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Acquired resistance to anti-MAPK targeted therapy confers an immune-evasive tumor microenvironment and cross-resistance to immunotherapy in melanoma

Nature Cancer volume 2, pages 693–708 (2021)Cite this article

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Abstract

How targeted therapies and immunotherapies shape tumors, and thereby influence subsequent therapeutic responses, is poorly understood. In the present study, we show, in melanoma patients and mouse models, that when tumors relapse after targeted therapy with MAPK pathway inhibitors, they are cross-resistant to immunotherapies, despite the different modes of action of these therapies. We find that cross-resistance is mediated by a cancer cell–instructed, immunosuppressive tumor microenvironment that lacks functional CD103+ dendritic cells, precluding an effective T cell response. Restoring the numbers and functionality of CD103+ dendritic cells can re-sensitize cross-resistant tumors to immunotherapy. Cross-resistance does not arise from selective pressure of an immune response during evolution of resistance, but from the MAPK pathway, which not only is reactivated, but also exhibits an increased transcriptional output that drives immune evasion. Our work provides mechanistic evidence for cross-resistance between two unrelated therapies, and a scientific rationale for treating patients with immunotherapy before they acquire resistance to targeted therapy.

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Fig. 1: TT resistance induces cross-resistance to immunotherapy.
Fig. 2: The TME in RTT tumors precludes a functional T cell response.
Fig. 3: The TME of RTT tumors shows reduced and dysfunctional CD103+ DCs.
Fig. 4: Restoration of a functional CD103+ DC compartment restores immunotherapy response.
Fig. 5: Cross-resistance is acquired during evolution of MAPKi resistance and is directly linked to a cell-intrinsic signaling program.
Fig. 6: The RTT signaling program predicts immunotherapy response in patients and is controlled by MAPK signaling.
Fig. 7: Inhibition of the reactivated MAPK pathway restores sensitivity to immunotherapy in RTT tumors.

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Data availability

Gene expression data (RNA-seq, Quant-seq, Smart-seq and SLAM-seq) and ATAC-seq data that support the findings of the present study have been deposited in the Gene Expression Omnibus under accession no. GSE132443. The human melanoma transcriptomic data were derived from the TCGA network (loaded from cBio portal, July 2019). Gene expression data for patients receiving checkpoint blockade are available from Gide et al. (accession no. PRJEB23709) and Liu et al. (dbGAP, accession no. phs000452.v3.p1)43,44. Source data for Extended Data Figs. 2b,e, 6c, 8e and 9d have been provided as Source data files. All other data supporting the findings of the present study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank the members of A.C.O.’s laboratory and J.Z.’s laboratory (Institute of Molecular Pathology (IMP)) for discussions, S. Rieser and M. Aichinger (IMP) for plasmid templates, the parental CT26 cell line, Lenti-X cells and mouse lines, E. Crespo-Rodriguez (ICR London) for support with set-up of the XRT experiments, J. Moon (University of Michigan) for providing OT-1 Luc Thy1.1 mice, M. Bosenberg for providing the parental (NTT) Braf and Braf/Pten cell lines, T. Burkhard and K. Froussios for next-generation sequencing analysis, P. Garin-Chesa, A. Pauli, F. Holstein and S. Cronin for proofreading the manuscript, and L. Formenti for contributing the western blot in Extended Data Fig. 8e. This work was supported by grants from the European Research Council (‘CombaTCancer’, grant no. 759590 to A.O.) and the Viennese Science and Technology Fund (grant no. LS-16-063 to A.O. and T.W.). S.V. is supported by the Medical Research Council (MC_UU_12022/7). This work was also supported by a National Health and Medical Research Council of Australia (NHMRC) Program grant (to R.A.S. and G.V.L.). G.V.L., R.A.S. and J.S.W. are supported by NHMRC Fellowships, and G.V.L. is also supported by the University of Sydney Medical Foundation and Melanoma Institute Australia. We also thank the Ainsworth Foundation and colleagues at Melanoma Institute Australia, Mater Hospital, the Royal Prince Alfred Hospital, Royal North Shore Hospital, Westmead Hospital and NSW Health Pathology for their support.

Author information

Author notes

  1. Mario Kuttke

    Present address: Institute of Vascular Biology and Thrombosis Research, Medical University of Vienna, Vienna, Austria

Authors and Affiliations

  1. Research Institute of Molecular Pathology, Vienna Biocenter, Vienna, Austria

    Lisa Haas, Anais Elewaut, Christian Umkehrer, Lukas Leiendecker, Izabela Krecioch, Maria Novatchkova, Mario Kuttke, Tobias Neumann, Johannes Zuber & Anna C. Obenauf

