Simvastatin overcomes the pPCK1-pLDHA-SPRINGlac axis-mediated ferroptosis and chemo-immunotherapy resistance in AKT-hyperactivated intrahepatic cholangiocarcinoma

Abstract

Background: Intrahepatic cholangiocarcinoma (ICC) is a challenging cancer with an increasing incidence. The Phase III TOPAZ-1/KEYNOTE-966 study demonstrated chemo-immunotherapy (CIT) as a significant advancement, potentially replacing traditional chemotherapy for advanced biliary tract cancer. Ferroptosis is a crucial process that affects cancer cell survival and therapy resistance. Although AKT hyperactivation is prevalent in numerous cancers, including ICC, its role in ferroptosis resistance remains unclear. This study explored whether targeting ferroptosis can enhance CIT response rates, specifically in ICC patients with AKT hyperactivation.

Methods: In vivo metabolic CRISPR screening in a Kras^G12D/Tp53^−/− ICC mouse model was used to identify primary regulators of ferroptosis during CIT (gemcitabine, cisplatin, and anti-mouse programmed cell death 1 ligand 1). Phosphoenolpyruvate carboxykinase 1 (PCK1) was assessed for its role in ferroptosis and treatment resistance in preclinical models under AKT activation levels. Molecular and biochemical techniques were used to explore PCK1-related resistance mechanisms in AKT-hyperactivated ICC.

Results: Under AKT hyperactivation condition, phosphorylated PCK1 (pPCK1) promoted metabolic reprogramming, enhancing ubiquinol and menaquinone-4 synthesis through the mevalonate (MVA) pathway. This cascade was mediated by the pPCK1-pLDHA-SPRINGlac axis. Inhibiting PCK1 phosphorylation or using simvastatin significantly augmented CIT efficacy in preclinical models. Clinical data further indicated that phosphorylated AKT (pAKT)-pPCK1 levels might serve as a biomarker to predict CIT response in ICC.

Conclusion: This study identified the pAKT-pPCK1-pLDHA-SPRINGlac axis as a novel mechanism driving ferroptosis resistance in AKT-hyperactivated ICC by associating glycolytic activation with MVA flux reprogramming. Targeting this axis, potentially through statin-based therapies, may offer a strategy to sensitize ICC cells to ferroptosis and improve treatment outcomes.

Keywords

Chemo-immunotherapy / Ferroptosis / Intrahepatic cholangiocarcinoma / Lactylation / Mevalonate pathway / PCK1

References

[1] Rizvi S, Khan SA, Hallemeier CL, Kelley RK, Gores GJ. Cholangiocarcinoma - evolving concepts and therapeutic strategies. Nat Rev Clin Oncol. 2018; 15(2): 95-111.

[2] Rauth S, Malafa M, Ponnusamy MP, Batra SK. Emerging Trends in Gastrointestinal Cancer Targeted Therapies: Harnessing Tumor Microenvironment, Immune Factors, and Metabolomics Insights. Gastroenterology. 2024; 167(5): 867-84.

[3] Oh D-Y, Ruth He A, Qin S, Chen L-T, Okusaka T, Vogel A, et al. Durvalumab plus gemcitabine and cisplatin in advanced biliary tract cancer. NEJM Evid. 2022; 1(8): EVIDoa2200015.

[4] Satriano L, Lewinska M, Rodrigues PM, Banales JM, Andersen JB. Metabolic rearrangements in primary liver cancers: cause and consequences. Nat Rev Gastroenterol Hepatol. 2019; 16(12): 748-66.

[5] Wang Y, Zheng L, Shang W, Yang Z, Li T, Liu F, et al. Wnt/beta-catenin signaling confers ferroptosis resistance by targeting GPX4 in gastric cancer. Cell Death Differ. 2022; 29(11): 2190-202.

[6] Chen Y, Li L, Lan J, Cui Y, Rao X, Zhao J, et al. CRISPR screens uncover protective effect of PSTK as a regulator of chemotherapy-induced ferroptosis in hepatocellular carcinoma. Mol Cancer. 2022; 21(1): 11.

[7] Wang W, Green M, Choi JE, Gijon M, Kennedy PD, Johnson JK, et al. CD8(+) T cells regulate tumour ferroptosis during cancer immunotherapy. Nature. 2019; 569(7755): 270-4.

