Abstract PR06: Identifying regulatory transcription factors in primary liver cancer cell lineage commitment using single cell ATAC sequencing
Authors: Craig Amanda J , Ma Lichun , Heinrich Sophia , Kelly Michael , Greten Tim , Wang Xin Wei 
Published: September 2022

Abstract
Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) are the two main types of primary liver cancer (PLC), a leading cause of cancer related mortality. The key mechanisms of transcription regulation that delineates a cell of origin to either HCC or iCCA remain underexplored. We hypothesize that chromatin accessibility patterns reflect transcriptional regulatory circuitry that controls PLC lineage development. We aim to trace developmental lineages of cells that compose tumors using single cell chromatin accessibility data. Single cell Assay for Transposase-Accessible Chromatin (ATAC) and RNA sequencing was done on biopsies collected from 16 patients, including 13 patients with HCC and 3 patients with iCCA tumors enrolled for interventional studies at the NIH Clinical Center. ATAC and RNA sequencing libraries were made using the 10X Genomics Chromium platform. Downstream analysis includes graph-based clustering, UMAP embedding, and cell type label transfer using Seurat v3.0. Transcription factor (TF) activity estimation was done using ChromVAR. In total, we collected high quality chromatin accessibility data from 18,609 cells and gene expression data from 32,127 cells. Using the gene expression data, we transferred malignant and non-malignant cell type labels to our chromatin accessibility data. Clustering based on the most variably accessible chromatin peaks successfully separated cells by cell type identity. Strikingly, further clustering of malignant cells alone using the most variable TF activity scores separated cells into clusters based on tumor type, suggesting variation in TF activity was driven by differences between HCC and iCCA tumors. Comparing TF activity between HCC and iCCA, we discovered 31 significant differentially activated TFs between the 2 lineages (FDR&lt;0.1). Gene set enrichment analysis revealed these 31 TFs fell into distinct families that regulate cell proliferation and differentiation. To further explore the role of these TFs in PLC, we utilized bulk transcriptome data collected from the TIGER-LC cohort, including samples from 62 HCC and 91 iCCA patients. Hierarchical clustering of the TIGER-LC cohort based on expression of the identified 31 TFs separated patients into 2 main clusters, 1 dominantly HCC and 1 dominantly iCCA. Several TFs strongly enriched in either HCC or iCCA showed mutual exclusivity in expression. To investigate the interplay between the microenvironment and activation of lineage associated TFs, we characterized the non-malignant cells for which we collected single cell chromatin accessibility data. Our data suggests proportions of certain T-cell and myeloid cell subtypes may be different between HCC and iCCA. We are further exploring transcriptional regulatory circuity of paracrine signaling between the surrounding microenvironment and malignant cells in the role of PLC lineage commitment. In conclusion, we hypothesize that activation of these TFs with interplay of the microenvironment may drive and sustain lineage commitment between HCC and iCCA tumors.
Citation Format: Amanda J Craig, Lichun Ma, Sophia Heinrich, Michael Kelly, Tim Greten, Xin Wei Wang. Identifying regulatory transcription factors in primary liver cancer cell lineage commitment using single cell ATAC sequencing [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PR06.