  2. Department of Oncology, Lausanne University Hospital, Lausanne, Switzerland

    Camille L. Gerard, Michel A. Cuendet & Olivier Michielin

  3. Institute of Cancer Research, London, UK

    Malin Pedersen, Harriet Witthock & Kevin J. Harrington

  4. Institute of Molecular Biotechnology, Vienna Biocenter, Vienna, Austria

    David Hoffmann

  5. Melanoma Institute Australia, University of Sydney, Sydney, New South Wales, Australia

    Ines Pires da Silva, Richard A. Scolyer, Georgina V. Long & James S. Wilmott

  6. Charles Perkins Centre, University of Sydney, Sydney, New South Wales, Australia

    Ines Pires da Silva, Richard A. Scolyer, Georgina V. Long & James S. Wilmott

  7. Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia

    Ines Pires da Silva, Richard A. Scolyer, Georgina V. Long & James S. Wilmott

  8. Molecular Modeling Group, Swiss Institute of Bioinformatics, UNIL Sorge, Lausanne, Switzerland

    Michel A. Cuendet & Olivier Michielin

  9. Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA

    Michel A. Cuendet

  10. Boehringer Ingelheim RCV GmBH & Co KG, Vienna, Austria

    Sebastian Carotta

  11. Royal Prince Alfred Hospital & NSW Health Pathology, Sydney, New South Wales, Australia

    Richard A. Scolyer

  12. Royal North Shore Hospital, Sydney, New South Wales, Australia

    Georgina V. Long

  13. Mater Hospital, North Sydney, New South Wales, Australia

    Georgina V. Long

  14. MRC Cancer Unit, University of Cambridge, Cambridge, UK

    Sakari Vanharanta

  15. Department of Dermatology, Medical University of Vienna, Vienna, Austria

    Thomas Wiesner

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Contributions

L.H. and A.C.O. conceived the study, designed the experiments and interpreted the results. A.C.O. supervised the study. L.H. developed experimental tools, performed in vitro experiments, in vivo treatment studies, flow-cytometry analysis, gene expression profiling and parts of the computational analysis, and analyzed the data. A.E. validated in vivo treatment studies in independent experiments, performed the in vitro co-culture in Figs. 3i, 4f and 7f, and performed the ATAC-seq library prep. C.U. performed large parts of the SLAM-seq experiment (Extended Data Fig. 9d–h) and generated matched CaTCH clones (Fig. 5f and Extended Data Fig. 8e). I.K. performed western blotting and IF staining (including quantifications) and helped with mouse colony maintenance. M.P. and H.W. performed experiments involving focal radiation. M.K. helped with flow-cytometry studies and in vivo studies (including CT26 flow-cytometry characterization displayed in Extended Data Fig. 6k,m). D.H. contributed to experimental design, computational analysis, in vivo studies and data interpretation. C.L.G. collected the patient information for the retrospective analysis in Fig. 1a,b. C.L.G., M.A.C. and O.V. analyzed and interpreted the collected patient data. I.S. and G.V.L. provided updated survival information for the Gide et al.43 dataset. J.S.W., G.V.L. and R.A.S. analyzed and interpreted VECTRA image analysis data of melanoma biopsies and related clinical data of patients treated with MAPKi therapies, and provided matched patient biopsies for the CLEC9a staining for Fig. 3g. L.L., M.N. and T.N. analyzed gene expression data, SLAM-seq data and whole-exome sequencing data. L.L. analyzed ATAC-seq data and contributed to experimental design and data interpretation. J.Z. provided conceptual input for experiment design and data interpretation of SLAM-seq. S.V. analyzed RNA-seq data, generated the ccIES and probed it in the RNA-seq data of TCGA and published melanoma patient datasets. S.C. and K.J.H. provided conceptual input to the experiment design and data interpretation. T.W. provided clinical expertize and input to experiment design, interpretation and presentation. L.H., T.W. and A.C.O. wrote the manuscript, and all authors read and approved it.

Corresponding author

Correspondence to Anna C. Obenauf.

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Competing interests

O.M. received fees for advisory roles from BMS, MSD, GSK, Novartis, Roche, Pierre-Fabre and Amgen and research funding from BMS, MSD and Amgen. R.A.S received fees for professional services from Qbiotics Group Limited, Novartis Pharma AG, MSD Sharp & Dohme (Australia), NeraCare, AMGEN Inc., Bristol-Myers Squibb, Novartis Pharmaceuticals Australia Pty Limited, Myriad Genetics GmbH and GlaxoSmithKline Australia. G.V.L. is consultant adviser for Aduro Biotech Inc, Amgen Inc., Array Biopharma Inc., Boehringer Ingelheim International GmbH, Bristol-Myers Squibb, Evaxion Biotech A/S, Hexel AG, Highlight Therapeutics S.L., Merck Sharpe & Dohme, Novartis Pharma AG, OncoSec, Pierre Fabre, QBiotics Group Limited, Regeneron Pharmaceuticals Inc., SkylineDX B.V. and Specialised Therapeutics Australia Pty Ltd. S.C. is an employee of Boehringer Ingelheim GmbH. J.Z. is a founder, shareholder and scientific adviser for Quantro Therapeutics GmbH. J.Z., A.C.O. and their labs received research support and funding from Boehringer Ingelheim. L.H. is currently employed as an Associate Editor of Nature Cancer. She was not involved in the decision-making process for the manuscript and did not have access to confidential information pertaining to peer review and editorial process. The other authors declare no conflicts of interest.