[8] Hänggi K, Ruffell B. Cell death, therapeutics, and the immune response in cancer. Trends Cancer. 2023; 9(5): 381-96.

[9] Doll S, Proneth B, Tyurina YY, Panzilius E, Kobayashi S, Ingold I, et al. ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition. Nat Chem Biol. 2017; 13(1): 91-8.

[10] Liao P, Wang W, Wang W, Kryczek I, Li X, Bian Y, et al. CD8(+) T cells and fatty acids orchestrate tumor ferroptosis and immunity via ACSL4. Cancer Cell. 2022; 40(4): 365-78.e6.

[11] Yi J, Zhu J, Wu J, Thompson CB, Jiang X. Oncogenic activation of PI3K-AKT-mTOR signaling suppresses ferroptosis via SREBP-mediated lipogenesis. Proc Natl Acad Sci U S A. 2020; 117(49): 31189-97.

[12] Hong X, Roh W, Sullivan RJ, Wong KHK, Wittner BS, Guo H, et al. The Lipogenic Regulator SREBP2 Induces Transferrin in Circulating Melanoma Cells and Suppresses Ferroptosis. Cancer Discov. 2021; 11(3): 678-95.

[13] Liang D, Minikes AM, Jiang X. Ferroptosis at the intersection of lipid metabolism and cellular signaling. Mol Cell. 2022; 82(12): 2215-27.

[14] Hill MA, Alexander WB, Guo B, Kato Y, Patra K, O'Dell MR, et al. Kras and Tp53 Mutations Cause Cholangiocyte- and Hepatocyte-Derived Cholangiocarcinoma. Cancer Res. 2018; 78(16): 4445-51.

[15] Wang W, Yuan T, Ma L, Zhu Y, Bao J, Zhao X, et al. Hepatobiliary Tumor Organoids Reveal HLA Class I Neoantigen Landscape and Antitumoral Activity of Neoantigen Peptide Enhanced with Immune Checkpoint Inhibitors. Adv Sci (Weinh). 2022; 9(22): 2105810.

[16] Tomlinson JL, Li B, Yang J, Loeuillard E, Stumpf HE, Kuipers H, et al. Syngeneic murine models with distinct immune microenvironments represent subsets of human intrahepatic cholangiocarcinoma. J Hepatol. 2024; 80(6): 892-903.

[17] Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29(1): 15-21.

[18] Liao Y, Smyth GK, Shi W. The R package Rsubread is easier, faster, cheaper and better for alignment and quantification of RNA sequencing reads. Nucleic Acids Res. 2019; 47(8): e47.

[19] Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015; 43(7): e47.

[20] Leek JT, Johnson WE, Parker HS, Jaffe AE, Storey JD. The sva package for removing batch effects and other unwanted variation in high-throughput experiments. Bioinformatics. 2012; 28(6): 882-3.

[21] Zhu XG, Chudnovskiy A, Baudrier L, Prizer B, Liu Y, Ostendorf BN, et al. Functional Genomics In Vivo Reveal Metabolic Dependencies of Pancreatic Cancer Cells. Cell Metab. 2021; 33(1): 211-21.e6.

[22] Wang X, Tokheim C, Gu SS, Wang B, Tang Q, Li Y, et al. In vivo CRISPR screens identify the E3 ligase Cop1 as a modulator of macrophage infiltration and cancer immunotherapy target. Cell. 2021; 184(21): 5357-74.e22.

[23] Li W, Xu H, Xiao T, Cong L, Love MI, Zhang F, et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 2014; 15(12): 554.

[24] Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, et al. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol. 2020; 18(7): e3000410.

[25] National Research Council Committee for the Update of the Guide for the C, Use of Laboratory A. The National Academies Collection: Reports funded by National Institutes of Health. Guide for the Care and Use of Laboratory Animals. Washington (DC): National Academies Press (US) Copyright © 2011, National Academy of Sciences; 2011.

[26] Liu F, Liao Z, Qin L, Zhang Z, Zhang Q, Han S, et al. Targeting VPS72 inhibits ACTL6A/MYC axis activity in HCC progression. Hepatology. 2023; 78(5): 1384-401.

