for example, Gerstberger S, Jiang Q, Ganesh K. Metastasis. Cell. 2023; 186(8): 1564-1579).

[0060] As used herein, the term "disseminated tumor cells (DTCs)" refers to tumor cells that have shed from the primary tumor site of a primary cancer and circulated in blood or lymphatic vessels, then colonized and survived in distant target organs, for example, tumor cells that have not yet expanded to form clinically detectable metastases. Therefore, the term "disseminated tumor cells (DTCs)" is in contrast to the concept of "circulating tumor cells (CTCs)" that have spread from the primary cancer and are circulating in the blood.

[0061] The cancer that causes / generates disseminated tumor cells (DTCs) can be any cancer, such as any primary cancer or a metastatic cancer caused by a primary cancer. As used herein, the terms "tumor" and "cancer" are used interchangeably to refer to cells known in the art and to the presence in a subject of cells that have characteristics characteristic of cancer cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, reduced cell death / apoptosis, and certain unique morphological features. Cancer cells are typically in the form of solid tumors. However, cancer also includes non-solid tumors, such as blood tumors, such as leukemias, in which cancer cells originate from the bone marrow. The term "cancer" as used herein includes precancerous lesions as well as malignant cancers and tumors. Many types of cancer are known to metastasize and shed circulating tumor cells or have metastatic properties, such as secondary cancers caused by a primary cancer that has metastasized. Other cancers may include, but are not limited to, the following organs or systems: brain, heart, lung, gastrointestinal, genitourinary tract, liver, bone, nervous system, gynecology, hematology, skin, breast, and adrenal glands.Examples of cancer include, but are not limited to, acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia (monocytic, myeloblastic, adenocarcinoma, angiosarcoma, astrocytoma, myelomonocytic and promyelocytic), acute T-cell leukemia, basal cell carcinoma, bile duct cancer, bladder cancer, brain cancer, breast cancer, bronchial cancer, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myeloid (granulocytic) leukemia, chronic bone marrow cancer, Leukemia, colon cancer, colorectal cancer, craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, proliferative abnormalities, dysplasia and metaplasia, embryonal carcinoma, endometrial cancer, endothelial sarcoma, ependymoma, epithelial cancer, erythroleukemia, esophageal cancer, estrogen receptor-positive breast cancer, essential thrombocythemia, Ewing's sarcoma, fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glioma, heavy chain disease, hemangioblastoma, liver cancer, hepatocellular carcinoma, hormone-insensitive prostate cancer, Leiomyosarcoma, liposarcoma, lung cancer, lymphangioendothelioma, lymphangiosarcoma, lymphoblastic leukemia, lymphomas (Hodgkin and non-Hodgkin), malignancies and hyperproliferative disorders of the bladder, breast, colon, lung, ovary, pancreas, prostate, skin, and uterus, lymphoid malignancies of T-cell or B-cell origin, leukemia, lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, myeloid leukemia, myeloma, myxosarcoma, neuroblastoma tumors, non-small cell lung cancer, oligodendroglioma, oral cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous gland carcinoma, seminoma, skin cancer, small cell lung cancer, solid tumors (tumors and sarcomas), small cell lung cancer, gastric cancer, squamous cell carcinoma, synovioma, sweat adenoma, thyroid cancer, primary macroglobulinemia, testicular tumors, uterine cancer, and Wilms' tumor.Other cancers include primary cancer, metastatic cancer, oropharyngeal cancer, hypopharyngeal cancer, liver cancer, gallbladder cancer, bile duct cancer, small intestine cancer, urethral cancer, kidney cancer, urothelial cancer, female genital tract cancer, uterine cancer, gestational trophoblastic disease, male genital tract cancer, seminal vesicle cancer, testicular cancer, germ cell tumors, endocrine gland tumors, thyroid cancer, adrenal cancer, pituitary cancer, hemangioma, sarcomas arising from bone and soft tissue, Kaposi's sarcoma, neural cancer, eye cancer, meningeal cancer, glioblastoma, neuroma, neuroblastoma, schwannoma, solid tumors from hematological malignancies such as leukemia, metastatic melanoma, recurrent or persistent epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, gastrointestinal stromal tumors, colorectal cancer, gastric cancer, melanoma, glioblastoma multiforme, non-squamous non-small cell lung cancer, malignant glioma, stromal tumors, epithelial ovarian cancer, primary peritoneal serous carcinoma, metastatic liver cancer, neuroendocrine cancer, refractory malignancies, triple-negative breast cancer, HER2-amplified breast cancer, nasopharyngeal cancer, oral cancer, bile duct cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck (SCCHN), non-thyroid medullary cancer, recurrent glioblastoma multiforme, neurofibromatosis type 1, CNS cancer, liposarcoma, leiomyosarcoma, salivary gland cancer, mucosal melanoma, acral lentiginous melanoma, paraganglioma, pheochromocytoma, advanced metastatic cancer, solid tumors, triple-negative breast cancer, colorectal cancer, sarcoma, melanoma, renal cancer, endometrial cancer, thyroid cancer, rhabdomyosarcoma, multiple myeloma, ovarian cancer, glioblastoma, gastrointestinal stromal tumor, mantle cell lymphoma, and refractory malignancies.

[0062] In some embodiments, the subject is a mammal, such as a human, a non-human primate (e.g., a gorilla, ape), a rodent (e.g., a rat, a mouse, a guinea pig), a pet (e.g., a cat, a dog), or a livestock (e.g., a horse, a cow, a sheep, a pig, a rabbit). In some embodiments, the subject is selected from the group consisting of: ■Subjects suspected of having primary cancer; ■ Patients who have primary cancer but have not received treatment for the primary cancer; ■ Patients who have primary cancer and have received treatment for the primary cancer; ■ Patients who have a primary cancer and have received treatment for the primary cancer but have not yet received preventive treatment for cancer metastasis; ■ Patients with primary cancer who have received treatment for that primary cancer and are receiving preventive treatment for cancer metastasis; and / or ■Objects with one or more of the above conditions.

[0063] In a preferred embodiment, the subject is preferably a subject suspected of having metastatic colonization based on the subject's medical history and imaging examination results (eg, CT, MRI, etc.).

[0064] In some embodiments, the subject has a primary cancer, for example, hepatocellular carcinoma, cholangiocarcinoma, prostate cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, gastric cancer, melanoma or cervical cancer; preferably hepatocellular carcinoma, breast cancer, colorectal cancer, lung adenocarcinoma. In some embodiments, the subject has the above-mentioned primary cancer and has a suspected metastasis caused by the primary cancer and / or is at risk of a suspected metastasis caused by the primary cancer, for example, metastasis to the lung (e.g., liver cancer lung metastasis), bone, brain or lymph node (e.g., liver cancer lymph node metastasis) caused by the primary cancer.

[0065] As used herein, the term "immune escape disseminated tumor cells (immune escape DTC)" refers to disseminated tumor cells (DTC) with immune escape ability, which can escape the body's immune system recognition and attack / killing through various mechanisms, or resist the surveillance and attack / killing of the immune system, thereby surviving, activating and proliferating in vivo. For example, immune escape DTC can downregulate the enrichment of immune response-related pathways through various mechanisms, significantly reduce or inhibit immune cell infiltration (e.g., CD45+ immune cell infiltration), inhibit chemokine expression, reduce immune cell recruitment, new antigen presentation defects, escape immune response, be in a quiescent state, and / or promote metastasis and colonization by shaping an immune desert microenvironment. In some embodiments, the immune escape DTC herein is a disseminated tumor cell (DTC) in which the PHGDH level in a sample obtained from the subject is higher than the reference level. In some embodiments, the PHGDH level herein is the gene expression level of PHGDH (e.g., nucleic acid expression level or mRNA expression level of PHGDH), protein expression level and / or protein activity level. In some embodiments, the PHGDH level in the sample obtained from the subject herein is the median, mean or standard value determined by statistical methods of the gene expression level, protein expression level and / or protein activity level measured in the sample obtained from the subject; and / or the reference level herein is (1) the median, mean or standard value determined by statistical methods of the gene expression level, protein expression level and / or protein activity level of PHGDH in the sample obtained from the subject at a previous time point; (2) the median, mean or standard value determined by statistical methods of the gene expression level, protein expression level and / or protein activity level of PHGDH in a reference population; or (3) a pre-specified PHGDH level, for example, a pre-specified immunohistochemistry (IHC) score value. Drugs or pharmaceutical compositions and their therapeutic applications

[0066] The present invention also provides a medicine or pharmaceutical composition comprising an effective amount of the PHGDH inhibitor of the present invention and a pharmaceutically or immunologically acceptable carrier.

[0067] As used herein, the terms "active agent" or "active agent herein" are used interchangeably to refer to a PHGDH inhibitor.

[0068] As used herein, the term "pharmaceutically / immunologically acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance with a reasonable benefit / risk ratio. As used herein, the term "effective amount" refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0069] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents. The term refers to pharmaceutical carriers that are not themselves essential active ingredients and are not unduly toxic upon administration. Suitable carriers are well known to those of ordinary skill in the art. A comprehensive discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0070] The pharmaceutically acceptable carrier in the composition may contain a liquid such as water, saline, glycerol, and ethanol. In addition, these carriers may also contain auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffering substances, etc. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably, about 6-8.

[0071] The active substance in the composition herein accounts for 0.001-99.9 wt % of the total weight of the composition, preferably 1-95 wt %, more preferably 5-90 wt %, and even more preferably 10-80 wt %, with the remainder being pharmaceutically acceptable carriers and other additives.

[0072] As used herein, the term "unit dosage form" refers to a dosage form for single administration of the composition of the present invention for ease of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, and sustained-release dosage forms.

[0073] In some embodiments, the composition is a unit dosage form or a multi-dosage form. As used herein, the term "unit dosage form" refers to a dosage form in which the product of the present disclosure is prepared into a single administration dose for ease of administration, including but not limited to various solid doses (such as tablets), liquids, capsules, and sustained-release agents. In some embodiments, the composition herein is administered at an appropriate frequency, for example, daily, every other day, weekly, every other week or two weeks, monthly, every other month or two months.

[0074] It should be understood that the effective dose of the active substance used may vary with the severity of the subject to be administered or treated. The specific situation is determined according to the individual circumstances of the subject (e.g., subject weight, age, physical condition, desired effect), which is within the scope of the judgment of a skilled physician.

[0075] The composition of the present invention may be in solid form (such as granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets) or liquid form (such as oral solution) or other suitable forms.

[0076] In some embodiments, in some aspects, the method further comprises administering one or more additional cancer treatments prior to, concurrently with, or after administering the PHGDH inhibitor.

[0077] In some embodiments, the other cancer treatments may include one or more treatments selected from the following groups: surgical resection, chemotherapy, radiotherapy, immune checkpoint inhibition therapy (e.g., PD-1 inhibitor therapy, PD-L1 inhibitor therapy, CTLA-4 inhibitor therapy), cellular immunotherapy (e.g., adoptive cell therapy), antigen chimeric receptor (CAR) cell therapy (e.g., CAR-T therapy, CAR-NK therapy), T cell receptor (TCR) T cell therapy, bispecific antibody therapy, tumor vaccine therapy, oncolytic virus therapy, etc. Example

[0078] The present application will be further described below in conjunction with specific embodiments and accompanying drawings. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. Those skilled in the art may make appropriate modifications and variations to the present invention, and these modifications and variations are all within the scope of the present invention.

[0079] In the following examples, experimental methods where specific conditions are not specified were generally performed under conventional conditions such as those described in Molecular Cloning: A Laboratory Manual, 4th edition, by Michael R. Green et al., Cold Spring Harbor Laboratory Press, New York, 2017, or according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are calculated by weight.

[0080] The mice used in the examples were purchased from Shanghai Lingchang Biotechnology Co., Ltd.; the experimental ethics involving clinical patients in the examples were approved by the Ethics Committee of Zhongshan Hospital Affiliated to Fudan University, and written informed consent signed by all included patients or their families was obtained.

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to this application. The preferred embodiments and materials described herein are for illustrative purposes only. Example 1: Characterizing the changing characteristics of disseminated tumor cells (DTCs) during metastasis and colonization

[0082] In order to characterize the changing characteristics of disseminated tumor cells (DTC) during metastasis and colonization, C57BL6 / J immune-competent mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were injected with Hepa1-6 liver cancer cells (50 μL cell suspension, 6 × 10 cells injected) through the tail vein. 6 Cells / mouse) were used to establish a mouse model of liver cancer with lung metastasis. The success of the model was determined by the following criteria: CT scans of mice three weeks after cell injection indicated the presence of lung metastases; 5 weeks after cell injection, mice were sacrificed, and lung tissue was fixed, embedded, and stained with hematoxylin and eosin (HE) to confirm the presence of lung metastases.

[0083] A mouse model of liver cancer with lung metastasis (n = 27) was established. Lung tissue was collected at nine time points after cell injection: T1 (0.25 hours), T2 (1 hour), T3 (4 hours), T4 (12 hours), T5 (24 hours), T6 (2 days), T7 (8 days), T8 (21 days), and T9 (35 days). Fresh lung tissue was collected after mice were sacrificed. Spatial transcriptome sequencing and single-cell transcriptome sequencing were performed using the BGI Stereo-seq platform (T1: n = 1; T2: n = 1; T3: n = 2, T4: n = 2, T5: n = 2, T6: n = 3, T7: n = 4, T8: n = 2, T9: n = 1, total n = 18) and the 10x genomics platform (n = 1 at each time point).

[0084] Spatial transcriptome sequencing was performed using the BGI Stereo-seq platform using the following steps: Sample preparation: Tissue samples were frozen, embedded in OCT, sectioned, and then plated onto the Stereo-seq chip. ■ Tissue permeabilization and library construction: After fixation and permeabilization of tissue sections, probes are used to capture mRNA molecules in situ on the chip, followed by cDNA synthesis, amplification, and library construction; ■Sequencing: Sequencing was completed using BGI's DNBSEQ series of high-throughput sequencers.

