Application of reagent targeting eIF2alpha / ATF4 pathway in treatment of hepatic fibrosis diseases

Abstract

The invention relates to the fields of biology, medicine and clinic. The invention specifically relates to application of a reagent targeting an eIF2alpha / ATF4 pathway in treatment of hepatic fibrosis diseases. Based on research of the inventor of the invention, on one hand, the invention discloses application of a reagent for targeted inhibition of an eIF2alpha / ATF4 pathway in preparation of drugs for treating hepatic fibrosis diseases, or application of the reagent in preparation of drugs for preventing liver cirrhosis and / or liver cancer; on the other hand, the invention discloses an application of the eIF2alpha / ATF4 pathway protein and / or the coding gene thereof in screening of drugs for treating hepatic fibrosis diseases, or an application in screening of drugs for preventing liver cirrhosis and / or liver cancer. Another aspect of the invention discloses a pharmaceutical composition comprising an agent for targeted inhibition of expression or activity of an eIF2alpha / ATF4 pathway protein; the invention has considerable application prospects in clinical treatment of hepatic fibrosis diseases.

Description

Technical Field

[0001] The present invention relates to the fields of biology, medicine, and clinical practice; specifically, it relates to the use of reagents targeting the eIF2α / ATF4 pathway in the treatment of liver fibrosis diseases. Background Art

[0002] Liver fibrosis is a common pathological outcome of liver injury caused by various etiologies, and its most prominent feature is the excessive accumulation of extracellular matrix (ECM). Liver metabolic syndromes (such as non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH)), alcohol abuse, and viral hepatitis are the main causes of liver fibrosis, all of which can cause hepatocyte damage and lead to acute or chronic liver injury. To cope with these injuries, the liver generates a large-scale inflammatory response to clear damaged hepatocytes. This response activates a population of "fibrogenic cells" resident in the liver to synthesize and secrete ECM proteins, and the progression of this process will lead to liver fibrosis and may further deteriorate into cirrhosis or primary hepatocellular carcinoma (HCC).

[0003] Compared with the cirrhosis and HCC stages, liver fibrosis is considered reversible because the liver structure remains relatively normal in the state of liver fibrosis and the liver retains its compensatory function (T. Kisseleva, D. Brenner, Molecular and cellular mechanisms of liver fibrosis and its regression. Nat Rev Gastroenterol Hepatol 18, 151-166 (2021)). Therefore, intervening in the progression of liver fibrosis before the occurrence of advanced liver diseases is clinically crucial and is also a key strategy for treating chronic liver injury diseases.

[0004] However, due to the lack of targeted therapies that can inhibit or reverse liver fibrosis, the most effective current treatment method is still to eliminate the etiologies that damage the liver.

[0005] Developing new strategies targeting liver fibrosis is very important, which requires an in-depth understanding of the mechanisms of this disease.

[0006] The most critical effector cells in liver fibrosis lesions are myofibroblasts that can secrete a large amount of ECM. So far, the academic community believes that the sources of liver myofibroblasts mainly include liver resident cells - hepatic stellate cells (HSC) and intrahepatic fibroblasts, as well as bone marrow-derived fibrocytes and mesenchymal stem cells, etc. Among them, HSC is considered the main source of liver myofibroblasts.

[0007] Hepatic stellate cells (HSCs) are a population of non-parenchymal cells resident in the liver and are the main fibrotic cell type generated in response to liver injury. In normal liver, HSCs maintain a non-proliferative quiescent state. After liver injury, HSCs are activated and transform from lipid-storing cells into myofibroblasts (or myofibroblast-like cells), which are proliferative, contractile, inflammatory, and most importantly, are characterized by enhanced ECM production (T. Higashi, S. L. Friedman, Y. Hoshida, Hepatic stellate cells as key target in liver fibrosis. Adv Drug Deliv Rev 121, 27-42 (2017)). Although a series of intracellular events and signaling pathways, including oxidative stress, autophagy, and metabolic reprogramming, can contribute to or mediate HSC activation in response to external triggers (e.g., TGFβ, PDGF, CTGF, Hedgehogs, etc.) (T. Tsuchida, S. L. Friedman, Mechanisms of hepatic stellate cell activation. Nat Rev Gastroenterol Hepatol 14, 397-411 (2017)), the master program regulating HSC activation remains unclear. Summary of the Invention

[0008] Based on the prior art theory, under classical stress conditions, ATF4 mainly regulates the transcription of UPR-related genes through TE.

[0009] However, the inventors of the present application unexpectedly found that under the conditions of inducing EMT, ATF4 tended to regulate the transcription of EMT-related genes by using super-enhancers; these results revealed different modes of action of ATF4 as a transcription factor and its non-classical regulatory mechanism under EMT conditions.

[0010] The inventors of the present application found that ATF4, as a major stress response factor, can directly promote the occurrence of EMT, thereby promoting the activation of HSCs.

[0011] In addition, the inventors of the present application used a combination of genetic intervention and chemical intervention to prove that inhibiting the eIF2α / ATF4 pathway can significantly alleviate the occurrence of liver fibrosis in multiple disease models, clarified the function of ATF4 in fibrotic diseases, and showed a new prospect of using ATF4 as an intervention target for treating liver fibrosis.

[0012] Accordingly, the first aspect of the present invention provides the use of a reagent for targeting and inhibiting the eIF2α / ATF4 pathway in the preparation of a drug for treating liver fibrosis diseases, or in the preparation of a drug for preventing liver cirrhosis and / or liver cancer, wherein the reagent is a reagent for targeting and inhibiting the expression or activity of proteins in the eIF2α / ATF4 pathway.

[0013] In some preferred embodiments of the present application, the reagent is a reagent for targeting the eIF2α / ATF4 pathway in hepatic stellate cells.

[0014] In some specific embodiments of the present application, the reagent is selected from any one of the following or a combination thereof: a reagent targeting ATF4, a reagent targeting p-eIF2-alpha. In some specific embodiments of the present application, there is provided the use of "ATF4 and / or p-eIF2-alpha" as a target in the preparation of a drug for treating liver fibrosis diseases, or in the preparation of a drug for preventing liver cirrhosis and / or liver cancer.

[0015] In some specific embodiments of the present application, the reagent targeting ATF4 refers to a reagent capable of regulating the expression level or activity of ATF4 or its expression product at the nucleic acid level or at the protein level; the "regulation" includes inhibition, reduction, inactivation, knockout, knockdown, or a combination thereof; optionally, the reagent targeting ATF4 includes a reagent for inhibiting, reducing, knocking out, knocking down, or knocking down the expression level of the ATF4 gene, or a reagent for inhibiting, reducing, or inactivating the activity of the expression product of the ATF4 gene.

[0016] In some specific embodiments of the present application, the reagent targeting ATF4 can selectively or specifically recognize and act on the ATF4 gene or its expression product; alternatively, the reagent targeting ATF4 can reduce the level or activity of the ATF4 gene or its expression product; alternatively, the reagent targeting ATF4 can knockout the level or activity of the ATF4 gene or its expression product; alternatively, the reagent targeting ATF4 can knockdown or knock down the level or activity of the ATF4 gene or its expression product; the reagent targeting ATF4 can inactivate the ATF4 gene or its expression product.

[0017] In some specific embodiments of the present application, the reagent targeting p-eIF2-alpha refers to a reagent that can regulate the expression level or activity of the EIF2S1 gene or its expression product at the nucleic acid level or at the protein level. The "regulation" includes inhibition, reduction, inactivation, knockout, knockdown, or a combination thereof; optionally, the reagent targeting p-eIF2-alpha is a reagent that inhibits, reduces, knocks out, knockdowns, or knocks down the expression level of the EIF2S1 gene, or inhibits, reduces, or inactivates the activity of the expression product of the EIF2S1 gene, or a reagent that inhibits, reduces, or inactivates the p-eIF2-alpha protein.

[0018] The p-eIF2-alpha protein is the product after phosphorylation modification of the eIF2-alpha protein; the eIF2-alpha protein is the expression product of the EIF2S1 gene.

[0019] In some specific embodiments of the present application, the reagent targeting p-eIF2-alpha can selectively or specifically recognize and act on the EIF2S1 gene or its expression product; or, the reagent targeting p-eIF2-alpha can reduce the level or activity of the EIF2S1 gene or its expression product; or, the reagent targeting p-eIF2-alpha can knockout the level or activity of the EIF2S1 gene or its expression product; or, the reagent targeting p-eIF2-alpha can knockdown or knock down the level or activity of the EIF2S1 gene or its expression product; the reagent targeting p-eIF2-alpha can inactivate the EIF2S1 gene or its expression product.

[0020] In some specific embodiments of the present application, the reagent targeting p-eIF2-alpha includes a reagent that inhibits, reduces, or inactivates the p-eIF2-alpha protein. The reagent targeting p-eIF2-alpha includes a reagent that inhibits, reduces, or inactivates the p-eIF2-alpha protein in a direct manner, such as a dephosphorylation reagent for the p-eIF2-alpha protein. The reagent targeting p-eIF2-alpha also includes a reagent that inhibits, reduces, or inactivates the p-eIF2-alpha protein in an indirect manner, such as a reagent that can activate another protein eIF2B related to p-eIF2-alpha, and a reagent that indirectly inhibits p-eIF2alpha by bypassing the function of p-eIF2alpha.

[0021] In some specific embodiments, the expression product of the ATF4 gene or the EIF2S1 gene refers to molecules in various forms of the ATF4 gene or the EIF2S1 gene at various stages, such as but not limited to molecules produced during amplification, replication, transcription, splicing, processing, translation, and modification of the ATF4 gene or the EIF2S1 gene, such as cDNA, mRNA, non-coding RNA, precursor protein, mature protein, and their fragments, or modified products of the protein.

[0022] In some preferred embodiments, the modified product of the protein includes the phosphorylated product of the protein. For example, the p-eIF2-alpha protein is the product of the phosphorylated modification of the expression product (eIF2-alpha protein) of the EIF2S1 gene. Therefore, the p-eIF2-alpha protein is also included in the expression product of the EIF2S1 gene.

[0023] In some specific embodiments, the "level" of the above-mentioned gene or its expression product can be at the protein level or at the nucleic acid level. Among them, the protein level can be achieved by inhibiting protein function with a specific inhibitor (such as an antibody or its antigen-binding fragment). Among them, the inhibition at the nucleic acid level can be accomplished by preventing mRNA from being translated into protein.

[0024] In some specific embodiments, the reagent targeting ATF4 or the reagent targeting p-eIF2-alpha is selected from: small molecule inhibitors, carbohydrates, lipids, proteins, polypeptides, nucleic acid molecules, or interfering viruses. Among them, the nucleic acid is selected from antisense oligonucleotides, siRNA, shRNA, dsRNA, microRNA, LncRNA, sgRNA, esiRNA, etc. Among them, the protein or polypeptide is selected from antibodies or their antigen-binding fragments. Among them, the antibody is selected from: chimeric antibodies, fully human antibodies, humanized antibodies; among them, the antigen-binding fragment includes but not limited to: Fab, Fab’, F(ab’)2, Fv fragment, ScFv, single-chain antibody, or domain antibody.

[0025] In some specific embodiments, the reagent targeting "ATF4" or the reagent targeting ATF4 refers to using the ATF4 gene or its expression product as a target to regulate the level or activity of the ATF4 gene or its expression product in hepatic stellate cells by the above-mentioned "regulation" method.

[0026] In some specific embodiments, the reagent targeting "p-eIF2-alpha" or the reagent targeting p-eIF2-alpha refers to using the EIF2S1 gene or its expression product as a target to regulate the level or activity of the EIF2S1 gene or its expression product in hepatic stellate cells by the above-mentioned "regulation" method.

[0027] In a specific embodiment of the present application, the reagent targeting p-eIF2-alpha is ISRIB.

[0028] In some specific embodiments, the reagent targeting ATF4 or the reagent targeting p-eIF2-alpha is selected from any one of the following:

[0029] a) siRNA, shRNA, dsRNA, microRNA, LncRNA, sgRNA or esiRNA that targets the ATF4 or EIF2S1 gene and knocks out, knockdowns or reduces the expression of the ATF4 or EIF2S1 gene;

[0030] b) An expression vector, the expression vector contains a gene fragment encoding the item a), and the expression vector can express the item a);

[0031] c) A recombinant viral vector, the recombinant viral vector is formed by cloning the gene fragment encoding the item a) into the coding region of the viral vector; preferably, the viral vector is any one of a lentiviral vector, an adeno-associated viral vector or a retroviral vector;

[0032] d) A virus, the virus is obtained by transfecting a eukaryotic cell with a virus packaging system; the virus packaging system contains the recombinant viral vector of the item c).

