eIF5B drives integrated stress response-dependent translation of PD-L1 in lung cancer

Abstract

Cancer cells express high levels of PD-L1, a ligand of the PD-1 receptor on T cells, allowing tumors to suppress T cell activity. Clinical trials utilizing antibodies that disrupt the PD-1/PD-L1 checkpoint have yielded remarkable results, with anti-PD-1 immunotherapy approved as first-line therapy for lung cancer patients. We used CRISPR-based screening to identify regulators of PD-L1 in human lung cancer cells, revealing potent induction of PD-L1 upon disruption of heme biosynthesis. Impairment of heme production activates the integrated stress response (ISR), allowing bypass of inhibitory upstream open reading frames in the PD-L1 5' UTR, resulting in enhanced PD-L1 translation and suppression of anti-tumor immunity. We demonstrated that ISR-dependent PD-L1 translation requires the translation initiation factor eIF5B. eIF5B overexpression, which is frequent in lung adenocarcinomas and associated with poor prognosis, is sufficient to induce PD-L1. These findings illuminate mechanisms of immune checkpoint activation and identify targets for therapeutic intervention.

Extended Data Fig. 1.. Validation of positive and negative regulators of PD-L1 identified in the CRISPR-Cas9 screen.

a, Flow cytometry analysis of cell surface PD-L1 in H358 cells expressing control sgRNA or sgRNAs targeting CMTM6, SMAD4, DPAGT1 or DNAJC13. b, Western blot analysis confirming loss of target protein and a decrease in PD-L1 in cells from (a). Experiments in a and b were repeated two independent times with similar results, data from a representative experiment are shown. Validation was performed with an independent sgRNA for CMTM6 and SMAD4. c, Western blot analysis confirming loss of target protein and an increase in PD-L1 in cells from Fig 1f. Experiments were performed three independent times with similar results, data from a representative experiment are shown.

Extended Data Fig. 2.. Effects of heme synthesis inhibition on PD-L1 mRNA and protein, and analysis of Pd-l1 mRNA and tumors from LLC cells.

a, Western blot analysis in Calu-6 and H2030 human lung cancer cell lines expressing the indicated sgRNAs. b, Western blot analysis in cells treated with 10 mM succinyl acetone (ALAD inhibitor) for 48h. c, qRT-PCR analysis of PD-L1 mRNA relative to ACTIN, in cells from (b). Error bars represent SDs from the mean for n=3 independent experiments. A student’s two-tailed t-test was used to assess statistical significance with ***p = 0.0002. d, Western blot analysis of PD-L1 in H1944 cells treated with 50 μM or 100 μM N-Methyl Protoporphyrin IX (FECH inhibitor) for 48h. e, qRT-PCR analysis of PD-L1 mRNA in cells from (d). Data represent mean PD-L1 mRNA expression normalized to ACTIN across n=3 technical replicates, shown as individual data points. All experiments in a-b and d-e were performed two independent times, with similar results. Data from representative experiments are shown. f, qRT-PCR analysis of Pd-l1 mRNA normalized to Actin in LLC cells expressing control or Urod shRNA. Bar graph represents mean normalized Pd-l1 mRNA from n=3 technical replicates, shown as individual data points from a representative experiment. Experiment were performed two independent times with similar results. g, Representative images of LLC tumors expressing control or Urod shRNA. This experiment was repeated twice with similar results. h, Gating strategy for TIL staining of tumors. Cells were gated based on SSC and FSC. Singlet cells were then gated for APC-Cy7-CD45⁺ and eFLUOR V500⁺ dead cells were excluded. i, Representative gating for CD4⁺ and CD8⁺ cells in one tumor from each experimental group. CD45⁺ live cells from each group was obtained by gating as in (h) and then gated for FITC-CD8⁺ and PE-CD4⁺ cells to obtain % CD8⁺ (of CD45⁺ cells).

