Insights into intestinal neuroendocrine tumors in relation to normal human EEC differentiation [ATACseq]

Mutationally silent small intestinal neuroendocrine tumors (SI-NETs) characterized by few recurrent alterations, such as those that inactivate the cyclin-dependent kinase (CDK) inhibitor CDKN1B in <10% of cases are not well defined at the molecular level. Since the native enteroendocrine cells (EECs), which these tumors resemble, are scarce; the lack of reference data from normal EECs limits identification of gene and cis-element dysregulation in SI-NETs. We established transcriptional landscapes (RNA-seq) in bulk cultures and at single-cell (sc) resolution during the trajectory of normal human EEC differentiation from ileal stem cells in vitro (PMID 38733993). We compared the findings in SI-NETs against the novel reference profiles of normal human EEC differentiation and maturation. SI-NETs correspond principally to mature EC cells, with discernible features of non-EC and progenitor cells; persistent expression of the latter genes likely contributes to malignant properties. Underlying this aberrant expression pattern we found diverse SI-NET enhancers. EECs normally differentiate through an intermediate HES6hi/ASCL1+ oscillatory state before NEUROD1+ pre-terminal cells emerge; ASCL1 expression then persists only in mature EC cells. In contrast, although SI-NETs express NEUROD1, they strictly lack ASCL1 and other genes normally co-expressed with ASCL1. Notably, ASCL1 and loci encoding transcription factors that define non-EC cell types (ISL1, ARX, PAX6, PDX1) are marked with H3K27me3, signifying epigenetic silencing. SI-NETs express CDKN1B but other CDK inhibitors, e.g., CDKN2A and CDKN1C, are epigenetically silenced, suggesting that tumor cells may especially depend on wild-type CDKN1B function. Chromatin and transcriptional features of SI-NETs, revealed for the first time with respect to normal EEC differentiation, will help identify tumorigenic pathways and candidate therapeutic targets.