[0033] In some embodiments, the interfering nucleic acid molecule of the above item a) (siRNA, shRNA, dsRNA, microRNA, LncRNA, sgRNA or esiRNA that targets the ATF4 or EIF2S1 gene and knocks out, knockdowns or reduces the expression of the ATF4 or EIF2S1 gene) can be designed and verified for its effect by using existing recognized technical methods.

[0034] In some embodiments, the expression vector of the above item b) is the expression vector of the interfering nucleic acid molecule of the above item a), and is obtained by cloning the gene fragment encoding the interfering nucleic acid molecule of the item a) into a known vector.

[0035] In some preferred embodiments, the expression vector of the interfering nucleic acid molecule of the above item a) is the recombinant viral vector of the above item c), and is formed by cloning the gene fragment encoding the interfering nucleic acid molecule of the above item a) into the coding region of the viral vector; preferably, the viral vector is any one of a lentiviral vector, an adeno-associated viral vector or a retroviral vector.

[0036] Preferably, the recombinant viral vector in item c) above becomes an infectious viral particle after viral packaging, infects cells, and then undergoes transcription. Subsequently, through steps such as enzymatic cleavage and processing in the cells, it becomes siRNA, ultimately achieving specific knockdown of the expression of the ATF4 or EIF2S1 gene.

[0037] More preferably, the expression vector in item b) and the recombinant viral vector in item c) further contain a promoter sequence and / or a nucleotide sequence encoding a marker that can be detected in cells; the detectable marker, for example, is green fluorescent protein (GFP).

[0038] More preferably, the recombinant viral vector in item c) is a lentiviral vector obtained by inserting the gene fragment encoding the interfering nucleic acid molecule in item a) above into the coding region between the two ITR sequences of the lentiviral vector. Preferably, the "lentiviral vector" can be pLKO, pSIH1, pLL3.7, pLenti, pGIPZ, etc.

[0039] More preferably, the recombinant viral vector in item c) is a recombinant adeno-associated viral vector obtained by inserting the gene fragment encoding the interfering nucleic acid molecule in item a) above into the coding region between the two ITR sequences of the adeno-associated viral vector.

[0040] The "adeno-associated viral vector" can be serotype AAV1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or can also be chimeric AAVs derived from these serotypes, such as AAV2-AAV3, AAVrh.10, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAVHSC15, AAV-HSC17, AAVhu.37, AAVrh.8, etc.

[0041] During transfection, AAV only causes a mild immune response in the host. In a preferred embodiment of the present invention, the adeno-associated viral vector is a serotype AAV2 or 8 or 9 vector.

[0042] In some preferred embodiments, an adeno-associated viral packaging system is used to obtain the virus in item d) above; the adeno-associated viral packaging system includes the recombinant adeno-associated viral vector containing the interfering nucleic acid of the ATF4 or EIF2S1 gene, the packaging plasmid of the adeno-associated virus, and the helper plasmid of the adeno-associated virus.

[0043] In a preferred embodiment of the present invention, the virus packaging system adopts an adeno-associated virus packaging system of a three-plasmid system, which includes a packaging plasmid pAAV-RC (containing the AAV2 capsid protein gene), a helper plasmid pHelper (containing genes that can assist in AAV replication), and the above-mentioned recombinant adeno-associated virus vector containing the ATF4 or EIF2S1 gene interfering nucleic acid. The adeno-associated virus packaging system transfects eukaryotic cells, and adeno-associated virus is obtained through virus packaging. This adeno-associated virus ultimately realizes specific knockdown of the expression of the ATF4 or EIF2S1 gene by infecting cells.

[0044] In a preferred embodiment of the present invention, the virus packaging system is a lentivirus packaging system, which includes the above-mentioned recombinant lentivirus vector containing the interfering nucleic acid molecule of item a) above, the packaging plasmid of lentivirus, and the helper plasmid.

[0045] In a preferred embodiment of the present invention, the virus packaging system adopts a lentivirus packaging system of a three-plasmid system, which includes a packaging plasmid pSPAX2, a helper plasmid pMD2.G, and the above-mentioned recombinant lentivirus vector containing the interfering nucleic acid molecule of item a) above. The lentivirus packaging system transfects eukaryotic cells, and lentivirus is obtained through virus packaging. This lentivirus ultimately realizes specific knockdown of the expression of the ATF4 or EIF2S1 gene by infecting cells.

[0046] In the second aspect of the present invention, there is provided the use of eIF2α / ATF4 pathway proteins and / or their coding genes in screening drugs for treating liver fibrosis diseases, or in screening drugs for preventing liver cirrhosis and / or liver cancer.

[0047] In some preferred embodiments of the present invention, the eIF2α / ATF4 pathway proteins are selected from ATF4 or p-eIF2-alpha.

[0048] Taking eIF2α / ATF4 pathway proteins and / or their coding genes (including the ATF4 gene and the EIF2S1 gene) as the objects / targets of action, drugs are screened to find drugs that can inhibit the expression of the ATF4 gene and / or the EIF2S1 gene as candidate drugs for treating liver fibrosis diseases, or as candidate drugs for preventing liver cirrhosis and / or liver cancer. The candidate drugs obtained by screening can be selected from, but are not limited to: small molecule inhibitors, carbohydrates, lipids, proteins, polypeptides, nucleic acid molecules, or interfering viruses.

[0049] The third aspect of the present invention provides a pharmaceutical composition, which includes an active ingredient and a pharmaceutically acceptable carrier or excipient, and the active ingredient includes a reagent that targets and inhibits the expression or activity of eIF2α / ATF4 pathway proteins.

[0050] In some embodiments of the present application, the reagent is a reagent targeting the eIF2α / ATF4 pathway in hepatic stellate cells.

[0051] In some preferred embodiments of the present application, the reagent is selected from any one of the following or a combination thereof: a reagent targeting ATF4, a reagent targeting p-eIF2-alpha;

[0052] Among them, the reagent targeting ATF4 is a reagent that inhibits, reduces, knocks out, knockdown or downregulates the expression level of the ATF4 gene, or inhibits, reduces or inactivates the activity of the expression product of the ATF4 gene; among them, the reagent targeting p-eIF2-alpha is a reagent that inhibits, reduces, knocks out, knockdown or downregulates the expression level of the EIF2S1 gene, or inhibits, reduces or inactivates the activity of the expression product of the EIF2S1 gene, or inhibits, reduces or inactivates the p-eIF2-alpha protein; among them, the expression level is at the nucleic acid level or the protein level.

[0053] In some preferred embodiments of the present application, the expression product is selected from any one of the following or a combination thereof: cDNA, mRNA, non-coding RNA, precursor protein, mature protein or its fragment, or a modified product of the protein; preferably, the modified product of the protein includes a phosphorylated product of the protein.

[0054] In some preferred embodiments of the present application, the reagent is selected from small molecule inhibitors, carbohydrates, lipids, proteins, polypeptides, nucleic acid molecules or interfering viruses.

[0055] In some more preferred embodiments of the present application, the reagent is selected from any one of the following: small molecule inhibitors, antisense oligonucleotides, siRNA, shRNA, dsRNA, microRNA, LncRNA, sgRNA, esiRNA, antibodies or antigen-binding fragments thereof; more preferably, the antigen-binding fragments are selected from any one of the following or a combination thereof: Fab, Fab’, F(ab’)2, Fv fragments, ScFv, single-chain antibodies or domain antibodies.

[0056] In some preferred embodiments of the present application, the reagent in the pharmaceutical composition comprises any one of the following:

[0057] a) siRNA, shRNA, dsRNA, microRNA, LncRNA, sgRNA or esiRNA that targets the ATF4 or EIF2S1 gene and knocks out, knockdown or downregulates the expression of the ATF4 or EIF2S1 gene;

[0058] b) An expression vector, which contains the gene fragment encoding the item a), and the expression vector is capable of expressing the item a);

[0059] c) A recombinant viral vector, which is formed by cloning the gene fragment encoding the item a) into the coding region of a viral vector; preferably, the viral vector is any one of a lentiviral vector, an adeno-associated viral vector or a retroviral vector;

[0060] d) A virus, which is obtained by transfecting a eukaryotic cell with a virus packaging system; the virus packaging system contains the recombinant viral vector of the item c).

[0061] In some embodiments of the present application, when preparing / formulating a pharmaceutical composition, the active ingredient is usually mixed with an excipient, or diluted with an excipient, or encapsulated in a pharmaceutical carrier. The pharmaceutical composition can be in an injection form, tablet form, pill form, powder form, capsule form or oral liquid form (such as syrup, etc.). Preferably, the injection form is adopted.

[0062] The above-mentioned pharmaceutical composition can be a candidate drug for treating liver fibrosis diseases, or for preventing liver cirrhosis and / or liver cancer, including administering an effective dose of the pharmaceutical composition to a subject.

[0063] The subject is selected from: human, mouse, rat, guinea pig, rabbit, horse, monkey, dog.

[0064] It should be understood that within the protection scope of the present invention, the above-mentioned various technical features of the present invention and the various technical features specifically described in the following examples can be combined with each other, so as to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Brief Description of the Drawings

[0065] Figure 1 It is demonstrated that ATF4 regulates genomic expression in a stimulus-dependent manner; Figure 1 a- Figure 1 For the specific explanations of f, please refer to

[0066] Figure 2 It is demonstrated that ATF4 regulates classical stress response genes under ER stress or amino acid deprivation conditions; Figure 2 a- Figure 2 For the specific explanations of e, please refer to the specific implementation manners;

[0067] Figure 3 It is demonstrated that ATF4 is essential for EMT induction in epithelial cells; Figure 3 a- Figure 3 For the specific explanations of d, please refer to the specific implementation manners;

[0068] Figure 4It is demonstrated that ATF4 positively regulates the EMT program in epithelial and mesenchymal cells; Figure 4 a- Figure 4 For the specific explanation of i, please refer to the specific implementation manner;

[0069] Figure 5 It is demonstrated that ATF4 positively regulates the EMT program in HSCs and is essential for HSC activation; Figure 5 a- Figure 5 For the specific explanation of h, please refer to the specific implementation manner;

[0070] Figure 6 It is demonstrated that HSC-specific knockout of Atf4 can alleviate liver fibrosis symptoms in mice; Figure 6 For the specific explanation of a-6f, please refer to the specific implementation manner;

[0071] Figure 7 It is verified that the expression of ATF4 in mouse HSCs is reduced after HSC-specific knockout of Atf4; Figure 7 For the specific explanation of a-7g, please refer to the specific implementation manner;

[0072] Figure 8 It shows the binding of ATF4 and H3K27ac to chromatin during HSC activation and ER stress; Figure 8 a- Figure 8 For the specific explanation of f, please refer to the specific implementation manner;

[0073] Figure 9 It shows the distribution of the binding regions of ATF4 and H3K27ac to chromatin under different conditions; Figure 9 a- Figure 9 For the specific explanation of f, please refer to the specific implementation manner;

[0074] Figure 10 It is demonstrated that ATF4 promotes the transcription of liver fibrosis genes through an enhancer-dependent mechanism; Figure 10 For the specific explanation of a-10d, please refer to the specific implementation manner;

[0075] Figure 11 It is demonstrated that pharmacological inhibition of the p-eIF2-alpha / ATF4 pathway can alleviate the progression of liver fibrosis in mice; Figure 11 a- Figure 11 For the specific explanation of l, please refer to the specific implementation manner;

[0076] Figure 12 It is demonstrated that pharmacological inhibition of the p-eIF2-alpha / ATF4 pathway can alleviate the progression of liver fibrosis in mice; Figure 12 a- Figure 12 For the specific explanation of g, please refer to the specific implementation manner;

[0077] Figure 13 It is demonstrated that pharmacological inhibition of p-eIF2-alpha / ATF4 can alleviate the symptoms of liver fibrosis induced by BDL or TAA; Figure 13 a- Figure 13 For the specific explanations of a-f, please refer to the specific implementation manners;

[0078] Figure 14 It shows the clinical relevance of ATF4 in HSCs to human liver fibrosis diseases; Figure 14 a- Figure 14 For the specific explanations of a-e, please refer to the specific implementation manners. Specific implementation manners

[0080] Previous studies have shown that epithelial-mesenchymal transition (EMT) plays an important role in fibrosis of various tissues, including the lung, kidney, and liver. The inventors of this application had an interesting discovery during the literature research: there are some common features between EMT and the activation of hepatic stellate cells (HSCs). For example, they both belong to the process of transdifferentiation. EMT allows epithelial cells to lose their original characteristics and acquire a mesenchymal phenotype, while the activation of HSCs essentially involves the transformation from quiescent, non-sinusoidal cells into mesenchymally differentiated myofibroblasts. In addition, the main result of HSC activation is the accumulation of extracellular matrix (ECM) proteins, which is exactly the typical product of EMT (T. Tsuchida, S. L. Friedman, Mechanisms of hepatic stellate cell activation. Nat Rev Gastroenterol Hepatol 14, 397-411 (2017)).

