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

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.

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 S1) receiving immunotherapy with checkpoint inhibitors (n = 54 patients). N^TT, targeted therapy (TT) naïve patients (n = 38); R^TT, TT (RAFi or RAFi/MEKi) resistant patients (n = 16). b, Summary of responses to immunotherapy in N^TT and R^TT patients in published patient cohorts (ORR = overall response rate, PFS = progression-free survival). c, CD3⁺CD8⁺ T cells in patient-matched N^TT and R^TT melanoma biopsies [scale bar pre-treatment: 2mm (left), 200 μm (right), 50μm (zoom-in); scale bar post progression: 5mm (left), 200 μm (right), 50μm (zoom-in)]. Experiment performed once on 10 matched biopsies. d, CD8⁺CD39⁺CD103⁺ T cells in patient-matched N^TT and R^TT melanoma biopsies [scale bar: 200 μm, 50μm (zoom-in)]. e, Quantification of tumour reactive (CD8⁺CD39⁺CD103⁺) T cells in patient-matched N^TT and R^TT melanoma biopsies, assessed by IF staining (n = 10 patients, Supplementary Table S2). Data analysis a two-sided log-rank (Mantel-Cox) test.

Extended Data Fig. 2. The Braf melanoma model responds to checkpoint inhibition in the N^TT state, but is resistant in the R^TT state.

a, Proliferation fold change (FC) of Braf melanoma cells after 72h at indicated drug conditions. Line indicating FC in proliferation of N^TT cells on lowest drug condition (n = RAFi: technical triplicates; RAFi/MEKi technical duplicates), (drug concentrations: RAFi: DMSO CTRL, 100nM, 300nM, 1μM, 3μM; RAFi/MEKi: DMSO CTRL, 10nM/3nM, 30nM/10nM, 100nM/30nM, 300nM/100nM). b, pERK status in N^TT and R^TT 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 72h at indicated drug conditions. Line indicating FC in proliferation of N^TT cells on lowest drug condition (n = RAFi: technical triplicates; RAFi/MEKi technical duplicates), (drug concentrations: RAFi: DMSO CTRL, 100nM, 300nM, 1μM, 3μM; RAFi/MEKi: DMSO CTRL, 10nM/3nM, 30nM/10nM, 100nM/30nM, 300nM/100nM). e, pERK status in N^TT and R^TT 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 N^TT and R^TT 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 N^TT and R^TT Braf melanoma (CTRL, RAFi n = 6 tumours; anti-PD-1/CTLA-4 ± RAFi, n = 10 tumours); arrows indicate therapy administration. R^TT 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.

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

a, Treatment response to immunotherapy in N^TT Braf/Pten melanoma tumour bearing mice, black arrows indicate therapy administration (N^TT CTRL, n = 2; N^TT anti-PD-1/CTLA-4, n = 4 mice). Experiment performed once. b, Generation of OVA antigen-expressing N^TT and R^TT 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 N^TT and R^TT Braf/Pten^OVA 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 R^TT Braf/Pten^OVA tumours; arrow indicating day of ACT (N^TT, RAFi/MEKi R^TT CTRL, n = 3 mice; N^TT, RAFi/MEKi R^TT ACT, n = 5 mice, P-value: **** 3.9E-11, ns 0.6) (left) and infiltration of OT-1^Luc 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/Pten^OVA tumours, R^TT 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 (N^TT, R^TT CTRL, n = 3 mice N^TT, R^TT ACT, n = 5 mice). g, Tumour infiltration of OT-1^Luc T cells into N^TT Braf/Pten^OVA tumours and R^TT Braf/Pten^OVA 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 N^TT and R^TT Braf melanoma cell lines (baseline and 24h post 10 ng/ml IFN-γ exposure). Experiment performed 3 times; representative example shown. k, Gene expression changes in N^TT and R^TT Braf melanoma cell lines treated with IFN-γ. Correlation between genes deregulated in N^TT (x-Axis) and R^TT (y-Axis) cell lines (P < 0.05), dots display individual genes. P-value: <1E−15. Supplementary Table S3. l, Braf^OVA 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 N^TT and R^TT Braf/Pten^OVA cell lines at indicated ratios; arrow indicating day of ACT (0.05% N^TT/R^TT CTRL, n = 4; ACT, n = 5; 0.05% R^TT/N^TT 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 (N^TT/ OVA⁺ n = 4; N^TT/ 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 R^TT tumours is strongly remodelled.