[0085] Single-cell transcriptome sequencing was performed using the following steps using the conditions, methods, and / or parameters recommended by the 10x Genomics platform: ■ Single-cell suspension preparation: Prepare a single-cell suspension using a grinding and enzymatic dissociation method. Mix the trypan blue staining solution with the cell suspension at a ratio of 1:9. Observe the staining under a microscope and count the cell number and viability. Repeat two times and only proceed with subsequent experiments if the average value meets the quality control values ​​recommended by the 10x Genomics platform. ■Cell capture and cDNA amplification: Resuspend cells in PBS containing 0.1% BSA and mix with gel beads and oil droplets. Add cells (approximately 20,000 cells / sample) to the Chromium Chip channel, where a water-in-oil system is formed through the microfluidics system. The gel beads release primer sequences after cell lysis, and mRNA is reverse-transcribed to generate cDNA with a 10x barcode and a unique molecular identifier (UMI). After the water-in-oil system is disrupted, the cDNA is purified and amplified by PCR to obtain stable cDNA. ■ Library Construction and Sequencing: The amplified cDNA undergoes enzyme fragmentation, end repair, and ligation with sequencing primers to construct a 3' end expression library. Paired-end sequencing is performed on the Illumina Novaseq 6000 sequencing platform.

[0086] To investigate the dynamic changes of the immune microenvironment around disseminated tumor cells (DTCs) during metastasis and colonization, a 50 × 50 μm area around tumor cells was defined based on spatial transcriptome data. 2 The area is a tumor microenvironment (TME) unit, and a TME unit is further divided into 25 10×10μm 2 The area is defined as 10 × 10 μm in the center. 2 The region is defined as the tumor location point ( Figure 1 ).

[0087] Subsequently, the TME units of all samples were clustered unsupervisedly, and the single-cell dimensionality reduction and clustering method was used to divide the TME units into 1 to 4 clusters along the pseudo-time axis, namely Cluster 1-Cluster 4 ( Figure 2B The specific steps are as follows: ■Identify highly variable genes in the single-cell data expression matrix and perform PCA dimensionality reduction; ■Unsupervised clustering of cells based on gene expression profiles; ■ Visualize single-cell clusters using the Uniform Manifold Approximation and Projection (UMAP) algorithm; ■Based on the clustering results, the data are divided into Cluster 1 to Cluster 4.

[0088] At the same time, pseudo-time series analysis was performed on all tumor cells. The Monocle2 R package was used to determine the pseudo-time series differentiation trajectory and transcriptome dynamics characteristics of all tumor cells collected at different sampling time points. Based on the pseudo-time series analysis results automatically generated by the R package, the tumor cells were divided into 1 to 4 stages along the pseudo-time series axis, namely, Phase 1-4 (Phase 1-4) ( Figure 2A ).

[0089] Table 1: TME cell clusters and tumor cells at different time points

[0090] Table 2: Changes in tumor cell gene expression along the pseudo-temporal axis

[0091] Finally, the gene expression information from the same tumor cell is matched with the information of its surrounding TME units to construct the relationship between the changes in tumor cell gene expression along the pseudo-temporal axis and the cell population in the TME unit on the timeline ( Figure 3 ).

[0092] Analysis found that tumor cell staging and TME unit clustering were synchronized on the timeline, suggesting that tumor cells and the immune microenvironment co-evolve during liver cancer metastasis and colonization. Example 2: Quiescent PHGDH+ tumor cells that evade immune killing are transiently enriched during cancer metastasis and colonization set

[0093] In order to analyze the percentage of tumor cells and the number of Phase 3 tumor cells at time points T1 (0.25 hours), T2 (1 hour), T3 (4 hours), T4 (12 hours), T5 (24 hours), T6 (2 days), T7 (8 days), T8 (21 days) and T9 (35 days), the inventors calculated the ratio of Phase 3 tumor cells to all tumor cells and the ratio of all tumor cells to all cells at each time point ( Figure 4A ) and the number of stage 3 tumor cells at each time point ( Figure 4B ).

[0094] Figure 4AThe proportion of stage 3 tumor cells to all tumor cells and the proportion of all tumor cells to all cells at each time point are shown; the left Y-axis represents the proportion of tumor cells with stage 3 tumor cell characteristics detected in the mouse lung tissue obtained at each sampling time point to the total number of tumor cells at that sampling time point; the right Y-axis represents the proportion of all tumor cells in the mouse lung tissue obtained at each sampling time point to the total number of cells. Figure 4B The number of stage 3 tumor cells at each time point is shown; the Y axis represents the absolute value of the number of tumor cells with the characteristics of stage 3 tumor cells detected in the mouse lung tissue obtained at each sampling time point. Figure 4A and 4B The results in the middle section show that stage 3 tumor cells are primarily concentrated at the T5-T7 time points, accounting for approximately half of all tumor cells surviving immune pressure, while the number and proportion of cells at other time points are lower. This result suggests that stage 3 tumor cells are transiently enriched in disseminated tumor cells (DTCs) that escape immune stress.

[0095] The inventors further performed differentially expressed gene analysis on stage 3 tumor cells and other tumor cells, using the FindMarkers function in the R package Seurat to calculate the up-regulated or down-regulated genes of the target cell subpopulation compared with other cell subpopulations. The gene expression levels were compared, and p = 0.05 was selected as the "significance" threshold to obtain differentially expressed genes ( Figure 5 ).

[0096] Figure 5 The differentially expressed gene analysis between stage 3 tumor cells and other tumor cells is shown, with the x-axis representing log2 (fold change) and the y-axis representing -log10 (adjusted p-value). This differentially expressed gene analysis of multiple genes showed that PHGDH was most significantly upregulated in stage 3 tumor cells (t-test), and PHGDH expression was highly consistent with that in stage 3 tumor cells. Therefore, stage 3 tumor cells were defined as PHGDH+ tumor cells for subsequent studies.

[0097] The inventors then performed pathway analysis on PHGDH+ tumor cells, using the online tool Metascape to analyze differentially expressed genes for GO and KEGG enrichment analysis. The R package GSVA was used to analyze pathway activity in individual cells. The R package GSEABase was used to analyze gene sets from MSigDB, KEGG, and canonical pathways.

[0098] The above pathway analysis results showed that the enrichment of PHGDH+ tumor cells in cell proliferation and immune response-related pathways was significantly downregulated ( Figure 6 ), and immune cell infiltration in the TME surrounding PHGDH+ tumor cells was significantly reduced ( Figure 7), suggesting that PHGDH+ tumor cells have characteristics such as escaping immune response and being in a quiescent state.

[0099] The above results indicate that PHGDH+ disseminated tumor cells (DTC) may be the key tumor cell population that escapes immune surveillance and killing during liver cancer metastasis and promotes the proliferation and expansion of metastatic lesions. Example 3: PHGDH+ tumor cells are significantly enriched in micrometastases and CD45+ immune cell infiltration is significantly reduced

[0100] The inventors collected paraffin-embedded lymph node metastasis samples from pan-cancer patients containing both macrometastases and micrometastases from the clinic, and performed multi-color immunofluorescence staining and cell analysis based on a digital pathology platform.

[0101] Specifically, based on the patients' medical histories and pathologists' diagnostic information, the inventors retrospectively screened the pathology specimen library of Zhongshan Hospital Affiliated to Fudan University, ultimately obtaining paraffin-embedded tissue samples of lymph node metastases from six patients with hepatocellular carcinoma, four with colorectal cancer, three with breast cancer, and three with lung cancer. Pathologists confirmed that all of these samples had macrometastases (small metastatic nests with more than 100 tumor cells) and micrometastases (single tumor cell metastasis or small metastatic nests with fewer than 100 tumor cells).

[0102] Each paraffin tissue sample was subjected to multicolor immunofluorescence staining using anti-PanCK antibody (Abcam, Catalog No. ab7753), anti-PHGDH antibody (Abcam, Catalog No. ab240744), and anti-CD45 antibody (CST, Catalog No. 47937). Multicolor immunofluorescence staining methods are known in the art and include dewaxing, hydrating, antigen retrieval, blocking, primary and secondary antibody incubation, and fluorescent dye reaction and antibody elution after incubation. Nuclei were stained after multiple rounds of repeated staining. After sealing, the entire slide was scanned using a fully automated pathology scanning system, and digital analysis was performed using HALO software (v3.2, IndicaLabs).

[0103] As shown in the results in Figure 8, in breast cancer (BRCA), colorectal cancer (CRC), lung adenocarcinoma (LUAD) and HCC lymph node metastases, PHGDH+ tumor cells were significantly enriched in micrometastases compared with macrometastases, while CD45+ immune cell infiltration was significantly reduced.

[0104] The above results further indicate that PHGDH+ disseminated tumor cells (DTCs) are a key tumor cell population that evades immune surveillance and killing during cancer metastasis and promotes the proliferation and expansion of metastatic lesions. Example 4: PHGDH+ tumor cells shape the immune desert microenvironment and promote cancer metastasis and colonization

[0105] To investigate the effects of PHGDH+ tumor cells on shaping the immune desert microenvironment and promoting cancer metastasis and colonization, PHGDH knockout (sgPHGDH) and control (sgCtrl) mouse cell lines were further constructed, including Hepa1-6 liver cancer cells, H22 liver cancer cells, B16-F10 melanoma cells, and MC38 colorectal cancer cells, and immunocompetent mice and immunodeficient mice were constructed by tail vein injection.

[0106] The method for constructing the above-mentioned PHGDH knockout and control mouse cell lines is known in the art. For example, lentiCRISPRv2-mCherry is enzymatically digested with the BsmB1 enzyme, and then four sgRNAs targeting PHGDH are cloned into lentiCRISPRv2-mCherry using standard molecular cloning methods. Hepa1-6 tumor cells are pre-plated in a 6-well plate, the required virus volume is calculated according to the cell MOI value and virus titer, and the virus suspension and transfection reagent Polybrene are added to infect the Hepa1-6 tumor cells. After 48 hours of transfection, the cells are transferred to a 96-well plate and clones with successful PHGDH knockout are selected to achieve PHGDH knockout in tumor cells. In the construction of the control group Hepa1-6 cell line, the sgRNA used did not knock out any gene.

[0107] After constructing immunocompetent mice and immunodeficient mice by tail vein injection, multicolor immunofluorescence was performed on the lung tissues of mice at the T5 time point. As shown in Figure 9, the results showed that the immune cell infiltration in the PHGDH knockout cell group was significantly increased compared with the control group.

[0108] In addition, lung tissues were collected at T8 time point for comparison of lung metastasis burden. Figure 10 The load of lung metastasis (Hepa1-6 liver cancer cells, H22 liver cancer cells, MC38 colorectal cancer cells, and B16-F10 melanoma cells) in mice at the T8 time point is shown, where sgPHGDH represents PHGDH knockout cells; sgCtrl represents control; C57BL6 / J and BALB / C represent immune-competent mice, and Nod / scid and BALB / C nude represent immunodeficient mice; t-test, NS represents no significant difference, * represents p<0.05, ** represents p<0.01, *** represents p<0.001. Therefore, it was found that in immune-competent mice, the lung metastasis load of the PHGDH knockout cell group was significantly lower than that of the control group; while in immunodeficient mice, there was no significant difference between the two groups ( Figure 10 ).

[0109] The inventors also constructed knockdown (shPHGDH) or overexpression (PHGDH) of PHGDH based on the human hepatoma cell line Huh7 cells. OE) cell line was used to recruit CD3+T cells and NK cells isolated from tumor tissue in vitro. The experimental results showed that compared with the control group (shCtrl), the PHGDH knockdown group significantly increased the recruitment of CD3+T cells and NK cells, while overexpression of PHGDH significantly inhibited the migration of CD3+T cells and NK cells ( Figure 11 ).

[0110] The above results indicate that PHGDH+ tumor cells can promote metastasis and colonization by shaping the immune desert microenvironment. Example 5: PHGDH downregulates chemokine expression through H3K27me3-mediated epigenetic silencing, promoting immune Formation of the immune desert microenvironment and tumor cell colonization

[0111] To elucidate the molecular mechanism by which PHGDH shapes the immune desert microenvironment, the inventors compared the chemokine expression and related transcription factor activity of PHGDH+ tumor cells (stage 3 tumor cells) compared with other tumor cells.

[0112] like Figure 12 and Figure 13 As shown, chemokine expression of PHGDH+ tumor cells (stage 3 tumor cells) ( Figure 12 ) and related transcription factor activities ( Figure 13 ) was significantly lower than that of other tumor cells.

[0113] At the same time, the inventors used PCR and ELISA to verify in human and mouse cell lines. The experimental results of PCR and ELISA suggest that PHGDH is a key factor in inhibiting the expression of tumor cell chemokines ( Figure 14 ).

[0114] In addition, since PHGDH itself is the most critical rate-limiting enzyme in serine metabolism and provides adenosylmethionine (SAM) for histone methylation, the inventors also performed mass spectrometry analysis on serine metabolites such as serine, methionine and SAM in PHGDH knockout mouse cells or PHGDH knockdown human cells. Figure 15 As shown in the figure, mass spectrometry analysis found that in PHGDH knockout mouse cells or PHGDH knockdown human cells, serine metabolites such as serine, methionine and SAM were significantly reduced, while serine metabolites of PHGDH overexpression were significantly increased.

[0115] It is known that SAM can regulate H3K27me3 (a histone methylation modification, catalyzed by EZH2, and associated with gene expression silencing). Therefore, we performed multicolor immunofluorescence and digital pathology analysis on mouse lung tissue and HCC patient lymph node micrometastasis samples at the T5 time point. Based on the multicolor fluorescence results and analyzed using the digital pathology platform, the number of H3k27me3+PHGDH+ disseminated tumor cells (DTC) compared with H3k27me3+PHGDH-DTC in each field of view was obtained, and a t-test was performed, where p < 0.05. Figure 16 As shown in the results, PHGDH+ disseminated tumor cells (DTC) co-expressed H3K27me3 significantly more than PHGDH- disseminated tumor cells (DTC), suggesting that the presence of H3k27me3+PHGDH+ tumor cell infiltration in micrometastatic lesions is a potential intervention point for tumor metastasis. These results indicate that PHGDH mediates the upregulation of H3K27me3 in disseminated tumor cells (DTC) by regulating SAM.