[0081] Although there are some commonalities between EMT and the activation of hepatic stellate cells (HSCs) from the perspective of literature analysis, it is not clear whether there is an interaction at the molecular level between the two (V. Masola, A. Carraro, S. Granata, L. Signorini, G. Bellin, P. Violi, A. Lupo, U. Tedeschi, M. Onisto, G. Gambaro, G. Zaza, In vitro effects of interleukin (IL)-1beta inhibition on the epithelial-to-mesenchymal transition (EMT) of renal tubular and hepatic stellate cells. J Transl Med 17, 12 (2019)).

[0082] The inventors of the present application attempted to conduct research: Can EMT regulate the activation of HSCs? If EMT can regulate the activation of HSCs, then how is it regulated? What is the mechanism behind this? The technology behind this may reveal new therapeutic opportunities.

[0083] Based on the inventors' previous research and literature research, it is known that after cancer cells undergo epithelial-mesenchymal transition (EMT) (hereinafter simply referred to as EMT cancer cells), they will exhibit an increased synthesis and secretion of extracellular matrix (ECM) proteins, which is similar to activated hepatic stellate cells (HSCs). To cope with the excessive endoplasmic reticulum burden caused by ECM secretion, that is, endoplasmic reticulum stress (ER stress), EMT cancer cells activate the PERK branch of the Unfolded Protein Response (UPR) pathway (Y.X. Feng, E.S. Sokol, C.A. Del Vecchio, S. Sanduja, J.H. Claessen, T.A. Proia, D.X. Jin, F. Reinhardt, H.L. Ploegh, Q. Wang, P.B. Gupta, Epithelial-to-mesenchymal transition activates PERK-eIF2αlpha and sensitizes cells to endoplasmic reticulum stress. Cancer Discov 4, 702-715 (2014)). Once activated, PERK phosphorylates eIF2α, and subsequently increases the translation of ATF4, which either promotes cell survival by regulating protein synthesis and folding or induces apoptosis when necessary (M. Schroder, R.J. Kaufman, The mammalian unfolded protein response. Annu Rev Biochem 74, 739-789 (2005)). In addition to its role in ER homeostasis, the activation of the PERK-eIF2α / ATF4 pathway is essential for cancer metastasis and drug resistance, two characteristics that are hallmarks of EMT cancer cells.

[0084] However, in a non-cancerous environment, there is no relevant research report on whether the PERK pathway, especially its downstream transcription factor ATF4, can regulate cell transdifferentiation and cell fate by controlling the EMT program.

[0085] The inventors of the present application unexpectedly found that under the conditions of inducing EMT, ATF4 tended to regulate the transcription of EMT-related genes by utilizing super-enhancers; these results revealed different modes of action of ATF4 as a transcription factor and its non-classical regulatory mechanism under EMT conditions.

[0086] The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited to the scope of the described embodiments. The process parameters not specified in the embodiments of the present application can be carried out according to conventional methods, and the raw materials used can be obtained through commercial channels.

[0087] Embodiment

[0088] 1. Design and screening of interfering sequences targeting the ATF4 gene

[0089] The mRNA sequence of the coding region (CDS region) of the human ATF4 gene was obtained by querying from NCBI (see https: / / www.ncbi.nlm.nih.gov / nuccore / NM_001675.4), and multiple suitable target sequences were selected using software.

[0090] The inventors designed siRNA interfering sequences targeting the human ATF4 gene based on the above target sequences, and through verification of the knockdown effect at the cellular level, finally screened the following 3 siRNA (siRNA-ATF4-1 to 3#) sequences, see Table 1 below:

[0091] Table 1

[0092]

[0093] The inventors designed appropriate stem-loop fragment sequences for the above 3 siRNA-ATF4-1 to 3# (in this embodiment, the DNA sequence corresponding to the stem-loop fragment is: CTCGAG), and thus obtained 3 corresponding shRNA (shRNA-ATF4-1 to 3#).

[0094] The sense strands of shRNA-ATF4-1 to 3# are the sense strands of the corresponding siRNA-ATF4-1 to 3#, and the antisense strands of shRNA-ATF4-1 to 3# are the antisense strands of the corresponding siRNA-ATF4-1 to 3#, which will not be elaborated here.

[0095] The inventors designed primer sequences for amplifying the DNA fragments corresponding to shRNA-ATF4-1 to 3#, see Table 2 below, sent them to Hangzhou Youkang Biotechnology Sequencing Company for artificial synthesis, and then annealed the synthesized single-strand fragments to form double-stranded structures.

[0096] Table 2

[0097]

[0098] 2. Construct the recombinant plasmid (pLKO.1-shATF4-puro / blast) for knocking down the ATF4 gene

[0099] The empty vector uses pLKO.1-puro / pLKO.1-blast (lentiviral vector), and the empty vector is digested with enzymes (AgeⅠ-HF endonuclease and EcoRⅠ-HF endonuclease).

[0100] Ligate the annealing products of shRNA-ATF4-1 to 3# obtained above with the digested empty vector (ligated by T4 ligase), transform the ligation products into the competent cell Escherichia coli Stbl3, select monoclonal colonies, amplify the bacteria by LB, and send them for sequencing to identify the inserted fragments.

[0101] The amplification, extraction, and purification of the recombinant plasmid with correct sequencing alignment are all carried out using conventional experimental operations, which will not be elaborated here.

[0102] The inventor verified the knockdown effects of multiple designed shRNA-ATF4 at the cellular level. Among them, the recombinant plasmids of shRNA-ATF4-1 to 3# had the best knockdown effect, which were named the knockdown plasmids ATF4-1 to 3# for specifically knocking down the human ATF4 gene.

[0103] 3. Construct the pGL3-promoter-COL1A1-SE reporter plasmid

[0104] Design the primer sequences (forward and reverse primer sequences are shown in SEQ ID NO: 12 and 13 respectively) for the co-binding region of ATF4 and the super-enhancer (hereinafter referred to as: COL1A1-SE fragment) on COL1A1.

[0105] Primer design method: First, use the IGV software to visualize the ChIP-Seq data, combine the results of peak calling, and record the positions of the entire super-enhancer on the COL1A1 gene and the binding positions of ATF4 within the super-enhancer range. Subsequently, search for the specific sequences of these positions through Pubmed-Gene. The selection of the COL1A1-SE fragment includes the co-binding sites of the COL1A1 super-enhancer and ATF4, with a length between 1 - 2 kb. At the same time, a negative control COL1A1-SE fragment (named COL1A1-SE-Pn) is selected, and its position is in the non-COL1A1-SE and non-ATF4 binding regions. Since the connection between the fragment and the vector uses homologous recombination, homologous sequences on the vector are added to the primers of the fragment. The primers are ordered and synthesized by Hangzhou Youkang Biotechnology Sequencing Company.

[0106] The genomic DNA of LX_2 cells (immortalized human hepatic stellate cell HSC cell line) was extracted using a genomic DNA extraction kit (Tiangen). The operation steps were strictly in accordance with the instructions. The concentration and purity of the product DNA were detected and stored at -20 °C for later use.

[0107] Using the genomic DNA of LX_2 cells as a template, a PCR reaction was performed using the KOD FX high-fidelity DNA polymerase kit (TOYOBO), and the COL1A1-SE fragment was amplified using the primers designed above.

[0108] The pGL3-promoter vector was digested with NheI-HF enzyme.

[0109] The COL1A1-SE fragment obtained by the above amplification and the digested pGL3-promoter vector were ligated by homologous recombination using a homologous recombination kit (Tiangen). The ligation product was transformed into competent Escherichia coli Stbl3 cells, and monoclonal colonies were selected. After amplifying the bacteria in LB, they were sent for sequencing to identify the inserted fragment.

[0110] The amplification, extraction, and purification of the recombinant plasmid with correct sequencing alignment were all carried out using conventional experimental operations, which will not be elaborated here. The expression intensity of the luciferase gene was verified in LX_2 cells.

[0111] 4. Preparation of lentivirus containing the above knockdown plasmid

[0112] The lentivirus packaging system used in this example includes two packaging helper plasmids, pSPAX2 and pMD2.G, and the above knockdown plasmids ATF4-1 to 3#.

[0113] The lentivirus packaging system was co-transfected into 293T cells to prepare a lentivirus solution containing the above knockdown plasmid. Specifically, 100 μl of OPTI-MEM medium, 750 ng of the core plasmid (knockdown plasmids ATF4-1 to 3#), packaging plasmids (562.5 ng of psPAX2, 187.5 ng of pMD.2G), and 3 μl of transfection reagent (Lipo8000) were added to a 1.5 ml centrifuge tube. They were thoroughly mixed with a pipette without incubation, and then evenly added to the culture plate in the form of drops. After gently shaking, the cells were continued to be cultured. The fresh medium of 293T cells was replaced 8 hours after transfection. Subsequently, the supernatant of 293T cells at 24 hours, 48 hours, and 72 hours after transfection was collected in sequence. The supernatant was filtered through a 0.45 μm filter membrane to obtain the lentivirus solution of this example, that is, the lentivirus solution containing the knockdown plasmids ATF4-1 to 3#, or expressed as: the lentivirus solution expressing shRNA-ATF4-1 to 3#.

[0114] The control group was a lentiviral solution of the pLKO.1-puro / pLKO.1-blast vector containing scramble shRNA.

[0115] Regarding the preparation of lentivirus, as well as the determination of the titer and purity of the obtained lentiviral solution, and the identification of integrity, etc., all were carried out by the inventor's laboratory according to routine experimental operations and will not be elaborated here.

[0116] 5. Construction of mice with specific knockout of Atf4 in hepatic stellate cells

[0117] The inventor team of this application established a mouse model Lrat-Cre; Atf4 with specific knockout of the Atf4 gene in hepatic stellate cells (HSC). fl / fl .

[0118] See Figure 6 a, the design schematic diagram of Lrat-Cre+ / -; Atf4 fl / fl (HSC-Atf4-KO) mice.

[0119] Lrat-cre + / - mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., and Atf4 loxp / WT mice were donated by the laboratory of Teacher Chen Jinghai of Zhejiang University. These two types of mice were crossed to obtain homozygous mice with specific knockdown of ATF4 in HSC, Atf4 loxp / loxp , Lrat-cre + / - (hereinafter referred to as Lrat-Cre; Atf4 fl / fl ) and control mice Atf4 loxp / loxp , Lrat-cre - / - (hereinafter referred to as Atf4 fl / fl ).

[0120] 1) Regarding the identification of mouse genotypes: Extract mouse tail DNA; identify the genotypes of Lrat-cre mice and Atf4 flox mice by PCR.

[0121] The identification primers for Lrat-cre mice and Atf4 flox mice are shown in Table 3 below; if differentiating between heterozygous and wild-type mice, only the "PCR No. ① 5'arm" reaction is required. If differentiating between homozygous and heterozygous mice, both the "PCR No. ① 5'arm" reaction and the "PCR No. ② WT" reaction are required to more accurately distinguish the genotypes of the mice.

[0122] Table 3

[0123]

[0124] In the previously constructed mouse model Lrat-Cre; Atf4 fl / fl the Cre expression is driven by the lecithin retinol acyltransferase (Lrat) promoter, ensuring specific knockout of the Atf4 gene in HSCs. 2)

[0126] In this model, to confirm the deletion of Atf4, we detected the expression of Atf4 in primary HSCs isolated from Atf4 fl / fl (WT) and Lrat-Cre; Atf4 fl / fl (HSC-Atf4-KO) mice.

[0127] See Figure 7 a. The morphology and lipid droplets of isolated HSCs were detected under a phase-contrast microscope, and the autofluorescence was detected by a fluorescence microscope under ultraviolet excitation light at a wavelength of 405 nm. Figure 7 a shows representative images and indicates a very high purity of the isolated HSCs.

[0128] See Figure 7 b. Results of the genotype identification experiment; representative images of DNA electrophoresis of PCR products.

[0129] See Figure 7 c. qPCR analysis of Lrat and Atf4 mRNA expression in HSCs and non-HSCs isolated from WT and HSC-Atf4-KO mice.

[0130] See Figure 7 d. Western blot showing the expression of ATF4 in HSCs and non-HSCs isolated from WT and HSC-Atf4-KO mice.

[0131] See Figure 7 e. Western blot showing the expression of ATF4 in four tissues (including liver, spleen, white adipose tissue (WAT), and skin) isolated from WT and HSC-Atf4-KO mice.

[0132] As described above Figure 7 from the results of a-7e, the expression of Atf4 was significantly reduced in Atf4-deficient HSCs, while other tissues were not affected.

