CRKL dictates anti-PD-1 resistance by mediating tumor-associated neutrophil infiltration in hepatocellular carcinoma

Background & Aims Immune checkpoint inhibitors (ICIs) resistance limits immunotherapy success in hepatocellular carcinoma (HCC). However, the mechanisms underlying immunotherapy resistance remain elusive. We aimed to identify the role of CT10 regulator of kinase-like (CRKL) in HCC against anti-PD-1 therapy. Methods Gene expression in HCC specimens from 10 patients accepted anti-PD-1 therapy was identified by RNA-sequencing. A total of 342 and 62 HCC samples from TMA1 and TMA2, respectively, were analyzed by immunohistochemistry. Transgenic mice (Alb-Cre/Trp53fl/fl) were given hydrodynamic tail vein injections (HDTVi) of adeno-associated virus serotype 8 (AAV8) vector to overexpress CRKL. Mass cytometry by time of flight (CyTOF) 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 resistant gene using a pooled genetic screen. CRKL overexpression nullifies anti-PD-1 therapy efficacy by mobilizing tumor-associated neutrophils (TANs) to block 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 adenomatous polyposis coli (APC)-mediated proteasomal degradation of β-catenin by competitively decreasing Axin1 binding, and thus fosters 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 (PDOTS) 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 may be developed for the treatment of HCC. Impact and implications Here, we found CRKL was overexpressed in anti-PD-1 resistant HCC and CRKL upregulation promotes anti-PD-1 resistance in HCC. We identified that CRKL/β-catenin/VEGFα and CXCL1 axis upregulation contributes to anti-PD-1 tolerance through promoting tumor-associated neutrophils (TANs) infiltration. These findings support the strategy of bevacizumab-based ICIs combination therapy, and CRKL inhibitors combined with anti-PD-1 therapy may be developed for the treatment of HCC.