CTLA4 blockade abrogates KEAP1/STK11-related resistance to PD-(L)1 inhibitors

Abstract

For patients with advanced non-small-cell lung cancer (NSCLC), dual immune checkpoint blockade (ICB) with CTLA4 inhibitors and PD-1 or PD-L1 inhibitors (hereafter, PD-(L)1 inhibitors) is associated with higher rates of anti-tumour activity and immune-related toxicities, when compared with treatment with PD-(L)1 inhibitors alone. However, there are currently no validated biomarkers to identify which patients will benefit from dual ICB^1,2. Here we show that patients with NSCLC who have mutations in the STK11 and/or KEAP1 tumour suppressor genes derived clinical benefit from dual ICB with the PD-L1 inhibitor durvalumab and the CTLA4 inhibitor tremelimumab, but not from durvalumab alone, when added to chemotherapy in the randomized phase III POSEIDON trial³. Unbiased genetic screens identified loss of both of these tumour suppressor genes as independent drivers of resistance to PD-(L)1 inhibition, and showed that loss of Keap1 was the strongest genomic predictor of dual ICB efficacy-a finding that was confirmed in several mouse models of Kras-driven NSCLC. In both mouse models and patients, KEAP1 and STK11 alterations were associated with an adverse tumour microenvironment, which was characterized by a preponderance of suppressive myeloid cells and the depletion of CD8⁺ cytotoxic T cells, but relative sparing of CD4⁺ effector subsets. Dual ICB potently engaged CD4⁺ effector cells and reprogrammed the tumour myeloid cell compartment towards inducible nitric oxide synthase (iNOS)-expressing tumoricidal phenotypes that-together with CD4⁺ and CD8⁺ T cells-contributed to anti-tumour efficacy. These data support the use of chemo-immunotherapy with dual ICB to mitigate resistance to PD-(L)1 inhibition in patients with NSCLC who have STK11 and/or KEAP1 alterations.

Fig. 1. Immunogenomic correlates and clinical outcomes with PCP chemo-immunotherapy in patients with STK11- and/or KEAP1-mutant advanced nsNSCLC.

a, PFS, OS and ORR with pemetrexed, carboplatin or cisplatin and pembrolizumab (PCP) in patients with STK11^MUT (n = 119) versus STK11^WT (n = 320) (top) or with KEAP1^MUT (n = 42) versus KEAP1^WT (n = 103) (bottom) advanced nsNSCLC. The comparison of ORR (partial response (PR)/complete response (CR)) in patients with STK11^MUT versus STK11^WT and KEAP1^MUT versus KEAP1^WT tumours was based on the chi-squared test. Log-rank test was used for comparisons of PFS and OS. Multivariate (MV) HRs (adjusted for age, ECOG performance status and presence of brain metastases) and 95% CIs were estimated using a Cox proportional hazards model. P ≤ 0.05 was considered statistically significant. NS, not significant. b, Prevalence of individual and combined STK11 and KEAP1 alterations in advanced LUAD (left; n = 8,592) or KRAS-mutated LUAD (right; n = 3,224) from the Foundation Medicine (FMI) database. c, TMB in single and double STK11^MUT and/or KEAP1^MUT and STK11^WT/KEAP1^WT LUAD in the FMI dataset. Median TMB (table insert) and fraction of tumours with a TMB of 10 or more mutations per Mb or fewer than 10 mutations per Mb (bar chart) in each subgroup are indicated. d, PD-L1 tumour proportion score (TPS) in single and double STK11^MUT and/or KEAP1^MUT and STK11^WT/KEAP1^WT LUAD in the FMI dataset (n = 8,836). The chi-squared test from a 2 × 4 contingency table was used to compare the distribution of PD-L1-positive (TPS ≥ 1%) and -negative (TPS < 1%) tumours across the four oncogenotypes. e, Volcano plot of enriched somatic genomic alterations in PD-L1-negative (TPS < 1%, grey circles) versus PD-L1-positive (TPS ≥ 1%, red circles) LUAD with an intermediate or high TMB (TMB^I/H; six or more mutations per Mb; n = 4,672). The size of individual circles is proportional to the prevalence of the corresponding alteration. Two-sided Fisher’s exact test was used for statistical comparisons and statistical significance was established at false discovery rate (FDR)-adjusted P ≤ 0.05.