Extended Data Fig. 3.. Urod depletion induces an immune suppressive and pro-tumorigenic transcriptional program in LLC cells.

a-b, Gene Set Enrichment Analysis highlighting expression of top gene sets significantly increased in LLC cells expressing Urod shRNA. c, Ingenuity Pathway Analysis demonstrating a decrease in expression of genes involved in immune cell recruitment and migration. Graph represents biological processes plotted against their bias adjusted z-scores. Transcriptional analysis was performed on n=3 biological (cell culture) replicates per group.

Extended Data Fig. 4.. Investigating the mechanism of PD-L1 regulation in UROD depleted cells.

a, qRT-PCR analysis of PD-L1 mRNA decay in H1944 cells expressing control or UROD sgRNA after treatment with 10 μM Actinomycin D in n=3 technical replicates per time point, across 4 time points. b, Quantification of PD-L1 levels, determined by western blot, in H1944 cells expressing control or UROD sgRNA after treatment with 20 μM cycloheximide across 5 time points. c, Immunoprecipitation of endogenous PD-L1 in H1944 cells transfected with control or UROD siRNA treated with 10 μM Velcade for 24 hours. Protein abundance and ubiquitylation monitored by western blot. Experiment in (c) was performed two independent times, with similar results. Data from a representative experiment are shown.

Extended Data Fig. 5.. The ISR pathway regulates PD-L1 expression independently of IFN-γ.

a, Western blot analysis in H1944 cells expressing control or UROD sgRNA after treatment with 200 nM ISRIB for 24 h. b, Western blot analysis of UROD in H358 and H1944 cells treated with 10ng/mL IFN-γ for 24h. c, Western blot analysis of PD-L1 in H1944 cells treated with DMSO or 100uM Salubrinal for 24h +/− 10ng/mL IFN-γ (left) or 10uM Ruxolitinib (right). d, Western blot analysis of PD-L1 in H1944 cells expressing a control or UROD sgRNA +/− 10ng/mL IFN-γ (left) or 10uM Ruxolitinib (right). All experiments were performed three independent times with similar results. Representative data are shown.

Extended Data Fig. 6.. ISR pathway activation enhances PD-L1 translation, and regulation of the PD-L1 5′ UTR reporter by UROD depletion.

a, qRT-PCR analysis of mean Pd-l1 mRNA, normalized to Actin, in S/S or A/A MEFs expressing either control or Urod shRNA with 3 technical replicates per sample. Experiment was performed two independent times with similar results. Data from a representative experiment are shown. b,c, qRT-PCR analysis of PD-L1 mRNA in 6 individual ribosomal fractions, with 3 technical replicates per sample for two independent primer pairs. Quantification of combined fractions (<3 and >3) from (b) are shown in main Fig. 5c. d,e, qRT-PCR analysis of PD-L1 mRNA in 6 individual ribosomal fractions, with 3 technical replicates per sample for two independent primer pairs. Quantification of combined fractions (<3 and >3) from (d) are shown in main Fig. 5d. f,g, qRT-PCR analysis of ATF4 mRNA in ribosomal fractions from main Fig. 5a (f) or main Fig. 5b (g). ATF4 mRNA abundance was calculated as described in main Fig. 5c and d. Error bars in f and g represent SDs from the mean from three independent fractions (<3 or >3 ribosomes). A student’s two-tailed t-test was performed to determine statistical significance. ***p=0.00062 (f); ***p=0.0008 (g). h,i, qRT-PCR analysis of ATF4 mRNA in 6 individual ribosomal fractions. j, qRT-PCR analysis of mean Luciferase mRNA, normalized to Actin, in MEF cells transfected with reporter constructs shown in main Fig. 5e, with 3 technical replicates, shown as individual data points. Experiment was performed two independent times, with similar results. Data from a representative experiment are shown. k, qRT-PCR analysis of mean Luciferase mRNA normalized to ACTIN in H358 cells from main Fig. 5g across n=3 technical replicates, shown as individual data points. l, qRT-PCR analysis of mean Luciferase mRNA normalized to Actin in MEF cells from main Fig. 5h across n=3 technical replicates, shown as individual data points. Experiment in (k) was performed in an independent cell line, shown in (l).

