CTC/cfDNA biomarker-guided precision medicine to overcome immune checkpoint inhibitor resistance in hepatocellular carcinoma

Background/Aim: Immune checkpoint inhibitors (ICs) have improved survival outcomes in advanced hepatocellular carcinoma (HCC), but most HCC patients experience treatment resistance. Thus, we aimed to stratify HCC patients to target therapy-resistant TICs and identify responders through stemness mRNA profiling. Our second aim was to evaluate novel CTC signatures to predict treatment efficacy in response to standard anti-VEGFA+anti-PD-L1 antibody immunotherapy with cancer-driver-targeted chemotherapies. Methods: The Genome-wide CRISPR-Cas9 knockout (GeCKO) method was used to identify genes required for killing tumors expressing TP53, CTNNB1, and ARID2. To confirm the drug efficacy, we used CRISPR/Cas9-mediated driver gene knock-ins and knockouts combined with alcohol Western diet feeding to promote HCC development in a humanized FNRG ( Fah - / - ;Nod;Rag1 - / - ;Il2rgc - / - ) mouse harboring ARID1A mutation or TP53 mutation in a PDX Model. Drugs paired with ICI were tested for treatment of HCC in genetically defined PDX and these humanized FNRG mice. Results: The GeCKO screening in ARID1A-mutant HCC showed that a combination of a Polycomb repressive complex 2 (PRC2) inhibitor, anti-PD-L1, and anti-VEGFA treatment reduced tumor size and extended survival in ARID1A-mutant PDX mice with humanized immune systems. The most successful combination from these approaches, comprising anti-EZH1/2 inhibitor + anti-PD-L1 + anti-VEGFA for ARID1A mutant HCC or all-trans retinoic acid (ATRA) and an HDAC inhibitor, along with anti-PD-L1, for TP53 mutant HCC-eradicated human patient-derived liver TICs grown ex vivo from tumors generated in mice. Single-cell RNA-seq analyses of patient HCCs identified 16 upregulated gene signatures with statistical significance. Principal component analyses provide a clear separation between Barcelona Clinic Liver Cancer (BCLC) stage categories A (benign) and B (malignant). Alcohol Western diet-associated TLR4-AKT signal leads to EZH2 phosphorylation of PRC2 complex to switch from histone suppressive activity to transactivation. The most effective targeting of PRC2 components, which eliminated human patient-derived liver TICs grown ex vivo in mice. Conclusions: Our innovative approach, using precision medicine to identify patients most likely to respond to specific drug combinations targeting TICs and cancer-driver mutations, will revolutionize cancer treatment by addressing a core component of therapeutic resistance in HCC.