Fig. 2. Clinical outcomes in molecularly defined patient subgroups in the phase III POSEIDON clinical trial.

a,b, Kaplan–Meier estimates of PFS according to blinded independent central review (BICR) per RECIST v.1.1 (a) and OS (b) with tremelimumab, durvalumab and platinum chemotherapy (TDCT, light blue curve), durvalumab plus platinum chemotherapy (DCT, dark blue curve) or platinum doublet chemotherapy alone (CT, red curve) in patients with (i) STK11^MUT and/or KEAP1^MUT (left) and (ii) STK11^WT and KEAP1^WT (right) metastatic nsNSCLC. Landmark 12-month PFS rates and 24-month and 36-month OS rates in each of the treatment arms are also shown (dotted lines). NE, not evaluable. c, Kaplan–Meier estimates of OS with TDCT, DCT or CT in patients with KRAS^MUT (left) and KRAS^WT (right) metastatic nsNSCLC. PFS and ORR analyses were based on a data cut-off date of 24 July 2019. OS analyses were based on a data cut-off date of 12 March 2021. HRs and 95% CIs were estimated using unstratified Cox proportional hazards models.

Fig. 3. Efficacy of anti-PD-1 monotherapy and dual anti-PD-1/anti-CTLA4 therapy in immune-competent models of STK11- and/or KEAP1-deficient NSCLC.

a, Left, in vivo TSG-focused CRISPR–Cas9 screening platform for identifying drivers of PD-1 inhibitor resistance. Right, the gradual decrease in tumour growth from untreated nude mice (n = 18) to C57BL/6 mice treated with anti-IgG (n = 18) to C57BL/6 mice treated with anti-PD-1 (n = 20) reflects increased anti-tumour immunity. Data are mean ± s.e.m. b, Volcano plot of relative sgRNA enrichment or depletion in tumours from C57BL/6 mice that were treated with anti-PD-1 (5 mg per kg; n = 20; 10 mg per kg; n = 25; total n = 45) versus anti-IgG isotype control (n = 18). c, Box plots of relative log₂-transformed fold change for sgRNAs targeting Stk11 (left) or Keap1 (right) across treatment groups (nude, n = 18; IgG, n = 20; anti-PD-1 5 mg per kg, n = 20). Individual sgRNAs are represented by coloured circles (10 sgRNAs per gene). Median (central line), interquartile range (box plot edges) and data range (whiskers) are indicated. d, PGx-Tuba-seq experimental strategy for in vivo multiplexed quantitative evaluation of the effect of TSG depletion on immunotherapy responses in an autochthonous, genetically engineered Kras^G12D-driven LUAD model (see Methods). IFU, infectious units. e, Relative tumour number (RTN) score reflecting differential sensitivity to dual anti-PD-1/anti-CTLA4 blockade compared with anti-PD-1 monotherapy. Significant effects are highlighted in colour (n = 16–19 mice per group). f, Sensitivity to dual ICB across several STK11 and/or KEAP1-deficient syngeneic models of Kras^G12D (KK, KLK) and Kras^G12C (KL2, KL5) mutant NSCLC (n = 8–10 mice per group). Comparison of tumour volume (TV) was performed at the time point at which the first mouse in any treatment arm reached end-point (tumour volume ≥ 1,500 mm³) and was based on the Mann–Whitney U test. Data are mean ± s.d. Time to tumour volume (TTV) ≥ 1,200 mm³ was used as a surrogate for survival. Comparison of TTV between treatment groups was based on the log-rank test. Statistical significance is indicated (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). Source Data

Fig. 4. Innate immune cells and CD4⁺ effectors are crucial mediators of dual anti-PD-1/anti-CTLA4 efficacy in Stk11- and/or Keap1-deficient models of KRAS-mutant NSCLC.

