02152nas a2200265   4500000000100000000000100001008004100002260001500043653002400058653002600082653002200108100001800130700002400148700002100172700003200193700001700225700002100242700002400263245013800287856005500425300000900480490000700489520137600496022001401872       2023                            d  c2023-05-0310aGenetic engineering10aMechanisms of disease10aPaediatric cancer1 aLaura Rüland1 aFrancesco Andreatta1 aSimone Massalini1 aSusana Chuva de Sousa Lopes1 aHans Clevers1 aDelilah Hendriks1 aBenedetta Artegiani00aOrganoid models of fibrolamellar carcinoma mutations reveal hepatocyte transdifferentiation through cooperative BAP1 and PRKAR2A loss  uhttps://www.nature.com/articles/s41467-023-37951-6  a23770 v143 aFibrolamellar 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.  a2041-1723