PI4k inhibitor and use thereof

Abstract

A PI4K inhibitor and use thereof, especially use in blocking or inhibiting autophagy caused by RAS mutations or treating tumors associated with RAS mutations. In addition, the present invention reveals that autophagy caused by RAS mutations is completely different in mechanism from existing starvation-induced autophagy. The pathway of autophagy caused by RAS mutations is P38-ULK1-PI4KB-WIPI2, especially the S256 and T263 sites of ULK1 phosphorylated PI4KB. It has been experimentally confirmed that the specificity of the pathway can be used as a diagnostic marker and a therapeutic target of tumors.

Description

PI4K inhibitors and their applications Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to P38-ULK1-PI4KB-WIPI2 pathway inhibitors and their application in blocking or inhibiting autophagy caused by RAS mutations or in treating RAS-mutant tumors. Background Technology

[0002] Macroautophagy (hereinafter referred to as autophagy) is a conserved degradative metabolic pathway in eukaryotic cells, crucial for maintaining cellular homeostasis and survival under stress. During autophagy, damaged organelles, invasive bacteria, and easily aggregated proteins are degraded via the lysosomal pathway. Autophagy is associated with the pathogenesis of various diseases; for example, autophagy dysregulation is linked to cancer. However, the specific molecular mechanisms leading to autophagy dysregulation, particularly the differences between cancer-associated autophagy and physiological autophagy (such as starvation-induced autophagy), remain unclear.

[0003] The RAS gene family, including HRAS, KRAS, and NRAS, encodes remarkably similar proteins of 188-189 amino acids and is the most common oncogene in human cancers. RAS genes encode monomeric GTPases, which act as molecular switches in signal transduction pathways regulating mammalian cell proliferation, differentiation, and survival. Constitutive activating RAS mutations occur in 20-25% of human cancers, with KRAS mutations being the most frequent and occurring in multiple cancer types. In many tumors, KRAS mutation activation is a key driver of cancer development and progression and is crucial for tumor growth. However, highly active RAS mutants are difficult to target due to the lack of drug-binding pockets on the RAS protein surface. To date, the only clinically effective inhibitors are AMG510 and MRTX849, which specifically target KRAS G12C.

[0004] Besides directly targeting RAS proteins, identifying drugs that inhibit downstream RAS effector proteins is a solution for treating cancers with RAS mutations. Studies have shown that activation of RAS mutations is associated with excessive activation of autophagy. RAS-activated autophagy has unique characteristics, such as providing nutrients for tumor growth, immune evasion, and remodeling the proteome through selective degradation, such as eliminating harmful components of inflammatory pathways to prevent cytokine-induced paracrine cell death. Therefore, RAS-mutant tumor cells are highly sensitive to autophagy inhibitors, suggesting that autophagy could serve as a therapeutic target for RAS-mutant tumors. Since autophagy can also maintain cellular homeostasis under physiological conditions, an autophagy inhibitor should be developed to specifically block RAS-induced autophagy without affecting physiological autophagy. However, the regulatory differences between RAS mutation-induced autophagy and physiological autophagy remain unclear, particularly regarding the different involvement of core autophagy factors, namely autophagy-associated genes (ATGs) and proteins.

[0005] The core steps of autophagy, including the biogenesis and maturation of autophagosomes, are regulated by ATG and proteins. Under starvation-induced autophagy (a physiological form of autophagy), components of the UNC-51-like kinase (ULK) complex, including ULK1 / 2, ATG13, and Rb1-inducible coiled protein 1 (RB1CC1 / FIP200 and ATG101), are activated by either inhibiting the mechanistic target of rapamycin complex 1 (mTORC1) or activating AMPK. Downstream of the ULK complex is the class III phosphatidylinositol 3-kinase (PI3K) complex, which consists of Beclin1, ATG14, PIK3 catalytic subunit 3 (PIK3C3 / VPS34), PIK3 regulatory subunit 4 (PIK3R4 / P150), and activating molecules in autophagy protein 1 regulated by Beclin-1. This secondary complex phosphorylates phosphatidylinositol (PI) lipids in the phagosome to recruit phosphatidylinositol-3-phosphate (PI3P) interacting proteins, such as WD repeat domain phosphatidylinositol interacting 2 (WIPI2). WIPI2 is an effector of PI3P, activating downstream events by recruiting lipid factors (ATG5-ATG12 conjugates that form complexes with ATG16, ATG3, and ATG7). The esterification mechanism catalyzes the binding of microtubule-associated protein 1 light chain 3 (MAP1LC3 / LC3 / ATG8) to phosphatidylethanolamine (PE) to construct the autophagosome. ATG9, acting as the seed of the autophagosome, along with ATG2, acts as a lipid scramblase to transfer lipids to the autophagic membrane. The ESCRT (Endosomal Sorting Complex for Transport) complex closes the phagosome, thus completing autophagosome biogenesis. Multiple SNARE complexes, along with membrane chain components, promote the fusion of the autophagosome with the lysosome. The cascade signaling and function of ATGs in physiological autophagy have been largely elucidated, but little is known about how ATGs are regulated under pathological conditions.

[0006] Summary of the Invention

[0007] To address the shortcomings of existing technologies, this application first clarifies the differences between RAS-mediated autophagy and physiological autophagy regulation, particularly the substitution of classical PI3K by PI4KB. It further reveals the P38-ULK1-PI4KB-WIPI2 signaling cascade in RAS-mediated autophagy, specifically the phosphorylation of ULK1 at a specific site, which leads to the selection of PI4KB as a substrate for phosphorylation, thereby initiating PI4P production and autophagy. More importantly, this application experimentally demonstrates the regulatory effects of ULK1-PI4K inhibitors on RAS-mediated autophagy and their therapeutic effects on tumors. For example, peptides competitively phosphorylated with PI4K or overexpression of phosphorylation-deficient PI4KB show comparable or better efficacy than chloroquine in inhibiting autophagy and the growth of KRAS-mutant tumors. Moreover, under normal conditions, peptide-1 phosphorylation has a negligible impact on PI4KB activity. This indicates that targeting peptide-1 phosphorylation minimizes potential side effects.

[0008] Furthermore, PI4KB phosphorylation levels are significantly elevated in RAS-mutant tumors, especially the levels of peptides containing specific site phosphorylation. Further analysis of the mechanistic characteristics of RAS-mediated autophagy suggests that PI4KB phosphorylation (especially specific site phosphorylation) can serve as a biomarker for the diagnosis or prognostic assessment of tumors, as well as a specific marker of RAS-mediated autophagy.

[0009] In a first aspect, the present invention provides the use of a PI4K inhibitor in the preparation of a medicament for the prevention and / or treatment of tumors, wherein the PI4K inhibitor blocks or inhibits PI4K phosphorylation.

[0010] PI4K is phosphatidylinositol 4-phosphokinase, which includes PI4K-IIα, PI4K-IIβ, PI4K-IIIα, and PI4KB.

[0011] Preferably, the PI4K inhibitor prevents and / or treats tumors by blocking or inhibiting autophagy caused by RAS mutations.

[0012] Preferably, the PI4K inhibitor prevents and / or treats tumors by blocking or reducing PI4P levels. More preferably, it prevents and / or treats tumors by blocking or reducing PI4P levels, thereby blocking or reducing its recruitment of interacting proteins.

[0013] Further optimization is needed to identify the number of interacting proteins that block or reduce PI4P recruitment or their expression levels.

[0014] The interacting proteins mentioned include WD repeat domain phosphoinositol interacting protein 2 (WIPI2).

[0015] Preferably, the PI4K inhibitor prevents and / or treats tumors by blocking or inhibiting ULK1 phosphorylation-induced PI4K phosphorylation.

[0016] In one specific embodiment of the present invention, the PI4K inhibitor blocks or inhibits PI4K phosphorylation induced by ULK1 phosphorylation, thereby blocking or reducing PI4P levels, blocking or reducing the levels of its recruitment interacting proteins, and thus blocking or inhibiting autophagy caused by RAS mutations, thereby achieving the prevention and / or treatment of tumors.

[0017] The ULK1 phosphorylation includes ULK1 phosphorylation produced via the P38 pathway. Preferably, the ULK1 phosphorylation sites include one or more of S317, S556, S758, or S479.

[0018] The sequence of ULK1 can be obtained from existing technologies, preferably SEQ ID NO: 82.

[0019] The PI4K mentioned is a PI4K complex or PI4KB.

[0020] The phosphorylation sites of PI4KB include S256 and / or T263.

[0021] The sequence of PI4KB can be obtained from existing technology, preferably SEQ ID NO: 81.

[0022] Preferably, the tumor is a RAS-mutant tumor.

[0023] Preferably, the tumor is selected from colorectal cancer, pancreatic cancer, bile duct cancer, lung cancer (e.g., non-small cell lung cancer), ovarian cancer, thyroid cancer, bladder cancer, breast cancer, liver cancer, melanoma, myelodysplastic syndrome, lymphoma, endometrial cancer, esophageal cancer, glioma, head and neck squamous cell carcinoma, urothelial carcinoma, neuroblastoma, renal cancer, leukemia, or multiple myeloma.

[0024] More preferably, the tumor is colonic adenocarcinoma, rectal adenocarcinoma, colorectal adenocarcinoma, lung adenocarcinoma, pancreatic cancer, or cholangiocarcinoma.

[0025] In one specific embodiment of the present invention, the tumor is colon cancer, lung cancer, or pancreatic cancer with RAS mutation.

[0026] Preferably, the RAS mutation is one or more of HRAS mutation, KRAS mutation, or NRAS mutation, preferably a KRAS mutation, such as one or more mutations of amino acids at positions 12, 13, 18, 59, 61, 62, 146, and 117, preferably a mutation of amino acid at position 12, and more preferably one or more of G12V, G12C, G12D, G12S, G12R, G13D, G13C, A18D, A59D, A59T, Q61H, Q61K, Q61L, Q61R, E62G, A146T, and K117N.

[0027] The PI4K inhibitors include reagents for gene knockout, reagents for gene silencing, antisense nucleic acids, reagents for gene mutation, small molecule compounds or their pharmaceutically acceptable salts, peptides or their expression promoters, peptide mutants or their expression promoters, fusion proteins, antibodies, traditional Chinese medicine or extracts of traditional Chinese medicine.

[0028] The gene knockout mentioned includes CRISPR or tissue-specific knockout.

[0029] The reagents required for gene silencing include interfering RNA, such as one or more of siRNA, dsRNA, shRNA, aiRNA, or miRNA.

[0030] Preferably, the siRNA targets one, two, or three of PI4KB, ULK1, or WIPI2.

[0031] Further preferably, the target site sequence of the siRNA targeting ULK1 includes SEQ ID NO: 2 and / or SEQ ID NO: 3;

[0032] Further preferably, the target site sequence of the siRNA targeting PI4KB includes one or more of SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27;

[0033] Further preferably, the target site sequence of the siRNA targeting WIPI2 includes one or more of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

[0034] Preferably, the shRNA targets PI4KB and / or ULK1.

[0035] Further preferably, the target site sequence of the shRNA targeting ULK1 includes SEQ ID NO: 29 and / or SEQ ID NO: 30;

[0036] Further preferably, the target site sequence of the shRNA targeting PI4KB includes one or more of SEQ ID NO: 37 and / or SEQ ID NO: 38.

[0037] Preferably, the reagents required for gene mutation include mutating the serine / threonine residues at the ULK1 phosphorylation and / or PI4KB phosphorylation sites. For example, mutating to a non-natural amino acid or to an amino acid that cannot be phosphorylated.

[0038] Preferably, the polypeptide is a polypeptide that competitively phosphorylates PI4K, such as an amino acid fragment of PI4KB containing the S256 and / or T263 sites.

[0039] Preferably, the length of the polypeptide is at least 8 aa, more preferably 8-60 aa, for example 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, 25 aa, 30 aa, 35 aa, 40 aa, 45 aa, 50 aa, 55 aa, 60 aa, and more preferably 8-20 aa or 8-30 aa.

[0040] Further preferably, the polypeptide includes at least positions 256 to 263 of the amino acid sequence of PI4KB, for example, including any one of SEQ ID NO: 1 or 83-88, or including an amino acid sequence having more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% identity with any one of SEQ ID NO: 1 or 83-88, or including a substituted, deleted, or inserted amino acid sequence having at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid.

[0041] The sequence of the polypeptide may include modifications, such as fatty acid modification, methylation, acetylation, hydroxylation, carboxylation, phosphorylation, etc.

[0042] The peptide expression promoters include peptides that can enhance the expression of peptides that competitively phosphorylate PI4K, such as overexpressing PI4K or its peptides or peptide modifications.

[0043] The peptide mutants include amino acid fragments of PI4KB that are mutated to one or more sites of S256, S258, T263 or S266 to be neither threonine nor serine, for example, to alanine or a non-natural amino acid.

[0044] More preferably, the polypeptide mutant includes a mutation of serine and threonine at positions 256 to 266 to alanine, such as a mutant of SEQ ID NO: 1 or any of 83-88.

[0045] The fusion protein includes a polypeptide that competitively phosphorylates PI4K, and preferably also includes an N-terminal Tat protein transduction domain. More preferably, the polypeptide and the N-terminal Tat protein transduction domain are linked by a GG linker. For example, the fusion protein may include SEQ ID NO: 57 or include an amino acid sequence that has more than 80% identity with SEQ ID NO: 57 or include an amino acid sequence that has at most 10 substituted, deleted or inserted amino acids with SEQ ID NO: 57.

[0046] Preferably, the small molecule compound is selected from arsenic oxyphenylene oxide or its derivatives, (aS)-5-(2-amino-4-oxo-3-(2-(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-6-yl)-N-(2,4-difluorophenyl)-2-methoxypyridine-3-sulfonamide or its analogues, 5-(2-amino-1-(4-(4-morpholinyl)phenyl)-1H-benzimidazol-6-yl)-N-(2-fluorophenyl)-2-methoxy-3-pyridinesulfonamide or its analogues, 2-amino-5-phenylthiazole, substituted 2-amino-5-pyridylthiazole, simeprevir or its analogues, T-00127_HEV1 (CAS: 900874-91-1), PIK93 (CAS: 593960-11-3), or Wollman penicillin.

[0047] Among them, the T-00127_HEV1 structure is

[0048] The PIK93 structure is as follows:

[0049] Preferably, the drug further includes a second active agent, including but not limited to EGFR inhibitors, RAS inhibitors, SHP2 inhibitors, SOS1 inhibitors, Raf inhibitors, MEK inhibitors, ERK inhibitors, PI3K inhibitors, PTEN inhibitors, AKT inhibitors, mTORC1 inhibitors, BRAF inhibitors, PD-L1 inhibitors, PD-1 inhibitors, CDK4 / 6 inhibitors, HER2 inhibitors, ULK1 inhibitors, or combinations thereof; preferably a MEK inhibitor.

[0050] In one specific embodiment of the present invention, the drug comprises a PI4K inhibitor and a MEK inhibitor, which may be a MEK1 and / or MEK2 inhibitor. The MEK inhibitor is selected from binimetinib, cobimetinib, trametinib, LNP-3794, HL-085, antroquinonol, E-6201, remetinib, mirdametinib, pimasertib, selumetinib, SHR-7390, CKI-27, GS-4875, ATR-001, ATR-002, ATR-006, ATR-004, ATR-005, CS-3006, FCN-159, CIP-137401, EB I-1051, SC-1-151, SRX-2626, EDV-2209, WX-554, GDC-0623, TAK-733, E-6201, RG- 7167, AZD-8330, PD-184352, GSK-2091976A, AS-703988, BI-847325, JTP-70902, CZ -775, RO4987655, RO5126766, RO-5068760, RDEA-436, MEK-300, AD-GL0001, SL-327, CI-1040, CInQ-03, G-573, PD184161, PD318088, PD98059, U0126, SL327, preferably trametinib.

[0051] The drug also includes a carrier, such as a viral vector or a non-viral vector.

[0052] The drug also includes pharmaceutically acceptable excipients.

[0053] The PI4K inhibitor and the second active agent can be administered simultaneously, separately, or sequentially. They can be formulated in the same formulation or in different formulations. They can be administered via corresponding methods (e.g., both by injection) or via different methods (e.g., one by oral administration and the other by injection).

[0054] A second aspect of the invention provides the use of PI4K inhibitors in blocking or inhibiting autophagy caused by RAS mutations, or in the preparation of medicaments for blocking or inhibiting autophagy caused by RAS mutations.

[0055] The PI4K inhibitors described herein block or inhibit PI4K phosphorylation.

[0056] Preferably, the PI4K inhibitor blocks or reduces PI4P levels, and more preferably blocks or reduces its recruitment of interacting proteins, thereby blocking or inhibiting autophagy induced by RAS mutations. More preferably, it blocks or reduces the number of interacting proteins recruited by PI4P or their expression levels.

[0057] The interacting proteins mentioned include WD repeat domain phosphoinositol interacting protein 2 (WIPI2).

[0058] Preferably, the PI4K inhibitor blocks or inhibits PI4K phosphorylation induced by ULK1 phosphorylation, thereby blocking or inhibiting autophagy caused by RAS mutations.

[0059] In one specific embodiment of the present invention, the PI4K inhibitor blocks or reduces PI4P levels by blocking or inhibiting ULK1 phosphorylation-induced PI4K phosphorylation, thereby blocking or reducing the level of its recruitment interacting proteins, and thus blocking or inhibiting autophagy caused by RAS mutations.

[0060] The ULK1 phosphorylation includes ULK1 phosphorylation produced via the P38 pathway. Preferably, the ULK1 phosphorylation sites include one or more of S317, S556, S758, or S479.

[0061] The PI4K mentioned is a PI4K complex or PI4KB.

[0062] The phosphorylation sites of PI4KB include S256 and / or T263.

[0063] Preferably, the RAS mutation is one or more of HRAS mutation, KRAS mutation, or NRAS mutation, preferably a KRAS mutation, such as one or more mutations of amino acids at positions 12, 13, 18, 59, 61, 62, 146, and 117, preferably a mutation of amino acid at position 12, and more preferably one or more of G12V, G12C, G12D, G12S, G12R, G13D, G13C, A18D, A59D, A59T, Q61H, Q61K, Q61L, Q61R, E62G, A146T, and K117N.

[0064] The PI4K inhibitors include reagents for gene knockout, reagents for gene silencing, antisense nucleic acids, reagents for gene mutation, small molecule compounds or their pharmaceutically acceptable salts, peptides or their expression promoters, peptide mutants or their expression promoters, fusion proteins, antibodies, traditional Chinese medicine or extracts of traditional Chinese medicine.

[0065] The gene knockout mentioned includes CRISPR or tissue-specific knockout.

[0066] The reagents required for gene silencing include interfering RNA, such as one or more of siRNA, dsRNA, shRNA, aiRNA, or miRNA.

[0067] Preferably, the siRNA targets one, two, or three of PI4KB, ULK1, or WIPI2.

[0068] Further preferably, the target site sequence of the siRNA targeting ULK1 includes SEQ ID NO: 2 and / or SEQ ID NO: 3;

[0069] Further preferably, the target site sequence of the siRNA targeting PI4KB includes one or more of SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27;

[0070] Further preferably, the target site sequence of the siRNA targeting WIPI2 includes one or more of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

[0071] Preferably, the shRNA targets PI4KB and / or ULK1.

[0072] Further preferably, the target site sequence of the shRNA targeting ULK1 includes SEQ ID NO: 29 and / or SEQ ID NO: 30;

[0073] Further preferably, the target site sequence of the shRNA targeting PI4KB includes one or more of SEQ ID NO: 37 and / or SEQ ID NO: 38.

[0074] Preferably, the reagents required for gene mutation include mutating the serine / threonine residue at the PI4KB phosphorylation site. For example, mutating it to a non-natural amino acid or to an amino acid that cannot be phosphorylated.

[0075] Preferably, the polypeptide is a polypeptide that competitively phosphorylates PI4K, such as an amino acid fragment of PI4KB containing the S256 and / or T263 sites.

[0076] Preferably, the length of the polypeptide is at least 8 aa, more preferably 8-60 aa, for example 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, 25 aa, 30 aa, 35 aa, 40 aa, 45 aa, 50 aa, 55 aa, 60 aa, and more preferably 8-20 aa or 8-30 aa.

[0077] Further preferably, the polypeptide includes at least positions 256 to 263 of the amino acid sequence of PI4KB, for example, including any one of SEQ ID NO: 1 or 83-88, or including an amino acid sequence having more than 80%, 85%, 90%, 95%, or 98% identity with any one of SEQ ID NO: 1 or 83-88, or including a substituted, deleted, or inserted amino acid sequence having at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid.

[0078] The peptide mutants include amino acid fragments of PI4KB that are mutated to one or more sites of S256, S258, T263 or S266 to be neither threonine nor serine, for example, to alanine or a non-natural amino acid.

[0079] More preferably, the polypeptide mutant includes a mutation of serine and threonine at positions 256 to 266 to alanine, such as a mutant of SEQ ID NO: 1 or any of 83-88.