[0133] Effect data

[0134] I. ATF4 regulates gene expression in a stimulus signal-dependent manner

[0135] According to the literature reports in this field,

[0136] EMT can be divided into three types; the EMT that occurs during embryonic formation, organ development, and organ transplantation is called type I EMT. Type II EMT mainly occurs during tissue injury repair and organ fibrosis. Type II EMT induced by inflammatory responses can promote tissue repair in the short term, but long-term chronic inflammation will lead to fibrosis. Type III EMT specifically refers to the changes in cell characteristics during tumorigenesis. During tumor progression, cancer cells will undergo some new molecular events based on gene mutations and epigenetic changes, leading to the activation of the EMT program, thereby obtaining stronger invasion and metastasis abilities. At the same time, tumor cells can also acquire the characteristics of stem cells.

[0137] Although previous studies have found that ATF4 may be involved in the regulation of type I EMT, the functions and specific mechanisms of ATF4 in type II and type III EMT are not clear.

[0138] 1. Using the immortalized mammary epithelial cell line HMLE overexpressing Twist_ER as the research model

[0139] We first used the classic EMT research model - the immortalized mammary epithelial cell line HMLE overexpressing Twist_ER (HMLE_Twist_ER) to explore the regulation of ATF4 on type III EMT.

[0140] Twist is one of the key transcription factors driving EMT, triggering EMT by inhibiting the transcription of CDH1 (the protein product is E-Cadherin). We stably knocked down ATF4 in HMLE_Twist_ER cells using the lentiviral solution containing the knockdown plasmids ATF4-1 to 3# prepared in Example 1 of this application. 1)

[0142] Obtaining a cell line stably transfected with lentivirus

[0143] The HMLE_Twist_ER mammary epithelial cells overexpressing Twist used in this laboratory are derived from the laboratory of Dr. Robert A. Weinberg; Resuscitate and seed the HMLE_Twist_ER cells in a 6-well plate. After the cells adhere to the wall, take out the packaged lentivirus (the lentivirus solution containing the knockdown plasmids ATF4-1 to 3# prepared in Example 1, the control lentivirus solution) and place it on ice to melt. Replace the fresh medium for the cells, and then add 400 μl of the lentivirus solution (leave one well of cells without adding the lentivirus solution as the screening control well). After shaking evenly, continue to culture in the incubator. Replace the fresh medium for the cells again after 24 hours. After 48 hours, according to the screening resistance of the core plasmid of the infected virus, replace the fresh medium containing the corresponding screening drug (puromycin or blasticidin). After about 2 - 3 days, the uninfected cells begin to die one after another. During this period, replace the fresh medium containing the screening drug for the cells every 1 - 2 days. After all the cells in the screening control well are dead (about 5 days for puromycin, about 10 - 14 days for blasticidin), the drug can be added for 1 - 2 days to screen and maintain the infected cells. Culture and expand the surviving cells in the infected group, and detect the expression of the corresponding protein as soon as possible to determine whether the construction of the stable transfected cell line is successful. Then freeze the cells and conduct subsequent experiments.

[0144] Experimental group HMLE (labeled as: shATF4 in the figure): HMLE_Twist_ER cells stably transfected with the lentivirus in Example 1 (the lentivirus containing the knockdown plasmids ATF4-#1 and #2);

[0145] Control group HMLE (labeled as: shCtrl in the figure): HMLE_Twist_ER cells stably transfected with the control lentivirus (the lentivirus containing the empty vector);

[0146] The HMLE_Twist_ER cells stably express Twist fused with ER (Twist-overexpression, abbreviated as Twist-OE), and induce EMT after treatment with 4-Hydroxytamoxifen (4-OHT).

[0147] In previous studies, ATF4 is usually a downstream pathway molecule in the ER stress process, regulating the expression of a series of stress factors to relieve ER stress or inducing apoptosis in cells with excessive damage to maintain the body's homeostasis. ATF4 has also been found to enhance the utilization rate of amino acids by regulating the expression of related genes during amino acid deprivation (AA-Dep). To study the mode of action of ATF4 in different pathophysiological processes, we used different stimulation methods, including control (DMSO), EMT (Twist-OE), ER stress (using its common inducer Thapsigargin, TG), and amino acid deprivation (AA-Dep) to treat cells, and detected the gene transcriptome affected by ATF4 knockdown under these conditions respectively.

[0148] 2) Conclusions

[0149] 2-1)

[0150] The expression of stress response genes (including ASNS, DDIT3, and SLC7A11) in HMLE cells under different treatment conditions was detected by bulk RNA sequencing.

[0151] See Figure 2 a, As expected, the expression of stress response genes (including ASNS, DDIT3, and SLC7A11) was strongly induced by endoplasmic reticulum stress or AA-Dep and was effectively inhibited when ATF4 was knocked down / downregulated.

[0152] 2-2)

[0153] See Figure 1 a, The heatmap shows genes with downregulated gene expression caused by ATF4 knockdown under Twist-OE (EMT) and Tg (ER stress) treatments, and these genes account for the top 2.5% under their respective conditions.

[0154] And, as can be seen from Figure 1 a, under EMT induction conditions (Twist-OE treatment), the genomic regions regulated by ATF4 are significantly different from those regulated under stress conditions (Tg and AA-Dep treatments), see Figure 1 a.

[0155] 2-3)

[0156] See Figure 1b. The inventors performed GO (Gene Ontology) enrichment analysis on genes whose expression was downregulated by more than 1.5-fold due to ATF4 knockdown under ER stress conditions. The upper panel shows the top 10 signaling pathways with the highest enrichment, and the lower panel shows the GSEA (Gene Set Enrichment Analysis) plot of the unfolded protein response (UPR) pathway.

[0157] See Figure 2 b. Under amino acid starvation induction conditions, GO (Gene Ontology) enrichment analysis was performed on genes whose expression was downregulated by more than 1.5-fold due to ATF4 knockdown. The upper panel shows the top 10 signaling pathways with the highest enrichment, and the lower panel shows the GSEA plot of the amino acid starvation signal pathway.

[0158] From Figure 1 b and Figure 2 The enrichment analysis experiments in b and b confirmed that ATF4 mainly regulates stress response pathways under stress conditions, such as gene sets of "unfolded protein response" and "amino acid starvation".

[0159] The hypergeometric distribution test was used for GO enrichment analysis, and the weighted Kolmogorov–Smirnov test was used for GSEA. These analyses were performed based on the average data of two biological replicates. This rule applies to GO enrichment analysis and GSEA in this article.

[0160] 2-4)

[0161] See Figure 1 c. When Twist was induced, GO enrichment analysis was performed on genes whose expression was downregulated by more than 1.5-fold due to ATF4 knockdown. The upper panel shows the top 10 signaling pathways with the highest enrichment, and the lower panel shows the GSEA plot of the epithelial-mesenchymal transition (EMT) pathway. Figure 1 As can be seen in c, in contrast, the most significantly regulated pathway by ATF4 under Twist-OE conditions is the EMT pathway.

[0162] See Figure 2 c. The volcano plot shows the differentially expressed genes (DEGs) of the above experimental group cells compared with the control group cells under Twist induction conditions. Upregulated or downregulated genes are highlighted in orange or blue, respectively. Examples of significantly regulated EMT genes, including FN1, VIM, COL1A1, COL3A1, and COL6A1, are marked.

[0163] From Figure 2It can be seen that after the knockdown of ATF4, the expression of many genes related to mesenchymal phenotypes, such as collagen (COL1A1, COL3A1, and COL6A1), FN1, and VIM, was downregulated.

[0164] The above results indicate that in response to stress or non-stress signals, ATF4 regulates gene transcription programs that are dependent on specific stimulus conditions.

[0165] 2. Using the immortalized human hepatic stellate cell line (HSCs) as a research model and inducing EMT with TGFβ

[0166] The above results demonstrate that ATF4 positively regulates the occurrence of type III EMT in epithelial cells and changes in cell-related characteristics.

[0167] In addition to the overexpression of EMT-TFs, the EMT program can also be triggered by extracellular factors, such as TGFβ, which is the most important driving signal for HSC activation.

[0168] To verify whether ATF4 also regulates type II EMT and HSC activation, we used LX-2 (an immortalized human HSC cell line) as a cell model and induced EMT in these cells with TGFβ. 1)

[0170] The lentiviral solutions containing the knockdown plasmids ATF4-#1 and #3 prepared in Example 1 above and the control lentiviral solution were used to transfect LX-2 cells, respectively. The experimental operations refer to the above "obtaining a cell line stably transfected with lentivirus" section, and will not be elaborated here.

[0171] Experimental group LX-2 (labeled as: shATF4 in the accompanying figure): LX-2 cells stably transfected with the lentivirus of Example 1 (lentivirus containing the knockdown plasmids ATF4-#1 and #3);

[0172] Control group LX-2 (labeled as: shCtrl in the accompanying figure): LX-2 cells stably transfected with the control lentivirus (lentivirus containing the empty vector).

[0173] The inventors of this application performed gene expression profiling (RNA sequencing) on the above experimental group and control group LX-2 cells after treatment under different conditions (DMSO, TGFβ, Tg). The stress condition was introduced by treatment with thapsigargin (Tg), and TGFβ induced EMT in the cells.

[0174] 2) Conclusion

[0175] 2-1)

[0176] See Figure 1d, The heatmap shows genes with downregulated gene expression caused by ATF4 knockdown under TGFβ (EMT) and Tg (ER stress) treatments, and these genes account for the top 2.5% under their respective conditions.

[0177] 2-2)

[0178] See Figure 1 e, GO enrichment analysis was performed on genes with more than 1.5-fold downregulation in expression caused by ATF4 knockdown under Tg treatment. The upper panel shows the top 10 signaling pathways with the highest enrichment, and the lower panel shows the GSEA (Gene Set Enrichment Analysis) plot of the unfolded protein response (UPR) pathway.

[0179] See Figure 2 d, GO enrichment analysis (upper panel) was performed on genes with more than 1.5-fold downregulation in expression in LX-2 cells under AA-Dep treatment by ATF4 knockdown, and GSEA analysis of amino acid deprivation by ATF4 knockdown compared with the control (lower panel).

[0180] From Figure 1 e and Figure 2 d, it can be seen that, as in HMLE, in LX-2 cells, when the cells are stimulated by Tg or AA-Dep, ATF4 mainly regulates the expression of stress-related genes.

[0181] 2-3)

[0182] See Figure 1 f, GO enrichment analysis was performed on genes with more than 1.5-fold downregulation in expression caused by ATF4 knockdown under TGFβ treatment. Figure 10 The upper panel shows the list of the top enriched gene sets, Figure 10 and the lower panel shows the GSEA plot of epithelial-mesenchymal transition (EMT).

[0183] See Figure 2 e, The volcano plot shows the DEGs of the experimental group cells compared with the control group cells under TGFβ treatment. Upregulated or downregulated genes are highlighted in orange or blue, respectively. Important EMT genes including COL1A1, COL1A2, COL3A1, COL4A1, and COL5A1 are marked in the figure.

[0184] From Figure 1 f and Figure 2 e, it can be seen that in LX-2 cells, under TGFβ treatment, the genes most significantly regulated by ATF4 belong to the EMT signaling pathway. This phenomenon is consistent with the role of ATF4 in regulating EMT gene expression during Twist induction in immortalized mammary epithelial cells HMLE.

[0185] Collectively, the above results indicate that the ATF4-driven transcriptional landscape is reprogrammed when confronted with different stimuli. More importantly, our findings suggest that ATF4 is not only a stress-responsive transcription factor but may also play an underappreciated role in cell state transitions.

[0186] II. In epithelial cells, ATF4 is required for inducing EMT. Reciprocal changes in the expression of epithelial and mesenchymal cell state marker genes are a hallmark of EMT induction.

[0187] In addition to demonstrating that ATF4 regulates the expression of type III EMT-related genes, we also explored the effect of ATF4 on the associated cellular properties induced by type III EMT.

[0188] Transfect HMLE_Twist_ER cells with the lentiviral solutions containing the knockdown plasmids ATF4-#1 and ATF4-#2 prepared in Example 1 above and the control lentiviral solution, referring to the content of point 1 in the first part above.

[0189] Experimental group HMLE-1 (labeled as: shATF4-1 in the figure): HMLE_Twist_ER cells stably transfected with the lentivirus of Example 1 (lentivirus containing the knockdown plasmid ATF4-1#);

[0190] Experimental group HMLE-2 (labeled as: shATF4-2 in the figure): HMLE_Twist_ER cells stably transfected with the lentivirus of Example 1 (lentivirus containing the knockdown plasmid ATF4-2#);

[0191] Control group HMLE (labeled as: shCtrl in the figure): HMLE_Twist_ER cells stably transfected with the control lentivirus (lentivirus containing the empty vector).

