Intrinsic Immunogenicity of Small Cell Lung Carcinoma Revealed by Its Cellular Plasticity

Abstract

Small cell lung carcinoma (SCLC) is highly mutated, yet durable response to immune checkpoint blockade (ICB) is rare. SCLC also exhibits cellular plasticity, which could influence its immunobiology. Here we discover that a distinct subset of SCLC uniquely upregulates MHC I, enriching for durable ICB benefit. In vitro modeling confirms epigenetic recovery of MHC I in SCLC following loss of neuroendocrine differentiation, which tracks with derepression of STING. Transient EZH2 inhibition expands these nonneuroendocrine cells, which display intrinsic innate immune signaling and basally restored antigen presentation. Consistent with these findings, murine nonneuroendocrine SCLC tumors are rejected in a syngeneic model, with clonal expansion of immunodominant effector CD8 T cells. Therapeutically, EZH2 inhibition followed by STING agonism enhances T-cell recognition and rejection of SCLC in mice. Together, these data identify MHC I as a novel biomarker of SCLC immune responsiveness and suggest novel immunotherapeutic approaches to co-opt SCLC's intrinsic immunogenicity. SIGNIFICANCE: SCLC is poorly immunogenic, displaying modest ICB responsiveness with rare durable activity. In profiling its plasticity, we uncover intrinsically immunogenic MHC Ihi subpopulations of nonneuroendocrine SCLC associated with durable ICB benefit. We also find that combined EZH2 inhibition and STING agonism uncovers this cell state, priming cells for immune rejection.This article is highlighted in the In This Issue feature, p. 1861.

©2021 American Association for Cancer Research.

Figures

Figure 1.

MHC Class I expression is increased in patient SCLC with non-neuroendocrine features, which is enriched in exceptional responders to immune checkpoint blockade. A, Frequency distribution of MHC Class I (MHC I) expression expressed as H-score in patient SCLC tumors (n=102). B, Representative micrographs of H&E and MHC I IHC in (top) MHC I low/negative (MHC Ilo/neg) and (bottom) MHC I uniformly high (MHC Ihi) patient SCLC tumors. Scale bar indicates 100 μm. C, Gene Set Enrichment Analysis (GSEA) based on the RNAseq expression fold-change values of MHC Ihi vs. MHC Ilo/neg patient SCLC tumors. Bar plot represents normalized enrichment scores (NES) for the top upregulated Hallmark gene. D, Representative micrographs of AXL IHC in (top) MHC Ilo/neg and (bottom) MHC Ihi patient SCLC tumors. Scale bar indicates 50 μm. E, (left) Representative multiplexed immunofluorescence images of MHC Ilo/neg and MHC Ihi patient SCLC tumors displaying expression of indicated proteins. (right) Quantification of ASCL1 and chromogranin expression by H-score in MHC Ihi and MHC Ilo/neg patient SCLC tumors. Error bars indicate mean ± s.e.m. F, (left) Representative multiplexed immunofluorescence images of MHC Ilo/neg and MHC Ihi patient SCLC tumors displaying intratumoral infiltration of immune cells marked by expression of indicated proteins. (right) Digital image quantification of indicated immune cell subsets in MHC Ihi and MHC Ilo/neg patient SCLC tumors intratumorally (tumor) or at the tumor/stroma interface (tumor/stroma). G, Immunophenotypic characterization of tumors and associated clinical courses of a SCLC patient with an exceptional response to immune checkpoint blockade (ICB) therapy. Clinical stage at diagnosis is indicated as limited stage (LS). Next-generation sequencing (NGS) results indicating loss-of-function variants or deletions of key SCLC tumor suppressor genes are listed. NED = no evidence of disease. H, Kaplan-Meier curves displaying overall survival of SCLC patients from initiation of immune checkpoint blockade (ICB), stratified by tumor MHC I IHC expression. P values were calculated using a two-tailed Mann-Whitney test (E, F), or log-rank test (H). *, P < 0.05; **, P < 0.01.

Figure 2.