[0080] The fusion protein includes a polypeptide that competitively phosphorylates PI4K, and preferably also includes an N-terminal Tat protein transduction domain. More preferably, the polypeptide and the N-terminal Tat protein transduction domain are linked by a GG linker. For example, the fusion protein may include SEQ ID NO: 57 or include an amino acid sequence that has more than 80% identity with SEQ ID NO: 57 or include an amino acid sequence that has at most 10 substituted, deleted or inserted amino acids with SEQ ID NO: 57.

[0081] Preferably, the small molecule compound is selected from arsenic oxyphenylene oxide or its derivatives, (aS)-5-(2-amino-4-oxo-3-(2-(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-6-yl)-N-(2,4-difluorophenyl)-2-methoxypyridine-3-sulfonamide or its analogues, 5-(2-amino-1-(4-(4-morpholinyl)phenyl)-1H-benzimidazol-6-yl)-N-(2-fluorophenyl)-2-methoxy-3-pyridinesulfonamide or its analogues, 2-amino-5-phenylthiazole, substituted 2-amino-5-pyridylthiazole, simeprevir or its analogues, T-00127_HEV1 (CAS: 900874-91-1), PIK93 (CAS: 593960-11-3), or Wollman penicillin.

[0082] A third aspect of the invention provides the use of a combination of a PI4K inhibitor and a second active agent (e.g., a MEK inhibitor) in the preparation of a medicament for the prevention and / or treatment of tumors.

[0083] The combination of the PI4K inhibitor and the second active agent (e.g., a MEK inhibitor) represents simultaneous, separate, or sequential administration. They can be formulated in the same or different formulations. They can be administered via corresponding methods (e.g., both by injection) or different methods (e.g., one by oral administration and the other by injection).

[0084] A fourth aspect of the invention provides the use of a combination of a PI4K inhibitor and a second active agent (e.g., a MEK inhibitor) in blocking or inhibiting autophagy caused by RAS mutations, or in the preparation of a medicament for blocking or inhibiting autophagy caused by RAS mutations.

[0085] The combination of the PI4K inhibitor and the second active agent (e.g., a MEK inhibitor) represents simultaneous, separate, or sequential administration. They can be formulated in the same or different formulations. They can be administered via corresponding methods (e.g., both by injection) or different methods (e.g., one by oral administration and the other by injection).

[0086] In a fifth aspect, the present invention provides the application of a ULK1-PI4K inhibitor, wherein the ULK1-PI4K is ULK1 phosphorylation-induced PI4K phosphorylation, and the ULK1-PI4K inhibitor blocks or inhibits ULK1 phosphorylation-induced PI4K phosphorylation.

[0087] The applications include:

[0088] A) Block or inhibit autophagy caused by RAS mutations;

[0089] B) Application in the preparation of drugs that block or inhibit autophagy caused by RAS mutations;

[0090] C) Use in the preparation of medicaments for the prevention and / or treatment of tumors. Preferably, ULK1-PI4K inhibitors prevent and / or treat tumors by blocking or inhibiting autophagy induced by RAS mutations.

[0091] The ULK1 phosphorylation sites include one or more of S317, S556, S758, or S479. Preferably, the ULK1 phosphorylation includes ULK1 phosphorylation generated via the P38 pathway.

[0092] The PI4K mentioned is a PI4K complex or PI4KB.

[0093] The phosphorylation sites of PI4KB include S256 and / or T263.

[0094] The ULK1-PI4K inhibitors include ULK1 inhibitors and / or PI4K inhibitors.

[0095] Preferably, the ULK1 inhibitor blocks or inhibits ULK1 phosphorylation; more preferably, it includes inhibiting at least one or more of the phosphorylations of S317, S556, S758 or S479.

[0096] Preferably, the PI4KB inhibitor blocks or inhibits PI4KB phosphorylation, and more preferably, it includes inhibiting at least the phosphorylation of S256 and / or T263.

[0097] The ULK1 inhibitors include reagents for gene knockout, reagents for gene silencing, antisense nucleic acids, reagents for gene mutation, small molecule compounds or their pharmaceutically acceptable salts, peptides or their expression promoters, antibodies, traditional Chinese medicine or extracts of traditional Chinese medicine.

[0098] The gene knockout mentioned includes CRISPR or tissue-specific knockout.

[0099] The reagents required for gene silencing include interfering RNA, such as one or more of siRNA, dsRNA, shRNA, aiRNA, or miRNA.

[0100] Preferably, the target site sequence of the siRNA targeting ULK1 includes SEQ ID NO: 2 and / or SEQ ID NO: 3.

[0101] Preferably, the target site sequence of the shRNA targeting ULK1 includes SEQ ID NO: 29 and / or SEQ ID NO: 30.

[0102] Preferably, the reagents required for gene mutation include mutating the phosphorylated serine / threonine of ULK1. For example, mutating to a non-natural amino acid or to an amino acid that cannot be phosphorylated.

[0103] Preferably, the polypeptide is a polypeptide that competitively phosphorylates ULK1, such as an amino acid fragment of ULK1 containing one or two of the sites S317, S556, S758 or S479.

[0104] Preferably, the length of the polypeptide is at least 8 aa, more preferably 8-60 aa, for example 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, 25 aa, 30 aa, 35 aa, 40 aa, 45 aa, 50 aa, 55 aa, 60 aa, and more preferably 8-20 aa or 8-30 aa.

[0105] The small molecule compound mentioned, such as SBI-0206965, has the following structure:

[0106] Preferably, the ULK1-PI4K inhibitor blocks or inhibits autophagy induced by RAS mutations by blocking or reducing PI4P levels, and more preferably by blocking or reducing its recruitment of interacting proteins. More preferably, it blocks or reduces the number of interacting proteins recruited by PI4P or their expression levels.

[0107] The interacting proteins mentioned include WD repeat domain phosphoinositol interacting protein 2 (WIPI2).

[0108] Preferably, ULK1-PI4K inhibitors block or inhibit PI4K phosphorylation induced by ULK1 phosphorylation, thereby blocking or inhibiting autophagy induced by RAS mutations.

[0109] In one specific embodiment of the present invention, the ULK1-PI4K inhibitor blocks or reduces PI4P levels by blocking or inhibiting ULK1 phosphorylation-induced PI4K phosphorylation, thereby blocking or reducing the levels of interacting proteins recruited by PI4P, and thus blocking or inhibiting autophagy caused by RAS mutations.

[0110] Preferably, the tumor is a RAS-mutant tumor.

[0111] Preferably, the tumor is selected from colorectal cancer, pancreatic cancer, bile duct cancer, lung cancer (e.g., non-small cell lung cancer), ovarian cancer, thyroid cancer, bladder cancer, breast cancer, liver cancer, melanoma, myelodysplastic syndrome, lymphoma, endometrial cancer, esophageal cancer, glioma, head and neck squamous cell carcinoma, urothelial carcinoma, neuroblastoma, renal cancer, leukemia, or multiple myeloma.

[0112] More preferably, the tumor is colon cancer, lung cancer, or pancreatic cancer with RAS mutation.

[0113] Preferably, the RAS mutation is one or more of HRAS mutation, KRAS mutation, or NRAS mutation, preferably a KRAS mutation, such as one or more mutations of amino acids at positions 12, 13, 18, 59, 61, 62, 146, and 117, preferably a mutation of amino acid at position 12, and more preferably one or more of G12V, G12C, G12D, G12S, G12R, G13D, G13C, A18D, A59D, A59T, Q61H, Q61K, Q61L, Q61R, E62G, A146T, and K117N.

[0114] In a sixth aspect, the present invention provides an application of a P38-ULK1-PI4KB-WIPI2 pathway inhibitor, the application including its use in blocking or inhibiting autophagy caused by RAS mutations; or, its use in the preparation of a medicament for blocking or inhibiting autophagy caused by RAS mutations; or, its use in the preparation of a medicament for the prevention and / or treatment of tumors.

[0115] The P38-ULK1-PI4KB-WIPI2 pathway inhibitors include one or more of the following: P38 pathway inhibitors, ULK1 inhibitors, PI4KB inhibitors, or WIPI2 inhibitors.

[0116] The aforementioned P38 pathway inhibitor blocks or inhibits the production of ULK1 phosphorylation. Preferably, the P38 pathway inhibitor includes reagents required for gene knockout, reagents required for gene silencing, antisense nucleic acids, reagents required for gene mutation, small molecule compounds or their pharmaceutically acceptable salts, peptides or their expression promoters, antibodies, traditional Chinese medicine or extracts of traditional Chinese medicine. For example, SB203580 has the following structural formula:

[0117] The WIPI2 inhibitors include reagents for gene knockout, reagents for gene silencing, antisense nucleic acids, reagents for gene mutation, small molecule compounds or their pharmaceutically acceptable salts, peptides or their expression promoters, antibodies, traditional Chinese medicine or extracts of traditional Chinese medicine.

[0118] P38-ULK1-PI4KB-WIPI2 generates ULK1 phosphorylation via the P38 pathway. ULK1 phosphorylation induces PI4KB phosphorylation. WIPI2 acts as a PI4P effector and further recruits liposomes containing the ATG16L1 / ATG12-ATG5 complex to promote autophagosome membrane formation.

[0119] P38-ULK1-PI4KB-WIPI2 pathway inhibitors prevent and / or treat tumors by blocking or inhibiting autophagy induced by RAS mutations.

[0120] P38-ULK1-PI4KB-WIPI2 pathway inhibitors block or inhibit RAS mutation-induced autophagy by blocking or inhibiting PI4P levels and recruited WIPI2. Further optimization aims to block or inhibit the level or content of WIPI2 recruited by PI4P.

[0121] P38-ULK1-PI4KB-WIPI2 pathway inhibitors block or inhibit PI4K phosphorylation induced by ULK1 phosphorylation, thereby blocking or inhibiting autophagy induced by RAS mutations.

[0122] P38-ULK1-PI4KB-WIPI2 pathway inhibitors block or inhibit ULK1 phosphorylation-induced PI4K phosphorylation, thereby blocking or reducing PI4P levels, blocking or reducing the level or content of WIPI2 recruited by ULK1, and thus blocking or inhibiting autophagy induced by RAS mutations.

[0123] In a seventh aspect, the present invention provides the use of a PI4K phosphorylated peptide as a biomarker in the preparation of products for the diagnosis and / or prognostic assessment of tumors, wherein the PI4K is PI4KB.

[0124] Preferably, the PI4KB phosphorylation sites include at least S256 and / or T263.

[0125] Preferably, the tumor is a RAS-mutant tumor.

[0126] The PI4K phosphorylated polypeptide is induced by ULK1 phosphorylation. Preferably, the ULK1 phosphorylation site includes one or more of S317, S556, S758, or S479.

[0127] Preferably, the length of the PI4K phosphorylated polypeptide is at least 8 aa, more preferably 8-60 aa, for example 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, 25 aa, 30 aa, 35 aa, 40 aa, 45 aa, 50 aa, 55 aa, 60 aa, and more preferably 8-20 aa or 8-30 aa.

[0128] More preferably, the PI4K phosphorylated polypeptide includes at least a polypeptide phosphorylated at position 256 of the amino acid sequence of PI4KB, for example, at least 8 aa, preferably 8-60 aa, as the N-terminus of position 256, or at least 8 aa, preferably 8-60 aa, as the C-terminus of position 256, or at least 8 aa, preferably 8-60 aa, of the upstream and downstream amino acids of position 256.

[0129] More preferably, the PI4K phosphorylated polypeptide includes at least a polypeptide phosphorylated at position 263 of the amino acid sequence of PI4KB, for example, at least 8 aa, preferably 8-60 aa, as the N-terminus of position 263, or at least 8 aa, preferably 8-60 aa, as the C-terminus of position 263, or at least 8 aa, preferably 8-60 aa, of the upstream and downstream amino acids of position 263.

[0130] In one specific embodiment of the present invention, the PI4K phosphorylated polypeptide includes at least positions 256 to 263 of the amino acid sequence of PI4KB, and may also include upstream and / or downstream amino acid sequences. For example, it includes any one of SEQ ID NO: 1 or 83-88.

[0131] Preferably, the PI4K phosphorylated polypeptide is a PI4K phosphorylated polypeptide found in tumor cells.

[0132] The diagnostic and / or prognostic assessment of tumors includes detecting the expression level of PI4K phosphorylated peptides, which can be qualitative or quantitative. Preferably, the expression level of PI4K phosphorylated peptides in tumor cells is detected. For example, if the expression level is higher than (statistically significant, e.g., significantly higher than) a threshold, it is considered a tumor or has a poor prognosis. Preferably, the threshold is obtained through experience or extensive experiments; for example, the threshold can be the average, median, or highest value of PI4K phosphorylated peptide expression levels in healthy individuals.

[0133] The detection can be based on Western blotting, ELISA, flow cytometry, mass spectrometry, ICC / IHC, Bio-Plex suspension array, 32 P Isotope radiolabeling, etc.

[0134] In one specific embodiment of the invention, the detection uses a phosphorylated antibody that specifically recognizes the phosphorylation state of the PI4KB peptide. Specifically, the antibody can be prepared, for example, by synthesizing the PI4KB peptide, immunizing animals (such as mice or rabbits), and antibody screening.

[0135] The product may be a reagent kit, a chip, or a device. Preferably, the device may be a mass spectrometer, chromatography instrument, PCR apparatus, sequencing instrument, etc.

[0136] In an eighth aspect, the present invention provides the application of a PI4K phosphorylated polypeptide as a marker in the preparation of a product for identifying autophagy caused by RAS mutations, wherein the PI4K is PI4KB.

[0137] Preferably, the PI4KB phosphorylation sites include at least S256 and / or T263.

[0138] The PI4K phosphorylated polypeptide is induced by ULK1 phosphorylation. Preferably, the ULK1 phosphorylation site includes one or more of S317, S556, S758, or S479.

[0139] Preferably, the length of the PI4K phosphorylated polypeptide is at least 8 aa, more preferably 8-60 aa, for example 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, 25 aa, 30 aa, 35 aa, 40 aa, 45 aa, 50 aa, 55 aa, 60 aa, and more preferably 8-20 aa or 8-30 aa.

[0140] More preferably, the PI4K phosphorylated polypeptide includes at least a polypeptide phosphorylated at position 256 of the amino acid sequence of PI4KB, for example, at least 8 aa, preferably 8-60 aa, of the 256th phosphorylation as the N-terminus, or at least 8 aa, preferably 8-60 aa, of the 256th phosphorylation as the C-terminus, or at least 8 aa, preferably 8-60 aa, of the upstream amino acid at position 256, the 256th phosphorylation, and the downstream amino acid at position 256.

[0141] More preferably, the PI4K phosphorylated polypeptide includes at least a polypeptide phosphorylated at position 263 of the amino acid sequence of PI4KB, for example, at least 8 aa, preferably 8-60 aa, of the 263rd phosphorylation as the N-terminus, or at least 8 aa, preferably 8-60 aa, of the 263rd phosphorylation as the C-terminus, or at least 8 aa, preferably 8-60 aa, of the upstream amino acid at position 263, the 263rd phosphorylation, and the downstream amino acid at position 263.

[0142] In one specific embodiment of the present invention, the PI4K phosphorylated polypeptide includes at least positions 256 to 263 of the amino acid sequence of PI4KB, and may also include upstream and / or downstream amino acid sequences. For example, it includes any one of SEQ ID NO: 1 or 83-88.

[0143] Preferably, the PI4K phosphorylated polypeptide is a PI4K phosphorylated polypeptide found in tumor cells.

[0144] Preferably, identifying autophagy induced by RAS mutations includes detecting the presence or expression level of PI4K phosphorylated peptides. For example, the presence of PI4K phosphorylated peptides or ULK1 phosphorylation-induced PI4K phosphorylated peptides indicates the presence of autophagy induced by RAS mutations.

[0145] In a ninth aspect, the present invention provides a PI4KB inhibitor, wherein the PI4KB inhibitor is one or more of a polypeptide, a polypeptide mutant, a fusion protein, or interfering RNA, and the interfering RNA is siRNA and / or shRNA.

[0146] The polypeptide comprises at least the amino acid sequence at position 256 and / or position 263 of the PI4KB amino acid sequence.

[0147] Preferably, the length of the polypeptide is at least 8 aa, more preferably 8-60 aa, for example 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, 25 aa, 30 aa, 35 aa, 40 aa, 45 aa, 50 aa, 55 aa, 60 aa, and more preferably 8-20 aa or 8-30 aa.

[0148] Preferably, the polypeptide comprises at least 8 amino acids, preferably 8-60 amino acids, at positions 256 and / or 263 of the PI4KB amino acid sequence. For example, position 256 may be the N-terminus of at least 8 amino acids, preferably 8-60 amino acids; or position 256 may be the C-terminus of at least 8 amino acids, preferably 8-60 amino acids; or, positions upstream of position 256 and downstream of position 256 may comprise at least 8 amino acids, preferably 8-60 amino acids. As another example, position 263 may be the N-terminus of at least 8 amino acids, preferably 8-60 amino acids; or position 263 may be the C-terminus of at least 8 amino acids, preferably 8-60 amino acids; or, positions upstream of position 263 and downstream of position 263 may comprise at least 8 amino acids, preferably 8-60 amino acids.

[0149] In one specific embodiment of the present invention, the polypeptide includes at least positions 256 to 263 of the amino acid sequence of PI4KB, and may also include upstream and / or downstream amino acid sequences. Preferably, it includes any one of SEQ ID NO: 1 or 83-88, or includes an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, or 98% or more identity with any one of SEQ ID NO: 1 or 83-88, or includes substituted, deleted, or inserted amino acid sequences having at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid.

[0150] The peptide mutants include amino acid fragments of PI4KB that are mutated to one or more sites of S256, S258, T263 or S266 to be neither threonine nor serine, for example, to alanine or a non-natural amino acid.

[0151] More preferably, the polypeptide mutant includes a mutation of serine and threonine at positions 256 to 266 to alanine, such as a mutant of SEQ ID NO: 1 or any of 83-88.

[0152] The fusion protein includes a polypeptide that competitively phosphorylates PI4K, and preferably also includes an N-terminal Tat protein transduction domain. More preferably, the polypeptide and the N-terminal Tat protein transduction domain are linked by a GG linker. For example, the fusion protein may include SEQ ID NO: 57 or include an amino acid sequence that has more than 80% identity with SEQ ID NO: 57 or include an amino acid sequence that has at most 10 substituted, deleted or inserted amino acids with SEQ ID NO: 57.

[0153] The target site sequence of the siRNA targeting PI4KB includes one or more of SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27.

[0154] The target site sequence of the shRNA targeting PI4KB includes one or more of SEQ ID NO: 37 and / or SEQ ID NO: 38.

[0155] In a tenth aspect, the present invention provides a ULK1 inhibitor, wherein the ULK1 inhibitor is an interfering RNA, and the interfering RNA is siRNA and / or shRNA;

[0156] The target site sequences of siRNAs targeting ULK1 include SEQ ID NO: 2 and / or SEQ ID NO: 3;

[0157] The target site sequences of shRNAs targeting ULK1 include SEQ ID NO: 29 and / or SEQ ID NO: 30.

[0158] In an eleventh aspect, the present invention provides a WIPI2 inhibitor, wherein the WIPI2 inhibitor is an interfering RNA, the interfering RNA is siRNA, and the target site sequence of the siRNA targeting WIPI2 includes one or more of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

[0159] In a twelfth aspect of the present invention, a vector is provided, the vector comprising a PI4KB inhibitor, a ULK1 inhibitor, and / or a WIPI2 inhibitor, wherein the PI4KB inhibitor, ULK1 inhibitor, and / or WIPI2 inhibitor are interfering RNA.

[0160] Preferably, the carrier can be a viral carrier or a non-viral carrier.

[0161] The viral vectors mentioned include lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, baculoviruses, herpes simplex viruses, etc.

[0162] The non-viral vectors mentioned include liposomes, polymers, peptides, antibodies, aptamers, and combinations thereof. Specifically, liposomes include, for example, lipid nanoparticles (LNPs), GalNac, LPP (lipopolyplexes), and especially LNPs. LNPs are prepared from four lipids in a certain proportion, typically comprising: cationic lipids, neutral lipids, steroidal lipids, and polyethylene glycol (PEG) lipids.

[0163] Specifically, examples of cationic lipids include, but are not limited to, octadecylamide (SA), lauryltrimethylammonium bromide, hexadecyltrimethylammonium bromide, myristyltrimethylammonium bromide, dimethyl di-octadecylammonium bromide (DDAB), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), 1,2-di-tetradecanoyl-3-trimethylammonium-propane (DMTAP), 1,2-di-octadecanoyl-3-trimethylammonium-propane (DOTAP), and DOTAP derivatives such as 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane and 1,2-di-hexadecanoyl-3-trimethylammonium-propane, 1,2-di-(9Z-octadecenoyl)-3-dimethylammonium-propane (DODAP), and DODAP. Derivatives such as 1,2-di-tetradecanoyl-3-dimethylammonium-propane, 1,2-di-hexadecanoyl-3-dimethylammonium-propane and 1,2-di-octadecanoyl-3-dimethylammonium-propane, 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-c-(4'-trimethylammonium)-butyryl-sn-glycerol (DOTB), di-octadecanoyl-alanylspermine, SAINT-2, polycationic lipid 2,3-dioleoyloxy-N-[2(spermine-carboxylamino)ethyl]-N,N-dimethyl-1-propanium trifluoroacetate (DOSPA), ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), etc.