a, FACS-based enumeration of immune cell subsets in Keap1-deficient (K7, K8), Stk11-deficient (L6, L9) or isogenic Keap1 and Stk11-proficient LKR10 (control, Con) allograft tumours reveals a myeloid-cell-enriched and CD8⁺ T-cell-depleted suppressive TIME with relative sparing of T_H1 CD4⁺ cells. Data are mean ± s.d. (n = 7–8 mice per group). TAMs, tumour-associated macrophages. b, FACS-based assessment of single and dual ICB-induced changes in the abundance of distinct T cell (left panels) and myeloid cell (right panels) subsets in the microenvironment of the Stk11- and Keap1-deficient KLK model. Data are mean ± s.d. (n = 7–8 mice per group). EffM, effector memory cells. c, Effect of CD4⁺ or CD8⁺ depletion on the in vivo growth kinetics of KL5 and KK models in the absence of treatment or with dual anti-PD-1/anti-CTLA4 therapy (n = 7–8 mice per group). Comparison of tumour volume was performed at the time point at which the first mouse in any treatment group reached a tumour volume ≥ 1,500 mm³. d, The anti-tumour activity of dual PD-1/CTLA4 ICB in the KL5 model is dependent on innate immune cells (n = 7–10 mice per group). Tumour volume is shown for the indicated treatment arms. e, iNOS inhibition curtails the anti-tumour efficacy of dual ICB in the KLK model (n = 7–8 mice per group). Comparison of tumour volume was performed at the time point at which the first mouse in any treatment group reached a tumour volume ≥ 1,200 mm³. Individual tumour growth trajectories in the anti-PD-1 + anti-CTLA4 (red) and L-NIL + anti-PD-1 + anti-CTLA4 (purple) treatment arms are also shown. Mann–Whitney U test was used for all pairwise statistical comparisons. Data are mean ± s.d. Statistical significance is indicated (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). Source Data

Extended Data Fig. 1. Clinical outcomes in patients with STK11- and/or KEAP1-mutated nsNSCLC treated with PCP or CP.

a. PFS and OS with PCP chemo-immunotherapy in patient subgroups with advanced (i) STK11^MUT vs STK11^WT (top panel) and (ii) KEAP1^MUT vs KEAP1^WT (bottom panel) nsNSCLC. HRs and 95% CIs were estimated using a Cox proportional hazards model. b. Best overall response with PCP in the indicated patient subgroups. The analysis was limited to the subset of response-evaluable patients with available comprehensive NGS profiling that included both STK11 and KEAP1 (N = 141). c. Kaplan–Meier estimates of PFS (left) and OS (right) with PCP in patients with advanced (i) STK11^WT;KEAP1^WT (WT,WT; indicated in blue); (ii) STK11^MUT;KEAP1^WT (MUT,WT; indicated in red); (iii) STK11^WT;KEAP1^MUT (WT,MUT; indicated in purple) and (iv) STK11^MUT;KEAP1^MUT (MUT, MUT; indicated in black) nsNSCLC. The analysis was limited to the subset of patients with available comprehensive NGS profiling that included both STK11 and KEAP1 (N = 145). d. Kaplan–Meier estimates of PFS (left) and OS (right) with CP in patients with advanced (i) STK11^MUT vs STK11^WT and (ii) KEAP1^MUT vs KEAP1^WT nsNSCLC. e. Kaplan–Meier estimates of PFS (left) and OS (right) with CP in patients with advanced (i) STK11^WT;KEAP1^WT (WT,WT; indicated in blue); (ii) STK11^MUT;KEAP1^WT (MUT,WT; indicated in red); (iii) STK11^WT;KEAP1^MUT (WT,MUT; indicated in purple) and (iv) STK11^MUT;KEAP1^MUT (MUT, MUT; indicated in black) nsNSCLC. The analysis was limited to the subset of patients with available comprehensive NGS profiling that included both STK11 and KEAP1 (N = 222). f. Best overall response with CP in the indicated patient subgroups. The analysis was limited to the subset of response-evaluable patients with available ORR data and comprehensive NGS profiling that included both STK11 and KEAP1 (N = 180).

