A novel allosteric driver mutation of β-glucuronidase promotes head and neck squamous cell carcinoma progression through STT3B-mediated PD-L1 N-glycosylation

Abstract

Head and neck squamous cell carcinoma (HNSCC) develops and advances because of the accumulation of somatic mutations located in orthosteric and allosteric areas. However, the biological effects of allosteric driver mutations during tumorigenesis are mostly unknown. Here, we mapped somatic mutations generated from 10 tumor-normal matched HNSCC samples into allosteric sites to prioritize the mutated allosteric proteins via whole-exome sequencing and AlloDriver, identifying the specific mutation H351Q in β-glucuronidase (GUSB), a lysosomal enzyme, as a novel allosteric driver mutation, which considerably encouraged HNSCC progression both in vitro and in vivo. Mechanistically, the allosteric mutation of H351Q remarkably attenuated protein trafficking from the endoplasmic reticulum (ER) to lysosomes, leading to ER retention, in which GUSB-H351Q facilitated the aberrant N-glycosylation of PD-L1 through increasing protein stability and mRNA transcripts of the STT3 oligosaccharyltransferase complex catalytic subunit B, an oligosaccharyltransferase complex. Moreover, GUSB-H351Q reshaped a more immunosuppressive microenvironment featuring increased infiltration of exhausted CD8⁺ T cells and remodeled tumor metabolism, characterized by increased activity of the purine metabolism pathways and pyruvic acid accumulation. This study provides a mechanism-driven approach to overcoming HNSCC progression and immune evasion and identifies novel druggable targets based on the presence of GUSB allosteric driver mutation.

Keywords

allosteric driver mutation / glycosylation / head and neck squamous cell carcinoma (HNSCC) / PD-L1 / STT3B / β-glucuronidase (GUSB)

References

[1] Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021; 71(3): 209-249.

[2] Cohen EEW, Soulieres D, Le Tourneau C, et al. Pembrolizumab versus methotrexate, docetaxel, or cetuximab for recurrent or metastatic head-and-neck squamous cell carcinoma (KEYNOTE-040): a randomised, open-label, phase 3 study. Lancet. 2019; 393(10167): 156-167.

[3] Ferris RL, Blumenschein GJ, Fayette J, et al. Nivolumab for recurrent squamous-cell carcinoma of the head and neck. N Engl J Med. 2016; 375(19): 1856-1867.

[4] Burtness B, Harrington KJ, Greil R, et al. Pembrolizumab alone or with chemotherapy versus cetuximab with chemotherapy for recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-048): a randomised, open-label, phase 3 study. Lancet. 2019; 394(10212): 1915-1928.

[5] Pickering CR, Zhang J, Yoo SY, et al. Integrative genomic characterization of oral squamous cell carcinoma identifies frequent somatic drivers. Cancer Discov. 2013; 3(7): 770-781.

[6] Su SC, Lin CW, Liu YF, et al. Exome sequencing of oral squamous cell carcinoma reveals molecular subgroups and novel therapeutic opportunities. Theranostics. 2017; 7(5): 1088-1099.

[7] Changeux JP, Christopoulos A. Allosteric modulation as a unifying mechanism for receptor function and regulation. Cell. 2016; 166(5): 1084-1102.

[8] Song K, Li Q, Gao W, et al. AlloDriver: a method for the identification and analysis of cancer driver targets. Nucleic Acids Res. 2019; 47(W1): W315-W321.

[9] Shen Q, Cheng F, Song H, et al. Proteome-scale investigation of protein allosteric regulation perturbed by somatic mutations in 7, 000 cancer genomes. Am J Hum Genet. 2017; 100(1): 5-20.

[10] Lu S, Shen Q, Zhang J. Allosteric methods and their applications: facilitating the discovery of allosteric drugs and the investigation of allosteric mechanisms. Acc Chem Res. 2019; 52(2): 492-500.

[11] Huang Z, Zhao J, Deng W, et al. Identification of a cellularly active SIRT6 allosteric activator. Nat Chem Biol. 2018; 14(12): 1118-1126.

[12] Huang M, Song K, Liu X, et al. AlloFinder: a strategy for allosteric modulator discovery and allosterome analyses. Nucleic Acids Res. 2018; 46(W1): W451-W458.

[13] Lu S, Ji M, Ni D, et al. Discovery of hidden allosteric sites as novel targets for allosteric drug design. Drug Discov Today. 2018; 23(2): 359-365.

[14] Ni D, Wei J, He X, et al. Discovery of cryptic allosteric sites using reversed allosteric communication by a combined computational and experimental strategy. Chem Sci. 2020; 12(1): 464-476.

[15] Lu S, He X, Yang Z, et al. Activation pathway of a G protein-coupled receptor uncovers conformational intermediates as targets for allosteric drug design. Nat Commun. 2021; 12(1): 4721-4736.

[16] Zhong J, Guo Y, Lu S, et al. Rational design of a sensitivity-enhanced tracer for discovering efficient APC-Asef inhibitors. Nat Commun. 2022; 13(1): 4961-4975.

[17] Song Q, Li M, Li Q, et al. DeepAlloDriver: a deep learning-based strategy to predict cancer driver mutations. Nucleic Acids Res. 2023; 51(W1): W129-W133.

[18] Wu L, Jiang J, Jin Y, et al. Activity-based probes for functional interrogation of retaining β-glucuronidases. Nat Chem Biol. 2017; 13(8): 867–873.