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Extended data

Extended Data Fig. 1 Targeted therapy resistant patients display reduced T cell infiltrate and cross-resistance to immunotherapy.

a, Overall survival (OS) of metastatic melanoma patients in the Lausanne Patient Cohort (Supplementary Table 1) receiving immunotherapy with checkpoint inhibitors (n = 54 patients). NTT, targeted therapy (TT) naïve patients (n = 38); RTT, TT (RAFi or RAFi/MEKi) resistant patients (n = 16). b, Summary of responses to immunotherapy in NTT and RTT patients in published patient cohorts (ORR = overall response rate, PFS = progression-free survival). c, CD3+CD8+ T cells in patient-matched NTT and RTT melanoma biopsies [scale bar pre-treatment: 2 mm (left), 200 µm (right), 50 µm (zoom-in); scale bar post progression: 5 mm (left), 200 µm (right), 50 µm (zoom-in)]. Experiment performed once on 10 matched biopsies. d, CD8+CD39+CD103+ T cells in patient-matched NTT and RTT melanoma biopsies [scale bar: 200 µm, 50 µm (zoom-in)]. e, Quantification of tumour reactive (CD8+CD39+CD103+) T cells in patient-matched NTT and RTT melanoma biopsies, assessed by IF staining (n = 10 patients, Supplementary Table 2). Data analysis (a) two-sided log-rank (Mantel-Cox) test.

Extended Data Fig. 2 The Braf melanoma model responds to checkpoint inhibition in the NTT state, but is resistant in the RTT state.

a, Proliferation fold change (FC) of Braf melanoma cells after 72 h at indicated drug conditions. Line indicating FC in proliferation of NTT cells on lowest drug condition (n = RAFi: technical triplicates; RAFi/MEKi technical duplicates), (drug concentrations: RAFi: DMSO CTRL, 100 nM, 300 nM, 1 µM, 3 µM; RAFi/MEKi: DMSO CTRL, 10 nM/3 nM, 30 nM/10 nM, 100 nM/30 nM, 300 nM/100 nM). b, pERK status in NTT and RTT Braf melanoma cells, 1-hour post drug exposure. Experiment performed twice; representative example shown. c, Treatment response of subcutaneously injected Braf/Pten melanoma (CTRL, n = 4 tumours; other groups, n = 6 tumours) continuously treated with TT; arrow indicating start of therapy. Experiment repeated 5 times; representative example shown. d, Proliferation FC of Braf/Pten melanoma cells after 72 h at indicated drug conditions. Line indicating FC in proliferation of NTT cells on lowest drug condition (n = RAFi: technical triplicates; RAFi/MEKi technical duplicates), (drug concentrations: RAFi: DMSO CTRL, 100 nM, 300 nM, 1 µM, 3 µM; RAFi/MEKi: DMSO CTRL, 10 nM/3 nM, 30 nM/10 nM, 100 nM/30 nM, 300 nM/100 nM). e, pERK status in NTT and RTT Braf/Pten melanoma cells, 1-hour post drug exposure. Experiment performed twice; representative example shown. f, Gating strategy highlighting successful CD8 T cell depletion in blood of mice treated with anti-CD8 versus CTRL antibody. g, Treatment response to anti-PD-1/CTLA-4 in combination with CD8 depletion in Braf melanoma. (CTRL, n = 6; all other groups, n = 10 tumours). Black arrows indicate anti-PD-1/CTLA-4 administration and blue arrows administration of CD8 depletion antibody. Experiment performed once. P-value: ** 0.0012, ns 0.9970. h, Spider plots indicating individual tumour growth curves of NTT and RTT Braf melanoma receiving checkpoint blockade (CTRL n = 6 tumours; anti-PD-1/CTLA-4, n = 8 tumours). Experiment repeated 9 times; representative example shown. i, Treatment response to anti-PD-1/CTLA-4 of NTT and RTT Braf melanoma (CTRL, RAFi n = 6 tumours; anti-PD-1/CTLA-4 ± RAFi, n = 10 tumours); arrows indicate therapy administration. RTT mice continuously treated with RAFi (5 mg/kg). Experiment performed once. P-value: **** 8.4e-6, ns 0.9924. Data in (a, c, d, g, i) displayed as mean ± SEM. Data analysis (g, i) two-way ANOVA. ** P < 0.01, **** P < 0.0001, ns = non-significant.