[0116] In addition, based on single-cell epigenetic and chromatin accessibility maps (scCPA-Tag), we found that chemokine and related transcription factor genes (e.g., inflammatory transcription factors such as RELA, IRF1, NFKB2, and STAT1) showed significantly lower H3K27me3 modification levels in PHGDH knockout tumor cells compared with the control group ( Figure 17 ), indicating that H3K27me3 can inhibit chemokine transcriptional expression.

[0117] In summary, PHGDH expression regulates H3K27me3-mediated transcriptional silencing of disseminated tumor cells (DTC) through SAM, inhibits chemokine expression, reduces immune cell recruitment, and thereby promotes the formation of a local immune desert microenvironment, which is beneficial to the metastasis and colonization of PHGDH+ disseminated tumor cells (DTC). Example 6: PHGDH inhibitors reverse the suppression of immune infiltration by PHGDH+ tumor cells and reshape high immune infiltration microenvironment

[0118] In order to reverse the inhibition of immune infiltration by PHGDH+ tumor cells and reshape the high immune infiltration microenvironment, the inventors used PHGDH inhibitors to intervene in the expression level of PHGDH and / or inhibit the upregulation of H3K27me3 caused by the PHGDH downstream pathway.

[0119] Specifically, the inventors used an inhibitor of enhancer of zeste homolog 2 (EZH2) as a PHGDH inhibitor. EZH2 is known to be a histone lysine methyltransferase and a catalytic component of the polycomb repressive complex 2 (PRC2), which also includes embryonic ectoderm development protein (EED) and zeste inhibitor 12 (SUZ12). EZH2 catalyzes the methylation of histone 3 at lysine 27 (H3K27).

[0120] More specifically, the inventors used the EZH2 inhibitor 3-deazenplanocin A (DZNep) and studied its effect on H3K27me3 levels in murine and human hepatoma cells, and determined the expression levels of chemokines.

[0121] As shown in Figure 18, the PCR and ELISA test results of mouse hepatocellular carcinoma cells (Hepa1-6) and human hepatocellular carcinoma cells (Huh7) show that after the EZH2 inhibitor DZNep was added to the control tumor cells, the mRNA expression levels of Ccl20, Cxcl2, and Cxcl10 were higher than those of the control group, and the secretion of CCL2, CXCL2, and CXCL10 was greater than that of the control group; and after the addition of DZNep to the PHGDH-overexpressing tumor cells, the mRNA expression levels of Ccl20, Cxcl2, and Cxcl10 were higher than those of the PHGDH-overexpressing tumor cells, and the secretion of CCL2, CXCL2, and CXCL10 was greater than that of the PHGDH-overexpressing tumor cells. Therefore, the above results indicate that EZH2 inhibitors can restore the expression levels of suppressed chemokines by reducing the level of H3K27me3 in mouse and human hepatocellular carcinoma cells (Figure 18).

[0122] At the same time, in order to verify whether EZH2 inhibitors can reverse the inhibition of immune infiltration by PHGDH+ tumor cells and reshape the high immune infiltration microenvironment, the inventors also administered the EZH2 inhibitor DZNep in immunocompetent mice and immunodeficient mice.

[0123] Figure 19 provides the results of in vivo experiments of EZH2 inhibitors in immunocompetent and immunodeficient mice; wherein, Figure 19A The results shown in the figure show that after collecting mouse lung tissue at the T5 time point and performing multi-color immunofluorescence, the number of immune cells surrounding tumor cells in the EZH2 inhibitor DZNep-treated group was significantly higher than that in the control group; Figure 19BThe results shown in Figure 19 show that after collecting mouse lung tissue at the T9 time point and performing enzyme-labeled immunohistochemistry, the number of CD45+, CD3, CD8, CD11c, CD68, and NK1.1-positive cells infiltrating the lung metastases in the EZH2 inhibitor DZNep-treated group was significantly higher than that in the control group. Therefore, the above results indicate that the EZH2 inhibitor DZNep can reverse the suppression of immune infiltration by PHGDH+ tumor cells in immune-competent mice and reshape the high immune infiltration microenvironment (Figure 19).

[0124] In addition, to further verify the effect of PHGDH inhibitors on lung metastasis burden, PHGDH knockout tumor cells (sgPHGDH) and controls (sgCtrl) were injected into immunocompetent mice (C57BL6 / J) and immunodeficient mice (Nod / scid), respectively. Then, each experimental group was treated with the EZH2 inhibitor DZNep. Figure 20 As shown in the results, in the immune-competent mice injected with control tumor cells, the EZH2 inhibitor DZNep-treated group had a significantly reduced number of lung metastases compared to the control group, while in the immune-competent mice injected with PHGDH-knockout tumor cells, there was no significant effect on lung metastases in either group. In the immunodeficient mice, there was no significant difference, suggesting that the EZH2 inhibitor DZNep can inhibit the lung metastasis burden in immune-competent mice. Therefore, the above results show that EZH2 inhibitors can significantly reduce the lung metastasis burden in immune-competent mice ( Figure 20 ).