Extended Data Fig. 2. Clinical outcomes in patient subgroups in the POSEIDON clinical trial.

a. PFS (left) and OS (right) with DCT in patient subgroups defined by clinical and molecular characteristics. HRs and 95% CIs were estimated using unstratified Cox proportional hazards models. The analysis of PFS was based on a data cut-off date of July 24, 2019 and the analysis of OS was based on a data cut-off date of March 12, 2021. b. Spider plots, depicting patient-level % change compared to baseline in the size of target lesion(s) (per RECIST v1.1) in patients with STK11 and/or KEAP1-mutated nsNSCLC treated with TDCT (top), DCT (middle) and CT (bottom). Individual trajectories are colour-coded based on best overall response. Only patients with both a baseline and at least one available post-baseline target lesion measurement are included. ORR and mDoR are based on confirmed objective responses by BICR. The analysis was based on a data cut-off date of July 24, 2019. c. OS in molecularly defined subgroups of patients with STK11^MUT and/or KEAP1^MUT metastatic nsNSCLC treated with TDCT vs CT (left) and TDCT vs DCT (right). HRs and 95% CIs were estimated using unstratified Cox proportional hazards models. The analysis was based on a data cut-off date of March 12, 2021.

Extended Data Fig. 3. Clinical outcomes in patients with STK11-mutated or KEAP1-mutated nsNSCLC in the phase III POSEIDON clinical trial.

a,b. Kaplan–Meier estimates of PFS according to BICR per RECIST v1.1 (a) and OS (b) with TDCT (light blue curve) vs DCT (dark blue curve) vs CT (red curve) in patients bearing STK11^MUT (left panel) and STK11^WT (right panel) metastatic nsNSCLC. Landmark 12-month PFS rates and 24-month OS rates in each of the treatment arms are also shown (dotted lines). PFS analyses were based on a data cut-off date of July 24, 2019. OS analyses were based on a data cut-off date of March 12, 2021. HRs and 95% CIs were estimated using unstratified Cox proportional hazards models. c. HR for OS and PFS with D + T + CT or D + CT versus CT in nsNSCLC subgroup with KEAP1 alterations.

Extended Data Fig. 4. Clinical outcomes in patients with KRAS-mutated nsNSCLC in the phase III POSEIDON clinical trial.

a. Kaplan–Meier estimates of PFS according to BICR per RECIST v1.1 with TDCT (light blue curve) vs DCT (dark blue curve) vs CT (red curve) in patients bearing KRAS^MUT (left panel) and KRAS^WT (right panel) metastatic nsNSCLC. Landmark 12-month PFS rates in each of the treatment arms are also shown (dotted lines). b,c. Kaplan–Meier estimates of PFS according to BICR per RECIST v1.1 (b) and OS (c) with TDCT vs DCT vs CT in patients bearing KRAS^MUT; STK11^MUT and/or KEAP1^MUT (left panel) and KRAS^MUT; STK11^WT and KEAP1^WT (right panel) metastatic nsNSCLC. PFS and ORR analyses were based on a data cut-off date of July 24, 2019. OS analyses were based on a data cut-off date of March 12, 2021. HRs and 95% CIs were estimated using unstratified Cox proportional hazards models.

Extended Data Fig. 5. Effect of distinct co-mutations on tumour growth and immune checkpoint inhibitor response in immune-competent models of KRAS-mutant NSCLC.

a. Therapeutic schedule and cohort size of ICB treatment study experimental groups. b. Mouse lung weight in the four treatment arms. Dots represent individual mice. c. Tumour growth effects of individual co-alterations. Tumours at the indicated percentiles of the tumour size distribution for each barcoded Lenti-sgRNA/Cre vector are shown, with 95% CIs. d,e. RTN score reflecting sensitivity to anti-PD-1 monotherapy (d) and dual anti-PD-1/anti-CTLA4 therapy (e) compared with isotype control IgG-treated mice. Significant effects are highlighted in colour. f. Experimental strategy to evaluate the anti-tumour activity of single or dual ICB with or without platinum doublet chemotherapy in the K and KLK (clone 17) isogenic allograft models. g. Efficacy of (chemo)-immunotherapy encompassing single (anti-PD-1) or dual (anti-PD-1/anti-CTLA4) ICB in the K and KLK isogenic models (N = 6-7 mice/group). Comparison of tumour volume (TV) between treatment arms in the KLK model was performed when the first mouse in any treatment group reached a TV of ≥ 1200 mm³. In the K model, comparison of tumour volume was performed when the second mouse across the entire cohort reached a TV of ≥ 1200 mm³, to account for the presence of a single allograft tumour with an atypical growth pattern in the IgG control group (rapid tumour growth over a 2-day interval – this mouse was included in the analysis and censored at the time of death). h. Evaluation of anti-tumour activity of distinct combination immunotherapies in the KL5 allograft model (N = 7 mice/group). Comparison of tumour volume was performed at the time point where the first mouse in any treatment group reached a TV ≥ 1200 mm³. The Mann–Whitney U test was used for pairwise statistical comparisons. Error bars represent standard deviation from the mean. Statistical significance is indicated at the P ≤ 0.05 (*), P ≤ 0.01 (**), and P ≤ 0.001 (***) levels. Source Data