[0164] Specifically, examples of neutral lipids include, but are not limited to, one or more of the following: 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE).

[0165] Specifically, examples of steroidal lipids include, but are not limited to, one or more of the following: alfalfa sterol, β-sitosterol, brassosterol, ergocalciferol, campesterol, cholesterol, coccidosterol, dehydrocholesterol, sterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, stigmasterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol; lanosterol, photosterol, phycosterol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid.

[0166] Specifically, examples of polyethylene glycol lipids include, but are not limited to, PEG-DMG and ALC-0159(M-DTDAM).

[0167] In a thirteenth aspect, the present invention provides a medicament comprising a PI4K inhibitor and a MEK inhibitor, and pharmaceutically acceptable excipients.

[0168] PI4K inhibitors and MEK inhibitors are as described in the first aspect.

[0169] The ratio of PI4K inhibitor to MEK inhibitor can be adjusted as needed, and can be greater than 1:1 or less than 1:1.

[0170] In a fourteenth aspect, the present invention provides a medicament comprising a P38-ULK1-PI4KB-WIPI2 pathway inhibitor and a pharmaceutically acceptable excipient.

[0171] In a fifteenth aspect, the present invention provides a method for treating and / or preventing tumors.

[0172] The method includes any one or more of the following:

[0173] A) Block or inhibit autophagy caused by RAS mutations;

[0174] B) Block or inhibit PI4K phosphorylation;

[0175] C) Block or inhibit ULK1 phosphorylation-induced PI4K phosphorylation;

[0176] D) Block or reduce PI4P levels, preferably block or reduce the levels of its recruitment interacting proteins, preferably WIPI2;

[0177] E) Block or inhibit ULK1 phosphorylation.

[0178] Preferably, the method includes knocking out the PI4K gene, silencing the PI4K gene, mutating the PI4K gene, or administering a polypeptide that competitively phosphorylates PI4K, or a mutant thereof, or a fusion protein containing the polypeptide.

[0179] Preferably, the PI4K is a PI4K complex or PI4KB.

[0180] Preferably, the method includes knocking out the ULK1 gene, silencing the ULK1 gene, or mutating the ULK1 gene.

[0181] Preferably, the method includes knocking out the WIPI2 gene, silencing the WIPI2 gene, or mutating the WIPI2 gene.

[0182] Preferably, the method comprises administering an effective amount of a P38-ULK1-PI4KB-WIPI2 pathway inhibitor or drug to a subject in need.

[0183] Preferably, the method comprises administering to a subject in need an effective amount of one or more of a P38 pathway inhibitor, a ULK1 inhibitor, a PI4KB inhibitor, or a WIPI2 inhibitor.

[0184] Preferably, the administration route can be gastrointestinal (e.g., oral) or non-gastrointestinal (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, intravenous, intracerebral, intrathecal, transdermal, rectal, intraperitoneal, intrapulmonary, vaginal, etc.).

[0185] In one embodiment of the invention, the drug is administered orally.

[0186] In one embodiment of the invention, the drug is administered by injection (e.g., subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, intraperitoneal injection).

[0187] Preferably, the method further includes administering a second active agent to a subject in need, preferably a P38-ULK1-PI4KB-WIPI2 pathway inhibitor, in combination with the second active agent. More preferably, a PI4K inhibitor is administered in combination with the second active agent.

[0188] The second active agent includes, but is not limited to, EGFR inhibitors, RAS inhibitors, SHP2 inhibitors, SOS1 inhibitors, Raf inhibitors, MEK inhibitors, ERK inhibitors, PI3K inhibitors, PTEN inhibitors, AKT inhibitors, mTORC1 inhibitors, BRAF inhibitors, PD-L1 inhibitors, PD-1 inhibitors, CDK4 / 6 inhibitors, HER2 inhibitors, ULK1 inhibitors, or combinations thereof; preferably, MEK inhibitors.

[0189] Preferably, the tumor is a RAS-mutant tumor.

[0190] In a sixteenth aspect of the invention, a method for treating and / or preventing tumors is provided, the method comprising administering a PI4K inhibitor and a MEK inhibitor in combination to a subject in need.

[0191] A seventeenth aspect of the present invention provides a method for diagnosing or prognostically assessing tumors, the method comprising detecting the levels of ULK1 phosphorylation and / or PI4KB phosphorylation.

[0192] Preferably, the tumor is a RAS-mutant tumor.

[0193] Preferably, the method includes determining whether ULK1 phosphorylation-induced PI4KB phosphorylation exists.

[0194] The phosphorylation sites of PI4KB include S256 and / or T263.

[0195] The phosphorylation sites of ULK1 include one or more of S317, S556, S758, or S479.

[0196] Preferably, the method includes detecting the levels of ULK1 phosphorylation and / or PI4KB phosphorylation. More preferably, the method includes detecting the levels of ULK1 phosphorylation and / or PI4KB phosphorylation in tumor cells.

[0197] Preferably, the method includes detecting the level of phosphorylation at S256 and / or T263 sites in PI4KB, for example, detecting the level of peptides including phosphorylation at S256 and / or T263 sites, preferably detecting the level of any one of the phosphorylations in SEQ ID NO: 1 or 83-88.

[0198] An eighteenth aspect of the present invention provides a method for blocking or inhibiting autophagy caused by RAS mutations, the method comprising any one or more of the following:

[0199] A) Block or inhibit PI4K phosphorylation;

[0200] B) Block or inhibit ULK1 phosphorylation-induced PI4K phosphorylation;

[0201] C) Block or reduce PI4P levels, preferably block or reduce the levels of its recruitment interacting proteins, preferably WIPI2;

[0202] D) Block or inhibit ULK1 phosphorylation.

[0203] Preferably, the method includes knocking out the PI4K gene, silencing the PI4K gene, or administering a polypeptide that competitively phosphorylates PI4K, or a mutant thereof, or a fusion protein containing the polypeptide.

[0204] Preferably, the method includes administering a P38-ULK1-PI4KB-WIPI2 pathway inhibitor or drug.

[0205] Preferably, the method comprises administering to a subject in need an effective amount of one or more of a P38 pathway inhibitor, a ULK1 inhibitor, a PI4KB inhibitor, or a WIPI2 inhibitor.

[0206] A nineteenth aspect of the present invention provides a method for identifying autophagy induced by RAS mutations, the method comprising detecting the levels of ULK1 phosphorylation and / or PI4KB phosphorylation.

[0207] Preferably, the method includes determining whether ULK1 phosphorylation-induced PI4KB phosphorylation exists.

[0208] The phosphorylation sites of PI4KB include S256 and / or T263.

[0209] The phosphorylation sites of ULK1 include one or more of S317, S556, S758, or S479.

[0210] Preferably, the method includes detecting the levels of ULK1 phosphorylation and / or PI4KB phosphorylation. More preferably, the method includes detecting the levels of ULK1 phosphorylation and / or PI4KB phosphorylation in tumor cells.

[0211] Preferably, the method includes detecting the level of phosphorylation at S256 and / or T263 sites in PI4KB, for example, detecting the level of peptides including phosphorylation at S256 and / or T263 sites, preferably detecting the level of any one of the phosphorylations in SEQ ID NO: 1 or 83-88.

[0212] The "medicine" of this invention may contain the inhibitor in a weight ratio of 0.01-99.5% (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%).

[0213] The "medicine" described in this invention can be a human medicine or a veterinary medicine.

[0214] The "medicine" described in this invention can be any suitable dosage form, such as a gastrointestinal or non-gastrointestinal dosage form, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder inhalers, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, drop pills, gels, etc.

[0215] The various dosage forms of the "drug" described in this invention can be prepared according to conventional pharmaceutical production methods.

[0216] The term "pharmaceutically acceptable" as used in this invention refers to the biological activity and properties of a substance that neither significantly stimulates the organism nor inhibits the active substance of the applied inhibitor or drug.

[0217] The "pharmaceutically acceptable salt" as described in this invention refers to a salt prepared from a pharmaceutically acceptable, non-toxic acid or base, wherein the acid or base includes inorganic acids or bases or organic acids or bases. The inorganic acid may be hydrochloric acid, hydrobromic acid, phosphoric acid, hydroiodic acid, or sulfuric acid. The inorganic base may be calcium, magnesium, lithium, sodium, zinc, aluminum, or potassium. The organic acid is selected from formic acid, glycolic acid, propionic acid, acetic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, maleic acid, glutamic acid, benzoic acid, stearic acid, alginic acid, benzenesulfonic acid, glucuronic acid, dihydroxynaphthyl acid, or galacturonic acid. The organic base may be diethanolamine, choline, procaine, lysine, or 1,2-ethylenediamine.

[0218] The "pharmaceuticalally acceptable excipients" described in this invention include, but are not limited to, one or more of the following: fillers, binders, wetting agents, disintegrants, lubricants, flavoring agents, fragrances, coloring agents, coating agents, acidity regulators, preservatives, diluents, and isotonic sterile salt solutions (such as sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc., or mixtures thereof).

[0219] The "inhibitor" or "drug" described in this invention can be administered via any suitable route, such as gastrointestinal administration (e.g., oral administration) or non-gastrointestinal administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, intravenous infusion, intracerebral, intrathecal, transdermal, rectal, etc.).

[0220] The term "tumor" as used in this invention can refer to any undesirable cell proliferation (or any disease that manifests as undesirable cell proliferation), vegetation, or an increased tendency or risk of undesirable cell proliferation, vegetation, or tumor. It can be benign or malignant, and can be primary or secondary (metastatic). A vegetation can be any abnormal growth or proliferation of cells and can be located in any tissue. Examples of tissues include the adrenal glands, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, mammary glands, cecum, central nervous system (including or excluding the cerebrum), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidneys, lacrimal glands, larynx, liver, lungs, lymph nodes, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary glands, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testes, thymus, thyroid gland, tongue, tonsils, trachea, uterus, vulva, and leukocytes.

[0221] The "antisense nucleic acid" described in this invention includes nucleotides that are perfectly complementary to the target sequence, as well as nucleotides with one or more nucleotide mismatches, provided that the antisense nucleic acid can specifically hybridize with the target sequence. For example, the antisense nucleic acid described herein includes polynucleotides having at least 70% or higher, preferably 80% or higher, more preferably 90% or higher, and even more preferably 95% or higher homology over a length of at least 15 consecutive nucleotides. Due to the formation of the hybrid, the transcription of the target gene and / or the translation of the target mRNA is reduced or blocked.

[0222] The "small molecule compound" mentioned in this invention refers to an organic compound with a molecular weight of less than 3000, 2500, 2000, 1500, 1000 or 500 Daltons, which may be natural or chemically synthesized.

[0223] The "interfering RNA" described in this invention includes single-stranded RNA (e.g., mature miRNA, ssRNA oligonucleotide, ssDNA oligonucleotide) or double-stranded RNA (e.g., siRNA, dsRNA, shRNA, aiRNA, or precursor miRNA). When the interfering RNA is in the same cell as the target gene or sequence, it can reduce or inhibit the expression of the target gene or sequence (e.g., by mediating degradation and / or inhibiting the translation of mRNA complementary to the interfering RNA sequence).

[0224] The siRNA is a small interfering RNA, and each strand of its molecule contains nucleotides of about 15 to about 60 in length (e.g., nucleotides of about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 in length, or nucleotides of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 in length). In one specific embodiment, the siRNA may be chemically synthesized. The siRNA molecules of the present invention are capable of silencing the expression of target sequences in vitro and / or in vivo. In some embodiments, the siRNA may not contain modified nucleotides; in other embodiments, the siRNA contains at least one modified nucleotide, for example, the siRNA contains one, two, three, four, five, six, seven, eight, nine, ten, or more modified nucleotides in the double-stranded region. dsRNA or precursor RNA molecules include any precursor molecule processed in vivo by a nuclease to produce active siRNA. shRNA is a small hairpin RNA or short hairpin RNA, including short RNA sequences that produce tight hairpin turns. These hairpin turns can be used to silence gene expression via RNA interference. The shRNA hairpin structure can be cleaved into siRNA by cellular machinery. miRNA is a single-stranded RNA molecule approximately 21-23 nucleotides in length that regulates gene expression.

[0225] The term "antibody" as used herein encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, or antibody capable of binding to a specific antigen. The term "antibody" as used herein is intended to broadly cover conventional four-chain antibodies as well as less conventional antibodies that do not have four chains (e.g., antibodies naturally lacking a light chain), such as nanobodies, single-domain antibodies, or single-chain antibodies. In some embodiments, the antibody is a murine antibody, rabbit antibody, chimeric antibody, humanized antibody, or fully human antibody.

[0226] The "non-natural amino acids" described in this invention are not naturally occurring amino acid types, but their functions are similar to those of naturally occurring amino acids. "Naturally occurring" amino acid residues refer to amino acid residues found in natural proteins or peptides. The naturally occurring amino acids include, but are not limited to, 20 standard amino acids, including glycine (Gly or G), alanine (Ala or A), valine (Val or V), leucine (Leu or L), isoleucine (Ile or I), serine (Ser or S), cysteine ​​(Cys or C), threonine (Thr or T), methionine (Met or M), proline (Pro or P), phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W), histidine (His or H), lysine (Lys or K), arginine (Arg or R), aspartic acid (Asp or D), glutamic acid (Glu or E), asparagine (Asn or N), and glutamine (Gln or Q), as well as their natural analogues, such as canavanine, pyrrolidone (PYL), selenocysteine, pyrrolidone-carboxy-lysine (PCL), sarcosine, β-alanine, phosphoserine, γ-carboxyglutamic acid, and ornithine. Examples of naturally occurring amino acid residues in their D stereoisomers include, for example, D-aspartic acid, D-serine, D-cysteine, D-alanine, and D-glutamic acid. The non-natural amino acids include, but are not limited to, 2-aminoisobutyric acid (Aib), imidazole-4-acetate (IA), imidazole propionic acid (IPA), α-aminobutyric acid (Abu), tert-butylglycine (Tle), 3-aminomethylbenzoic acid, anthranilic acid, deaminohistidine (abbreviated as DesaminoHis, also known as imidazole propionic acid, abbreviated as lmpr), and β-analytes of amino acids such as β-alanine, 2-aminohistidine, β-hydroxyhistidine, homohistidine, and N-aminobutyric acid. α-Acetyl-histidine, α-fluoro-methyl-histidine, α-methyl-histidine, α,α-dimethyl-glutamic acid, m-CF3-phenylalanine, α,β-diaminopropionic acid (abbreviated as Dap), 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine, (1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic acid and (1-aminocyclooctyl)carboxylic acid.

[0227] The term "comprising" or "including" as used in this invention is an open-ended expression. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0228] The term "treatment" as used in this invention refers to slowing down, interrupting, preventing, controlling, stopping, alleviating, reducing, or reversing a sign, symptom, disorder, condition, or progression or severity of a disease after it has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0229] The term "effective amount" as used in this invention refers to the amount or dose of the inhibitor or drug of this invention that provides the desired treatment or prevention after being administered to a subject or organ in one or more doses.

[0230] The term "prevention" as used in this invention refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in the body.

[0231] The term "diagnosis" as used in this invention refers to determining whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or determining the progression of a disease or its possible future progression.

[0232] The "prognostic assessment" described in this invention refers to assessing a patient's response to treatment and the risk of developing the disease in the future.

[0233] The "subject" described in this invention can be a human or a non-human mammal, or a cell, tissue, or organ of a human or non-human mammal. The non-human mammal can be a wild animal, a zoo animal, an economically important animal, a pet, a laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), or monkeys, etc.

[0234] The term "and / or" as used in this invention encompasses all combinations of items connected by the term, and should be considered as if each combination had been individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". As another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C". Attached Figure Description

[0235] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0236] Figure 1: KRAS(G12V)-induced autophagy system and dependence on various autophagy factors.

[0237] (A) Immunoblot analysis of KRAS(G12V)293T cells (treated with different concentrations of Dox for 36 hours or untreated).

[0238] (B) Immunoblotting analysis was performed on the KRAS expression levels of the different cell lines shown.

[0239] (C) Immunoblotting analysis of LC3 lipidation in control cells and KRAS(G12V) cells cultured for 1.5 hours with or without 500 nM bafloxacin A1.

[0240] (D) Immunofluorescence and confocal microscopy imaging were performed to analyze the colocalization of KRAS(G12V)-induced LC3 cells in 293T cells. The figures show representative cell images. Scale bars are included in the figures.

[0241] (E) Quantitative analysis of the LC3 point (mean ± standard error (SEM)) in control cells and KRAS (G12V) cells shown in (D). Three experiments were conducted (50 cells per group / experiment) and statistical analysis was performed (two-tailed t-test). **, p < 0.01.

[0242] (F) Electron microscopic analysis of autophagic vesicle structures in control cells and KRAS(G12V) cells. Scale bars are shown in the figure.

[0243] (G) Number of autophagic vesicles in each cell type analyzed in quantification (F). Data are expressed as mean ± SEM. Three experiments (30 cells per group / experiment) were conducted to obtain statistical data (two-tailed t-test). **, p < 0.01.

[0244] (H) Tandem fluorescent LC3 system was used to detect KRAS(G12V)-induced autophagy in control cells stably expressing mCherry-pHluorin-LC3B and KRAS(G12V) cells. The figure shows representative cell images. Scale bars are included in the figure.

[0245] (I)RFP + GFP + and RFP + GFP - Quantitative analysis of LC3 points. Data are expressed as mean ± SEM. Three experiments (50 cells per group / experiment) were conducted to obtain statistical data (two-tailed t-test). *, p < 0.05; **, p < 0.01.

[0246] (J) Immunoblotting and in-gel fluorescence detection were performed on control cells and KRAS (G12V) cells that were stably expressed with HaloTag (Halo)-LC3B after being pulse-labeled with 100 nM tetramethylrhodamine conjugate ligand for 20 minutes in culture medium. The cells were cultured for 2 hours with (or without) 500 nM bafloxacin A1.

[0247] (K) Immunoblotting was performed on control cells and KRAS(G12V) cells cultured for 1.5 hours with or without 500 nM bafloxacin A1 to assess the degradation of exogenous p62.

[0248] (L) The ratio of P62 to tubulin analyzed in quantitative (K) assays, with the control set at 1.00 (control cells not treated with bafloxacin A1) (mean ± SEM). Three experiments were performed (two-tailed t-test) to obtain statistical data. **, p < 0.01; ****, p < 0.0001.

[0249] (M) FACS analysis was performed on control cells and KRAS (G12V) cells co-expressing mt-Keima and Parkin using V610 and Y610-mCherry detectors (Beckman CytoFLEX LX). FACS results represent at least three independent experiments.

[0250] (N) Percentage of mitophagic cells as shown in (M) based on Y610-mCherry / V610. Data are expressed as mean ± SEM. Three experiments were performed (two-tailed t-test) to obtain statistical data. ****, p < 0.0001.

[0251] (O) The heatmap shows LC3 lipidation in knockdown-symmetric proteins or control cells overexpressing ATG4B-C74S and KRAS(G12V)293T cells (control set to 1.00, showing the ratio of lipidated LC3 to tubulin, see Figures 2D and E), autophagic fluxes of the tandem fluorescent LC3 system (AG and AL, control set to 1.00, see Figures 2F and 3A), and changes in HaloTag-LC3B treatment analysis (Halo, Halo-TMR band intensity normalized by summing the band intensities of Halo-TMR-LC3B and Halo-TMR, control (KRAS(G12V)293T cells treated with ligand but not with bafloxacin A1) set to 1.00, see Figures 3B and C), as well as mitophagy (MtP, control set to 1.00, see Figures 3D and E). Color intensity represents the log2 (fold) of the count for each sample.

[0252] (P) The heatmap shows changes in LC3 lipidation (control set to 1.00, showing the ratio of lipidated LC3 to tubulin, see Figures 3F and G) and autophagic flux of the tandem fluorescent LC3 system (AG and AL, control set to 1.00, see Figures 3H and I) in the control and EBSS groups (Beclin1, ATG14, ATG2A and 2B or ATG9A knocked down, respectively). Color intensity represents the log2 (fold) of the count for each sample.

[0253] Figure 2: Characterization of KRAS(G12V)-induced autophagy.

[0254] (A) Immunoblotting analysis of LC3 lipidation in control cells and cells transfected with KRAS(G12D) or KRAS(G12C) plasmids.

[0255] (B) Immunoblotting analysis of LC3 lipidation in control cells or KRAS(G12V), KRAS(G12D), and KRAS(G12C) cells (with or without 500 nM bafloxacin A1 cultured for 1.5 hours) treated with 10 μM AMG510 for 1.5 hours.

[0256] (C) Relative RNA expression (mean ± SEM) of cells transfected with control or anti-VPS13A, VPS13B, VPS13C, VPS13D, VPS4A, and VPS4B shRNA. Three experiments were performed (two-tailed t-test) to obtain statistical data. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

[0257] (D) Immunoblot analysis of LC3 lipidation in KRAS(G12V) cells. The cells were transfected as controls, or knocked down with ULK1, FIP200, ATG5, ATG16L1, WIPI2, Beclin1, ATG14, STX17, SNAP29, VAMP8, VPS4A and 4B, VPS13A, VPS13B, VPS13C, VPS13D, ATG9A or ATG2A and B, or overexpressed ATG4B-C74S (with or without 500 nM bafloxacin A1 and cultured for 1.5 h).