a, T cell influx into N^TT and R^TT 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, N^TT n= 15, R^TT n= 16 ROI). P-value: * 0.0204, ns 0.0756. c, PCA plot displaying top 500 most variable genes for T cells sorted from N^TT and R^TT Braf/Pten^OVA 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/Pten^OVA melanoma both groups, n= 4 tumours; Braf/Pten melanoma N^TT= 5 tumours, R^TT= 8 tumours). Experiment performed once. P-value: * 0.0185, ** 0.0083. j, k CD103⁺MHCII⁺ DCs in N^TT and R^TT 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 N^TT and R^TT Braf/Pten^OVA melanoma quantified separately at tumour margin and centre (n = 2 tumours per condition, all groups 10 ROI, except N^TT 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 R^TT vs. N^TT tumours (Braf/Pten N^TT, Braf N^TT, BRAF R^TT n = 3; Braf/Pten R^TT n = 4 tumours). b, Mean fluorescence intensity (MFI) of maturation markers on CD103⁺ DCs from N^TT and R^TT Braf/Pten melanoma (N^TT, n = 5; R^TT, 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 N^TT and R^TT ± Poly I:C Braf/Pten^OVA tumours. d, GSEA of IFN-alpha response in CD103⁺ DCs sorted from R^TT vs. N^TT Braf/Pten^OVA 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/Pten^OVA 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-1^Luc T cells measured by BLI at 24h post ACT in Braf/Pten^OVA 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/Pten^OVA tumours treated with ACT or anti-GR-1 plus ACT (N^TT ACT+ISO, n = 6; R^TT ACT+ISO, n = 4; N^TT ACT+anti-GR-1, n = 6; R^TT ACT+anti-GR-1, n = 4 mice). j, DC maturation in Poly I:C injected Braf/Pten^OVA tumours assessed by CD40 expression using flow cytometry. k, CD103⁺ DC influx in R^TT 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 R^TT Braf/Pten^OVA tumours. (R^TT CTRL, R^TT + ACT, R^TT FLT3L + ACT, n = 3; R^TT + ACT + Poly I:C, R^TT FLT3L + ACT+ Poly I:C, n = 4 mice). Experiment performed twice; representative example shown. P-value: ** 0.0100. m, Treatment response of R^TT 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 72h at indicated drug conditions. Line indicating FC in proliferation of N^TT cells on lowest drug condition (n = technical triplicates) (drug concentration: DMSO CTRL, 10nM, 30nM, 100nM, 300nM MEKi). c, pERK status in N^TT and R^TT CT26 colon carcinoma cell lines, 1-hour post drug exposure. Experiment performed twice; representative example shown. d, Treatment response in mice bearing N^TT and R^TT CT26 (Kras^G12D/G12D Cdkn2a ^-/-) tumours (N^TT and R^TT CTRL, n = 6; N^TT and R^TT 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 N^TT and R^TT cell lines (baseline and 24h post 10 ng/ml IFN-γ exposure). Experiment performed 3 times; representative example shown. f, Gene expression changes in N^TT and R^TT CT26 colon carcinoma cell lines treated with IFN-γ. Correlation between genes deregulated in N^TT (x-Axis) and R^TT (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 N^TT and R^TT 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 R^TT 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 N^TT and R^TT 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 N^TT and R^TT 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 N^TT and R^TT 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 N^TT and R^TT 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 N^TT and R^TT CT26 colon carcinoma (N^TT, n = 6; R^TT, 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.

