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 5

SI-NETs express NEUROD1 but not ASCL1, and epigenetic features account for repression of the ASCL1+ state.

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SI-NETs express NEUROD1 but not ASCL1, and epigenetic features account f...
See also Supplemental Figure 6. (A) In EEC precursors, HES6hi and ASCL1+ cell states oscillate before NEUROD1+NKX2-2+ preterminal EECs emerge. (B) SI-NET (scRNA-seq primary [Pri] and metastatic [Met] cells) expression of genes enriched at successive steps in EEC differentiation, illustrating persistence of progenitor genes and notable paucity of EEC precursor (ASCL1+HES6hi) markers. (C) Specific genes ordinarily expressed in oscillating ASCL1+HES6hi cells are excluded from Pri or Met SI-NET cells, unlike genes that characterize ISCs, secretory/early EEC precursors, and mature EECs. (D) ASCL1 is absent and HES6 is barely detected in scattered cells in 3 SI-NETs (scRNA-seq). NKX2-2 and NEUROD1 are robustly expressed in mature normal EECs and SI-NET cells. (E) HES6, ASCL1, and genes coexpressed in normal EEC precursors — SOX2 and MYCL — are notably absent from group A SI-NETs (n = 85, bulk RNA-seq, normalized DeSeq2 counts), while NEUROD1 and NKX2-2 are expressed in all cases. Some group B tumors express ASCL1 and lack NEUROD1 or NKX2-2. Bar colors refer to institutional sources of samples. (F) Immunohistochemical validation of ASCL1 absence and NEUROD1 expression in SI-NET tissue microarrays. NEUROD1 immunostaining, seen in 87% of 79 SI-NETs, was typically strong (mean H-score, 177; median, 190); ASCL1 did not stain any of the 79 SI-NETs. Scale bars: 50 μm. (G) Chromatin accessibility and H3K27 marking in normal EEC differentiation and SI-NETs at key representative loci. Precursor markers HES6 and ASCL1 lack H3K27ac; instead, ASCL1 carries broad H3K27me3, indicating epigenetic silencing. Among ASCL1-synexpressed TF loci, SOX2 resembles ASCL1, while MYCL, also silent, has accessible H3K4me3+ promoter DNA but elsewhere lacks H3K27ac or H3K27me3. Similar to normal EECs, NEUROD1 has accessible chromatin and H3K27ac but not H3K27me3 marking. (H) Schematic summaries. Left: normal EEC differentiation. Right: SI-NETs, where ISC and Sec-pro states are less represented than the mature EEC state and absence of the ASCL1+ state may explain accelerated EC differentiation and presence of non-EC elements.