Extended Data Fig. 7.. eIF5B is necessary for PD-L1 upregulation in UROD depleted human lung cancer cells and the KLN205 syngeneic mouse model.

a, Flow cytometry analysis of cell-surface PD-L1 in independent human lung cancer cells. Bar graph represents Mean Fluorescence Intensity of cell-surface PD-L1 in PD-L1 Low (Calu-6, H2030) and PD-L1 High (H358, H441, Hop62) cell lines. b, Western blot analysis of PD-L1 and eIF5B in Calu-6 and H2030 cells expressing a control or UROD sgRNA transfected with control or EIF5B siRNA. c, Western blot analysis of PD-L1 and eIF5B in H441, H358 and Hop62 cells expressing a control or UROD sgRNA transfected with control or EIF5B siRNA. Experiments in b-c were performed two independent times with similar results. Data from a representative experiment are shown. d, Western blot analysis of PD-L1 in KLN205 cells expressing a Scrambled shRNA or two independent Urod shRNAs. e, Western blot analysis of PD-L1, UROD, and eIF5B in KLN205 cells expressing a control shRNA or Urod shRNA with and without Eif5b shRNA. Experiments in d and e were performed two independent times with similar results. Data from a representative experiment are shown. f, Quantification of tumor volumes of KLN205 cells shown in (e) transplanted to syngeneic DBA/2 mice (n=8 mice for Scrambled shRNA, n=5 for Eif5b shRNA, n=9 for Urod shRNA and n=8 for Urod + Eif5b shRNA). Linear mixed model was used to assess statistical significance, as described in Methods. p value (Scrambled vs Urod shRNA) = 0.0002, p value (Urod shRNA vs Urod + Eif5b shRNA) = 4.9351e-05.

Extended Data Fig. 8.. EIF5B depletion reduces cell proliferation without affecting global translation of human lung cancer cells.

a, MTS cell proliferation assay measuring proliferation of H358, H1944, H2030, Calu-6 and H441 cells transfected with control or EIF5B siRNA with n=3 biological replicates. Student’s t-test with Holm-Sidak adjustment was performed per time point to assess statistical significance. ** = p<0.01; *** = p<0.001; **** = p<0.0001. Adjusted p values were as follows for Day 1, 2, 3, 4 and 5 respectively. H358: 0.74, 0.096, 0.064, 0.013 and 0.0001, H1944: 0.495, 0.044, 0.042, 0.0449, 0.018, H2030: 0.801, 0.068, 0.06, 0.039, 0.008, Calu6: 0.57, 0.42, 0.002, 0.016 and 0.008, H441: 0.258, 0.079, 0.01, 0.0002 and 0.012. b, Autoradiography analysis of newly synthesized S-35 labeled proteins in cells from (a). Experiments were performed two independent times for H358 and H1944 cells with similar results. Data from a representative experiment are shown.

Extended Data Fig. 9.. Meta-analysis of EIF5B expression correlated with survival across lung cancer datasets.

a, Oncoprint plot depicting mRNA upregulation, amplifications, and mutations in EIF5B in human lung adenocarcinoma samples from the TCGA, Firehose Legacy. b, Kaplan-Meier survival analysis of patients with or without EIF5B alterations shown in (a) from the TCGA Firehose Legacy lung adenocarcinoma study using cBioPortal. n= 230 patients, Log rank test p-value = 0.0238. c, Summary of meta analyses results. d, Forest plot of meta-analysis showing association between EIF5B mRNA expression and patient survival outcome in all lung cancer patients across different studies (26 studies with 4,528 patients in total). e, Forest plot showing association between EIF5B expression and patient outcome in lung adenocarcinoma patients across different studies (20 studies with 2,882 patients in total). f, forest plot showing no association between EIF5B and patient survival in lung squamous cell carcinoma patients (26 studies with 1,277 patients in total). The confidence interval was calculated based on two-sided tests.

Extended Data Fig. 10.. Luciferase mRNA levels from the PD-L1 5′ UTR reporter with EIF5B overexpression.

a, qRT-PCR analysis of mean Luciferase mRNA normalized to ACTIN in cells from Fig. 6i across three technical replicates, shown as individual data points. b, qRT-PCR analysis of Luciferase mRNA in cells from Fig. 6j, normalized to Actin across three technical replicates, shown as individual data points.