Extended Data Fig. 7. Modulation of the CD103⁺ DC compartment restores immunotherapy response in R^TT 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 R^TT 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 R^TT 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-L^D MuLV gp70 specific T cells into N^TT and R^TT CT26 colon carcinoma treated with anti-PD-1 and Poly I:C (injected and contralateral tumour displayed separately) (N^TT CTRL, n = 4; R^TT CTRL, n = 3; R^TT 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 R^TT 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 R^TT 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 N^TT 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 72h at indicated drug conditions. Line indicating FC in proliferation of N^TT cells on lowest drug condition (n = technical triplicates), (drug concentration: DMSO CTRL, 100nM, 300nM, 1μM, 3μM RAFi). d, Proliferation FC of Braf melanoma cell lines after 72h in indicated drug conditions of N^TT and N^{TT-Dacarbazine} cell lines (n = technical duplicates), (drug concentration: CTRL, 10 μg, 50 μg, 100 μg, 500 μg Dacarbazine). e, pERK status in CaTCH-isolated N^TT and R^TT Braf/Pten cell lines, 1-hour post drug exposure. Experiment performed twice; representative example shown. f, Treatment response to ACT in matched CaTCH isolated N^TT and R^TT Braf/Pten^OVA 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 N^TT and R^TT Braf/Pten^OVA melanoma cells (left, tumours were not exposed to RAFi) (all groups n = 3 tumours) and in sorted RAFi/MEKi R^TT 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.

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

a, HOMER motif enrichment analysis of upregulated genes comparing R^TT vs. N^TT 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 N^TT and R^TT (RAFi resistant) Braf/Pten^OVA melanoma. d, pERK status in N^TT and R^TT Braf/Pten^OVA 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 N^TT (left) or R^TT (right) Braf/Pten^OVA melanoma treated with MEKi for 2 hours. Significant targets genes identified in SLAM-seq in N^TT cells (black), R^TT 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 N^TT and R^TT Braf/Pten^OVA melanoma ± MEKi. Target genes are grouped according to their expression change upon MEKi in both cell lines. (N^TT: genes that change expression upon MEKi only in N^TT cell line (R^TT FC <1.5), R^TT: genes that change expression upon MEKi only in R^TT cell line (R^TT FC <1.5), Common: gene expression FC upon MEKi exceeds ±1.5 in both cell lines). h, Expression of selected immune-related genes in N^TT, R^TT and R^TT + MEKi (72h) sorted Braf/Pten^OVA melanoma cells from Rag2-/-mice (N^TT, n = 3 R^TT, n = 8; R^TT+ MEKi n = 6 tumours). i, PCA plot displaying top 500 most variable genes for Braf/Pten^OVA melanoma cells sorted from N^TT and R^TT tumours after 72h of MEKi or CTRL treatment.

Extended Data Fig. 10. Inhibition of the reactivated MAPK pathway in RAFi resistant R^TT 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 R^TT Braf/Pten^OVA cancer cell line (left) and BLI image and quantification of TGL⁺ R^TT Braf/Pten^OVA 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 R^TT Braf/Pten^OVA 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 R^TT Braf/Pten^OVA 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 R^TT 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 N^TT Braf/Pten^OVA 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-1^Luc T cells measured by BLI at 48h post ACT in Braf/Pten^OVA 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/Pten^OVA 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.

Fig. 1. Targeted therapy resistance induces cross-resistance to immunotherapy.