[27] Liu Y, Fang Y, Chen X, Wang Z, Liang X, Zhang T, et al. Gasdermin E-mediated target cell pyroptosis by CAR T cells triggers cytokine release syndrome. Sci Immunol. 2020; 5(43): eaax7969.

[28] Xiong YX, Zhang XC, Zhu JH, Zhang YX, Pan YL, Wu Y, et al. Collagen I-DDR1 signaling promotes hepatocellular carcinoma cell stemness via Hippo signaling repression. Cell Death Differ. 2023; 30(7): 1648-65.

[29] Doman JL, Sousa AA, Randolph PB, Chen PJ, Liu DR. Designing and executing prime editing experiments in mammalian cells. Nat Protoc. 2022; 17(11): 2431-68.

[30] Hsu JY, Grünewald J, Szalay R, Shih J, Anzalone AV, Lam KC, et al. PrimeDesign software for rapid and simplified design of prime editing guide RNAs. Nat Commun. 2021; 12(1): 1034.

[31] Kelstrup CD, Bekker-Jensen DB, Arrey TN, Hogrebe A, Harder A, Olsen JV. Performance Evaluation of the Q Exactive HF-X for Shotgun Proteomics. J Proteome Res. 2018; 17(1): 727-38.

[32] Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008; 26(12): 1367-72.

[33] Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics. 2014; 13(9): 2513-26.

[34] UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 2021; 49(D1): D480-9.

[35] Song JW, Lam SM, Fan X, Cao WJ, Wang SY, Tian H, et al. Omics-Driven Systems Interrogation of Metabolic Dysregulation in COVID-19 Pathogenesis. Cell Metab. 2020; 32(2): 188-202.e5.

[36] Lam SM, Zhang C, Wang Z, Ni Z, Zhang S, Yang S, et al. A multi-omics investigation of the composition and function of extracellular vesicles along the temporal trajectory of COVID-19. Nat Metab. 2021; 3(7): 909-22.

[37] Claessens AJ, Yeung CK, Risler LJ, Phillips BR, Himmelfarb J, Shen DD. Rapid and sensitive analysis of reduced and oxidized coenzyme Q10 in human plasma by ultra performance liquid chromatography-tandem mass spectrometry and application to studies in healthy human subjects. Ann Clin Biochem. 2016; 53(Pt 2): 265-73.

[38] Usui Y, Nishimura N, Kobayashi N, Okanoue T, Kimoto M, Ozawa K. Measurement of vitamin K in human liver by gradient elution high-performance liquid chromatography using platinum-black catalyst reduction and fluorimetric detection. J Chromatogr. 1989; 489(2): 291-301.

[39] Jemal M, Schuster A, Whigan DB. Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard. Rapid Commun Mass Spectrom. 2003; 17(15): 1723-34.

[40] Xu D, Wang Z, Xia Y, Shao F, Xia W, Wei Y, et al. The gluconeogenic enzyme PCK1 phosphorylates INSIG1/2 for lipogenesis. Nature. 2020; 580(7804): 530-5.

[41] Ye B, Liu B, Hao L, Zhu X, Yang L, Wang S, et al. Klf4 glutamylation is required for cell reprogramming and early embryonic development in mice. Nat Commun. 2018; 9(1): 1261.

[42] Obenauer JC, Cantley LC, Yaffe MB. Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res. 2003; 31(13): 3635-41.

[43] Hornbeck PV, Zhang B, Murray B, Kornhauser JM, Latham V, Skrzypek E. PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res. 2015; 43(Database issue): D512-20.

[44] Cho KF, Branon TC, Udeshi ND, Myers SA, Carr SA, Ting AY. Proximity labeling in mammalian cells with TurboID and split-TurboID. Nat Protoc. 2020; 15(12): 3971-99.

[45] Li X, Jiang Y, Meisenhelder J, Yang W, Hawke DH, Zheng Y, et al. Mitochondria-Translocated PGK1 Functions as a Protein Kinase to Coordinate Glycolysis and the TCA Cycle in Tumorigenesis. Mol Cell. 2016; 61(5): 705-19.