[0258] (E) Quantitative analysis of the ratio of lipid-modified LC3 to tubulin in (D), with a control set at 1.00 (control cells not treated with bafloxacin A1) (mean ± SEM). Three experiments were performed (two-tailed t-test) to obtain statistical data. ***, p < 0.001; ****, p < 0.0001.

[0259] (F)RFP + GFP + and RFP + GFP - Quantitative analysis of LC3 points was performed. Data are expressed as mean ± SEM. Three experiments (50 cells per group / experiment) were conducted to obtain statistical data (two-tailed t-test). *, p < 0.05.

[0260] Figure 3: Characterization of KRAS(G12V)-induced autophagy.

[0261] (A) Immunofluorescence analysis of mCherry-pHluorin-LC3B cells expressing KRAS(G12V). These cells were transfected with controls, or with knockdown of ULK1, FIP200, Beclin1, ATG14, WIPI2, ATG5, ATG16L1, ATG2A and B, VPS13A, VPS13B, VPS13C, VPS13D, VPS4A and 4B, STX17, SNAP29, VAMP8, ATG9A, or overexpression of ATG4B-C74S. The figure shows a representative cell image. Scale bars are included in the figure.

[0262] (B) Immunoblotting and in-gel fluorescence detection were performed on KRAS (G12V) cells and control cells that were stably expressed with HaloTag (Halo)-LC3B after being pulse-labeled with 100 nM tetramethylrhodamine conjugate ligand for 20 min in culture medium. The cells were transfected with controls, or knocked down ULK1, FIP200, Beclin1, ATG14, WIPI2, ATG5, ATG16L1, ATG2A and B, VPS13A, VPS13B, VPS13C, VPS13D, VPS4A and 4B, STX17, SNAP29, VAMP8, ATG9A, or overexpressed ATG4B-C74S, and cultured with (or without) 500 nM bafloxacin A1 for 1.5 h.

[0263] (C) Quantitative analysis of the results shown in (B). Halo-TMR band intensity was normalized by summing the band intensities of Halo-TMR-LC3B and Halo-TMR, with the control (KRAS(G12V)293T cells treated with ligand but not with bafloxacin A1) set at 1.00. Three experiments (two-tailed t-tests) were performed to obtain statistical data. ***, p < 0.001; ****, p < 0.0001.

[0264] (D) FACS analysis was performed on control cells and KRAS (G12V) cells co-expressing mt-Keima and Parkin using V610 and Y610-mCherry detectors (Beckman CytoFLEX LX). Cells were transfected with controls or knocked down ULK1, FIP200, Beclin1, ATG14, WIPI2, ATG5, ATG16L1, ATG2A and B, VPS13A, VPS13B, VPS13C, VPS13D, VPS4A and 4B, STX17, SNAP29, VAMP8, ATG9A, or overexpressed ATG4B-C74S. FACS results represented at least three independent experiments.

[0265] (E) Quantitative results shown in (D). Percentage of mitophagic cells calculated based on Y610-mCherry / V610. Data are expressed as mean ± SEM. Three experiments were performed (two-tailed t-test) to obtain statistical data. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0266] (F) After 1.5 hours of EBSS treatment (with or without 500 nM bafloxacin A1), LC3 lipidation in starvation-induced autophagy was analyzed by immunoblotting in cells transfected with controls or with knockdown of Beclin1, ATG14, ATG9A, or ATG2A and B.

[0267] (G) The ratio of lipid-modified LC3 to tubulin analyzed in quantitative (L) assays, with a control set at 1.00 (KRAS(G12V) cells treated with bafloxacin A1) (mean ± SEM). Three experiments were performed (two-tailed t-test) to obtain statistical data. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0268] (H) After EBSS treatment, immunofluorescence was performed on 293T cells that were transfected with controls or had Beclin1, ATG14, ATG9A, and ATG2A and B knocked down and expressed mCherry-pHluorin-LC3B in starvation-induced autophagy. The figure shows a representative cell image. Scale bars are included in the figure.

[0269] (I)RFP + GFP + and RFP + GFP - Quantitative analysis of LC3 points. Data are expressed as mean ± SEM. Three experiments (50 cells per group / experiment) were conducted to obtain statistical data (two-tailed t-test). *, p < 0.05, **, p < 0.01.

[0270] Figure 4: PI4KB regulates KRAS(G12V)-induced autophagy.

[0271] (A) The heatmap shows the changes in LC3 lipidation (the ratio of lipidated LC3 to tubulin, with controls set at 1.00) in the KRAS(G12V) and starvation groups using different inhibitors (see Figures 5A-F). The color intensity represents the log2 (fold) of the count for each sample.

[0272] (B) The heatmap shows changes in LC3 lipidation (the ratio of lipid-treated LC3 to tubulin, with a control set at 1.00, see Figure 6B), autophagic flux (AG and AL, see Figures 6C and D), and mitophagy (MtP, see Figures 6E and F) in PI4K knockdown or PI4KB inhibitor-treated KRAS(G12V)293T cells. Color intensity represents the log2 (fold) of the count for each sample.

[0273] (C) Dotted immunoblotting analysis of PI4P generated by KRAS(G12V) in control cells with knocked-down PI4KB and KRAS(G12V)293T cells.

[0274] (D) Quantitative results shown in (C) (mean ± SEM), with a control set at 1.00. Three experiments (two-tailed t-tests) were conducted to obtain statistical data. **, p < 0.01.

[0275] (E) Immunofluorescence analysis of PI4P spots in KRAS(G12V) FHC cells with or without PI4KB knockdown. The figure shows a representative cell image. A scale bar is included in the figure.

[0276] (F) Quantitative analysis of the results shown in (E) (mean ± SEM). Three experiments were conducted (50 cells per group / experiment) and statistical analysis was performed (two-tailed t-test). **, p < 0.01.

[0277] (G) Co-localization analysis of KRAS(G12V)-induced LC3 sites in FHC cells with or without PI4KB knockdown was performed using immunofluorescence and confocal microscopy. The figures show representative cell images. Scale bars are included in the figures.

[0278] (H) Quantitative analysis of LC3 points in control cells and KRAS(G12V)FHC cells as shown in (G). Data are expressed as mean ± SEM. Three experiments (50 cells per group / experiment) were performed to obtain statistical data (two-tailed t-test). **, p < 0.01.

[0279] Immunoblot analysis was performed on PI4P-rescued LC3 lipidation in KRAS(G12V)293T cells where PI4KB was suppressed by knockdown (I, J) or inhibitor treatment (K, L). Results in (I, K) are quantified in (J, L) (mean ± SEM). Three experiments were performed (two-tailed t-test) to obtain statistical data. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0280] (M) Immunofluorescence analysis of PI4P-rescued LC3 spots in KRAS(G12V)293T cells where PI4KB was suppressed by knockdown or inhibitor treatment. The figure shows a representative cell image. Scale bars are included in the figure.

[0281] Results (N) Quantitative (M) are shown (mean ± SEM). Three experiments were conducted (50 cells per group / experiment) and statistical analysis was performed (two-tailed t-test). **, p < 0.01.

[0282] (O) FACS analysis of PI4P-rescued mitophagy in KRAS(G12V)293T cells where PI4KB was suppressed by knockdown or inhibitor treatment. FACS results represent at least three independent experiments.

[0283] (P) Percentage of mitophagic cells as shown in (O) based on Y610-mCherry / V610. Error bars represent the standard deviation (mean ± SEM) of three experiments. Three experiments were conducted (two-tailed t-test) to obtain statistical data. ***, p < 0.001; ****, p < 0.0001.

[0284] (Q)RFP + GFP + and RFP + GFP - Quantification of LC3 sites (see Figure 7). KRAS(G12V)293T cells with PI4KB inhibition by knockdown or inhibitor treatment were rescued with PI4P. Data are presented as mean ± SEM. Three experiments (50 cells per group / experiment) were performed to obtain statistical data (two-tailed t-test). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0285] Figure 5: The PI3K and PI5K complex cannot regulate KRAS(G12V)-induced autophagy, but it can regulate starvation-induced autophagy.

[0286] (A) Immunoblot analysis of LC3 lipidation in KRAS (G12V) cells treated with SAR405 (40 nM), PIK-III (40 nM), VPS34-IN1 (40 nM), Wollman penicillin (20 nM), or YM201636 (100 nM) and control cells (cultured for 1.5 hours with or without 500 nM bafloxacin A1).

[0287] (B) Quantitative analysis of the results shown in (A) (mean ± SEM). Three experiments were conducted (two-tailed t-test) to obtain statistical data. **, p < 0.01.

[0288] (C) After 1.5 hours of EBSS treatment (with or without 500 nM bafloxacin A1), Western blot analysis was performed on LC3 lipidation in starvation-induced autophagy in cells treated with SAR405 (40 nM), PIK-III (40 nM), VPS34-IN1 (40 nM), or Wollman penicillin (20 nM).

[0289] (D) After culturing for 1.5 hours with or without 500 nM bafloxacin A1, LC3 lipidation in cells treated with YM201636 (100 nM) in low glucose-induced autophagy was analyzed by immunoblotting.

[0290] (E) Quantitative results (C, D) are shown (mean ± SEM). Three experiments (two-tailed t-tests) were conducted to obtain statistical data. **, p < 0.01; ***, p < 0.001.

[0291] (F) Immunoblotting analysis of LC3 lipidation in KRAS (G12V) cells treated with Woman penicillin (200 nM) and control cells (cultured for 1.5 h with or without 500 nM bafloxacin A1).

[0292] Figure 6: The PI3K and PI5K complex cannot regulate KRAS(G12V)-induced autophagy, but it can regulate starvation-induced autophagy.

[0293] (A) Relative RNA expression in cells after transfection with shRNA of the control group or PI4KIIA, PI4KIIB, PI4KA, or PI4KB (mean ± SEM). Three experiments were performed to obtain statistical data (two-tailed t-test). **, p < 0.01; ***, p < 0.001.

[0294] (B) Immunoblotting analysis of LC3 lipidation in KRAS (G12V) cells treated with PI4K knockdown or PI4KB inhibitor and control cells (cultured for 1.5 hours with or without 500 nM bafloxacin A1).

[0295] (C) Immunofluorescence was performed on KRAS(G12V) cells expressing mCherry-pHluorin-LC3B and control cells after PI4K knockdown or treatment with a PI4KB inhibitor.

[0296] (D) Quantitative analysis of the results shown in (C) (mean ± SEM). Three experiments were conducted (50 cells per group / experiment) and statistical analysis was performed (two-tailed t-test). **, p < 0.01; ***, p < 0.001.

[0297] (E) FACS analysis was performed on control cells co-expressing mt-Keima and Parkin and KRAS (G12V) cells (transfected with controls or knocked down PI4K) using V610 and Y610-mCherry detectors (Beckman CytoFLEX LX). FACS results represent at least three independent experiments.

[0298] (F) Quantitative results shown in (E). Percentage of mitophagic cells calculated based on Y610-mCherry / V610. Data are expressed as mean ± SEM. Three experiments were performed (two-tailed t-test) to obtain statistical data. ***, p < 0.001; ****, p < 0.0001.

[0299] Figure 7: Immunofluorescence of 293T cells rescued by PI4P after treatment with PI4KB to inhibit mCherry-pHluorin-LC3B expression via knockdown or inhibitor therapy. The figure shows a representative cell image. A scale bar is included in the figure.

[0300] Figure 8: PI4KB inhibitors delay the growth of RAS-mutant xenograft tumors.

[0301] (A, B) HCT116 and SW620 cells were treated with trametinib (100 nM) and PIK93 (1 μM) for 96 hours, and cell viability (mean ± SEM) was analyzed. Three experiments were performed (two-tailed t-test) to obtain statistical data. ****, p < 0.0001.

[0302] (C, D) Images of xenograft tumors of HCT116 and SW620 cells were obtained from mice treated with: (1) control group (Ctrl); (2) trametinib (Tra); (3) PIK93; or (4) a combination of both (Tra+PIK93) (n=12 per group). The tumors were removed and photographed 24 days after treatment.

[0303] Weights of xenograft tumors (mean ± SD) are shown in (E, F) and (C, D). Statistical analysis was performed using a two-tailed t-test. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

[0304] Growth curves (mean ± SD) of xenograft tumors are shown in (G, H) and (C, D). Statistical analysis was performed using two-way ANOVA; ****, p < 0.0001.

[0305] Figure 9: PI4KB inhibitors delay the growth of RAS-mutant xenograft tumors.

[0306] Immunofluorescence and immunohistochemical analyses of xenograft tumor sections shown in (A, B) (Figures 8C, D) are presented. As shown, the sections were stained with antibodies against LC3, p-ERK1 / 2, or Ki67. A scale bar is included in the figures.

[0307] (C, D) Statistical analysis was performed on the number of LC3 spots, p-ERK positivity rate, and Ki67 expression level shown in (A, B) (mean ± SD). A two-tailed t-test was used for statistical analysis; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0308] Figure 10: WIPI2 acts as a PI4P effector in KRAS-induced autophagy.

[0309] (A) Immunofluorescence and confocal microscopy imaging were performed to analyze the colocalization of WIPI2 and LC3 points in control cells and KRAS(G12V) cells. The figure shows a representative cell image. A scale bar is included in the figure.

[0310] (B) Percentage of WIPI2 points co-located with LC3 in (A) (mean ± SEM). Three experiments were conducted (50 cells per group / experiment) and statistical analysis was performed (two-tailed t-test). ***, p<0.001.

[0311] (C) Immunofluorescence and confocal microscopy imaging were performed, and colocalization analysis of WIPI2 and OSBP-PH points in control and KRAS(G12V) cells was conducted. The figures show representative cell images. Scale bars are included in the figures.

[0312] (D) Percentage of WIPI2 points co-localized with OSBP-PH points in (C) (mean ± SEM). Three experiments were conducted (50 cells per group / experiment) and statistical analysis was performed (two-tailed t-test). ***, p<0.001.

[0313] (E) Immunofluorescence analysis of WIPI2 spots induced by KRAS(G12V) with or without PI4KB knockdown. The figure shows representative cell images. Scale bars are included in the figure.

[0314] (F) Quantitative analysis of the number of WIPI2 points shown in (E) (mean ± SEM). Three experiments were conducted (50 cells per group / experiment) and statistical analysis was performed (two-tailed t-test). **, p < 0.01.

[0315] Immunoblot analysis was performed on membrane-bound WIPI2 in control cells and KRAS (G12V) cells treated with the (G)PI4KB inhibitor T-00127-HEV1 (10 μM).

[0316] (H) Quantitative analysis of the membrane-bound WIPI2 levels shown in (G) (mean ± SEM). Three experiments were conducted (two-tailed t-test) to obtain statistical data. **, p < 0.01.

[0317] (I) Immunoblotting analysis of membrane-bound WIPI2 in control cells and KRAS(G12V) cells overexpressing OSBP-PH.

[0318] (J) Quantitative analysis of the membrane-bound WIPI2 levels shown in (I) (mean ± SEM). Three experiments were conducted (two-tailed t-test) to obtain statistical data. **, p < 0.01.

[0319] (K) Liposome flotation assays showed that WIPI2 binds to PI3P, PI4P, and PI5P. The total lipid level of liposomes was normalized by PI.

[0320] (L)PIP test strips show WIPI2 combined with PI3P, PI4P and PI5P.

[0321] (M) Dotted immunoblotting analysis of PI4P generated by KRAS(G12V) in control and knockdown of ATG9A, ATG2A and 2B or PI4KB KRAS(G12V)293T cells.

[0322] (N) Results shown in Quantitative (M) (mean ± SEM). Three experiments (two-tailed t-tests) were conducted to obtain statistical data. ***, p < 0.001.

[0323] (O) Immunofluorescence analysis of KRAS(G12V)-induced WIPI2 spots in FHC cells with knockdown of ATG9A, ATG2A, and 2B or PI4KB. The figure shows a representative cell image. A scale bar is included in the figure.

[0324] (P) Quantitative analysis of the number of WIPI2 points shown in (O) (mean ± SEM). Three experiments were conducted (50 cells per group / experiment) and statistical analysis was performed (two-tailed t-test). ***, p < 0.001.

[0325] (Q) Quantitative analysis of the area of ​​WIPI2 points shown in (O) (mean ± SEM). Three experiments were conducted (50 cells per group / experiment) and statistical analysis was performed (two-tailed t-test).

[0326] Figure 11: P38-ULK1-PI4K-WIPI2 cascade regulation of KRAS(G12V)-induced autophagy.

[0327] (A) Schematic diagram of RAS signal path.

[0328] (B) The heatmap shows changes in LC3 lipidation (see Fig. 12B and C), autophagic flux (AG and AL, see Fig. 12D and E), and mitophagy (MtP, see Fig. 12F and G) in control cells and KRAS(G12V)293T cells using different inhibitors in the RAS signaling pathway. The color intensity represents the log2 (fold) of the count for each sample.

[0329] Immunofluorescence analysis was performed on the regulation of the p-ULK1-S556 site induced by KRAS(G12V) using (C)SB203580. The image shows a representative cell image. A scale bar is included in the figure.

[0330] (D) Quantitative analysis of the number of WIPI2 points shown in (C) (mean ± SEM). Three experiments were conducted (50 cells per group / experiment) and statistical analysis was performed (two-tailed t-test). *, p < 0.05; **, p < 0.01.

[0331] (E) Immunoblot analysis of the regulation of p-ULK1-S556 levels induced by SB203580 on KRAS(G12V).

[0332] (F) Results shown in Quantitative (H) (mean ± SEM). Three experiments (two-tailed t-tests) were conducted to obtain statistical data. ***, p < 0.001.

[0333] Figure 12: KRAS(G12V)-induced autophagy is achieved through the P38 pathway.

[0334] (A) Immunoblot analysis of p-ERK, p-AKT, p-P38 or p-JN levels in control cells and KRAS(G12V) cells treated with FR180204 (10 μM), MK2206 (10 μM), SB203580 (10 μM) or JNK-IN-8 (10 μM).

[0335] (B) Immunoblotting analysis of LC3 lipidation in KRAS (G12V) cells treated with FR180204 (10 μM), MK2206 (10 μM), SB203580 (10 μM), or RBC8 (10 μM) (with or without 500 nM bafloxacin A1 and cultured for 1.5 h).

[0336] (C) Quantitative analysis of the results shown in (B) (mean ± SEM). Three experiments were conducted (50 cells per group / experiment) and statistical analysis was performed (two-tailed t-test). **, p < 0.01; ***, p < 0.001.

[0337] (D) Immunofluorescence was performed on 293T cells expressing mCherry-pHluorin-LC3B after treatment with FR180204 (10 μM), MK2206 (10 μM), SB203580 (10 μM), or RBC8 (10 μM). The image shows a representative cell image. A scale bar is included in the figure.

[0338] (E) Quantitative (D) RFP shown + GFP + and RFP + GFP - The LC3 point was determined. Data are expressed as mean ± SEM. Three experiments (50 cells per group / experiment) were conducted to obtain statistical data (two-tailed t-test). *, p < 0.05; **, p < 0.01.

[0339] (F) FACS analysis was performed on control cells and KRAS (G12V) cells co-expressing mt-Keima and Parkin, treated with FR180204 (10 μM), MK2206 (10 μM), SB203580 (10 μM), or RBC8 (10 μM) using V610 and Y610-mCherry detectors (Beckman CytoFLEX LX). FACS results represent at least three independent experiments.

[0340] (G) Quantitative (F) results. Percentage of mitophagic cells calculated based on Y610-mCherry / V610. Data are expressed as mean ± SEM. Three experiments were performed (two-tailed t-test) to obtain statistical data. ****, p < 0.0001.

[0341] Figure 13: P38-ULK1-PI4K-WIPI2 cascade regulation of KRAS(G12V)-induced autophagy.

[0342] (A) Immunoblotting analysis of the rescue effect of WT-ULK1 and ULK1-S556A on KRAS(G12V)-induced LC3 lipidation when ULK1 is knocked down.

[0343] (B) Quantitative RFP + GFP + and RFP + GFP - The LC3 point (see Figure 14A) was used to analyze the rescue effect of WT-ULK1 and ULK1-S556A on KRAS(G12V)-induced LC3 flux in 293T cells. Data are presented as mean ± SEM. Three experiments (50 cells per group / experiment) were performed to obtain statistical data (two-tailed t-test). **, p < 0.01.

[0344] (C) Results shown in the quantitative plot (see Figure 14B). Percentage of mitophagic cells calculated based on Y610-mCherry / V610. Data are expressed as mean ± SEM. Three experiments were performed (two-tailed t-test) to obtain statistical data. ***, p < 0.001; ****, p < 0.0001.

[0345] (D) Immunofluorescence analysis of SB203580-induced KRAS(G12V)-induced WIPI2 site regulation in FHC cells. The figure shows a representative cell image. Scale bars are included in the figure.