a, Clinical response rate and b, Progression-free survival (PFS) of metastatic melanoma patients in the Lausanne Patient Cohort (Supplementary Table S1) receiving immunotherapy with checkpoint inhibitors (n = 54 patients). N^TT, targeted therapy (TT) naïve patients (n = 38); R^TT, TT (RAFi or RAFi/MEKi) resistant patients (n = 16). c, Quantification of T cells in tumours of N^TT and R^TT melanoma patients receiving MAPK pathway inhibitors (RAFi or RAFi/MEKi combination), assessed by immunofluorescence (IF) staining of matched patient biopsies (n = 10 patients, Supplementary Table S2). d, Scheme outlining (1) generation of N^TT and R^TT cell lines, before therapy or after tumour relapse on treatment and (2) testing immunotherapy response in tumours derived from N^TT and R^TT cell lines. e, Treatment response of subcutaneously injected Braf melanoma (Braf^V600E/WT Cdkn2a^-/-) tumours continuously treated with indicated TT; arrow indicating start of therapy (CTRL, n = 6 tumours; other groups, n = 8). Experiment performed 3 times; representative example shown. f, Treatment response in mice bearing N^TT and R^TT Braf melanoma; arrows indicate immunotherapy administration (all groups, n = 8 tumours). No targeted therapy was administered. Experiment performed 9 times; representative example shown. P-value: **** 4.8E-13, ns 0.9963, ns 0.7039. g, T cells in N^TT and R^TT tumours of Braf melanoma, treated with CTRL or anti-PD-1/CTLA-4 assessed by IF staining (scale bar 20 μm). Experiment performed twice; representative example shown. h, T cells in N^TT and R^TT tumours of Braf melanoma, treated with CTRL or anti-PD-1/CTLA-4 assessed by flow cytometry (N^TT CTRL, n = 6; N^TT anti-PD-1/CTLA-4, n = 9; R^TT CTRL, n = 6; R^TT anti-PD-1/CTLA-4, n = 9 tumours). Experiment performed 3 times; representative example shown. P-value: * 0.0196, *** 0.0009, ns >0.9999. Data in e, f, h displayed as mean ± standard error of mean (SEM). Data analysis a one-sided chi-square test b two-sided log-rank (Mantel-Cox) test f two-way ANOVA h one-way ANOVA. * P < 0.05, *** P < 0. 001, **** P < 0. 0001, ns = non-significant.

Fig. 2. The tumour microenvironment in R^TT tumours precludes a functional T cell response.

a, Scheme outlining experiments to assess infiltration and treatment response to adoptive cell transfer (ACT) of pre-activated OT-1^Luc effector T cells in N^TT and R^TT (RAFi resistant) Braf/Pten^OVA melanoma (Braf^V600E/WT Cdkn2a^-/- Pten^-/-) tumours. b, Tumour infiltration of OT-1^Luc T cells in N^TT and R^TT Braf/Pten^OVA melanoma, measured by bioluminescence imaging (BLI) at indicated days (all groups, n = 7 tumours). Experiment performed 7 times; representative example shown. P-value: ** 0.0030, *** 0.0005, * 0.0210. c, Treatment response to ACT in Braf/Pten^OVA tumours; arrow indicating day of ACT (N^TT, R^TT CTRL, n = 3; N^TT, R^TT ACT, n = 5 mice). No targeted therapy was administered. Experiment performed 7 times; representative example shown. P-value: **** 2E-6, ns 0.4934. d, MHC-I surface level of N^TT and R^TT Braf/Pten^OVA melanoma (baseline and 24h post 10 ng/ml IFN-γ exposure). Experiment performed 3 times; representative example shown. e, Gene expression changes in N^TT and R^TT Braf/Pten^OVA melanoma cell lines treated with IFN-_γ. Correlation between genes deregulated in N^TT (x-Axis) and R^TT (y-Axis) cell lines (P < 0.05), dots display individual genes. P-value: <1E−15 (Supplementary Table S3). f, Scheme outlining the in vitro killing assay using pre-activated OT-1 T cells (left) and quantification of viability of Braf/Pten^OVA melanoma cell lines after 24 h of co-culture (right) (CTRL, n = 2; N^TT-OVA R^TT-OVA, n = 3 independent clones). g, Scheme outlining experiments to assess ACT response in tumours comprised of 99.95% Luc^- (majority, defines the TME) and 0.05% Luc⁺ (for BLI read-out of minority population) N^TT or R^TT Braf/Pten^OVA melanoma at indicated ratios. h, BLI of Luc⁺ Braf/Pten^OVA cells at day 0 and day 6 post ACT/CTRL injection (left) and quantified fold change (FC) in BLI signal on day 6 (right) (99.95% R^TT/ 0.05% N^TT + ACT; n = 5, all other groups, n = 4 mice). Experiment performed 3 times; representative example shown. Data in b, c, f, h displayed as mean ± SEM. Data analysis b two-tailed unpaired t-test with Welch correction for unequal variance c two-way ANOVA e two-tailed Pearson Correlation. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = non-significant.