Extended Data Fig. 6. Characterization of the STK11^MUT and/or KEAP1^MUT NSCLC TIME.

a. FACS-based enumeration of immune cell subsets in Keap1- deficient (K7,K8), Stk11-deficient (L6,L9) or isogenic Keap1 and Stk11-proficient LKR10 allograft tumours. Error bars indicate standard deviation from the mean (N = 7-8 mice/group). b,c. FACS-based assessment of T cell subsets in the immune microenvironment of syngeneic Keap1-deficient (KK) (b) or Keap1- and Stk11-deficient (KLK) (c) Kras^G12D-mutant allograft tumours compared with isogenic Keap1 and Stk11-proficient (K) tumours (all models were derived from the LKR13 mouse LUAD cell line, with CRISPR/Cas9-mediated editing of the corresponding genomic loci). Error bars indicate standard deviation from the mean (N = 8 mice/group). The Mann–Whitney U test was used for comparison of the abundance of immune cell subsets. Statistical significance in all panels is indicated at the P ≤ 0.05 (*), P ≤ 0.01 (**), and P ≤ 0.001 (***) levels. d. Uniform manifold approximation and projection (UMAP) plot of immune cells derived from untreated K, KK and KLK allograft tumours and processed for scRNA-seq (N = 2 mice/group). e. Bar plot depicting the relative abundance of distinct immune cell subsets (as a % of all non-tumour cells) in each isogenic model. f. Representation of scRNA-seq-derived neutrophil/T cell (top panel), myeloid/T cell (middle panel) and T_H1 CD4+/CD8+ T cell (bottom panel) ratios across the K, KK and KLK isogenic models. g. Proportions and average M1 or M2 scores (top panel) and N1 or N2 scores (bottom panel) in the monocyte/macrophage or neutrophil compartments in the K, KK and KLK models. h,i. UMAP visualization (h) and average expression levels (i) of Fcgr4 mRNA expression in the myeloid compartment demonstrating enrichment in Stk11 and/or Keap1-deficient models. Source Data

Extended Data Fig. 7. Characterization of the STK11^MUT and/or KEAP1^MUT NSCLC TIME in clinical cohorts.

a. Multicolour immunofluorescence (mIF) analysis of surgically resected early-stage human nsNSCLC confirms a higher ratio of CD11b+/CD8+ cells (N = 13, STK11^MUT and/or KEAP1^MUT and N = 19, STK11^WT and KEAP1^WT) and lower abundance of CD8+ T cells (N = 14; STK11^MUT and/or KEAP1^MUT and N = 27; STK11^WT and KEAP1^WT) in STK11^MUT and/or KEAP1^MUT NSCLC. The Mann–Whitney U test was used for statistical comparisons. b. Inferred neutrophil: T lymphocyte and macrophage: T lymphocyte ratios in STK11 and/or KEAP1-mutant LUAD in the TCGA dataset. The Kruskal–Wallis test was used for the three-group statistical comparisons. c. Immune contexture of LKB1-deficient (N = 22) versus LKB1-proficient (N = 35) (top row) and NRF2^High (N = 8) versus NRF2^Low (N = 49) (bottom row) nsNSCLC in the ICON cohort, based on previously validated gene expression signatures. The Mann–Whitney U test was used for statistical comparisons and P ≤ 0.05 was considered statistically significant. d. xCell-based digital deconvolution of the tumour immune microenvironment in the ICON cohort of surgically resected nsNSCLC (N = 8, STK11^MUT;KEAP1^MUTorWT; N = 10, STK11^WT;KEAP1^MUT;N = 39, STK11^WT;KEAP1^WT). Each box indicates the interquartile range (IQR) with the median and whiskers indicate the upper and lower values within 1.5 times the IQR. The Kruskal–Wallis test was used for the three-group statistical comparison and P ≤ 0.05 was considered statistically significant. e. RNA-seq-based deconvolution of the STK11^MUT and or KEAP1^MUT TIME in the TCGA PanCancer Atlas lung adenocarcinoma cohort. Left to right: ImmuneScore (assessed by xCell); CD8+ T cells (evaluated by QuanTIseq); non-Treg CD4+ T cells (QuanTIseq); and T_H1 signature (xCell). Each box indicates the interquartile range (IQR) with the median and whiskers indicate the upper and lower values within 1.5 times the IQR. The Kruskal–Wallis H test was used for statistical comparisons. Source Data

