弥漫性大B细胞淋巴瘤对EBV癌蛋白的依赖性

Background : Up to 14% of diffuse large B-cell lymphomas (DLBCL) are Epstein-Barr virus-positive (EBV+), and their growth and survival are dependent on EBV latency gene products. DLBCL is a heterogeneous group of aggressive lymphomas, and we previously found that EBV is associated with the BN2 LymphGen subtype. Additionally, multiple genes were mutated at lower frequencies in EBV+ tumors relative to EBV-, suggesting viral oncogenes functionally replace host driver mutations. To identify the EBV oncogenes essential for EBV+ DLBCL and the functional consequences of their inactivation, we are conducting CRISPR/Cas13d screens in EBV+ DLBCL cell lines. Methods : EBV+ DLBCL cell lines (Farage, BCKN1, IBL1, and IBL4) were characterized using whole-exome sequencing (WES), RNA sequencing (RNA-seq), and immunoblotting. We compared the mutational and transcriptomic profiles of EBV+ tumors and cell lines to inform CRISPR/Cas13d functional screens. These screens aim to identify EBV gene product dependencies in EBV+ DLBCL and the impact on cell growth and survival. Results : WES analysis revealed the genetic subtype of each EBV+ DLBCL cell line: Farage was classified as ST2, and the rest were classified as “Other”. RNA-seq and immunoblotting revealed that all expressed an atypical latency III program, characterized by the absence of LMP2A expression. This novel latent gene program was also observed in the EBV+ DLBCL tumors. All cell lines were infected with EBV1, except IBL1, which, to our knowledge, is the only known cell line infected with an EBV1/EBV2 intertypic recombinant. By employing Cas13d for our knockdown screen, we can determine the specific transcripts within the complex EBV latent transcriptome that are essential for the growth and survival of EBV+ DLBCL. Targeting RNA also avoids excessive DNA damage and low sensitivity that arises from targeting 10-20 copies of EBV DNA per cell with Cas9. We have stably expressed two different Cas13d orthologs in each cell line and are optimizing their RNA knockdown efficiency using a FACS-based transient knockdown strategy. Use of clones achieving the most efficient knockdown will allow us to identify essential EBV latent genes and determine the phenotypic consequences (growth arrest, apoptosis, etc.) of inactivating each essential viral oncogene. Conclusions : EBV+ DLBCL exhibits distinct host mutation and gene expression profiles, including the replacement of several subtype-defining mutations with EBV oncogenes. The available EBV+ DLBCL cell lines faithfully model the ST2 or “Other” LymphGen subtypes and exhibit the same atypical EBV latent gene expression pattern as tumors. By determining EBV oncogene dependencies and the effects of their inactivation, we aim to uncover how EBV cooperates with host driver mutations in the pathogenesis of EBV+ DLBCL. Understanding these therapeutic vulnerabilities will provide a foundation for precision therapy.