A p300/GATA6 axis determines differentiation and Wnt dependency in pancreatic cancer models
Zheng Zhong, Nathan Harmston, Kris C. Wood, Babita Madan, David M. Virshup
Zheng Zhong, Nathan Harmston, Kris C. Wood, Babita Madan, David M. Virshup
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Research Article Oncology

A p300/GATA6 axis determines differentiation and Wnt dependency in pancreatic cancer models

Abstract

Wnt signaling regulates the balance between stemness and differentiation in multiple tissues and in cancer. RNF43-mutant pancreatic cancers are dependent on Wnt production, and pharmacologic blockade of the pathway, e.g., by PORCN inhibitors, leads to tumor differentiation. However, primary resistance to these inhibitors has been observed. To elucidate potential mechanisms, we performed in vivo CRISPR screens in PORCN inhibitor–sensitive RNF43-mutant pancreatic cancer xenografts. As expected, genes in the Wnt pathway whose loss conferred drug resistance were identified, including APC, AXIN1, and CTNNBIP1. Unexpectedly, the screen also identified the histone acetyltransferase EP300 (p300), but not its paralog, CREBBP (CBP). We found that EP300 is silenced due to genetic alterations in all the existing RNF43-mutant pancreatic cancer cell lines that are resistant to PORCN inhibitors. Mechanistically, loss of EP300 directly downregulated GATA6 expression, thereby silencing the GATA6-regulated differentiation program and leading to a phenotypic transition from the classical subtype to the dedifferentiated basal-like/squamous subtype of pancreatic cancer. EP300 mutation and loss of GATA6 function bypassed the antidifferentiation activity of Wnt signaling, rendering these cancer cells resistant to Wnt inhibition.

Authors

Zheng Zhong, Nathan Harmston, Kris C. Wood, Babita Madan, David M. Virshup

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Figure 6

p300 maintains GATA6 expression via activating its promoter and an intronic enhancer.

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p300 maintains GATA6 expression via activating its promoter and an intro...
(A) The UCSC conservation track (100 vertebrate species), ENCODE p300 ChIP-seq track (upper: K562 cells, ENCSR000EGE; lower: HepG2 cells, ENCSR000BLW), and H3K27Ac ChIP-Seq track (HPAF-II cells, extracted from GSE64557) in the GATA6 locus. The potential intronic enhancer region is highlighted. (B) EP300 knockout diminished H3K27Ac in the GATA6 promoter region and intronic enhancer region. H3K27Ac was assessed by the CUT&RUN assay in HPAF-II NTC, EP300 knockout, and Panc10.05 cells. Data are represented as mean ± SD (n = 3 technical replicates). (C) Schematic representation of the luciferase reporter constructs. The GATA6 promoter region and enhancer region (highlighted in A) were cloned into the pGL4.10(luc2) vector. MCS, multiple cloning site. Luc2 encodes a firefly luciferase. (D) GATA6 intronic enhancer sequence and p300 boosted GATA6 promoter-driven luciferase expression. Dual-luciferase reporter assay using firefly luciferase reporter constructs shown in C and a constitutively expressed Renilla luciferase performed in HEK293 cells (n = 2-3 biological replicates). WT p300 or a p300 mutant harboring inactivating mutations in its HAT domain (p300HAT–) were cotransfected. pro-Luc-en#1/2 are clones with 2 SNVs near a microsatellite site in the enhancer region.