ROS1 mutations promote an immunosuppressive tumor microenvironment via MYC to confer immune evasion in head and neck cancer

Abstract

Aim: Immune checkpoint inhibitors (ICIs) have transformed cancer therapy; however, their efficacy in head and neck cancer (HNC) remains limited, with only a minority of patients achieving durable responses. Understanding the molecular mechanisms underlying ICI resistance in HNC is therefore crucial.

Methods: We conducted an integrative analysis of genomic, transcriptomic, and clinical data from 139 ICI-treated HNC patients (MSKCC cohort) and 502 treatment-naïve HNC cases (TCGA cohort). ROS1 mutation status, tumor mutational burden (TMB), neoantigen load, immune cell infiltration (via CIBERSORT), and immune-related gene expression were evaluated. Gene set enrichment analysis (GSEA) was performed to identify dysregulated pathways. Survival outcomes were assessed using Kaplan-Meier analysis and Cox regression, with statistical significance defined as P < 0.05.

Results: Patients harboring ROS1 mutations exhibited significantly poorer outcomes following ICI therapy, with shorter median overall survival [OS: 5.0 vs. 11.0 months, hazard ratio (HR) = 3.22, 95%CI: 1.26-8.19, P = 0.011] compared to ROS1 wild-type counterparts. Multivariate analysis confirmed ROS1 mutation as an independent predictor of poor OS in ICI-treated patients (HR = 4.78, 95%CI: 1.70-13.43, P = 0.003). In contrast, ROS1 mutations showed no prognostic significance in the treatment-naïve TCGA-HNC cohort (P = 0.26), confirming their role as a predictive (not prognostic) biomarker for ICI response. Interestingly, despite exhibiting higher TMB and neoantigen levels, ROS1-mutant patients showed inferior survival, underscoring the context-dependent limitations of TMB as a predictive biomarker. Mechanistically, ROS1-mutant tumors displayed an immunosuppressive tumor microenvironment characterized by diminished CD8⁺ T cell infiltration, attenuated interferon-γ signaling, and downregulation of immune-related genes (CXCL9, CXCL10, IFNG, PD-L1). GSEA revealed enrichment of MYC pathway activity in ROS1-mutant tumors, which suppressed antigen presentation and T cell activation pathways.

Conclusion:ROS1 mutations drive ICI resistance in HNC by promoting an immunosuppressive TME via MYC-mediated transcriptional reprogramming, impairing antigen presentation and T cell function. Incorporating ROS1 status into biomarker panels may improve patient stratification and guide combinatorial therapies targeting both immune evasion and oncogenic pathways.

Keywords

Head and neck cancer / ICI resistance / ROS1 mutation / tumor microenvironment

References

[1] Bray F,Sung H.Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J Clin2024;74:229-63

[2] Petrelli F,Paderno A.Treatment of primary tumor in metastatic head and neck carcinoma: a systematic review and meta-analysis.Oral Oncol2025;163:107248

[3] Szturz P,Guo Y.Treatment decision-making factors and sequencing in recurrent and/or metastatic squamous cell carcinoma of the head and neck.Cancer Treat Rev2025;135:102910

[4] Liu X,Varvares MA,Peng G.Immunotherapeutic strategies in head and neck cancer: challenges and opportunities.J Clin Invest2025;135:e188128 PMCID:PMC11996880

[5] Ferris RL,Fayette J.Nivolumab for recurrent squamous-cell carcinoma of the head and neck.N Engl J Med2016;375:1856-67 PMCID:PMC5564292

[6] Seiwert TY,Mehra R.Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial.Lancet Oncol2016;17:956-65

[7] Burtness B, Harrington KJ, Greil R, et al; KEYNOTE-048 Investigators. 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;394:1915-28.

[8] Johnson DE,Leemans CR,Bauman JE.Head and neck squamous cell carcinoma.Nat Rev Dis Primers2020;6:224 PMCID:PMC7944998

[9] Samstein RM,Shoushtari AN.Tumor mutational load predicts survival after immunotherapy across multiple cancer types.Nat Genet2019;51:202-6 PMCID:PMC6365097

[10] Sharma P.The future of immune checkpoint therapy.Science2015;348:56-61

[11] Rizvi NA,Snyder A.Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer.Science2015;348:124-8 PMCID:PMC4993154

[12] Snyder A,Merghoub T.Genetic basis for clinical response to CTLA-4 blockade in melanoma.N Engl J Med2014;371:2189-99 PMCID:PMC4315319

[13] Drilon A, Siena S, Dziadziuszko R, et al; trial investigators. Entrectinib in ROS1 fusion-positive non-small-cell lung cancer: integrated analysis of three phase 1-2 trials. Lancet Oncol. 2020;21:261-70. PMCID:PMC7811790

[14] Waliany S.Taletrectinib: TRUST in the continued evolution of treatments for ROS1 fusion-positive lung cancer.J Clin Oncol2024;42:2622-7 PMCID:PMC11286343

[15] Li W,Yang N.Efficacy and safety of taletrectinib in Chinese patients with ROS1+ non–small cell lung cancer: the Phase II TRUST-I study.J Clin Oncol2024;42:2660-70 PMCID:PMC11272140