[0346] (E) Quantitative analysis of the number of WIPI2 points shown in (D) (mean ± SEM). Three experiments were conducted (50 cells per group / experiment) and statistical analysis was performed (two-tailed t-test). **, p < 0.01.

[0347] (F) Immunofluorescence analysis of KRAS(G12V)-induced WIPI2 spots in ULK1 knockdown FHC cells. The figure shows a representative cell image. A scale bar is included in the figure.

[0348] (G) Quantitative analysis of the number of WIPI2 points shown in (F) (mean ± SEM). Three experiments were conducted (50 cells per group / experiment) and statistical analysis was performed (two-tailed t-test). ***, p < 0.001.

[0349] (H) Dotted immunoblotting analysis of PI4P induced by KRAS(G12V) by knocking down PI4KB or ULK1 in 293T cells.

[0350] (I) Results shown in Quantitative (H) (mean ± SEM). Three experiments (two-tailed t-tests) were conducted to obtain statistical data. ****, p < 0.0001.

[0351] Figure 14: KRAS(G12V)-induced autophagy is achieved through the P38 pathway.

[0352] (A) Immunofluorescence was performed on 293T cells expressing mCherry-pHluorin-LC3B after treatment with FR180204 (10 μM), MK2206 (10 μM), SB203580 (10 μM), or RBC8 (10 μM). The figure shows a representative cell image. A scale bar is included in the figure.

[0353] (B) FACS analysis was performed on control cells and KRAS(G12V) cells co-expressing mt-Keima and Parkin to analyze the rescue effect of WT-ULK1 and ULK1-S556A on KRAS(G12V)-induced mitophagy. The percentage of mitophagic cells was calculated based on Y610-mCherry / V610. FACS results represent at least three independent experiments.

[0354] (C) Immunoblotting analysis of LC3 lipidation in different cancer cell lines treated with (or not treated with) SB203580 (10 μM) (with or without 500 nM bafloxacin A1 cultured for 1.5 h).

[0355] (D) Perform FACS analysis on different cancer cell lines co-expressing mt-Keima and Parkin, treated (or not treated) with SB203580 (10 μM). Calculate the percentage of mitophagic cells based on Y610-mCherry / V610. FACS results represent at least three independent experiments.

[0356] (E) Quantitative results shown in (D). Percentage of mitophagic cells calculated based on Y610-mCherry / V610. Data are expressed as mean ± SEM. Three experiments were performed (two-tailed t-test) to obtain statistical data. ****, p < 0.0001.

[0357] Figure 15: PI4KB-peptide-1 phosphorylation required to mediate KRAS(G12V)-induced autophagy by different ULK1 phosphorylation.

[0358] (A) Immunoblot analysis of PI4KB phosphorylation in control cells and KRAS(G12V)293T cells using Phos-tag gel.

[0359] (B) Immunoblot analysis of PI4KB phosphorylation in control cells and KRAS(G12V)293T cells (ULK1 knocked down or not knocked down) using Phos-tag gel.

[0360] (C) Immunoprecipitation analysis was performed to investigate the interaction between PI4KB and ULK1.

[0361] (D) Mass spectrometry analysis of the percentage of the five phosphorylated PI4KB peptides.

[0362] (E) Immunoblotting analysis of PI4KB phosphorylation of anti-p-PI4KB antibody in control cells and KRAS(G12V)293T cells.

[0363] (F) Immunoblotting analysis of PI4KB phosphorylation of anti-p-PI4KB antibody in CIP-treated or untreated KRAS(G12V)293T cells.

[0364] (G) ULK1 PI4KB phosphorylation was analyzed by immunoprecipitation and in vitro kinase assays. HEK293T cells were transfected with an HA-labeled ULK1 construct, lysed, and used for immunoprecipitation. Immunoprecipitates were cultured in an in vitro kinase reaction mixture containing ATP and the substrate GFP-PI4KB (treated with or untreated with SBI-0206965 (10 μM) and peptide-1 (Tat-Pep.1) at indicated concentrations). The reaction products were separated on an SDS-PAGE gel. Anti-HA immunoblotting was used as a control to quantify the amount of ULK1 protein precipitated. PI4KB phosphorylation was detected using an anti-p-PI4KB antibody. This experiment was repeated three times.

[0365] (H) After knocking down PI4KB in 293T cells (with or without 500 nM bafloxacin A1 and culturing for 1.5 h), the rescue effect of PI4KB-WT or PI4KB-SA on KRAS(G12V)-induced LC3 lipidation was analyzed by immunoblotting.

[0366] (I) Dotted immunoblotting analysis was performed to investigate the effect of the PI4KB mutant on KRAS(G12V)-induced PI4P production in 293T cells.

[0367] (J) Immunoblotting analysis was performed on KRAS(G12V)-induced LC3 lipidation after overexpression of PI4KB-SA in 293T cells (with or without 500 nM bafloxacin A1 for 1.5 h).

[0368] (K) Immunofluorescence analysis of KRAS(G12V)-induced LC3 spots in FHC cells with PI4KB-SA overexpression. The figure shows a representative cell image. A scale bar is included in the figure.

[0369] (L) Quantitative (K) LC3 points (mean ± SEM). Three experiments were conducted (50 cells per group / experiment) and statistical analysis was performed (two-tailed t-test). ***, p < 0.001.

[0370] Figure 16: Validation of PI4KB-peptide-1 phosphorylation antibody, the inhibitory effect of PI4KB-SA on the proliferation of RAS mutant cells, and the precise localization of PI4KB-peptide-1 and ULK1 phosphorylation.

[0371] (A) Immunoblotting analysis of PI4KB phosphorylation of anti-p-PI4KB antibody in HCT116 cells and SW620 cells with or without RAS knockdown.

[0372] (B) Immunoblotting analysis of PI4KB phosphorylation of anti-p-PI4KB antibody in MiaPaCa-2 cells (with or without 10 μM AMG510 cultured for 1.5 h).

[0373] (C) Immunohistochemical analysis of sections of MiaPaCa-2 cells (cultured for 1.5 hours with or without 10 μM AMG510). As shown in the figure, the sections were stained with antibodies against PI4KB and p-PI4KB. A scale bar is shown in the figure.

[0374] (D) Immunoblotting analysis was performed on the inhibitory effect of PI4KB-SA on LC3 lipidation in control cells and RAS mutant cancer cell lines (with or without 500 nM bafloxacin A1 cultured for 1.5 hours).

[0375] (E) Quantitative analysis of the ratio of lipid-modified LC3 to tubulin in (D), with the control set at 1.00 (control cells treated with bafloxacin A1) (mean ± SEM). Three experiments were performed (two-tailed t-test) to obtain statistical data. ***, p < 0.01; ****, p < 0.0001.

[0376] (F) CCK8 analysis (mean ± SEM) was performed on the proliferation of control cells and Ras-mutant cell lines treated with GFP-peptide-1 alone or in combination with trametinib (Tra, 100 nM) for 96 hours. Three experiments (two-tailed t-tests) were performed to obtain statistical data. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0377] Figure 17: PI4KB-peptide-1 phosphorylation required for different ULK1 phosphorylations to mediate KRAS(G12V)-induced autophagy.

[0378] (A) The changes in p-PI4KB caused by different site mutations of PI4KB-peptide-1 in control cells and ULK1-overexpressing 293T cells were analyzed by immunoblotting.

[0379] (B) PI4P levels (mean ± SEM) were quantified by different site mutations in PI4KB-peptide-1 in KRAS(G12V)293T cells (see Figure 19A). Three experiments were performed (two-tailed t-test) to obtain statistical data. **, p < 0.01.

[0380] (C) Statistical analysis (mean ± SEM) was performed on LC3 lipidation resulting from different site mutations in PI4KB-peptide-1 overexpression in control cells and PI4KB knockdown KRAS(G12V)293T cells (see Figure 19B). Three experiments were conducted to obtain statistical data (two-tailed t-test). **, p < 0.01.

[0381] (D) Immunoblotting analysis of changes at different sites of p-ULK in KRAS(G12V) and starvation-induced 293T cells.

[0382] (E) Immunoblotting analysis was performed on the changes of p-PI4KB and p-Beclin1-S30 in 293T cells overexpressing ULK1-WT, K46I or 3E (S317E / S556E / S758E) mutants.

[0383] (F) Mass spectrometry analysis of phosphorylation sites of ULK1 in KRAS(G12V) and starvation-induced 293T cells.

[0384] (G) The heatmap shows the changes in p-PI4KB and p-Beclin1 with different ULK mutations in ULK-3E-overexpressing 293T cells (see Figure 19C-E). The color intensity represents the log2 (fold) of the count for each sample.

[0385] (H) Immunoblotting analysis of the specificity of p-ULK-S479 antibody in ULK1 KO MEF cells (reexpressing Myc-ULK1 WT or S479A mutant or not expressed).

[0386] (I) Immunoblotting analysis of the specificity of p-ULK-S479 antibody in KRAS(G12V) and starvation-induced 293T cells.

[0387] Figure 18: Validation of PI4KB-peptide-1 phosphorylation antibody, the inhibitory effect of PI4KB-SA on the proliferation of RAS mutant cells, and the precise localization of PI4KB-peptide-1 and ULK1 phosphorylation.

[0388] (A) Images of xenograft tumors of HCT116 and SW620 cells were obtained from mice treated with: (1) control group (Ctrl); (2) trametinib (Tra); (3) PI4KB-SA overexpression (SA); or (4) a combination of both (Tra+SA) (n=12 per group). The tumors were removed and photographed 24 days after treatment.

[0389] (B) and (A) show the weights of xenograft tumors (mean ± SD). Statistical analysis was performed using a two-tailed t-test. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

[0390] Growth curves (mean ± SD) of xenograft tumors are shown in (C)(A). Statistical analysis was performed using two-way ANOVA; ****, p < 0.0001.

[0391] Immunofluorescence and immunohistochemical analyses of xenograft tumor sections shown in (D)(A). As shown, sections were stained with antibodies against LC3, P62, p-ERK1 / 2, or Ki67. A scale bar is included in the figure.

[0392] (E) Statistical analysis was performed on the number of LC3 spots, P62 positivity rate, p-ERK positivity rate, and Ki67 expression level shown in (D) (mean ± SD). Two-tailed t-tests were used for statistical analysis; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0393] Figure 19: Validation of PI4KB-peptide-1 phosphorylation antibody, the inhibitory effect of PI4KB-SA on the proliferation of RAS mutant cells, and the precise localization of PI4KB-peptide-1 and ULK1 phosphorylation.

[0394] (A) Dotted immunoblotting analysis of the effect of the PI4KB mutant on KRAS(G12V)-induced PI4P production in 293T cells.

[0395] (B) After knocking down PI4KB in 293T cells (with or without 500 nM bafloxacin A1 and cultured for 1.5 h), the rescue effect of PI4KB-WT, PI4KB-SA and PI4KB mutants on KRAS(G12V)-induced LC3 lipidation was analyzed by immunoblotting.

[0396] (CE) Immunoblotting analysis was performed on changes in p-PI4KB and p-Beclin1-S30 in 293T cells overexpressing mutants at designated sites of ULK1-3E (S317E / S556E / S758E) or 3E (S317E / S556E / S758E).

[0397] Figure 20: PI4KB-peptide-1 inhibits autophagy and growth in RAS mutant cells.

[0398] (A) Immunohistochemical analysis was performed on HCT116, SW620, A549, H1299, and MiaPaCa-2 cell sections treated with or without SBI-0206965 (10 μM) or peptide-1 (Tat-Pep.1, 25 μM) for 2 hours. As shown in the figure, the sections were stained with antibodies against PI4KB and p-PI4KB. A scale bar is included in the figure.

[0399] (B) Immunoblotting analysis of LC3 lipidation in HCT116, SW620, A549, H1299 and MiaPaCa-2 cells overexpressing (or not overexpressing) GFP-peptide-1 (GFP-Pep.1) (cultured with or without 500 nM bafloxacin A1 for 1.5 h).

[0400] (C) Quantitative analysis of the ratio of lipid-modified LC3 to tubulin in (B), with the control set at 1.00 (control cells treated with bafloxacin A1) (mean ± SEM). Three experiments were performed (two-tailed t-test) to obtain statistical data. ***, p < 0.01; ****, p < 0.0001.

[0401] (D) FACS analysis was performed on HCT116, SW620, A549, H1299, and MiaPaCa-2 cells (expressing or not expressing GFP-peptide-1 (GFP-Pep.1)) co-expressing mt-Keima and Parkin using V610 and Y610-mCherry detectors (Beckman CytoFLEX LX). FACS results represent at least three independent experiments.

[0402] (E) Percentage of mitophagic cells calculated based on Y610-mCherry / V610, as shown in (D). Data are expressed as mean ± SEM. Three experiments were performed (two-tailed t-test) to obtain statistical data. ****, p < 0.0001.

[0403] (F) Immunofluorescence analysis of WIPI2 spots on different cancer cell lines treated with HEV1 (10 μM), SBI-209626 (10 μM), SB203580 (10 μM) for 1.5 hours or overexpressing GFP-peptide-1 (GFP-Pep.1). The figure shows representative cell images. Scale bars are included in the figure.

[0404] (G) Quantitative analysis of WIPI2 points shown in (F). Data are expressed as mean ± SEM. Three experiments (50 cells per group / experiment) were conducted to obtain statistical data (two-tailed t-test). **, p < 0.01; ***, p < 0.01; ****, p < 0.0001.

[0405] (H) CCK8 assay was performed on the cell proliferation of HCT116, SW620, A549, H1299, and MiaPaCa-2 cells. The cells were treated for 96 hours with the following substances: control group (Ctrl); trametinib (Tra, 100 nM); Tat-peptide-1 (Pep.1, 5 μM); a combination of trametinib and Tat-peptide-1 (Tra+Pep.1); chloroquine (CQ, 10 μM); and a combination of trametinib and chloroquine (Tra+CQ) (mean ± SEM). Three experiments were performed (two-tailed t-test) to obtain statistical data. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0406] Figure 21: PI4KB-peptide-1 phosphorylation is increased in RAS-mutant cancers.

[0407] (A) Immunoblotting analysis of p-PI4KB levels in different cancer cell lines.

[0408] (B) Tissue microarray of CRC tissue (control and RAS mutation) and representative immunohistochemical staining of p-PI4KB in adjacent normal tissue.

[0409] (C) p-PI4KB levels of tissue microarrays from patients with colon cancer (adjacent to cancer: n=119; cancer: n=60 (Ctrl) and n=59 (RAS-mt)) and rectal cancer (adjacent to cancer: n=37; cancer: n=22 (Ctrl) and n=15 (RAS-mt)) (mean ± SD). Statistical analysis was performed using Wilcoxon's signed-rank test. **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0410] Figure 22: ULKl-mediated PI4KB-peptide-1 phosphorylation may be a therapeutic target for RAS-mutant cancers.

[0411] (A) Immunoblot analysis of PI4KB phosphorylation of anti-p-PI4KB antibody in 293T cells expressing Myc-ULK1. The 293T cells were either overexpressing or not overexpressing GFP-peptide-1 (GFP-Pep.1), or treated or untreated with SBI-0206965 (10 μM).

[0412] (B) Dotted immunoblotting analysis was performed to investigate the effect of GFP-peptide-1 on KRAS(G12V)-induced PI4P production.

[0413] (C) Immunoblotting analysis of KRAS(G12V)-induced LC3 lipidation when GFP-peptide-1 (GFP-Pep.1) was overexpressed (with or without 500 nM bafloxacin A1 and cultured for 1.5 hours).

[0414] (D) Immunofluorescence analysis of KRAS(G12V)-induced LC3 spots with or without overexpression of GFP-peptide-1 (GFP-Pep.1). The figure shows representative images. Scale bars are included in the figure.

[0415] (E) Quantitative analysis of LC3 points shown in (D) (mean ± SEM). Three experiments were conducted (50 cells per group / experiment) and statistical analysis was performed (two-tailed t-test). **, p < 0.01.

[0416] (F) The effect of GFP-peptide-1 (GFP-Pep.1) on autophagic flux was detected in KRAS(G12V) cells stably expressing mCherry-pHluorin-LC3B using a tandem fluorescent LC3 system. The figure shows a representative cell image. Scale bars are included in the figure.

[0417] (G)RFP + GFP + and RFP + GFP - Quantitative analysis of LC3 points was performed. Data are expressed as mean ± SEM. Three experiments (50 cells per group / experiment) were conducted to obtain statistical data (two-tailed t-test). **, p < 0.01.

[0418] (H) FACS analysis was performed on control cells co-expressing mt-Keima and Parkin and KRAS (G12V) cells (expressing or not expressing GFP-peptide-1 (GFP-Pep.1)) using V610 and Y610-mCherry detectors (Beckman CytoFLEX LX). FACS results represent at least three independent experiments.

[0419] (I) Percentage of mitochondrial autophagy cells as shown in (H) based on Y610-mCherry / V610. Data are expressed as mean ± SEM. Three experiments were performed (two-tailed t-test) to obtain statistical data. ****, p < 0.0001.

[0420] (J) Truncation of Pep.1, immunoblotting analysis of the inhibitory effect of truncated Pep.1-6, Pep.1-5, Pep.1-4, Pep.1-3, Pep.1-2, and Pep.1-1 on ULK1-promoted p-PI4KB.

[0421] Figure 23: ULKl-mediated PI4KB-peptide-1 phosphorylation may be a target for the treatment of RAS-mutant cancers.

[0422] (A) Images of xenograft tumors of HCT116 and SW620 cells were obtained from mice treated with the following substances: (1) control group (Ctrl, n=14); (2) trametinib (Tra, n=12); (3) GFP-peptide-1 (GFP-Pep.1, n=14); or (4) a combination of trametinib and GFP-peptide-1 (Tra+Pep.1, n=12); (5) chloroquine (CQ, n=14); (6) a combination of trametinib and chloroquine (Tra+CQ, n=12).

[0423] (B) and (A) show the weights of xenograft tumors (mean ± standard deviation (SD)). Statistical analysis was performed using a two-tailed t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0424] (C)(A) show the growth curves of xenograft tumors (mean ± SD). Statistical analysis was performed using two-way ANOVA; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0425] Immunofluorescence and immunohistochemical analysis of xenograft tumor sections shown in (D)(A). As shown, sections were stained with antibodies against LC3, P62, p-PI4KB, PI4KB, p-ERK1 / 2, or Ki67. The scale bar is located in the lower right corner of the image.

[0426] (E) Statistical analysis was performed on the number of LC3 points, P62 positivity rate, p-PI4KB positivity rate, t-PI4KB positivity rate, p-ERK positivity rate, and Ki67 expression level shown in (D) (mean ± SD). A two-tailed t-test was used for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0427] Figure 24: Inhibitory effect of PI4KB-peptide-1 on tumors in a KPC mouse pancreatic cancer model.

[0428] (A) Schematic diagram of KPC mouse experimental design.

[0429] (B) Representative images of primary pancreatic tumors in the treatment groups during endpoint analysis: Control group (Ctrl); Trametinib (Tra); Tat-peptide-1 (Pep.1); Trametinib and Tat-peptide-1 combination (Tra+Pep.1); Chloroquine (CQ); Trametinib and chloroquine combination (Tra+CQ).

[0430] The weights (mean ± SD) of primary pancreatic tumors shown in (C) and (B) (n = 10 tumors) were analyzed using a two-tailed t-test. *, p < 0.05; **, p < 0.01.

[0431] Immunofluorescence and immunohistochemical analyses of xenograft tumor sections shown in (D) and (B) are presented. Tissue morphology under different conditions is shown by H&E staining. As shown, sections were stained with antibodies against LC3, P62, p-PI4KB, t-PI4KB, p-ERK, Ki67, MHC-I, or CD8a. The scale bar is located in the lower right corner of the image.

[0432] (E) Statistical analysis was performed on the tumor area, number of LC3 points, P62 positivity rate, p-PI4KB positivity rate, t-PI4KB positivity rate, p-ERK positivity rate, Ki67 expression, MHC-I expression, and CD8a positivity rate shown in (D) (mean ± SD). Two-tailed t-tests were used for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0433] (F) The figure shows the survival curves of KPC mice. Mice were treated with the following substances: control group (Ctrl, n = 10); trametinib (Tra, n = 11); Tat-peptide-1 (Pep.1, n = 10); combination of trametinib and Tat-peptide-1 (Tra+Pep.1, n = 12); chloroquine (CQ, n = 11); combination of trametinib and chloroquine (Tra+CQ, n = 13).

[0434] (G)RAS-induced specific autophagy schematic diagram. Detailed Implementation

[0435] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0436] Cells, animals, and experimental methods used in the examples

[0437] 1. Cells

[0438] Cells (HEK293T, HCT116, SW620, RKO, HT29, A549, H1299, MiaPaCa2, and SW1990) were cultured in Duchenne Modified Eagle Medium (DMEM) or RPMI-1640 (FHC) containing 10% fetal bovine serum at 37°C and 5% CO2. After lentiviral infection, HEK293T cells stably expressing Flag-KRAS (G12V) were selected using antibiotics (Flag-KRAS(G12V)-293T stable). For lentiviral transduction, HEK293T cells (pMD2.G and psPAX2) were transfected using plasmids. The virus was harvested 60-72 hours after transfection. The viral supernatant was centrifuged for 5 minutes (600×g) to remove cell debris. The virus supernatant (30% virus supernatant) was diluted with fresh medium containing 10 μg / mL agglutinin and used to infect the designated cells. After 24 hours, the medium was replaced with growth medium. Transduced cells were selected and cultured in DMEM supplemented with 10% fetal bovine serum, 150 μg / mL genomic trimethoprim (Sigma), and 15 μg / mL cyprodinil (Sigma). Target gene expression was confirmed by SDS-PAGE electrophoresis and Western blotting.