Fig. 3. The tumour microenvironment of R^TT tumours shows reduced and dysfunctional CD103⁺ DCs.

a, T cell influx into N^TT and R^TT Braf/Pten^OVA melanoma 5 days post ACT assessed by IF (scale bar 100 μm and 20 μm). Experiment performed twice; representative image shown. b, T cell influx into N^TT and R^TT Braf/Pten^OVA melanoma 5 days post ACT quantified separately at tumour margin and centre (n = 2 tumours per condition; all groups n = 10 ROI, except R^TT centre, n = 11 ROI). P-value: **** 1.08E-5, 5.7E-6. c, Gene set enrichment analysis (GSEA) of IFN-γ response in T cells sorted from R^TT vs. N^TT Braf/Pten^OVA melanoma (N^TT, R^TT, n = 4 tumours per condition). d, Heat map displaying T cell effector genes in T cells sorted from N^TT and R^TT tumours (N^TT, R^TT, n = 4 tumours per condition). e, Suppressive myeloid cells (left) and CD103⁺ DCs (right) in N^TT and R^TT tumours of Braf/Pten^OVA melanoma, assessed by flow cytometry (n = 9 tumours each). Experiment performed 5 times; displayed is a pool of 2 representative experiments. P-value: **** 8E-5, 9.4E-8. f, CD103⁺MHCII⁺ DCs in N^TT and R^TT Braf/Pten^OVA melanoma, assessed by IF (scale bar 100 μm and 20 μm). Experiment performed twice; representative image shown. g, Quantification of CLEC9a⁺ cells in tumours of N^TT and R^TT melanoma patients receiving MAPK pathway inhibitors (RAFi or RAFi/MEKi combination), assessed by IF staining of matched patient biopsies (n = 10 patients, Supplementary Table S2). h, Gene expression changes in sorted CD103⁺ DCs from N^TT and R^TT Braf/Pten^OVA melanoma assessed by Smart-seq (N^TT, n = 4; R^TT, n = 5 sorted tumours; in technical triplicates). Supplementary Table S5. i, Scheme outlining co-culture of CD103⁺ DCs isolated from N^TT and R^TT Braf/Pten^OVA melanoma using magnetic cell sorting (MACS) with CFSE labelled naïve OT-1 T cells to assess T cell activation potential (left) and representative histogram illustrating CFSE signal in T cells (right). Experiment performed 6 times; representative example shown. Data in b and e displayed at mean ± SEM and analysed using two-tailed unpaired t-test with Welch correction for unequal variance or Mann-Whitney U test if not normal distributed. **** P < 0.0001.

Fig. 4. Restoration of a functional CD103⁺ DC compartment restores immunotherapy response.

a, Scheme outlining experiments to assess impact of DC maturation (intratumoural Poly I:C) and DC expansion (FLT3L overexpression from tumour cells) on ACT using effector T cells. b, c Tumour infiltration of OT-1^Luc T cells in N^TT and R^TT Braf/Pten^OVA melanoma measured by BLI 24 h post ACT (b) representative image and (c) quantified 24h post ACT (n = 4, 4, 3, 3, 4, 4 mice from left to right). Experiment performed twice; representative example shown. P-value: *** 0.0004, * 0.0376, * 0.0351. d, Spiderplot indicating response to ACT ± Poly I:C ± FLT3L in R^TT Braf/Pten^OVA tumours. (R^TT CTRL, R^TT CTRL + FLT3L, R^TT + ACT, R^TT FLT3L + ACT, n = 3; R^TT + ACT + Poly I:C, R^TT FLT3L + ACT+ Poly I:C, n = 4 mice). Experiment performed twice; representative example shown. e, Gene expression changes in CD103⁺ DCs sorted from Braf/Pten^OVA N^TT, R^TT or R^TT tumours 24h post Poly I:C injection, assessed by Smart-seq and normalized to N^TT (N^TT, R^TT + Poly I:C, n = 4; R^TT, n = 5 sorted tumours; in technical triplicates). f, Representative histogram illustrating CFSE signal in T cells co-cultured with CD103⁺ DCs isolated from N^TT, R^TT and R^TT Braf/Pten^OVA melanoma treated with Poly I:C. g, Quantification of T cell proliferation based on CFSE dilution in DC co-culture assays displayed in Fig. 4f (n = 3 tumours per condition). Experiment performed once. P-value: **** 5E-5, ** 0.0013. h, Scheme illustrating experiments to address immunological memory in mice that were bearing R^TT tumours and had complete regression in response to Poly I:C + FLT3L + ACT. i, Tumour growth curve in mice that previously had complete response to ACT, Poly I:C and FLT3L reinjected with R^TT Braf/Pten^OVA melanoma compared to naïve mice (Naïve, n = 5; Reinjected, n = 8 mice). Experiment performed twice; representative example shown. Data in c, g displayed as mean ± SEM and analysed using one-way ANOVA. * P <0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001, ns= non-significant.

