Our research focuses on the Epstein-Barr virus (EBV) host-pathogen relationship. We use genetic and proteomic techniques to interrogate how EBV reprograms key growth, survival, metabolic and immune-evasion pathways to enable immortalized B-cell growth. EBV is an oncogenic gamma-herpesvirus that persistently infects >95% of adults worldwide. EBV is associated with multiple human malignancies, with nearly 200,000 cancers attributable to EBV each year worldwide. These include Burkitt lymphoma, Hodgkin lymphoma, HIV-associated lymphomas, post-transplant lymphoproliferative diseases, immune-senescence-associated lymphomas, nasopharyngeal and gastric carcinomas. EBV’s association with cancer is an outgrowth of its relationship with host cells. Upon infection of primary B lymphocytes, EBV enters a state of viral latency, but is hardly quiescent. Rather, EBV expresses oncogenic membrane proteins, transcription factors and microRNAs that efficiently transform resting B-lymphocytes into rapidly growing lymphoblasts. In vitro, EBV converts primary resting B lymphocytes into immortalized lymphoblastoid cell lines (LCLs), which provide an excellent tissue culture model for studies of EBV latency proteins and lymphoproliferative disorders. Key EBV oncoproteins mimic signaling from CD40 and the B-cell receptor. We also study related B-cell activation pathways in primary human B-cells, in particular CD40/NF-kB.
Current research includes the following areas:
CRISPR/Cas9 screens for synthetic lethal targets in EBV-transformed B-cells
We performed the first genome-wide CRISPR/Cas9 loss-of-function systematic genetic analysis for host B-cell factors critical for EBV-transformed B-cell growth and survival. Our studies have identified multiple druggable lymphoblastoid B-cell and Burkitt lymphoma targets. We are using CRISPR genetics to further define newly-identified B-cell dependencies, including novel viral strategies to evade tumor suppressor responses. We are using parallel CRISPR/Cas9 screens of B-cell immune receptor pathways to identify factors uniquely and commonly important for EBV oncoprotein function.
The LCL and primary human B-cell NF-κB Genomic Binding Landscape
The EBV oncoprotein LMP1 mimics CD40 signaling to constitutively activate the canonical and non-canonical NF-κB pathways, each of which are critical for EBV-mediated B-cell transformation. Yet, little is known about NF-kB pathway-specific roles in EBV-transformed B-cells, or in primary human B-cells. We used ChIP-seq to identify the LCL NF-κB genomic binding landscape, providing the first analysis of all NF-κB transcription factor subunits in a human cell. Our analysis identified a fascinating but complex NF-κB genomic binding landscape. We are using CRISPR to further identify NF-κB subunit-specific roles. To enable comparison of LMP1 and CD40 B-cell effects, we are using ChIP-seq to identify NF-κB pathway roles in ex vivo CD40-stimulated primary human B-cells.
Quantitative proteomic analysis of EBV B-cell lytic replication
EBV B-cell replication enables lifelong B-cell colonization and seeding of oropharyngeal epithelial cells. Yet, systematic proteomic or genetic analysis of EBV lytic replication has not been performed. In collaboration with Michael Weekes (University of Cambridge) and Steve Gygi (Harvard Medical school), we are using multiplexed tandem-mass spectrometry to identify B-cell proteome-wide and cell-surface remodeling at three time points after lytic reactivation. Our approach is providing insights into known and novel B-cell pathways subverted by EBV. These included significant remodeling of innate and adaptive immune, metabolic and cap-dependent protein translation pathways.
Proteomic Analysis of EBV of primary human B-cell transformation
EBV transformation of a resting, short-lived human B-cell into an immortalized lymphoblast is one of the most fascinating, yet incompletely understood aspects of EBV biology. We are using proteomic, CRISPR and chemical genetic approaches to study key metabolic pathways exploited by EBV at distinct stages of B-cell transformation.
Novel human immunodeficiencies manifest by susceptibility to EBV and EBV-associated malignancy
Rare primary immunodeficiencies highlight mechanisms that control EBV and result in markedly elevated EBV loads, hematological disorders and B-cell cancers. We are using immunologic and whole exome approaches to identify and characterize novel primary human immunodeficiency diseases that result in the inability to control EBV infection and EBV-driven cancers.