Extended Data Fig. 8. Dual anti-PD-1/anti-CTLA4 blockade reshapes the immune contexture of Stk11- and/or Keap1-deficient models of KRAS-mutant NSCLC.

a. FACS-based assessment of single and dual ICB-induced changes in the abundance of distinct T cell (left panels) and myeloid cell (right panels) subsets in the microenvironment of Stk11-deficient (KL5) and Keap1-deficient (KK) models. Error bars indicate standard deviation from the mean (N = 7-8 mice/group). The Mann–Whitney U test was used for pairwise comparisons of the abundance of immune cell subsets. Statistical significance is indicated at the P ≤ 0.05 (*), P ≤ 0.01 (**), and P ≤ 0.001 (***) levels. b. ICB-induced changes in the abundance of distinct T cell and dendritic cell subsets in the microenvironment of KLK, KK and KL5 models assessed by FACS. Error bars indicate standard deviation from the mean (N = 7-8 mice/group). c. FACS-based quantification of lymphoid and myeloid cell subsets in KL2 allograft tumours in response to single or dual ICB. Error bars indicate standard deviation from the mean (N = 7-8 mice/group). The Mann–Whitney U test was used for comparison of the abundance of immune cell subsets. Statistical significance in all panels is indicated at the P ≤ 0.05 (*), P ≤ 0.01 (**), and P ≤ 0.001 (***) levels. Source Data

Extended Data Fig. 9. Treatment-induced immune modulation in the TIME of Stk11- and/or Keap1-deficient models of KRAS-mutant NSCLC.

a,b, FACS-based enumeration of lymphoid (a) and myeloid cell (b) subsets in isogenic K (LKR13 Kras^G12D-mutant; Stk11/Keap1 WT) and KLK (Kras^G12D-mutant, Stk11/Keap1 knockout, clone 17) allograft models as well as in the KL5 (Kras^G12C-mutant Stk11-deficient) allograft model (N = 3-5 mice/group for the K and KLK models and N = 6-7 mice/group for the KL5 model). The Mann–Whitney U test was used for pairwise statistical comparisons. Error bars represent standard deviation from the mean. Statistical significance is indicated at the P ≤ 0.05 (*), P ≤ 0.01 (**), and P ≤ 0.001 (***) levels. Source Data

Extended Data Fig. 10. Immune depletion studies.

a. Effective immune depletion of CD4+ or CD8+ T cells with anti-CD4+ and anti-CD8+ antibodies, respectively. Error bars indicate standard deviation from the mean (N = 3 mice/group). b. Spider plots indicating individual LKR13 KLK allograft tumour volume trajectories in response to the indicated therapies (N = 7-8 mice/group). Source Data

Extended Data Fig. 11. The randomized phase III POSEIDON clinical trial.

a, Clinicogenomic characteristics of mutation-evaluable patients with advanced nsNSCLC in the POSEIDON clinical trial. b, POSEIDON clinical trial schema. c, Mutation-evaluable population among patients with nsNSCLC. d, Venn diagram indicating overlap of somatic mutations in KRAS, STK11 and KEAP1 in the POSEIDON dataset.