Fig. 1.. A genome-wide CRISPR-Cas9 screen identifies regulators of PD-L1 in lung cancer cells.

a, Overview of CRISPR-Cas9 screening approach. b,c, Detection of loss (b) or gain (c) of PD-L1 in H358 cells by flow cytometry in cells expressing a sgRNA targeting PD-L1 (b) or cells treated with 10ng/mL IFN-γ for 24h (c). Data from a single experiment are shown in (b) and (c) and are representative of two independent experiments with 3 independent sgRNA with similar results. RIGER analysis identified positive (d) and negative (e) regulators of PD-L1. CD274 (PD-L1) is highlighted in red. Samples subject to RIGER analysis had cell culture replicates (n=2) as well as library replicates (GeCKO A and B). Genes with bold diamonds were selected for validation. Blue diamond indicates genes involved in mitochondrial function. RIGER analysis is described in Methods. f, Flow cytometry analysis of cell-surface PD-L1 in H1944 cells expressing Cas9 and a control sgRNA or sgRNA targeting MUL1, UQCR10, MRPS12, or UROD.

Fig. 2.. Impairment of heme biosynthesis induces PD-L1 in NSCLC cells.

a, Western blot analysis in H1944 cells with control or UROD sgRNA. For this and all subsequent westerns, Tubulin served as a loading control. Data from a single experiment are shown in (a) and are representative of three independent experiments with two independent sgRNAs with similar results. b, Quantitative real-time PCR (qRT-PCR) analysis of PD-L1 mRNA in cells shown in (a). Bar graphs represent PD-L1 mRNA expression normalized to ACTIN and error bars represent SDs from the mean across n=3 independent experiments. A student’s two-tailed t-test was performed to determine statistical significance. ***p =0.0008. c, Schematic of the heme biosynthesis pathway. d, Levels of heme synthesis in control or UROD siRNA-treated H1944 cells 72 hours after transfection. Bar graphs represent heme levels normalized to total protein as pmol/mg and error bars represent SDs from the mean. This experiment was performed one time with n=3 three biological replicates. A student’s two-tailed t-test was performed to determine statistical significance. *** p = 0.004. e, Western blot analysis in H1944 cells after transfection with the indicated siRNAs. f, Western blot analysis in H1944 cells stably expressing Cas9 and the indicated sgRNAs with and without exogenous heme supplementation for 48h. Data from a single experiment are shown in e and f and are representative of two independent experiments with similar results.

Fig. 3.. Loss of UROD accelerates tumorigenesis in vivo by suppressing CD8⁺ T-cells.

a, Western blot analysis in Lewis Lung Carcinoma (LLC) cells expressing doxycycline-inducible control shRNA or two independent shRNAs targeting Urod. Data from a single experiment are shown and are representative of two independent experiments with similar results. b,c, Quantification of tumor volumes of LLC cells expressing the indicated shRNAs in immunodeficient NSG mice (n=12 mice per group, p value = 0.3542) (b) or in syngeneic C57BL/6 mice (c) (n=7 mice Scrambled shRNA, n=12 mice Urod shRNA-1 and n=11 mice Urod shRNA-2). p value (Scrambled vs Urod shRNA-1) = 1.016513e-06, p value (Scrambled vs. Urod shRNA-2) = 0.0063. d, Quantification of CD8⁺ tumor infiltrating lymphocytes (TILs, expressed as a percentage of CD45⁺ cells). A Two-Sample Fisher-Pitman Permutation Test was used to compare CD8⁺ TILs between groups. n= 6 Scrambled shRNA and Urod shRNA tumors each, n=4 Scrambled + αPD-1 tumors and n= 5 Urod shRNA+ αPD-1 tumors. p value (Scrambled shRNA vs Urod shRNA) = 0.0038, p value (Urod shRNA vs Urod shRNA+ αPD-1) = 0.0029. e,f, Quantification of tumor volumes in syngeneic C57BL6 mice after treatment with a CD8⁺ depleting antibody (200ug, IP every 3 days) (n= 8 Scrambled mice, n= 6 Urod shRNA mice, p-value = 0.1491) (e) or αPD-1 antibody (200ug, IP every 3 days) (n = 8 Scrambled mice, n= 7 Scrambled + αPD-1 mice, n= 12 Urod shRNA mice, n=12 Urod shRNA+ + αPD-1 mice). p value (Scrambled shRNA vs Urod shRNA) =1.533e-06, p value (Urod shRNA vs Urod shRNA + αPD-1) = 1.7034e-05. (f). For all tumor growth experiments (b-c and e-f), graphs represent mean tumor volumes and error bars represent SDs from the means. A linear mixed effect model was used to determine statistical significance for all tumor growth assays, as described in Statistics and Reproducibility.