[16] Canon J,Saiki AY.The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity.Nature2019;575:217-23

[17] Gainor JF,Sequist LV.EGFR mutations and ALK rearrangements are associated with low response rates to PD-1 pathway blockade in non-small cell lung cancer: a retrospective analysis.Clin Cancer Res2016;22:4585-93 PMCID:PMC5026567

[18] Ambrosini M,Manca P.Immune checkpoint inhibitors for POLE or POLD1 proofreading-deficient metastatic colorectal cancer.Ann Oncol2024;35:643-55

[19] Hoadley KA, Yau C, Hinoue T, et al; Cancer Genome Atlas Network. Cell-of-origin patterns dominate the molecular classification of 10,000 tumors from 33 types of cancer. Cell. 2018;173:291-304.e6. PMCID:PMC5957518

[20] Angelova M,Hackl H.Characterization of the immunophenotypes and antigenomes of colorectal cancers reveals distinct tumor escape mechanisms and novel targets for immunotherapy.Genome Biol2015;16:64 PMCID:PMC4377852

[21] Nielsen M,Blicher T.NetMHCpan, a method for quantitative predictions of peptide binding to any HLA-A and -B locus protein of known sequence.PLoS One2007;2:e796 PMCID:PMC1949492

[22] Thorsson V, Gibbs DL, Brown SD, et al; Cancer Genome Atlas Research Network. The immune landscape of cancer. Immunity. 2018;48:812-30.e14. PMCID:PMC5982584

[23] Love MI,Anders S.Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol2014;15:550 PMCID:PMC4302049

[24] Danaher P,Dennis L.Gene expression markers of tumor infiltrating leukocytes.J Immunother Cancer2017;5:18 PMCID:PMC5319024

[25] Yu G,Han Y.clusterProfiler: an R package for comparing biological themes among gene clusters.OMICS2012;16:284-7 PMCID:PMC3339379

[26] Drilon A,Iyer S,Keddy C.ROS1-dependent cancers - biology, diagnostics and therapeutics.Nat Rev Clin Oncol2021;18:35-55 PMCID:PMC8830365

[27] Kato S,Walavalkar V,Sharabi A.Hyperprogressors after immunotherapy: analysis of genomic alterations associated with accelerated growth rate.Clin Cancer Res2017;23:4242-50 PMCID:PMC5647162

[28] Zaretsky JM,Shin DS.Mutations associated with acquired resistance to PD-1 blockade in melanoma.N Engl J Med2016;375:819-29 PMCID:PMC5007206

[29] Casey SC,Li Y.MYC regulates the antitumor immune response through CD47 and PD-L1.Science2016;352:227-31 PMCID:PMC4940030

[30] Fan Y,Li Y.Galectin-3 cooperates with CD47 to suppress phagocytosis and T-cell immunity in gastric cancer peritoneal metastases.Cancer Res2023;83:3726-38 PMCID:PMC10843008

[31] Krenz B,Ade CP.MYC- and MIZ1-dependent vesicular transport of double-strand RNA controls immune evasion in pancreatic ductal adenocarcinoma.Cancer Res2021;81:4242-56 PMCID:PMC7611539

[32] Dhanasekaran R,Park J.MYC overexpression drives immune evasion in hepatocellular carcinoma that is reversible through restoration of proinflammatory macrophages.Cancer Res2023;83:626-40 PMCID:PMC9931653

[33] Dou Y, Katsnelson L, Gritsenko MA, et al; Clinical Proteomic Tumor Analysis Consortium. Proteogenomic insights suggest druggable pathways in endometrial carcinoma. Cancer Cell. 2023;41:1586-605.e15. PMCID:PMC10631452

[34] Spranger S,Gajewski TF.Melanoma-intrinsic β-catenin signalling prevents anti-tumour immunity.Nature2015;523:231-5

[35] Gao J,Zhao H.Loss of IFN-γ pathway genes in tumor cells as a mechanism of resistance to anti-CTLA-4 therapy.Cell2016;167:397-404.e9 PMCID:PMC5088716

[36] Li T,Xu J.TGFBR2 mutation predicts resistance to immune checkpoint inhibitors in patients with non-small cell lung cancer.Ther Adv Med Oncol2021;13:17588359211038477 PMCID:PMC8366138

[37] Xiao W,Huang T.TP53 mutation as potential negative predictor for response of anti-CTLA-4 therapy in metastatic melanoma.EBioMedicine2018;32:119-24 PMCID:PMC6020711

[38] Yang L,Liu X.DYNC2H1 mutation as a potential predictive biomarker for immune checkpoint inhibitor efficacy in NSCLC and melanoma.Invest New Drugs2025;43:199-213

[39] Kortlever RM,Wilson CH.Myc cooperates with Ras by programming inflammation and immune suppression.Cell2017;171:1301-15.e14 PMCID:PMC5720393

[40] Dienstmann R,Guinney J,Tejpar S.Consensus molecular subtypes and the evolution of precision medicine in colorectal cancer.Nat Rev Cancer2017;17:79-92