[0125] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application. Sequence information PHGDH genomic DNA sequence (NCBI reference sequence: NG 009188.1) (SEQ ID NO: 1) aatactatgcagccataaaaaatgatgagttcatgtcctttgtagggacatggatgaaattggaaatcatcattctcagtaaactatcgcaaggacaaaaaaa ccaaacaccgcatgttcccactcataggtgggaattgaacaatgagaacacatggacacaggaaggggaacatcgcacaccgggaccttttgtggggtgg ggggaggtggggggatagcattagggatatacctaatgttaaatgacgagttaatgggtgcagcaccagcatggcacatgtatacatatgtaaca aa cctg cacgttgtg ca catgta ccataaa a cttaaaatataata aa aa aataataaataaataaa gta ctgaag g atg ggg ctg ccttg ccctcccca g ca aaaaa aaaa aa aaaa aaa a aaaa gttaattcttccgtgatttttgtgccgtttatgtttg cttttacatttcacatgta aattatttgaaatttgtcttg gca ta a aattcag atttg a cttattttctag atg a ctgtccacttgttcaaacaatattattg aatgattctctatg ga gtgtatttatg gta ata g ccaaatgtaa gga attgtcg acg ggg ag a aaa gtgaa gttta atcaagattgtcagtg aa gatatg cttg g ctaag ccatttctttttgtcctctctccctccttctttctctg ctttc ttcccctctcattcttctgttctttgtttttctttttgtcgtattcatatgtaacttgattcattctctttg ggg ata g aaacattttta a atttcttacg aattaaaaa attctttaaatttgta aaaaaaaaaaa g ttccctctga gta ggga ctacatgtgacttgttca gcattgttattccagtg cctg gc acata g tca gtactatag a a atatttg gg cag gttctttg aatgtg ccacacttgttctgtttttccttctg cctcg aattctttttcttta attctttg catag ccagtttctttcttatttca gtctcaa ccca ag ctccaccttttcag gaa gg cctttcctcatcataaa gtctatgattcctctcccctaacctgtctttca catcatctggtttta cattatttattatg ccct gag attatcca gttcaatttttgtgttatttatttattctctctca aacgcaatgta a gctccatg ag ag cag aga cg gtcttaatcaaa g cttcatttccaa ccc cctcag cagtg cctg a cacattacagtttca ataa atatacactg attgagtg g atg aatg aag g aaagtcaag cactag ag atta cactca cttgtatg cttt g ccttatttaatcccta ca aaa gccttatg aagtaag cactatcata ctattcattccatattacatag gag aaaacta ag tcttag g gg gg ca atgta atttg cc caaaatcacattg ttcataacttgag aag ctg aaattcaa atca a gatatg cctgttttacg gatgaca gttcattttctgcatcttaaaattcattttatttcttttt tattatactttaagttcta gg gta catgtg cacg a catg caggtttgttacatattttatttcttatg attc agtttgg atatcaca gctg g tctcca ag cag aaaa agag agttcctactctcctccccttaaccccattaactttatcctca ga gttaca gg cgga a cag ctg ga catcttg caaccttag gta ggg gtag gtcttg cctt ga ca gattcactaca ca ag gaag ca gga gtgtgta cattatttta g gcttccag cttcg aaag aga ga gcca aa ag ggg ctg g cctg gg cacgca cta gga ccttttccccaa acttca ctaatcag agtg gttttctcttg a ag gg gttggg g ccag ag gaa gg gagag aag gag gg gtctg agg ag aag g gca gaa ctgttttcctactacttccaag ctg g ccaaa gttcctgttttatgg atg g atg caataacacag ggagagg gag aaatttccccaactccaaccctca gtta tcaaag aaaaaccctta gtttg caattg atgttg gag attctg gg gtgtagtg gtg aggg atatatctgtattg g cagtatca ggtctcag cccg acca gcaa cacatttgatgatatcg cttctaaaaatca ctcg gtacctctg tactaactttg gagtcaagtttaaactcctcctcag gg caag acacttcatg ag cag cccctg g cccctctg cagtctcacctcctg gtatcccactgg catcccccaca gccatg gta g agaa gccatg aa catgttg caactcctcaactctaag cctttg ga a atgtcatttctctctcccccg g ctaattcatattcag ctctcaa gattag g ca gttaaccatg ag ca g cttcaag g cag gttatg ttttacctatctctgtatccttg a g cctctcaaa gta g ctg cttga atg agttcaaaag gttag ctg aaa ggcaaccttg cttg g aaatcctcctg gca cctca gg ag atg g ttctcctccttg ctc ccaaag ccctccag ca caccttatttataattcttaaactataccttttaatttttttattatttttta g ag acag gtctca ctctgttg cccag gttg g agtg cagt gg cacaatcatg gttcactg cag cctcg aa ctcctg gg ctcaag caatcctcctgcctca gctccca ag tag atag g acta ca g caatgtgcca ccatg ccg gg gtactcttcttaaa atg ttgttctttttctttttgag g aaag aa g gctgtgtcttatgtgtcacag g gccta g cccatag g ag gta ctg g ataaattaatttttg a atcaaa cctcttctga gcctctgtg atgtg gaa gg catg gtg ctag g cactactg gg ggtataca a aatcgtgtctctg attaa gg aaatg ag a gttcatg ggg aacaag acatg aaaagttcataatatattg cttaag g ca ctgtga gataacttgaagttaattctata gaa atttaaaag gagg gaa cattctg a ctta tgta a caattatcaca caccctcctttca cttg ga aatg gactcttaatctgttaattttcttaa cg gttta gaagtgtcttttattgtgtg g cttg g atctgttta aaaa gta ggtg g gtcataaataataaata aag aaaaca ggtg atca gg cttagga gg ccctctg g gttgtgcgttca ga cagtatccaa g aagtttg acttg g tg tg aaag g gtg gg atta catgttaatg ata g cag g gtttag attctacactgaaa gg a ag cagttagtg ag ccctg gg ag g agttg aagg aaag ca gt ta caaaaatg g atg cttcccaa gaccctccattaa gcctcctg gg ata ggtcccctgtctgtccctag actcca gg gtactgctgg g cacttcaa ga gag aca gag aatttgaatactcatg agtcttg ga atgta g gaataa ca attcatccgaa caag ca aa catg atag a caatttttttttta cccactg ag g acattgtgtct aaa aatctg atttgtg ccca gg aattatctgtata g aattttcattactttata gaattgatattg catagttctgtag g ataa gg atctag cacttg a gttatg aat ag ctcag gatga cataa cacacaatggtta gttg g actg g atatgtccataaagctgaattctaattcaactatg ag catctctgattctgcttctg attcta g gt ga ctttaatcccaaattg aatttctga catttctgtaaaatg ggg catga gag ag cacgtcg gttatgg gg tttaaatataaaa ga gttaa gag ctga gaagt g cctccttg gtcaatta aa ccacactgacatcccgttttccttctttctttttcataatta ga g ag gaa gtg g ag gg aaag gaaataa ata a ctttaacaa agtat g cctagtttaaatg ca cca ggtccccg ctcttttataaactcata attctcattgcatcactaatttga atta atcacag ctccttg g tgtctcag aaag cttcctat tg cttttca ctacgtaactttccaa gtctgtgtgtttg gta ctctag g atgttag gccaccag ag gaa agag gatgtatctttttttttttttactttaa g ttttag gg ta catgtg cacaatg cg cag gttagtta catatgtatacatgtg ccatg ctg gtgtgctg cacccattaactcgtcatttag cattag gtatatctcctaatg ctat cccccccccctccccccaccccaca a ca gtccccag agtgtg atgttccccttctgtgtccatgtgttctcattg ttca attcccacctatg agtga gaatatg c g gtgtttggttttttgttcttg cg atagttta ctg ag aatg atgattccaatttcatccatgccctacaaag ga catgaa ctcatcattttttatg g ctg catagtat tccatg gtgtatatgtg ccatattttcttaatcca gtctatcattg ttg g acattttctttttg ata gtcgaattcccttca acgtctag ca ca g tg cctt g cacattta c ccactcaaa aaaa atttcgtaag g aatta ataaatg aatcctta gg ag g aaaag tga aaatgaagtttttctctca gg atg ag g tgtatttctccgttcatttcag atatg catcag cta gtca g cgtaaattg tg ctttttatatg ctca ccag gtgtaggtaag ag ctttg g ctg ag atg g ag aaattcatcg cgg g ag gataata a ag cg gg cag g gatttg g caacctcag ag ccg cga gg ag gag g cg g agtcg cg g aga gtttg agtatttccgtccaatcaaaa gg ag a ctgtaag ag ga ggag ga g gag g ag atg a ctgg gg ag cg gg ag ctg ga gaatactgcccagttactcta g cgcgccag gccg aa ccgca gcttcttg g cttag gtacttct a ctca cag cgg ccg attccgag g ccaa ctccag caatg g cttttg caaatctg cgg aaa gtg ctcatcagtg a ca gcctg g acccttg ctgccg ga ag atct tg ca agatg gag gg ctg ca ggtg gtg gaaaa g cag aaccttag ca aa gag gag ctg ata g cgg ag ctg cag gta ag g cg ag ag agag aaaattg ag g tctctag g g caacctccatg gaaaaag g ctg g ctg cg cccag gccag cg cgcccccctcg catg caccccgtatcaatta gttccgg g g cctcctgag att g g g g ggtag ag aag a a cg gg g g cgg g a g g a g g cag aaag ag g gaa gaa caaa cg g cg gcga gatg caaacttttctttta gtttg caaccg cgtcttt cacgttg g catg cctccgcta gcattg caaagtg cg gg ctg ctcca actg gtcctgcag gctg ctcg cg g atgccag cgcg g g atg ccag cg cg g cg ccc cag cg ccttag cg cg caattgttctg g ca g cctcg cg ccg cctctcccccaacccccaaccgcctccg cg cg g gg cg atcg g g ag ag g g gccccaaagtg g cttctttgcg g g g aaccca gg g a ctg g cgattcttccca accaagttctg g gccg ccccgccgattcta g cctg ccttgg cg gtg gcgg g gttg g g g gg g tcg g gg g g cg gg g g g aag ctg g cg g ag a cccaaccagtctg gtg g ctgctg g g g cag ctg ga g gg g a a g g cag ccctcgtaag gca gcaaaca cgt a cccg cccctcgtctg atg caag actg ctccg tg ctttcg ccg cccctctg cctcctgg g a g ccttcg g a g g aaa gg g ag gcg g g cg gg g ag cg ctg g g g tccag atttag cctcctccccacctttg gtttatcgctg gttg g ga gtgaccag actcgactag aatccg atcccaaacg ctccca g atgatttatagtcttag c a a atttttatctccttttgttgtgatatataattaatctacttcaa atctttatccaccgtgtttgaaaag g cctg ctg g gcctg gtg g ccttgtccg g aatatttatttt g tg aactcttca ctccagtg cctg ca ctttcg acctctg ta g tcgacccag ctgcccagtctctacctca cctg atcca gtatattctcccccaccctccaaatcgc a caaccg ccttctg cccg cctcag ccctgtccaccaccttca g ccgtctcctcctccg a gga ccccttag a ccgcaga gg ctg ctgttgttg ctttagttgtg cc a caaccacgtggtcctg g aaactg cctcg ctcactttgtctg cctttgtatctg ctg gcgtg g atcttgtcaccatg g catgacactatatccaa gtatccag ctc tg ccctgtactcag a a g aatttg gtccctg ccttcaa gaagtttgtaag ctg gttag gg ag catgg ctaata cgca gg ag acagttg a gcagatga caca a g a g catag ccccgaatta g a ccatccag ctccctaacctttg cg tgttccccttctg g aa a ag cca ga gaaag gtccaatcatcatg cacag aattg gtg g g ca aggg ctta gg acttga gta gg attg ga cg ggtg g ca gg ctcctcacatctgtcacctcctttcttgtgtgaaattgtatcagtgg ctctg ggga acaacca gg aaa gg cagtg gg gtgtcctg cagaaca g cctctg actctca cctcca ccag agca cca g gttg g gaag agg gtg g caatcaa gtg ctttcacctctcta aa gg aaatta gaaacctg ata cccca gtg ag ggg gtg g gg atag ggag ggatg ag gg cag aattg ag ag gaatg ggagg cctcatccata atgag gg gt tgttccatg aagtca gg aatcag ctg g gtg g atg ctg gg agtctg g tgctgaaa cttg gag aacattttaaa gg cg ctgaaaa ctcttg g cag ga gag ga gga gtgtttgttgg ctaa cttgactg ctg gg catttttg ga ctgtttg ggag gg ctcag ctttcttgtctgtctttg ca a cacatttg gttca ga atgccaattaat ctccttg gtca gcccactg aag g gtcctcatttctacactatg cctttttattctg catgaa gag gtgtctg g cata gtgtctcctg ccctcccccactgaa gtctct ttaatg ctg aag ga ag cttcg gca gcgttg cta g aaccgg g cctg gccttggttccca ctg ccttgtattctg gg cag g cg gtctcctctcactca ccccctg g tcag gaa gta caatcattcctccctgccctttca gtg gtg cttttca gacttg ga gcaagtcata gttca gtttaag g ggtcatg acg ag cattttaaa aa acaa a gta gg aaatatcag a ca g caatgcag ata g ata gtaa ggttgtaagtattgtttcatg a attttgtttcatttatgttgtgtagttgtatgtgtgtgg catgtg a g tatgaaattacaacttaaaaatgtgtttctg g ccg gg cg cg gtg g ctcacg cctgtaatcccaaca ctttg gg ag ggg cca aag g ag gc agatcatttg a a ctg ag gag gttg aga ccaga ctg gg caa catgg caaaacctgtctcta caaaaa atacg aacatta g cta ag catcttg ca caa g cctgtagtctca g ct a ctg g gg g atg gg gtg ga gg g ctg ag g ccctg gttg ctgtcactctcccaacctg gtg ctctagtg gg agg atcgcttg ag ccta gga gttg g gctgc a gta a gccatg atctcg cactg caatcca g cctg gg caaca ga gctagaccctgtcccacccctccaccaacaa ag atgtgtttcttacaatg aacctcagtc aaa aaa gtttg gaaa a cgttgg cttaaa ataataaag catcttg tttg gaactcacatcttaaatatacctaatatgccg gaaaattatttaaataatatta ccatgt atca gtg gcttttaaa ctttga ctgtg accca ca ataagaaatttgttttatgtcataacttaa a cactcatacatatgtgtacaca cag a ca cacacaaattttatg aa father ctcttttta catatg atg cattctg gtattttcttctgtttcattaagta g cattg ctggtca gg acca cta aa ctaaacactg gtaatg gtatttaa ttta ctg aag cacatccataccttcatatca gtacttgaa gaaa g attaag aag gg aataaatctttccatg ccaaatttctttca aaatttctttaaata gg aagt tcccag cctttacacaa gg atttagtttg caaa a gttaaataaccatca a attaatcagg atcacacatcttgtg ca a aa cagg gtag a cctg ccttccctatag ctg g gg aacatgacctg g atagtttcattgtcttg ccctatg atttaagtcag ag gcatg g caatg ctag aatta cctcctag g cca ag cttgtccccttg gga aagg aag catgtgtg aa cctg ag g aatg g ca atg gattcccattg ctgagttag ca gtttg tct cg aaca gccata a atcaag cagttttcta g ctgaa cag c ag ctaag gaca gg ggag cg gg ag ga cttgta gg aagtgtg gg aa gtgcctgttcccg cca cagtg cag acaca g aag ccaca cg ca gagg gcagtga a catcag aatttggtg ctgtgtag agag ag a cttg cttcctcaccca a g gaatgacaaa g a g ag g gg ag g atctctg g acactgg ag a a gttttaatcgca a a ctg cag ag gatgtg ag cag atg g gtg catttg gtta g g gctgtggtttccataagtag ag ga ca g g a g cag acag cttg g g g caaagtctttg a a g g g g gtg cgttgtg g cg tg a g g ag gg aag g g g cttg g gtg cag g g gctttg ag g gtcca g ca gg ag gtag gttg g attcag gctgtctgtgaatatgtg ct g attgaatga gtgg atag a gtgg gtagttg a g gttgtaag ag g ccttg gaagctcattaa ctctga ctg atgtg g g a tgtctg gttcta atg tg g g accaag g tctcctgagtg acagtttctgcccttg g g a g ag gtga gacctg atg ctcatctgtag g a gtctg actg g g g actcttagtttccctg g ca ccaaag aag g ct g gtttgag g g aaa a g tg aag ag acta ca g cataaatcttta ca a g g gagatccatcca g ag agtccatgctcatg aaa gaaa caata gtgctgtttctgtg c ttgttcag g g gctaca a g aatgg gtg g actttg g g g agtctgcatg ctctccagcta gtgttg cctgtgg g g ag ca g gtttg caag gtttg g gg ag catttt gg cag ctttg ga gag aa cattg aag aag g atg g acccttaag g cccaag ggagtg attg g tag ag cctctg gag cag g actg gg cctg a gatgtgg gc tctg gaa cttg atg cta g ag a gaca ag gtg ata ag atg atg actctg g gtggtcccttctcctcttg ctccatg cctg cttcttg tca gttgaa ctttg acttttg g tcctg ag ctg acca cttg a aa gg ag gttgtgtttcctcagtgtgttaccaattttctaag g tg tca gtgctgg ctg caa ag aaatg accactaaaaatgtg ctta ccca cccactg ccca g ttg ccctccaaaaa g gtcatg cagtgtacactcctgtttccctccatgctcaccaatg ctg aatattattcattttctttctg cca aaatg a tg cccaaaaatatcatattta aaaatctatatttccttg atatctag cgttg aa gagtctataaa cta gtg aaactagtg a cca ctttgtagttcttctg tg cattgcc tatttatatatttatctattttttattgtgctctttatccttaaaaaa gtg gatttgtg gtgcatg cctataatccca g cta ctcg gg ag g ctg aa gca g gag aatcg cttg aacccgg gagg cg g ag gttg cagtg ag ccaa gatcg cg ccattgcactccag cctg gg caataag