Identification of wild-type Kirsten rat sarcoma (KRAS) and its signalling pathway in immune evasion in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the third most lethal cancer and sixth most commonly diagnosed cancer worldwide. The landscape of advanced HCC treatments has been evolving rapidly in recent years. Systemic therapies have emerged from single-agent treatment combined with immune checkpoint inhibitors (ICIs) and molecular-targeted therapies such as atezolizumab-bevacizumab or durvalumab-tremelimumab. Although ICIs demonstrate tremendous promise for the treatment of HCC patients, their response rates are modest (~15%). This highlights the urgent need for identifying effective biomarkers to predict patient response. Therefore, understanding the immune evasive mechanism of HCC is urgently needed. Increasing evidence indicates that oncogenic pathway activation is associated with generating an unfavourable tumour microenvironment with consequent immunotherapy resistance. Our study identified a critical oncogenic KRAS (Kirsten rat sarcoma viral oncogene homolog) signalling pathway involved in immune evasion in an antigen-expressing c-MycOE /Tp53KO HCC mouse model by data-independent acquisition mass spectrometry (DIA-MS) proteomics. We identified that wild-type Kras was highly upregulated in immune-escaped tumours, with concurrent activation of its ligand-driven EGFR and its downstream MEK/ERK signalling. Likewise, endogenous Kras overexpression in this model led to an increase in tumour burden with shorter survival time of mice, implicating the regulatory role of KRAS signalling in immune evasion. Clinically, wild-type Kras was overexpressed in HCC at both mRNA and protein levels and associated with tumour recurrence and poorer patients’ survival. Using single-cell RNA sequencing (scRNA-seq) analysis, we demonstrated that wild-type KRAS hampered the recruitment of dendritic cells, leading to defective T-cell activity via suppression of the interferon (IFN) responses. Activation of KRAS/MEK/ERK signalling impaired HCC recognition by T cells via downregulation of major histocompatibility complex class I (MHC-I)-driven antigen presentation. To elucidate the underlying molecular mechanisms, we employed a novel in vivo CRISPR/Cas9 system targeting MEK1/2, a downstream effector of KRAS. MEK1/2 knockout reversed the immunosuppressive effects of wild-type KRAS, enhancing IFN responses, antigen presentation, and CD8⁺ T cell recruitment. Moreover, our study proposed a new combinational therapy targeting wild-type KRAS to enhance immunotherapy efficacy in HCC. The combination therapy involving the KRAS inhibitor MRTX0902, MEK inhibitor trametinib, and anti-programmed cell death protein 1 (PD-1) treatment demonstrated increased intra-tumoral CD8⁺ T cell infiltration and improved survival outcomes. This data, together with the observation showing upregulation of this signalling pathway in PD-1-treated tumours, suggest a rational therapeutic strategy with a combination of KRAS inhibitor with ICIs.