The effectiveness of immune checkpoint inhibitor (ICI) therapy for hepatocellular carcinoma (HCC) is limited by treatment resistance. However, the mechanisms underlying immunotherapy resistance remain elusive. We aimed to identify the role of CT10 regulator of kinase-like (CRKL) in resistance to anti-PD-1 therapy in HCC. Gene expression in HCC specimens from 10 patients receiving anti-PD-1 therapy was identified by RNA-sequencing. A total of 404 HCC samples from tissue microarrays were analyzed by immunohistochemistry. Transgenic mice (Alb-Cre/) received hydrodynamic tail vein injections of a -overexpressing vector. Mass cytometry by time of flight was used to profile the proportion and status of different immune cell lineages in the mouse tumor tissues. was identified as a candidate anti-PD-1-resistance gene using a pooled genetic screen. CRKL overexpression nullifies anti-PD-1 treatment efficacy by mobilizing tumor-associated neutrophils (TANs), which block the infiltration and function of CD8 T cells. PD-L1 TANs were found to be an essential subset of TANs that were regulated by CRKL expression and display an immunosuppressive phenotype. Mechanistically, CRKL inhibits APC (adenomatous polyposis coli)-mediated proteasomal degradation of β-catenin by competitively decreasing Axin1 binding, and thus promotes VEGFα and CXCL1 expression. Using human HCC samples, we verified the positive correlations of CRKL/β-catenin/VEGFα and CXCL1. Targeting CRKL using CRISPR-Cas9 gene editing ( knockout) or its downstream regulators effectively restored the efficacy of anti-PD-1 therapy in an orthotopic mouse model and a patient-derived organotypic tumor spheroid model. Activation of the CRKL/β-catenin/VEGFα and CXCL1 axis is a critical obstacle to successful anti-PD-1 therapy. Therefore, CRKL inhibitors combined with anti-PD-1 could be useful for the treatment of HCC. Here, we found that CRKL was overexpressed in anti-PD-1-resistant hepatocellular carcinoma (HCC) and that CRKL upregulation promotes anti-PD-1 resistance in HCC. We identified that upregulation of the CRKL/β-catenin/VEGFα and CXCL1 axis contributes to anti-PD-1 tolerance by promoting infiltration of tumor-associated neutrophils. These findings support the strategy of bevacizumab-based immune checkpoint inhibitor combination therapy, and CRKL inhibitors combined with anti-PD-1 therapy may be developed for the treatment of HCC.

中文翻译：

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CRKL 通过介导肝细胞癌中肿瘤相关中性粒细胞浸润来决定抗 PD-1 耐药性

免疫检查点抑制剂（ICI）治疗肝细胞癌（HCC）的有效性受到治疗耐药性的限制。然而，免疫治疗耐药的机制仍然难以捉摸。我们的目的是确定 CT10 激酶样调节因子 (CRKL) 在 HCC 抗 PD-1 治疗耐药中的作用。通过 RNA 测序鉴定了 10 名接受抗 PD-1 治疗的患者的 HCC 样本中的基因表达。通过免疫组织化学分析了来自组织微阵列的总共 404 个 HCC 样本。转基因小鼠(Alb-Cre/)接受水动力尾静脉注射α-过表达载体。采用飞行时间质谱流式技术来分析小鼠肿瘤组织中不同免疫细胞谱系的比例和状态。使用汇总基因筛选将其鉴定为候选抗 PD-1 抗性基因。 CRKL 过表达通过动员肿瘤相关中性粒细胞 (TAN) 来阻止 CD8 T 细胞的浸润和功能，从而使抗 PD-1 治疗效果无效。 PD-L1 TAN 被发现是 TAN 的一个重要子集，受 CRKL 表达调节并表现出免疫抑制表型。从机制上讲，CRKL 通过竞争性降低 Axin1 结合来抑制 APC（腺瘤性结肠息肉病）介导的 β-catenin 蛋白酶体降解，从而促进 VEGFα 和 CXCL1 表达。使用人类 HCC 样本，我们验证了 CRKL/β-catenin/VEGFα 和 CXCL1 的正相关性。使用 CRISPR-Cas9 基因编辑（敲除）或其下游调节剂靶向 CRKL，可有效恢复原位小鼠模型和患者来源的器官型肿瘤球体模型中抗 PD-1 疗法的功效。 CRKL/β-catenin/VEGFα 和 CXCL1 轴的激活是成功抗 PD-1 治疗的关键障碍。因此，CRKL抑制剂联合抗PD-1可能有助于治疗HCC。在这里，我们发现 CRKL 在抗 PD-1 耐药性肝细胞癌 (HCC) 中过度表达，并且 CRKL 上调促进 HCC 中的抗 PD-1 耐药性。我们发现 CRKL/β-连环蛋白/VEGFα 和 CXCL1 轴的上调通过促进肿瘤相关中性粒细胞的浸润而有助于抗 PD-1 耐受。这些发现支持了基于贝伐珠单抗的免疫检查点抑制剂联合治疗策略，CRKL抑制剂联合抗PD-1疗法可能被开发用于HCC的治疗。