Fig. 4.. Integrated Stress Response (ISR) pathway activation induces PD-L1 protein in NSCLC.

a, Western blot analysis of peIF2α and total eIF2α in H1944 cells expressing a control sgRNA or a sgRNA targeting UROD. b, Autoradiography analysis of newly synthesized S-35 labeled proteins in H1944 cells transfected with control or UROD siRNAs, with three biological replicates per sample. Wildtype (WT) cells treated with 20uM cycloheximide (CHX) for 1 hour prior to metabolic labeling served as positive control. c, Quantification of S-35 Met/Cys signal from (b) normalized to control siRNA cells. Error bars represent SDs from the mean, a two-tailed student t test was performed to assess statistical significance. ***p value = 0.0008. d, Western blot analysis in H1944 cells treated with Salubrinal (in μM) for 48h. e, Western blot analysis in H1944 cells treated with 100 μM Salubrinal and/or 200 nM ISRIB for 24 hours. f, Western blot analysis of peIF2α and PD-L1 in cells cultured in normoxic or hypoxic conditions for 24h and 48h. g, Quantitative real-time PCR analysis of PD-L1 mRNA in cells shown in (f). h, Western blot analysis of peIF2α, total eIF2α, and PD-L1 in cells treated with Arsenite (100uM for H1944 cells, 50uM for Calu-6 and 5uM for H358 cells) for 24h. i, Quantitative real-time PCR analysis of PD-L1 mRNA in cells shown in (h). Bar graph represents PD-L1 mRNA expression normalized to ACTIN across three technical replicates, shown as individual data points. j, Western blot analysis in eIF2α wildtype (S/S) or ser51Ala mutant (A/A) cells expressing control or Urod shRNA. Experiments in a-j were performed two independent times with similar results, and data from a representative experiment are shown.

Figure 5.. ISR activation enhances PD-L1 translation.

a,b, Polysome analysis of siRNA-treated (a) or Salubrinal-treated (100 μM, 24 hours) (b) H1944 cells. c, qRT-PCR analysis of PD-L1 mRNA in ribosomal fractions from (a). Experiment was performed with two independent primer pairs with similar results. d, qRT-PCR analysis of PD-L1 mRNA in ribosomal fractions from (b). Fractions associated with <3 ribosomes were grouped together to represent poorly translated mRNAs and fractions with >3 ribosomes were grouped as efficiently translated mRNAs. PD-L1 mRNA expression in each fraction was normalized to Luciferase and PD-L1 mRNA abundance was calculated as the percent of total PD-L1 in all fractions. Luciferase mRNA control was added to each fraction prior to RNA extraction to control for variability in total RNA in fractions during the RNA isolation and reverse transcription reactions. Error bars in c and d represent SDs from the mean from three independent fractions (<3 or >3 ribosomes). A student’s two-tailed t-test was performed to determine statistical significance. *p=0.017; **p= 0.001. Data from individual ribosomal fractions for two independent primer pairs are provided in Extended Data Fig. 6b-e. e, Schematic of the wildtype human PD-L1 5´ UTR with five upstream CTGs cloned upstream of a Firefly luciferase reporter. f, Dual-luciferase assay of MEF cells transfected with the indicated firefly luciferase reporter constructs normalized to co-transfected control Renilla luciferase reporter. Error bars represent SDs from the mean for n=3 biological replicates. A student’s two-tailed t-test was performed to determine statistical significance. **** = p<0.0001 for all constructs except M1 (p=0.14). Data from a single experiment are shown in (f) and are representative of three independent experiments with similar results. g, Dual luciferase reporter analysis of the PD-L1 5′ UTR in H358 cells expressing a control or UROD sgRNA. Error bars represent SDs from the mean relative luciferase activity (Firefly/Renilla) across n=3 biological replicates per group. A student’s two-tailed t-test was performed to assess statistical significance with *p = 0.015. h, Dual luciferase reporter analysis of PD-L1 5’ UTR in MEFs cells with a control or Urod shRNA. Error bars represent SDs from the mean relative luciferase activity (Firefly/Renilla) across n=3 biological replicates per group. A student’s two-tailed t-test was performed to assess statistical significance with **p = 0.0075. Experiments in (g) and (h) were performed two independent times with similar results. Data from a representative experiment are shown.

