Identifying regulators of aberrant stem cell and differentiation activity in colorectal cancer using a dual endogenous reporter system

Sandor Spisak; David Chen; Pornlada Likasitwatanakul; Paul Doan; Zhixin Li; Pratyusha Bala; Laura Vizkeleti; Viktoria Tisza; Pushpamali De Silva; Marios Giannakis; Brian Wolpin; Jun Qi; Nilay S Sethi

doi:10.1038/s41467-024-46285-w

Identifying regulators of aberrant stem cell and differentiation activity in colorectal cancer using a dual endogenous reporter system

Nat Commun. 2024 Mar 12;15(1):2230. doi: 10.1038/s41467-024-46285-w.

Authors

Sandor Spisak^#^{1

2}, David Chen^#^{1

3}, Pornlada Likasitwatanakul^#^{1

4

5

6}, Paul Doan^#^{1

5}, Zhixin Li^{1

5}, Pratyusha Bala^{1

5}, Laura Vizkeleti⁷, Viktoria Tisza², Pushpamali De Silva¹, Marios Giannakis^{1

4

5

8}, Brian Wolpin^{1

4

8}, Jun Qi⁹, Nilay S Sethi^{10

11

12

13}

Affiliations

¹ Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.
² Institute of Molecular Life Sciences, HUN-REN Research Centre for Natural Sciences, Budapest, Hungary.
³ Temerty Faculty of Medicine, University of Toronto, Toronto, ON, Canada.
⁴ Department of Medicine, Harvard Medical School, Boston, MA, USA.
⁵ Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard University, Cambridge, MA, USA.
⁶ Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
⁷ Department of Bioinformatics, Faculty of Medicine, Semmelweis University, 1094, Budapest, Hungary.
⁸ Gastrointestinal Cancer Center, Dana-Farber Cancer Institute, Boston, MA, USA.
⁹ Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.
¹⁰ Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. nilay_sethi@dfci.harvard.edu.
¹¹ Department of Medicine, Harvard Medical School, Boston, MA, USA. nilay_sethi@dfci.harvard.edu.
¹² Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard University, Cambridge, MA, USA. nilay_sethi@dfci.harvard.edu.
¹³ Gastrointestinal Cancer Center, Dana-Farber Cancer Institute, Boston, MA, USA. nilay_sethi@dfci.harvard.edu.

^# Contributed equally.

Abstract

Aberrant stem cell-like activity and impaired differentiation are central to the development of colorectal cancer (CRC). To identify functional mediators of these key cellular programs, we engineer a dual endogenous reporter system by genome-editing the SOX9 and KRT20 loci of human CRC cell lines to express fluorescent reporters, broadcasting aberrant stem cell-like and differentiation activity, respectively. By applying a CRISPR screen targeting 78 epigenetic regulators with 542 sgRNAs to this platform, we identify factors that contribute to stem cell-like activity and differentiation in CRC. Perturbation single cell RNA sequencing (Perturb-seq) of validated hits nominate SMARCB1 of the BAF complex (also known as SWI/SNF) as a negative regulator of differentiation across an array of neoplastic colon models. SMARCB1 is a dependency and required for in vivo growth of human CRC models. These studies highlight the utility of biologically designed endogenous reporter platforms to uncover regulators with therapeutic potential.

MeSH terms

Cell Differentiation / genetics
Colorectal Neoplasms* / genetics
Humans
RNA, Guide, CRISPR-Cas Systems*
Stem Cells / metabolism

Substances

RNA, Guide, CRISPR-Cas Systems

Abstract

MeSH terms

Substances

Grants and funding