[0192] Cells of the experimental groups HMLE-shATF4-1 and HMLE-shATF4-2 and cells of the control group HMLE-shCtrl were treated with solvent control or 4-OHT (20 nM) for 10 days, and then the expression of EMT marker genes was analyzed by Western blotting, and the mRNA expression of EMT marker genes was analyzed by qPCR.

[0193] Conclusion

[0194] See Figure 3a. In the HMLE cell model, the expression of mesenchymal cell markers such as FN1, N-Cad (N-Cadherin, gene name CDH2), Vimentin (gene name VIM), and Slug (gene name SNAI2) driven by Twist (Twist is induced by 4-OHT) was significantly downregulated by knockdown of ATF4.

[0195] See Figure 3 a and Figure 4 a. When ATF4 was knocked down, in contrast, the expression of epithelial markers Claudin-1 and CDH1 (gene name of E-Cadherin) was significantly upregulated.

[0196] The experimental group HMLE-1 and 2 and the control group HMLE cells were treated with 4-OHT for 10 days and then stained with Calcein-AM to observe the proportion of dispersed cells representing the mesenchymal state of the cells. See the morphology of the experimental group HMLE-1 and 2 and the control group HMLE cells in Figure 3 b. Representative fluorescence images (left) and quantitative analysis of the relative area occupied by dispersed cells (right).

[0197] After treatment with increasing concentrations of 4-OHT for 10 days, see the morphology and distribution of the experimental group HMLE-1 and 2 and the control group HMLE cells in Figure 4 c. Which shows representative phase-contrast microscope images.

[0198] From Figure 3 b and Figure 4 c, it can be seen that Twist-OE induced HMLE cells to become spindle-shaped and disperse, reflecting their transition to the mesenchymal state, while knockdown of ATF4 effectively prevented this change in Twist-OE cells and maintained the typical epithelial cell morphology - tightly connected cylindrical shape.

[0199] The migration ability of the experimental group 1-2 and the control group cells after treatment with 4-OHT was evaluated by transwell assay. See Figure 3 c. Representative images (left) and quantitative analysis (right) of Calcein-AM-labeled cells that passed through the transwell chamber.

[0200] From Figure 3 c, it can be seen that knockdown of ATF4 inhibited cell migration, which is another characteristic of EMT.

[0201] Another important role in the occurrence of EMT is the appearance of CD24 in epithelial cells low CD44 high (CD24 lo CD44 hi)Enrichment of stem cell populations (S.A. Mani, W. Guo, M.J. Liao, E.N. Eaton, A. Ayyanan, A.Y. Zhou, M. Brooks, F. Reinhard, C.C. Zhang, M. Shipitsin, L.L. Campbell, K. Polyak, C. Brisken, J. Yang, R.A. Weinberg, The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133, 704 - 715 (2008)).

[0202] See Figure 3 Representative flow cytometry plots of CD24 lo CD44 hi cells in experimental group HMLE-1 and 2 and control group HMLE cells after treatment with control reagent or 4-OHT for 10 days (left), and quantitative analysis measured by flow cytometry (right).

[0203] From Figure 3 the results of d, we found that Twist-OE increased the CD24 lo CD44 hi population, while ATF4 knockdown effectively blocked the enrichment of this group of stem cell-like cells.

[0204] These above results indicate that ATF4 indeed regulates the EMT program and is essential for the occurrence of EMT in epithelial cells.

[0205] III. In hepatic stellate cells (HSCs), ATF4 positively regulates the EMT program and is essential for HSC activation.

[0206] The above results prove that ATF4 positively regulates the occurrence of type III EMT and the changes in cell-related characteristics in epithelial cells. As described in the background art, slightly different from classical EMT, the EMT that occurs in HSCs during liver fibrosis is the transformation of quiescent interstitial cells into myofibroblasts with the ability to migrate and secrete collagen. During the EMT that occurs in this process, in addition to highly expressing interstitial cell genes such as SNAI1, SNAI2, ZEB1, CDH2, FN1, and VIM, cells also highly express ACTA2 (actin alpha 2, smooth muscle, the protein product is α-SMA), DES (desmin), and the most important type I collagen - COL1A1 (collagen type I alpha 1 chain) and COL1A2 (collagen type I alpha 2 chain) and other type II EMT-specific genes that mark HSC activation.

[0207] To verify whether ATF4 also regulates type II EMT and the activation of HSCs, we used immortalized HSC cells - LX_2 and induced EMT in these cells with TGFβ.

[0208] Experimental group LX-2 (labeled in the attached figure as: shATF4): LX-2 cells stably transfected with the lentivirus of Example 1 (the lentivirus containing the knockdown plasmids ATF4-#1 and #3);

[0209] Control group LX-2 (labeled in the attached figure as: shCtrl): LX-2 cells stably transfected with the control lentivirus (the lentivirus containing the empty vector).

[0210] Conclusion

[0211] 1) Expression of interstitial marker genes

[0212] The EMT program is also involved in the process of HSC activation because a significant feature of activated HSCs is the upregulation of interstitial marker genes.

[0213] See Figure 5 a, Western blot shows the expression of interstitial marker genes in LX-2 cells of the experimental group and the control group after treatment with or without TGFβ for 2 days.

[0214] See Figure 4 d, After treatment of LX-2 cells in the experimental group and the control group with TGFβ for 2 days, the mRNA expression of EMT marker genes was analyzed by qPCR.

[0215] From Figure 5 a and Figure 4As can be seen in Fig. 6d, although TGFβ treatment increased the expression of mesenchymal marker genes including FN1, N-Cad, and Slug in LX-2 cells, knockdown of ATF4 significantly inhibited this upregulation.

[0216] It has been reported in the literature (J.C. Tsai, L.E. Miller-Vedam, A.A. Anand, P. Jaishankar, H.C. Nguyen, A.R. Renslo, A. Frost, P. Walter, Structure of the nucleotide exchange factor eIF2B Reveals mechanism of memory-enhancing molecule. Science 359, (2018)) that ISRIB is a chemical activator that inhibits the function of p-eIF2-alpha by activating eIF2B, thereby reducing the translation of its downstream ATF4.

[0217] See Figure 5 Fig. 6b, Western blotting showed the expression of mesenchymal marker genes in LX-2 cells after treatment with different concentrations of TGFβ (0, 1, and 5 ng / ml) with or without ISRIB (1 mM).

[0218] As Figure 5 can be seen in Fig. 6b, consistent with ATF4 knockdown, ISRIB also inhibited the expression of TGFβ-responsive genes.

[0219] See Figure 5 Fig. 6c, Western blotting showed the expression of mesenchymal marker genes in LX-2 cells after overexpressing ATF4 and treating with different concentrations of TGFβ (0, 0.5, 1, and 5 ng / ml) for 2 days; see Figure 4 Figs. 6e and 6d, qPCR analysis showed the mRNA expression of mesenchymal marker genes in LX-2 cells after overexpressing ATF4 and treating with different concentrations of TGFβ (0, 0.5, 1, and 5 ng / ml) for 2 days.

[0220] As Figure 5 compared in Figs. 6c, 6e, and 6d, overexpression of ATF4 (ATF4-OE) strongly enhanced the response of LX-2 cells to TGFβ-induced mesenchymal gene expression. For example, Figure 5 in Fig. 6d, in the case of ATF4 overexpression, only 0.5 ng / ml of TGFβ was required to induce the expression levels of SNAI1 and FN1 to reach the levels induced by 5 ng / ml of TGFβ in normal LX-2 cells.

[0221] 2) Expression of α - smooth muscle actin (α - SMA) and collagen

[0222] In addition to increasing classical interstitial genes, upregulation of α - smooth muscle actin (α - SMA, encoded by the ACTA2 gene) and collagen is a hallmark of HSC activation, according to previous reports (C.R. Gandhi, Hepatic stellate cell activation and pro - fibrogenic signals. J Hepatol 67, 1104 - 1105 (2017)).

[0223] 2 - 1)

[0224] See Figure 5 e. The results of Western blot experiments showed the expression of fibrogenic genes in LX - 2 cells of the experimental group and the control group after treatment with different concentrations of TGFβ (0, 5, and 10 ng / ml) for 2 days. See 5f. The mRNA expression of fibrogenic genes in LX - 2 cells of the experimental group and the control group after treatment with TGFβ for 2 days was analyzed by qPCR.

[0225] From Figure 5 e and Figure 5 f, it can be seen that although TGFβ treatment increased the expression of α - SMA, knockdown of ATF4 effectively blocked this up - regulation of expression. In addition to α - SMA, the deletion of ATF4 also inhibited the expression of type I collagen induced by TGFβ, which is an important marker gene for liver fibrosis.

[0226] 2 - 2)

[0227] See Figure 5 g. The mRNA expression of fibrogenic genes in LX - 2 cells after overexpressing ATF4 and then treating with different concentrations of TGFβ (0, 0.5, and 1 ng / ml) for 2 days was analyzed by qPCR.

[0228] See Figure 5 h. The mRNA expression of fibrogenic genes in LX - 2 cells after treatment with solvent control (Ctrl), TGFβ (5 ng / ml) for 2 days (TGFβ), or pretreated with ISRIB (1 mM) for 3 days and then treated with both TGFβ (5 ng / ml) and ISRIB (1 mM) for 2 days (TGFβ + ISRIB) was analyzed by qPCR.

[0229] In contrast to the above Figure 5 e and Figure 5 f results, in Figure 5 g, overexpression of ATF4 (ATF4 - OE) strongly enhanced the expression of TGFβ - driven fibrogenic markers; inFigure 5 In h, the downregulation of ATF4 translation by ISRIB effectively inhibited the expression of α-SMA and type I collagen.

[0230] 2-3)

[0231] PDGF-BB is another important extracellular stimulator for HSC activation. To further verify the role of ATF4 in the process of HSC activation, we also used PDGF-BB as an inducer for HSC activation.

[0232] See Figure 4 In f, after LX-2 cells were treated with solvent control, PDGF-BB, ISRIB, or the combination of PDGF-BB and ISRIB for two days, Western blot showed the expression of fibrosis-related genes.

[0233] See Figure 4 In g, after LX-2 cells were treated with solvent control, PDGF-BB, or the combination of PDGF-BB and ISRIB for two days, collagen gene expression was analyzed by qPCR.

[0234] In Figure 4 In f and 4g, a similar regulatory effect of ISRIB was also observed in the PDGF-BB-triggered HSC activation model.

[0235] 2-4)

[0236] See 4h. In LX-2 cells or AML12 cells (an immortalized mouse hepatocyte cell line), the control vector or ATF4 overexpression plasmid was overexpressed, and the cells were treated with TGFβ or the combination of TGFβ and ISRIB. Collagen gene expression was analyzed by qPCR.

[0237] See 4i. Western blot showed the expression of FN1 after LX-2 or AML12 cells overexpressed the control vector or ATF4 overexpression plasmid and were treated with TGFβ or the combination of TGFβ and ISRIB.

[0238] From Figure 4 It can be seen from h and 4i that overexpression of ATF4 in LX-2 cells can completely restore the effect of ISRIB, indicating that the anti-fibrotic effect of ISRIB is mediated by ATF4. As a control, the regulatory effect of p-eIF2-alpha-ATF4 on ECM gene expression was significantly weaker in the liver cell AML12.

[0239] Overall, these above results indicate that ATF4 promotes the activation of HSCs by promoting the EMT molecular program in HSCs.

[0240] IV. Specific knockout of Atf4 in vivo can alleviate liver fibrosis.

[0241] Activation of hepatic stellate cells (HSCs) is a key event in liver fibrosis. Next, we investigated the role of ATF4 in the pathological progression of liver fibrosis in vivo.

[0242] In Example 1, the inventors of the present application established a mouse model Lrat-Cre; Atf4 with specific knockout of the Atf4 gene in HSCs fl / fl .

[0243] Then, the inventors used carbon tetrachloride (CCl4), a commonly used liver fibrosis inducer, to induce liver fibrosis. After five weeks of induction, liver tissues were collected to evaluate the progression of fibrosis.

[0244] Conclusion 1)

[0246] See Figure 6 b, Western blot shows the expression of EMT and fibrosis marker genes in liver tissues of WT and HSC-Atf4-KO mice.

[0247] See Figure 6 c, qPCR analysis shows Figure 6 the expression of fibrosis marker genes in the liver tissues in b.

[0248] See Figure 7 f, qPCR analysis shows the expression of Col1a2 in liver tissues of WT and HSC-Atf4-KO mice, with or without CCl4 treatment.

[0249] See Figure 7 g, qPCR analysis shows the expression of Atf4, Fn1, and Col1a1 in HSCs isolated from liver tissues of WT and HSC-Atf4-KO mice treated with control solvent or CCl4.