[0439] 2. Mice

[0440] The Tsinghua University Laboratory Animal Use and Management Committee has approved the conduct of mouse experiments.

[0441] To conduct xenotransplantation research, nude mice purchased from Charles River (Beijing) were housed in light- and temperature-controlled enclosures and ventilated cages in an SPF-grade facility. The mice had free access to food and water. Two × 10⁶ cells were resuspended in 100 μL of an artificial basement membrane (Yisheng Biotechnology, 40183ES10). 6 HCT116 or SW620 cells were subcutaneously injected into the peritoneum of 4-week-old male NOD / SCID mice via the thigh to induce xenograft tumors and establish a xenograft tumor model. Treatment was then initiated with intraperitoneal injections of control (corn oil), 1 mg / kg trametinib, 40 mg / kg Tat-peptide-1 (Tat-Pep.1), 50 mg / kg chloroquine, or a combination of the above doses of trametinib with Tat-peptide-1 or chloroquine, twice weekly. Tumors were measured twice weekly using a diameter gauge, and tumor volume was calculated using the following formula: Volume = 4 / 3 × π × (((length + width) / 2) / 2) 3 Each group analyzed more than 12 tumors. Tumors were removed, photographed, and weighed, and the average tumor weight was calculated. The significance of differences in tumor size was calculated using a two-tailed t-test.

[0442] LSL-Kras G12D p53 F / FPdx1-Cre mice (KPC) were donated by Charles J. David (Tsinghua University). In this genotype, age- and sex-matched (unless otherwise stated) experimental males and females were littermates. Treatment was then initiated intraperitoneally with control (corn oil), 1 mg / kg trametinib, 40 mg / kg Tat-peptide-1 (Tat-Pep.1), a combination of the above doses of trametinib and Tat-Pep.1, 40 mg / kg chloroquine, or a combination of the above doses of trametinib and chloroquine, three times a week.

[0443] 3. Experimental Methods

[0444] 1) Plasmids, siRNA oligonucleotides, and transfection

[0445] Human KRAS (G12V), KRAS (G12D), or KRAS (G12C) was cloned into the pLenti-CMV vector. EGFP-PI4KB and HA-PI4KB plasmids were generated by PCR and ligation. HA-PI4KB and HA-PI4KB-D656A plasmids were generated by site-directed mutagenesis PCR. siRNA and shRNA are shown in Tables 1-2. The EGFP-peptide-1 plasmid was cloned by PCR and ligation. PI4P (Avanti Polar Lipids) was transfected using unlabeled shuttle PIP vector 3 (Echelon Biosciences). HEK293T cells were transfected with plasmids using PEI (Polysciences) according to the manufacturer's protocol, and FHC cells were transfected with plasmids using X-tremeGENE HP (Roche). siRNA transfection was performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocol.

[0446] Table 1

[0447] Table 2

[0448] 2) Peptide synthesis

[0449] The L-amino acid peptide was synthesized by Beijing Zhongke Yaguang Biotechnology Co., Ltd. The Tat-peptide-1 (Tat-Pep.1) peptide sequence YGRKKRRQRRRGGELPSLSPAPDTGLSPSK (SEQ ID NO: 57) consists of 11 amino acids from the N-terminal Tat protein transduction domain, a flexible GG linker, and 17 amino acids (253-269) derived from the C-terminal P4KB. The control peptide (Tat control) consists of the Tat protein transduction domain, a GG linker, and a scrambling sequence (YGRKKRRQRRRGGVGNDFFINHETTGFATEW (SEQ ID NO: 58)).

[0450] 3) Immunofluorescence

[0451] Cells were cultured at room temperature with 4% low-temperature paraformaldehyde for 20 minutes. Cells were then further permeabilized for 10 minutes at room temperature with 0.1% Triton X-100 diluted in PBS, followed by blocking with 10% fetal bovine serum diluted in PBS for 1 hour, and incubated with primary antibody for 1 hour at room temperature. Cells were washed three times with PBS and then incubated with secondary antibody for 1 hour at room temperature.

[0452] In tissue immunofluorescence, paraffin-embedded tumors were prepared into 5 μm sections and dried in an oven at 60°C for 24 hours. They were then dewaxed with xylene and hydrated with a gradient of ethanol (100-70%). After incubation with antigen retrieval solution (ZSGB Biotechnology Co., Ltd., Beijing, China) and 3% H2O2 for 30 minutes each, the slides were rinsed with water and incubated overnight with the primary antibody at 4°C. The next day, the slides were rinsed again and incubated with the secondary antibody at room temperature for 1 hour.

[0453] Fluorescence images were obtained using an Olympus FV3000 confocal microscope. Quantification was performed using ImageJ.

[0454] 4) HaloTag-LC3B Processing Analysis

[0455] Autophagy flux analysis was performed using HEK293T cells stably expressing the Tet-on KRAS (G12V) system and HaloTag-LC3B. After induction with doxycycline (final concentration 1 μg / ml) for 36 hours, cells in the culture medium were pulse-labeled for 20 minutes with 100 nM tetramethylrhodamine (TMR) conjugate ligand (Promega, G8251). Cells were washed twice with phosphate-buffered saline (PBS), and then further cultured for 2 hours in fresh medium containing only doxycycline (final concentration 1 μg / ml) or in combination with 100 nM bafloxacin A1 (BafA1). After culture, cells were lysed, and 20 μg of protein was collected for SDS-PAGE electrophoresis of each sample. For in-gel fluorescence imaging, gel imaging was performed immediately after SDS-PAGE using a ChemiDoc imaging system (Bio-Rad Laboratories). When using Western blotting, proteins were transferred from SDS-PAGE gels to polyvinylidene fluoride (PVDF) membranes (Millipore, IPVH00010). After incubation with a suitable antibody, the signal was detected using a chemiluminescent HRP substrate and a ChemiDoc imaging system (Bio-Rad Laboratories). Band intensity was quantified using the "Gel Analyzer" tool in the open-source image processing software Fiji.

[0456] 5) Co-immunoprecipitation (co-IP) and Western blotting

[0457] Cells were lysed on ice for 30 minutes in IP buffer containing a mixture of protease inhibitors (50 mM Tris / HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP40, 10% glycerol), and the lysates were removed by centrifugation. The resulting supernatant was incubated with agarose microspheres conjugated to a specified antibody and vortexed at 4°C for 3 hours. The agarose microspheres were then washed five times with IP buffer and examined by Western blotting.

[0458] 6) Immunoprecipitation and in vitro kinase assay

[0459] Cells expressing the specified protein were lysed in ice-cold lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.3% Triton X-100, 5 mM EDTA, supplemented with Complete protease inhibitor mixture (EDTA-free) (Roche) and PhosStop phosphatase inhibitor mixture (Roche). After centrifugation of the lysate, the resulting supernatant was incubated with the specified antibody-conjugated agarose microspheres at 4°C for 1 hour. After incubation, the microspheres were washed three times with lysis buffer (as described above) and once with kinase reaction buffer (20 mM HEPES, pH 7.5, 20 mM MgCl2, 25 mM β-glycerophosphate, 2 mM dithiothreitol, 100 μM sodium orthovanadate). The microspheres were then placed in a final volume of 20 μl of reaction buffer and incubated at 30°C for 15 minutes. The buffer contained 20 μM ATP and 5 μg of purified GFP-PI4KB protein. Add 5 times the amount of SDS sample buffer and heat to 65°C for 5 minutes to stop the reaction. Then transfer the protein onto a PVDF membrane and analyze it by Western blotting.

[0460] 7) Dotted immunoblot

[0461] To determine PI4P, total lipids were extracted from HEK293T cells. Cell suspensions (300 mL per aliquot) were incubated with 1.2 mL of chloroform / methanol solution (chloroform:methanol = 1:2), vortexed for 30 seconds, and then shaken at 180 rpm for 1 hour at 37°C. The chloroform phase was collected, the lipid solution was evaporated by a nitrogen stream, and further dried in a 37°C incubator for 1 hour. The dried lipids were resuspended in anhydrous ethanol. The lipid solution was measured and used as a standard (PC) to normalize the lipid concentration. The lipids were coated onto a nitrocellulose membrane (Millibor) and air-dried for 1 hour. The membrane was incubated overnight at room temperature with PI4P antibody. The next day, the membrane was washed and incubated for 1 hour with anti-mouse antibody (diluted 1:1000 in blocking buffer). Finally, chemiluminescence was used to reveal the PI4P levels recognized by the antibody.

[0462] To determine the binding of WIPI2 to lipids, a phospholipid membrane was immobilized using a purified WIPI2-coated membrane (Echelon PIP test paper, catalog number P-6001). Proteins were detected using anti-WIPI2 and anti-mouse secondary antibodies and scanned using a ChemiDoc imaging system.

[0463] 8) qRT-PCR method

[0464] Total RNA was isolated from different cell lines using TRIzol reagent (Beyotime, R0016) according to the manufacturer's instructions. An equal volume of RNA was reverse transcribed into cDNA using the Revert Aid First-Strand cDNA Synthesis Kit (Abclonal, E047-01B) according to the manufacturer's instructions. Quantitative PCR was performed using the ABI Step One Plus system. PCR reactions were performed using the SYBR Green PCR master mix kit (Abclonal, RK20429) and 0.5 μM specific primers in 10 μL of reaction buffer. The PCR primers used are shown in Table 3.

[0465] Table 3

[0466] 9) Electron microscopy (EM) analysis

[0467] After inducing 293T-KRAS (G12V) and control cells, they were treated with Dox for 36 hours. Cells were fixed with 2.5% glutaraldehyde at room temperature for 1 hour and washed three times with 0.1M PB (0.02M NaH2PO4, 0.08M Na2HPO4, pH 7.4) for 15 minutes each time. After fixation, they were stained with 1% osmium tetroxide (SPI, 1250423) on ice for 0.5 hours. Cells were washed three times with ultrapure water for 15 minutes each time and then placed in 1% uranium acetic acid aqueous solution (EMS, 22400) overnight at 4°C. The samples were then washed three times with ultrapure water for 15 minutes each time and dehydrated using a low-temperature gradient of ethanol (50%, 70%, 80%, 90%, 100%, 100%, 100%; 2 minutes each time). EPON 812 resin was impregnated with a 1:1 (volume ratio) resin and ethanol mixture for 8 hours, a 2:1 (volume ratio) resin and ethanol mixture for 8 hours, and a 3:1 (volume ratio) resin and ethanol mixture for 8 hours. Then, pure resin was impregnated twice, each time for 8 hours. Finally, fresh resin was impregnated, and the samples were polymerized in a 60°C oven for 48 hours. The embedded samples were cut into 80 nm thick sections and stained with uranyl acetate and lead citrate (C1813156). The samples were imaged using an H-7800 120 kV transmission electron microscope.

[0468] 10) Mass spectrometry (MS) and Phos-Tag gel analysis

[0469] 293T cells expressing HA-PI4KB (with or without Myc-ULK1) were cultured in three 10 cm culture dishes until 100% confluence. The small molecule kinase inhibitor SBI-0206965 was added to the culture to inhibit ULK1. HA-PI4KB was then immunosorbed using agarose conjugated with an anti-HA antibody and eluted with HA peptides. The purified HA-PI4KB was analyzed by SDS-PAGE and colocalization analysis was performed using Coomassie Brilliant Blue staining. Mass spectrometry analysis of specific HA-PI4KB bands was performed to identify post-translational modifications, including phosphorylation. Mass spectrometry analysis was performed by the Center for Protein Chemistry and Proteomics, Tsinghua University. PI4KB phosphorylation was confirmed using Phos-Tag gel analysis (Wako, 199-17391) according to the manufacturer's instructions.

[0470] 11) Liposome flotation and granulation test

[0471] POPC (1-palmitoyl-2-oleoyl-glycerol-3-choline phosphate), POPS (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine), DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine), cholesterol, PI3P, PI4P, and PI5P were purchased from Avanti Polar Lipids. A lipid mixture was formed by mixing POPC:POPS:DOPE:cholesterol with 1% PI3P, PI4P, or PI5P in a ratio of 4:2.5:2.5:10. The method for preparing small single-chamber liposomes (SUVs) using the above lipid mixture is described above. The lipid mixture was dried with a nitrogen stream and further dried at 37°C for 1 hour. Then, HEPES buffer (20 mM HEPES, pH 7.4, 150 mM NaCl) was added to fully hydrate the lipid membrane, followed by freezing in liquid nitrogen and thawing in a 42°C water bath, repeated 10 times. Finally, the liposomes were extruded 20 times from a polycarbonate membrane with specific pore sizes to generate an SUV.

[0472] In the flotation experiment, purified WIPI2 was added to 120 μL of SUV solution to achieve the stated concentration and incubated at room temperature for 30 minutes. Membrane flotation was performed to remove free proteins as described above. Briefly, 480 μL of 50% OptiPrep was added to the above 120 μL solution. The mixture was then covered sequentially with 480 μL of 30% OptiPrep and 90 μL of HEPES buffer, and centrifuged at 100,000 x g for 2 hours. A 60 μL top fraction was collected. 1 μL of the top fraction was taken and the PC concentration was determined using a microplate spectrophotometer. The remaining fraction was added with 5x SDS loading buffer and analyzed by Western blotting.

[0473] 12) Cell proliferation detection

[0474] Cell proliferation was assessed using a CCK-8 assay kit (Dojindo Molecular Technologies). In short, cells were seeded in 96-well plates. 10 μL of CCK-8 solution was added to each well (each well containing 100 μL of culture medium), and the plates were incubated at 37°C for 2 hours. Absorbance was measured at 450 nm using an ELISA plate analyzer. Cell growth was analyzed on days 1 and 4 in the cell proliferation assay.

[0475] 13) Immunohistochemical (IHC) staining

[0476] The pancreas or tumor was removed, fixed in PBS solution with 4% paraformaldehyde, and then embedded in paraffin. 5μm sections were prepared and stained with hematoxylin and eosin (H&E). IHC preparation: Paraffin-embedded tumors or pancreas were prepared into 5μm sections and dried in an oven at 60°C for 24 hours, followed by dewaxing with xylene and gradient hydration with ethanol (100-70%). After incubation for 30 minutes each with antigen retrieval solution (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., China) and 3% H2O2, the slides were rinsed with water and incubated overnight with the primary antibody at 4°C. The next day, the slides were rinsed again and incubated with the secondary antibody (Beijing ZSGB Biotechnology Co., Ltd., China) at room temperature for 30 minutes, followed by staining with 3,3′-diaminobenzidine (DAB) and hematoxylin, respectively.

[0477] Tissue microarrays containing tumor tissues from colorectal cancer and rectal cancer, along with adjacent normal tissues, were obtained from Shanghai Zhuoli Biotechnology Co., Ltd. The samples were paraffin-embedded. These clinical materials were used for research with patient consent and approval from the institution's research ethics committee. Paraffin-embedded tissue sections (4 mm) were prepared according to standard methods, and p-PI4KB expression was detected by IHC staining (1:1000 dilution). Pathologists evaluated the sections, and the experimental results and patient outcomes were blinded to the relevant pathologists.

[0478] 14) Immunohistochemistry (IHC) scoring method

[0479] This is a modified label score (H score) calculated based on the percentage of positively stained cancer cells and their intensity in each tissue core. Staining intensity is assessed using an immunostaining score, which is the sum of the proportion and intensity of stained tumor cells. In short, a proportion score representing the estimated proportion of positively stained tumor cells is first obtained (0, none; 1, <1 / 100; 2, >1 / 100 to <1 / 10; 3, >1 / 10 to <1 / 3; 4, >1 / 3 to <2 / 3; 5, >2 / 3). Then, an intensity score representing the average intensity of specified positively stained tumor cells is obtained (0, none; 1, weak; 2, moderate; 3, strong). The proportion score and intensity score are then summed to obtain a total score ranging from 0 to 8.

[0480] 15) Statistical Analysis

[0481] Statistical information for each experiment, including statistical methods, p-values, and number of experiments (n), is shown in the attached figures and corresponding legends. Statistical analysis was performed using GraphPad Prism.

[0482] The reagents used in the examples or their sources are shown in Table 4.

[0483] Table 4

[0484] Example 1: Establishing a cellular system for analyzing KRAS-induced autophagy

[0485] Constitutively activated RAS mutants can trigger autophagy. This example aims to determine whether RAS-induced autophagy and starvation-induced autophagy employ the same ATG cascade.

[0486] 1. KRAS activation triggers autophagy upregulation

[0487] 293T cells were modified with doxycycline (Dox)-induced G12V mutant KRAS gene (KRAS(G12V)) to activate autophagy. KRAS(G12V) expression was dose-dependent; to facilitate subsequent autophagy analysis, the concentration and duration of induction were systematically adjusted to achieve expression levels comparable to endogenous RAS in various cancer cell lines (Figures 1A-1B). Lipid-modified LC3 (LC3-II), an autophagosome membrane marker, was further examined. Western blot analysis showed that Dox treatment increased LC3-II levels regardless of the presence of the lysosomal inhibitor bafloxacin A1 (used to block autophagosome maturation), indicating that KRAS(G12V) expression activates autophagy (Figure 1C). This is similar to results obtained in 293T cells expressing KRAS(G12D) or KRAS(G12C) alleles (Figure 2A). Compared to the control, the LC3 point (a marker of autophagosomes) was continuously increased in Dox-treated KRAS(G12V) cells (Fig. 1D, Fig. 1E). Electron microscopy analysis showed that KRAS(G12V)-expressing cells contained more autophagic vesicles compared to the control (Fig. 1F, Fig. 1G).

[0488] Using a dual-fluorescent (mCherry-pHluorin) LC3 reporter gene, KRAS(G12V) enhanced autophagic flux. Fluorescence analysis of autophagosomes and autolysosomes was performed using this system. Consistent with its effect on autophagosome biogenesis, Dox-induced KRAS(G12V) increased the number of autophagosome and autolysosome sites (Fig. 1H, Fig. 1I), indicating that KRAS(G12V) enhances autophagosome formation and autophagic flux. Furthermore, this study used a HaloTag (Halo)-based reporter gene treatment method to verify the KRAS(G12V)-induced enhancement of autophagic flux (Fig. 1J). Subsequently, p62 clearance and mitophagy were assessed using a mito-keima assay (Fig. 1K-1N) to confirm autophagic degradation. These results again consistently demonstrate that autophagic flux increases after KRAS(G12V) expression.

[0489] Several KRAS (G12C) inhibitors, including AMG510 (Amgen), have entered clinical trials and are currently being evaluated for their efficacy in treating non-small cell lung cancer, colorectal cancer (CRC), and other solid tumors carrying this mutation. To determine whether mutant KRAS-induced autophagy depends on KRAS activation in the system described in this application, the effect of AMG510 on LC3-II was examined. AMG510 treatment reduced LC3-II levels in cells expressing KRAS (G12C) but failed to reduce LC3-II levels in cells expressing KRAS (G12V) or KRAS (G12D) (Figure 2B), suggesting that KRAS activation triggers the upregulation of autophagy.

[0490] 2. Similarities and differences in the use of autophagy factors in KRAS (G12V) and starvation-induced autophagy.

[0491] To elucidate the roles of autophagy factors in KRAS-induced autophagy, we targeted key autophagy factors involved in various regulatory steps of starvation-induced autophagy by knocking down genes in a Dox-induced KRAS (G12V) expression system on 293T cells. Key autophagy factors involved in starvation-induced autophagy included the ULK kinase complex (ULK1 and FIP200), the PI3K complex (ATG14 and Beclin-1), the PI3P effector WIPI2, lipidation factors (ATG5, ATG16L1, and ATG4B-C74S overexpression), lipid transfer factors ATG2A and ATG2B, ATG9 vesicles (ATG9A), the ESCRT complex (VPS4A & B), and the SNARE complex (STX17, SNAP29, and VAMP8). The knockdown efficiency of each gene was verified by Western blotting or qPCR (Figures 2C-2E). Autophagy-related experiments were then used to comprehensively evaluate the roles of these autophagy factors in KRAS-induced autophagy.

[0492] Consistent with the blocking effects observed in starvation-induced autophagy, inhibition of the ULK kinase complex, the PI3P effector WIPI2, and lipidation factors significantly attenuated KRAS(G12V)-induced LC3 lipidation (Fig. 10, 2D, 2E) and autophagic flux (Fig. 10, 2F, 3A-3E). Furthermore, inhibition of the SNARE complex also interfered with autophagic flux, consistent with observations in starvation-induced autophagy (Fig. 10, 2D-F, 3A-3E). Interestingly, while the PI3K complex, ATG9A, and various ATG2s are indispensable for starvation-induced autophagy, knockdown of them did not affect KRAS-induced autophagy (Fig. 10, 1P, 3F-3I). Moreover, KRAS-induced autophagy did not require ESCRT (Fig. 10, 2D-2F, 3A-3E). Notably, two lipid transfer proteins, VPS13A and VPS13B, used to transfer ATG2-like lipids, are essential in KRAS-induced autophagy (Fig. 1O, Fig. 2D-2F, Fig. 3A-3E). These findings collectively indicate that KRAS-induced autophagy and starvation-induced autophagy employ different autophagy factors, including both the same and different autophagy factors.