Source data

Extended Data Fig. 3 Cross-resistance is mediated via the tumour microenvironment.

a, Treatment response to immunotherapy in NTT Braf/Pten melanoma tumour bearing mice, black arrows indicate therapy administration (NTT CTRL, n = 2; NTT anti-PD-1/CTLA-4, n = 4 mice). Experiment performed once. b, Generation of OVA antigen-expressing NTT and RTT Braf/Pten cell lines using indicated expression vector (top) and quantification of processed MHC-I loaded ovalbumin peptide (SIINFEKL) by flow-cytometry (bottom). c, Spider Plots indicating individual tumour growth curves of NTT and RTT Braf/PtenOVA tumours receiving ACT (CTRL n = 3; ACT, n = 5 tumours). Experiment performed 7 times; representative example shown. d, Treatment response to ACT in RAFi/MEKi RTT Braf/PtenOVA tumours; arrow indicating day of ACT (NTT, RAFi/MEKi RTT CTRL, n = 3 mice; NTT, RAFi/MEKi RTT ACT, n = 5 mice, P-value: **** 3.9E-11, ns 0.6) (left) and infiltration of OT-1Luc T cells measured by bioluminescence imaging (BLI) at indicated days (all groups, n = 5 tumours P-value: * 0.021, ** 0.0079, ns 0.0952) (right). Experiment performed twice with two independent clones; representative example shown. e, f, Treatment response to ACT in Braf/PtenOVA tumours, RTT tumours assessed (e) off RAFi for the entire experiment [P-value: **** 1.7E-6, ns 0.33] and (f) under continuous exposure to RAFi (10 mg/kg) [P-value: ****5.8E-8, ns 0.35]; arrow indicating day of ACT (NTT, RTT CTRL, n = 3 mice NTT, RTT ACT, n = 5 mice). g, Tumour infiltration of OT-1Luc T cells into NTT Braf/PtenOVA tumours and RTT Braf/PtenOVA tumours ± RAFi (10 mg/kg), (all groups, n = 3 mice). Experiment performed once. P-value: *** 0.0004, *** 0.0003. h, i Principal Component Analysis (PCA) plots displaying top 500 most variable genes for (h) Braf/Pten and (i) Braf melanoma treated with IFN-γ. j, MHC-I surface expression of NTT and RTT Braf melanoma cell lines (baseline and 24 h post 10 ng/ml IFN-γ exposure). Experiment performed 3 times; representative example shown. k, Gene expression changes in NTT and RTT Braf melanoma cell lines treated with IFN-γ. Correlation between genes deregulated in NTT (x-Axis) and RTT (y-Axis) cell lines (P < 0.05), dots display individual genes. P-value: <1E-15. Supplementary Table 3. l, BrafOVA melanoma cell viability after 24 h of co-culture in in vitro killing assay using pre-activated OT-1 T cells at indicated effector:target ratios (all groups, n = 2 replicates). Experiment performed twice; representative example shown. m, Treatment response to ACT in tumours consisting of NTT and RTT Braf/PtenOVA cell lines at indicated ratios; arrow indicating day of ACT (0.05% NTT/RTT CTRL, n = 4; ACT, n = 5; 0.05% RTT/NTT CTRL, n = 4; ACT, n = 4 tumours). Experiment performed twice; representative example shown. n, Scheme outlining experiments to test antigen-specificity of T cell killing in vivo (left) and BLI signal at day 6 post ACT for tumours containing 0.05% OVA+ Luc+ or 0.05% OVA– Luc+ CTRL cells (NTT/ OVA+ n = 4; NTT/ OVA- CTRL n = 5 tumours) (right). Data in (a, d-g, m, n) displayed as mean ± SEM. Data analysis (d-f) two-way ANOVA (d) two-tailed unpaired t-test (g) one-way ANOVA (k) two-sided Pearson correlation. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = non-significant.

Extended Data Fig. 4 The tumour microenvironment of RTT tumours is strongly remodelled.