a gtgaaactctgtctcaaaaaaa aaa aaaaa aaa aaa aaaaaa aa gttgatttgtag ga attctatca cttttg gtgatgaatccttgttctgttacatatggtg aaa gg agttttctcca catatcag ga ccacta cttg cttttgtttatg gg atctttttccatg ca gaa gttta aaatttg aatgtaactg g atttgtcaatattttcttccag a gtttctaag atttgtg ccttgctt atg aa agg ttttcccaatccg a gtcataaaaatatcccctga cattttcttttaatgcttatattttta a aaattctttaattcatctg g atttttaa a aatctg aagtg ag gtag caatctattatttttcttcttg aattttcataa cattatttcttgtttagttcttcctttccgcatcaatttg agttctg ctttttattttctaaattctca catattctg tttgtct a gtgtctatttagttctg ttga ctg atttgtca atttctg aacgtttttcctgctttttttcaacttctttta ata agatttttg aaaatccttga gca ctctta g aagtaga ccttatgcctccttatca cagtcaaag g aa ggg atttctgatga g ca attttcag gcatgtg aa gtgttaaaa gttatttg g gaaa gag g gtg a ag gtgtg ag g atg gg g ga ctg atg agg gtg gtttcta ccg g cctctcatcactg aattcatttattcaatg ga agtgtataaaa ca cttcattgtg aagtatcaactgtgag ctt g cctacag g ccaca gg caacttgctcttctcg acag ag g catttgtgcctatttctattatcccccagtag g ca gg ctg g ctg ggagga gcag gtcaccag a ag atccag g gacctttgttag g gag cttctg ccatcttgg ag aa g ccacaggatttctg g caatgtctctcctg gtttag gtgtctaaatg ctgtatttg gaa gg ta gttg gaa ctcaacag atttg g acttg attcca g cttctg ccatatcctagctatgtg acttca g caagttctctaaattctttaagtgtca gtttg cttgtctgta c father g gataattg ta atatctgtcttag g attgttgtg aag cacagtg ctg gatgtaaa gta cttg ctcaaaaaatg gtag ctattgtta cta gtaaatctctcc aaggg atttta cagtttg gtttttg cacctaa ctg g ccatg gccttg gctctg gccctatcttcttg acatcttgg cag gtcta ccaactcttcttca gtta ca gtctc agag tta gccagtg g caatg actttcca gttcttg gctg gttttg atctgtaagagtg accttg g gca g gtta cctctcaaag cctctgtttctttatctg g aa ac tg ggg atg attataccccttctccttg ga gttgtgaa gag aataatgtaaag caactggtgtaatg cctagtaccca gca ggtg ctcaataaatg gtttatgtg ctg gtgag g ctg gta ctatg g atatatg gtata g atagtaaagttttaatataaatccctg catta gg cata g aaaatataa ctaa ggag accaaa actg cag gtctaag aga gga caacta gatatgaaa catattatattggtgatcacaacaaaaatg gtg g ggtgtag ggag agtg acagttca gg gttgttg gcaa atgtttcctgg aag ataactttattg caccttg aa aa gtca gta aa gtgtgtcca ggca gg gaaatccttttctttg ga gcctgtgtg g tg g acttgaatgtg ga aaa gatgccagtatcctgtttctg cctg g cttg gctgatatttaactag g ctg ctatgg g gg g aaatcag aatacctccag gtg ctca cctg gtgttg g cctca g ca ctg g gg cagtca g ca ctgag gtg g cttctgtttg attcctctaa g ccctgggtg cccatgtgacttgtata caccca gg tcataatcagttta g cata atg c cag atacgtacata g cg g gcatccg ataa aatg aag gaa gaaaagtacag gg ga gg gg aag g ga aag ta gg cag gcttaactttg ttgg g cttca gttt ttcctctgtaaaatg ga gttg g acca g atg ctg gtg aacatttcttcaaatctcccactctga cgttctg cttca g aacctcag acataaaag gatttg gag tg tc ttccag g ccg cctg catgtttctctctg ctg gta gg caca gcccccaa g ca gtgag gatgtgtg atg cataaacacctccag ag gaatcgttcta ctattg ctc ttg aa caattcttccagtgtttag aag ctcttaa gg cttaaatattctatg ctg cagcctaa g catcattcctcttctcttcttagtg ga g ataaaatta cccactg ct ctcctta cattta ctttgtccatatttg ctcctatgctctag g ctcgtg cacaacaaaca cagtgtg gg cccttaccctag aag ccaacttctcatg a cctttctctat ctccag aatccatg ca gtgg g aatg aag g ta aaag aag gttttcatgg g atcca gctg ag ag ctctacg ggg aaaatgg atctg agg ag ccatgtg ctcc atctcttttattttaca gg tag ag actag gg gtatag agtg ag gtg aattaccgcagtg a cccacacattg ttg g cag acctag g atta ga actctgtcttcctg g ttcccag cttg g tg cttttg aaag cata cttg ctg ctttcttaccg g cctggtgtctg ccactttg gg acag a gtgtgg acttg ctcacctgccccatttcttag g g attctcattctgtgtttg a gcaa gaatattcttattctg g aaag aa ccacatacca cag g attctgg gtg ag cataag gaag attgtcttg gg g atctg actta g ctcacgtatagtg g ctatg atg aattca gtgtcttattttttg catatgtatatttttagtctaatattg cctg g gtgtctg ag ca a gtctag atg aatttaattg ctc tcatttttcccctg cccctcttcctttg gtctctctttta gg aaatgtttttctttcaacattcgtttcattcattatttactcattcg gcca to ccaacatttattg to gtg cctt ccctgtatcag gg a cag g gg ctta caaa gta g aatttg atccca cctctgccctcagtag ctcagtgtctaatg g ag gta gtgatgtt catta ag cgtcgcca g ata ctgtg ctag gtg ctgtgcctgttctctctcgcttgttcctca cacacttga gaa ggccg aag ctg attcata g cttgg aag g cag ggg ccttg g atttg a a cccag g cctg acca atgg cag aacctatca g atgtgtgg acag atg a cattgcctttctttctttg gatatatcaaaatcag cca g ca gg cag gaactcccat tttg ag caag caatgtgca g gaatgata gg gta taca gag agg aaca gg agatg g cccctg acttccag catgtgtctg atg g acatccag g ctg cag gc atcatg gtg ctgtctag aga gatgag cca g gtg ccca gag cccatgg g ccaatgctg ccctttcttg ag catg ccaaa caaag cg g ttg gtgtgttag agg cacagtctcctccactctaagtaaaaatcag catg actctag cccacatttccctagtgagtatacca aa acttctg aactg gc acttcct aagg ttccg gaaatg catctcttactcag ga gtaag caatg atgtg cctg cg gcttta cg a gttctcacag aatg a ctttctgg accca a atgttttttctg cttc agga ctgtgaag g ccttattgttcg ctctg ccaccaag gtga ccgctgatgtcatcaacg cag ctg ag aaa ctccag gtg gtgg g cag gg ctgg caca g gt g tg g acaatgtgg atctg g ag gccg caa caa gg aag gg catcttg gttatgaa gta a gtcatg ga gg ctg cg g gcggtttg ggg gtag gg gg gtg agt g cg g ag actg acca ca cctag g ga gaaaaaa ctcacttg ag ag aa agctgtccattg g aag gg cttcca gg actgcctg g tctag gg cctg catg g tca acacaca cag cag gtggtttcag g tttttg gaa gg cag ctatgctcaccactatatcaccaatgccatcag ggtgttaca ca gt ttttg aa attga ga g tccctg cataatctcaaaatgtttcacg ag cccaccccatg cg ca gttgcttg cacctctgtgacctg gttca gg aatcg gaa g gtcagtg agttcatctg cat ttcctgctcccaccca g ccccctctg cctctattaatg ctgtttgtg g cag gttttgtcagctcta ctttactgtg cttgta cagag g cacaacctttg ctag caga ctaatg actag aatccttgccctccccacttccctg cca ccttctg gaa cta ag acactagttctcttttca gtg ctcta g gg caa g ag ga gaag gg tcccattta aag ctgtttctg ca g aaacacag gg gcag ag gtcatca gca cg ccagtgctgta ctgtacccgtctgtcactta g atg gtatg g caag g ccatccccag gc ctctttgttcttaag a cttttctcttcccttg ggg acttcattgtcctta agaccttccccctccctg cactg cacttccccctgta gg gta gag gttaattg gtca cc tg a ctga agtcaatattcaaca gca gaaatgttaaacgataa cccatcccacattcttgccttg g accca g ag gca gccagg cccca atctctgcacctctact tg cgcccccataca g cctgtttg ctgtg ggagg atga gaa gccag gtg gttttgcag g ca ga ca gactctg ag ag tccgtttatcttataca g gatctcttga cttttcttcttgta accttattaaccttcattccag ag atg aaaa aga ca gacccagta gggg aataatcag g gtg a acacgtatatg aa attctttcaaaa acc ta a aaag cattaa g aaag aaaaata gtttgtg g gttgccacctatttttttttataaaaatg gg a ag gg tctg gattg ccctgtgacttag g gtgtg ag gagg ctttctcagttca g aacttgttgag ag atag gaa gagtagtca g acag gtggag att a ctaataaaag cta g gttgg gg ctg cgtgtgg ggg ctcac a cctgtaatcccag ca ctttg g aag g ccaag gtgg g ag gatca cttg g gcccagga gtttg ag acca gcctg gg caacaaa gtgag a cccccatctac aaa atacaaaaaaaaaaaaag aag gaa ag aaag cca g gtatgttg g cacca gcctgta gtctcag ctactg ag aag gttg ag ctgg aag g attg ctt gaa ccctg g gaa gtca agg ctg cagtg ag ctgtg atcatg ccagtacactcagcctg g gtg a ca gag cg ag g ccttgtcaaaaaaaaaaaaaaaag c cag gttgg gtaacttg atg aag attgtag g cattg gactga gccctg a attcg agaga ctctg accttggtaag atcaatg gtag g ag ca g ca gg g aatt tgtg ctttctg ag g ca g gtag atcctg aa ata gg ag aaag aaa agg gctg aacca caca caaatctg atcat g tg ag acactctctg ccttg gg ag ga g tgtttg gataga gag aag ccag atatgtttctctaatg g ag gtccctctg ca gg gaaacataaag ccag ggg aag g gg gttctcttcca g cccttg gctta g gg cg ag agtacatca g ag aactctgg acagtg gcgtggtca gttcatgg ag cag gagtg ag agaa cacag cctg ca ctcaag gctttctcttgg gg aag gag tg g gaatactg gg tctgtgcccattgatgtccccctttctttgatctttagcacccca atg gg aaca gcctca gtgccg cag aactca cttgtgg aatg a tcatgtg cctgg cca g gtaagtccctg a cttctcag caaag cta gtctctccg atatgccaattattacca cttg ccaag caaatg g acccag aaaa a cttag a aa cattcgtctcag cg a cttg cag actg cta ag aag g cga catg ca g actg caaag aacctttaa gg ccatca g gtcctctattcaacattcatta cacag gt tcccta gtgca caaa g gtg cacttctcttta ctcatgttctattctctgctg caga caatctg ccttctctag cctcca actcttg ca a atgttctccata ccctg ct g cctg ga gataa caa gg aag gatgttcca gg cag cag ggtatg ga cagtgtctag gtaccctctgcttacttttcaagattaag cttca gtatta attatcca aga ag ctctctg g cttttccag cta gg ctatg tccctcactg attcccctggccctca ggg cttatctctgtca ctg cccttacaaatctgtattgtaatag a atg g a gta a gaacctg cttca gg gtccttgtgtgttctgtg g gttcataa ca caatg gaactca aatg g gcaa gactcttg ctcttctag ca gga gttcca g gcctctt gag tg g atg g gg ca g cctag aaatcaaatg gttgtg g cacag a atgtg cttgagtctca gtgtgttg gtta gtaattg gcaaaa ctgattg catg g ag ca c ttctgttg aaca gg caattcaa gcatttatgtg g tcaa g gg g aa ga aaca ggctcccaataaaca gg gtag g aa gttgttctag gtccctttg aag ctccatg g attccctaaa cca g cg g acctag gcatca agg actccttgtttctctactca ggcctg aaaccttccg gga cca a gaaattacttttcccatg g cg ag acctg ctttccctcctgctg ga ggagg atctgg gg gaatttacctctgctctaactcctccctgca gtttccatctgag ctctctg gtattcactg atattca ctg gtag gt gaa ag g ag g cag tg gg gg g aaag ga g aaacag g gatag ctta cacagcagtag g gtctcag cctag aag ggg cccccag gctg g ca g ccg g gttct ccca g aacatctag g ctg agtgtctctccccctg gag aagtaca g aag cctg gcaccctg gtttcacttcag ctattaccttcag gg atttttttttttttttttgag ta a g gg aaaag atctca atgtgg ctttctg attaattacctcactttttttctg gaggtg g aa g ga aag gattg g g a g ccagcaatactttccctccttttcca g g a cca g ctttgtttaaa g g aatcggtg aaatg ctgtg gtttaaaa g a a gctccccattg tca a ctg ctcaccatg a cctcactcatcaa a g gtg catca aaa cta g g aaacacgca caccctattg gg aag g a atgcgttcgtttcatcag actgttccta gca g g ga gg acg gtag g a g a gtgtg g gtgcctatatgtag actct a cag gattg gg caatgtg g gtgg ag ag ca g g cgtg g ctg g g cctcacagtag g g actttg ag cag atg g cg g g gg ctcctg ag g tg cag aag aacag cacacag aaag g aa g g aag ga gag aatcaa caaatgta ggtttca ccatttttgcctg ctttaccatctcctcctcta gta agtca g gccag ca a ccatttaatt attgtga gg accctcg cccacattag ag tcctctccagtctg g cctg atg aggattccaaag cattg aaa cctg g ag ag gctccatg atcag ctg g g a g cag and ccag agaa aag g gttag g gatttcttgtgttctgtggtg ccg ca cgcatacatcttagatag cactgtg ga gtctg gtg ggaag aaaata gg atgtg a ggg aga g gag ag ag ggg ccag gtgg gg ctctgtctcactag gtca a aggcattttatca gg caag ctttcccaag aa gtg aaca gttcctcaag g caagt cccgtccttattacctg cctg atg g gtca gag cagtg ag gtg a atgcagcttgtcccccatgtg gcttg gta ctg aa gttcttctgagttta cccataag atcgt g cg cctctgtg atg gagg acag attcca ccttcag ccttaccctca gatg g agatcaatgtcctagtgatgtgta gag atg aga gaatgtttttgtgtg aa g ttagtgaa gtgaaa g ca cg g aagtag cag cttg ag atttctcttttg cacgctcgctcactctga ggtttttg g catcag gttaa ag g cctacatttaagtactc aggagg ctg g ccgtg ctg gg gttg actttg ctg g ctttgaatctgttcaagtgcctcca g ttgg gtg atctg gggag aactgtttaa cctcca gtg cctccctc cgtgca gctgtgaatg tta g ag g agttaatgtgtgtctaaacatttcttttag gatgtctaaaataaagtg ctccttaaacattgtctctcaatcttcctttttg cct cca g agatgtgttgatg a cccatg ccca cag g acatatatcg ccctttgacctctaatgttg ccctaattg cca cttcctctgtctcctcca g aaaaca cag atgt a g a ctcag acttccatctg ga ctaatag g a g g a ga cag g ctgtg aagagtgtg tg tg tgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgttg gtg gtg g tg g g acag g aag gtg gtg cg gatgtttaaa a atgttg gg acca cg cattcag gacag a atg g aagtgtctgg g ctttg g catcca cccaccca ctcta gg g tcca g actagtctaa g a ccctcg g g gttaa g aaactctcaag g a a g cataggtaatta g gtgtcctcttcata ata gta gaa ga cgtcata g gttatg gtg ct g g atg ag atag gtag atca a aataattccttg a g cttctg g agtactcgatgtcatcag a gttg cccagtaccccactcagttatcccta caga ccctgg ag g attg gcgatcg cttaaagtaaaaaactca ctctg gttg atg cctcagtg g ctg ag gtgg aatccatacctcctcccag atg cctg g ctg gtcacag a a atccc a g g ctttctaagtca g atcttgccaagg gtttctctcttctgttttccctttatcccccatcagtgttcctaaacctgaacccccag accca gttcttg gg g aagtg g ctgtcatg gg cagtg a ctgtgcaaa cctg atgttg catctccttcctg gg ctg g cgg g agtccg aatg g accctctgaa cctgtgtctatccttg cag g ca g at tcccca gg cg acg g cttcg atg aag ga cg g caaatg g ga g cg gaag a aggtga gca gcg g ccttg actcg cccca cctg gg ctca g gg cccg gg gtc cactcatgttgctg a cttcag cttctttccttttg cctgtttg gttg cagttcatg g gaacag a gctgaatg gaaa g accctg gg aattcttg gcctg g gca gga ttg gg ag ag ag gta g cta cccg gatgcagtcctttg g gatgaa g gtaagatgttg ctg gaa ccctgtg atgtg gga ctttctg cag caattttg gg aaag g cag catgtctg gg ca g aag cca gaag ctttgttcta gga gggtctgaccctctcttgg ag cccccatcta aataa gtgttaaa gtcaag g ag gg ag a gaac a ctg gcctg ctg atctg g actcaaaa gctg g aaatacttg gtg ggg gtcctttagctctctggtg agtg aata g ccctg agtcccag tg aacca g gtgttg at gg ctcttttg ag actttg gttcctgtcttctta g ttta aaagaattta aacaag agacacg gtgcag cattg ag ga gttt attgcaaag gaaaaa g aatattttg aaa gttaagtg cag a gta g acagtaca cctcg gg ag ag ag agaattca gggtg gg ctg ctcataag agtga gg ca gtgttg g ccg gg cg cg gtg g ctc a cg cctgtaatccag ca ctttg g ga gg ccg ag gca gg cag atcatg ag gtcagga gatca ag accatcctgg ataacatg gtga aaccg catcg ctacta aaa aaa aaaaaa aa aaaattag ccg gg