[46] Xue R, Zhang Q, Cao Q, Kong R, Xiang X, Liu H, et al. Liver tumour immune microenvironment subtypes and neutrophil heterogeneity. Nature. 2022; 612(7938): 141-7.

[47] Ma L, Hernandez MO, Zhao Y, Mehta M, Tran B, Kelly M, et al. Tumor Cell Biodiversity Drives Microenvironmental Reprogramming in Liver Cancer. Cancer Cell. 2019; 36(4): 418-30.e6.

[48] Zhang M, Yang H, Wan L, Wang Z, Wang H, Ge C, et al. Single-cell transcriptomic architecture and intercellular crosstalk of human intrahepatic cholangiocarcinoma. J Hepatol. 2020; 73(5): 1118-30.

[49] Yang C, Geng H, Yang X, Ji S, Liu Z, Feng H, et al. Targeting the immune privilege of tumor-initiating cells to enhance cancer immunotherapy. Cancer Cell. 2024; 42(12): 2064-81.e19.

[50] Schubert M, Klinger B, Klünemann M, Sieber A, Uhlitz F, Sauer S, et al. Perturbation-response genes reveal signaling footprints in cancer gene expression. Nat Commun. 2018; 9(1): 20.

[51] Wagner A, Wang C, Fessler J, DeTomaso D, Avila-Pacheco J, Kaminski J, et al. Metabolic modeling of single Th17 cells reveals regulators of autoimmunity. Cell. 2021; 184(16): 4168-85.e21.

[52] Fu L, Li S, Xiao W, Yu K, Li S, Yuan S, et al. DGKA Mediates Resistance to PD-1 Blockade. Cancer Immunol Res. 2021; 9(4): 371-85.

[53] Li J, Pan C, Boese AC, Kang J, Umano AD, Magliocca KR, et al. DGKA Provides Platinum Resistance in Ovarian Cancer Through Activation of c-JUN-WEE1 Signaling. Clin Cancer Res. 2020; 26(14): 3843-55.

[54] Liu J, Wei Y, Wu Y, Li J, Sun J, Ren G, et al. ATP2C2 Has Potential to Define Tumor Microenvironment in Breast Cancer. Front Immunol. 2021; 12: 657950.

[55] Huang D, Wang Y, Thompson JW, Yin T, Alexander PB, Qin D, et al. Cancer-cell-derived GABA promotes β-catenin-mediated tumour growth and immunosuppression. Nat Cell Biol. 2022; 24(2): 230-41.

[56] Ma R, Zhang W, Tang K, Zhang H, Zhang Y, Li D, et al. Switch of glycolysis to gluconeogenesis by dexamethasone for treatment of hepatocarcinoma. Nat Commun. 2013; 4: 2508.

[57] Li Y, Luo S, Ma R, Liu J, Xu P, Zhang H, et al. Upregulation of cytosolic phosphoenolpyruvate carboxykinase is a critical metabolic event in melanoma cells that repopulate tumors. Cancer Res. 2015; 75(7): 1191-6.

[58] Tang K, Zhu L, Chen J, Wang D, Zeng L, Chen C, et al. Hypoxia Promotes Breast Cancer Cell Growth by Activating a Glycogen Metabolic Program. Cancer Res. 2021; 81(19): 4949-63.

[59] Gou D, Liu R, Shan X, Deng H, Chen C, Xiang J, et al. Gluconeogenic enzyme PCK1 supports S-adenosylmethionine biosynthesis and promotes H3K9me3 modification to suppress hepatocellular carcinoma progression. J Clin Invest. 2023; 133(13).

[60] Montal ED, Dewi R, Bhalla K, Ou L, Hwang BJ, Ropell AE, et al. PEPCK Coordinates the Regulation of Central Carbon Metabolism to Promote Cancer Cell Growth. Mol Cell. 2015; 60(4): 571-83.

[61] Gaschler MM, Andia AA, Liu H, Csuka JM, Hurlocker B, Vaiana CA, et al. FINO(2) initiates ferroptosis through GPX4 inactivation and iron oxidation. Nat Chem Biol. 2018; 14(5): 507-15.

[62] Bian X, Jiang H, Meng Y, Li YP, Fang J, Lu Z. Regulation of gene expression by glycolytic and gluconeogenic enzymes. Trends Cell Biol. 2022; 32(9): 786-99.

