Use of nucleotide synthetic pathway inhibitors and macropinocytosis inhibitors for treatment of tumors

Abstract

The present invention relates to the use of nucleotide synthesis pathway inhibitors and macropinocytosis inhibitors for the treatment of tumors. The present disclosure provides the use of nucleotide synthetic pathway inhibitors and macropinocytosis inhibitors in the preparation of medicaments or kits for the treatment of tumors, where the nucleotide synthetic pathway inhibitors may include pyrimidine and / or purine synthetic pathway inhibitors such as orotic dehydrogenase (DHODH) inhibitors and phosphoribose pyrophosphate synthetase 1 (PRPS) inhibitors, the macropinocytosis inhibitor can comprise a macropinocytosis substrate uptake inhibitor, a PAK1 inhibitor, an EGFR signal pathway inhibitor, an ErbB signal pathway inhibitor, an NF-KB signal pathway inhibitor, an EREG inhibitor, a p65 inhibitor and an AREG inhibitor. The invention discloses a new way for activating macropinocytosis by using intracellular signals, provides a new insight for a regulation mechanism in tumor cells, and provides a new method for tumor treatment.

Description

Technical Field

[0001] This application relates to the field of tumor treatment. Specifically, this application relates to the use of nucleotide synthesis pathway inhibitors, such as DHODH inhibitors and macropinocytosis inhibitors, for the treatment of tumors. Background Technology

[0002] Digestive system tumors are currently the most prevalent systemic malignant tumors worldwide, mainly including hepatocellular carcinoma (HCC), colorectal cancer (CRC), cholangiocarcinoma (CHOL), pancreatic adenocarcinoma (PAAD), and stomach adenocarcinoma (STAD) [1]. With the continuous deepening of research on precision oncology, finding key targets that affect tumor growth, metastasis, and drug resistance, as well as conducting precise screening of existing drugs and developing new combination drug regimens, has important clinical significance for individualized precision oncology [2].

[0003] Metabolic reprogramming is one of the important characteristics of tumor cells[3], in which the upregulation of the de novo pyrimidine synthesis pathway can meet the bioenergy required for rapid cell proliferation[4]. The de novo pyrimidine synthesis pathway is highly dependent in tumors of different types and genetic backgrounds, so targeting the de novo pyrimidine synthesis is an important target for inhibiting tumor growth[5]. Since dihydroorotate dehydrogenase (DHODH) is the rate-limiting enzyme of the de novo pyrimidine synthesis pathway, it plays a key role in the synthesis of uridine monophosphate (UMP)[6] and is associated with oxidative phosphorylation (OXPHOS)[7], so inhibiting DHODH has become a promising target in the treatment strategy of CRC and HCC[8,9].

[0004] Despite the development of numerous DHODH-targeting inhibitors, such as brequinar (BRQ) and leflunomide (Lef), phase II clinical trials have yielded limited results [10-12]. The discrepancy between preclinical and clinical trial results suggests that DHODH inhibition may involve mechanisms for maintaining intracellular nucleotides, thereby leading to drug resistance in tumor cells [13, 14]. Recent studies have shown that nucleotide transporters may be involved in resistance to BRQ. Combining BRQ with dipyridamole, an inhibitor of equilibrative nucleoside transporter 1 (ENT1) and ENT2, synergistically inhibits tumor growth in vivo and in vitro

[13] . However, the clinical efficacy of this combination therapy strategy is only slightly improved compared to BRQ alone [5, 14]. These results suggest that nucleotide transporters are not the primary mechanism of resistance. In addition to de novo pyrimidine synthesis and nucleotide transport, cells can also acquire nucleotides through autophagy

[15] . However, autophagy plays an ambivalent role in tumors, hindering the development of preclinical drugs. There is still a need in this field to overcome tumor drug resistance and to provide effective methods and drugs for treating tumors.

[0005] References

[0006] 1.Sung, H., et al., Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36Cancers in 185Countries.CA Cancer JClin, 2021.71(3): p.209-249.

[0007] 2. Plana, D., ACPalmer, and PKSorger, Independent Drug Action in Combination Therapy: Implications for Precision Oncology. Cancer Discov, 2022.12(3): p.606-624.

[0008] 3.Hanahan, D., Hallmarks of Cancer: New Dimensions.Cancer Discov, 2022.12(1): p.31-46.

[0009] 4.Pavlova,N.N.,J.Zhu,and C.B.Thompson,The hallmarks of cancermetabolism:Still emerging.Cell Metab,2022.34(3):p.355-377.

[0010] 5.Mullen,N.J.and P.K.Singh,Nucleotide metabolism:a pan-cancermetabolic dependency.Nat Rev Cancer,2023.23(5):p.275-294.

[0011] 6.Madak,J.T.,et al.,Revisiting the role of dihydroorotatedehydrogenase as a therapeutic target for cancer.Pharmacol Ther,2019.195:p.111-131.

[0012] 7.Wu,H.,et al.,Cyclin D1 extensively reprograms metabolism to supportbiosynthetic pathways in hepatocytes.J Biol Chem,2023.299(12):p.105407.

[0013] 8.Yamaguchi,N.,et al.,PCK1 and DHODH drive colorectal cancer livermetastatic colonization and hypoxic growth by promoting nucleotidesynthesis.Elife,2019.8.

[0014] 9.Zhu,Z.,et al.,UBE2T-mediated Akt ubiquitination and Akt / beta-catenin activation promotes hepatocellular carcinoma development byincreasing pyrimidine metabolism.Cell Death Dis,2022.13(2):p.154.

[0015] 10.Moore,M.,et al.,Multicenter phase II study of brequinar sodiuminpatients with advanced gastrointestinal cancer.Invest New Drugs,1993.11(1):p.61-5.

[0016] 11.Maroun,J.,et al.,Multicenter phase II study of brequinar sodiuminpatients with advanced lung cancer.Cancer Chemother Pharmacol,1993.32(1):p.64-6.

[0017] 12.Urba,S.,et al.,Multicenter phase II trial of brequinar sodium inpatients with advanced squamous-cell carcinoma of the head and neck.CancerChemother Pharmacol,1992.31(2):p.167-9.

[0018] 13.Mullen,N.J.,et al.,ENT1 blockade by CNX-774overcomes resistance toDHODH inhibition in pancreatic cancer.Cancer Lett,2023.552:p.215981.

[0019] 14.Yu,Y.,et al.,Therapeutic targeting of both dihydroorotatedehydrogenase and nucleoside transport in MYCN-amplified neuroblastoma.CellDeath Dis,2021.12(9):p.821.

[0020] 15.Mimura,K.,et al.,Genome-wide CRISPR screening reveals nucleotidesynthesis negatively regulates autophagy.J Biol Chem,2021.296:p.100780.

[0021] 16.Zhou,Z.,et al.,Acetyl-Coenzyme A Synthetase 2PotentiatesMacropinocytosis and Muscle Wasting Through Metabolic Reprogramming inPancreatic Cancer.Gastroenterology,2022.163(5):p.1281-1293e1.

[0022] 17.Lee,S.W.,et al.,EGFR-Pak Signaling Selectively Regulates GlutamineDeprivation-Induced Macropinocytosis.Dev Cell,2019.50(3):p.381-392e5.

[0023] 18.Zhang,M.S.,et al.,Hypoxia-induced macropinocytosis represents ametabolic route for liver cancer.Nat Commun,2022.13(1):p.954.

[0024] 19.Commisso,C.,et al.,Macropinocytosis of protein is an amino acidsupply route in Ras-transforrned cells.Nature,2013.497(7451):p.633-7.

[0025] 20.Chung,J.J.,et al.,Albumin-associated free fatty acids inducemacropinocytosis in podocytes.J Clin Invest,2015.125(6):p.2307-16.

[0026] 21.Palm,W.,et al.,The Utilization of Extracellular Proteins asNutrients Is Suppressed by mTORC1.Cell,2015.162(2):p.259-270.

[0027] 22.Li,Y.,et al.,Macropinocytosis-mediated membrane recycling drivesneural crest migration by delivering F-actin to the lamellipodium.Proc NatlAcad Sci U S A,2020.117(44):p.27400-27411.

[0028] 23.Desai,A.S.,M.R.Hunter,andA.N.Kapu stin,Using macropinocytosis forintracellular deliVery of therapeutic nucleic acids to tumour cells.PhilosTranis R Soc Lond B Biol Sci,2019.374(1765):p.20180156.

[0029] 24.Jayashankar, V.and A.L.Edinger,Macropinocytosis confers resistanceto therapies targeting cancer anabolism.Nat Commun,2020.11(1):p.1121.

[0030] 25.Su,H.,et al.,Cancer cells escape autophagy inhibition via NRF2-induced macropinocytosis.Cancer Cell,2021.39(5):p.678-693e11.

[0031] 26.Chhabra,E.S.and H.N.Higgs,The many faces of actin:matchingassembly factors with cellular structures.Nat Cell Biol,2007.9(10):p.1110-21.

[0032] 27.Liu,T.,et al.,Nucleus-exported CLOCK acetylates PRPS to promote denovo nucleotide synthesis and liver tumour growth.Nat Cell Biol,2023.25(2):p.273-284.

[0033] 28.Nazemi,M.,et al.,The extracellular matrix supports breast cancercell growth under amino acid starvation by promoting tyrosine catabolism.PLoSBiol,2024.22(1):p.e3002406.

[0034] 29.Redelman-Sidi,G.,et al.,The Canonical Wnt Pathway DrivesMacropinocytosis in Cancer.Cancer Res,2018.78(16):p.4658-4670.

[0035] 30.Levantini,E.,et al.,EGFR signaling pathway as therapeutic targetin human cancers.Semin Cancer Biol,2022.85:p.253-275.

[0036] 31.Sahu,U.,et al.,Pyrimidines maintain mitochondrial pyruvateoxidation to support de novo lipogenesis.Science,2024.383(6690):p.1484-1492.

[0037] 32. Morrissey, SM, et al., Tumor-derived exosomes driveimmunosuppressive macrophages in a pre-metastatic niche through glycolytic dominant metabolic reprogramming. Cell Metab, 2021.33(10): p.2040-2058e10.

[0038] 33.Boyd, AS, Leflunomide in dermatology. J Am Acad Dermatol, 2012.66(4): p.673-9. Summary of the Invention

[0039] Inhibiting nucleotide synthesis pathways, such as their key enzyme dihydronicotinic acid dehydrogenase (DHODH), has become a promising anti-cancer strategy. However, nucleotide synthesis pathway inhibitors, such as DHODH inhibitors, have shown limited efficacy in clinical trials, suggesting potential drug resistance mechanisms. In this study, the inventors discovered that endogenous nucleotide deficiency resulting from nucleotide synthesis inhibition activates macropinocytosis in tumor cells via a novel signaling axis involving the upregulation of lactate from glycolysis, activation of the transcription factor p65, and subsequent NF-κB-dependent expression of the epidermal growth factor receptor (EGFR) ligand EREG, which is involved in EGFR-PAK1 signaling. In humans, activated macropinocytosis internalizes albumin, which can competitively bind to DHODH inhibitors, thereby partially restoring DHODH activity and pyrimidine synthesis. Differences in albumin concentration may explain the inconsistencies in cell culture medium and clinical trial efficacy. In various cancer models, the combined use of nucleotide synthesis inhibitors, such as DHODH inhibitors, and macropinocytosis inhibitors synergistically inhibited tumor proliferation and growth. In summary, these findings reveal a novel pathway for activating macropinocytosis using intracellular signals and provide a theoretical basis for combining nucleotide synthesis pathway inhibitors and macropinocytosis inhibitors as an improved treatment approach.

