Integrative epigenome and transcriptome analyses reveal transcriptional programs differentially regulated by ASCL1 and NEUROD1 in small cell lung cancer

Small cell lung cancer (SCLC), an aggressive neuroendocrine carcinoma, has an extremely poor prognosis. ASCL1 and NEUROD1 are key regulators of neuroendocrine features, and previous studies have suggested that SCLC plasticity occurs during the transition from ASCL1-positive (SCLC-A) to NEUROD1-positive (SCLC-N) subtypes. In this study, we attempted to understand the transcriptional programs governed by ASCL1 and NEUROD1 to identify markers of SCLC plasticity. Immunohistochemistry and epigenome and transcriptome analyses in ASCL1/NEUROD1 double-positive SCLC cells (SCLC-A/N) revealed co-expression of ASCL1 and NEUROD1 in almost half of SCLC cases. Genome-wide profiling of histone modifications, ASCL1 and NEUROD1 binding sites, and gene co-expression patterns revealed that both ASCL1 and NEUROD1 are active in SCLC-A/N and regulate partially distinct target genes. Furthermore, SCLC-A/N exhibited characteristics that were intermediate between SCLC-A and SCLC-N subtypes. NEUROD1 knockout, followed by RNA-seq, suggested an association between NEUROD and NHLH transcription factors that might shape the NEUROD1-mediated regulatory network. Small RNA-seq further indicated that miR-139-5p is specifically expressed in NEUROD1-positive SCLC, and transcriptomic studies suggested that miR-139-5p might regulate an array of pathologically relevant genes in collaboration with other NEUROD1-associated miRNAs. Our integrative analyses provide deeper insights into SCLC heterogeneity and multi-layered transcriptional programs differentially governed by ASCL1 and NEUROD1.