a, T cell influx into NTT and RTT Braf melanoma, assessed by IF (scale bar 100 µm and 20 µm). Experiment performed twice; representative image shown. b, T cell quantified separately at tumour margin and centre (n = 3 tumours per condition, NTT n = 15, RTT n = 16 ROI). P-value: * 0.0204, ns 0.0756. c, PCA plot displaying top 500 most variable genes for T cells sorted from NTT and RTT Braf/PtenOVA tumours. d, e Gating strategy highlighting identification of CD3+ CD8+ T cells, CD103+ CD11c+ DCs and CD11b+ GR-1+ suppressive myeloid cells. f, Suppressive myeloid cells in Braf melanoma, assessed by flow cytometry (n = 7 tumours per condition). P-value: * 0.023. Experiment performed 3 times; data represents pool of 2 experiments. g, CD103+ DCs in Braf melanoma, assessed by flow cytometry (n = 8 tumours per condition). P-value: *** 0.0005. Experiment performed 3 times; data represents pool of 2 experiments. h, Gating strategy highlighting the identification of CD103 DCs in an alternative gating strategy (Lineage negative (CD11b−,Gr-1-, NK1.1-, CD3−, B220−, F480−) MHCII + CD103+ cells). i, Quantification of CD103+MHCII+ DCs with alternative gating strategy. (Braf/PtenOVA melanoma both groups, n = 4 tumours; Braf/Pten melanoma NTT = 5 tumours, RTT = 8 tumours). Experiment performed once. P-value: * 0.0185, ** 0.0083. j, k CD103+MHCII+ DCs in NTT and RTT Braf melanoma, assessed by IF staining in (j) displayed as a representative picture (scale bar 100 µm and 20 µm) (Experiment performed twice) and (k) quantified separately at tumour margin and centre (n = 3 tumours per condition, all groups 15 ROIs). P-value: *** 0.0002, *** 0.0010. l, CD103+MHCII+ DCs in NTT and RTT Braf/PtenOVA melanoma quantified separately at tumour margin and centre (n = 2 tumours per condition, all groups 10 ROI, except NTT margin n = 11). P-value: **** 5.7E-6, 1.1E-5. Data in (b, f, g, i, k, l) displayed as mean ± SEM and analysed by two-tailed unpaired t–test with Welch correction for unequal variance or with Mann-Whitney-U-test if not normal distributed. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = non-significant.

Extended Data Fig. 5 Modulation of the myeloid cell compartment restores immunotherapy response.

a, DC maturation score comparing gene expression profiles of RTT vs. NTT tumours (Braf/Pten NTT, Braf NTT, BRAF RTT n = 3; Braf/Pten RTT n = 4 tumours). b, Mean fluorescence intensity (MFI) of maturation markers on CD103+ DCs from NTT and RTT Braf/Pten melanoma (NTT, n = 5; RTT, n = 6 tumours), assessed by flow cytometry. Experiment performed twice with independent cell lines; representative example shown. P-value: **** 6.9E-5, ** 0.0043, * 0.0149. c, PCA plot displaying top 500 most variable genes for CD103+ DCs sorted from NTT and RTT ± Poly I:C Braf/PtenOVA tumours. d, GSEA of IFN-alpha response in CD103+ DCs sorted from RTT vs. NTT Braf/PtenOVA melanoma. e, Quantification of T cell proliferation based on CFSE dilution in DC co-culture assays displayed in Fig. 3i (n = 4 tumours per condition). P-value: * 0.029. f, Scheme outlining experiment to assess impact of depleting suppressive myeloid cells on ACT in Braf/PtenOVA tumours. g, Depletion of Ly6C+CD11b+ and Ly6G+CD11b+ cells in blood 3 days post anti-GR-1 administration. h, Tumour infiltration of effector OT-1Luc T cells measured by BLI at 24 h post ACT in Braf/PtenOVA tumour bearing mice treated with Isotype CTRL or anti-GR-1 antibody (n = 9, 9, 8, 8 mice from left to right). Experiment performed 3 times; representative example shown. P-value: ns 0.53, * 0.0128. i, Treatment response of Braf/PtenOVA tumours treated with ACT or anti-GR-1 plus ACT (NTT ACT + ISO, n = 6; RTT ACT + ISO, n = 4; NTT ACT + anti-GR-1, n = 6; RTT ACT + anti-GR-1, n = 4 mice). j, DC maturation in Poly I:C injected Braf/PtenOVA tumours assessed by CD40 expression using flow cytometry. k, CD103+ DC influx in RTT tumours overexpressing FLT3L, assessed by flow cytometry (n = 3 tumours). P-value: * 0.0118. l, Survival in response to ACT ± Poly I:C ± FLT3L in RTT Braf/PtenOVA tumours. (RTT CTRL, RTT + ACT, RTT FLT3L + ACT, n = 3; RTT + ACT + Poly I:C, RTT FLT3L + ACT + Poly I:C, n = 4 mice). Experiment performed twice; representative example shown. P-value: ** 0.0100. m, Treatment response of RTT Braf melanoma in WT mice (left) and BATF3-/- mice (right) treated with indicated therapies; black arrows indicate anti-PD-1/CTLA-4 administration, red arrows indicate Poly I:C injection (CTRL, anti-PD-1/CTLA-4, Poly I:C, n = 6 tumours; Poly I:C + anti-PD-1/CTLA-4, n = 4 tumours). Experiment performed twice; representative example shown. P-value: ** 0.01, ns 0.1931. Data in (b, e, h, k, m) is displayed as mean ± SEM. Data analysis (b, h, k) two-tailed unpaired t-test with Welch correction for unequal variance or with Mann-Whitney-U-test if not normal distributed l two-sided log-rank test (Mantel-Cox) test (m) two-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = non-significant.