[0250] From Figure 6 b, it can be seen that in wild-type animals, mesenchymal cell markers, including FN1 and Vimentin, were significantly increased after CCl4 treatment, which was consistent with the upregulation of the epithelial-mesenchymal transition program.

[0251] Figure 6 In b, 6c, and 7f, fibrosis genes (including Acta2, Col1a1, and Col1a2) were highly induced, indicating that HSCs were in an activated state. In contrast, this CCl4-triggered induced expression of mesenchymal and fibrosis-related genes was significantly inhibited in HSC-Atf4-KO animals, indicating that ATF4 is essential for the activation of HSCs in vivo.

[0252] Figure 7 In group g, the expression of these fibrosis-related genes in quiescent HSCs was also inhibited by ATF4 deletion, which was consistent with the above results. 2)

[0254] As the most important marker of fibrosis, type I collagen accumulates in the liver during fibrosis progression.

[0255] See Figure 6 d and 6e, respectively, are Figure 6 Sirius red staining and Masson trichrome staining of liver tissues in b.

[0256] It can be seen that the deletion of Atf4 significantly reduced the level of hepatic collagen.

[0257] The inventor team used a hydroxyproline-based assay to quantify the collagen content, and the results are shown in Figure 6 f, and it can be confirmed that HSC-specific Atf4 deletion significantly reduced the collagen level in the liver.

[0258] Similarly, judged by the levels of liver metabolic enzymes and bilirubin in serum, see Figure 6 g, measuring Figure 6 the serum ALT, AST, and TBIL levels of the experimental animals in b, it can be seen that the progression of pathological fibrosis was effectively alleviated by HSC-specific Atf4 deletion.

[0259] In summary, these results all indicate that the expression of ATF4 in HSCs is essential for the activation of HSCs and the progression of liver fibrosis.

[0260] V. ATF4 activates HSCs by promoting the epigenetic features of EMT

[0261] To reveal the potential mechanism by which ATF4 regulates the EMT program to promote HSC activation, we investigated the binding of ATF4 to chromatin.

[0262] We used immortalized HSC cells - LX_2 and induced EMT in these cells with TGFβ.

[0263] Conclusion 1)

[0265] By performing ChIP-seq, we found that in LX-2 cells treated with TGFβ, ATF4 bound to more than 2,800 genomic loci. See Figure 8 a, the distribution of ATF4 binding peaks across the genome under TGFβ (TGFβ-ATF4) or Tg (Tg-ATF4) treatment.

[0266] GO enrichment analysis was performed on the genes closest to the ATF4 binding sites under TGFβ or Tg treatment, see Figure 8 b, which shows the top 10 signaling pathways with the highest enrichment. After annotating these ATF4 binding peaks, it was found that under TGFβ induction, the genes most significantly enriched were mainly genes related to the EMT signaling pathway.

[0267] In LX-2 cells treated with DMSO or Tg: see Figure 9 a, which shows the genome-wide distribution of ATF4 ChIP-seq peaks under control conditions; see Figure 9 b, the GO enrichment analysis plot of the genes closest to the ATF4 binding sites under control conditions, which shows the most abundant pathways.

[0268] By comparison, the most significant ATF4 binding gene sets are genes related to the regulation of protein translation (mTORC1 signaling) and the UPR pathway.

[0269] Considering that ATF4 regulates the expression of specific gene sets in a stimulus signal-dependent manner as demonstrated in the first part of the above argument, see Figure 1 ; combined with the above ChIP-seq results, it shows that ATF4 achieves the specificity of gene transcription regulation by binding to different genomic regions in response to different signals.

[0270] The DNA binding motifs of ATF4 under TGFβ or Tg treatment were identified by HOMER, Figure 8 c shows the top 3 most significant motifs. As can be seen from Figure 8 c, despite the binding selectivity, ATF4 seems to bind to its classical DNA binding motif regardless of whether LX-2 cells are treated with TGFβ or Tg.

[0271] These results reveal a model in which upstream triggering signals - fibrosis induction or endoplasmic reticulum stress - can selectively direct ATF4 to specific chromatin regions to regulate gene transcription. 2)

[0273] To further validate this hypothesis, we examined the epigenomic features in LX-2 cells under different treatment conditions by performing ChIP-seq analysis, and detected the acetylation of lysine 27 on histone H3 (H3K27ac), a known active histone marker representing enhanced gene transcriptional activity (T.H. Beacon, G.P.Delcuve, C.Lopez, G.Nardocci, I.Kovalchuk, A.J.van Wijnen, J.R.Davie, The dynamic broad epigenetic (H3K4me3, H3K27ac) domain as a mark of essential genes.Clin Epigenetics 13, 138 (2021); B.Pelham-Webb, A.Polyzos, L.Wojenski, A.Kloetgen, J.Li, D.C.Di Giammartino, T.Sakellaropoulos, A.Tsirigos, L.Core, E.Apostolou, H3K27ac bookmarking promotes rapid post-mitotic activation of the pluripotent stem cell program without impacting 3D chromatin reorganization.Mol Cell 81, 1732-1748e1738 (2021)).

[0274] Figure 8 The Venn diagram in panel d summarizes the number of peaks identified by ATF4 and H3K27ac ChIP-seq under TGFβ treatment, Figure 9 The Venn diagram in panel c shows the overlap between the chromatin regions bound by ATF4 and the chromatin regions marked by H3K27ac under Tg treatment. Overall, the number of H3K27ac-marked peaks was comparable in LX-2 cells treated with TGFβ or Tg.

[0275] Figure 8 The heatmap in panel e shows the normalized ChIP-seq signals for three sets of data: TGFβ-specific ATF4 binding sites, TGFβ / Tg common ATF4 binding sites, or Tg-specific ATF4 binding sites (from top to bottom), and simultaneously analyzes the signal intensity of H3K27ac near the corresponding binding sites.

[0276] Combined Figure 8As can be seen from the results of d and 8e, in TGFβ-treated LX-2 cells, most ATF4 binding peaks overlap with H3K27ac marked sites.

[0277] Typical enhancers (TEs) and super enhancers (SEs) of regulatory genes were determined according to the binding intensity and binding range of H3K27ac under ER stress and EMT conditions. Briefly, all TEs with an interval of no more than 12.5 kb were regarded as an enhancer unit, and the signals of H3K27ac on all enhancer units were sorted from weak to strong, so as to find a group of enhancer units with significantly higher H3K27ac signal intensity, which were SEs (Hnisz D, Abraham B J, Lee T I, et al. Super-enhancers in the control of cell identity and disease[J]. Cell, 2013, 155(4): 934-47.). See Figure 9 d, by sorting the binding intensity of H3K27ac on the enhancer and using the inflection point of the binding intensity as the segmentation point, the enhancers above this inflection point were determined as SEs, while those below this inflection point were TEs.

[0278] Although both SEs and TEs are very important for transcriptional activation, the genes regulated by SEs more often encode key factors that determine cell fate (W.A. Whyte, D.A. Orlando, D. Hnisz, B.J. Abraham, C.Y. Lin, M.H. Kagey, P.B. Rahl, T.I. Lee, R.A. Young, Master transcription factors and mediators establish super-enhancers at key cell identity genes. Cell 153, 307-319(2013)、D. Hnisz, B.J. Abraham, T.I. Lee, A. Lau, V. Saint-Andre, A.A. Sigova, H.A. Hoke, R.A. Young, Super-enhancers in the control of cell identity and disease. Cell 155, 934-947(2013)).

[0279] See Figure 8f, Left panel: Overlap distribution of ATF4 binding peaks with super-enhancers (SEs), typical enhancers (TEs), or other types of H3K27ac-marked elements (Others) under TGFβ treatment; Right panel: EMT pathway enrichment analysis using genes driven by SEs or TEs.

[0280] From Figure 8 As can be seen from the left panel of f, under TGFβ treatment, more than 6% of the ATF4 binding sites are located in SEs (ATF4-SE), while approximately 30% are located in TEs (ATF4-TE). An interesting result found in the right panel is that ATF4-SE sites show a stronger enrichment level for EMT genes than ATF4-TE sites ( Figure 5 f, right panel), indicating that ATF4 preferentially regulates EMT genes through SEs.

[0281] See Figure 8 g, Left panel: Overlap distribution of ATF4 binding peaks with super-enhancers (SEs), typical enhancers (TEs), or other types of H3K27ac-marked elements (Others) under Tg treatment. Right panel: UPR pathway enrichment analysis using genes driven by TEs; genes driven by SEs are not enriched in the UPR pathway.

[0282] By comparing Figure 8 the results of g with Figure 8 those of f, it can be found that for regulating Tg-induced UPR genes, although the overlap ratios of its binding peaks with SEs or TEs are similar (ATF4-SE 4% and ATF4-TE 16%), ATF4 does not seem to act preferentially through SEs.

[0283] These above results suggest that ATF4 may utilize the changes in epigenetic features during the EMT induction process to activate the gene expression related to HSC activation. 3)

[0285] We next attempted to identify the candidate genes bound and directly regulated by ATF4 during the EMT induction process and to dissect the functional link between epigenetic modifications and ATF4-driven transcription.

[0286] See Figure 10 a, By analyzing the ChIP-Seq results, the co-binding regions of ATF4 and SEs on the COL1A1 and LOXL2 genes under different stimulation conditions (Ctrl, TGFβ, and Tg treatments). Super-enhancers with ATF4 binding sites are marked with gray rectangles. Black arrows indicate the ATF4 binding sites used for ChIP-qPCR tests.

[0287] See Figure 9e, Snapshots of normalized ATF4 and H3K27ac and input ChIP-seq tracks at the CDH2 locus under different conditions (Ctrl, TGFβ, and Tg treatments). A typical enhancer containing an ATF4 binding site is highlighted with a gray rectangle.

[0288] From Figure 10 a and Figure 9 e, it can be seen that many mesenchymal and HSC activation genes are bound by ATF4, including COL1A1, LOXL2, and CDH2. By using the H3K27ac signal, the regions of COL1A1 and LOXL2 near the ATF4 binding regions can be defined as SEs, while the region associated with CDH2 is defined as a TE.

[0289] See Figure 10 b, ChIP-qPCR analysis shows the recruitment of ATF4 to the corresponding regions of COL1A1 and LOXL2 under solvent control, TGFβ, or combined TGFβ and JQ1 treatments. Figure 10 The ChIP-qPCR in b confirmed that TGFβ treatment induced the binding of ATF4 to these regions, while blocking the enhancer program by JQ1 (JQ1 is an inhibitor that inhibits enhancer activity by targeting BRD4) inhibited the interaction of ATF4 with its binding regions.

[0290] See Figure 9 f, ChIP-qPCR analysis shows the recruitment of ATF4 to the corresponding regions under solvent control, TGFβ, or combined TGFβ and C646 treatments. Figure 9 Similar results to those in Figure 10 b can also be seen in f. Interfering with histone acetylation by C646 (C646 is a chemical inhibitor of the H3K27ac transferase p300) can also inhibit the binding of ATF4 to COL1A1 and LOXL2. Figure 10 b and Figure 9 f further confirmed that TGFβ-induced epigenetic modifications are necessary for the repositioning of ATF4 on the genome. 4)

[0292] To verify the functional consequences of ATF4 binding, we established a luciferase-based reporter system by inserting the ATF4 binding site into the regulatory region upstream of the reporter gene. This reporter system can respond to TGFβ, and overexpression of ATF4 can further promote the transcription of the reporter gene ( Figure 6 c). This indicates that ATF4 binding promotes the transcription of COL1A1 in the presence of TGFβ.

[0293] See Figure 10c, A luciferase reporter gene containing the upstream genomic region (-9108 to -8708) of the COL1A1 gene was constructed to measure the transcriptional activity of the co-binding region of ATF4 and H3K27ac. The COL1A1 reporter gene was co-transfected with the Renilla luciferase reporter gene into LX-2 cells transduced with a control vector (Vec) or an ATF4 overexpression construct (ATF4-OE). After transfection, the cells were treated with solvent control, TGFβ (1 ng / ml) for 2 days, JQ1 (250 nM) for 12 hours, or a combination of TGFβ (1 ng / ml) for 2 days and JQ1 (250 nM) for 12 hours, and then analyzed by dual luciferase assay.

[0294] See Figure 10 d, qPCR showed the expression levels of COL1A1 and LOXL2 in control or ATF4-overexpressing LX-2 cells treated with solvent control, TGFβ, JQ1, or a combination of TGFβ and JQ1.

[0295] From Figure 10 As can be seen from the results of c and 10d, consistent with the role of JQ1 in regulating the binding of ATF4 to the COL1A1 gene, the transcriptional activity of the COL1A1-reporter gene was also abolished by JQ1, and JQ1 treatment effectively inhibited TGFβ-induced, ATF4-dependent gene expression.

[0296] Therefore, in summary, the inventor team determined that ATF4 promotes EMT gene expression in an enhancer-dependent manner.