Modeling plastic SCLC cell states in vitro reveals immunologically distinct SCLC subpopulations. A, (left) Phase contrast images of suspension H69 and adherent H69M SCLC cell lines. (right) Bivariate plot of normalized H3K27Me3 and H3K27Ac signal intensity in H69M cells at all gene loci derived from RNAseq data set. Scale bars indicate 100 μm. B, Volcano plot for genes showing significantly increased H3K27Ac and decreased H3K27Me3 indicated in red in (A) in MHC Ihi vs. MHC Ilo/neg patient SCLC tumors. C, Flow cytometric analysis of HLA-ABC and PD-L1 expression on H69 and H69M (representative of n=3 independent experiments). D, E, Gene Set Enrichment Analysis (GSEA) based on the expression fold-change values in H69M PD-L1/MHC Ihi vs. H69M PD-L1/MHC Ilo samples. Bar plot (D) represents normalized enrichment scores (NES) for the top Hallmark gene sets and neuroendocrine gene set (27), and GSEA plots for IFN-γ response and neuroendocrine (27) gene sets are shown in (E). Arrows mark rank positions of indicated genes. F, (top) Schematic of pan-HLA I immunopeptidome analysis. (bottom) Mass spectrum of HLA I-complexed peptides recovered from H69 cells at the point in the chromatogram where the signal peptide LLDVPTAAV elutes (molecular ion at m/z 449.76 marked by yellow arrow). Minor co-eluting ions are chemical background. G, Mass spectrum of HLA I-complexed peptides recovered from H69M and H69M PD-L1/MHC Ihi cells shown at the elution position of the LLDVPTAAV signal peptide (yellow arrow). Red arrows indicate positions of doubly charged ions corresponding to distinct co-eluting HLA-I complexed endogenously processed (TAP-dependent) peptides. H, Paired elution positions of shared peptides detected in different samples forms an elution line, a mark of correct identification. Elution positions of a reference set of HLA-A2.01-restricted peptides in H69M PD-L1/MHC Ihi cells as a function of the elution positions of this set detected in an HLA-A2.01+ epithelial line and H69M PD-L1/MHC Ihi cells (top) vs. H69 cells (bottom). Yellow arrows indicate paired elution positions of known signal peptides. Adjusted P values were calculated using the al Benjamini & Hochberg (BH) method (E).

Figure 3.

EZH2 inhibition promotes the non-neuroendocrine antigenic SCLC state. A, (left) Flow cytometric analysis of HLA-ABC expression on the indicated suspension, neuroendocrine (blue) and adherent, non-neuroendocrine (red) SCLC cell lines (representative of n=2 independent experiments) and (right) associated qRT-PCR relative quantification (RQ) values of TAP1 expression normalized to H69 cells. B, Immunoblot of TAP1, H3K27Me3, and B-actin levels in indicated suspension, neuroendocrine (blue) and adherent, non-neuroendocrine (red) SCLC cell lines. C, Bivariate plot of Log2 fold change in mRNA expression (normalized by robust multi-array analysis [RMA]) of TAP1 and EZH2 in suspension and adherent SCLC cell lines from the CCLE. D, Flow cytometric analysis of HLA-ABC and AXL expression in H69 cells treated with GSK126 (5 μM) +/− 24 h IFN-γ. E, (left) Schematic of non-neuroendocrine cell state change induced by transient EZH2 inhibition in neuroendocrine SCLC via 6 d GSK126 (5 μM) pulse followed by long term sub-culture without drug. Schematic created with BioRender.com. (right) Flow cytometric analysis of HLA-ABC and AXL expression in indicated stable cell lines at basal conditions (representative of n=2 independent experiments). F, (top) qRT-PCR of TAP1 in H69EZ-G normalized to H69 cells (RQ), representative of n=2 independent experiments. Error bars are mean ± s.e.m. of n=2 biological replicates. (bottom) Immunoblot of TAP1, H3K27Me3, and B-actin levels in H69 cells and HLA-ABC-positive (+) or negative (–) fractions sorted from H69EZ-G cells. G, Gene Set Enrichment Analysis (GSEA) based on the expression fold-change values of MHC Ihi vs. MHC Ilo/neg patient SCLC tumors. Bar plot represents normalized enrichment scores (NES) for the top Hallmark gene sets, SPARCS gene set (13), neuroendocrine gene set (27). H, CXCL10 secretion by indicated suspension, neuroendocrine (blue) and adherent, non-neuroendocrine (red) SCLC cell lines upon 24 h treatment with indicated concentrations of ADU-S100. Error bars are mean ± s.e.m of n=3 biological replicates. I, (left) Histogram of percent HLA+ cells in indicated H69M cell lines, as measured by flow cytometry. Error bars are mean ± s.e.m of n=3 biological replicates. (right) Representative flow cytometric analysis of HLA-ABC expression in indicated cell lines. J, Schematic of postulated de-repression of MHC Class I expression in non-neuroendocrine SCLC cells, and proposed therapeutic activation of this pathway in neuroendocrine SCLC cells with combined EZH2 inhibition and STING agonism. Schematic created with BioRender.com. P values were calculated using an unpaired two-tailed Student’s t test (F, I) or Bonferroni corrected pairwise comparisons after an ANOVA global test (H). **, P < 0.01; ****, P < 0.0001; n.s., not significant. R2 and associated p value (C) represents Pearson correlation coefficient.