[0040] Macropinocytosis is a non-selective endocytic process in which extracellular fluid and solutes are taken up through membrane folds and cup-shaped processes. PAK1 (p21-Activated Kinase 1) plays a crucial role in the formation of membrane folds. Studies have shown that in glutamine-deficient conditions, pancreatic cancer cells can upregulate macropinocytosis by activating the EGFR-PAK1 signaling axis, thereby taking up extracellular albumin and breaking it down into amino acids in lysosomes to maintain their survival. Currently, the main factors inducing macropinocytosis are external factors of the tumor microenvironment, such as amino acid deficiency (especially glutamine), serum starvation, hypoxia, and glucose deficiency. Besides maintaining tumor cell survival in nutrient-deprived environments, macropinocytosis also participates in tumor drug resistance and metastasis. Compared to normal tissues, tumor cells have a macropinocytosis capacity several to hundreds of times greater; therefore, targeting macropinocytosis may be a promising strategy for cancer treatment. Research suggests that macropinocytosis may be an important pathway for tumor cells to acquire nucleotides. This invention has demonstrated that inhibitors of nucleotide synthesis pathways, including DHODH inhibitors, induce resistance through macropinocytosis, and elucidated their molecular mechanisms and explored their applications in tumor treatment, including, for example, colorectal cancer and liver cancer.

[0041] In some embodiments, the present invention provides the use of nucleotide synthesis pathway inhibitors and macropinocytosis inhibitors in the preparation of medicaments or kits for treating tumors.

[0042] In some embodiments, nucleotide synthesis pathway inhibitors may include pyrimidine and / or purine synthesis pathway inhibitors, such as inhibitors of pyrimidine and / or purine synthesis pathway enzymes, such as inhibitors of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme of pyrimidine synthesis pathway enzymes (e.g., buquina, leflunomide, teriflunomide, ASLAN003, BAY2402234, IMU-838, and PTC299), such as inhibitors of phosphoribosyl pyrophosphate synthase 1 (PRPS1), the rate-limiting enzyme of purine and pyrimidine nucleotide precursor synthesis (e.g., siRNA).

[0043] In some implementations, macropinocytosis inhibitors may include macropinocytosis substrate (e.g., albumin) uptake inhibitors (e.g., EIPA), macropinocytosis signaling pathway inhibitors such as PAK1 inhibitors (e.g., IPA3, G-5555, ZMF-10, FRAX1036), EGFR signaling pathway inhibitors (e.g., Gefitinib, Erlotinib, Icotinib, and Afatinib), ErbB signaling pathway inhibitors (e.g., Dacomitinib, afatinib), NF-κB signaling pathway inhibitors (e.g., SC75741, Bortezomib, Scutellarin, Shikonin, and Triptolide), and p65 inhibitors (e.g., Maslinic acid and Mangiferin).

[0044] In some embodiments, the inhibitor may include inhibitors that suppress gene and / or protein expression in signaling pathways, such as gene knockout or knockdown agents, such as siRNA, antisense RNA, and gene editing agents, including, for example, ZFN, TALENs, and CRISPER gene editing agents. In some embodiments, the inhibitor may also include known small molecule compounds, chemotherapeutic agents, etc.

[0045] In some implementations, the tumor may include a tumor resistant to treatment with inhibitors of nucleotide synthesis pathways, for example, to make it sensitive to treatment with inhibitors of nucleotide synthesis pathways.

[0046] In some implementations, the tumor may include tumors sensitive to nucleotide synthesis pathway inhibitors, for example, to prevent the development of drug resistance.

[0047] In some implementations, tumors may include colorectal cancer, liver cancer, pancreatic cancer, breast cancer, head and neck tumors, lung cancer, melanoma, ovarian cancer, and esophageal cancer.

[0048] In some embodiments, the present invention provides the use of macropinocytosis inhibitors in the preparation of medicaments or kits for treating tumors resistant to nucleotide synthesis pathway inhibitors, for example, to modify them to be sensitive to nucleotide synthesis pathway inhibitors.

[0049] In some embodiments, the present invention provides the use of macropinocytosis inhibitors in the preparation of medicaments or kits for treating tumors sensitive to nucleotide synthesis pathway inhibitors, for example, to prevent drug resistance.

[0050] In some embodiments, the present invention provides a medicament or kit for treating tumors, which may contain inhibitors of nucleotide synthesis pathways and macropinocytosis inhibitors.

[0051] In this paper, the inventors, through GSEA analysis based on transcriptome sequencing, determined that inhibition of nucleotide synthesis pathways such as DHODH significantly activates macropinocytosis pathways, such as the EGFR signaling pathway. Furthermore, nucleotide synthesis pathway inhibitors, such as DHODH inhibitors, and macropinocytosis inhibitors, such as the TKI inhibitor gefitinib, successfully inhibited the development of tumors such as colorectal cancer and liver cancer in vivo and in vitro, providing new insights into targeted cancer therapy. The inventors also discovered that endogenous nucleotide deficiency can lead to macropinocytosis in tumor cells, regulated through the lactate-p65-EREG / EGFR-PAK1 signaling axis, providing novel insights into the regulatory mechanisms of macropinocytosis in tumors. Attached Figure Description

[0052] Figure 1 DHODH inhibitors significantly upregulated macropinocytosis and lysosome-related pathways. Enrichment analysis of differentially expressed gene sets in Huh7 cells after 48 h of treatment with Lef (100 μM) or BRQ (50 μM). NES, normalized enrichment scores.

[0053] Figure 2 DHODH inhibitor-induced macropinocytosis. (A) qRT-PCR analysis of macropinocytosis-related mRNAs in Huh7, HepG2, Hepa1-6, KYSE510, SW620, SW480, HT1080, and NCIH358 cells treated with BRQ (50 μM) or Lef (100 μM) for 24 h. (BC) Representative confocal images of dextran uptake / macropinocytosis bodies (red) and nuclei (DAPI, blue) in Huh7, HepG2, Hepa1-6, KYSE510, SW620, SW480, HT29, and NCIH358 cells treated with BRQ (50 μM) or Lef (100 μM) for 48 h. Scale bar, 10 μm. Quantitative results are shown in Figure C. (DE) Representative confocal images of actin filaments (phalloidin, red) and nuclei (DAPI, blue) in Huh7 cells treated with BRQ (50 μM) or Lef (100 μM) for 48 h. White asterisks represent cell membrane folds. Scale bar, 10 μm. Quantitative results are shown in Figure E. (F) Representative images of Huh7 cells treated with BRQ (50 μM), Lef (100 μM), or control (DMSO) for 48 h by transmission electron microscopy (TEM). Red asterisks represent macrocytic pinocytosis of Huh7 cells. Scale bar, 1 μm. (GH) Representative confocal images of degraded FITC-BSA (green) and nuclei (DAPI, blue) in Huh7 cells treated with BRQ (50 μM) or Lef (100 μM) for 48 h. Scale bar, 10 μm. Quantitative results are shown in Figure H. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0054] Figure 3 Knockdown or knockout of DHODH significantly activates macropinocytosis. (A) Representative immunoblot images of DHODH protein in Huh7 and SW620 cells after transfection with siNC or siDHODH. (B) Representative immunoblot images of DHODH protein in Huh7 cells after DHODH knockout. (C) qRT-PCR analysis of macropinocytosis-related mRNAs in Huh7 and SW620 cells after transfection with siNC or siDHODH. (D) qRT-PCR analysis of macropinocytosis-related mRNAs in Huh7 cells after DHODH knockout. (EF) Representative confocal images of dextran uptake / macropinocytosis (red) and nuclei (DAPI, blue) in Huh7 and SW620 cells after transfection with siNC or siDHODH. Quantitative results are shown in Figure F. Scale bar, 10 μm. (GH) Representative confocal images of dextran uptake / macropinocytosis (red) and nuclei (DAPI, blue) in Huh7 cells after DHODH knockout. The quantitative results are shown in Figure H. Scale bar: 10 μm. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0055] Figure 4 Replenishment of uridine and OA partially reversed macropinocytosis induced by DHODH inhibitors. (A) qRT-PCR analysis of macropinocytosis-related mRNAs in Huh7 cells treated with BRQ (50 μM), Lef (100 μM), or uridine (50 μM) for 24 h. **p<0.01, ***p<0.001, ****p<0.0001 vs. control group; #p<0.05, ##p<0.01, ###p<0.001 vs. BRQ or Lef group. (B) qRT-PCR analysis of macropinocytosis-related mRNAs in Huh7 cells treated with BRQ (50 μM), Lef (100 μM), or orotate (100 μM) for 24 h. **p<0.01, ***p<0.001, ****p<0.0001 vs. control group; ###p<0.001, ###p<0.0001 vs. BRQ or Lef group. (CD) Representative confocal images of dextran uptake / macrocytic pinocytosis (red) and nuclei (DAPI, blue) in Huh7 cells treated with BRQ (50 μM), Lef (100 μM), orotate (100 μM), or uridine (50 μM) for 48 h. Scale bar, 10 μm. Quantitative results are shown in Figure D. **p<0.01, ***p<0.001, ****p<0.0001 vs. control group; #p<0.05, ##p<0.01, ###p<0.001 vs. BRQ or Lef group.

[0056] Figure 5 Knockdown of PRPS1 activates macropinocytosis. (A) Representative immunoblot images of PRPS1 protein in Huh7 cells transfected with siNC or siPRPS1. (B) qRT-PCR analysis of macropinocytosis-related mRNAs in Huh7 cells transfected with siNC or siPRPS1. (CD) Representative confocal images of dextran uptake / macropinocytosis (red) and nuclei (DAPI, blue) in Huh7 cells transfected with siNC or siPRPS1. Scale bar, 30 μm. Quantitative results are shown in Figure D. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0057] Figure 6 Exogenous nucleotide deficiency cannot activate macropinocytosis. qRT-PCR analysis of macropinocytosis-related mRNAs in Huh7 cells treated with complete medium containing or without nucleotides for 48 h. ***p<0.001.

[0058] Figure 7 Nucleotide transporters and autophagy do not participate in tumor cell resistance to DHODH inhibitors. (A) qRT-PCR analysis of nucleotide transporter-related mRNAs in HT29 xenografts from nude mice after treatment with BRQ, Lef, or control. (B) qRT-PCR analysis of nucleotide transporter-related mRNAs in Huh7 cells treated with BRQ (50 μM) or Lef (100 μM) for 24 h. ****p<0.0001. (C) Representative protein immunoblot images of autophagy-related proteins in AOM / DSS orthotopic colorectal tumors after treatment with BRQ, Lef, or control. (D) Representative protein immunoblot images of autophagy-related proteins in HT29 xenografts from nude mice after treatment with BRQ, Lef, or control. (E) Representative protein immunoblot images of autophagy-related proteins in Huh7 cells after treatment with BRQ (50 μM) or Lef (100 μM) for 48 h or after DHODH knockdown. (F) Huh7 cells were treated with BRQ (50 μM), Lef (100 μM), or the autophagy inhibitor Bafilomycin A1 (BafA1) for 72 h, and relative cell viability was detected by CCK8 assay. ***p<0.001.