[0493] Example 2: PI4KB regulates oncogenic KRAS-induced autophagy

[0494] Our interest was piqued by the observational independence of PI3K in KRAS-induced autophagy, as the PI3K complex has historically been considered crucial in starvation-induced autophagy, linking protein signaling and membrane remodeling. Although PI3K is dispensable in KRAS-induced autophagy, the PI3P effector WIPI2 remains indispensable (Fig. 1O, Fig. 2D-2F, Fig. 3A-3E). Given that WIPI2 binds to various PI-phosphates, this example first hypothesizes that another PI-kinase plays a role similar to PI3K in KRAS-induced autophagy. To determine the specific PI-kinase and PI-phosphate responsible for this role, this example evaluated the effects of different phosphokinase inhibitors on LC3-II levels in KRAS(G12V)-induced 293T cells.

[0495] Notably, treatment with the PI3K inhibitors SAR405, PIK-III, VPS34-IN1, and wamancozeb (20 nM) failed to reduce LC3-II levels in Dox-induced cells compared to controls, as did the PI5K inhibitor YM201636 (Fig. 4A, 5A, 5B). Conversely, these inhibitors effectively delayed starvation-induced and glucose starvation-induced autophagy, serving as positive controls (Fig. 4A, 5C-5E). Interestingly, treatment with 200 nM wamancozeb (inhibiting type III PI4K) resulted in a decrease in LC3-II levels during Dox treatment (Fig. 4A, 5F), suggesting that PI4K and PI4P may play a role in KRAS(G12V)-induced autophagy.

[0496] In the system of this application, shRNA-mediated PI4K isoform knockdown was used to assess LC3-II levels and autophagic flux assays to determine which PI4K factor primarily regulates oncogenic KRAS-induced autophagy. Knockdown efficiencies for all isoforms were greater than 80% (Figure 6A). Knockdown of PI4KIIA, PI4KIIB, and PI4KA did not affect KRAS(G12V)-induced LC3-II accumulation, while knockdown of PIK4B significantly reduced LC3-II and autophagic flux (Figures 4B, 6B-6F).

[0497] The effect of PI4KB inhibitors on KRAS(G12V)-induced autophagy was further investigated to confirm the influence of PI4KB on KRAS(G12V)-induced autophagy. Tests showed that T-00127_HEV1 and PIK93 strongly inhibited KRAS(G12V)-induced LC3 lipidation and autophagic flux (Figures 4B, 6B-6F, and 7). Furthermore, PI4P levels were assessed using dotted immunoblotting and PI4P staining, revealing that KRAS(G12V) expression increased PI4P levels, while PI4KB knockdown eliminated this effect (Figures 4C-4F). Similarly, PI4KB silencing or T-00127-HEV1 treatment significantly reduced KRAS(G12V)-induced LC3 lipidation and point count (including KRAS(G12V)-expressing FHC and Dox-induced KRAS(G12V)HEK293T), as well as autophagic flux in Dox-induced KRAS(G12V)HEK293T (Fig. 4G-4Q). The addition of exogenous PI4P reversed these effects in Dox-induced KRAS(G12V)HEK293T (Fig. 4I-4Q). The data suggest that oncogenic KRAS induces autophagy through a PI4P-dependent mechanism, and in KRAS(G12V)-induced autophagy, PI4KB-mediated PI4P can replace the function of classical PI3K.

[0498] Example 3: Reducing the proliferation of RAS-mutant tumor cells by inhibiting PI4KB

[0499] To determine the role of PI4KB in regulating autophagy and tumor cell proliferation, two CRC cell lines with different KRAS mutations, HCT116 (KRAS-16), were treated with PIK93 alone or in combination with the MEK inhibitor trametinib (Tra). G13D ) and SW620 (KRAS G12VConsistent with previous experience, trametinib reduced the proliferation of HCT116 and SW620 cells in vitro. Furthermore, PIK93 alone and in combination with trametinib were observed to inhibit cell proliferation (Fig. 8A, Fig. 8B). Next, a subcutaneous xenograft model and these two KRAS mutant cell lines were used in NOD / SCID mice. Tumors were treated with controls, trametinib, PIK93, or combinations thereof. Animals were monitored to track tumor size. Consistent with cellular data, treatment with trametinib and PIK93 alone delayed tumor growth in KRAS mutant tumors, and combined treatment further inhibited tumor growth (Fig. 8C-8H). Immunofluorescence and immunohistochemical analyses showed that PIK93 treatment reduced LC3 points and increased P62 in mice; trametinib treatment reduced tumor p-ERK in mice. The cell proliferation marker Ki67 was reduced in the monotherapy group and further reduced in the dual-drug treatment group (Fig. 9). These data indicate that inhibiting PI4K can reduce tumor cell autophagy and proliferation, and this strategy can be combined with MEK inhibition to enhance tumor suppression, consistent with the findings of several studies. These studies show that inhibiting autophagy and MEK can produce beneficial effects in cancer treatment.

[0500] Example 4: WIPI2 acts as a PI4P effector in KRAS-induced autophagy

[0501] WIPI2, a mammalian ortholog of yeast Atg18, is a PI3P-binding protein containing a WD40 repeat sequence. It was initially reported to promote LC3 lipidation and subsequent growth of phagocytic vesicles by recruiting the ATG12-ATG5 / ATG16L1 complex. Other literature indicates that, in addition to binding PI3P, WIPI2 also interacts with PI4P and PI5P. Since the data from the above examples suggest that PI4KB, PI4P, and WIPI2 are indispensable for KRAS(G12V)-induced autophagy, this example attempts to determine whether WIPI2 is a PI4P effector. The results show that the WIPI2 formation site co-localizes with LC3 under KRAS(G12V) induction, indicating that WIPI2 is localized on the autophagosome membrane in KRAS-induced autophagy (Figures 10A and 10B). The WIPI2 site also overlaps with the PH domain of OSBP (OSBP-PH bound to PI4P) and has the best expression level, indicating an association between PI4P, WIPI2 and autophagosome membrane (Fig. 10C, Fig. 10D).

[0502] Further experiments were conducted to determine whether the location of WIPI2 on the autophagosome membrane is affected by PI4P. In one experiment, knockdown of PI4KB resulted in a reduction in the number of WIPI2 sites induced by KRAS(G12V) (Fig. 10E, Fig. 10F). In two other experiments, PI4P was reduced by treatment with T-00127-HEV1, or its accessibility was restricted by overexpression of OSBP-PH. As shown in the membrane separation assay, in both cases, reducing the level or accessibility of PI4P reduced the amount of membrane-bound WIPI2 (Fig. 10G-10J). In summary, these data indicate that WIPI2 must target the autophagosome membrane via PI4P.

[0503] To confirm the binding activity of WIPI2, its ability to bind PI4P was analyzed using lipid flotation assays. The results showed that WIPI2's binding capacity for PI4P was higher than its binding capacity for PI3P or PI5P (Figure 10K). Furthermore, PIP strip assays showed that WIPI2 consistently bound PI4P more strongly than it bound PI3P and PI5P (Figure 10L). Consistent with the autophagy analysis, ATG9A and ATG2 are not required for the generation of PI4P via KRAS (G12V) and for the formation of WIPI2 sites (Figures 10M-10Q). In conclusion, these data indicate that WIPI2 acts as a PI4P effector in oncogenic KRAS-induced autophagy.

[0504] Example 5: P38-ULK1-PI4K-WIPI2 cascade regulation of KRAS(G12V)-induced autophagy

[0505] RAS activates the ERK, PI3K / AKT, P38, JNK, and Ral pathways, leading to different cellular responses (Fig. 11A). To examine which of these downstream pathways is involved in autophagy induced by oncogenic KRAS (G12V), this study tested the effects of a series of specific inhibitors on KRAS (G12V)-induced autophagy. The effects of the respective inhibitors were validated by target (Fig. 12A). Compared to controls, the P38-specific inhibitor SB203580 reduced LC3 lipidation in Dox-induced cells, while the ERK inhibitor (FR180204), PI3K / AKT inhibitor (MK2206), and Ral inhibitor (RBC8) had no such effect (Fig. 11B, Fig. 12B, Fig. 12C). The effect of P38 inhibition on autophagosome biogenesis was further confirmed by autophagy flux-related assays, including dual-fluorescence LC3 assays and mito-keima assays (Fig. 11B, Fig. 12D-12G). The JNK inhibitor JNK-IN-8 increases LC3 lipidation when KRAS(G12V) is expressed, but decreases LC3 lipidation after treatment with bafloxacin A1, indicating that it can affect autophagosome maturation. Autophagy flux-related assays confirmed this. In dual-fluorescence LC3 assays, JNK inhibition did not affect autophagosomes, but it reduced autolysosomes (Figs. 12D-12E). In mito-keima assays, JNK inhibition also reduced mitophagy (Figs. 12F-12G).

[0506] Because inhibiting p38 blocks autophagosome biogenesis, this pathway was the focus of subsequent research. The p38-MAPK pathway mediates autophagy activation by activating mouse ULK1 through phosphorylation at serine (S)555. Observations showed that the ULK1-S556 site (a marker of ULK1 phosphorylation and early autophagosome formation; human ULK1 S556 corresponds to mouse ULK1 S555) forms after KRAS (G12V) expression, and inhibition of p38 alleviates its formation (Fig. 11C, Fig. 11D). Immunoblot analysis confirmed increased ULK-S556 phosphorylation, which could be inhibited by SB203580 (Fig. 11E, Fig. 11F). Next, a rescue experiment was conducted using ULK1-deficient cells. Wild-type ULK1 can restore LC3 lipidation induced by KRAS(G12V) but the ULK1-S556A point mutant cannot (Figs. 13A-13C, 14A-14B). This indicates that P38-mediated activation of ULK1 at the S556 site plays a crucial role in KRAS(G12V)-induced autophagy. In various cancer cell lines with endogenous RAS mutations (HCT116, SW620, H1299, A549, MiaPaCa2, and SW1990), RAS-driven autophagy also requires P38 activation (Figs. 14C-14E).

[0507] Next, the relationship between P38, ULK1, PI4P, and WIPI2 was determined. SB203580 treatment or ULK1 knockdown inhibited KRAS(G12V) expression-induced WIPI2 site formation (Figs. 13D-13G). ULK1 knockdown blocked KRAS(G12V) expression-induced PI4P growth, similar to the effect of PI4KB knockdown (Figs. 13H, 13I). These data indicate that P38 activates ULK1 via S556 phosphorylation, and ULK1 activates PI4P biogenesis and targets WIPI2, an early autophagosome, in an oncogenic KRAS signaling environment. Example 6: KRAS(G12V)-induced autophagy and KRAS-mutant tumor growth require ULK1-mediated PI4KB phosphorylation.

[0508] To understand how ULK1 activation affects PI4KB, Phos-tag gel electrophoresis was performed in this study. Overexpression of KRAS (G12V) led to a shift in PI4KB gel migration towards higher molecular weights, indicating a higher degree of phosphorylation (Figure 15A). Interestingly, ULK1 knockdown reversed the PI4KB migration shift after KRAS (G12V) expression (Figure 15B). Furthermore, ULK1 was correlated with PI4KB in the immunoprecipitation assay (Figure 15C). Therefore, these data indicate that ULK1 is involved in PI4KB phosphorylation. Mass spectrometry analysis confirmed five phosphorylated PI4KB peptides, with peptide-1 phosphorylation showing regulation by ULK1 activity (Figure 15D). S256, S258, T263, and S266 in peptide-1 are potential ULK1 phosphorylation sites.

[0509] To further elucidate peptide-1 phosphorylation, a phosphorylation-specific antibody targeting four potential phosphorylation sites was developed. Enhanced phosphorylation signaling was observed after induction of KRAS (G12V) expression, but this signal was eliminated by treatment with calf intestinal alkaline phosphatase, thus confirming the antibody's specificity (Figures 15E and 15F). The antibody's specificity was further validated in HCT116, SW620, and MiaPaCa2; in Western blotting or immunostaining, the phosphorylation signal was inhibited by RAS knockdown or AMG510, respectively (Figures 16A-16C). Using this antibody, the direct phosphorylation of peptide-1 by ULK1 in the PI4KB environment was investigated. Immunoisolated ULK1 and PI4KB were reacted in vitro with ATP. The results showed that peptide-1 phosphorylation occurs in the presence of ULK1, and this phosphorylation effect was completely eliminated by treatment with the ULK1 inhibitor SBI-0206965. In addition, excess peptide-1 (which may compete with PI4KB during ULK1 phosphorylation) also eliminates peptide-1 phosphorylation (Figure 15G).

[0510] To determine the effect of PI4KB phosphorylation on peptide-1, four potential phosphorylation sites were mutated to alanine (A), resulting in a phosphorylation-deficient mutant (SA mutant). Re-expression of WT PI4KB in PI4KB knockdown cells restored LC3 lipidation following KRAS (G12V) expression, while the SA mutant did not (Fig. 15H), indicating that PI4KB requires peptide-1 phosphorylation to regulate KRAS (G12V)-induced autophagy. To analyze the effect of the SA mutation on kinase activity, a PI4P dot blot assay was performed. Knockdown of PI4KB in control cells reduced PI4P, while WT PI4KB and SA PI4KB restored PI4P, but the kinase death mutant (D656A) did not restore PI4P. However, in KRAS (G12V)-expressing cells, WT PI4KB restored upregulated PI4P, but the SA mutant failed to generate further PI4P after PI4KB knockdown (Fig. 15I). In summary, these data suggest that peptide-1 phosphorylation may be a form of PI4KB kinase activity that specifically regulates KRAS(G12V)-induced autophagy.

[0511] In addition to loss of function, overexpression of the PI4KB-SA mutant exhibits a dominant-negative effect on KRAS(G12V)-induced autophagy, as demonstrated by LC3 lipidation and dot formation (Figs. 15J-15L). Overexpression of PI4KB-SA consistently inhibits autophagy and cell proliferation in RAS mutant cell lines (HCT116, SW620, H1299, A549, MiaPaCa2, and SW1990) (Figs. 16D-16F). Therefore, the antitumor effect of PI4KB-SA expression was subsequently determined using a subcutaneous xenograft model. Stable PI4KB-SA overexpressing HCT116 and SW620 cell lines were generated and used in xenograft experiments. Consistent with the effect of PI4K inhibition, PI4KB-SA expression reduced autophagy (LC3 and P62), cell proliferation (Ki67), and tumor growth. This effect has been demonstrated in combination with MEK inhibitors (Fig. 18). These findings indicate that both oncogenic KRAS-induced autophagy and tumor growth require ULKl-mediated PI4KB phosphorylation.

[0512] The mass spectrometry analysis described above did not precisely identify the phosphorylation sites on peptide-1. However, experiments with various combinations of phosphorylation site mutations revealed that simultaneous mutations at sites 1 and 3 (S256 and T263) eliminated PI4KB phosphorylation, while the other two potential sites did not (Figure 17A). This mutation also weakened PI4KB-mediated PI4P growth and impaired autophagy during KRAS (G12V) expression (Figures 17B-17C, 19A, and 19B). These results indicate that S256 and T263 are key PI4KB phosphorylation sites crucial for autophagy activation. Preferred amino acid sequences for ULK1 substrates have been identified in the prior art, consisting of a Leu or Met residue at position -3 and aliphatic and aromatic hydrophobic residues at positions +1 and +2. The adjacent residues of S256 and T263 contain L at positions +1 and +2, respectively, thus partially matching this sequence.

[0513] Example 7: Specific ULK1 phosphorylation pattern determines that PI4KB is the major substrate

[0514] ULK1 activity is finely regulated through phosphorylation and dephosphorylation events at specific sites to initiate autophagy. In starvation-induced autophagy, AMPK phosphorylates ULK1 at S317 and S556, while mTORC1 inhibition induces dephosphorylation at S758. Our experimental results validate this ULK1 phosphorylation pattern, which facilitates ULK1 phosphorylation of Beclin-1 at S30, a key step in PI3K activation during starvation-induced autophagy. Under these conditions, PI4KB peptide-1 remains unphosphorylated (Fig. 17D). Interestingly, during KRAS (G12V) expression, ULK1 exhibits a different phosphorylation pattern, with the aforementioned phosphorylation sites (even the inhibitory S758 site) remaining phosphorylated, while PI4KB peptide-1 (but not Beclin-1) is phosphorylated (Fig. 17D). This simultaneous phosphorylation of these three sites may be attributed to the co-activation of mTORC1, P38, and other kinases during KRAS (G12V) expression. These findings highlight the crucial role that different ULK1 phosphorylation patterns play in determining their substrate specificity.

[0515] Next, this embodiment attempts to determine the phosphorylation pattern controlling ULK1's preference for PI4KB as a substrate. One possible scenario is that simultaneous phosphorylation at sites S317, S556, and S758 prompts ULK1 to preferentially use PI4KB. Therefore, these three sites were mutated to glutamate (E) to mimic their phosphorylation state. However, compared to wild-type ULK1, this mutant exhibited weaker PI4KB phosphorylation ability, indicating that another phosphorylation site, along with the aforementioned three sites, jointly determines ULK1's substrate selectivity for PI4KB (Figure 17E). To further explore this, mass spectrometry analysis was performed on ULK1 phosphorylation under KRAS (G12V) expression or starvation conditions. The analysis results showed that there were 33 common phosphorylation sites under KRAS expression or starvation conditions, while there were 16 and 2 specific phosphorylation sites, respectively (Figure 17F).

[0516] Mutation analysis was then performed to identify other phosphorylation sites controlling ULK1's specific selection of PI4KB. Among the 16 serine / threonine residues mutated to aspartic acid (D), a significant increase in PI4KB phosphorylation was observed when the S479 site was mutated to aspartic acid, while Beclin-1 S30 phosphorylation decreased accordingly (Fig. 17G, Fig. 19C-E). This indicates a pattern of simultaneous phosphorylation at four sites (S317, S556, S758, and S479), which is key to ULK1's preferential selection of PI4KB over Beclin-1. To verify phosphorylation at the S479 site, a ULK1 S479 phosphorylation antibody was generated and confirmed by ULK1 knockdown and the S479A mutation (Fig. 17H). Using this antibody, ULK1 phosphorylation at the S479 site was observed to increase after KRAS (G12V) expression and moderately decrease after starvation, consistent with mass spectrometry analysis results (Fig. 17F and Fig. 17I). In summary, the conclusion is that phosphorylation at sites S317, S556, S758, and S479 in KRAS(G12V)-induced autophagy collectively determines ULK1's preference for PI4KB as a substrate.

[0517] Example 8: ULK1-regulated PI4KB phosphorylation is a target for the diagnosis or treatment of RAS-mutant cancers.

[0518] The above examples demonstrate that ULK1-regulated PI4KB-peptide-1 phosphorylation is a marker of KRAS overactivation and autophagy induction. In Western blot analysis, RAS-mutant cancer cells showed significantly higher levels of PI4KB-peptide-1 phosphorylation compared to cancer cells without RAS mutations (Figure 20A). Furthermore, in mutant RAS tissue microarrays from CRC patients, the overall PI4KB-peptide-1 phosphorylation level in RAS-mutant colon or rectal cancer was significantly higher than in other types (Figure 21). Based on these data, it is concluded that upregulated PI4KB-peptide-1 phosphorylation is a marker of RAS-mutant cancers.

[0519] The effect of phosphorylated substrate PI4KB-peptide-1 on ULK1-regulated PI4KB phosphorylation was investigated, and the results were consistent with in vitro ULK1 kinase assays. Multiple experiments demonstrated that expression of PI4KB-peptide-1 or treatment of cells with PI4KB-peptide-1 fused to Tat inhibited PI4KB phosphorylation in the peptide-1 region, leading to reduced PI4P production and inhibition of autophagy (Fig. 15G, Fig. 22A). Furthermore, PI4KB phosphorylation blocked by PI4KB-peptide-1 specifically inhibited KRAS-promoted PI4P production and autophagy (Fig. 22B-22I). Further truncating Pep.1 sequentially to obtain Pep1-6, Pep1-5, Pep1-4, Pep1-3, Pep1-2, and finally Pep.1-1 containing only 8 amino acids (sequences shown in Table 5) significantly inhibited ULK-activated PI4KB phosphorylation (Fig. 22J). Of course, PI4KB-peptide-1 inhibits LC3 lipidation, autophagic flux and WIPI2 site formation in various RAS mutant cancer cell lines (Figures 20B-20G).

