Genome-Scale ORF Screen for Mediators of NF-κb Activation in DLBCL

Cancer genome and transcriptome sequencing can identify novel oncogenes and tumor suppressors, discover distinct cancer subtypes, and predict therapeutic responses. Analysis of the coding genome of diffuse large B cell lymphoma (DLBCL) has identified various genetic mutations in the ABC and GCB molecular subtypes. Notably, ABC DLBCLs have recurrent activating mutations involving the canonical NF-kB pathway. Although an oncogenic role for constitutive NF-kB activity has been demonstrated in ABC DLBCL, the molecular mechanisms of various oncogenic mutations are still elusive. To understand the pathogenesis of DLBCL, we used high-throughput exome and transcriptome sequencing of more than 500 DLBCL biopsies.

To enable functional screening of mutant alleles identified by exome-seq and RNA-seq, we are constructing an inducible retroviral expression library of over 200 open reading frames (ORFs) of genes mutated in DLBCL. We have cloned the mutant and wildtype forms of these genes together with a unique 26-base-pair 'bar code' to facilitate screening with high-throughput sequencing.

We have transduced a subset of this ORF library into DLBCL cell lines, induced ORF expression by doxycycline, and FACS sorted into high and low populations based on expression the known NF-κB target gene CD83. We then performed barcode sequencing to assay relative enrichment of ORFs that confer higher or lower NF-κB activity. ORF screening revealed that MYD88^L265P, CARD11^L232LI, both well-characterized gain-of-function mutants, were among the highest ranking genes for induction of NF-κB activity. Interestingly, we observed enrichment for USP7^D271E in NF-κB high populations, though these genes have not been shown to have a tumorigenic role in DLBCL.

Ubiquitin Specific Protease-7 (USP7) is a regulator of NF-κB transcriptional activity, in part by de-ubiquitinating p65. To test how USP7 mutations contribute to NF-κB activity, we engineered the ABC DLBCL cell line TMD8 to express an NF-κB reporter consisting of the NF-κB transcriptional response element (TRE) fused to GFP. NF-κB activity upon overexpression of wildtype or mutant USP7 was monitored by flow cytometry (FACS). We observed that the mutated form of USP7 significantly increased NF-κB activity over that seen with wildtype, suggesting this is a gain-of-function mutant.

We next used the CRISPR gene-targeting system to probe the function of USP7 genetically. Single guide RNAs (sgRNAs) targeting the USP7 coding regions were coexpressed in TMD8 cells with the endonuclease Cas9. SgRNA-expressing, GFP+ cells were monitored over time among total live cells by flow cytometry, and a relative reduction of the GFP+ population was observed in the sgUSP7 population. These results suggest that USP7 may function as an oncogene in DLBCL.

This ongoing work demonstrates the efficacy of a high-throughput ORF expression screen to characterize mutations found in the genomic landscape of the DLBCL.