[0059] Figure 8Macropinocytosis and albumin uptake can partially attenuate the inhibitory effect of DHODH inhibitors on tumor cell proliferation. (AB) Representative images of clonogenic assays in Huh7, SW620, and HT29 cells treated with BRQ (5 μM), Lef (10 μM) w / wo 2% albumin for 14 days. Quantification is shown in Figure B. **P<0.01, ***P<0.001, ****p<0.0001 vs. control group; ##P<0.01, ###P<0.001, ####P<0.0001 vs. BRQ or Lef alone. (C) Proliferation numbers of Huh7, SW620, and HT29 cells treated with BRQ (5 μM), Lef (10 μM) w / wo 2% albumin. ****p<0.0001. (D) Representative images of 3D spheroidization assays of Huh7 and HT29 cells treated with BRQ (5 μM), Lef (10 μM) w / wo 2% albumin for 14 days. Scale bar, 100 μm. (E) Relative amount of Sytox Green+ per square millimeter after Huh7 cells were treated with BRQ (5 μM), Lef (10 μM) w / wo 2% albumin for 48 hours. RSL3 group is a positive control.

[0060] Figure 9 Albumin can partially reverse cell cycle arrest induced by Lef or BRQ. (A) Cell cycle of Huh7 and SW480 cells treated with BRQ (50 μM), Lef (100 μM), 2% albumin, or control (DMSO) after G0 / G1 cell cycle arrest. (B) Top part: Flowchart of induction of G0 / G1 cell cycle arrest. Bottom part: Cell cycle of Huh7 cells after G0 / G1 cell cycle arrest.

[0061] Figure 10 Mass spectrometry results. (AD) Mass spectrometry results of dihydroorotic acid, orotic acid, UMP and UDP in Huh7 cells treated with BRQ (50 μM), Lef (100 μM) w / wo EIPA (10 μM) or 2% albumin for 48 hours. * p<0.05, ** p<0.01, *** p<0.001, ****p<0.0001 vs. vehicle group. #p<0.05, ##p<0.01 vs. BRQ or Lef group alone. $p<0.05, $$p<0.01, $$$p<0.001 vs. BRQ with albumin or Lef with albumin group alone. (EL) Mass spectrometry results of alanine, glutamate, glutamine, glycine, ornithine, phenylalanine, proline and serine in Huh7 cells treated with BRQ (50 μM), Lef (100 μM) w / w EIPA (10 μM) or 2% albumin for 48 hours. *p<0.05, **p<0.01, ***p<0.001, vs.BRQ or Lef group alone group. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 vs.BRQ with albumin or Lef with albumin groupalone.

[0062] Figure 11 Molecular docking and SPR results. (AD) Molecular docking analysis of the binding sites of DHODH enzyme or albumin with DHODH inhibitors (Lef or BRQ). DHODH enzyme (blue), DHODH inhibitor (Lef or BRQ) (yellow), Albumin (green). (EF) Molecular docking analysis of the binding sites of collagen with DHODH inhibitors (Lef or BRQ). DHODH inhibitor (Lef or BRQ) (yellow), albumin (green). (GL) DHODH protein was immobilized on a chip, and its binding with gradient concentrations of BRQ or Lef (containing 1% Albumin) was tested by SPR experiments. The figure shows the binding kinetics and KD values ​​of DHODH protein.

[0063] Figure 12DHODH inhibitors combined with macropinocytosis inhibitors synergistically inhibit the proliferation of colorectal cancer and liver cancer in vitro. (AB) Representative images of clonogenic assays of Huh7, SW620, and HT29 cells treated with BRQ (5 μM), Lef (10 μM), or EIPA (2 μM) for 14 days in the presence of albumin. Quantitative results are shown in Figure B. *p<0.05, ***p<0.001, ****p<0.0001 vs. BRQ or Lef group. (C) Proliferation numbers of Huh7, SW620, and HT29 cells treated with BRQ (50 μM), Lef (100 μM), or EIPA (10 μM) in the presence of albumin. **p<0.01, ****p<0.0001. (D) Representative confocal images of dextran uptake / macrocytic pinocytosis (red) and nuclei (DAPI, blue) in Huh7 cells treated with BRQ (50 μM), Lef (100 μM), or EIPA (10 μM) for 48 h. Scale bar, 10 μm.

[0064] Figure 13DHODH inhibitors combined with macropinocytosis inhibitors synergistically inhibit the proliferation of colorectal and hepatocellular carcinoma in vivo. (A) Top part: HT29 cells were subcutaneously xenografted into 6-8 week old male nude mice and treated for 21 days with control, Lef, BRQ, EIPA, Lef+EIPA, or BRQ+EIPA. Bottom part: Representative images of HT29 xenograft tumors. (B) Tumor weight of HT29 xenograft tumors in nude mice treated for 21 days with control, Lef, BRQ, EIPA, Lef+EIPA, or BRQ+EIPA, n=5 mice. ***p<0.001, ****p<0.0001 vs. BRQ or Lef group. (C) Top part: Flowchart of primary cell culture for HT29 xenograft tumors. Bottom section: Representative confocal images of dextran uptake / macropinoplasty (red), EPCAM (green), and nuclei (DAPI, blue) in primary cells derived from HT29 xenograft tumors. Scale bar, 10 μm. (D) Tumor volume of HT29 xenograft tumors in nude mice treated for 21 days with control, Lef, BRQ, EIPA, Lef+EIPA, or BRQ+EIPA. **p<0.01, ****p<0.0001. (E) Body weight changes in HT29 xenograft nude mouse models during treatment with control, Lef, BRQ, EIPA, Lef+EIPA, or BRQ+EIPA. (F) Representative images of Ki-67 immunohistochemistry in HT-29 xenograft experiments. Scale bar, 50 μm. (G) Top section: AOM / DSS-induced colitis-associated colorectal cancer model and treatment flowchart. Lower section: Representative images of colorectal tumors in mice treated with control, Lef, BRQ, EIPA, Lef+EIPA, or BRQ+EIPA in an AOM / DSS-induced colorectal tumorigenesis model. Black asterisks represent colorectal tumors, n = 8 mice. (H) Number of colorectal tumor nodules in mice treated with control, Lef, BRQ, EIPA, Lef+EIPA, or BRQ+EIPA in an AOM / DSS-induced colorectal tumorigenesis model. *p<0.05, ***p<0.001 vs. BRQ or Lef group. (I) Colorectal tumor size distribution ratio in mice treated with control, Lef, BRQ, EIPA, Lef+EIPA, or BRQ+EIPA in an AOM / DSS-induced colorectal tumorigenesis model. (J) Colorectal weight in mice treated with control, Lef, BRQ, EIPA, Lef+EIPA, or BRQ+EIPA in an AOM / DSS-induced colorectal tumorigenesis model. *p<0.05, **p<0.01. (K) HE-stained images of colon tumors in mice treated with control, Lef, BRQ, EIPA, Lef+EIPA, or BRQ+EIPA in an AOM / DSS-induced colon tumorigenesis model. Scale bar, 2000 μm.(L) Representative immunohistochemical image of Ki-67 in a LOM / DSS-induced colon tumorigenesis model. Scale bar, 50 μm.

[0065] Figure 14 Endogenous nucleotide deficiency can activate PAK1. (A) Representative immunoblot images of p-PAK1 and PAK1 proteins in Huh7, SW620, SW480, and HT29 cells treated with BRQ (50 μM) and Lef (100 μM) for 48 h. (B) Representative immunoblot images of p-PAK1 and PAK1 proteins in Huh7 cells after transfection with siNC or siDHODH. (C) Representative immunoblot images of p-PAK1 and PAK1 proteins in Huh7 cells after transfection with siNC or siPRPS1. (D) Representative immunoblot images of p-PAK1 protein in Huh7 cells treated with BRQ (50 μM), Lef (100 μM), orotic acid (100 μM), or uridine (50 μM) for 48 h.

[0066] Figure 15 Inhibition of PAK1 suppresses macropinocytosis induced by endogenous nucleotide deficiency. (A) Representative immunoblot images of p-PAK1 protein in Huh7 cells treated with BRQ (50 μM), Lef (100 μM), or IPA3 (10 μM) for 48 h. (BC) Representative confocal images of dextran uptake / macropinocytosis (red) and nuclei (DAPI, blue) in Huh7 cells after treatment with BRQ (50 μM), Lef (100 μM), or IPA3 (10 μM) for 48 h or after PAK1 knockdown. Quantitative results are shown in Figure C. **p<0.01, ***p<0.001 vs. control group.

[0067] Figure 16 PAK1 inhibitors and DHODH inhibitors synergistically inhibit cell proliferation in vitro. (AB) Representative images of clonogenic assays of Huh7, SW480, SW620, and HT29 cells treated with BRQ (5 μM), Lef (10 μM), or IPA3 (2 μM) for 14 days in the presence of albumin. Quantitative results are shown in Figure B. **p<0.01, ***p<0.001, ****p<0.0001 vs. BRQ or Lef group. (C) Proliferation of Huh7 and HT29 cells treated with BRQ (50 μM), Lef (100 μM), or IPA3 (10 μM) in the presence of albumin. ****p<0.0001 vs. BRQ or Lef group. (D) Representative images of 3D spheroidization assays of Huh7 and HT29 cells treated with BRQ (5 μM), Lef (10 μM), or IPA3 (2 μM) for 14 days in the presence of albumin. Scale bar, 100μm.

[0068] Figure 17 Knockdown of PAK1 and treatment with DHODH inhibitors synergistically inhibit cell proliferation in vitro. (A) Representative immunoblot images of PAK1 protein in Huh7 cells treated with BRQ (50 μM) or Lef (100 μM) after transfection with siNC or siPAK1. (B) qRT-PCR analysis of PAK1 mRNA in Huh7 cells after transfection with siNC or siPAK1. ****p<0.0001. (CD) Representative images of clonogenic assays of Huh7 cells treated with BRQ (5 μM) or Lef (10 μM) in the presence of albumin after transfection with siNC or siPAK1. Quantitative results are shown in Figure D. **p<0.01, ****p<0.0001. (E) The proliferation of Huh7 and SW620 cells treated with Lef (100 μM) or BRQ (50 μM) in the presence of albumin after transfection with siNC or siPAK1 was detected by direct counting method. ****p<0.0001.

[0069] Figure 18 Inhibition of DHODH activation of EGFR. (A) Gene enrichment analysis of the ErbB signaling pathway in Huh7 cells after treatment with Lef (100 μM) or control (DMSO) for 48 hours. (B) Representative protein immunoblot images of p-EGFR and EGFR proteins in Huh7, SW480, SW620, and HT29 cells after treatment with BRQ (50 μM) or Lef (100 μM) for 48 hours. (C) Representative protein immunoblot images of p-EGFR protein in Huh7 cells after transfection with siNC or siDHODH. (D) Representative protein immunoblot images of p-EGFR protein in Huh7 cells after treatment with BRQ (50 μM), Lef (100 μM), orotate (100 μM), or uridine (50 μM) for 48 hours.

