Multiomic analyses delineate human neuroendocrine tumor cell states in relation to normal enteroendocrine cell ontogeny
Pratik N.P. Singh, Elsa Hadj Bachir, James R. Howe, Andrew M. Bellizzi, Paloma Cejas, Shariq Madha-Krause, Charles B. Epstein, Jennifer A. Chan, Bradley Bernstein, Matthew H. Kulke, Qiao Zhou, Ramesh A. Shivdasani
Pratik N.P. Singh, Elsa Hadj Bachir, James R. Howe, Andrew M. Bellizzi, Paloma Cejas, Shariq Madha-Krause, Charles B. Epstein, Jennifer A. Chan, Bradley Bernstein, Matthew H. Kulke, Qiao Zhou, Ramesh A. Shivdasani
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Research Article Development Gastroenterology Oncology

Multiomic analyses delineate human neuroendocrine tumor cell states in relation to normal enteroendocrine cell ontogeny

Abstract

Cancers reflect aberrant growth and differentiation of normal cell populations. Biological understanding of small intestine neuroendocrine tumors (SI-NETs) is hampered because their closest normal counterparts, enteroendocrine cells (EECs), constitute tiny fractions of intestinal epithelium. Recent characterization of adult human EEC ontogeny from intestinal stem cells can help overcome that limitation. Transient expression of the transcription factor gene ASCL1 normally ensures proper timing and fidelity of well-differentiated EECs, which express NEUROD1. Here, we report that SI-NETs resembled mature enterochromaffin cells; however, individual tumor cells coexpressed stem/progenitor genes, harboring each differentiation state along the EEC trajectory except ASCL1+ precursors. We found that enhancers normally active, and others inactive, during EEC differentiation underlie aberrant SI-NET gene activity. SI-NETs uniformly expressed NEUROD1 but lacked ASCL1, owing to inaccessible chromatin and repressive H3K27me3 marking at the ASCL1 locus. Multiple cyclin-dependent kinase inhibitor (CDKi) genes were similarly silenced, other than CDKN1B, the only gene recurrently mutated in SI-NETs. Deletion of CDKN1B altered cell cycle kinetics during human EEC differentiation, and deletions of ASCL1 or CDKN1B activated certain genes that are expressed in SI-NETs but not in the normal EEC trajectory. We propose that a limited CDKi repertoire and absence of ASCL1-dependent constraints on EEC maturation together explain unique SI-NET characteristics.

Authors

Pratik N.P. Singh, Elsa Hadj Bachir, James R. Howe, Andrew M. Bellizzi, Paloma Cejas, Shariq Madha-Krause, Charles B. Epstein, Jennifer A. Chan, Bradley Bernstein, Matthew H. Kulke, Qiao Zhou, Ramesh A. Shivdasani

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Figure 4

Distinct classes of enhancers associated with SI-NET expression of stem/early precursor cell genes and absence of non-EC genes.

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Distinct classes of enhancers associated with SI-NET expression of stem/...
See also Supplemental Figure 5. (A) H3K27ac signals at enhancers, clustered based on chromatin accessibility (ATAC-seq, left, n = 3) and H3K27ac marking (right, n = 10). During normal EEC differentiation, type I sites (ISC-enriched, 7.1%) progressively lose accessibility and H3K27ac; type II sites (20.1%) are accessible throughout, but lack H3K27ac in any phase we examined by ChIP-seq; type III sites (35.3%) are inaccessible and lack H3K27ac; type IV sites (15.1%) open and acquire H3K27ac progressively; and type V sites (22.4%) carry marks of enhancer activity throughout. DFCI, Dana-Farber Cancer Institute. (B) IGV tracks illustrating enhancers of types I, II, and III near genes enriched in normal ISCs (D0-1, compared with mature normal EECs, D4-8) that are expressed in SI-NETs. ATAC-seq data represent all samples (n = 3); H3K27ac data represent 3 of 10 samples. (C) Chromatin accessibility and H3K27ac marks in reference EECs and SI-NETs at PITX2 and CDH17, representative genes highly expressed in SI-NETs. The PITX2 locus has enhancers of types II (accessible but lacking H3K27ac in normal EEC differentiation) and III (de novo recruitment in SI-NETs); the CDH17 locus has a type II enhancer. (D) Features of active SI-NET enhancer types in ISCs and mature EECs. (E) Integrated and merged UMAP of scATAC-seq derived cells (n = 18,954) 24 hours, 72 hours, 96 hours, 120 hours, and 144 hours after NEUROG3 activation. The 11 cell clusters correspond to scRNA-seq defined states (Supplemental Figure 2E) by transfer of gene anchors. Right and below: type IV sites accessible in SI-NETs are not specific to any EEC subtype differentiated in vitro. (F) Chromatin accessibility and H3K27ac marks in normal EEC differentiation and SI-NETs at representative non-EC loci. TF genes ISL1, ARX, and PAX6 carry H3K27me3 (3 samples representing n = 5 are shown) and lack H3K27ac in SI-NETs, whereas normal cells show progressively increased accessibility and H3K27ac over the course of EEC differentiation.