目的:免疫检查点抑制剂(ICIs)耐药性限制了肝细胞癌(HCC)的免疫治疗成功。然而,免疫治疗耐药的潜在机制仍然难以捉摸.我们旨在确定CT10调节激酶样(CRKL)在HCC对抗抗PD-1治疗中的作用。
方法:通过RNA测序鉴定10例接受抗PD-1治疗的HCC标本中的基因表达。TMA1和TMA2的HCC样本分别为342和62,通过免疫组织化学进行分析。转基因小鼠(Alb-Cre/Trp53fl/fl)给予腺相关病毒血清型8(AAV8)载体的流体动力学尾静脉注射(HDTVi)以过表达CRKL。通过飞行时间(CyTOF)的质量细胞计数用于描绘小鼠肿瘤组织中不同免疫细胞谱系的比例和状态。
结果:使用合并遗传筛选将CRKL鉴定为候选抗PD-1抗性基因。CRKL过表达通过动员肿瘤相关嗜中性粒细胞(TAN)以阻断CD8+T细胞的浸润和功能而使抗PD-1治疗功效无效。发现PD-L1+TAN是由CRKL表达调节并显示免疫抑制表型的TAN的重要子集。机械上,CRKL通过竞争性降低Axin1结合抑制腺瘤性结肠息肉病(APC)介导的β-catenin蛋白酶体降解,并因此促进VEGFα和CXCL1表达。使用人类肝癌样本,我们验证了CRKL/β-catenin/VEGFα与CXCL1的正相关。使用CRISPR-Cas9基因编辑(CRKL敲除)或其下游调节因子靶向CRKL有效地恢复了在原位小鼠模型和患者来源的器官型肿瘤球体(PDOTS)模型中的抗PD-1疗法的功效。
结论: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可用于HCC的治疗。
■这里,我们发现CRKL在抗PD-1耐药HCC中过表达,CRKL上调可促进抗PD-1耐药.我们确定CRKL/β-连环蛋白/VEGFα和CXCL1轴上调通过促进肿瘤相关中性粒细胞(TAN)浸润促进抗PD-1耐受性。这些发现支持基于贝伐单抗的ICIs联合治疗策略,CRKL抑制剂联合抗PD-1治疗可用于HCC的治疗。
OBJECTIVE: 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.
METHODS: 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/Trp53fl/fl) received hydrodynamic tail vein injections of a CRKL-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.
RESULTS: CRKL 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 (CRKL 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.
CONCLUSIONS: 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.
UNASSIGNED: 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.