Probing the Tumor Suppressor Function of BAP1 in CRISPR-Engineered Human Liver Organoids

Abstract

The deubiquitinating enzyme BAP1 is a tumor suppressor, among others involved in cholangiocarcinoma. BAP1 has many proposed molecular targets, while its Drosophila homolog is known to deubiquitinate histone H2AK119. We introduce BAP1 loss-of-function by CRISPR/Cas9 in normal human cholangiocyte organoids. We find that BAP1 controls the expression of junctional and cytoskeleton components by regulating chromatin accessibility. Consequently, we observe loss of multiple epithelial characteristics while motility increases. Importantly, restoring the catalytic activity of BAP1 in the nucleus rescues these cellular and molecular changes. We engineer human liver organoids to combine four common cholangiocarcinoma mutations (TP53, PTEN, SMAD4, and NF1). In this genetic background, BAP1 loss results in acquisition of malignant features upon xenotransplantation. Thus, control of epithelial identity through the regulation of chromatin accessibility appears to be a key aspect of BAP1's tumor suppressor function. Organoid technology combined with CRISPR/Cas9 provides an experimental platform for mechanistic studies of cancer gene function in a human context.

Experimental identification of cancer driver alterations in the era of pan‐cancer genomics

“…They are amenable to NGS‐based mutation analysis, and organoids derived from normal human adult stem cells showed mutations in several known cancer driver genes . In analogy to 2D cell cultures, targeted alteration of candidate genes using the CRISPR‐Cas9 system has been successfully implemented in organoids, and targeted deletion of tumor suppressor genes alters cellular properties in vitro and heightens the transformation potential of organoids in vivo . Over the last two to three years, a series of functional validation screens for VUS were developed based on the generation of barcoded expression clones for large numbers of variants .…”

Novel patient-derived preclinical models of liver cancer

“…54 A similar strategy has also been employed with human cholangiocyte organoids to study the role of the tumour suppressor BAP1 in CCA. 55 BAP1 deletion by CRISPR/Cas9 technology led to increased motility but not full tumoural transformation. Additional loss of tumour suppressors, including TP53, PTEN, SMAD4, and NF1, led to full malignant transformation.…”

Fast and efficient generation of knock-in human organoids using homology-independent CRISPR/Cas9 precision genome editing

“…Liver ductal organoids are composed of a simple epithelium, with cells expressing early cholangiocyte markers such as KRT19 29 . To test HDR-mediated knock-in at the KRT19 locus, we transfected human liver ductal organoids with the HDR targeting plasmid, a plasmid expressing Cas9 and a fluorescent marker as transfection readout, and a plasmid expressing a sgRNA inducing a DSB directly upstream of the KRT19 stop codon, using our recently described method 31 . To test NHEJ, we employed the same transfection approach using instead the universal NHEJ self-cleaving targeting plasmid, the same KRT19 sgRNA plasmid and the appropriate frame selector, which also expresses Cas9 and a fluorescent marker (mCherry) as transfection readout.…”

“…Two days after electroporation, transfected organoids were picked based on transient mCherry expression from the frame selector plasmid. These were subsequently dissociated into single cells to grow clonal lines 31 (Fig. 3a).…”

“…Transfection of human liver ductal organoids was performed using an electroporation-based protocol described in 31 . Briefly, the organoids were co-electroporated with the appropriate sgRNA plasmid, the frame selector plasmid and the targeting construct.…”