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 3

Lineage infidelity and nonphysiologic gene and enhancer activity in SI-NETs.

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Lineage infidelity and nonphysiologic gene and enhancer activity in SI-N...
See also Supplemental Figures 3 and 4. (A) Relative expression of EC and non-EC marker genes (averaged across respective scRNA-seq clusters, left 2 columns) in bulk RNA-seq data averaged across SI-NETs (right column) from group A (n = 82 after excluding outliers with overt non-EC features). (B) Examples of EC (e.g., NR5A2, ARC) and non-EC (e.g., ISL1, ARX) markers that are absent and non-EC genes that are expressed (e.g., VSTM2L and CPLX2, shown in the feature plot, others in the dot plot) in scRNA-seq data from SI-NET cells. (C) Differential gene expression distinguishes normal EC from non-EC cells. Although the EC phenotype dominates in SI-NETs, the tumors express many mRNA features of non-EC cells. (D) Relative expression of SI-NET enriched markers (Supplemental Figure 3C) in bulk RNA-seq data during normal EEC differentiation and median expression across 82 group A tumors. (E) Histone modifications H3K27ac (ChIP-seq, n = 10 SI-NET samples) and H3K4me3 (ChIP-seq, n = 3 SI-NETs) and open chromatin (ATAC-seq, n = 3 SI-NETs) at H3K27ac-marked genomic sites identified in ≥3 of 10 tumors. Signals are plotted ±3 kb from ATAC summits. (F) Sorting of H3K27ac-marked sites by signal rank reveals enrichment of enhancers near genes ordinarily expressed in normal ISCs (red; KLF2, KCNK5, etc., as represented below during normal EEC differentiation and in group A SI-NETs), Sec-pro (black; HES1, SOX4, ETS2, IRF1, etc.), and terminal EECs (blue; CHGA, RFX6, NKX2-2, etc.). The box-and-whisker plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median.