cgtg gtgg cg gg cgcctgtagtcccag ctacttgg g ag g ctg agg cag ga gaatg g cgtgaa cccg ggag g cg g ag cttgca gtg ag ccg agatcatg ccactg cactccag cctg gg cga cagag cg actccgtcttaaaaaacag aaacaaaaacaaaaa cg a at gagg cagtgttg attattg ctgg ag aaactccctttatag g agttttacatgattattcataag gag gtg g gaa g ag ctgtta ctagta ag ca tgttttg g gtg g tcctctgg gtg ca catg tagged ctgta cattcttgttcatttg gcacatgtcttatta g catcttaaatctcca cccag g agtgtg ttttttactattataatg a g ccag gg g gtcagtttg ag gaca gg aaa a atcaaagtg caca gg ctgtctagatgg gaa gttccctactg aa g atag ctttg cttg a atgag ctcag tta g a atatg aataccg ag g cttattgtgttg a ctatag gatca cca ag gttgctgcacttccttgattacctatcctg cctcaca ggg ag ggtg cttgtgtcctg ccc attctttctttccttctg cttttgttttgtctg cttcctgtgatgattta gaaaa cag g aacaagtttatg gcctcacagtag ag cctta catccattgtccatctctc cttcca gtttccctccatatttca gaaaag atttaaaa gtg cttg tatgtaca ctatgatatg attgtag ag cct atg gtg g ctg gaa g acctcacaccggtctca gaa atcacacctaa cctctgtctctcccca g gtctctg ccttttcctactctgg ga gctcagtag gcttctg g ctactcccttag cctg attgcaa ccctctg gttg c catcagtag cagtg ccaccca cttg g gta atccag ga ga ccttg caaatg ag agag gcaattcag ctaag ag aggg gatctg cag gg cttcccag atag g ccaaaga gatgaa ccca cta ag gcta ctatg gg acatctctctgttcactttctgttttag g gaatta ca gg g attatg ggg tca aactg cag cca ggaa gt ctttaaacattag ga g atcagttttga caacca g atcatgtca aatctagaaaagtttacata g ag ctg ctatg gaa ctctccctttttctacatccctcctaccttc ttag a gttagaa ggg atcttaata gattaacccttaattttacattg a catctagaatttgt aa gcttatg cctaa gga ctcatag caaatg g acagtag ccta ga g cctg gtga gcatcccattgtacacattg a atattatcctctccctcctg cctccactccccatcttatttatttaagtcag cca g atatctttcctaag cctctg cact gag aag gg ccag g tgtattcttg atccctg g actcctg aga gag gcagatgtag ccca g ca g gatcattg atcatg ta aacaaaccaca g ccttgccctctg tg ca g gg atg gtg gg atg ccataa gccaca aa gagcactccctcctccatcctcttttgtaa g gg atcaa gg gtcaag gtttagtccatg g ag aattttgata for a aag gca ga ggtca gga gttaagtaaaa ctaag ag cactcata g ccaaataggtg ag catttcattg atg aa g ca gtaa ctg g gag a cag gctccag a cca gtg gccgttccaa gg ctcctgcccccttccccag gg ggtcctcttcccca tgactccccttccccag g cttccttctccg tg g caccactacacatca atatt cctg g caatattcttcatcatgg ag acttcg g cag cg acttcaa ccagatga aaagcccca cctcattcctg a atgattga ggtg cta g gtaag cttgtctgtcc atctg g cactg ccca a ctctcta ccctg gg atg cttccaag ag gtg catg ggtacctg gg accttg ag catca gtgtcag g aaatg g ctgaca cctct cgtg c atgtgtttg attg aa agag atgtcta cg g cctg ctcatttcagtgttaatctgtgcctca gag cag gtcaccca ctga atgttttattcccattcccag catg gtgt ggagg gtgccagtg ccttcactttg catttcttctgtccag atgtctctgg ag ggtcattctta actgtcttg cttccag aacatg ctctttgtaaacccctg aa ac atg gcttg gatcattccgtctcccacctcag cccctccgg ag ctg cctg gacctcatcattccg g aga gtcta a gtgg cctcttctcgtg ctcag ctg cttacctc aa ccacgtcattatcata cttttg agtctg g atg aattttgtcttag gg cgg ag agctcatca gttctaccg gtcttg g agaa caag attttctaaag gg aa agg aag attcttg aacatattgtg aaag ag atgtataag caccca ga ca gg aaatg aagata ag tgtttattaaacacaa gttat ttcata ctag cctcatcctctttt tg g aaaccattattaaa ctggtag agtatg ga aatccctag atgtcag attackcg g actatttaaagttatgttttaatcatgtaataaa g gaatg cattctcata ta a attaaaacttg g aca ag g ctatacttg attaccatctg a accctagtctcctcacg gtaaacatcagttta gcatgtcatccg ag a cttttctatg catttg ca and aatg catacagttccatg cctgtgg atgg attctttg gctgcaa acaatagaatattgtttgtg atgttaag aaaaa aa catgtttatctca g aaca actaa atctag catatttttaaacagttgtgtg g aattttattgtgca gataa attgctttttattgg ccattctctcaatg atgg gtatttag attgtgtatagatatg ga ct ttg gtaa gacatctttcatgattccctcg gg gttcg gatg gag ccccttga gcttg gtgctattg aag g gatta g tg cttg gtgctg ttgtg g aatg gg cca cc tcagtttg g ag gg ag atagtatg caata gtg ctccaccttg cccaccattctcatca acag cctg aaccatgtgtcatttgtgg atg g cag gaa gcaa ggaga g gtagttaata cttaag ggtg cag aatca g gtccaaaaa g acatatgaa gg ctagaatg gttg g gca ccctca ca gg aaattta g cta g aatacatttctg c a ccctacttgtg g gtccaaaa ag ca gcta ga caagttttag atgtagaagcatttgtcaaaag cttttctg g gg g ag cag gg cttttggttta caaa agag cag tgtg gttgtcagtg g catag ctg ata gaaaa g gtaacctgacatttg gcagcattaaaag aa gtatg gcttccg ga gcaag gg ag gtg acattcctcaca ct g tg g cctgacctg ca g cctacctgtggtctg agtcataca ggtctta acgctctatactgttgtgtctgcg ag a ga ctttttctcaata gtgttg ctg aatg a cag g tcgtgacctcag aga ctcag ctcattcatg ctg gtgtgttcctta g aatgtgttaagag g ca caaaccaatca aga ag cg ag ccttg tg ctttatg atcaag g ttttccttg ag gg aa ca g acaag catctctta a cagtttctttgtag cca gcgttgtgatg a atccaactttag cccaga gg gttg g atg ttgaacctg ag g ca c cagttatcaccgattttttcag gtcca gctaatg tttgctga g gtcta ctgtgtcggttcctgtg cttg aaatg a ctctg ca gg gtcag aattattatcccag gttta cag ataag gaaactg agg ccca gg aag g ttctatg atatg ctg aag ataatgcaactatg a actggtg aatttcg g ctcag ccttcca gg ctg g cattctttg ccca gtacca cttaactttatcatg atccatttta g gtaaaca gtccctgtg ccatg ccggtatg ctta gctattccaaa ga ca gata ctctccag g atg g gcag ctcctatctcagtgtgttacctgg aatg ctta ctgcccag ag ca g tg attctcaaactcaag ag cctcg ggttctg g caag gtg cctta ggg aca cag gtg gg ag ca a gg ag gg gccca ggg ag acag cag g tg gtg g cacattgagcctttta cctctg cag ctttgaattg acctatttttatatttgg gtct ctgtttaa g atttc attg ccgtg gtag ggaga gttccaatg cta aa cta aaaattca g ctgtg g ggca gag ag ag ca cgtga cg gtgag gg ttcttcttcag ag cctctttaa ctg ggagg cctccttg ctctcctctttgctctg ctg a gccag caag g ccctg cagccag cataccag cctctcag gg ctgtcttctgtg acctca gg ca ggtag ga g ccaccag ctg ccctg g agtcctg ctta gaa agtg ag gcttcctctgttgcctcaacatca gg agtctg ccctg gg gctgg g attg ag gtgag g ga acccct tcatgtttctcccca agg cttctctccgg gag tagtctttg aatg g tg ggtacccag ag gtgctccaatgtctgtatattag ga catctga ccctttccag g aca c a cca g ga ccctgaca gg cacgtgtgtcctg gg cctgttgtcaccctttccatcccctcagtgttgtctcctagtgtcg g aaccccaa ccccca g ctg agttca g ctcttctctg ccctg gttatctcaaatg gaaa atga gaacag ag gtttg ctcagccag attccttccctg cagtcatctg cag ataaatg agatactctctgtg aa aga ag g aag ctg agtcagtgcttcg ctca cccctgag g aag atg ctcacctgctcctctccccaaccccctccctccag cccca gtca cctgtctgtg g ag gg gg cca ggttca gcctg ca g ggtccttcctcctg ccag ccccca g gttgttcttggctcctgag catg ga gta gacgtca g tca gg ctgg accttg tg atctcta ttg gtgttcccttcctctccccactccatatg ag aaaaaaaa atacatctgg atta ga gatg aacag g gccattgttctg g ccg g cctcag gg ccag cag a cg aag aag acttgtg a ca a cgatgg ca gg g atcccatctcctcg cctg gacactttctcctccccaccag aaatg agta g gttacag ccgag gggag cag cag tgtctg aaaaag accaccag cgttctg ag ctg g ag gttcttata gg gcgtgg g aga gg cag catctttcccca a aa cctg cctg atg gttg gtgtg gg g cc a cttg g ctttccttattcca caag gctaatg atgaaaaaa ggg atccag ag gcttcaccttaatcataaccattttag cttctgccctag aattactgaatttg aag tcctctttcctattacgttcttttttattttgttttatttttttttttttttctgagacatcttgttctgtcaccca g g ctgg agtg cagtg g ca caatcttgg ctca ctgcag cctcaacctccta g g cccaa gtgatcctcctg cctcag ccttctg g gtg gca cag gtgcatg ccaccaa a cttg g ctagtttttta a attttttgta gg g g g g g g g gtctgactatgtttctccag ctagtctctg actcctg g ggtcaaatg atcctcctg cctcag cctcccaaa gtg ctg g gattatag gtg tg ag caatgg caccc atctttcccattatattctaaagtatctatactgtatccctaatttca a aagtaatttatg a ataatgtagtag a a aacttg aaag ca ca gg aaag caaaaag ga g a a a aataattccacta cccag ag acaaccg ctgttaaagta ctatgctgtgtattctttgtttttgtctaatcctg g g ctcactcttta cattctgtctcg g g gtt aa g g g g gatttctcttcaaataattcccagtg attg aatgtg ag ag tg a g agatggctgca atcatctg cccag ctcctttatttag g gttg a g g a cccag gtcca g g g g aactg acctgta cctttcca cacagtg g gca gtgactg g g g g ag g acacaggtca g g cttcctgatttag a g g g catg cagtcttg ctcccactctg g g tcctg ccttgta cccctgtgtgtcctcctcttcg cttctg ctca ctctgttttagtacctgatg cacctgtttcctg ga ctg ga ccctccag ctgca gctg ccatttct ccttctccctttcctg g ctgtccg ctcccta catgactgccctccctccctcag g actccctgctttctaag g acttccaa gctcca ccttgtccaga ctatatg ag ctattgttg catctgtgtgtg atca cagcatg gagg caga cg ca ccttcttttg agctctg g ctgtg cccctcctcattgtgg aag ccctg cttccctcag ctctc aag ca gaca agaa ca g tcccag catcattg tg tg gtcatgag aattaattaagctgatcatg gtacttagtatatg gtaaatagtactta gtatg tg g aacatg g ta cttagtgtatg gta a attaactg g ag a atta attaag ctg ag catg gtagttagtatatg gtaa atg ctcaaaaaatgtttgccattcttaataataacaata ctaataattaa gaatg acacttcctccctctcttg cttccaacca ga ctata g ggtatgaccccatcatttcccca gag gtctcg g cctcctttg gtgttcag ca g ctgcccctgg ag gag atctgg cctctctgtg atttcatcactgtgcacactcctctctg ccctccacg aca g gta ggtgtgtcctta cattgtg gattg g tca c aga ag cca cag accagttaacaaatg gg ccctccatctgg g ccttccccag acagtg gtaaccag ctgt ggg ga gag gtcca cctg ggtcctg cag aggc tg g tgttttgttag aca ccccta cgttg g ata ggg ga g ggtg ag ccag ctagaagtg cttg gg gtcta ggtaag ctg g gcacag g gacaa cca gtgaccc ccatg g atg ctttctg cag cccctg cag gg ctttctgattcccaga cccacttg a aa cagtaatatctca ag aatttctaag atgtttctaaactg a gtctgg ccc ag atccataaca gg ga ctcctg ccccag ag catctg agg ag gaa gag atg aga gcaaa ga gg ctg ccgtcca gca ggag ag ag gctctg gg aaa g ag ctg gctcaag g aaa gg ga aga cctctg gaag ccag g gatgag cgtg gg gatcctg gtg ctg ccccag ca gg aag atgcttcg ctttcttccag g cttg ct ga atgacaa cacctttg cccagtg caa ga ag ggg gtg cgtgtg gtgaactgtgccccgtg ga gg g atcgtg gacgaa gg cg ccctg ctccg gg ccctg ca g tctg gccagtgtg ccg ggg ctg cactg g acgtgtttacgg aa gtaa gtgcctgg ca g cctcag cgtcag gagg acg gg aga g ata gg ga gca gag a gg cccatg g cag ggaa ag cctg g cgtttta ca gaaag cctcta gcttattgtctctttcatccatg gtaaaa ggag aaaactgtgttg ccaaaaa cacaccttt g tg gtttgag g agtccttg cg g ag cctg gtatg g ag g cag ctatgtggttttctg caaaa ctcg gg cttta aaggg ga cccagttcctggttccg gg cttctc aga ctg ctaacatattg a caaa gataaa g ccacaaatcagttctg aa gttg agggtctg g ag cag gaa ga gag gggg ctttagtgtata gttg ag g gacc ctg atg gaaatg ca g tcccattg ccaag aaaatgtccttttca gtgtccatgg gcattctttttcccttcttctcttg ctg g gtg ga cag aag cccga g gata ctg g ca gtattccctcag g gtcctcg gg gtg gg ctg ctttccca gctg g ag caga ggctca g cctcacaccag atccagtagg g aag a gtg catttg cactcg a atccttttg gctcctctcctg aaagatcctgtccccaacattg g ag cttcccctg gtg a gg aag g gg aaa gga catg gactttg g atta ggtagttgtg gttcg cag gtta ctag ctgtgtgtccttgg aag gtca ctg g ccctctctg ag cccctg ta gca ctcactgtgta ctcccag ccca gg g caca cagtg ccttttg gtttgt a a ccttg gtcatctttctcag ctcttag atcataag ctccctg a a g g cag g gcaag ccttgtccatctttgtatcaccatcctccctaacaca gcaccttctcctg ct ctcttca gcatccttg aa cca cttg ctg gtg gta cttacatg gttacatgtg g ccg ggactca cag gattacatg gtta catgtg g cca g g a ctcacag g gtta a atg gtcaaaattcaatg g ga ccaacag ag ccctg ag g a g a atg g ccg g aagatg cctaaataacata cccaag g g caatcttctcagtga gcag aa g g g tg g aaaag acactg g ag ggtg g ggtg g a g gtg cca cagatg g gcaacctgtcctg atgag ag ccaa g atg cagttacctgcgtg g a gtgtcttg g aa g a g cag g acactgtg ctaaaagtcca g ca a g g cacatcg acag caagtg ctgcatg gatgcag g g g g gtg agtttcgg g g aaaag ccca gta aca a ca a caacagtag ctcctattg gtcaag gg cctcccatgttaaag gtg cataatccccacagtaatgttatg ag g g a g atag gatcaacccatttcacag ataaag a a a ctg ag gcttag atcaag gttcaccccca ag gca cctag gtg gttaa gtg gtgaa g g tg a g atttg aatctg gttca cacttt gtg ctgtgtccactctg ct gag atg g aacattcatgtg g aag cccattattga gtccagtg accaccctgacaaaatag aagtg g aag g ca cca gaactcctg actg ccttccctcca a ctt tcctgttg cctg gg gtg g cccaag agg gtgtg gccagtccatg g ca gccaacttag ag gtatctctttctg gg cag g agccg cca cg g gaccg gg ccttg g tg g accatga gaatgtcatca gctgtcccca cctgg gtg ccag caccaag gag gctca ga gccg ctgtg gg ga gg aaattg ctgttcagttcgtg ga cat g gtg aag g gg aaatctctcacg gg ggttgtaa gtatca ccacctg ggg ctg ggg g ccag g agtca ga gg g ag gag ag gaa gg aag g catcttgtag g gg ctggtg g cag cgtg g gtg aatag attca g ccctg gg ag ctg aag aatagg g aaatctg cttgagtca gca ctctccg g ag cag gtg gg cg g gag cct cccgtctccag ccttg atag cag ag g ccttg g cag cag aga gcccg g ctcaggcctg ttatatcgtagtcttg ctg cag ag attgtg g cccttcccag g ccc ag cctctag ag aaag g ctcctttgttctccacatg ccgtgg ga gtg a ag g agtgctg cttg g gtgccag ctg ga cg cag ccg ca g ca g gtg gg g atgtg g ttg g g g acg g ccatgta g aaatttg caccctgtaag ctcccca g accctg ccttga ca g cctgccctaccta ctcccaaatga g ccctctgtg ctg g ctg a ccc ccttg ctttccccaa atcaag gcataag a cccccacttcttgtctttgcttccatcaa gcccttcctg gcatg tg cgtctcctacg a g cttaacctg acttacacttc a a gtcctgtctcattcattca gtctgtg g atattccttaagtttca ctg g gtaccagacattgtcata gctcttg g g a g a cacgg gtg cat g ag g g a g gcata g ctctccttcca gg g g cttg ctg cctccttctg a agtcttg cttg g cctttccagccccttctca attctg agaa ccatgttctttcttgttatta ag gttcagttccatg g g gtgtttttttttttccctaatctttctactta a cctaagttctaa gttccctgag g acaa a aaa acatg acttaa g cctctctg ca