Organoid models of fibrolamellar carcinoma mutations reveal hepatocyte transdifferentiation through cooperative BAP1 and PRKAR2A loss

Abstract

Fibrolamellar carcinoma (FLC) is a lethal primary liver cancer, affecting young patients in absence of chronic liver disease. Molecular understanding of FLC tumorigenesis is limited, partly due to the scarcity of experimental models. Here, we CRISPR-engineer human hepatocyte organoids to recreate different FLC backgrounds, including the predominant genetic alteration, the DNAJB1-PRKACA fusion, as well as a recently reported background of FLC-like tumors, encompassing inactivating mutations of BAP1 and PRKAR2A. Phenotypic characterizations and comparisons with primary FLC tumor samples revealed mutant organoid-tumor similarities. All FLC mutations caused hepatocyte dedifferentiation, yet only combined loss of BAP1 and PRKAR2A resulted in hepatocyte transdifferentiation into liver ductal/progenitor-like cells that could exclusively grow in a ductal cell environment. BAP1-mutant hepatocytes represent primed cells attempting to proliferate in this cAMP-stimulating environment, but require concomitant PRKAR2A loss to overcome cell cycle arrest. In all analyses, DNAJB1-PRKACAfus organoids presented with milder phenotypes, suggesting differences between FLC genetic backgrounds, or for example the need for additional mutations, interactions with niche cells, or a different cell-of-origin. These engineered human organoid models facilitate the study of FLC.

Recruitment of BAG2 to DNAJ-PKAc scaffolds promotes cell survival and resistance to drug-induced apoptosis in fibrolamellar carcinoma

“…Future investigation of the effects of etoposide/navitoclax combinations in FLC tumor PDX mice and hepatocyte organoid models expressing an allele of the fusion kinase will undoubtedly reveal additional details regarding the oncogenic action of DNAJ-PKAc. 100 …”

Mapping of mitogen and metabolic sensitivity in organoids defines requirements for human hepatocyte growth

“…3b ). Of note, while FSK promotes growth of cholangiocyte organoids 21 , FSK addition to the expansion medium of hepatocyte organoids slows down their growth and induces some traits of maturity 39 (Supplementary Fig. 5b ).…”

“…Human fetal liver tissues were derived from healthy abortion material from anonymous donors, under informed consent and ethical approval (Commission of Medical Ethics, Leiden University Medical Center, Leiden). FH organoid lines were established from tissue (FH donors 1–4, GW10, GW12, GW15, GW12, respectively) and cultured, largely as described previously 25 , 39 , with some modifications as follows. Liver cell suspensions were obtained by a short collagenase IV (Sigma–Aldrich) incubation with subsequent mincing of the tissue.…”

cAMP-PKA/EPAC signaling and cancer: the interplay in tumor microenvironment

“…Therefore, the type of oncogenic alteration is a determinant of the complex roles for cAMP signaling in cancer. Tumors with the DNAJB1 – PRKACA gene fusion or BAP1 -mutation share a molecular hallmark, namely aberrant activation of PKA [ 416 ]. Certainly, there are other driving events that lead to hyperactivation of cAMP signaling pathways during the development of some types of cancer.…”