Fig. 6.. ISR-dependent translation of PD-L1 requires the alternative translation initiation factor eIF5B.

a,b,c, Western blots of H1944 cells expressing the indicated sgRNAs and transfected with siRNAs targeting EIF2D (a), EIF2A (b), or EIF5B (c). d, Quantification of western blots shown in a-c normalized to protein levels in control siRNA-treated cells. e, Western blots of LLC cells expressing the indicated shRNAs targeting Urod, or Eif5b. All experiments (a-c and e) were performed two independent times with similar results, data shown are from a representative experiment. f, Quantification of tumor volumes of LLC cells expressing the indicated shRNAs in syngeneic C57BL/6 mice. Tumor volumes of individual mice are shown (n=12 Scrambled shRNA, n=7 Eif5b shRNA, n=11 Urod shRNA and n=6 Eif5b + Urod shRNA mice). p value (Scrambled shRNA vs Eif5b shRNA = 0.0251, p value (Scrambled shRNA vs Urod shRNA) = 4.075e-10, p value (Urod shRNA vs Urod + Eif5b shRNA) = 8.666e-12. A linear mixed effect model was used to determine statistical significance, as described in Statistics and Reproducibility. g, Western blot analysis in H1944 cells expressing eGFP or EIF5B cDNA. h, qRT-PCR analysis of EIF5B and PD-L1 in H1944 cells shown in g. Bar graph represents mean relative expression of EIF5B and PD-L1 from 3 technical replicates, with individual data points plotted. Experiments in g and h were performed two independent times with similar results, data shown are from a representative experiment. i, Dual luciferase reporter analysis of the PD-L1 5′ UTR in H358 cells with transient overexpression of a control eGFP or EIF5B cDNA. Error bars represent SDs from the mean relative luciferase activity (Firefly/Renilla) across n=3 biological replicates/group. A student’s two-tailed t-test was performed to assess statistical significance with **p = 0.005. j, Dual luciferase reporter analysis of the PD-L1 5′ UTR in MEFs expressing a control eGFP or EIF5B cDNA. Error bars represent SDs from the mean relative luciferase activity (Firefly/Renilla) across n=3 biological replicates/group. A student’s two-tailed t-test was performed to assess statistical significance with ****p = 0.000083. Experiments in i and j were performed two independent times with similar results. Data from representative experiments are shown. k, Model of translational control of PD-L1 in response to ISR activation. Under normal conditions, a functional ternary complex consisting of eIF2α bound to GTP and Met-tRNA initiates translation of PD-L1 at the canonical AUG. Inhibitory upstream open reading frames (uORFs) suppress translation at the canonical AUG, limiting PD-L1 translation. Phosphorylation of eIF2α and ISR activation induce bypass of inhibitory uORFs in the human PD-L1 5′ UTR. In the absence of a functional ternary complex, eIF5B can substitute for eIF2α, culminating in enhanced PD-L1 translation and suppression of anti-tumor immunity.