g cttg tgtg g gtg gg cccag g ccatg ga gtcaaa g g gtcag g aaa atg g g ctg g gtgttcttg gttgtcctggctg accctcag ctg g gtgattttcg ctggtg ag g a ca g ca ctgtg g ca g g a g ag acg g gg attttg gttctgccctccca cctctgg attgag a g cccagccctcca g g cctctctgccccctccatcctg ag g aa a g aag g gtg cctcct g ctgcccag cag cccca ca cagtccatg gaa gtcag cag g gctatg a cca gcagcatg cg aggag gtca gcaga g actctg a cctgtctg catcctctgtc ctctatgctgtgtg gg ctcctca g gg ca g ag cacacttcactcatcttg cacctggtcgg cctctga gca ggttcgttcctcccag g ag atg ctg ctcgtttccc agg ctgag gtttg ag ctcatca ccattg ccag ccaatctg g gcttcagggttttaccctttca g ccttctcag aaa g ca g ctgtctg ccttccccatcg cag cctt g ca atttattgccatta ccatta g gtagcagtga cattcca g ag cttttcctg aa agg g actcctg a atataa gctctg g ca gag cgag ggg gtg gggg ag ggagg gg g accttg ca gag ag atg gg g ag gaggg ggtg ag aggtgtatg gg ctctg ccctg ctgg gtgtatctg ctg ca gg acaca ga gttccatcaa atg gaccacacagtgtccccatctta gg ag gtgaaacctcttg gtcaaaataa ctacccttagcaaattg aa ctgttc a cccacatca gcaa ata gtcttag ag ta g ccatttgg aaaa ag a gacatttttgtca cataagaaatatattttcctaattcttccctg cattttcccca gttcaattctgttcta a cctca gaca cag aataa c attag g gtca g gttg gtgtcccaaa gtg cctg a ctcctccctg g aattctcccacctgtcca ccag gg aag a ctg agaatcctcctttta cttgg g ag ccctgt g atgg acacctccccg gg cttg gg ctctg cag g ccca cacag ag ga ca g agag atgtg ccg agg tg cctgcgttatg gg ccctctta gttg g actttcctct g ctgctg cag gccctca g ccccg gcagtg g cag catg gtg ctccagatctcccccag cag cta gctgctccaccccaccacctcttgtctg actccttg gag ag g atccag gag ccatg cag caag aag cctg cagacctgtcacctcccactg g ag ag g ctcccgtg aag cccgg cctca gca gtcattcccata ccatg attctttctctg ttta aaaaaaa a aaattctccaatg atatcttga aacag a aa ga gaag catctgttctctcta atta catcttgtag ctg cct g tttgatttg catctttctaa g atg gttg acttta caa gttatctcaata a aa gtg gcca gatg cctaactcag aa cca g ag cattttg aa cca caaaatg ga att gaa attttg g cccag aacaa gctg gttctgttatcag g cccctg g gtgg gg cag ggg gcca gcca g cca g gtcctag acata acttttg gg g gatatg ggg cttgtgtcccctcagtgtcacaacatg cctca cagtg gacttcg catg cgttg atatttgaag cacg atcatcaaaactttgtg ataattgatcgta gtgtttagta a caatgtaaacactta a aaaaattcaag ata g aaaataaaaatg aag gcaa gttg g ga ctg cca ga gaag a cccgtca ctcctcatccaagttatctg cg ac tcccatatgttttgtgtcaaag actcacctttattgtg ctgtcca atcccttcccca gtg ca gaaacaagtctcccatg ga gg ggg ctg ggg caga caca gtttg ctg a aag gag caattttga gtg gttgtg g cattctgtgtccatttctg g ctccacagctttcttcatttgtag gaa caagtccttgtcctgttgttagg g ctg at g gaa gttgtca cccacca gg cacca agg ca gga gtgaccctata ctgtctttcttgtg gag ctg ggtcttg gca g cca g atcttg attca gg atctg ccatgcct cttcctgaccacag ccccg ctcctccatcctctgca ggtg aatg cccag g ccctta ccagtg ccttctctcca ca caccaag ccttg g attggtctgg ca g aag ctctg gg gaca ctg atg cgag cctg gg ctg g gtcccccaa aggg accatccaggtg father cag ggtg ag ctg gg g accttg ca gagg ga gg gg ga ggagg gg atg ag gga gtgtg g gatctg ccctg ctg g gtgtatttg ctgcag g acacag gg tta gtgag ag g ca gtg a gg gtg ccttg g accctgccctg a gtata g ctcccttactactg gtg g gag g gtg gtaaag gg ag gg gttaaaaaaagtcgttg gaaa gatgta ctg aatattcata a atcatgtttatgtag cat tttta aga ccta gatattttg g gtgcaag ag aa a cctctacg aga gag ag agttcatgcg ag ag gtcgta g gatg g ctg g ag gg ag g ccctaattaa gca a cag gatgctg gattctggtttttg g ccctgtctcttccctgctg tg tgactctcccttctg ag g cttgg attcttcacttgtaaagtg ag a gttag gg g ca g atg ag ccag gag cg gtg agga cactttgtg ctctgtaactta ctaag gtg gtaccttgg gccgtctg a ca g ccctcgag ag ag agtttgctcag ccgtg gagtg gg aatg ag aacagtg ccctctga ccacccctcg gg ctgctg a gtg g gctgtgg ccacctttg cagtg g atcca gtactgtg cg g atgtg gttga gtg acca aggagg gctccagtttccttaaccctgtag a cgtgtatctttccccatg ga ctcttg ggtgtctcacagttgg g ca ga gattg ca gg g ag aggg cag ctaaa cccta gg cattgacaataccag gg aag gg cag ag cca g gg g gtccca ctg gcctgg gtgtccaa g cg ccg aag gaaa caag gca gg gag ctg gg ca g ca gttgcttcg gtta cttctttctg ctttg atttcctg ag g ctg g ca a ggctctg agtg ca g gaag gg gta aga gtag gg aattttcctgtgtcccctg gga ga ataatag gta gg cca gg atg gccaag ccag gtgca gg gttg ggg ag cgga gtgg a gttg ag ctg agttttgg ag agg agttctcag g gtg gttaa aa catcctgg gtttcctg g cttg gg ccca g attatgcctcttctg ag cccactg aag gg cg agtg gacctg cctg ga gcaa gctg cctttg g ggtcccca gg g ca g cgag g ag cccata gtcca ga gctag gtg ccagtg g cttcctgccccctcctgtagtg ctcaacaaa ca g tg acctcatg gta g cttctctctgtcccca gg aacatccctg aa g aatg ctgg g aactg cctaag ccccg ca gtcattgtcg gcctcctg aaa ga gg cttcca a gca g gcg gatgtg aacttg gtg aacg ctaa g ctg ctg gtg aaag ag g ctg g cctcaatgtg cgcccctctcccccacgctg cctccccatccctgtca g cacta gtcttctcccccacatttcca g ag ccc g ttctctg ag cg g ag g ccta ggtcccag ccttg catcgg cctgtcta cctgtg agg g gtag ctg ca gtttcttca actg ca a aatgaa gata ctg cctg g ccc cg a gtgttgctaatg g cactg ctttgtgtatg a gtgctgtg gg aatg ga ggcagtag aagtgtccccatttcaca g cca aag aaaatg acg ag ctagtgtgt ttg a ctctg cccga catg gctgccag gccatgtttg actctg ccta a ctcccctcag gg ctcctcatg ccgta g ca cccg g gttcttg attcacttg caag ctcta ggag ccctg ctg ccttgca cg gcttccccgttg g cg ccttcccctctggttccctgtttag atcaaa gtctgtttcaaag cctgttg ctcag cca gtgg gag ctg g cag aa gg g atag g ca gta g ag ctgccatg tcctcacccctctgctcccctccactcctg catgccagtcatg ccactg atg ccgtg cag g ag gctgtgtca g ag ca gg ag gg gccag a gtg ga gtctcctca ca g ccctg cctccctgcttttctttcctccctgttttcctccaa g ccttcg cctgtg cctg g cag atctctttg ccc tccctttaag ga gatcattgg ctgttcca gg aa g ctg atg ccgaag g gca cacagcttg g cccatttgccctctcccttctggtcctg a attactg ag cacatta tcca gg ctg ga gccctacatcctaccaatg g gtgatttg g cca agag ag g agggtg ga cgtggtg cag cca ggag gtgtaa cag tca ccttg ccttctcca caca g gtcaccacctccca ca gccctg ctg caccag gg ga gcaa gg cttcgg ggaatg cctcctg g ccgtg g ccctg g cag g cg cccctta ccag g ctgt ggg cttg gtccaa gg ca ctacgcctgtactg ca gg gg ctcaatg ga gctgtcttcag g ccaga gtgcctctccg cag gg acctg cccctg ctcctattccg ga ctcag acctctg a ccctg caatg ctg ccta ccatg attg gtg agg ag ggccctg tag gg ctg g ctg g tg tccttg ag g ctg gg gtg gg gtctg ccctgg a attga actcta cccaccttccttta gcccctcttcatgtccca gg gtgtctctgg atctgcaccatacag ag g gtctg atg cca gttttcagaa ccttca gggag tg g atactcagttcaaa g ag g gaaagtg ccttatccag g gtca ca gag cag catgg ca gg ggtg gg gccata gcctctattcctg ccca gctgtgg atcct cagcttgccatgttaggtacactggaccagcttgtggagccatagcccaggagctcagggacattgagtgcaggtttcttactcctacctgctggccctgtgg ctgtccctggtggccagcccagctgcagcaaaacctacaaagcctccagccatggtaggcgtcttggacctgccccagtcagctggggcttgggctgctag gggttttggcacacgtccatgtttggcggagggtgtgccttcaaaccctgaagggcctaatttcaccattctttctggctgcccaagggaacttccctgcttttct cccttgctgttggctggataaaactggcaatcagaaagtcaagagctacagctgatggtcatggtgttcccagagagtcaggaatatccatggaagctgag cagatgccctgttgctctcccatctcagctctttgattctgagaccatcatccgctcattgcacctttgatcacaaaagctttgaacttctgattctgctcccaa tccc tcgctcctttttcccctatcccctgtgccaaccaggagtttcttctatttccaggcctcctggcagaggcaggcgtgcggctgctgtcctaccagacttcactggt gtcagatggggagacctggcacgtcatgggcatctcctccttgctgcccagcctggaagcgtggaagcagcatgtgactgaagccttccagttccacttcta accttggagctcactggtccctgcctctggggcttttctgaagaaacccacccactgtgatcaatagggagagaaaatccacattcttgggctgaacgcggg cctctgacactgcttacactgcactctgaccctgtagtacagcaataaccgtctaataaagagcctacccccaactccttctgcacttttgtgtggtcattatccta aagcgccaccagagggcgtccaaaggcagacgtagggtttggtttagactgcgggagcggagcgggtgtgggggaagatggggatgagcaa atggc ttggttgagttctttgaaggtgatccctctcttgtctgccgaaggttactcagaggcacttttacaggagcaaagctcaatgtatttcacagtgctacggtatttc agaccccttccatctgggaatatacatgcacgttaataagtaagattcaacacacaagcccagcattatgtaccaggcactgggctaggtgctttactttaaga ggagaattcaaacctgaccctttttccatagaagtctggtgggagggacagagcatgcagaggtgactggaagcagtgagtgatgctacaacagaggtgt ataggaaattctctagagtccaaaggaggaagtaatttgagctgaggatattggtgtctcaggagatgtttagaaggatttcataagggaaagaaagtgta aaattatgggggtatgagactatttggcaagttcacaaagcaaggtcagtgtgggttggtgaacatgtcacttttttttgaaagatgattttcatg caaaataca gctgaacggcactctacccaaaagattcacttcagggaatttttttccccattaataagggatgatccattcttactgtgtgcccagtagtgtctgtttctgagatt cctggcctggctaagaaggcttgacacagggcagctgcttggcactggcaggggcgagtccagcactctggcctccttagttttgtgatggagctaagcat caataagaactccagcagtaagggctgtgttagtgtctgcagtgaactcagaggggttggccttgctgtagctcaccttgaagggaatgggcctggttgtga atgtcatggtcaactctggacagctgtctgactgggaactcagtgtttaattatcctagacctttgtttcctcatctgtgggagggctaatggtgcctctcacaga actgcaatgagaatttactggattaaacagtgacttacgggaagtgccaggagcacttggtgaatgttggtttcttcatcctatgttagtctgagagcagacag gcaggtctcaattctttacatagaaccaaccccatgaaccaaatagttctcaacatgacctcattgcaattatgttcagccagatctagacactgggtgtcctga catcagacaacccattctctccaactggaaatatacctgtgcctcacatggcatccactgaggtcacttgagtgcactgatggatagaaaaacaggctgaaatt tatgaagttaaaaattcagttaaaaattgagctcaatgtttagcctctcagcctccttcctcataagcccctaaacaaatcatgttctgtgcactaaggtcttcgga acagtactagaaacgcagattacttgagcatctcaaaatatcttcctagactgggtcatgaaagagggcatgggacgatcttatcgtatcacgtctcccatggc tgtccacatgacctctcccaaacttaaagggcaggattcgttgtaaaattcagctggtttctttaggaaactctgtaatatttttcataatagctgcatcaatttaca ttcccatcaacagtctagaaagtttctccttttctccacatctttaccaacacttgctatctcttgtctttttggtaatagccatcttaacaggtgtgaggtggtatctc accgtggttttgatttgcatttccctgatgactaagtaaataggccagacacaaaaagaaaattattgcacttactcatttatatgtggaatccccccccaaaaaa gaggtcaaatatattgacataggaactagaaaagtagttgaggggggtgtctagggagatacaggtcaaagaatgtaaagtagaaaatacatagggtga gta PHGDH mRNA sequence (NCBI Reference Sequence: NM_006623.3) (SEQ ID NO:2): cacctttccgcgggccgcggggatggcggcgcagggcgtagggcctgggccggggtcggcggcgcccccggggctggaggcggcccggcagaagc tggcgctgcggcggaagaaggtgctgagcaccgaaggagatggagctgtacgagctggcgcaggcggcgggcggcgctatcgaccccgacgtgttca agatcctggtggacctgctgaagctgaacgtggcccccctcgccgtcttccagatgctcaagtccatgtgtgccgggcagaggctagcgagcgagcccca ggaccctgcggccgtgtctctgcccacgtcgagcgtgcccgagacccgagggagaaacaaaggcagcgctgccctcgggggagcattggccctggcgg aacgcagcagccgcgaaggatccagccagaggatgccacgccagcccagcgctaccaggctgcccaaggggggcgggcctgggaagagccctacac ggggcagcacctaggatggggcagagacttgttgcatctttgtccccagcaaaggctacatgttacctccttcaattgataataaacctttctgagatgcaaac tcgagaatactgcccagttactctagcgcgccaggccgaaccgcagcttcttggcttaggtacttctactcacagcggccgattccgaggccaactccagcaa tggcttttgcaaatctgcggaaagtgctcatcagtgacagcctggacccttgctgccggaagatcttgcaagagg gagggctgcaggtggtggaaaagca gaaccttagcaaagaggagctgatagcggagctgcaggactgtgaaggccttattgttcgctctgccaccaaggtgaccgctgatgtcatcaacgcagctg agaaactccaggtggtgggcagggctggcacaggtgtggacaatgtggatctggaggccgcaacaaggaagggcatcttggttatgaacacccccaatg ggaacagcctcagtgccgcagaactcacttgtggaatgatcatgtgcctggccaggcagattccccaggcgacggcttcgatgaaggacggcaaatggg agcggaagaagttcatgggaacagagctgaatggaaagaccctgggaattcttggcctgggcaggattgggagagaggtagctacccggatgcagtcc tttgggatgaagactatagggtatgaccccatcatttccccagaggtctcggcctcctttggtgttcagcagctgcccctggaggagatctggcctctctgtga tttcatcactgtgcacactcctctcctgccctccacgacaggcttgctgaatgacaacacctttgcccagtgcaagaagggggtgcgtgtggtgaactgtgccc gtggagggatcgtggacgaaggcgccctgctccgggccctgcagtctggccagtgtgccggggctgcactggacgtgtttacggaagagccgccacgg gaccgggccttggtggaccatgagaatgtcatcagctgtccccacctgggtgccagcaccaaggaggctcagagccgctgtggggaggaaattgctgttc agttcgtggacatggtgaaggggaaatctctcacgggggttgtgaatgcccaggcccttaccagtgccttctctccacacaccaagccttggattggtctggc agaagctctggggacactgatgcgagcctgggctgggtcccccaaagggaccatccaggtgataacacagggaacatccctgaagaatgctgg gaactg cctaagccccgcagtcattgtcggcctcctgaaagaggcttccaagcaggcggatgtgaacttggtgaacgctaagctgctggtgaaagaggctggcctca atgtcaccacctcccacagccctgctgcaccaggggagcaaggcttcggggaatgcctcctggccgtggccctggcaggcgccccttaccaggctgtggg cttggtccaaggcactacacctgtactgcaggggctcaatggagctgtcttcaggccagaagtgcctctccgcagggacctgcccctgctcctattccggact cagacctctgaccctgcaatgctgcctaccatgattggcctcctggcagaggcaggcgtgcggctgctgtcctaccagacttcactggtgtcagatggggag acctggcacgtcatgggcatctcctccttgctgcccagcctggaagcgtggaagcagcatgtgactgaagccttccagttccacttctaaccttggagctcact ggtccctgcctctggggcttttctgaagaaacccacccactgtgatcaatagggagagaaaatccacattcttgggctgaacgcgggcctctgacactgctta cactgcactctgaccctgtagtacagcaataaccgtctaataaagagcctacccccaaaaaaaaaaa PHGDH polypeptide sequence (NCBI reference sequence: NP_006614.2) (SEQ ID NO: 3): MAFANLRKVLISDSLDPCCRKI LQDGGLQVVEKQNLSKEELIAELQDCEGLIVRSATKVTADVINAAEKLQVVGRAGTGVDNV DLEAATRKGI LVMNTPNGNSLSAAELTCGMIMCLARQIPQATASMKDGKWERKKFMGTELNGKTLGILGLGRIGREVATRMQ SFGMKTIGYDPIISPEVSASFGVQQLPLEEIWPLCDFITVHTPLLPSTTGLLNDNTFAQCKKGVRVVNCARGGIVDEGALLRALQ SGQCAGAALDVFTEEPPRDRALVDHENVISCPH LGASTKEAQSRCGEEIAVQFVDMVKGKSLTGVVNAQALTSAFSPHTKPW IGLAEALGTLMRAWAGSPKGTIQVITQGTSLKNAGNCLSPAVIVGLLKEASKQADVNLVNAKLLVKEAGLNVTTSHSPAAPGE QGFGECLLAVALAGAPYQAVGLVQGTTPVLQGLNGAVFRPEVPLRRDLPLLLFRTQTSDPAMLPTMIGLLAEAGVRLLSYQTS LVSDGETWHVMGISSLLPSLEAWKQHVTEAFQFHF Forward primer (SEQ ID NO: 4): ATGGCCTTCGCAAATCTGC Reverse primer (SEQ ID NO: 5): AGTTCAGCTATCAGCTCCTCC.

