Diffuse Large B-cell Lymphoma (DLBCL) is the most common aggressive lymphoma. It is a highly heterogenous disease, subsets of which have unique prognostic and therapeutic features. Up to 15% of DLBCLs are EBV-positive (EBV+), and their continued growth and survival are dependent on viral products; however, the specific viral and host dependencies of EBV+ DLBCL remain undefined — in part due to disease heterogeneity and the lack of knowledge regarding the role of EBV across various DLBCL subtypes.
Our lab aims to address this gap in knowledge by addressing the following questions:
- How does EBV contribute to DLBCL subtype classification?
- What are the host gene dependencies of DLBCL and how does EBV contribute to tumorigenesis?
The recently defined genetic classification of DLBCL includes at least six subtypes but does not consider EBV status, despite EBV’s known associate with and role in B cell lymphomagenesis. We analyzed published whole exome and RNA sequencing of DLBCL tumors (n=481) and identified 19 EBV-positive DLBCLs.
We found that EBV-positive DLBCL is significantly enriched within the BN2 subtype and comprised ~1/3 of EBV-positive DLBCLs. Most of the remaining ~2/3 are distinct from currently defined subtypes. Several differentially mutated genes within the BN2 tumors were found to correlate with EBV status. These include subtype-defining genes and genes involved in oncogenic processes. This finding supports our hypothesis that EBV oncogenes can substitute for specific cellular mutations in DLBCL.
Overall, we find that EBV-positive DLBCL is enriched within, and likely has a shared pathogenesis with, the BN2 subtype of DLBCL. The remaining, ‘Unclassified’ DLBCLs may constitute novel subtypes. EBV status correlates with distinct mutation and transcriptomic profiles, including subtype-defining features. Thus, integrating EBV status into the classification of DLBCL is likely to improve tumor subtyping and molecular studies, and helps us resolve how EBV contributes to DLBCL tumorigenesis.
~ Quincy Rosemarie
This project aims to elucidate the viral and host oncogenic dependencies of EBV+ DLBCL through two complementary CRISPR/Cas functional screens across several DLBCL subtypes.
First, we will determine which latency genes are essential for EBV+ DLBCL growth and survival by performing a Cas13 RNA knockdown screen, an RNA-targeting Cas. Latency genes will be screened at 500x coverage, and longitudinal guide RNA depletion will identify which viral latency genes are required for tumor cell survival.
In parallel, a whole-genome screen utilizing CRISPR/Cas9 will interrogate host gene dependencies across subtypes. Stably expressing Cas9 DLBCL cell line clones will be generated and tested for knockout efficiency before performing the genome-wide screen at 500x coverage.
By determining EBV and host dependencies, we expect to determine how EBV cooperates with host driver mutations in EBV+ DLBCL pathogenesis. Understanding these therapeutic vulnerabilities would lay the foundation for precision therapy in patients.
~ Lily Wenger
