Functional genomic screens for molecular targets in cancer

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

We used genome-wide CRISPR-Cas9 screens as a discovery tool to identify determinants of lymphoma oncogenic signaling and survival. Traditionally, we have used loss-of-function "drop out" screens to identify essential genes in lymphoma cell line models. More recently, we have been engineering lymphoma cell lines with fluorescent reporter constructs to read out activity of key signaling pathways and have used FACS to sort cells into subpopulations with relatively high or low signaling. In one implementation, we knocked GFP into the IRF4 locus, creating and IRF4-GFP fusion protein that is upregulated by oncogenic NF-kB signaling in ABC DLBCL. CRISPR-Cas9 screening of these cells revealed that that N-linked glycosylation by the OST-B enzyme is required to maintain constitutive B cell receptor (BCR) signaling, a hallmark of the ABC DLBCL subtype. We showed that OST-B inhibition decreased BCR glycosylation, thereby decreasing the assembly of the BCRs into active signaling clusters in the plasma membrane. As a result, deglycosylated BCRs interacted more readily with CD22, a negative regulator of BCR signaling. Thus, OST-B represents a potential therapeutic target in ABC DLBCL. In another recent study, we used CRISPR-Cas9 screens to address a 70-year-old conundrum, namely why glucocorticoids are effective in lymphoid but not myeloid malignancies. We discovered that glucocorticoids themselves reduce oncogenic BCR signaling in all lymphoma types tested. By studying the genomic target genes of the glucocorticoid receptor, we showed that glucocorticoids induce lysosomal and proteasomal degradation of the BCR as well as degradation of Src-family kinases necessary for proximal BCR signaling. With this, glucocorticoids can now be rationally included in multi-drug regimens such as the ViPOR regimen that we have developed.