Extended Data Fig. 6 The CT26 colon carcinoma model displays cross-resistance and an immune-evasive TME.

a, Treatment response of subcutaneously injected CT26 colon carcinoma (CTRL or MEKi, n = 8 tumours) continuously treated with TT; arrow indicating start of therapy. Experiment performed twice; representative example shown. b, Proliferation FC in CT26 colon carcinoma cell lines after 72 h at indicated drug conditions. Line indicating FC in proliferation of NTT cells on lowest drug condition (n = technical triplicates) (drug concentration: DMSO CTRL, 10 nM, 30 nM, 100 nM, 300 nM MEKi). c, pERK status in NTT and RTT CT26 colon carcinoma cell lines, 1-hour post drug exposure. Experiment performed twice; representative example shown. d, Treatment response in mice bearing NTT and RTT CT26 (KrasG12D/G12D Cdkn2a -/-) tumours (NTT and RTT CTRL, n = 6; NTT and RTT anti-PD-1, n = 14 tumours) treated with anti-PD-1; arrows indicate therapy administration. Experiment performed 5 times; representative example shown. P-value: **** 1.5E-8, ns 0.2838. e, MHC-I surface expression of NTT and RTT cell lines (baseline and 24 h post 10 ng/ml IFN-γ exposure). Experiment performed 3 times; representative example shown. f, Gene expression changes in NTT and RTT CT26 colon carcinoma cell lines treated with IFN-γ. Correlation between genes deregulated in NTT (x-Axis) and RTT (y-Axis) cell lines (P < 0.05), dots display individual genes. P-value: <1E-15. g, PCA plot displaying top 500 most variable genes for CT26 colon carcinoma cell lines treated with IFN-γ. h, i T cells in untreated NTT and RTT CT26 colon carcinoma tumours assessed by IF staining and (h) quantified separately at tumour margin and centre (n = 3 tumours per condition; all 15 ROI, except RTT centre n = 16) and (i) displayed as a representative picture (scale bar 100 µm and 20 µm). Experiment performed twice. P-value: ** 0.0017, **** 3E-5. j, CD103+ DCs in untreated NTT and RTT CT26 colon carcinoma tumours assessed by IF staining and quantified separately at tumour margin and centre (n = 3 tumours per condition, all 15 ROI). P-value: *** 0.0002, * 0.046. k, CD103+ DC infiltration NTT and RTT tumours of CT26 colon carcinoma, assessed by flow cytometry (n = 16, 14 tumours each). Data represents pool of 2 independent experiments. P-value: * 0.016. l, CD103+ DC infiltration NTT and RTT tumours of CT26 colon carcinoma, alternative gating strategy displayed in Extended Data Fig. 4h (n = 5, 8 tumours). P-value: ** 0.0081. m, Suppressive myeloid cell infiltration in NTT and RTT tumours of CT26 colon carcinoma, assessed by flow cytometry (n = 16, 14 tumours each). Data represents pool of 2 independent experiments. P-value: **** 7E-6. n, MFI of indicated maturation markers on CD103+ DCs from NTT and RTT CT26 colon carcinoma (NTT, n = 6; RTT, n = 8 tumours) assessed by flow cytometry. P-value: * 0.0426, ns 0.1419, * 0.0293. Experiment performed once. Data in (b, d, h, j-n) displayed as mean ± SEM. Data analysis (d) two-way ANOVA (f) two-sided Pearson correlation (h, j-n) two-tailed unpaired t–test with Welch correction for unequal variance or with Mann-Whitney-U-test if not normal distributed. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = non-significant.

Source data

Extended Data Fig. 7 Modulation of the CD103+ DC compartment restores immunotherapy response in RTT CT26 colon carcinoma.

a, Scheme outlining experiment to assess impact of maturation (intratumoural Poly I:C) and expansion (FLT3L overexpression from tumour cells) of DCs on anti-PD-1 treatment in mice bearing RTT CT26 colon carcinoma (left) and survival curve of mice (right, CTRL, n = 3; all other groups, n = 5 mice). Experiment performed twice; representative example shown. P-value: ** 0.0067. b, Treatment response of RTT CT26 colon carcinoma to anti-PD-1 (day 6, 9, 12) in combination with intratumoural Poly I:C injection (day 5, 9, 12) for intratumoural (left) or contralateral tumour. (CTRL, n = 8, anti-PD-1 n = 10; contralateral and intratumoural, n = 5 tumours each). c, Influx of H2-LD MuLV gp70 specific T cells into NTT and RTT CT26 colon carcinoma treated with anti-PD-1 and Poly I:C (injected and contralateral tumour displayed separately) (NTT CTRL, n = 4; RTT CTRL, n = 3; RTT anti-PD-1, n = 4; anti-PD-1 + Poly I:C intratumoural, anti-PD-1 + Poly I:C contralateral, n = 5 tumours). Experiment performed once. P-value: all ns. d, Gating strategy highlighting the identification of gp70 Tetramer positive T cells. e, Treatment response to anti-PD-1 (day 6, 9, 12) in combination with intratumoural Poly I:C injection (day 5, 9, 12) ± CD8 depletion (day 3, 5, 10, 14), injected and contralateral tumour displayed separately. (CTRL, anti-PD-1, anti-PD-1 + Poly I:C + CD8 depletion n = 6; anti-PD-1 + Poly I:C, n = 7 mice). Experiment performed once. P-value: **** 1.9E-6, ****2.5 E-5, ns 0.9989. f, Scheme outlining experiment to assess impact of focal radiation ± anti-PD-1 ± FLTL3L in mice bearing RTT CT26 colon carcinoma (left) and survival curve of mice treated with indicated therapies (CTRL, n = 9; anti-PD-1, n = 8; XRT + anti-PD-1, n = 8; XRT + anti-PD-1 + FLT3L, n = 8 mice). Experiment performed once. P-value: *** 0.0006, ** 0.0389. g, Treatment response of RTT CT26 colon carcinoma to anti-PD-1 (day 15, 18, 21, 24) in combination with focal radiation (9 Gy, day 14) and FLT3L administration (10 consecutive doses, initiated on day 7). Number of responding mice indicated in graph. Data in (b, c, e) displayed as mean ± SEM. Data analysis (a, f) two-sided log rank (Mantel-Cox) test (e) two-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = non-significant.