[0070] Figure 19 EGFR is an upstream regulatory gene of PAK1. (A) Representative immunoblot images of p-EGFR and p-PAK1 proteins in Huh7 cells treated with BRQ (50 μM), Lef (100 μM), or gefitinib (10 μM) for 48 h. (B) Representative immunoblot images of p-EGFR and p-PAK1 proteins in Huh7 cells transfected with siNC or siEGFR and treated with BRQ (50 μM) or Lef (100 μM) for 48 h.

[0071] Figure 20Inhibition of EGFR suppresses macropinocytosis induced by endogenous nucleotide deficiency. (AB) Representative confocal images of dextran uptake / macropinocytosis (red) and nuclei (DAPI, blue) in Huh7 cells after treatment with BRQ (50 μM), Lef (100 μM), or gefitinib (10 μM) for 48 h or after EGFR knockdown. Scale bar, 10 μm. Quantitative results are shown in Figure B. **p<0.01, ***p<0.001 vs. control group.

[0072] Figure 21 EGFR inhibitors and DHODH inhibitors synergistically inhibit tumor cell proliferation in vitro. (AB) Representative images of clonogenic assays of Huh7, SW480, SW620, and HT29 cells treated with BRQ (5 μM), Lef (10 μM), or gefitinib (2 μM) for 14 days in the presence of albumin. Quantitative results are shown in Figure B. **p<0.01, ***p<0.001, ****p<0.0001 vs. BRQ or Lef group. (C) Number of Huh7 and HT29 cells proliferated in the presence of albumin after treatment with Lef (100 μM), BRQ (50 μM), or gefitinib (10 μM). ****p<0.0001. (D) Representative images of 3D spheroidization assays of Huh7 and HT29 cells treated with BRQ (5 μM), Lef (10 μM), or gefitinib (2 μM) for 14 days in the presence of albumin. Scale bar, 100μm.

[0073] Figure 22 Knockdown of EGFR and treatment with DHODH inhibitors synergistically inhibited tumor proliferation in vitro. (AB) Representative images of clonogenic assays of Huh7 cells treated with BRQ (5 μM) or Lef (10 μM) for 14 days in the presence of albumin after transfection with siNC or siEGFR. Quantitative results are shown in Figure B. **p<0.01, ****p<0.0001 vs. BRQ or Lef group. (C) qRT-PCR analysis of EGFR mRNA in Huh7 cells after transfection with siNC or siEGFR. ****p<0.0001. (D) Detection of the proliferation of Huh7 and HT29 cells treated with Lef (100 μM) or BRQ (50 μM) in the presence of albumin after transfection with siNC or siEGFR using direct counting method. ****p<0.0001.

[0074] Figure 23EGFR ligand expression was significantly increased in the presence of DHODH inhibitors. (A) qRT-PCR analysis of BTC, AREG, EREG, and HBEGF mRNA in Huh7 and HT29 cells treated with BRQ (50 μM) or Lef (100 μM) for 24 h. (B) Representative protein immunoblot images of EREG, BTC, HBEGF, and AREG proteins in Huh7 cells treated with BRQ (50 μM) or Lef (100 μM) for 24 h. (C) Extracellular content of EGFR ligands (EREG, BTC, HBEGF, and AREG) detected by ELISA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs. control group.

[0075] Figure 24 EREG activates macropinocytosis via the EGFR-PAK1 pathway. (A) Representative immunoblot images of p-EGFR and p-PAK1 proteins in Huh7 cells treated with recombinant EREG or BTC for 48 h. (B) Representative confocal images of dextran uptake / macropinocytosis (red) and nuclei (DAPI, blue) in Huh7 cells treated with recombinant EREG or BTC for 48 h. (C) qRT-PCR analysis of EREG mRNA in Huh7 cells transfected with siNC or siEREG. ****p<0.0001. (D) Representative immunoblot images of p-EGFR and p-PAK1 proteins in Huh7 cells transfected with siNC or siEREG and treated with BRQ (50 μM) or Lef (100 μM) for 48 h. (E) Representative confocal images of dextran uptake / macrocytic pinocytosis (red) and nuclei (DAPI, blue) in Huh7 cells after 48 h of treatment with BRQ (50 μM) or Lef (100 μM) following transfection with siNC or siEREG. Scale bar, 100 μm.

[0076] Figure 25 DHODH inhibition regulates EREG transcriptionally. (A) qRT-PCR analysis of BTC and EREG mRNA in Huh7 cells treated with BRQ (50 μM), Lef (100 μM), or actinomycin D (10 μM) for 24 h. **p<0.01, ****p<0.0001 vs. control group; #p<0.05, ####p<0.0001 vs. BRQ or Lef group.

[0077] Figure 26Endogenous nucleotide deficiency induces p65 activation. (A) Genomic enrichment analysis of the NF-κB signaling pathway in Huh7 and SW480 cells treated with Lef (100 μM) for 48 h. (B) Representative immunoblot images of p-p65, p-IKK, and Iκb proteins in Huh7, SW480, and SW620 cells treated with BRQ (50 μM) or Lef (100 μM) for 1 h. (C) Representative immunoblot images of p65 proteins in the nucleus and cytoplasm of Huh7 cells treated with BRQ (50 μM) or Lef (100 μM) for 1 h. (D) Representative confocal images of p-p65 (red) and nucleus (DAPI, blue) in Huh7 cells after transfection with siNC, siPRPS1, or siDHODH. Scale bar, 10 μm. (E) Dual-luciferase reporter gene assay. Huh7 cells were treated with Lef (100 μM), BRQ (50 μM), or uridine (50 μM) for 48 h, and the NF-κB luciferase reporter gene was detected. ****p<0.0001 vs. control group; ####p<0.0001 vs. BRQ or Lef group.

[0078] Figure 27 p65 transcriptional regulation of EREG. (A) Bioinformatics analysis of p65 binding to the EREG region promoter. (B) Dual-luciferase reporter gene assay. The binding between p65 and the EREG promoter was detected in HEK-293T cells using a dual-luciferase reporter assay. ***p<0.001 vs. EREG WT Group; ####p<0.0001 vs. EREG WT +RELA group. (C) qRT-PCR analysis of EREG mRNA in Huh7 and SW480 cells treated with BRQ (50 μM), Lef (100 μM), or SC75741 (10 μM) for 24 h. ***p<0.001, ****p<0.0001 vs. control group; ##p<0.01, ###p<0.001, ####p<0.0001 vs. BRQ or Lef group. (D) qRT-PCR analysis of RELA and EREG mRNA in Huh7 and SW480 cells transfected with siNC or siRELA and treated with BRQ (50 μM) or Lef (100 μM) for 24 h. **p<0.01, ***p<0.001, ****p<0.0001 vs. “RELA” siNC+ control, BRQ or Lef group; ###p<0.001, ####p<0.0001 vs. “EREG” siNC+ control, BRQ or Lef group.

[0079] Figure 28p65 regulates macropinocytosis induced by endogenous nucleotide deficiency via the EREG / EGFR-PAK1 signaling axis.

[0080] (A) Representative immunoblot images of p65, p-EGFR, and p-PAK1 proteins in Huh7 cells treated with BRQ (50 μM) or Lef (100 μM) for 48 h after transfection with siNC or siRELA. (B) Representative immunoblot images of p-p65, p-EGFR, and p-PAK1 proteins in Huh7 cells treated with BRQ (50 μM), Lef (100 μM), or SC75741 (10 μM) for 48 h. (CD) Representative confocal images of dextran uptake / macrocytic pinocytosis (red) and nuclei (DAPI, blue) in SW480 cells treated with BRQ (50 μM), Lef (100 μM), or SC75741 (10 μM) for 48 h after transfection with siNC or siRELA. Scale bar: 10 μm. Quantitative results are shown in Figure D. *p<0.05, **p<0.01 vs. control group.

[0081] Figure 29 NF-κB inhibitors and DHODH inhibitors synergistically inhibit cell proliferation in vitro. (A) Representative images of 3D spheroidization assays of Huh7 and HT29 cells treated with BRQ (5 μM), Lef (10 μM), or SC75741 (2 μM) for 14 days in the presence of albumin. Scale bar, 100 μm. (BC) Representative images of colony formation assays of Huh7, SW480, and HT29 cells treated with BRQ (5 μM), Lef (1 μM), or SC75741 (2 μM) for 14 days in the presence of albumin. Quantitative results are shown in Figure C. ***p<0.001, ****p<0.0001 vs. BRQ or Lef group. (D) Proliferation numbers of Huh7 and SW620 cells treated with BRQ (50 μM), Lef (100 μM), or SC75741 (10 μM) in the presence of albumin. **p<0.01, ****p<0.0001 vs. BRQ or Lef group.

[0082] Figure 30Knockdown of p65 and treatment with DHODH inhibitors synergistically inhibit cell proliferation in vitro. (AB) Representative images of clonogenic assays of Huh7 cells treated with BRQ (5 μM) or Lef (10 μM) for 14 days after transfection with siNC or siRELA in the presence of albumin. Quantitative results are shown in Figure B. **p<0.01 vs. BRQ or Lef group. (C) Number of Huh7 cells proliferated after transfection with siNC or siRELA and treatment with Lef (100 μM) or BRQ (50 μM). *p<0.05, ****p<0.0001 vs. BRQ or Lef group.

[0083] Figure 31 DHODH inhibitors can enhance lactate-induced NF-κB activation driven by glycolysis.

[0084] (A) via Lactate-Glo TM Lactate content in different models and cell lines was detected by assay. Mean ± SD (n = 2–3). *Compared with siNC or vector groups alone; #Compared with BRQ, Lef, siDHODH, or siPRPS1 groups alone. (B) Representative confocal images showing p-p65 (red) and nuclei (DAPI, blue) in Huh7 and SW480 cells treated with lactate (20 μM), pyruvate (20 μM), and UK5099 (5 μM) for 1 hour. (C) Representative Western blots of p-p65, p-IKK, and Iκb proteins in Huh7 cells treated with lactate (20 μM), pyruvate (20 μM), and UK5099 (5 μM) for 1 hour. (D) qRT-PCR analysis of EREG mRNA in Huh7 and SW480 cells treated with lactate (20 μM), pyruvate (20 μM), and UK5099 (5 μM) for 24 hours. *Compared with the control group. #Compared with the lactate, pyruvate, or UK5099 groups alone. (E) Representative Western blots of p-EGFR, EREG, and EGFR after Huh7 cells were treated with lactate (20 μM), pyruvate (20 μM), and UK5099 (5 μM) for 36 hours. (F) Representative confocal images showing dextran uptake / macrocytic pinocytosis (red), lysosomes (green), and nuclei (DAPI, blue) in SW480 cells treated with lactate (20 μM), pyruvate (20 μM), and UK5099 (5 μM) for 48 hours.