[19] Naz H, Islam A, Waheed A, et al. Human beta-glucuronidase: structure, function, and application in enzyme replacement therapy. Rejuvenation Res. 2013; 16(5): 352-363.

[20] Kong X, Zheng Z, Song G, et al. Over-expression of GUSB leads to primary resistance of anti-PD1 therapy in hepatocellular carcinoma. Front Immunol. 2022; 13: 876048-876061.

[21] Wang F, Sun N, Li Q, et al. Self-referenced synthetic urinary biomarker for quantitative monitoring of cancer development. J Am Chem Soc. 2023; 145(2): 919-928.

[22] Schjoldager KT, Narimatsu Y, Joshi HJ, et al. Global view of human protein glycosylation pathways and functions. Nat Rev Mol Cell Biol. 2020; 21(12): 729-749.

[23] Li CW, Lim SO, Xia W, et al. Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity. Nat Commun. 2016; 7: 12632-12643.

[24] Li CW, Lim SO, Chung EM, et al. Eradication of triple-negative breast cancer cells by targeting glycosylated PD-L1. Cancer Cell. 2018; 33(2): 187-201.

[25] Bramwell KK, Mock K, Ma Y, et al. β-Glucuronidase, a regulator of lyme arthritis severity, modulates lysosomal trafficking and MMP-9 secretion in response to inflammatory stimuli. J Immunol. 2015; 195(4): 1647-1656.

[26] Chakraborty A, Dimitroff CJ. Cancer immunotherapy needs to learn how to stick to its guns. J Clin Invest. 2019; 129(12): 5089-5091.

[27] Marsico G, Russo L, Quondamatteo F, et al. Glycosylation and integrin regulation in cancer. Trends Cancer. 2018; 4(8): 537-552.

[28] Davicino RC, Elicabe RJ, Di Genaro MS, et al. Coupling pathogen recognition to innate immunity through glycan-dependent mechanisms. Int Immunopharmacol. 2011; 11(10): 1457-1463.

[29] Hsu JM, Xia W, Hsu YH, et al. STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion. Nat Commun. 2018; 9(1): 1908-1925.

[30] Nussinov R, Tsai CJ. Allostery in disease and in drug discovery. Cell. 2013; 153(2): 293-305.

[31] Ogasawara D, Ichu TA, Vartabedian VF, et al. Selective blockade of the lyso-PS lipase ABHD12 stimulates immune responses in vivo. Nat Chem Biol. 2018; 14(12): 1099-1108.

[32] Oshima A, Kyle JW, Miller RD, et al. Cloning, sequencing, and expression of cDNA for human beta-glucuronidase. Proc Natl Acad Sci USA. 1987; 84(3): 685-689.

[33] Tomatsu S, Montano AM, Dung VC, et al. Mutations and polymorphisms in GUSB gene in mucopolysaccharidosis VII (Sly syndrome). Hum Mutat. 2009; 30(4): 511-519.

[34] Zhou W, Zhang S, Cai Z, et al. A glycolysis-related gene pairs signature predicts prognosis in patients with hepatocellular carcinoma. PeerJ. 2020; 8: e9944-9962.

[35] Xu J, Guo Y, Ning W, et al. Comprehensive analyses of glucose metabolism in glioma reveal the glioma-promoting effect of GALM. Front Cell Dev Biol. 2021; 9: 717182-717196.

[36] Yellajoshyula D, Pappas SS, Rogers AE, et al. THAP1 modulates oligodendrocyte maturation by regulating ECM degradation in lysosomes. Proc Natl Acad Sci USA. 2021; 118(31): e2100862118.

[37] Mori D, Shibata K, Yamasaki S. C-type lectin receptor dectin-2 binds to an endogenous protein beta-glucuronidase on dendritic cells. PLoS One. 2017; 12(1): e0169562.

[38] Lu H, Fermaintt CS, Cherepanova NA, et al. Mammalian STT3A/B oligosaccharyltransferases segregate N-glycosylation at the translocon from lipid-linked oligosaccharide hydrolysis. Proc Natl Acad Sci USA. 2018; 115(38): 9557-9562.

[39] Cherepanova NA, Venev SV, Leszyk JD, et al. Quantitative glycoproteomics reveals new classes of STT3A-and STT3B-dependent N-glycosylation sites. J Cell Biol. 2019; 218(8): 2782-2796.

[40] Sato T, Sako Y, Sho M, et al. STT3B-dependent posttranslational N-glycosylation as a surveillance system for secretory protein. Mol Cell. 2012; 47(1): 99-110.

[41] Liu J, Lu J, Li W. A comprehensive prognostic and immunological analysis of a six-gene signature associated with glycolysis and immune response in uveal melanoma. Front Immunol. 2021; 12: 738068-738082.

[42] Cob-Calan NN, Chi-Uluac LA, Ortiz-Chi F, et al. Molecular docking and dynamics simulation of protein beta-tubulin and antifungal cyclic lipopeptides. Molecules. 2019; 24(18): 3387–3397.

[43] Li W, Zhang S, Zhao Y, et al. Molecular docking and molecular dynamics simulation analyses of urea with ammoniated and ammoxidized lignin. J Mol Graph Model. 2017; 71: 58-69.

RIGHTS & PERMISSIONS

2025 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.