Molecular and Pathologic Characterization of YAP1-Expressing Small Cell Lung Cancer Cell Lines Leads to Reclassification as SMARCA4-Deficient Malignancies

Abstract

Purpose: The classification of small cell lung cancer (SCLC) into distinct molecular subtypes defined by ASCL1, NEUROD1, POU2F3, or YAP1 (SCLC-A, -N, -P, or -Y) expression, paves the way for a personalized treatment approach. However, the existence of a distinct YAP1-expressing SCLC subtype remains controversial.

Experimental design: To better understand YAP1-expressing SCLC, the mutational landscape of human SCLC cell lines was interrogated to identify pathogenic alterations unique to SCLC-Y. Xenograft tumors, generated from cell lines representing the four SCLC molecular subtypes, were evaluated by a panel of pathologists who routinely diagnose thoracic malignancies. Diagnoses were complemented by transcriptomic analysis of primary tumors and human cell line datasets. Protein expression profiles were validated in patient tumor tissue.

Results: Unexpectedly, pathogenic mutations in SMARCA4 were identified in six of eight SCLC-Y cell lines and correlated with reduced SMARCA4 mRNA and protein expression. Pathologist evaluations revealed that SMARCA4-deficient SCLC-Y tumors exhibited features consistent with thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT). Similarly, the transcriptional profile SMARCA4-mutant SCLC-Y lines more closely resembled primary SMARCA4-UT, or SMARCA4-deficient non-small cell carcinoma, than SCLC. Furthermore, SMARCA4-UT patient samples were associated with a YAP1 transcriptional signature and exhibited strong YAP1 protein expression. Together, we found little evidence to support a diagnosis of SCLC for any of the YAP1-expressing cell lines originally used to define the SCLC-Y subtype.

Conclusions: SMARCA4-mutant SCLC-Y cell lines exhibit characteristics consistent with SMARCA4-deficient malignancies rather than SCLC. Our findings suggest that, unlike ASCL1, NEUROD1, and POU2F3, YAP1 is not a subtype defining transcription factor in SCLC. See related commentary by Rekhtman, p. 1708.

Figure 1.

SMARCA4 mutations are enriched within SCLC-Y cell lines. A, Venn diagram showing co-occurring and subtype-specific mutations associated with each SCLC molecular subtype. Genetic mutations that are present in at least 50% of samples within each SCLC subtype based on Rudin and colleagues (2) were identified through cBioportal. There are 26 mutations exclusive to SCLC-Y cell lines (Supplementary Table S7). B, Of all 26 mutations identified, SMARCA4 mutations were present in six of eight SCLC-Y cell lines, together with TP53 mutations on an RB1 wild-type background. *, H157DM was previously annotated in CCLE as H1339. C, All cell lines in the CCLE (lung and nonlung cancer cell lines) that had complete proteomic and RNA-seq profiles were interrogated for the correlation between SMARCA4 protein and mRNA. There is a positive correlation (Pearson correlation = 0.53; P = 1.5e−27) between low SMARCA4 mRNA and loss of SMARCA4 protein. D, SCLC cell lines with SMARCA4 mutations have a significantly lower NE-score compared with wild-type (WT) SMARCA4 SCLC cell lines (Mann–Whitney test; **, P = 0.0068). SCLC cell lines in the CCLE were binned into SMARCA4 mutant and WT groups. NE scoring was performed using a previously published 50-gene transcriptomic signature (16).

Figure 2.

SMARCA4-deficient SCLC cell lines are morphologically and immunophenotypically similar to SMARCA4-UT. A, Outline of the methodology employed for assessing SMARCA4-deficient SCLC cell lines by a panel of five anatomical pathologists. B, Representative H&E images of cell line xenografts diagnosed as high-grade NE carcinoma (SCLC) and other malignant neoplasms (non-SCLC). Scale bar = 100 μm. C, Heatmap of re-evaluated diagnosis of SCLC cell line xenografts (row) and each pathologists’ classification (column). Abbreviations: LCNEC, large cell neuroendocrine cancer; SMARCA4-UT, SMARCA4-deficient undifferentiated tumor; SCLC/LCNEC, combined SCLC and LCNEC components; Other, undifferentiated tumor or small blue round cell tumor.

Figure 3.

Distinct immunophenotype of SMARCA4-mutant compared with SMARCA4-WT SCLC cell lines. Representative IHC images of SMARCA4, synaptophysin (SYN), CD56, TTF1, INSM1, and RB1 in SMARCA4-WT, NE-high SCLC cell line (H1092) compared with SMARCA4-mutant SCLC cell lines. Scale bar = 100 μm.

Figure 4.

SCLC-Y cell lines with SMARCA4 loss share a similar transcriptome to SMARCA4-UT. A, Unsupervised hierarchical clustering, based on differentially expressed genes in primary SMARCA4-UT compared with primary SMARCA4-mutant NSCLC identified by Le Loarer and colleagues (; n = 758 genes), was performed on SCCOHT and lung carcinoma samples [cell lines from the CCLE and bulk-RNA sequenced patient samples from Le Loarer and colleagues (24)]. Inset: SMARCA4-UT cluster from A with cell line and sample annotation. B, mRNA expression of SMARCA4, SMARCA2, and CLDN4 (claudin-4) in cell lines of the SMARCA4-UT cluster, SMARCA4 mutant NSCLC, SMARCA4 proficient NSCLC, and SCLC-A, -N, -P (Supplementary Table S5). Kruskal–Wallis test with a Dunn multiple comparison test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, P ≥ 0.05. C, IHC of SMARCA2 and claudin-4 in SMARCA4-WT and SMARCA4-mutant cell line xenografts. Scale bar = 100 μm.

Figure 5.

YAP1 is expressed in SMARCA4-UT primary samples. A, Unsupervised hierarchical clustering, based on a SCLC-specific YAP1 signature (n = 49 genes) identified by Pearsall and colleagues (10), was performed on SCCOHT and lung carcinoma samples [cell lines from the CCLE and bulk-RNA sequenced patient samples from Le Loarer and colleagues (24)]. Cell lines that cluster with the SMARCA4-UT samples are highlighted in the gray box. B, YAP1 IHC on SCLC-A/N/P and SMARCA4-mutant cell line xenografts. Scale bar = 100 μm. C, H&E, SMARCA4, and YAP1 IHC in primary SMARCA4-UT samples (n = 6) together with an SCLC patient sample. Scale bar = 100 μm.