Claims

1. Use of a PHGDH inhibitor or a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph or solvate thereof as an active substance in the preparation of a medicament for treating a subject having and / or suspected of having immune-escape disseminated tumor cells (immune-escape DTC).

2. The use according to claim 1, wherein The PHGDH inhibitor inhibits the expression of PHGDH or inhibits the activity of PHGDH; Wherein, the inhibition of PHGDH expression is to inhibit the transcription or translation of the PHGDH gene, including: preventing the PHGDH gene from being transcribed, or reducing the transcription activity of the PHGDH gene, or preventing the PHGDH gene from being translated, or reducing the translation level of the PHGDH gene; and / or The inhibition of PHGDH activity is to weaken the biological effect of PHGDH, including: inhibiting the synthesis of serine synthesis pathway and its downstream products, inhibiting the upregulation of H3K27me3 caused by the downstream pathway of PHGDH, etc.

3. The use according to claim 1, wherein The PHGDH inhibitor is a peptide, a nucleic acid molecule, an aptamer, a small molecule compound or a polysaccharide; For example, the nucleic acid molecules include antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA (siRNA), short hairpin RNA (shRNA), single guide RNA (sgRNA), or microRNA; and / or For example, the small molecule compound includes NCT-503, CBR-5884, disulfiram, galactone A, Zeste enhancer homolog 2 (EZH2) inhibitor or a small molecule compound having a molecular formula of C 20 H 23 A compound of F3N4S (CAS No. 1916571-90-8).

4. The use according to claim 4, wherein The Zeste enhancer homolog 2 (EZH2) inhibitor is selected from the group consisting of BIX-01294, chaetocin, CPI-169, CPI-905, CPI-360, CPI-209, CPI-1205, tazepam, EPZ005687, EPZ011989, 3-deazenplanocin A A, DZNep), EI1, GSK503, GSK126, GSK926, GSK343, JQEZ5, MC3629, OR-S0, OR-S1, PF-06821497, PF-06726304 acetate, SAH-EZH2, SHR2554, sinefungin, UNC1999, UNC2399, ZLD1039, EPZ-6438, CPI-169, CPI-1205, GSK-126, GSK343, and GSK503.

5. The use according to claim 2, wherein The immune escape DTC is a disseminated tumor cell (DTC) in which the PHGDH level in a sample obtained from a subject is higher than a reference level; Wherein, the PHGDH level is the gene expression level of PHGDH (such as the nucleic acid expression level or mRNA expression level of PHGDH), protein expression level and / or protein activity level; The PHGDH level in the sample obtained from the subject is the median value, mean value or standard value determined by statistical methods of the gene expression level, protein expression level and / or protein activity level measured in the sample obtained from the subject; and / or The reference level is (1) the median, mean or standard value determined by statistical methods of the gene expression level, protein expression level and / or protein activity level of PHGDH in a sample obtained from the subject at a previous time point; (2) the median, mean or standard value determined by statistical methods of the gene expression level, protein expression level and / or protein activity level of PHGDH in a reference population; or (3) a pre-specified PHGDH level, for example, a pre-specified immunohistochemistry (IHC) score value.

6. The use according to claim 2, wherein The subject is a mammal, such as a human, a non-human primate (e.g., an ape), a rodent (e.g., a rat, a mouse, a guinea pig), a pet (e.g., a cat, a dog), or a livestock (e.g., a horse, a cow, a sheep, a pig, a rabbit); and / or The subject is selected from the following groups: a subject suspected of having primary cancer; a subject having primary cancer but not yet receiving treatment for the primary cancer; a subject having primary cancer and having received treatment for the primary cancer; a subject having primary cancer and having received treatment for the primary cancer but not yet receiving treatment for cancer metastasis; A subject who has primary cancer and has received treatment for the primary cancer and preventive treatment for cancer metastasis; or a subject with one or more of the above conditions; preferably a subject suspected of metastasis and colonization based on the subject's medical history and imaging examination results (e.g., CT, MRI, etc.); and / or The sample is a tissue of the subject, such as a fresh sample, a frozen sample, a fixed sample (such as a formalin-fixed sample, a paraffin-embedded sample); preferably, it is a biopsy tissue suspected of metastasis and colonization based on the subject's previous medical history and imaging examination results (such as CT, MRI, etc.).

7. The use according to claim 1, wherein The subject has a primary cancer, for example, hepatocellular carcinoma, cholangiocarcinoma, prostate cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, gastric cancer, melanoma or cervical cancer; preferably hepatocellular carcinoma, breast cancer, colorectal cancer, lung adenocarcinoma; and / or The subject has a primary cancer and has and / or is at risk of cancer metastasis suspected to be caused by the primary cancer, e.g., metastasis to the lung (e.g., lung metastasis from liver cancer), bone, brain, or lymph node (e.g., lymph node metastasis from liver cancer) caused by the primary cancer.

8. The use according to claim 1, wherein The PHGDH inhibitor is contained in a pharmaceutical composition or a kit, for example, a pharmaceutical composition or a kit in a form suitable for administration by a method selected from the group consisting of oral administration, injection (e.g., direct naked DNA or protein injection, liposome-encapsulated DNA, RNA or protein injection), gold-coated gene gun bombardment, replication-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene encoded protein, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, transdermal administration; and / or The pharmaceutical composition or kit may further include other anti-tumor active ingredients or be used in combination with other anti-tumor active ingredients, for example, the other anti-tumor active ingredients are selected from: tumor immunotherapy agents (such as PD-1 antibodies, PD-L1 antibodies, adoptive cell therapy agents, antigen chimeric receptor T cell therapy agents), chemotherapy drugs; for example, the other anti-tumor active ingredients are selected from: ipilimumab and nivolumab.

9. A product for treating a subject having and / or suspected of having disseminated tumor cells (DTC), comprising (A) a therapeutically effective amount of a PHGDH inhibitor; (B) a pharmaceutically or immunologically acceptable carrier or excipient; (C) optionally, one or more additional cancer therapeutic agents.

10. Use of PHGDH and its biological effects as therapeutic targets for disseminated tumor cells (DTC) in drugs that inhibit the activation and proliferation of disseminated tumor cells (DTC).

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