[0297] VI. Pharmacological inhibition of the eIF2α / ATF4 pathway alleviates liver fibrosis

[0298] According to existing reports, ISRIB can block the translation of ATF4 in vivo (A.A. Anand, P. Walter, Structural insights into ISRIB, a memory-enhancing inhibitor of the integrated stress response. FEBS J 287, 239-245 (2020)), but the key role of ATF4 in fibrosis has not been revealed in the prior art.

[0299] The inventors of this application discovered the key role of ATF4 in fibrosis (see the description in the above Part IV), and this discovery provides an opportunity to treat liver fibrosis by targeting this transcription factor. The inventor team also tried to further confirm it through pharmacological approaches.

[0300] The inventor team attempted to use ISRIB to treat carbon tetrachloride (CCl4)-induced liver fibrosis in mice.

[0301] Male C57BL / 6 mice at 8 weeks of age (purchased from Shanghai Slack Experimental Animal Co., Ltd.) were treated with carbon tetrachloride (0.5 ml / kg) or solvent control (corn oil) twice a week for 5 weeks, with or without ISRIB (5 mg / kg). There were 5 - 7 mice in each group (n = 5 - 7).

[0302] Conclusion 1)

[0304] See Figure 11 a, Western blot showed the expression of EMT and fibrosis marker genes in the liver tissues of mice in different treatment groups. See Figure 12 a, qPCR analysis showed the expression of EMT and fibrosis marker genes in the liver tissues of mice treated as indicated.

[0305] From Figure 11 a, it can be seen that although ISRIB treatment had no effect on the expression of interstitial genes in the control group, it significantly reduced the expression of the interstitial gene product Vimentin in the CCl4-induced group.

[0306] Combined with Figure 11 a and Figure 12 a, it can be seen that ISRIB treatment also reduced the expression of the HSC activation gene α-SMA and the liver fibrosis gene type I collagen in the liver tissues after CCl4 treatment.

[0307] To examine whether the changes in fibrosis gene expression occurred in hepatic stellate cells HSCs, the inventor team isolated HSCs and hepatocytes from the treated animals and examined their gene expression.

[0308] See Figure 11 b, qPCR analysis showed the expression of fibrosis genes in hepatocytes and HSCs isolated from the liver tissues in Figure 11 a. See Figure 12 b, Western blot results showed the expression of ATF4 in HSCs isolated from the liver tissues of mice treated with corn oil, CCl4, or CCl4 combined with ISRIB.

[0309] From Figure 11From the results of b and 12b, it was found that ISRIB inhibited the induced expression of ATF4 in HSCs, and at the same time, ISRIB also down-regulated the expression of fibrotic genes in HSCs. In contrast, the expression of fibrotic genes in hepatocytes was much lower than that in HSCs, indicating that the antifibrotic effect of ISRIB was mainly achieved by targeting HSCs. 2)

[0311] See Figure 11 c: Figure 11 Sirius red staining results of liver tissue in a; see Figure 11 d: Quantitative determination by hydroxyproline Figure 11 Liver collagen content of liver tissue in a; see Figure 12 c, Figure 11 Masson's trichrome staining results of liver tissue in a.

[0312] From Figure 11 the results of c, 11d and 12c, it can be seen that consistent with the changes in gene expression, the accumulation of collagen in the liver was significantly reduced after ISRIB treatment. 3)

[0314] To test whether ISRIB inhibits fibrosis by directly reducing CCl4-induced hepatocyte death, we examined the expression of a panel of cell death markers.

[0315] See Figure 12 d, Western blot shows the expression of cleaved PARP and phosphorylated RIP3 in liver tissues of animals treated with the indicated reagents for 24 hours. From Figure 12 the results of d, it was found that ISRIB did not change necrosis or apoptosis induced by acute CCl4 treatment.

[0316] See Figure 12 e, Figure 12 Representative images of TUNEL staining of the liver collected in d. It can be seen that in the TUNEL assay, we found that ISRIB not only did not change the percentage of hepatocytes with DNA fragmentation, but also the area of necrotic hepatocytes (lightly stained cells indicated by swollen morphology and nuclear condensation) was not changed by ISRIB treatment.

[0317] In addition, see Figure 12 f, Western blot shows Figure 12 the expression of GSDMD and cleaved Caspase-1 in liver tissues collected in d; see Figure 12 g, Western blot shows Figure 12 the expression of ATF4 pathway genes in the indicated tissues of animals in a.

[0318] Figure 12 Results of f and 12g showed that CCl4 treatment did not induce apoptosis, and ISRIB did not alter the eIF2α pathway in other tissues including adipose tissue, muscle, and skin.

[0319] Collectively, the above results indicate that ISRIB inhibits CCl4-induced liver fibrosis by targeting HSC activation. 3)

[0321] In addition to CCl4-induced fibrosis, we also tested the effect of ISRIB in two other fibrosis models: one triggered by bile duct ligation (BDL) surgery and the other induced by thioacetamide (TAA).

[0322] See Figure 11 e, 8-week-old male C57BL / 6 mice were subjected to sham operation (Sham) or common bile duct ligation (BDL), and then received ISRIB (5 mg / kg) or vehicle control twice a week for 4 weeks (n = 5 mice per group). Western blot showed the expression of EMT and fibrosis marker genes in the liver tissues of mice in different treatment groups.

[0323] See Figure 11 f, 8-week-old male C57BL / 6 mice were treated with vehicle control, TAA (150 mg / kg), or a combination of TAA and ISRIB (5 mg / kg) twice a week for 5 weeks (n = 5 mice per group). Western blot showed the expression of EMT and fibrosis marker genes in the liver tissues of mice in different treatment groups.

[0324] See Figure 13 a, qPCR analysis was performed on the livers of mice with or without bile duct ligation (BDL) and with or without ISRIB treatment to detect the expression of EMT and fibrosis marker genes; see Figure 13 b, qPCR analysis was performed on the livers of mice treated with vehicle control, TAA, or a combination of TAA and ISRIB to detect the expression of EMT and fibrosis marker genes.

[0325] Collectively Figure 11 e and 11f, Figure 13 Combined with the results of a and 13b, consistent with the findings in the above CCl4 model, ISRIB treatment effectively reduced the expression of Vimentin, α-SMA, and type I collagen induced by BDL or TAA, respectively.

[0326] See Figure 11 g, The hepatic collagen content of the liver tissues in e was determined by hydroxyproline quantification Figure 11 in e.Figure 11 h, by quantitative determination of hydroxyproline Figure 11 The hepatic collagen content of the liver tissue in f. See Figure 13 c: Figure 13 The Sirius red staining result of the liver tissue in a; Figure 13 d: Figure 13 The Sirius red staining result of the liver tissue in b; Figure 13 e: Figure 13 The Masson's trichrome staining result of the liver tissue in a; Figure 13 f: Figure 13 The Masson's trichrome staining of the liver tissue in b.

[0327] Comprehensively Figure 11 g and 11h, and Figure 13 The results of c - f, it can be seen that the production and aggregation of intrahepatic collagen caused by BDL or TAA are also significantly reduced.

[0328] Generally speaking, the above results all indicate the role of ISRIB in inhibiting HSC activation during the process of liver fibrosis, and strongly highlight the potential of ATF4 - targeted therapy in the treatment of liver fibrosis. 4)

[0330] Finally, the inventor team investigated the clinical relevance of HSC-ATF4 in patients with cirrhosis. For this purpose, a recently published dataset (P. Ramachandran, R. Dobie, J. R. Wilson-Kanamori, E. F. Dora, B. E. P. Henderson, N. T. Luu, J. R. Portman, K. P. Matchett, M. Brice, J. A. Marwick, R. S. Taylor, M. Efremova, R. Vento-Tormo, N. O. Carragher, T. J. Kendall, J. A. Fallowfield, E. M. Harrison, D. J. Mole, S. J. Wigmore, P. N. Newsome, C. J. Weston, J. P. Iredale, F. Tacke, J. W. Pollard, C. P. Ponting, J. C. Marioni, S. A. Teichmann, N. C. Henderson, Resolving the fibrotic niche of human liver cirrhosis at single-cell level. Nature 575, 512-518 (2019)) was consulted, in which the liver non-parenchymal cells (NPCs) of healthy donors and patients with cirrhosis were analyzed by single-cell RNA sequencing (scRNA-seq).

[0331] See Figure 11 i, t-SNE representation of single-cell RNA sequencing (scRNA-seq) analysis of human hepatic stellate cells (HSCs) from healthy donors (n = 5) and patients with cirrhosis (n = 5). Cells in healthy or cirrhotic samples are highlighted in cyan or red, respectively.

[0332] See Figure 14 a, UMAP embedding analysis of single-cell RNA sequencing data showing 81,714 cells from healthy (n = 5) and cirrhotic (n = 5) human livers, including 11 major cell types (B cells; circulating cells; endothelial cells; epithelial cells; ILC: innate lymphoid cells; MP: mononuclear phagocytes; mast cells; stromal cells; plasma cells; T cells; pDC: plasmacytoid dendritic cells) shown in the left panel; the middle panel shows 2,715 stromal cells annotated by subtype (qHSC: quiescent HSC; aHSC: activated HSC; VSMC: vascular smooth muscle cells); the right panel shows the UMAP embedding of HSCs stratified by sample origin (above) or HSC activation status (below).

[0333] See Figure 11 j, the violin plot shows Figure 11 the ATF4 mRNA expression in HSCs in i; the inventor team found that the RNA expression of ATF4 in HSCs of cirrhotic patients increased compared with that of the healthy control group.

[0334] To explore the changes of ATF4 at the protein level using scRNA-seq data, we examined the expression of ATF4 target genes, which could faithfully represent the protein level and transcriptional activity-promoting ability of ATF4.

[0335] See Figure 11 k, on the dataset comparing the fold change of HSCs expression between cirrhotic patients and healthy donors in Figure 11 i, gene set enrichment analysis (GSEA) was performed on two gene sets of ATF4 target genes. The "ATF4 targets under TGFβ" gene set on the left refers to the ATF4 target genes regulated by TGFβ treatment in LX-2 cells, and these data are from the above-mentioned bulk RNA sequencing; the "ATF4 targets under Tg" gene set on the right refers to the ATF4 target genes regulated by Tg treatment in LX-2 cells, also from the above-mentioned bulk RNA sequencing.

[0336] By performing GSEA analysis on the ATF4-regulated gene set obtained from LX-2 cells (see Figure 1 d), see Figure 11 the left panel of k, we found by comparison that the ATF4-regulated genes (ATF4-TGFβ) under TGFβ treatment were significantly upregulated in the HSCs of cirrhotic patients compared with healthy donors.

[0337] In contrast, see Figure 11 the right panel of k, the classical ATF4-regulated genes (ATF4-Tg) triggered by endoplasmic reticulum stress were not enriched in the context of liver cirrhosis.

[0338] These results not only showed the increase in ATF4 activity in HSCs, but also highlighted the upstream and downstream-dependent role of ATF4 in liver fibrosis.

[0339] In addition, the inventor team also examined the evolutionary relationship between quiescent and activated HSCs (qHSCs and aHSCs) through pseudotime trajectory analysis to study the relationship of ATF4 in fibrosis.

[0340] See Figure 14b, UMAP embedding analysis of single-cell RNA sequencing data for qHSC and aHSC subsets in pseudotime trajectories (left panel); the magnified UMAP embedding plot is colored by qHSC and aHSC subtypes. Arrows indicate the covered RNA velocity streamlines, showing the cell state transitions inferred from the shown cells by scVelo dynamic modes (right panel). See Figure 14 c, Heatmap showing the dynamically expressed genes arranged in the predicted pseudotime order, illustrating the fluctuations in gene expression during the transition from qHSC to aHSC. See Figure 14 d, Clinical features of patient liver tissues stained with ATF4. See Figure 14 e, Two representative IHC staining images for ATF4 staining in fibrotic liver tissues and the Fib-4 scores of the patients (scale bar: 30 μm).

[0341] VII. Summary

[0342] Activation of hepatic stellate cells (HSCs) is the major biological event during the development of liver fibrosis. Compared with advanced liver diseases such as liver cirrhosis and hepatocellular carcinoma (HCC), liver fibrosis is considered controllable and reversible if the key cellular pathways driving HSC activation can be effectively targeted.

[0343] 1. In the study of this application, the inventor team determined that ATF4 is a key factor required for HSC activation. Although in the prior art, ATF4 is generally considered the master regulator of the endoplasmic reticulum stress response; unexpectedly, the inventor team of this application found that ATF4 promotes the activation and transdifferentiation of HSCs by regulating the epithelial-mesenchymal transition (EMT) program through a pathway independent of endoplasmic reticulum stress, and EMT is a key pathway for cell state determination.