Figure 4.

Non-neuroendocrine SCLC cells are immunogenic in vitro and in vivo. A, Flow cytometric analysis of MHC Class I allele (H2-Kb and H2-Db) expression on indicated SCLC cell lines (representative of n=5 independent experiments). B, Specific lysis of RPP.OVA or RPP-A.OVA cells after 48 h co-culture with activated CD8+ T cells from OT-I mice at indicated effector:target (E:T) ratios (representative of n=2 independent experiments). Each data point represents mean ± s.e.m. of triplicate wells. C, Tumor volume measurements of RPP and RPP-A cells after s.c. inoculation into syngeneic C57/BL6 mice. Each data point represents mean ± s.e.m. tumor volumes (n=5 in RPP group and n=5 in RPP-A group). D, UMAP plots displaying unsupervised clustering of single-cell RNA sequencing data for CD45+ cells from RPP and RPP-A tumors formed from (C) at day 14. Labels indicate cell assignment based on comparison of cluster gene expression signatures from Han et al.(59) CD8 and CD4 T cells are indicated in each tumor. E, Frequency of top 10 TCR clonotypes in RPP and RPP-A tumors. The α/β chains of the immunodominant TCR in RPP-A tumors, as well as the β chain shared among clonotypes, are indicated by arrows. F, (top) Schematic of tumor cell-T cell co-culture, performed at a stimulator:responder (S:R) ratio of 1:1. (bottom) IL-2 secretion from 72 h co-cultures of RPP (blue bars) or RPP-A (red bars) cells +/− 24 h IFN-γ (100 ng/mL) stimulation and no (nil), parental untransduced BW5147.3 (P), or Trav14D-3-DV8; Trbv29 transgenic BW5147.3 (TCR) T cells. Black bars represent P or TCR T cells alone without (–) or with (+) PMA (50 nM) and Ionomycin (1 μg/mL). ^ = above assay threshold of detection. Error bars are mean ± s.e.m of n=4 independent experiments. G, (top) Phase contrast image of RPP-EZ SCLC cell line. Scale bar indicates 100 μm. (bottom) IL-2 secretion from 72 h co-cultures of RPP-EZ cells +/− 24 h IFN-γ (100 ng/mL) or IFN-β (100 ng/mL) stimulation and no (nil), parental untransduced BW5147.3 (P), or Trav14D-3-DV8; Trbv29 transgenic BW5147.3 (TCR) T cells. Black bars represent P or TCR T cells alone without (–) or with (+) PMA (50 nM) and Ionomycin (1 μg/mL). ^ = above assay threshold of detection. Error bars are mean ± s.e.m of n=3–5 independent experiments. H, Tumor volume measurements of RPP-EZ or RPP cells after subcutaneous inoculation into syngeneic C57/BL6 mice followed by intratumoral vehicle or ADU-S100 treatments (first treatment, 50 μg; second treatment, 35 μg) on indicated days (day 19/23 for Cohort 1 [n=7, darker shade shade with squares]; day 21/24 for Cohort 2 [n=3 mice per group; lighter shade with triangles]). The number of responders, defined as minimal residual tumors that are not resolved for the duration of the study and/or no measurable tumors for more than 7 doubling times, is indicated for each group. I, Kaplan-Meier curves displaying survival of mice from (H). P values were calculated using a log-rank test (C, I) and chi-square test (H). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.