ASCL1 is a lineage oncogene providing therapeutic targets for high-grade neuroendocrine lung cancers

Abstract

Aggressive neuroendocrine lung cancers, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), represent an understudied tumor subset that accounts for approximately 40,000 new lung cancer cases per year in the United States. No targeted therapy exists for these tumors. We determined that achaete-scute homolog 1 (ASCL1), a transcription factor required for proper development of pulmonary neuroendocrine cells, is essential for the survival of a majority of lung cancers (both SCLC and NSCLC) with neuroendocrine features. By combining whole-genome microarray expression analysis performed on lung cancer cell lines with ChIP-Seq data designed to identify conserved transcriptional targets of ASCL1, we discovered an ASCL1 target 72-gene expression signature that (i) identifies neuroendocrine differentiation in NSCLC cell lines, (ii) is predictive of poor prognosis in resected NSCLC specimens from three datasets, and (iii) represents novel "druggable" targets. Among these druggable targets is B-cell CLL/lymphoma 2, which when pharmacologically inhibited stops ASCL1-dependent tumor growth in vitro and in vivo and represents a proof-of-principle ASCL1 downstream target gene. Analysis of downstream targets of ASCL1 represents an important advance in the development of targeted therapy for the neuroendocrine class of lung cancers, providing a significant step forward in the understanding and therapeutic targeting of the molecular vulnerabilities of neuroendocrine lung cancer.

Keywords: ASCL1 transcriptome; personalized therapy; target discovery.

Fig. 1.

ASCL1 is highly expressed in a subset of NSCLC cell lines. (A) Unsupervised clustering analysis of genome-wide expression analysis performed on 206 lung cell lines including NSCLCs, SCLCs, and normal immortalized HBECs/HSAECs. (B) Known TP53, KRAS, RB1, and EGFR mutations for the putative NE-NSCLC cohort. (C) Log2 expression differences between NE-NSCLC and NSCLC lines plotted against –log10(P value). ASCL1 is noted. A total of 69 genes fall within the cutoff of ± log2 >5.0 and P < 0.001. (D) Supervised clustering of NSCLC cell lines using the 69 most significantly differentially expressed genes. (E) Log10 relative mRNA expression of ASCL1 in lung cancer cell lines measured by qRT-PCR. (F) Protein expression of ASCL1 in lung cancer cell lines. These data were collected from three separate gels.

Fig. 2.

ASCL1 is required for the survival of NE-NSCLC cell lines. (A) siRNA-mediated knockdown of ASCL1 reduces relative mRNA expression of ASCL1 and DLL1 in NCI-H1755 and HCC1833 cells, as measured by qRT-PCR. n.s., not significant. n = 3. *P < 0.05; **P < 0.01; ***P < 0.005. (B) siRNA-mediated knockdown reduces ASCL1 protein expression in NCI-H1755 and HCC1833 cells compared with controls. (C) Long-term stable shRNA-mediated ASCL1 knockdown inhibits the colony-forming ability of NE-NSCLC cell lines compared with typical NSCLC lines. Quantification of colony-forming ability after 14 d in culture is shown. n = 3 wells for shNTC and shASCL1. ***P < 0.005; ****P < 0.0001. (D) ASCL1 knockdown induces apoptosis in NCI-H1755 and HCC1833 cell lines, as measured by cell cycle analysis of the sub-G1 population. NCI-H1993 is unaffected by siASCL1. n = 3 for each group; t test performed between SCR and siASCL1-1, -2, and -3. *P < 0.05; **P < 0.01; ***P < 0.005. (E) Loss of ASCL1 in NCI-H1755 increases Annexin-V positivity, as measured by flow cytometry. (F) ASCL1 knockdown induces cleavage of PARP in NCI-H1755 and HCC1833 cells compared with controls. (G) siRNA screen comparing cell death phenotype in NE-NSCLC, NSCLC, and normal HBEC lung cell lines after knockdown of ASCL1, SOX2, and TTF1. The z-scores were calculated based on control experiments and color-coded to visualize cell death and growth, respectively. NE, NE-NSCLC lines; NL, normal immortalized HBEC line.

Fig. 3.

IHC analysis for ASCL1 performed on adenocarcinoma and squamous cell carcinoma samples. (A) Representative slides showing ASCL1 nuclear staining in adenocarcinoma and squamous cell carcinoma samples from tissue microarray and whole- section IHC. Negative samples are displayed as a reference. (B) Table displaying the number of tumor samples staining positive for ASCL1. (C) Overall survival was not significantly different between ASCL1(+) tumors and ASCL1(−) tumors.

Fig. 4.

ASCL1 ChIP-Seq analysis delineates downstream targets. (A) Base coverage plot of the 912 consensus peaks in all five ASCL1(+) cell lines. The base coverage in a 1,000-bp window is centered on the summits of the 912 consensus peaks collected at a 10-bp resolution and averaged for each cell line. The vertical axis is the normalized average base coverage of the consensus peaks, and the horizontal axis is the position relative to the summit. (B) Venn diagram showing peak-level overlap for all five ASCL1(+) samples along with the E-box binding site for each sample, as well as the consensus E-box binding site. Peak-level heterogeneity between samples is evident. (C) The 72-gene ASCL1 expression signature clustering analysis groups 9/10 of the NE-NSCLC cell lines separate from the typical NSCLC lines. (D) The ASCL1-associated 72-gene signature correlates with poor survival in retrospective analyses of lung adenocarcinoma patient datasets. Patients more likely to express the gene signature have poorer 5-y outcomes. (Upper) P < 0.0001, Gehan–Breslow–Wilcoxon test. (Lower) P < 0.0302, Gehan–Breslow–Wilcoxon test.

Fig. 5.

BCL2 is a conserved ASCL1 target with therapeutic implications. (A) Consensus E-box binding sites for ASCL1 are present in all ASCL1(+) samples within the BCL2 second intronic region. (B) Knockdown of ASCL1 by siRNA reduces BCL2 mRNA in NCI-H1755 and HCC1833 cell lines. n = 3. *P < 0.05; **P < 0.01; ***P < 0.005. (C) siRNA-mediated reduction of ASCL1 reduces BCL2 protein in NCI-H1755 and HCC1833. (D) Knockdown of BCL2 does not affect ASCL1 protein expression. (E) BCL2 knockdown by siRNA induces cell death in NE-NSCLCs, as measured by cell cycle analysis of the sub-G1 phase. n = 3 for each group. **P < 0.01; ****P < 0.0001). (F) Treatment of cell lines with BCL2 inhibitor ABT-263 for 24 h demonstrates specificity for ASCL1(+) NE-NSCLC cell lines. n = 3 for each group. *P < 0.05; **P < 0.01; ***P < 0.005. (G) ABT-263 treatment induces dose-dependent cleavage of PARP and Caspase 3 after 12 h of treatment in NCI-H1755. (H) Xenografts established from NCI-H1993 and NCI-H1755 were treated with 100 mg/kg ABT-263 or vehicle control for 14 consecutive days after tumors reached an average size of ∼250 mm³. n = 3 per treatment group per cell line. ***P < 0.001. (I) Photograph of resected s.c. tumors from ABT-263–treated xenografts. (J) Weight of H1993 and H1755 xenograft tumors treated with ABT-263 after resection. ***P < 0.001.