ASCL1 and NEUROD1 Reveal Heterogeneity in Pulmonary Neuroendocrine Tumors and Regulate Distinct Genetic Programs

Abstract

Small cell lung carcinoma (SCLC) is a high-grade pulmonary neuroendocrine tumor. The transcription factors ASCL1 and NEUROD1 play crucial roles in promoting malignant behavior and survival of human SCLC cell lines. Here, we find that ASCL1 and NEUROD1 identify heterogeneity in SCLC, bind distinct genomic loci, and regulate mostly distinct genes. ASCL1, but not NEUROD1, is present in mouse pulmonary neuroendocrine cells, and only ASCL1 is required in vivo for tumor formation in mouse models of SCLC. ASCL1 targets oncogenic genes including MYCL1, RET, SOX2, and NFIB while NEUROD1 targets MYC. ASCL1 and NEUROD1 regulate different genes that commonly contribute to neuronal function. ASCL1 also regulates multiple genes in the NOTCH pathway including DLL3. Together, ASCL1 and NEUROD1 distinguish heterogeneity in SCLC with distinct genomic landscapes and distinct gene expression programs.

Figure 1. ASCL1 and NEUROD1 define heterogeneity in hSCLC

(A) Microarray expression for ASCL1, NEUROD1 and neuroendocrine genes in hSCLC cell lines that define ASCL1^High and NEUROD^High subtypes (see Table S1). (B) Pearson correlation analysis with respect to ASCL1 or NEUROD1 expression in hSCLC cell lines. (C) RNA-Seq showing ASCL1 and NEUROD1 in hSCLC cell lines (see Table S2). (D) Known mutations in cell lines used in this study. (E) Heat map and dendrograms using ASCL1^High and NEUROD1^High gene signatures from hSCLC cell lines stratify human primary tumors by gene expression (see Table S3). Dendrogram on top reflects clustering of the primary tumors, dendrogram on left reflects the ASCL1^High and NEUROD1^High gene signatures. Blue shading indicates NEUROD1 related, red shading indicates ASCL1 related, and black identifies genes expressed in both subtypes of hSCLC.

Figure 2. ASCL1 and NEUROD1 binding are largely non-overlapping in hSCLC cell line genomes

(A) ChIP-Seq signal for ASCL1 (common to NCI-H889, NCI-H2107, and NCI-H128), NEUROD1 (common to NCI-H524 and NCI-H82), and H3K27Ac at these sites (see Table S4). Signals were quantified +/− 5 kb around the peak summits. (B) Stitched H3K27Ac enhancers plotted in increasing rank order based on ChIP-Seq signal minus the input signal. Super-enhancers (SE) are labeled in blue. (C) Venn diagram comparing NCI-H128 and NCI-H82 SE overlap (see Table S5, S6). (D) Bar plot showing the number of NCI-H128 H3K27Ac defined SE or enhancers (Enh) with at least one ASCL1 binding site, and NCI-H82 SE or Enh with at least one NEUROD1 binding site. (E) DNA motifs found in ASCL1 or NEUROD1 bound regions (HOMER). (F) Plots showing the distribution of motifs +/− 500 bp in ASCL1 and NEUROD1 unique binding sites and shared sites. (G) DNA motifs found in H3K27Ac bound regions in NCI-H128 and NCI-H82. Also see Fig. S1.

Figure 3. ASCL1 directly regulates known oncogenes in hSCLC

ChIP-Seq showing ASCL1 (A) or NEUROD1 (B) binding near oncogenes and lineage-specific genes in hSCLC cell lines. (C) qRT-PCR of ASCL1 targets in NCI-H889 cells infected with lentivirus containing shControl, or one of two shASCL1. Asterisk is significance to p<0.05, error bar is SEM by standard student T-test. (D) Quantification of the protein decrease from Western analysis of the knockdown experiment in (C).