[63] Yang X, Wang Z, Zandkarimi F, Liu Y, Duan S, Li Z, et al. Regulation of VKORC1L1 is critical for p53-mediated tumor suppression through vitamin K metabolism. Cell Metab. 2023; 35(8): 1474-90.e8.

[64] Gao R, Li Y, Xu Z, Zhang F, Xu J, Hu Y, et al. Mitochondrial pyruvate carrier 1 regulates fatty acid synthase lactylation and mediates treatment of nonalcoholic fatty liver disease. Hepatology. 2023; 78(6): 1800-15.

[65] Chen Y, Wu J, Zhai L, Zhang T, Yin H, Gao H, et al. Metabolic regulation of homologous recombination repair by MRE11 lactylation. Cell. 2024; 187(2): 294-311.e21.

[66] Chen H, Li Y, Li H, Chen X, Fu H, Mao D, et al. NBS1 lactylation is required for efficient DNA repair and chemotherapy resistance. Nature. 2024; 631(8021): 663-9.

[67] Hendrix S, Kingma J, Ottenhoff R, Valiloo M, Svecla M, Zijlstra LF, et al. Hepatic SREBP signaling requires SPRING to govern systemic lipid metabolism in mice and humans. Nat Commun. 2023; 14(1): 5181.

[68] Loregger A, Raaben M, Nieuwenhuis J, Tan JME, Jae LT, van den Hengel LG, et al. Haploid genetic screens identify SPRING/C12ORF49 as a determinant of SREBP signaling and cholesterol metabolism. Nat Commun. 2020; 11(1): 1128.

[69] Bayraktar EC, La K, Karpman K, Unlu G, Ozerdem C, Ritter DJ, et al. Metabolic coessentiality mapping identifies C12orf49 as a regulator of SREBP processing and cholesterol metabolism. Nat Metab. 2020; 2(6): 487-98.

[70] Aregger M, Lawson KA, Billmann M, Costanzo M, Tong AHY, Chan K, et al. Systematic mapping of genetic interactions for de novo fatty acid synthesis identifies C12orf49 as a regulator of lipid metabolism. Nat Metab. 2020; 2(6): 499-513.

[71] Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature. 1990; 343(6257): 425-30.

[72] Zhang HL, Hu BX, Ye ZP, Li ZL, Liu S, Zhong WQ, et al. TRPML1 triggers ferroptosis defense and is a potential therapeutic target in AKT-hyperactivated cancer. Sci Transl Med. 2024; 16(753): eadk0330.

[73] Tu RH, Wu SZ, Huang ZN, Zhong Q, Ye YH, Zheng CH, et al. Neurotransmitter Receptor HTR2B Regulates Lipid Metabolism to Inhibit Ferroptosis in Gastric Cancer. Cancer Res. 2023; 83(23): 3868-85.

[74] Song X, Liu J, Kuang F, Chen X, Zeh HJ, Kang R, et al. PDK4 dictates metabolic resistance to ferroptosis by suppressing pyruvate oxidation and fatty acid synthesis. Cell Rep. 2021; 34(8): 108767.

[75] Li Z, Xu ZM, Chen WP, Du XJ, Ou CX, Luo ZK, et al. Tumor-repopulating cells evade ferroptosis via PCK2-dependent phospholipid remodeling. Nat Chem Biol. 2024; 20(10): 1341-52.

[76] Shi L, An S, Liu Y, Liu J, Wang F. PCK1 Regulates Glycolysis and Tumor Progression in Clear Cell Renal Cell Carcinoma Through LDHA. Onco Targets Ther. 2020; 13: 2613-27.

[77] Cheung KS, Yeung YWM, Wong WS, Li B, Seto WK, Leung WK. Statins associate with lower risk of biliary tract cancers: A systematic review and meta-analysis. Cancer Med. 2023; 12(1): 557-68.

[78] Wijarnpreecha K, Aby ES, Ghoz H, Cheungpasitporn W, Lukens FJ, Harnois DM, et al. Statins and Risk of Cholangiocarcinoma: A Systematic Review and Meta-analysis. J Gastrointestin Liver Dis. 2020; 29(4): 629-35.

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