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 9bar code9 to facilitate screening with high-throughput sequencing. We have transduced a subset of this ORFlibrary into DLBCL cell lines, induced ORFexpression by doxycycline, and FACS sorted into high and low populations based on expression the known NF-κBtarget gene CD83 . We then performed barcode sequencing to assay relative enrichment of ORFsthat confer higher or lower NF-κBactivity. ORFscreening revealed that MYD88 L265P , CARD11L232LI , both well-characterized gain-of-function mutants, were among the highest ranking genes for induction of NF-κBactivity. Interestingly, we observed enrichment for USP7 D271E in NF-κBhigh 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-κBtranscriptional activity, in part by de-ubiquitinating p65. To test how USP7 mutations contribute to NF-κBactivity, we engineered the ABC DLBCL cell line TMD8 to express an NF-κBreporter consisting of the NF-κBtranscriptional response element (TRE) fused to GFP. NF-κBactivity upon overexpression of wildtype or mutant USP7 was monitored by flow cytometry (FACS). We observed that the mutated form of USP7 significantly increased NF-κBactivity 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 ORFexpression screen to characterize mutations found in the genomic landscape of the DLBCL. Disclosures No relevant conflicts of interest to declare.