Figure 4. ASCL1 and NEUROD1 have largely distinct transcriptional targets

Candidate direct targets of ASCL1 and NEUROD1 in SCLC, identified through gene expression profiling and ChIP-Seq (see Fig. S2, Table S7). (A) Fold change expression values of ASCL1 and NEUROD1 direct targets in ASCL1^High vs NEUROD1^High hSCLC cell lines (x-axis) and primary SCLC tumors (y-axis). (B) Spearman correlation values of ASCL1 and NEUROD1 direct target gene expression with respect to ASCL1 (x-axis) and NEUROD1 (y-axis) across 81 primary SCLC tumors. (C) Fold change expression values of ASCL1 and NEUROD1 direct target genes in 27 ASCL1^High vs 5 neutral (x-axis) and 6 NEUROD1^High vs 5 neutral SCLC cell lines (y-axis). Neutral SCLC are those with ASLC1^Low and NEUROD1^Low. ASCL1 unique targets (red), NEUROD1 unique targets (blue), and shared targets (green) are shown. (D) 640 ASCL1 and 443 NEUROD1 targets were identified with 49 of them shared. (E) There are 141 ASCL1 targets conserved between mouse and human SCLC models (see Fig. S2, Table S8, S9). (F) Examples of ASCL1, NEUROD1, and shared target genes in SCLC models highlighting TF, Notch pathway, and neuronal related genes (see Table S7 for details on ontology and pathway analysis). Genes highlighted in red are ASCL1 targets conserved between mouse and human SCLC (see Table S9).

Figure 5. ASCL1 but not NEUROD1, is required for tumor formation in a mouse model of high grade pulmonary NE tumors

(A) Alleles used for the mSCLC model referred to as the triple knockout, TCKO, after Cre recombination. CKO alleles of Ascl1 and Neurod1 are also shown. (B) Timeline for tumor induction. (C-E) H&E histology of lung sections from TCKO (C), TCKO;Ascl1^CKO (D) and TCKO;Neurod1^CKO (E) mice infected intratracheally with Adenoviral-Cre 25 weeks earlier. Note the lack of tumor when Ascl1 is absent. (C’-E’) High magnification of tissue in C-E. (F-G) Graphs show tumor load (F) and tumor count (G) in lungs from TCKO mice with Ascl1 and Neurod1 genotypes as indicated. N=10-16 as shown. Error bars indicate mean+/−SEM and ** indicates P<0.01 by one-way ANOVA. (H) PCR genotype for Neurod1 verifying tumors are mutant for Neurod1. Deleted allele is 600 bp, Flox allele is 850 bp and wild type is 750 bp. Also see Fig. S3.

Figure 6. ASCL1 but not NEUROD1 in normal lung NE cells and in mouse SCLC

(A-C) Adjacent lung sections from a TCKO mouse infected intratracheally with Adenoviral-CMV::Cre 25 weeks earlier. Neuroendocrine tumors are evident by H&E histology (A), expression of ASCL1 (B) and SYP (C). (A’-C’) High magnification of tissue in A-C. (D-F”) Immunofluorescence showing tumor cells identified by SYP (blue) or CGRP (blue) co-express ASCL1 (red) and but not NEUROD1 (red). (G) Immunofluorescence showing NEUROD1 in olfactory epithelium as a positive control for the antibody. (H) Western blots showing ASCL1 but not NEUROD1 in mouse SCLC. (I) RNA-Seq genomic tracks showing expression of Ascl1, Syp, Calca but not Neurod1 in mouse SCLC. (J) P40 Neurod1::Cre;Rosa^LSLtdTom mouse lung showing Neurod1-lineage cells (TOM+) are detected in neurons (TUJ1+) but not neuroendocrine cells (CGRP+) in NEBs. (K) TOM+ cells are detected in the cerebellum (Cb), a known Neurod1-lineage. (L) Quantification of the number of Neurod1-lineage (TOM+) cells detected in neuroendocrine cells in lungs of P0 and P40 mice.