[0520] Table 5

[0521] Notably, PI4KB-peptide-1 also inhibited the proliferation of these cancer cells, exhibiting a proliferation-inhibiting effect comparable to chloroquine when used in combination with trametinib. Chloroquine is a lysosomal inhibitor used clinically to inhibit autophagy (Figure 20H). Furthermore, in xenograft tumor models, expression of PI4KB-peptide-1 inhibited PI4KB-peptide-1 phosphorylation, autophagy, cell proliferation, and tumor growth, and its tumor-inhibiting effect when used in combination with trametinib was comparable to or better than that of chloroquine (SW620) (HCT116) (Figure 23).

[0522] The therapeutic potential of peptide-1 phosphorylation was further validated using the KPC mouse pancreatic cancer model, a well-known model that faithfully reproduces the biological mechanisms of human pancreatic cancer. Consistent with results from studies in cancer cell lines and xenograft experiments, treatment with PI4KB peptide-1 blocked PI4KB-peptide-1 phosphorylation and autophagy in tumor tissue. Combined with trametinib, it effectively inhibited tumor growth and prolonged survival (Figures 24A-24G). Compared to chloroquine (CQ), PI4KB peptide-1 alone could inhibit tumor growth and prolong survival. Furthermore, the combination of PI4KB peptide-1 and trametinib showed superior efficacy compared to chloroquine in inhibiting tumor growth, protecting normal pancreatic tissue, and prolonging survival (Figures 24D, E, F).

[0523] PI4KB peptide-1 exhibited superior tumor-suppressive activity compared to chloroquine in immunocompetent mice, while its tumor-suppressive activity was similar to that of chloroquine in immunodeficient mice, suggesting that PI4KB peptide-1 is more effective than chloroquine in positively modulating the immune response. Recent studies have shown that autophagy in pancreatic cancer (approximately 98% of RAS mutations) hinders immune recognition of cancer cells by degrading MHC-I. To explore the potential role of immune regulation in the superior function of PI4KB peptide-1, we analyzed the levels of MHC-I in tumors (Figs. 24D-24F). Both chloroquine and PI4KB peptide-1 increased MHC-I, consistent with previous findings on autophagy-induced MHC-I degradation. However, only PI4KB peptide-1 enhanced CD8+ T cell infiltration (Figs. 24D-24F). The lack of enhancement of CD8+ T cell infiltration by chloroquine may be due to its general inhibition of T cell autophagy, which is crucial for T cell function. Furthermore, chloroquine also inhibits T cell function.

[0524] Therefore, the inhibition of tumor-specific autophagy by PI4KB peptide-1 provides a better tumor-killing effect, which is achieved by blocking metabolic remodeling of cancer cells, enhancing MHC-I-mediated tumor antigen presentation, and minimally inhibiting CD8+ T cell activation. These data further support targeting PI4KB peptide-1 as a promising strategy for treating RAS mutation-driven cancers.

[0525] This application investigated the potential molecular mechanisms of autophagy dysphagia in cancer using various oncogenic RAS-induced autophagy models. The results revealed that KRAS(G12V)-induced autophagy is independent of several autophagy factors typically associated with starvation-induced autophagy, such as PI3K, ATG9A, ATG2, and ESCRT. Therefore, the dependence on PI3K was particularly emphasized, revealing that RAS signaling-induced activation of PI4KB via specific phosphorylation states of ULK1 (simultaneous phosphorylation at S317, S479, S556, and S758 sites) is crucial. Notably, ULK1 activation via RAS leads to phosphorylation of peptide-1 of PI4KB (including S256 and T263 sites), enhancing PI4KB activity and generating PI4P. Furthermore, similar to starvation-induced autophagy, WIPI2 acts as a downstream effector, specifically as a PI4P rather than a PI3P effector, to initiate downstream events in autophagosome biogenesis. Combined inhibition of MEK1 / 2 and PI4KB-peptide-1 phosphorylation effectively suppressed autophagy, while also inhibiting the proliferation of mutant KRAS tumor cell lines in vitro and xenografts in mouse and pancreatic KPC models. These results elucidate the mechanistic framework of another oncogenic KRAS-induced autophagy pathway and the unique phosphorylation regulation of ULK1, through which PI4KB can be activated in KRAS-mutant cancers.

[0526] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0527] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. The use of PI4K inhibitors in the preparation of drugs for the prevention and / or treatment of tumors, characterized in that, The PI4K inhibitor blocks or inhibits PI4K phosphorylation, and preferably, the tumor is a RAS-mutant tumor.

2. The application according to claim 1, characterized in that, The PI4K inhibitors described herein prevent and / or treat tumors by blocking or inhibiting autophagy induced by RAS mutations.

3. The application according to claim 1, characterized in that, The PI4K inhibitors described herein prevent and / or treat tumors by blocking or reducing PI4P levels; Preferably, tumors are prevented and / or treated by blocking or reducing PI4P levels, thereby blocking or reducing its recruitment of interacting proteins. Preferred methods include blocking or reducing the number of interacting proteins recruited by PI4P or their expression levels.

4. The application according to claim 3, characterized in that, The interacting proteins mentioned include WD repeat domain phosphoinositol interacting protein 2 (WIPI2).

5. The application according to claim 1, characterized in that, The PI4K inhibitors described herein prevent and / or treat tumors by blocking or inhibiting ULK1 phosphorylation-induced PI4K phosphorylation.

6. The application according to claim 1, characterized in that, The PI4K inhibitor blocks or inhibits ULK1 phosphorylation-induced PI4K phosphorylation, thereby blocking or reducing PI4P levels, blocking or reducing the levels of its recruitment interacting proteins, and thus blocking or inhibiting autophagy caused by RAS mutations, thereby preventing and / or treating tumors.

7. The application according to claim 5 or 6, characterized in that, The ULK1 phosphorylation mentioned includes ULK1 phosphorylation generated via the P38 pathway.

8. The application according to any one of claims 1-7, characterized in that, The PI4K mentioned is a PI4K complex or PI4KB.

9. The application according to claim 8, characterized in that, The phosphorylation sites of PI4KB include S256 and / or T263.

10. The application according to any one of claims 5-9, characterized in that, The phosphorylation sites of ULK1 include one or more of S317, S556, S758, or S479.

11. The application according to any one of claims 1-10, characterized in that, The RAS mutation is one or more of HRAS mutation, KRAS mutation or NRAS mutation, preferably KRAS mutation, such as one or more of G12V, G12C, G12D, G12S, G12R, G13D, G13C, A18D, A59D, A59T, Q61H, Q61K, Q61L, Q61R, E62G, A146T, K117N.

12. The application according to claim 1 or 11, characterized in that, The tumor is selected from colorectal cancer, pancreatic cancer, bile duct cancer, lung cancer (e.g., non-small cell lung cancer), ovarian cancer, thyroid cancer, bladder cancer, breast cancer, liver cancer, melanoma, myelodysplastic syndrome, lymphoma, endometrial cancer, esophageal cancer, glioma, squamous cell carcinoma of the head and neck, urothelial carcinoma, neuroblastoma, renal cancer, leukemia, or multiple myeloma. Preferably, the tumor is colon cancer, lung cancer, or pancreatic cancer with RAS mutation.

13. The application according to any one of claims 1-12, characterized in that, The PI4K inhibitors include reagents for gene knockout, reagents for gene silencing, reagents for gene mutation, antisense nucleic acids, small molecule compounds or their pharmaceutically acceptable salts, peptides or their expression promoters, peptide mutants or their expression promoters, fusion proteins, antibodies, traditional Chinese medicine or extracts of traditional Chinese medicine.

14. The application according to claim 13, characterized in that, The gene knockout mentioned includes CRISPR or tissue-specific knockout.

15. The application according to claim 13, characterized in that, The reagents required for gene silencing include interfering RNA, such as one or more of siRNA, dsRNA, shRNA, aiRNA, or miRNA.

16. The application according to claim 15, characterized in that, The siRNA targets one, two, or three of PI4KB, ULK1, or WIPI2. Preferably, the target site sequence of the siRNA targeting ULK1 includes SEQ ID NO: 2 and / or SEQ ID NO: 3; Preferably, the target site sequence of the siRNA targeting PI4KB includes one or more of SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27; Preferably, the target site sequence of the siRNA targeting WIPI2 includes one or more of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO:

16.

17. The application according to claim 15, characterized in that, The shRNA targets PI4KB and / or ULK1; Preferably, the target site sequence of the shRNA targeting ULK1 includes SEQ ID NO: 29 and / or SEQ ID NO: 30; Preferably, the target site sequence of the shRNA targeting PI4KB includes one or more of SEQ ID NO: 37 and / or SEQ ID NO:

38.

18. The application according to claim 13, characterized in that, The polypeptide is a polypeptide that competitively phosphorylates PI4K, such as an amino acid fragment of PI4KB containing the S256 and / or T263 sites. Preferably, the length of the polypeptide is at least 8 aa, more preferably 8-60 aa, and even more preferably 8-30 aa; More preferably, the polypeptide includes at least positions 256 to 263 of the amino acid sequence of PI4KB, preferably including any one of SEQ ID NO: 1 or 83-88, or including an amino acid sequence having more than 80% identity with any one of SEQ ID NO: 1 or 83-88, or including a substituted, deleted, or inserted amino acid sequence having up to 10 amino acids with any one of SEQ ID NO: 1 or 83-88.

19. The application according to claim 13, characterized in that, The peptide mutants include amino acid fragments of PI4KB that are mutated to one or more sites of S256, S258, T263 or S266 to be neither threonine nor serine, for example, to alanine or a non-natural amino acid. More preferably, the polypeptide mutant includes a mutation of serine and threonine at positions 256 to 266 to alanine, such as a mutant of SEQ ID NO: 1 or any of 83-88.

20. The application according to claim 13, characterized in that, The fusion protein includes a polypeptide that competitively phosphorylates PI4K, and preferably also includes an N-terminal Tat protein transduction domain. More preferably, the polypeptide and the N-terminal Tat protein transduction domain are linked by a GG linker. For example, the fusion protein may include SEQ ID NO: 57 or include an amino acid sequence that has more than 80% identity with SEQ ID NO: 57 or include an amino acid sequence that has at most 10 substituted, deleted or inserted amino acids with SEQ ID NO:

57.

21. The application according to claim 13, characterized in that, The small molecule compound is selected from arsenic oxyphenylene oxide or its derivatives, (aS)-5-(2-amino-4-oxo-3-(2-(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-6-yl)-N-(2,4-difluorophenyl)-2-methoxypyridine-3-sulfonamide or its analogues, 5-(2-amino-1-(4-(4-morpholinyl)phenyl)-1H-benzimidazol-6-yl)-N-(2-fluorophenyl)-2-methoxy-3-pyridinesulfonamide or its analogues, 2-amino-5-phenylthiazole, substituted 2-amino-5-pyridylthiazole, simeprevir or its analogues, T-00127_HEV1 (CAS: 900874-91-1), PIK93 (CAS: 593960-11-3), or Wollman penicillin.

22. The application according to claim 1, characterized in that, The drugs also include EGFR inhibitors, RAS inhibitors, SHP2 inhibitors, SOS1 inhibitors, Raf inhibitors, MEK inhibitors, ERK inhibitors, PI3K inhibitors, PTEN inhibitors, AKT inhibitors, mTORC1 inhibitors, BRAF inhibitors, PD-L1 inhibitors, PD-1 inhibitors, CDK4 / 6 inhibitors, HER2 inhibitors, ULK1 inhibitors, or combinations thereof; preferably MEK inhibitors. More preferably, the drug comprises a PI4K inhibitor and a MEK inhibitor, wherein the MEK inhibitor is selected from pimettinib, comettinib, trametinib, LNP-3794, HL-085, antroquinol, E-6201, remettinib, midamettinib, pimaserte, sermetinib, SHR-7390, CKI-27, GS-4875, ATR-001, ATR-002, ATR-006, ATR-004, ATR-005, CS-3006, FCN-159, and CIP-13740.

1. EBI-1051, SC-1-151, SRX-2626, EDV-2209, WX-554, GDC-0623, TAK-733, E-6201, RG-7167, AZD-8330, PD-184352, G SK-2091976A, AS-703988, BI-847325, JTP-70902, CZ-775, RO4987655, RO5126766, RO-5068760, RDEA-436, MEK-300, AD-GL0001, SL-327, CI-1040, CInQ-03, G-573, PD184161, PD318088, PD98059, U0126, and SL327 are preferred, with trametinib being the preferred choice.

23. Application of PI4K inhibitors in blocking or inhibiting autophagy induced by RAS mutations.

24. The application according to claim 23, characterized in that, The PI4K inhibitors described herein block or inhibit PI4K phosphorylation.

25. The application according to claim 23 or 24, characterized in that, The PI4K inhibitors described above block or reduce PI4P levels, thereby blocking or inhibiting autophagy caused by RAS mutations. Preferably, by blocking or reducing PI4P levels, the levels of its recruited interacting proteins are blocked or reduced, thereby blocking or inhibiting autophagy induced by RAS mutations.

26. The application according to any one of claims 23-25, characterized in that, The PI4K inhibitors described herein achieve the blocking or inhibition of autophagy caused by RAS mutations by blocking or inhibiting PI4K phosphorylation induced by ULK1 phosphorylation.

27. The application according to any one of claims 23-26, characterized in that, The PI4K inhibitor blocks or reduces PI4P levels by blocking or inhibiting ULK1 phosphorylation-induced PI4K phosphorylation, thereby blocking or reducing the levels of its recruiting interacting proteins, and thus blocking or inhibiting autophagy induced by RAS mutations.

28. The use of PI4K inhibitors and MEK inhibitors in combination in the preparation of drugs for the prevention and / or treatment of tumors.

29. Application of combined PI4K inhibitors and MEK inhibitors in blocking or inhibiting autophagy induced by RAS mutations.

30. The application of ULK1-PI4K inhibitors, characterized in that, The ULK1-PI4K is PI4K phosphorylation induced by ULK1 phosphorylation, and the ULK1-PI4K inhibitor blocks or inhibits PI4K phosphorylation induced by ULK1 phosphorylation. The applications include blocking or inhibiting autophagy caused by RAS mutations or in the preparation of drugs for the prevention and / or treatment of tumors.

31. The application according to claim 30, characterized in that, The ULK1-PI4K inhibitors include ULK1 inhibitors and / or PI4K inhibitors.

32. The application according to claim 30, characterized in that, The PI4K mentioned is a PI4K complex or PI4KB.

33. The application according to claim 32, characterized in that, The phosphorylation sites of PI4KB include S256 and / or T263.

34. The application according to any one of claims 30-33, characterized in that, The phosphorylation sites of ULK1 include one or more of S317, S556, S758, or S479.

35. The use of a P38-ULK1-PI4KB-WIPI2 pathway inhibitor, said use including blocking or inhibiting autophagy caused by RAS mutations or in the preparation of medicaments for the prevention and / or treatment of tumors.

36. The application according to claim 35, characterized in that, The P38-ULK1-PI4KB-WIPI2 pathway inhibitors include one or more of the following: P38 pathway inhibitors, ULK1 inhibitors, PI4KB inhibitors, or WIPI2 inhibitors. Preferably, the ULK1 inhibitor blocks or inhibits ULK1 phosphorylation; more preferably, it includes inhibiting at least one or more of the phosphorylation of S317, S556, S758 or S479. Preferably, the PI4KB inhibitor blocks or inhibits PI4KB phosphorylation, and more preferably, it includes inhibiting at least the phosphorylation of S256 and / or T263.

37. The use of a PI4K phosphorylated peptide as a biomarker in the preparation of products for the diagnosis and / or prognostic assessment of tumors, wherein the PI4K is PI4KB; Preferably, the PI4KB phosphorylation sites include at least S256 and / or T263; Preferably, the tumor is a RAS-mutant tumor.

38. The application of a PI4K phosphorylated polypeptide as a marker in the preparation of products that identify autophagy caused by RAS mutations, wherein the PI4K is PI4KB; Preferably, the PI4KB phosphorylation sites include at least S256 and / or T263.

39. The application according to claim 37 or 38, characterized in that, The PI4K phosphorylated polypeptide includes at least positions 256 to 263 of the amino acid sequence of PI4KB, preferably including any one of SEQ ID NO: 1 or 83-88.

40. A PI4KB inhibitor, characterized in that, The PI4KB inhibitor is one or more of a polypeptide, a polypeptide mutant, a fusion protein, or interfering RNA, wherein the interfering RNA is siRNA and / or shRNA; wherein: The polypeptide comprises at least the amino acid sequence at position 256 and / or position 263 of the amino acid sequence of PI4KB; Preferably, the polypeptide includes at least positions 256 to 263 of the amino acid sequence of PI4KB, and more preferably includes any one of SEQ ID NO: 1 or 83-88; The peptide mutants include amino acid fragments of PI4KB that are mutated to one or more sites of S256, S258, T263 or S266 to be neither threonine nor serine, for example, to alanine or a non-natural amino acid. Preferably, the polypeptide mutant includes a mutation of serine and threonine at positions 256 to 266 to alanine, such as a mutant of SEQ ID NO: 1 or any of 83-88; The fusion protein includes a polypeptide that competitively phosphorylates PI4K, and preferably also includes an N-terminal Tat protein transduction domain. More preferably, the polypeptide and the N-terminal Tat protein transduction domain are linked by a GG linker. For example, the fusion protein may include SEQ ID NO: 57 or include an amino acid sequence that has more than 80% identity with SEQ ID NO: 57 or include an amino acid sequence that has at most 10 substituted, deleted or inserted amino acids with SEQ ID NO:

57. The target site sequence of the siRNA targeting PI4KB includes one or more of SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27; The target site sequence of the shRNA targeting PI4KB includes one or more of SEQ ID NO: 37 and / or SEQ ID NO:

38.

41. A ULK1 inhibitor, wherein the ULK1 inhibitor is an interfering RNA, and the interfering RNA is siRNA and / or shRNA; The target site sequences of siRNAs targeting ULK1 include SEQ ID NO: 2 and / or SEQ ID NO: 3; The target site sequences of shRNAs targeting ULK1 include SEQ ID NO: 29 and / or SEQ ID NO:

30.

42. A WIPI2 inhibitor, characterized in that, The WIPI2 inhibitor is an interfering RNA, and the interfering RNA is siRNA. The target site sequence of the siRNA targeting WIPI2 includes one or more of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO:

16.

43. A method for treating and / or preventing tumors, characterized in that, The method includes any one or more of the following: A) Block or inhibit autophagy caused by RAS mutations; B) Block or inhibit PI4K phosphorylation; C) Block or inhibit ULK1 phosphorylation-induced PI4K phosphorylation; D) Block or reduce PI4P levels, preferably block or reduce the levels of its recruitment interacting proteins, preferably WIPI2; E) Block or inhibit ULK1 phosphorylation.

44. The method according to claim 43, characterized in that, The method includes knocking out the PI4K gene, silencing the PI4K gene, or administering a peptide that competitively phosphorylates PI4K, or a mutant thereof, or a fusion protein containing the peptide.

45. The method according to claim 43 or 44, characterized in that, The PI4K mentioned is a PI4K complex or PI4KB.

46. ​​The method according to any one of claims 43-45, characterized in that, The method involves administering an effective amount of a P38-ULK1-PI4KB-WIPI2 pathway inhibitor to a subject in need; Preferred administration is one or more of the PI4KB inhibitor of claim 40, the ULK1 inhibitor of claim 41, or the WIPI2 inhibitor of claim 42.

47. A method for diagnosing tumors, characterized in that, The method includes detecting the levels of ULK1 phosphorylation and / or PI4KB phosphorylation; Preferably, the tumor is a RAS-mutant tumor; Preferably, the method includes determining whether ULK1 phosphorylation-induced PI4KB phosphorylation exists.

48. The method according to claim 47, characterized in that, The phosphorylation sites of PI4KB include S256 and / or T263; the phosphorylation sites of ULK1 include one or more of S317, S556, S758 or S479.

49. The method according to claim 47 or 48, characterized in that, The method includes detecting the level of phosphorylation at S256 and / or T263 sites in PI4KB, preferably detecting the level of any one of the phosphorylations in SEQ ID NO: 1 or 83-88.

50. The method according to any one of claims 47-49, characterized in that, The method involves detecting the levels of ULK1 phosphorylation and / or PI4KB phosphorylation in tumor cells.

51. A method for blocking or inhibiting autophagy caused by RAS mutations, characterized in that, The method includes any one or more of the following: A) Block or inhibit PI4K phosphorylation; B) Block or inhibit ULK1 phosphorylation-induced PI4K phosphorylation; C) Block or reduce PI4P levels, preferably block or reduce the levels of its recruitment interacting proteins, preferably WIPI2; D) Block or inhibit ULK1 phosphorylation; Preferably, the method includes knocking out the PI4K gene, silencing the PI4K gene, mutating the PI4K gene, or administering a polypeptide that competitively phosphorylates PI4K, or a mutant thereof, or a fusion protein containing the polypeptide.

52. The method according to claim 51, characterized in that, The method involves administering a P38-ULK1-PI4KB-WIPI2 pathway inhibitor; Preferred administration is one or more of the PI4KB inhibitor of claim 40, the ULK1 inhibitor of claim 41, or the WIPI2 inhibitor of claim 42.

53. A drug, characterized in that, The drugs mentioned include PI4K inhibitors and MEK inhibitors, as well as pharmaceutically acceptable excipients; Preferably, the PI4K inhibitor is the PI4KB inhibitor of claim 40.

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