Fig. 5. Cross-resistance is acquired during evolution of MAPKi resistance and is directly linked to a cell-intrinsic signalling program.

a, Scheme outlining experiments to test ACT or anti-PD-1/CTLA-4 response in mice bearing R^TT-vitro tumours, of cell lines made resistant in vitro. b, Tumour infiltration of OT-1^Luc T cells into R^TT-vitro Braf/Pten^OVA tumours, measured by BLI at indicated days (N^TT, n = 3; R^TT-vitro n = 3 mice). Experiment performed twice; representative example shown. P-value: * 0.0382, * 0.0340, ** 0.0074. c, Tumour infiltration of OT-1^Luc T cells into R^TT-NSG Braf/Pten^OVA tumours, measured by BLI at indicated days (N^TT, R^TT-NSG, n = 4 mice). Experiment performed twice; representative example shown. P-value: * 0.0168, ** 0.0058, ns 0.2687. d, Treatment response to anti-PD-1/CTLA-4 in (d) N^TT R^TT-NSG (left) and (e) R^TT-vitro (right) Braf melanoma; arrows indicating therapy administration (N^TT and R^TT CTRL, n = 6; N^TT or R^TT IT, n = 8). Experiment performed 3 times; representative example shown. P-value: **** 4.8E-11, **** 7.3E-14. e, Treatment response to anti-PD-1/CTLA-4 in N^TT and N^{TT-Dacarbazine} Braf melanoma, arrows indicate therapy administration. (N^TT, N^TT-Dac CTRL, n = 6 tumours; N^TT, N^TT-Dac anti PD-1/CTLA-4, n = 10 tumours). Experiment performed once. P-value: **** 7.5E-5, *** 0.0008. f, Scheme illustrating the use of the lineage tracing system CaTCH employing a barcode guided inducible GFP reporter to isolate the treatment naïve (N^TT) founding clone of a targeted therapy resistant (R^TT) clone. Experimental steps: 1) Injection of barcoded N^TT cell line into mice. 2) Resistance generation using RAFi/MEKi. 3) Isolation of R^TT cell line. 4) Next-generation sequencing of barcodes in R^TT cell line and isolation of matching N^TT and R^TT clones. 5) OVA engineering. 6) ACT experiment. g, Infiltration of OT-1^Luc T cells into matched CaTCH isolated N^TT and R^TT Braf/Pten^OVA tumours (all groups, n = 5 mice). Experiment performed twice; representative example shown. P-value: ns 0.9367, * 0.0298, * 0.0133. Data in b, c, d, e, g displayed as mean ± SEM. Data analysis b, c, g two-tailed unpaired t-test with Welch correction for unequal variance or Mann-Whitney U test if not normal distributed and d, e two-way ANOVA. * P <0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001, ns = non-significant.

Fig. 6. The R^TT signalling program is predictive for immunotherapy response in patients and controlled by MAPK signalling.