Extended Data Fig. 8 Cross-resistance to immunotherapy is cell intrinsic, acquired during resistance formation and specific to MAPK pathway inhibition.

a, Active response to RAFi in NTT Braf/Pten tumours (7 doses) and resistance formation upon 27 doses (CTRL, n = 6; 7 doses n = 8, 27 doses n = 10 tumours). b, Characterization of suppressive myeloid cells, T cells and CD103+DCs in Braf/Pten tumours actively responding to RAFi (7 doses) and in relapsing tumours, fully resistant to RAFi (27 doses) (n = 8 tumours per group; except CD3+ 7 doses, n = 7; CD3+ 27 doses, n = 9; CD11b+ Gr-1, CD103+ 27 doses, n = 10). Experiment performed twice; representative example shown. P-value top row: ** 0.0011, ** 0.0085, ** 0.0014; bottom row: ns 0.08, ** 0.0018, *** 0.0003. c, Proliferation FC in Braf/Pten and Braf melanoma cell lines (made resistant to TT in vitro) after 72 h at indicated drug conditions. Line indicating FC in proliferation of NTT cells on lowest drug condition (n = technical triplicates), (drug concentration: DMSO CTRL, 100 nM, 300 nM, 1 µM, 3 µM RAFi). d, Proliferation FC of Braf melanoma cell lines after 72 h in indicated drug conditions of NTT and NTT-Dacarbazine cell lines (n = technical duplicates), (drug concentration: CTRL, 10 µg, 50 µg, 100 µg, 500 µg Dacarbazine). e, pERK status in CaTCH-isolated NTT and RTT Braf/Pten cell lines, 1-hour post drug exposure. Experiment performed twice; representative example shown. f, Treatment response to ACT in matched CaTCH isolated NTT and RTT Braf/PtenOVA tumours (CTRL, n = 3 mice, ACT, n = 5 mice). Experiment performed twice; representative example shown. P-value: **** 3E-5, ns 0.9871. g, h PCA plot displaying top 500 most variable genes for (g) Braf/Pten and (h) Braf melanoma tumours. i, Expression of genes comprising the ccIES in sorted NTT and RTT Braf/PtenOVA melanoma cells (left, tumours were not exposed to RAFi) (all groups n = 3 tumours) and in sorted RAFi/MEKi RTT melanoma cells (all groups n = 3 tumours) (right). j, Overall survival stratified based on ccIES expression in TCGA melanoma patients (n = 469 patients). k, l Progression-free survival stratified based on ccIES expression in patients receiving (k) anti-PD-1/CTLA-4 combination therapy (n = 32 patients) or (l) anti-PD-1 monotherapy (n = 121 patients). m, Correlation of ccIES with CD103 score and T cell score in TCGA melanoma patients (n = 469 patients). Data in (a, b, c, d, f) displayed as mean ± SEM. Data analysis (b) two-tailed unpaired t–test with Welch correction for unequal variance or with Mann-Whitney-U-test if not normal distributed (f) two-way ANOVA. P-value in (j-l) derived from a Cox proportional hazards model using gene score as a continuous variable and analysis in (m) two-sided Pearson Correlation coefficient (PCC). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = non-significant.

