ATF4 programs proline-dependent immune evasion in β-Catenin-driven hepatocellular carcinoma
Background & AimsHepatocellular carcinoma (HCC) frequently exhibits resistance to immune checkpoint inhibitors (ICIs), particularly in {beta} -catenin-driven tumors characterized by immune exclusion. While the Unfolded Protein Response (UPR) and the Integrated Stress Responses (ISR) enable tumor adaptation to metabolic stress their role in shaping tumor immunogenicity remains incompletely understood. We investigated whether ATF4, a central effector of the integrated stress response, couples metabolic reprogramming to suppression of anti-tumor immunity in HCC. MethodsWe combined transcriptomic analyses across three independent human HCC cohorts with mechanistic studies using an immunotherapy-resistant MYC/{beta}-catenin-driven murine HCC model. We integrated CRISPR/Cas9-mediated deletion of Atf4 with RNA-sequencing and targeted metabolomics. The impact of tumor-derived metabolites on macrophage differentiation and polarization was evaluated using primary bone marrow-derived cells. Therapeutic responses were evaluated in orthotopic and subcutaneous models treated with anti-PD-1 and anti-VEGFA. ResultsATF4 and XBP1 transcriptional signatures are selectively enriched in human HCC and associate with poor prognosis, vascular invasion, and an immunosuppressive myeloid-enriched tumor microenvironment. Genetic ablation of Atf4 markedly suppressed tumor growth in immunocompetent but not immunodeficient hosts, establishing a requirement for immune-mediated tumor control. Mechanistically, Atf4 loss downregulated Aldh18a1 and disrupted proline biosynthesis, resulting in extracellular proline depletion. This proline-deficient environment abrogated monocyte-to-macrophage differentiation and decreased M2 polarization, thereby reshaping the tumor microenvironment toward enhanced T cell infiltration and activation. Functionally, Atf4-deficient tumors exhibited restored sensitivity to anti-PD-1 monotherapy and showed pronounced responses to combined anti-PD-1/anti-VEGFA treatment in aggressive orthotopic models. ConclusionATF4 programs a proline-dependent metabolic axis that sustains macrophage-mediated immunosuppression and immune evasion in {beta}-catenin-driven HCC. Disruption of this pathway converts immune-excluded tumors into T cell-inflamed states and restores responsiveness to immunotherapy. By governing proline homeostasis and macrophage-mediated immunosuppression, ATF4 is a key metabolic checkpoint for immune evasion, linking stress adaptation to immune escape and a candidate therapeutic target in HCC. Impact and implicationsWe identify ATF4 as a crucial metabolic-immune orchestrator that sustains myeloid-driven immune evasion in {beta}-catenin-dependent HCC through proline-dependent circuitry. Disrupting the ATF4-proline axis converts immune-desert tumors into T cell-inflamed lesions by blocking macrophage differentiation, thereby sensitizing tumors to immune checkpoint therapy. This work positions ATF4 as a tractable therapeutic target to overcome immunotherapy resistance in HCC. Graphical abstract Highlights- ATF4 orchestrates an immunosuppressive tumor microenvironment in HCC by coupling metabolic stress adaptation to immune evasion. - Ablation of ATF4 disrupts proline biosynthesis, leading to a marked depletion of extracellular proline. - Cancer cell-derived proline availability contributes to macrophage differentiation and M2 polarization; its loss restores T cell-mediated anti-tumor surveillance and sensitizes beta-catenin-driven HCC to immune checkpoint blockade.