a, Scheme outlining the generation of the cancer cell-intrinsic immune evasion signature (ccIES, 106 genes, Supplementary Table S7) by overlapping bulk-transcriptomic data from both melanoma models (Immune evasion signature, 279 genes Supplementary Table S6) with genes deregulated in sorted Braf/Pten tumour cells. b, Expression of genes comprising the ccIES in N^TT and R^TT cancer cells sorted from Braf/Pten^OVA tumours derived from Rag2-/-mice (R^TT tumours in this experiment were continuously exposed to 10 mg/kg RAFi). (n = 3 mice). c, Progression-free survival of patients receiving anti-PD-1 monotherapy (n = 41 patients) stratified based on ccIES expression. d, Clinical response in patients receiving anti-PD-1 (n = 41 patients) stratified based on ccIES expression. e, Correlation of ccIES with CD103 score and T cell score (Supplementary Table S8, S9) in patients receiving anti-PD-1 (n = 41 patients). f, Upstream regulator analysis (Ingenuity) on ccIES-genes, sorted based on z-score. g, HOMER motif enrichment analysis of upregulated ccIES genes, comparing R^TT vs. N^TT. h, Volcano plot of differential TF activity (weighted mean difference) in ATAC-Seq data comparing R^TT vs. N^TT Braf/Pten^OVA melanoma cells sorted from tumours (n= 3 tumours per condition). TFs with a TF activity >= abs (0.25) are highlighted in black. Corresponding significance is computed using the analytical approach and adjusted for multiple testing by Benjamini-Hochberg procedure (y axis). i, Line plots highlighting ATAC-Seq profile for indicated TF motifs in N^TT and R^TT Braf/Pten^OVA cell lines. j, Top: Heatmap of normalized (reads per genomic content; RPGC) gene accessibility tracks (ATAC-seq). Depicted are accessibility profiles for peaks uniquely identified in N^TT and R^TT cell lines. Profile plots and heatmaps represent accessibility around unique peak centre (± 1KB). Heatmaps sorted by descending accessibility. Bottom: HOMER Motif enrichment analysis of peaks uniquely identified in N^TT and R^TT cell lines. Top 3 predicted motifs for each condition displayed. P-value in c derived from a Cox proportional hazards model using gene score as a continuous variable and analysis in e two-sided Pearson Correlation.

Fig. 7. Inhibition of the re-activated MAPK pathway restores sensitivity to immunotherapy in R^TT tumours.

a, Scheme illustrating the rationale and experiment to inhibit MAPK signalling in R^TT cells and profile the contribution of MAPK signalling to cross-resistance. b, Gene expression changes in ccIES-genes in response to 72h of MEKi in R^TT Braf/Pten^OVA melanoma cells sorted from Rag2-/-mice (R^TT, n = 5; R^TT+ MEKi n = 6 tumours). c, Gene expression changes analysed via SMART-Seq in ccIES genes (divided into up and downregulated genes) in N^TT and R^TT Braf/Pten^OVA melanoma cells sorted from tumours after 72h of MEKi treatment (n = 3 tumours per condition, in technical triplicates). Black line highlights Mean. d, Suppressive myeloid cells and CD103⁺ DC levels in N^TT and R^TT Braf/Pten^OVA tumours 3 days post MEK inhibition (N^TT, R^TT n = 9 tumours; R^TT+ MEKi, n = 10 tumours). Experiment performed twice; data represents pool of both experiments. P-value: CD11b⁺GR-1⁺ **** 2.5E-6, 5.2E-8; CD103⁺CD11c⁺ **** 2.3E-9, * 0.048. e, Tumour infiltration of OT-1^Luc T cells in R^TT Braf/Pten^OVA melanoma, measured by BLI at indicated days post ACT, MEKi started 24h before ACT (R^TT, n = 4; R^TT+ MEKi, n = 5 tumours). Experiment performed 3 times; representative example shown. P-value: ** 0.0087, * 0.0159. f, Co-culture of CD103⁺ DCs isolated from N^TT, R^TT and R^TT Braf/Pten^OVA melanoma treated with MEKi (0.5mg/kg for 3 doses) with CFSE labelled naïve OT-1 T cells displayed as representative histogram of CFSE signal in T cells. g, Spider plot indicating tumour volume in R^TT Braf/Pten^OVA tumour bearing mice treated with indicated therapies (all groups n = 4; except MEKi + ACT, n = 5 mice). Experiment performed twice; representative example shown. h, Summary scheme highlighting the core findings. Data in d, e displayed as mean ± SEM. Data analysis d one-way ANOVA e two-tailed unpaired t-test with Welch correction for unequal variance or Mann-Whitney U test if not normal distributed. * P < 0.05, ** P < 0.01, **** P < 0.0001, ns = non-significant.