Source data

Extended Data Fig. 9 The reactivated MAPK pathway in RTT tumours has a qualitatively and quantitatively different output.

a, HOMER motif enrichment analysis of upregulated genes comparing RTT vs. NTT tumours of indicated models. b, Heatmap of normalized (RPGC) gene accessibility tracks. Depicted are accessibility profiles for peaks containing motifs of MAPK effectors (left, containing any of the following motifs: AP-1, Fosl2, Fra1, Fra2, Jun-AP-1, c-Jun-CRE, JunB, JunD, ATF2, ATF3) or peaks without MAPK motifs (right). c, Scheme illustrating workflow of SLAM-seq experiment in NTT and RTT (RAFi resistant) Braf/PtenOVA melanoma. d, pERK status in NTT and RTT Braf/PtenOVA melanoma, 1-hour post exposure to MEKi. Experiment performed twice; representative example shown. e, PCA Plot highlighting the Top 500 most variable genes (based on reads containing TC conversions) in SLAM-seq dataset. f, Changes in abundance of newly synthesized mRNA (detected in SLAM-seq based on T > C conversions) in NTT (left) or RTT (right) Braf/PtenOVA melanoma treated with MEKi for 2 hours. Significant targets genes identified in SLAM-seq in NTT cells (black), RTT cells (red) or both (blue) are labelled. Only genes with >2RPMu in CTRL or MEKi conditions displayed. g, Expression of newly synthesized mRNA (RPMu) of 488 target genes identified with SLAM-seq (log2FC < −1, >1, padj<0.1, >2 RPMu) in NTT and RTT Braf/PtenOVA melanoma ± MEKi. Target genes are grouped according to their expression change upon MEKi in both cell lines. (NTT: genes that change expression upon MEKi only in NTT cell line (RTT FC < 1.5), RTT: genes that change expression upon MEKi only in RTT cell line (RTT FC < 1.5), Common: gene expression FC upon MEKi exceeds ±1.5 in both cell lines). h, Expression of selected immune-related genes in NTT, RTT and RTT + MEKi (72 h) sorted Braf/PtenOVA melanoma cells from Rag2-/- mice (NTT, n = 3 RTT, n = 8; RTT + MEKi n = 6 tumours). i, PCA plot displaying top 500 most variable genes for Braf/PtenOVA melanoma cells sorted from NTT and RTT tumours after 72 h of MEKi or CTRL treatment.

Source data

Extended Data Fig. 10 Inhibition of the reactivated MAPK pathway in RAFi resistant RTT tumours restores immunotherapy response.

a, Quantification of T cell proliferation based on CFSE dilution in DC co-culture assays displayed in Fig. 7f (n = 3 tumours per condition). Experiment performed once. P-value: ns 0.21, **** 3E-5. b, Scheme illustrating the use of the ‘thymidinekinase’ (HSV-TK) suicide gene (activated by ganciclovir [GCV]) to induce apoptosis in the RTT Braf/PtenOVA cancer cell line (left) and BLI image and quantification of TGL+ RTT Braf/PtenOVA cancer cells at day 0 and 3 post GCV/MEKi administration (n = 5 mice) (right). Experiment performed twice; representative example shown. P-value: * 0.0188, * 0.0154. c, CD103+ DCs (left) and suppressive myeloid cells (right) in RTT Braf/PtenOVA tumours in response to GCV or MEKi administration (all groups, n = 5 tumours), assessed by flow cytometry. Experiment performed twice; representative example shown. P-value: ns 0.3766, * 0.0303, ns; ns 0.9350, **** <E-15. d, Survival curve illustrating treatment response in RTT Braf/PtenOVA tumour bearing mice treated with indicated therapies (all groups n = 4; except MEKi + ACT, n = 5 mice). Experiment performed twice; representative example shown. P-value: ** 0.0029. e, Survival curve and corresponding spider plot illustrating treatment response in RTT Braf tumour bearing mice treated with indicated therapies (n = 6 mice per group, 2 tumours each). Black arrows indicate immunotherapy administration, continuous MEKi was initiated on Day 5. Experiment performed twice; representative example shown. P-Value: *** 0.0005. f, Scheme illustrating experiments where mice bearing established NTT Braf/PtenOVA tumours were treated with a short run-in phase (4 doses) of RAFi or RAFi/MEKi and subsequently switched to ACT. g, Tumour infiltration of effector OT-1Luc T cells measured by BLI at 48 h post ACT in Braf/PtenOVA tumour bearing mice (n = 5 mice per group). Experiment performed once with two independent clones. Data represents one representative clone. P-value: ns 0.07, 0.071. h, Treatment response of Braf/PtenOVA tumours to ACT (all groups: CTRL n = 3 mice, ACT n = 5 mice per group). Data in (a - c, g, h) displayed as mean ± SEM. Data analysis (a) two-tailed unpaired t-test (b, c) one-way ANOVA (d, e) two-sided log-rank (Mantel-Cox) test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = non-significant.

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Unprocessed western blots.

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Haas, L., Elewaut, A., Gerard, C.L. et al. Acquired resistance to anti-MAPK targeted therapy confers an immune-evasive tumor microenvironment and cross-resistance to immunotherapy in melanoma. Nat Cancer 2, 693–708 (2021). https://doi.org/10.1038/s43018-021-00221-9

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