Abstract C057: Fibrolamellar carcinoma single-nucleus RNA sequencing reveals alteration of mitochondrial energetic pathways
Authors: Bharath Nihal , Isaac Biju , Sherman Marc S. , Smith Colton J. , Karski Michael , Sun Liang , Pepe-Mooney Brian J. , Kycia Ina , Rogers Michael , Goessling Wolfram , Vakili Khashayar 
Published: December 2023

Background: Fibrolamellar carcinoma (FLC) is a primary liver cancer, which arises in a background of normal liver. The DNAJB1-PRKACA (DP) fusion gene is present in the majority of FLC and is an oncogenic driver in animal models. The oncogenic mechanism of DP is unclear. To date, transcriptomic analyses of FLC have been based on bulk RNA sequencing. Given the lack of cell-specific data in bulk transcriptomic analysis, we hypothesize that using single nucleus RNA sequencing (snRNA-Seq) will allow for identification of significantly dysregulated signaling pathways specific to tumor cells.

Methods: Single nuclei were isolated from 7 FLC tumors and 4 normal liver (NL). The Chromium Next GEM Single Cell 3’ Reagent Kit was used to generate sequencing libraries. All samples passing QC were submitted for sequencing by GeneWiz with a read depth of 20,000 reads/cell. An R-based pipeline for the single-cell profiling was built using Seurat R toolkit. Cells exhibiting extremely low or high library sizes, gene features outside the 95% confidence interval, or displaying high mitochondrial content were filtered out. Clusters from snRNA-Seq analysis were annotated using singleR, enrichR, and publicly available datasets.

Results: FLC and NL nuclei counts were 98664 and 56644, respectively. We generated thirty one clusters and annotated 21 cell types. The FLC tumor group was annotated based on previously published FLC bulk transcriptomic data. We identified increased expression of SLC16A14, TMEM163, PDE10A, OAT, and GALNTL6 in FLC tumor cells. The predominant cells types and percentage of cells (%NL/%FLC) per group were: hepatocytes (43/57), endothelial cells (18/82), FLC tumor cells (15/85), stellate cells (28/72), Kupffer cells (50/50), cholangiocytes (62/38), Other 1 (22/78), T cells (36/64), and fibroblasts (69/31). FLC tissue contained only 3% of the liver sinusoidal endothelial cells. Comparison of FLC tumor cells demonstrated differential upregulation of 1879 and downregulation of 1579 genes in tumor vs. NL. Gene ontology (GO) enrichment identified the following top processes in FLC tumor cells: ATP metabolic process, cellular respiration, and response to peptide hormone. GO analysis of hepatocytes identified: ribonucleotide metabolic process, energy derivation by oxidation of organic compounds, and response to peptide hormone as significant pathways.

Conclusions: FLC tumor cells demonstrate alterations in mitochondrial processes and energetics. FLC cells are known to contain abundant mitochondria, which is not observed in other primary liver epithelial cancers. The PRKACA component of DP fusion protein is the catalytic subunit of protein kinase A (PKA). Since PKA is known to regulate mitochondrial function, these data support the possibility that DP may directly alter mitochondrial function. Another interesting finding is the presence of the FLC tumor cell type in NL group (15%), which may suggest its dedifferentiation status or its cell-of-origin as a subpopulation of liver cells distinct from mature hepatocytes or cholangiocytes.

Citation Format: Nihal Bharath, Biju Isaac, Marc S. Sherman, Colton J. Smith, Michael Karski, Liang Sun, Brian J. Pepe-Mooney, Ina Kycia, Michael Rogers, Wolfram Goessling, Khashayar Vakili. Fibrolamellar carcinoma single-nucleus RNA sequencing reveals alteration of mitochondrial energetic pathways [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C057.