RESUMEN
Recognition of Cas9 and other proteins encoded in delivery vectors has limited CRISPR technology in vivo. Here, we present a protocol for genome engineering using selective CRISPR antigen removal (SCAR) lentiviral vectors in Renca mouse model. This protocol describes how to conduct an in vivo genetic screen with a sgRNA library and SCAR vectors that can be applied to different cell lines and contexts. For complete details on the use and execution of this protocol, please refer to Dubrot et al. (2021).1.
Asunto(s)
Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas , Ratones , Animales , Sistemas CRISPR-Cas/genética , Biblioteca de Genes , Genoma , Línea CelularRESUMEN
Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking1,2. Here we identify the innate immune kinase TANK-binding kinase 1 (TBK1)3 as a candidate immune-evasion gene in a pooled genetic screen4. Using a suite of genetic and pharmacological tools across multiple experimental model systems, we confirm a role for TBK1 as an immune-evasion gene. Targeting TBK1 enhances responses to PD-1 blockade by decreasing the cytotoxicity threshold to effector cytokines (TNF and IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumour models, with concordant findings in matched patient-derived organotypic tumour spheroids and matched patient-derived organoids. Tumour cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNF and IFNγ in a JAK-STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is an effective strategy to overcome resistance to cancer immunotherapy.
Asunto(s)
Resistencia a Antineoplásicos , Evasión Inmune , Inmunoterapia , Proteínas Serina-Treonina Quinasas , Humanos , Evasión Inmune/genética , Evasión Inmune/inmunología , Inmunoterapia/métodos , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Organoides , Factores de Necrosis Tumoral/inmunología , Interferón gamma/inmunología , Esferoides Celulares , Caspasas , Quinasas Janus , Factores de Transcripción STATRESUMEN
The immune system can eliminate tumors, but checkpoints enable immune escape. Here, we identify immune evasion mechanisms using genome-scale in vivo CRISPR screens across cancer models treated with immune checkpoint blockade (ICB). We identify immune evasion genes and important immune inhibitory checkpoints conserved across cancers, including the non-classical major histocompatibility complex class I (MHC class I) molecule Qa-1b/HLA-E. Surprisingly, loss of tumor interferon-γ (IFNγ) signaling sensitizes many models to immunity. The immune inhibitory effects of tumor IFN sensing are mediated through two mechanisms. First, tumor upregulation of classical MHC class I inhibits natural killer cells. Second, IFN-induced expression of Qa-1b inhibits CD8+ T cells via the NKG2A/CD94 receptor, which is induced by ICB. Finally, we show that strong IFN signatures are associated with poor response to ICB in individuals with renal cell carcinoma or melanoma. This study reveals that IFN-mediated upregulation of classical and non-classical MHC class I inhibitory checkpoints can facilitate immune escape.
Asunto(s)
Linfocitos T CD8-positivos , Neoplasias , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inhibidores de Puntos de Control Inmunológico , Evasión Inmune , Interferón gamma/genética , Interferón gamma/metabolismo , Subfamília C de Receptores Similares a Lectina de Células NKRESUMEN
CRISPR-Cas9 genome engineering has increased the pace of discovery for immunology and cancer biology, revealing potential therapeutic targets and providing insight into mechanisms underlying resistance to immunotherapy. However, endogenous immune recognition of Cas9 has limited the applicability of CRISPR technologies in vivo. Here, we characterized immune responses against Cas9 and other expressed CRISPR vector components that cause antigen-specific tumor rejection in several mouse cancer models. To avoid unwanted immune recognition, we designed a lentiviral vector system that allowed selective CRISPR antigen removal (SCAR) from tumor cells. The SCAR system reversed immune-mediated rejection of CRISPR-modified tumor cells in vivo and enabled high-throughput genetic screens in previously intractable models. A pooled in vivo screen using SCAR in a CRISPR-antigen-sensitive renal cell carcinoma revealed resistance pathways associated with autophagy and major histocompatibility complex class I (MHC class I) expression. Thus, SCAR presents a resource that enables CRISPR-based studies of tumor-immune interactions and prevents unwanted immune recognition of genetically engineered cells, with implications for clinical applications.
