IDDF2024-ABS-0410 In vivo screens identify FZD4 as a druggable cholesterol regulator to overcome sorafenib and immune resistance in hepatocellular carcinoma

<h3>Background</h3> In advanced-stage hepatocellular carcinoma (HCC) management, sorafenib remains an important option for patients who are ineligible for alternative treatments. Despite clinical evidence indicating that sorafenib may affect the efficacy of subsequent anti-PD-1 therapy, nivolumab is currently administered after sorafenib tolerance in various regions. Indeed, the lack of sequential biopsies hinders the comprehension of how sorafenib alters the immune microenvironment, thereby limiting the identification and characterization of biomarkers in patients receiving second-line immunotherapy. <h3>Methods</h3> Multiple sorafenib therapy-response models were developed to conduct <i>in vivo</i> transcriptome-wide screening of druggable molecules that affect the efficacy of sorafenib and subsequent immunotherapy. Genetic mechanisms of FZD4 were investigated by RNA sequencing, mass spectrometry-based protein identification and sterolomics. Multiplex immunohistochemistry, flow cytometry, and integrated scRNA-seq analyses were applied to describe and confirm the alterations in FZD4 expression, cholesterol homeostasis, and the associated CD8⁺ T cell anti-tumor immunity in humans and mice. <h3>Results</h3> Clinically, high FZD4 expression was correlated with HCC recurrence and metastasis, predicted poor prognosis (IDDF2024-ABS-0410 Figure 1, IDDF2024-ABS-0410 Figure 2), and functionally conferred characteristics of LCSCs, rendering them resistant to sorafenib-induced cytotoxicity (IDDF2024-ABS-0410 Figure 3). We discovered compromised effector functions in FZD4-rich tumor-infiltrating CD8⁺ T cells and in antigen-specific FZD4 HCC-CAR-T coculture settings. Notably, improved <i>in vivo</i> antitumor immune responses were observed with regimens involving AAV8-mediated gene delivery of shFZD4, sorafenib, and anti-PD-1, either separately or in combination (IDDF2024-ABS-0410 Figure 4). Mechanistically, the interaction between FZD4 and ROR2, in the presence of WNT5A, finely controlled JNK-mediated cholesterol biosynthesis and uptake in tumor cells, resulting in increased cellular cholesterol levels and tumor progression (IDDF2024-ABS-0410 Figure 5). Additionally, FZD4 regulated excess cellular cholesterol via the efflux pathway, potentially leading to elevated cholesterol levels within CD8⁺ T cells via XBP1 splicing, thereby contributing to their exhaustion (IDDF2024-ABS-0410 Figure 6). <h3>Conclusions</h3> Our work finds a novel mechanism behind FZD4-induced cross-resistance to sorafenib and anti-PD-1 and proposes that targeting the noncanonical WNT5A-FZD4-ROR2 axis may enhance the efficacy of systemic therapies by inhibiting cholesterol metabolic reprogramming in HCC.