[0344] 2. In the study of this application, the inventor team of this application adopted a method based on HSC-specific knockout animals and clearly demonstrated that the expression of ATF4 in HSCs is crucial for HSC activation. Combining the finding that pharmacological inhibition of ATF4 production can effectively alleviate liver fibrosis, our study provides new opportunities for the clinical treatment of liver fibrosis;

[0345] 3. The study of this application reveals a novel, stress-independent function of the eIF2α / ATF4 stress response pathway in the progression of liver fibrosis disease.

[0346] Liver fibrosis is a pathological wound healing process that occurs after chronic or acute liver injury; thus, the activation of HSCs is intuitively regarded as a cell biological event that inevitably encounters various stress factors. For example, the increase and accumulation of reactive oxygen species (ROS) are a recognized hallmark of liver fibrosis. The profibrotic function of ROS is directly related to the oxidative stress of HSCs because the depletion of certain antioxidant proteins (such as cytoglobin) makes animals more prone to fibrosis.

[0347] Similarly, endoplasmic reticulum stress is also frequently observed in liver diseases, including chronic hepatitis and HCC, which can be judged by the activation of the UPR pathway in hepatocytes and HSCs during the progression of liver diseases. Almost all previous studies have attributed the role of UPR in liver fibrosis to its role in regulating ER homeostasis. And, in the prior art, ATF4 is generally considered the master regulator of the endoplasmic reticulum stress response.

[0348] A recent paper (M.P. Ye, W.L. Lu, Q.F. Rao, M.J. Li, H.Q. Hong, X.Y. Yang, H. Liu, J.L. Kong, R.X. Guan, Y. Huang, Q.H. Hu, F.R. Wu, Mitochondrial stress induces hepatic stellate cell activation in response to the ATF4 / TRIB3 pathway stimulation. J Gastroenterol 58, 668 - 681 (2023)) suggests that ATF4 may be involved in HSC activation by regulating mitochondrial UPR.

[0349] After reading this paper, the inventor team of this application believes that its conclusion is also controversial: because according to the experimental design of this paper, an AAV8 - mediated method was used to inhibit the expression of ATF4 in the liver, but this mainly blocked the expression of ATF4 in hepatocytes. AAV8 does not have HSC affinity and cannot target the expression of ATF4 in HSCs; therefore, although this paper mentions that ATF4 may be involved in HSC activation by regulating mitochondrial UPR, judging from its experimental method (AAV8 - mediated) and experimental content, it cannot prove / support the conclusion that "ATF4 may be involved in HSC activation by regulating mitochondrial UPR". This conclusion is controversial / uncertain for those skilled in the art.

[0350] However, the discovery of the inventors of the present application shows for the first time that UPR factors can drive the process of liver fibrosis, beyond their classical functions in sensing unfolded proteins and resetting the ER environment. Specifically, the present application discovers that ATF4, as a major stress response factor, can directly promote the occurrence of EMT, thereby promoting the activation of HSCs. The new discovery of the present application implies that the role of stress response pathways in disease progression may need to be further evaluated and investigated, because the pathological background may profoundly affect the actual functions of these stress pathways.

[0351] As is well known, stress response pathways are usually bifunctional. For example, the UPR pathway can both promote "anti-stress" adaptation for cell survival and trigger cell death pathways to eliminate irreversibly stressed cells. The research of the present application elevates the functional complexity to another level - stress response factors can determine cell fate by regulating EMT, which is an important cell state transition program.

[0352] Although there are many differences between the typical EMT program and the process of HSC activation, these two cell events have a very significant common feature - the induction of ECM production. The inventors of the present application identified another interesting similarity between EMT and HSC activation through the research of the present application - both are driven by ATF4-mediated signal transduction. The discovery of the inventors of the present application reveals that UPR factors play a more fundamental function by determining the EMT program.

[0353] The discovery of the inventors of the present application reveals a new mode of action of ATF4 in gene transcription. As a typical UPR factor, ATF4 is known to be able to directly bind to a conserved motif to drive gene transcription during ER stress. Several classical UPR genes, including GADD34, CHOP, and ATF3, are transcriptionally regulated through the ATF4 motif in the promoter regions of these genes. Surprisingly, we found that although the DNA motif bound by ATF4 remains almost unchanged, the transcriptome driven by ATF4 during HSC activation changed significantly compared to ER stress. After further analyzing the histone modifications of the new genes targeted by ATF4, we found that ATF4 tended to bind to enhancer regions during HSC activation. Therefore, we propose a model: the activation of HSCs or the induction of the EMT program can reset the chromatin accessibility landscape, enabling ATF4 to turn to key genes for HSC activation (such as Col1A1 and LOXL2), thereby promoting their transcription.

[0354] Fibrosis remains a reversible and potentially curable stage during the progression of liver disease. The inventor team of this application adopted a method based on specifically knocking out animals of HSCs, and clearly demonstrated that the expression of ATF4 in HSCs is crucial for HSC activation, revealing that ATF4 is an important target for the treatment of liver fibrosis; the inventor team of this application also verified through chemical intervention means that the upstream factor p-eIF2-alpha of ATF4 is a feasible target.

[0355] Chemical inhibition of p-eIF2-alpha can effectively inhibit the development of liver fibrosis in mice. The chemical inhibitor of p-eIF2-alpha, ISRIB, was developed by the Peter Walters group several years ago and has been applied in a series of tests for neurological syndromes. Studies have shown that ISRIB has a neuroprotective effect. It can improve the memory function of aged and traumatic brain injury mice, prevent neurodegeneration in prion disease mice, reduce neuropathic pain caused by diabetes, and restore the impaired social and anxiety-like behaviors in adult cacna1c-deficient mice. The research of this application first discovered that the known chemical drug ISRIB also has the use of inhibiting liver fibrosis.

[0356] In addition, the safety of targeting the eIF2α / ATF4 pathway has been reported not only in many animal studies but also verified in a recent phase I clinical trial for amyotrophic lateral sclerosis (ALS) (W. Cho, A. Jeong, P. Malik, J. Boiser, X. Huang, M. Rosebraugh, A Phase 1 First-in-human Study to Investigate the Safety, Tolerability and Food Effect of ABBV-CLS-7262 (P6-4.002). Neurology 100(17 Supplement 2) (2023)). Considering several key liver fibrosis drivers reported currently (such as the TGFβ signaling pathway), the significant / severe toxicity existing in blocking the pathways of these key drivers has led to none of them being applicable to the clinical development of fibrosis treatment. However, the clinical application of the method of inhibiting the eIF2α / ATF4 signaling pathway has the advantages of safety and effectiveness.

[0357] In summary, the inventors of this application believe that inhibiting the eIF2α / ATF4 signaling pathway is a promising and feasible strategy for intervening in liver fibrosis.

[0358] The present invention is not limited to the above embodiments. The protection scope of the present invention is defined by the appended claims. Those skilled in the art can make various changes, modifications, substitutions, combinations, and simplifications to these embodiments without departing from the principles and essence of the present invention, and all of them should be equivalent replacement methods and fall within the protection scope of the present invention.

Claims

1. Use of a reagent for targeted inhibition of the eIF2α / ATF4 pathway in the preparation of a medicament for treating liver fibrosis diseases, or in the preparation of a medicament for preventing liver cirrhosis and / or liver cancer, characterized in that: The reagent is a reagent that targets and inhibits the expression or activity of proteins in the eIF2α / ATF4 pathway.

2. The use according to claim 1, wherein the reagent is a reagent that targets the eIF2α / ATF4 pathway in hepatic stellate cells; preferably, the reagent is selected from any one of the following or a combination thereof: a reagent targeting ATF4, a reagent targeting p-eIF2-alpha; the reagent targeting ATF4 is a reagent that inhibits, reduces, knocks out, knocks down or reduces the expression level of the ATF4 gene, or inhibits, reduces or inactivates the activity of the expression product of the ATF4 gene; the reagent targeting p-eIF2-alpha is a reagent that inhibits, reduces, knocks out, knocks down or reduces the expression level of the EIF2S1 gene, or inhibits, reduces or inactivates the activity of the expression product of the EIF2S1 gene, or inhibits, reduces or inactivates the p-eIF2-alpha protein; the expression level is at the nucleic acid level or the protein level; preferably, the expression product is selected from any one of the following or a combination thereof: cDNA, mRNA, non-codingRNA, precursor protein, mature protein or a fragment thereof, or a modified product of the protein; preferably, the modified product of the protein includes a phosphorylated product of the protein; preferably, the reagent is selected from small molecule inhibitors, carbohydrates, lipids, proteins, polypeptides, nucleic acid molecules or interfering viruses.

3. The use according to claim 2, characterized in that, The reagent is selected from any one of the following: small molecule inhibitors, antisense oligonucleotides, siRNA, shRNA, dsRNA, microRNA, LncRNA, sgRNA, esiRNA, antibodies or antigen-binding fragments thereof; preferably, the antigen-binding fragment is selected from any one of the following or a combination thereof: Fab, Fab’, F(ab’)2, Fv fragment, ScFv, single-chain antibody or domain antibody.

4. The use according to claim 3, characterized in that, The reagent targeting p-eIF2-alpha is ISRIB.

5. The use according to claim 2, wherein the reagent comprises any one of the following: a) siRNA, shRNA, dsRNA, microRNA, LncRNA, sgRNA or esiRNA that targets the ATF4 or EIF2S1 gene and knocks out, knocks down or reduces the expression of the ATF4 or EIF2S1 gene; b) an expression vector that contains a gene fragment encoding the item a) and is capable of expressing the item a); c) a recombinant viral vector formed by cloning the gene fragment encoding the item a) into the coding region of a viral vector, and the viral vector is any one of a lentiviral vector, an adeno-associated viral vector or a retroviral vector; d) a virus obtained by transfecting a eukaryotic cell with a virus packaging system; the virus packaging system contains the recombinant viral vector of the item c).

6. Use of eIF2α / ATF4 pathway proteins and / or their encoding genes in screening drugs for treating liver fibrosis diseases, or in screening drugs for preventing liver cirrhosis and / or liver cancer.

7. The use according to claim 6, characterized in that: The eIF2α / ATF4 pathway proteins are selected from ATF4 or p-eIF2-alpha.

8. A pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier or excipient, wherein the active ingredient comprises a reagent that targets and inhibits the expression or activity of eIF2α / ATF4 pathway proteins.

9. The pharmaceutical composition according to claim 8, wherein: The reagent is a reagent that targets the eIF2α / ATF4 pathway in hepatic stellate cells; Preferably, the reagent is selected from any one of the following or a combination thereof: a reagent that targets ATF4, a reagent that targets p-eIF2-alpha; The reagent that targets ATF4 is a reagent that inhibits, reduces, knocks out, knocks down or knockdowns the expression level of the ATF4 gene, or inhibits, reduces or inactivates the activity of the expression product of the ATF4 gene; The reagent that targets p-eIF2-alpha is a reagent that inhibits, reduces, knocks out, knocks down or knockdowns the expression level of the EIF2S1 gene, or inhibits, reduces or inactivates the activity of the expression product of the EIF2S1 gene, or inhibits, reduces or inactivates the p-eIF2-alpha protein; The expression level is at the nucleic acid level or the protein level; Preferably, the expression product is selected from any one of the following or a combination thereof: cDNA, mRNA, non-codingRNA, precursor protein, mature protein or its fragment, or a modified product of the protein; preferably, the modified product of the protein includes a phosphorylated product of the protein; Preferably, the reagent is selected from small molecule inhibitors, carbohydrates, lipids, proteins, polypeptides, nucleic acid molecules or interfering viruses; more preferably, the reagent is selected from any one of the following: small molecule inhibitors, antisense oligonucleotides, siRNA, shRNA, dsRNA, microRNA, LncRNA, sgRNA, esiRNA, antibodies or antigen-binding fragments thereof; more preferably, the antigen-binding fragment is selected from any one of the following or a combination thereof: Fab, Fab’, F(ab’)2, Fv fragment, ScFv, single-chain antibody or domain antibody.

10. The pharmaceutical composition according to claim 9, wherein the reagent comprises any one of the following: a) siRNA, shRNA, dsRNA, microRNA, LncRNA, sgRNA or esiRNA that targets the ATF4 or EIF2S1 gene and knocks out, knocks down or knockdowns the expression of the ATF4 or EIF2S1 gene; b) An expression vector comprising a gene fragment encoding the item a) and capable of expressing the item a); c) A recombinant viral vector formed by cloning the gene fragment encoding the item a) into the coding region of a viral vector; preferably, the viral vector is any one of a lentiviral vector, an adeno-associated viral vector or a retroviral vector; d) A virus obtained by transfecting a eukaryotic cell with a virus packaging system; the virus packaging system contains the recombinant viral vector of item c).

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