[0085] Figure 32(A) Representative confocal images showing p-p65 (red) and nuclei (DAPI, blue) in Huh7 and SW480 cells treated with BRQ (50 μM), Lef (100 μM) / oxamate (50 μM) for 1 hour. (B) Representative Western blots of p-p65, p-IKK, and Iκb proteins in Huh7 cells treated with BRQ (50 μM), Lef (100 μM), or oxamate (50 μM) for 1 hour. (C) qRT-PCR analysis of EREG mRNA in Huh7 and SW480 cells treated with BRQ (50 μM), Lef (100 μM), and oxamate (50 μM) for 24 hours. *Compared with control group. #Compared with BRQ or Lef alone. (D) Representative Western blots of p-EGFR, EGFR, EREG, p-PAK1 / 2, and PAK1 proteins in Huh7 cells treated with BRQ (50 μM), Lef (100 μM), or oxamate (50 μM) for 36 h. (E) Representative confocal images showing dextran uptake / macrocytic pinocytosis (red), lysosomes (green), and nuclei (DAPI, blue) in SW480 cells treated with BRQ (50 μM), Lef (100 μM), and oxamate (50 μM) for 48 h. (FG) ECAR of SW480 cells treated with BRQ (50 μM), Lef (100 μM) w / wouridine (50 μM) for 48 h, measured using a Seahorse XFe96 analyzer. Mean ± SD (n = 2, 3, or 4). (HI) ECAR of HT29 cells transfected with siDHODH or siPRPS1 measured using a Seahorse XFe96 analyzer. Mean ± SD (n = 2, 3, or 4). (J) OCR of SW480 cells after treatment with BRQ (50 μM) and Lef (100 μM) for 48 hours (including 50 μM uridine) was measured using a Seahorse XFe96 analyzer. (K) OCR of HT29 cells transfected with siDHODH or siPRPS1 was measured using a Seahorse XFe96 analyzer.

[0086] Figure 33Synergistic antitumor effects of EGFR and DHODH inhibitors in HT29 xenograft tumors in vivo. (A) Top: HT29 cells were subcutaneously xenografted into 6-8 week old male nude mice and treated for 27 days with control, Lef, BRQ, gefitinib, Lef + gefitinib, or BRQ + gefitinib. Bottom: Representative images of HT29 xenograft tumors. (B) Tumor volume of HT29 xenograft tumors in nude mice treated for 27 days with control, Lef, BRQ, gefitinib, Lef + gefitinib, or BRQ + gefitinib. ****p<0.0001. (C) Top: Flowchart of primary cell culture for HT29 xenograft tumors. Bottom: Representative confocal images of dextran uptake / macropinoplasts (red), EPCAM (green), and nuclei (DAPI, blue) in primary cells derived from HT29 xenograft tumors. Scale bar, 10 μm. (DE) Tumor weight and body weight of nude mice with HT29 xenografts after 27 days of treatment with control, Lef, BRQ, gefitinib, Lef + gefitinib, or BRQ + gefitinib. (F) Representative immunohistochemical images of p-EGFR, p-PAK1, and Ki67 in HT29 xenografts after 27 days of treatment with control, Lef, BRQ, gefitinib, Lef + gefitinib, or BRQ + gefitinib. Scale bar, 100 μm. ***p<0.001, ****p<0.0001 vs. BRQ or Lef group.

[0087] Figure 34 Synergistic in vivo antitumor effects of EGFR inhibitors and DHODH inhibitors in Huh7 xenograft tumors

[0088] (A) Representative images of Huh7 xenograft tumors, treated for 9 weeks with control, Lef, BRQ, gefitinib, Lef + gefitinib, or BRQ + gefitinib, respectively. (B) Tumor volume of Huh7 xenograft tumors in nude mice treated for 9 weeks with control, Lef, BRQ, gefitinib, Lef + gefitinib, or BRQ + gefitinib. **p<0.01, ****p<0.0001. (C) Representative confocal images of dextran uptake / macropinoplasty (red), EPCAM (green), and nuclei (DAPI, blue) in primary cells derived from Huh7 xenograft tumors. Scale bar, 10 μm.

[0089] (DE) Tumor weight and body weight of Huh7 xenografts in nude mice after 9 weeks of treatment with control, Lef, BRQ, gefitinib, Lef + gefitinib, or BRQ + gefitinib. **p<0.01, ***p<0.001 vs. BRQ or Lef group. (F) Immunohistochemical representative images of p-EGFR, p-PAK1, and Ki67 in Huh7 xenografts after 9 weeks of treatment with control, Lef, BRQ, gefitinib, Lef + gefitinib, or BRQ + gefitinib. Scale bar, 50 μm.

[0090] Figure 35 Synergistic in vivo antitumor effects of EGFR inhibitors and DHODH inhibitors in the AOM / DSS model

[0091] (A) Upper part: AOM / DSS-induced colitis-associated colorectal cancer model and treatment flowchart.

[0092] Lower section: Representative images of colon tumors in mice treated with control, Lef, BRQ, gefitinib, Lef + gefitinib, or BRQ + gefitinib in an AOM / DSS-induced colon tumorigenesis model. Black asterisks represent colon tumors, n = 8 mice. (B) Number of tumor nodules in the colon of mice treated with control, Lef, BRQ, gefitinib, Lef + gefitinib, or BRQ + gefitinib in an AOM / DSS-induced colon tumorigenesis model. *p<0.05, ***p<0.001 vs. BRQ or Lef group. (C) Tumor size distribution ratio in mice treated with control, Lef, BRQ, gefitinib, Lef + gefitinib, or BRQ + gefitinib in an AOM / DSS-induced colon tumorigenesis model. (DE) Colon weight and length in an AOM / DSS-induced colitis-associated colorectal cancer model after treatment with control, Lef, BRQ, gefitinib, Lef + gefitinib, or BRQ + gefitinib. ***p<0.001 vs. BRQ or Lef group. (F) Immunohistochemical representative images of p-EGFR, p-PAK1, and Ki67 in an AOM / DSS-induced colitis-associated colorectal cancer model after treatment with control, Lef, BRQ, gefitinib, Lef + gefitinib, or BRQ + gefitinib. Scale bar, 50 μm.

[0093] Figure 36Validation of the DHODH-p65-EGFR-PAK1 signal axis in clinical samples. (A) Intergenetic correlation analysis of DHODH and EGFR ligands (EREG / AREG / HBEGF) in colorectal cancer and hepatocellular carcinoma from the TCGA database. (B) Intergenetic correlation analysis of RELA and EGFR ligands (EREG / AREG / HBEGF) in colorectal cancer and hepatocellular carcinoma from the TCGA database. (C) qRT-PCR analysis of DHODH and EGFR ligands (EREG / AREG / HBEGF) in 24 colorectal cancer patients. ***p<0.001, ****p<0.0001. (D) qRT-PCR analysis of PRPS1 and EGFR ligands (EREG / AREG / HBEGF) in 24 colorectal cancer patients. **p<0.01. (E) Immunohistochemical images of colorectal cancer patient tissue microarrays. Scale bar, 500 μm. (F) Immunohistochemical image of a microarray chip in liver cancer patient tissue. Scale bar, 500 μm.

[0094] Figure 37 Schematic diagram of the signaling pathway by which DHODH inhibitors regulate cell proliferation. Detailed Implementation

[0095] Unless otherwise specified, the terminology used herein has the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Unless otherwise specified, the materials, reagents, etc., described below are commercially available, and the relevant experimental methods can be performed using any suitable materials with reference to methods known to one of ordinary skill in the art. Details of the materials and reagents used in the experiments of this invention are provided in the appended tables below.

[0096] 1. The effects of DHODH inhibition on nucleotide transporters and autophagy were investigated using qRT-PCR, Western blotting, and cell proliferation assays; GSEA analysis was used to study the effects of DHODH inhibitors on gene enrichment in colorectal and hepatocellular carcinoma cells; phalloidin staining, transmission electron microscopy, DQ-BSA uptake assays, qRT-PCR, and 70 kDa dextran uptake assays were performed using the method of Su, H. et al. (Su, H., et al., Cancer cells escape autophagy inhibition via NRF2-induced macropinocytosis. Cancer Cell, 2021, 39(5): p.678-693e11.) The effects of DHODH inhibitors on macropinocytosis in tumor cells were studied; the effects of knockdown or knockout of DHODH and knockdown of PRPS1 on macropinocytosis in tumor cells were studied using qRT-PCR, 70kDa dextran uptake assay and WB assay; the effects of adding DHODH inhibitors and simultaneously supplementing uridine and OA on macropinocytosis in tumor cells were studied using qRT-PCR and 70kDa dextran uptake assay.

[0097] 2. The effects of DHODH inhibitors combined with albumin on tumor cell proliferation were investigated using clonogenic assays, cell counting assays, 3D spheroid assays, and cell death assays. The effects of DHODH inhibitors combined with albumin on tumor cell cycle were investigated using cell cycle detection assays and a G1 phase cell cycle synchronization model. Mass spectrometry was used to explore the mechanism by which macropinocytosis alleviates the inhibitory effects of DHODH inhibitors on cell cycle and growth. Molecular docking analysis was used to explore the mechanism by which albumin maintains intracellular nucleotide levels while inhibiting DHODH. The effects of DHODH inhibitors combined with macropinocytosis inhibitors on tumor cell proliferation were investigated using clonogenic assays, cell counting assays, and 70kDa dextran uptake assays. HT29 subcutaneous xenograft tumor models and AOM / DSS-induced primary colon tumor mouse models were constructed to study the effects of DHODH inhibitors combined with macropinocytosis inhibitors on in vivo tumor proliferation.

[0098] 3. The Western blot assay was used to detect the effect of endogenous nucleotide deficiency on PAK1; the 70kDa dextran uptake assay was used to study the effect of PKA1 inhibition on tumor cell macropinocytosis; and the colony formation assay, cell counting assay, and 3D spheroid assay were used to study the effect of PAK1 inhibitors or PAK1 knockdown combined with DHODH inhibitors on tumor cell proliferation.

[0099] 4. The effects of endogenous nucleotide deficiency on the EGFR pathway were investigated using GSEA analysis and Western blotting. Western blotting was used to demonstrate that EGFR is an upstream regulatory gene of PAK1. A 70kDa dextran uptake assay was used to investigate the effect of EGFR inhibition on macropinocytosis in tumor cells. Colony formation assays, cell counting assays, and 3D spheroidization assays were used to investigate the effects of EGFR inhibitors or EGFR knockdown combined with DHODH inhibitors on tumor cell proliferation. qRT-PCR, Western blotting, and ELISA were used to investigate the effects of DHODH inhibitors on EGFR ligands. Western blotting and a 70kDa dextran uptake assay were used to confirm that EGFR activates macropinocytosis via the EGFR-PAK1 pathway.

[0100] 5. The enrichment of the NF-κB signaling pathway in endogenous nucleotide deficiency was investigated using qRT-PCR and GSEA analysis; the effect of endogenous nucleotide deficiency on p65 was investigated using Western blotting, immunofluorescence staining, and dual-luciferase reporter assay; the binding of transcription factor p65 to the promoter region of EREG was investigated using dual-luciferase reporter assay and qRT-PCR; the effect of p65 on the regulation of macropinocytosis induced by endogenous nucleotide deficiency by the EREG / EGFR-PAK1 signaling axis was investigated using Western blotting and 70kDa dextran uptake assay; and the effects of NF-κB inhibitors or p65 knockdown combined with DHODH inhibitors on tumor cell proliferation were investigated using colony formation assays, cell counting assays, and 3D spheroidization assays.

