Development of a CRISPR/Cas9 based RiboTag strategy to identify regulators of melanoma immune interaction

Abstract

The development of immunotherapy approaches has revolutionised the field of melanoma treatment by prolonging the survival of patients. Nevertheless, a significant proportion of patients either do not respond to immunotherapy or relapse. To understand the mechanisms of the melanoma immune crosstalk resulting in resistance to immunotherapy, we aim to dissect pathways and gene expression signatures in melanoma to identify new potential therapeutic targets or prognostic biomarkers. <br /> As a highly heterogeneous tissue melanoma lesions not only contain transformed melanocytes, but also cells of the tumour microenvironment. Therefore, most of sequencing approaches rely on mechanical processes in order to dissect tissues and isolate the target cells to study melanoma cell specific expression patterns. But the extensive processing can introduce stress related artifacts in the analysis. In order to find an alternative approach which avoids the extensive processing but still gives access to tumour cell-specific transcripts, a murine RiboTag melanoma model was generated. An adapted CRISPR/Cas9 approach was used to endogenously fuse the ribosomal protein L8 (RPL8) with a triple HA peptide tag. The incorporation of the tagged RPL8 into translating polyribosomes enabled the isolation ribosome-associated mRNAs by immunoprecipitation. <br /> We used RiboTag melanoma cells in a C57BL/6 mouse model for immune checkpoint blockade (ICB) therapy and found the expression of Cnot9, a subunit of the CCR4-NOT complex, significantly downregulated in ICB-treated melanomas. In previous exome sequencing studies analysing cutaneous melanoma CNOT9 has been described to harbour a hotspot mutation, but the consequences for progression or successes of therapy approaches remained unknown. To investigate the impact of these mutations on melanoma-immune interactions, we performed a Gene Set Enrichment Analysis (GSEA) of a cutaneous melanoma dataset and observed an enrichment of immune activation and antigen presentation signatures in tumours bearing CNOT9 mutations. A knockdown of CNOT9 in human MZ7 melanoma cells induced similar expression patterns, suggesting that this immune-activated status of CNOT9 mutated melanoma may be due to a partially impaired functionality. To further evaluate possible functional effects of the mutation, the protein structure of CNOT9 was analysed by modelling and mutations were localised. This structural study revealed that the S87P and P131L point mutations occur in the concave surface of CNOT9, which is described to be the binding pocket of Roquin and CNOT4. In literature, both proteins are described to be involved in translational regulation of immune response-associated mRNA. To further investigate the functional effects of the mutations and potential impact on interactions with binding partners, melanoma cells expressing CNOT9 variants bearing either the S87P or P131L point mutation were established. <br /> The use of RiboTag in combination with an experimental model for ICB therapy facilitated a comprehensive characterisation of melanoma-specific gene expression patterns during therapy, revealing a downregulated expression of Cnot9 in inflamed melanomas. Melanoma-associated mutations in CNOT9 as well as a CNOT9 knockdown induced inflammatory response pathways, indicating an involvement of CNOT9 in immune regulatory processes in melanoma. <br /> Thus, our work can contribute to a better understanding of the interactions between melanoma and the immune system, which in turn offers the opportunity of improving the tailored treatment of malignant melanoma.