Background and Aims: Major genomic drivers of hepatocellular carcinoma (HCC) are nowadays well recognized, although models to establish their roles in human HCC initiation remain scarce. Here, we used human liver organoids in experimental systems to mimic the early stages of human liver carcinogenesis from the genetic lesions of TP53 loss and L3 loop R249S mutation. In addition, chromatin immunoprecipitation sequencing (ChIP‐seq) of HCC cell lines shed important functional insights into the initiation of HCC consequential to the loss of tumor‐suppressive function from TP53 deficiency and gain‐of‐function activities from mutant p53. Approach and Results: Human liver organoids were generated from surgical nontumor liver tissues. CRISPR knockout of TP53 in liver organoids consistently demonstrated tumor‐like morphological changes, increased in stemness and unrestricted in vitro propagation. To recapitulate TP53 status in human HCC, we overexpressed mutant R249S in TP53 knockout organoids. A spontaneous increase in tumorigenic potentials and bona fide HCC histology in xenotransplantations were observed. ChIP‐seq analysis of HCC cell lines underscored gain‐of‐function properties from L3 loop p53 mutants in chromatin remodeling and overcoming extrinsic stress. More importantly, direct transcriptional activation of PSMF1 by mutant R249S could increase organoid resistance to endoplasmic reticulum stress, which was readily abrogated by PSMF1 knockdown in rescue experiments. In a patient cohort of primary HCC tumors and genome‐edited liver organoids, quantitative polymerase chain reaction corroborated ChIP‐seq findings and verified preferential genes modulated by L3 mutants, especially those enriched by R249S. Conclusions: We showed differential tumorigenic effects from TP53 loss and L3 mutations, which together confer normal hepatocytes with early clonal advantages and prosurvival functions.

Hepatocellular Carcinoma (HCC) represents the third leading cause of cancer-related mortality worldwide. TP53 mutations are pivotal genomic drivers for HCC development through frequent concurrent loss-of-function (LOF) aberrations and protein-altering missense mutations. Over 90% of the TP53 missense mutations are distributed within the core DNA binding domain (DBD), where R249S is the most common. Cumulative studies have demonstrated the cancer-promoting properties of TP53R249S mutation in HCC, but its biological impact on tumor initiation remains to be defined. To mimic the early stages of liver carcinogenesis, we generated normal hepatic organoids from human liver tissues of 3 individuals. CRISPR-Cas9 mediated knockout of TP53 in liver organoids resulted in pleomorphic malignant features, including dysplasia, hyperchromasia, atypical and frequent mitosis, loss of polarity, and increased nuclear to cytoplasm ratio. To recapitulate TP53R249S genotype in HCC, we ectopically expressed R249S mutant in liver organoids through lentiviral infection. TP53R249S liver organoids displayed tumorigenic properties as evidenced by increased lesion forming incidence (37.5%) in subcutaneous xenografts when compared with TP53KO (17%) and TP53 wild-type (WT) organoids (0%). Chromatin immunoprecipitation sequencing (ChIP-seq) analysis with HCC cell lines substantiated the specific gain-of-function (GOF) transcriptional activities of R249S mutant. A unique subset of transcription start site-proximal peaks was exclusively found in R249S mutant cells when compared with other TP53 missense mutants and WT. Integration of ChIP-seq and RNA-seq analysis identified transcription factor ZMIZ2 as a direct transcription target of R249S mutant. Our results showed that ZMIZ2 is preferentially overexpressed in HCC patients carrying TP53R249S mutation and exerts a vital role for proliferation of R249S mutant HCC cells. Knockdown of ZMIZ2 profoundly suppressed global H3K27 acetylation (H3K27ac) and H3K4 trimethylation (H3K4me3) in HCC cells. Transcriptome profiling of ZMIZ2 knockdown cells identified multiple downstream targets enriched in chromatin binding and interaction with histone deacetylase, further reinforcing its involvement in epigenetic regulation. In summary, our study revealed that TP53R249S mutation confers distinct advantages in increased tumorigenicity to human liver organoids through GOF transcriptional activities. ZMIZ2 serves as a direct downstream effector contributing to the oncogenic growth arising from R249S mutation plausibly through altering chromatin remodelling. Citation Format: Mingjing Xu, Yin Kau Lam, Jianqing Yu, Kelvin Kwok Chai Ng, Nathalie Wong. TP53 R249S mutation confers hepatic organoids with gain-of-function (GOF) tumorigenic features through transcriptional activation of ZMIZ2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2590.