[0101] 6. The glycolysis level of cells after DHODH inhibition was detected using the seahorse assay; the lactate level of cells after DHODH inhibition was detected using a lactate assay kit.

[0102] 7. The in vivo synergistic antitumor effects of EGFR inhibitors and DHODH inhibitors were investigated using HT29 and Huh7 subcutaneous xenograft nude mouse models and AOM / DSS-induced colorectal cancer models.

[0103] 8. Validation of the DHODH-p65-EREG / EGFR-PAK1 signal axis in clinical samples using bioinformatics analysis, qRT-PCR experiments, and immunohistochemical staining experiments on tumor patient tissue microarray chips.

[0104] Experimental results

[0105] 1. Inhibition of DHODH significantly induces macropinocytosis

[0106] 1.1 DHODH inhibitors significantly upregulated macropinocytosis and lysosome-related pathways

[0107] To clarify the mechanism of tumor cell resistance to DHODH inhibitors, we first treated Huh7 cells with the DHODH inhibitor leflunomide (Lef) for 48 h, followed by transcriptome sequencing. Differentially expressed genes were then subjected to gene set enrichment analysis (GSEA). The results showed that inhibition of DHODH significantly activated macropinocytosis and lysosomal pathways, including cellular response to nutrient levels, amino sugar and nucleotide sugar metabolism, fastdigestion and absorption, and lysosome metabolism. Figure 1 A).

[0108] 1.2 DHODH inhibitors induce macropinocytosis

[0109] To clarify whether DHODH inhibition can activate macropinocytosis, we first examined the expression levels of macropinocytosis-related mRNAs in patients treated with the DHODH inhibitors leflunomide (Lef) or brequinar (BRQ). qRT-PCR results showed that various tumor cells, including colorectal cancer, liver cancer, esophageal cancer, and fibrosarcoma, exhibited upregulation of macropinocytosis-related genes (see Su, H., et al., Cancer cells escape autophagy inhibition via NRF2-induced macropinocytosis. Cancer Cell, 2021, 39(5): p.678-693e11.). Figure 2 A). Since macropinocytosis involves the formation of membrane folds

[26] , membrane folds increase when macropinocytosis levels rise. We used phalloidin to stain the cytoskeleton of Huh7 cells, and the results showed that the membrane folds of cells increased significantly under treatment with the DHODH inhibitors Lef or BRQ. Figure 2 DE). Furthermore, transmission electron microscopy and dextran uptake experiments showed that the number of intracellular macrocytic pinocytosis significantly increased under treatment with the DHODH inhibitors Lef or BRQ. Figure 2 BC, Figure 2 F).

[0110] The fusion of macropinocytoplasm and lysosome is essential for the degradation and reuse of macromolecules. We added DQ-BSA (65kDa) fluorescent dye to observe whether the increased macropinocytoplasm could be degraded by lysosomes. This dye only emits green fluorescence when degradation occurs inside the lysosome. The results showed that under DHODH inhibitor treatment, the increased macropinocytoplasm in Huh7 and SW620 cells could be hydrolyzed by lysosomes. Figure 2 GH).

[0111] 1.3 Knockdown or elimination of DHODH significantly activates macropinocytosis

[0112] To further clarify the phenomenon that inhibiting DHODH can induce macropinocytosis in tumor cells, we constructed transient and stable knockout cell lines of DHODH (the stable knockout cell lines were constructed using the method of Su, H. et al., Su, H., et al., Cancer cells escape autophagy inhibition via NRF2-induced macropinocytosis. Cancer Cell, 2021, 39(5): p.678-693e11). Figure 3 AB), and then the level of macropinocytosis was measured. The results showed that after knocking down or knocking out DHODH in Huh7 and SW620 cells, the expression level of macropinocytosis-related genes (AB) was significantly reduced. Figure 3 The intake of CD and glucan was significantly increased. Figure 3 EH).

[0113] 1.4 Replenishing uridine and OA can partially reverse macropinocytosis induced by DHODH inhibitors.

[0114] Given the importance of DHODH in the pyrimidine synthesis pathway, we wanted to investigate whether macropinocytosis was induced by pyrimidine deficiency. To clarify this hypothesis, we conducted uridine and orotate (OA) supplementation experiments. The results showed that under DHODH inhibitor treatment (Lef and BRQ), uridine and orotate supplementation could partially reverse the increase in macropinocytosis-related gene expression caused by endogenous nucleotide deficiency. Figure 4 AB) and the increase in macropinocytosis levels ( Figure 4 CD).

[0115] 1.5 knockdown of PRPS1 activates macrocell drinking

[0116] To further confirm the activation of macropinocytosis by the lack of endogenous nucleotides in cells, we knocked down another key enzyme in de novo pyrimidine synthesis, phosphoribosyl pyrophosphate synthetase 1 (PRPS1). Figure 5 A)

[27] . The results showed that knocking down PRPS1 also activated the expression of macropinocytosis-related genes ( Figure 5 B) and macrophage levels ( Figure 5 CD).

[0117] 1.6 Exogenous nucleotide deficiency cannot activate macropinocytosis

[0118] We also examined macropinocytosis levels under conditions of exogenous nucleotide deficiency, and the results showed that the expression levels of macropinocytosis-related genes were not significantly upregulated under conditions of exogenous nucleotide deficiency. Figure 6 In summary, these results indicate that a lack of endogenous nucleotides in tumor cells can induce macropinocytosis.

[0119] 1.7 Summary

[0120] In this section of the results, we found that inhibiting the de novo pyrimidine synthesis pathway significantly induced macropinocytosis by using DHODH inhibitors, transient knockdown, or stable knockdown of DHODH or transient knockdown of PRPS1, another key enzyme in pyrimidine synthesis. More importantly, the increase in macropinocytosis induced by DHODH inhibition was reversed upon rehydration with uridine or orotic acid. These results suggest that DHODH-induced endogenous nucleotide deficiency can induce macropinocytosis.

[0121] 2. Nucleotide transporters and autophagy are not involved in resistance to DHODH inhibitors.

[0122] The above results indicate that endogenous nucleotide deficiency induced by DHODH inhibitors can activate macropinocytosis. To rule out the influence of nucleotide transporters and autophagy on DHODH inhibitor resistance, we first examined the expression of nucleotide transporter-related genes and autophagy in HT29 xenograft nude mouse models and Huh7 cells after treatment with Lef or BRQ. The results showed that after Lef or BRQ treatment, the expression of nucleotide transporter mRNAs (ENT1, ENT2, CNT1, CNT2, and UCK2) was not significantly upregulated. Figure 7 (AB). This suggests that treatment with DHODH inhibitors did not activate the nucleotide transporter uptake pathway.

[0123] Furthermore, in mouse models of AOM / DSS-induced colorectal cancer and nude mouse models of HT29 xenograft tumors, treatment with the DHODH inhibitors Lef or BRQ significantly activated the autophagy-related pathway (LC II / I). Figure 7 CD). Inhibition of DHODH in Huh7 cells also resulted in the observation of activation of autophagy-related pathways. Figure 7 E) To elucidate the role of autophagy in DHODH inhibitor resistance, we combined the DHODH inhibitor BRQ with the autophagy inhibitor bafilomycin A1 (BafA1). The results showed that the combination group had more surviving cells compared to the single-drug group. These results suggest that BRQ-induced autophagy may induce cell death rather than maintain survival. Figure 7 F).

[0124] 3. Macrocytic endocytosis can partially weaken the inhibitory effect of DHODH inhibitors on tumor cell proliferation by ingesting albumin.

[0125] 3.1 Macrocytic pinocytosis can partially weaken the inhibitory effect of DHODH inhibitors on tumor cell proliferation by ingesting albumin.

[0126] To date, our results indicate that inhibition of DHODH enhances macropinocytosis in tumor cells; however, the impact of induced macropinocytosis on tumor progression remains unclear. To elucidate the effect of macropinocytosis on tumor growth, we used clonogenic assays, cell counting assays, and 3D spheroidization assays to evaluate the effects of Lef or BRQ on tumor cell proliferation in vitro. The results showed that both Lef and BRQ significantly inhibited the proliferation of colorectal cancer and liver cancer cells, rather than inducing cell death. Figure 8 AE). Notably, the presence of albumin (a major macropinocytosis substrate) partially reversed the proliferative inhibitory effect of DHODH inhibitors. Figure 8 This suggests that macropinocytosis induced by DHODH inhibitors can take up albumin, thereby partially maintaining cell proliferation. This finding may partially explain the discrepancy between the preclinical and clinical therapeutic effects of BRQ, because albumin in human plasma at a concentration of 35-55 g / L is the main substrate for macropinocytosis and can partially reverse nucleotide deficiency; however, the concentration of albumin in in vitro culture medium is 0.1-0.3 g / L.

[0127] 3.2 Albumin can partially reverse cell cycle arrest induced by Lef or BRQ.

[0128] We further investigated the effects of DHODH inhibitors on the cell cycle, and the results showed that DHODH inhibitors blocked the S phase of the cell cycle. Figure 9A). To investigate the effect of albumin on the S phase of cells, we applied a G1 phase cell cycle synchronization model ( Figure 9 B), we found that Lef- or BRQ-induced cell cycle arrest could be partially reversed in the presence of albumin. Figure 9 A).

[0129] 3.3 Mass Spectrometry Results

[0130] To investigate the mechanism by which macropinocytosis alleviates the inhibitory effects of DHODH inhibitors on cell cycle and growth, we performed mass spectrometry experiments. The results showed that Lef and BRQ significantly reduced the content of nucleotide synthesis intermediates (lactoacid, UMP, and UDP), while increasing the content of DHODH enzyme metabolic precursor (dihydroorotate). Figure 10 In the presence of albumin, the reduction of orotic acid, UMP, and UDP was largely reversed; while EIPA significantly reduced the recovery effect of albumin. Figure 10 (AD). Simultaneously, compared to the addition of a DHODH inhibitor in the presence of albumin, the use of EIPA significantly reduced intracellular amino acid levels (Figure 10E-L), suggesting that EIPA may inhibit macropinocytosis. These results indicate that tumor cells may take up albumin through macropinocytosis, thereby partially offsetting the growth-inhibiting effects of DHODH inhibitors and providing the necessary amino acids for cell proliferation.

[0131] 3.4 Molecular docking and SPR results

[0132] To investigate the mechanism by which albumin maintains intracellular nucleotide levels when inhibiting DHODH, we performed molecular docking analysis. DHODH inhibitors Lef or BRQ were docked with DHODH or Albumin. A binding energy less than 0 indicates that the ligand and receptor can bind spontaneously without external force. Generally, a binding energy less than -5 kcal / mol is considered to indicate easy interaction between the ligand and receptor. The results showed that the docking energy between Lef or BRQ and DHODH or Albumin was less than -5 kcal / mol, demonstrating good binding affinity and stability. The best docking result was visualized, indicating that Lef or BRQ binds to DHODH through hydrogen bonding and strong electrostatic interactions. Figure 11 AB). Furthermore, Lef or BRQ can bind to albumin ( ). Figure 11This means that albumin can competitively bind to Lef or BRQ with DHODH. Therefore, we hypothesize that after the addition of albumin, albumin can competitively bind to Lef or BRQ with the DHODH enzyme, the inhibitory effect of DHODH inhibitors is weakened, the de novo pyrimidine biosynthesis pathway is partially restored, and cell proliferation is partially restored. SPR results further demonstrate that in the presence of DHODH, albumin can competitively bind to Lef / BRQ (CD), meaning that albumin can competitively bind to Lef or BRQ in the presence of DHODH. Figure 11 GL). Although type I collagen is one of the substrates for macropinocytosis

[28] , the binding energy of type I collagen to Lef / BRQ is very weak ( Figure 11 These results suggest that albumin may play a unique role in resisting Lef / BRQ, distinct from other macrocytic pinocytosis substrates. Therefore, we hypothesize that the inhibitory effect of DHODH inhibitors is alleviated due to the specific binding of albumin to Lef / BRQ, and the de novo pyrimidine synthesis pathway is partially restored.

[0133] 4. DHODH inhibitors combined with macropinocytosis inhibitors synergistically inhibit tumor growth.

[0134] 4.1 DHODH inhibitors combined with macropinocytosis inhibitors can synergistically inhibit the proliferation of colorectal cancer and liver cancer cells in vitro.

[0135] So far, our experimental results suggest that inhibition of DHODH can induce macropinocytosis. To clarify whether activated macropinocytosis is involved in tumor cell growth in the presence of DHODH inhibitors, we first used EIPA, a macropinocytosis-specific inhibitor. The results showed that when EIPA and a DHODH inhibitor were used in combination, the combination group significantly inhibited tumor cell proliferation in vitro compared to single-agent therapy. Figure 12 Furthermore, EIPA can effectively inhibit macropinocytosis induced by Lef and BRQ. Figure 12 D).

[0136] 4.2 DHODH inhibitors combined with macropinocytosis inhibitors can synergistically inhibit the growth of colorectal cancer and liver cancer in vivo.

[0137] To further investigate the effects of combined use of DHODH inhibitors and macropinocytosis inhibitors on tumor growth, we constructed a nude mouse HT29 subcutaneous xenograft tumor model and an AOM / DSS-induced primary colon tumor mouse model to test the efficacy of combined treatment with EIPA and DHODH inhibitors in vivo.

[0138] In the HT29 subcutaneous xenograft model, the combination therapy groups (EIPA+Lef and EIPA+BRQ) significantly reduced tumor volume and burden compared to inhibitors alone. Figure 13AB, DE). More importantly, the detection of macropinocytosis levels using primary cells isolated from HT29 subcutaneous xenografts revealed that Lef or BRQ alone significantly increased macropinocytosis levels. Compared to the monotherapy group, the combination therapy group showed a significant reduction in macropinocytosis levels (AB, DE). Figure 13 C). Furthermore, compared to the single-treatment group, the expression of the cell proliferation-related surface marker Ki67 was significantly reduced after combination therapy. Figure 13 F).

[0139] Similar results were obtained in AOM / DSS-induced colorectal cancer models. Compared with monotherapy, the combination therapy groups (EIPA+Lef and EIPA+BRQ) showed a significant reduction in tumor number, weight, and size. Figure 13 GJ). Immunohistochemistry also showed that, compared with the single-treatment group, the expression of the cell proliferation-related surface marker Ki67 was significantly reduced after combination therapy. Figure 13 L).

[0140] 4.3 Summary

[0141] In this section of the results, we demonstrated that DHODH inhibitor-induced macropinocytosis can maintain tumor growth by using nude mouse subcutaneous xenografts, AOM / DSS tumor-inducing models, and in vitro proliferation experiments.

[0142] 5. PAK1-mediated macropinocytosis induced by DHODH inhibition

[0143] 5.1 Inhibiting DHODH activation of PAK1

[0144] Since macropinocytosis mediates resistance to DHODH in tumor cells, how does inhibition of DHODH induce macropinocytosis? To clarify this question, we first examined the executor of macropinocytosis activation—p21 activated kinase 1 (PAK1)

[29] . The results showed that PAK1 protein was significantly activated in various tumor cell lines after treatment with DHODH inhibitors, knockdown of DHODH, or knockdown of PRPS1. Figure 14 AC). When uridine or orotic acid is replenished, it can partially reverse the activation of PAK1 caused by DHODH inhibition. Figure 14 D).

[0145] 5.2 Inhibition of PAK1 can suppress macropinocytosis induced by endogenous nucleotide deficiency.

[0146] To further investigate the effect of PAK1 on inhibiting DHODH-induced macropinocytosis, we first used the PAK1 inhibitor IPA3 to detect the activation level of PAK1. We found that IPA3 significantly inhibited the activation of PAK1 induced by Lef or BRQ treatment.Figure 15 A). More importantly, knocking down PAK1 or using IPA3 significantly inhibited macropinocytosis induced by DHODH inhibitors. Figure 15 BC).

[0147] 5.3 PAK1 inhibitors and DHODH inhibitors can synergistically inhibit cell proliferation in vitro.

[0148] Given that PAK1 is involved in nucleotide deficiency-induced macropinocytosis, targeting PAK1 may offer a potential strategy for inhibiting tumor growth. Results showed that, compared to single-agent therapy, the combination of IPA3 and DHODH inhibitors significantly inhibited tumor cell proliferation in vitro. Figure 16 AD).

[0149] 5.4 Knockdown of PAK1 and treatment with DHODH inhibitors synergistically inhibit cell proliferation in vitro.

[0150] To further clarify the role of PAK1-mediated macropinocytosis in maintaining the presence of DHODH inhibitors, we transiently knocked down PAK1 ( Figure 17 AB). Compared with monotherapy with the DHODH inhibitor Lef / BRQ, treatment with a DHODH inhibitor after PAK1 knockdown significantly inhibited tumor growth in vitro. Figure 17 CE).

[0151] 5.5 Summary

[0152] In summary, these results suggest that PAK1 plays a key role in mediating macropinocytosis activated by the lack of endogenous nucleotides due to DHODH inhibition, and that the combined use of DHODH inhibitors and PAK1 inhibitors can synergistically inhibit tumor growth.

[0153] 6. DHODH inhibitors activate PAK1-induced macropinocytosis via EGFR.

[0154] 6.1 Inhibiting DHODH activation of EGFR

[0155] To locate the upstream pathway of PAK1, we first performed GSEA analysis, which showed that the ErbB signaling pathway was significantly activated in the absence of endogenous nucleotides. Figure 18 A). In addition, under glutamine deficiency, pancreatic cancer cells activate PAK1 by activating EGFR signaling, thereby activating macropinocytosis

[17] . Therefore, we first examined the activation level of EGFR after inhibiting DHODH, and the results showed that inhibiting DHODH significantly upregulated EGFR phosphorylation ( Figure 18 More importantly, uridine and OA supplementation can partially reverse the upregulation of p-EGFR expression. Figure 18D). These results suggest that a lack of endogenous nucleotides can activate the EGFR signaling pathway.

[0156] 6.2 EGFR is an upstream regulatory gene of PAK1.

[0157] To further clarify whether EGFR is upstream of PAK1, we treated and knocked down EGFR with gefitinib (an EGFR inhibitor) and then examined PAK1 activation after DHODH inhibition. The results showed that EGFR inhibition significantly reversed PAK1 activation induced by DHODH inhibitors. Figure 19 AB).

[0158] 6.3 Inhibition of EGFR can suppress macropinocytosis induced by endogenous nucleotide deficiency.

[0159] Since EGFR is upstream of PAK1, can inhibiting EGFR reverse the upregulation of macropinocytosis induced by DHODH inhibitors? To answer this question, we measured macropinocytosis levels after EGFR inhibition. The results showed that inhibiting or knocking down EGFR with Gefitinib significantly suppressed DHODH inhibitor-induced macropinocytosis. Figure 20 AB).

[0160] 6.4 EGFR inhibitors and DHODH inhibitors synergistically inhibit tumor cell proliferation in vitro.

[0161] The results above suggest that inhibiting DHODH can activate EGFR, which in turn activates PAK1, thereby activating macropinocytosis and maintaining survival. So, can the combination of the EGFR inhibitor gefitinib and the DHODH inhibitor inhibit tumor cell proliferation? The results showed that compared with the single-drug group, the combination group significantly inhibited tumor growth. Figure 21 AD).

[0162] 6.5 EGFR knockdown combined with DHODH inhibitors synergistically inhibits tumor proliferation in vitro.

[0163] To further clarify the role of EGFR-mediated macropinocytosis in maintaining the presence of DHODH inhibitors, we transiently knocked down EGFR ( Figure 22 A). Compared with monotherapy with the DHODH inhibitor Lef / BRQ, EGFR knockdown significantly inhibited cell proliferation ( Figure 22 BD).

[0164] 6.6 EGFR ligand expression was significantly increased in the presence of DHODH inhibitors.

[0165] EGFR activation requires binding to extracellular ligands, which in turn activates intracellular signaling pathways

[30] . To clarify the mechanism by which DHODH inhibitors induce EGFR activation, we first examined the mRNA and protein levels of EGFR ligands. The results showed that, in addition to EGF, the expression levels of four other ligands—BTC, AREG, EREG, and HBEGF—were significantly increased. Figure 23 AB). ELISA analysis revealed that treatment with DHODH inhibitors significantly increased the levels of EREG and BTC. Figure 23 C).

[0166] 6.7 DHODH inhibition activates EGFR-PAK1-induced macropinocytosis by upregulating EREG.

[0167] Given the significant increase in EREG and BTC after treatment with DHODH inhibitors, we wanted to further clarify which ligand plays a crucial role in EGFR-PAK1-mediated macropinocytosis. We first examined the activation of EGFR and PAK1 after stimulation with recombinant proteins EREG and BTC. The results showed that both ligands could activate EGFR and PAK1. Figure 24 A). However, only EREG can significantly induce macropinocytosis (A). Figure 24 B). To further clarify whether DHODH inhibition mediated macropinocytosis via EGFR-PAK1 activated by EREG, we transiently knocked down EREG ( Figure 24 C) The activation levels of EGFR and PAK1 induced by DHODH inhibitors were then measured. Results showed that knockdown of EGFR partially reversed the activation of EGFR and PAK1 induced by DHODH inhibitors. Figure 24 D). More importantly, knocking down EREG can partially reverse the inhibition of DHODH-induced upregulation of macropinocytosis levels (D). Figure 24 E).

[0168] 6.8 Summary

[0169] In summary, we demonstrated that inhibition of DHODH enables tumor cells to activate EGFR-PAK1-mediated macropinocytosis by upregulating EREG mRNA and protein levels. Combined inhibition of EGFR and DHODH significantly suppressed tumor growth.

[0170] 7. DHODH inhibitors promote EGFR-PAK1 activation by NF-κB transcription of EREG to activate macropinocytosis.

[0171] 7.1 DHODH inhibition regulates EREG transcriptionally.

[0172] To clarify the regulatory level of EREG after DHODH inhibition, we pretreated with the transcriptional inhibitor actinomycin D. The results showed that actinomycin D could reverse the EREG mRNA upregulation induced by Lef and BRQ. Figure 25 A). This suggests that DHODH inhibition regulates EREG through transcription.

[0173] 7.2 Endogenous nucleotide deficiency induces p65 activation

[0174] Since EREG is regulated at the transcriptional level, our next step is to identify the transcription factors that regulate EREG. We first performed GSEA analysis, and the results showed that Lef treatment significantly activated the NF-κB signaling pathway. Figure 26 A). p65 is a key component of NF-κB signaling. We first examined p65 activation and nuclear translocation. The results showed that endogenous nucleotide deficiency caused by DHODH inhibition and PRPS1 knockdown led to p65 activation and increased nuclear translocation. Figure 26 (BD), and at the same time, upregulation of p-IKK expression upstream of p65 and downregulation of IκB protein expression can also be observed. Figure 26 B). NF-κB luciferase reporter gene assays showed that in the presence of Lef or BRQ, NF-κB luciferase reporter gene expression was significantly upregulated, while uridine supplementation reversed this phenomenon. Figure 26 E). This suggests that endogenous nucleotide deficiency can activate the NF-κB signaling pathway.

[0175] 7.3p65 transcriptional regulation of EREG

[0176] To clarify that EREG is a transcriptional target of p65, we first analyzed the promoter region of EREG using bioinformatics. The results showed that the EREG promoter region contains potential p65 binding sites. Figure 27 A). Next, we performed a dual-luciferase reporter gene assay, which showed that p65 could bind to the promoter region of EREG. When the predicted binding site was deleted, p65 could not bind to the promoter region of EREG. Figure 27 B). These results suggest that the -421 to -412 region of the EREG promoter is the p65 binding site. Furthermore, knocking down p65 or using SC75741 (an NF-κB inhibitor) can significantly reduce EREG mRNA levels. Figure 27 CD).

[0177] 7.4p65 regulates macropinocytosis induced by endogenous nucleotide deficiency via the EREG / EGFR-PAK1 signaling axis.

[0178] After confirming that transcription factor p65 can bind to the promoter region of EREG, to clarify the relationship between transcription factor p65 and the EREG / EGFR-PAK1-macropinocytosis signaling axis, we knocked down RELA (encoding p65 protein) and inhibited p65 activation using SC75741, respectively, and then measured the activation levels of EGFR and PAK1. The results showed that inhibiting p65 activation significantly reversed DHODH inhibitor-induced EGFR and PAK1 activation. Figure 28 AB). More importantly, inhibition of p65 significantly inhibited macropinocytosis induced by Lef or BRQ ( ). Figure 28 CD).

[0179] 7.5NF-κB inhibitors and DHODH inhibitors can synergistically inhibit cell proliferation in vitro.

[0180] Since p65 is involved in EREG-EGFR-PAK1-induced macropinocytosis, could the combination of NF-κB inhibitors and DHODH inhibitors inhibit tumor growth? Results showed that, compared with single-agent therapy, the combination of NF-κB inhibitor SC75741 and DHODH inhibitors significantly and synergistically inhibited tumor cell proliferation in vitro. Figure 29 AD).

[0181] 7.6 Knockdown of p65 and treatment with DHODH inhibitors synergistically inhibit cell proliferation in vitro.

[0182] To further clarify the role of p65 in DHODH inhibitor-induced macropinocytosis, we transiently knocked down the RELA gene and then examined the effect on cell proliferation. The results showed that, compared with LeffBRQ alone, knocking down RELA and inhibiting DHODH significantly suppressed in vitro tumor growth. Figure 30 AC).

[0183] 7.7 Summary

[0184] In summary, these findings clarify that p65 acts as a key transcription factor in inducing macropinocytosis, and under conditions of endogenous nucleotide deficiency, it transcriptionally regulates EREG, thereby activating EGFR-PAK1 signaling-mediated macropinocytosis.

[0185] 8. DHODH inhibitors enhance glycolysis-induced lactate activation of NF-κB.

[0186] When DHODH is inhibited, pyruvate accumulates excessively

[31] . Lactic acid produced by pyruvate glycolysis can activate p65

[32] . To investigate whether lactate activates p65 when DHODH is inhibited, we first examined lactate levels in vivo and in vitro when DHODH was inhibited. Inhibition of DHODH significantly increased intracellular lactate levels ( Figure 31A). It is worth noting that supplementation with uridine or orotate (OA) can reverse excess lactate ( Figure 31 A). More importantly, lactate or pyruvate can significantly induce p65 activation and drive EREG-EGFR-PAK1-induced macropinocytosis (A). Figure 31 BF). Furthermore, MPC1 / 2 inhibitors, which prevent pyruvate from entering mitochondria for TCA metabolism, also significantly activate p65 and macropinocytosis (BF). Figure 31 BF).

[0187] To further confirm that DHODH inhibitors activate p65 through lactate accumulation, we treated cells with and without LDHA inhibitors in the presence of Lef / BRQ. When LDHA was inhibited, the p65-EREG-EGFR-PAK1-induced macropinocytosis triggered by DHODH inhibitors was eliminated. Figure 32 This indicates that DHODH-induced lactate accumulation activates NF-κB. Although DHODH inhibition leads to pyruvate overload, it remains uncertain whether the accumulated pyruvate will be converted to lactate via glycolysis. By detecting ECAR, we found enhanced aerobic glycolysis, leading to excessive lactate production from pyruvate when DHODH was inhibited. Figure 32 FK).

[0188] 9. Synergistic antitumor effects of EGFR inhibitors and DHODH inhibitors in vivo

[0189] Given that the EGFR inhibitor gefitinib is commonly used clinically to treat cancer patients

[30] , and the DHODH inhibitor leflunomide is an FDA-approved drug for treating rheumatic diseases

[33] , we wanted to investigate the effect of Lef combined with gefitinib. We first constructed a nude mouse model of HT29 subcutaneous xenograft tumor and set up different treatment groups. The results showed that the combined (Lef + gefitinib, BRQ + gefitinib) treatment group significantly reduced tumor volume and weight ( Figure 33 (AB, D) The body weight of the mice did not change significantly. Figure 33 E). Immunohistochemical analysis further revealed a significant decrease in the levels of the proliferation markers Ki67 and p-EGFR and p-PAK1. Figure 33 F). More importantly, compared with the Lef or BRQ monotherapy groups, the combination therapy groups (Lef + gefitinib, BRQ + gefitinib) showed a significant reduction in macropinocytosis in tumor cells. Figure 33 C).

[0190] The efficacy of the combined treatment was further validated in the Huh7 subcutaneous tumorigenesis experiment. The Huh7 subcutaneous tumorigenesis experiment showed that the combined treatment group had a significant reduction in tumor volume and weight. Figure 34AB, D). Immunohistochemical analysis showed a significant decrease in the expression levels of p-EGFR, p-PAK1, and the proliferation marker Ki67 in the combined treatment group. Figure 34 F), furthermore, compared with the Lef or BRQ monotherapy groups, the combination therapy groups (Lef + gefitinib, BRQ + gefitinib) showed a significant reduction in macropinocytosis in tumor cells. Figure 34 C).

[0191] We further extended our findings to an AOM / DSS-induced colorectal cancer model. The combination therapy group showed a significant reduction in both the number and size of tumors. Figure 35 Immunohistochemical analysis also showed a significant decrease in the expression levels of p-EGFR, p-PAK1, and the proliferation marker Ki67 in the combination therapy group. Figure 35 F)

[0192] 10. Validation of the DHODH-p65-EGFR-PAK1 signal axis in clinical samples

[0193] To broaden our conclusions, we first analyzed the correlation between DHODH gene expression and EGFR ligands (EREG, AREG, and HBEGF) in the TCGA database. The results showed a significant negative correlation between them. Figure 36 A). A significant positive correlation exists between the expression of RELA (the p65-encoding gene) and the expression of these EGFR ligands (EREG, AREG, and HBEGF). Figure 36 B). Furthermore, we examined the correlation between DHODH / PRPS1 and EGFR ligands (EREG / AREG / HBEGF) in tumor samples from 24 colorectal cancer patients (patient information is shown in Table 1). Similar to the results from the TCGA dataset, a significant negative correlation existed between DHODH / PRPS1 and EGFR ligands (EREG / AREG / HBEGF). Figure 36 CD). More importantly, DHODH expression was negatively correlated with p-EGFR expression in patients with colorectal cancer and liver cancer. Figure 36 EF).

[0194] Table 1

[0195]

[0196]

[0197] Table 2 siRNA sequences

[0198]

[0199] Table 3. Primers for human qRT-PCR

[0200]

[0201]

[0202] Table 4. Primers for mouse qRT-PCR

[0203]

[0204] Table 5. Reagents or materials used in the experiment

[0205]

[0206]

[0207]

[0208]

[0209]

Claims

1. Use of nucleotide synthesis pathway inhibitors and macropinocytosis inhibitors in the preparation of drugs or kits for the treatment of tumors.

2. The use according to claim 1, wherein the nucleotide synthesis pathway inhibitor includes pyrimidine and / or purine synthesis pathway inhibitors, such as inhibitors of pyrimidine and / or purine synthesis pathway enzymes, such as inhibitors of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme of pyrimidine synthesis pathway enzymes, such as inhibitors of phosphoribosyl pyrophosphate synthase 1 (PRPS1), the rate-limiting enzyme of purine and pyrimidine nucleotide precursor synthesis.

3. The use as described in claim 1 or 2, wherein the macropinocytosis inhibitor comprises macropinocytosis substrate (e.g., albumin) uptake inhibitor, macropinocytosis signaling pathway inhibitor such as PAK1 inhibitor, EGFR signaling pathway inhibitor, ErbB signaling pathway inhibitor, NF-κB signaling pathway inhibitor, EREG inhibitor, p65 inhibitor, and AREG inhibitor.

4. The use according to any one of claims 1-3, wherein the inhibitor comprises an inhibitor of gene and / or protein expression in a signaling pathway, such as gene knockout or knockdown agents, such as siRNA, antisense RNA, gene editing agents, including, for example, ZFN, TALENs, CRISPER gene editing agents.

5. The use according to any one of claims 1-4, wherein the tumor includes a tumor resistant to treatment with a nucleotide synthesis pathway inhibitor, for example, to make it sensitive to treatment with a nucleotide synthesis pathway inhibitor.

6. The use according to any one of claims 1-5, wherein the tumor includes tumors sensitive to treatment with inhibitors of nucleotide synthesis pathways, for example, to prevent the development of drug resistance.

7. The use according to any one of claims 1-6, wherein the tumor includes colorectal cancer, liver cancer, pancreatic cancer, and breast cancer.

8. Use of the macropinocytosis inhibitor as defined in any one of claims 1-7 in the preparation of a medicament or kit for treating tumors resistant to nucleotide synthesis pathway inhibitor therapy, for example, to modify it to be sensitive to nucleotide synthesis pathway inhibitor therapy.

9. Use of the macropinocytosis inhibitor as defined in any one of claims 1-7 in the preparation of a medicament or kit for treating tumors sensitive to nucleotide synthesis pathway inhibitors, for example, to prevent drug resistance.

10. A medicament or kit for treating tumors, comprising an inhibitor of nucleotide synthesis pathways and a macropinocytosis inhibitor as defined in any one of claims 1-7.

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