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Nfkbiz 3′ UTR Mutations Confer Selective Growth Advantage and Affect Drug Response in Diffuse Large B-Cell Lymphoma

Introduction: The activated B-cell-like (ABC) molecular subgroup of diffuse large B-cell lymphoma (DLBCL) is characterized by activation of NF-κB signaling and increased mortality. Although recurrent mutations affecting genes such as MYD88, CD79A/B and TNFAIP3 contribute to this phenomenon in some cases, there remain tumors with no known genetic basis for NF-κB-activation. Previously, amplification (AMP) of the NFKBIZ locus has been reported in 10% of ABC DLBCLs and it was demonstrated that this contributes to activation of NF-κB signaling. We recently described a novel pattern of non-coding mutations affecting the 3′ untranslated region (UTR) of NFKBIZ resulting in an overall mutation rate of ~30% (UTR or AMP) in ABC DLBCL. NFKBIZ mutations are mutually exclusive with MYD88 mutations, suggesting they may also act as driver mutations. The NFKBIZ protein interacts with NF-κB transcription factors and is thought to regulate canonical NF-κB signaling. We hypothesized that NFKBIZ 3′ UTR mutations affect post-transcriptional regulation of the mRNA by disrupting the conserved secondary structure of the UTR. The binding of regulatory proteins is abrogated by the structural changes induced by mutations, which consequently lead to stabilization of the mRNA. This causes an accumulation of protein and may be a novel mechanism to promote cell growth and survival in ABC DLBCL.
 Methods: NFKBIZ 3′ UTR mutations were introduced into the WSU-DLCL2 DLBCL cell line using the CRISPR-Cas9 system, producing eight different CRISPR-mutant lines. Custom droplet digital PCR assays and western blotting were used to asses mRNA and protein levels, respectively. Competitive growth assays with wild-type (WT) and CRISPR-mutant lines were performed to assess whether UTR mutations provide a growth advantage in vitro. A similar study was performed in vivo by engrafting a mix of WT and mutant cells into NSG mice. We separately compared gene expression profiles (generated by RNA-Seq) of the parental cell line and a subset of CRISPR-mutant lines. Genes up- and down-regulated by NFKBIZ 3' UTR mutations were identified and analyzed for pathway enrichment. Finally, the IC50 of drugs relevant to DLBCL was determined by WST-1 assays after drug treatment on WT and mutant lines.
 Results: Introduction of NFKBIZ mutations into DLBCL cells confirmed that UTR mutations lead to varying degrees of increased NFKBIZ mRNA and protein levels. NFKBIZ UTR deletions afforded DLBCL cells a selective growth advantage over WT both in vitro and in vivo. In an assay containing all mutants and WT, mutants with the highest NFKBIZ expression had the largest advantage, suggesting NFKBIZ expression drives this growth advantage. In assays comparing individual mutants to WT, each mutant out-competed WT over time despite varying degrees of NFKBIZ expression, suggesting that all of these mutations act as drivers. Analysis of differentially expressed genes revealed some known NF-κB targets as well as overlap with multiple targets of MYD88, which supports our hypothesis that NFKBIZ and MYD88 regulate a common set of genes. We also discovered potential novel targets of NFKBIZ, including CD274 (PD-L1) and the src kinase HCK. Western blot confirmed that HCK protein is highly expressed in NFKBIZ CRISPR-mutant lines. HCK is a potentially relevant therapeutic target as it has been shown to be overexpressed in multiple cancer types and has been associated with poor overall survival. Expression of PD-L1 in NFKBIZ mutant cases could suggest that immunotherapies may be useful in patients with these mutations, as immunotherapies have had limited success in DLBCL, this may be a way to select patients likely to respond. Mutant cell lines had significantly higher IC50 compared to WT for the drugs Ibrutinib, Idelalisib and Masitinib, but not Bortezomib, suggesting that NKFBIZ UTR mutations confer resistance to drugs specifically targeting the NF-κB pathway.
 Conclusions: This work directly establishes a role for NFKBIZ amplifications and 3′ UTR mutations in driving ABC DLBCL through NF-κB signaling. We demonstrate that these mutations can cause over-expression of NFKBIZ and provide a selective growth advantage to tumor cells. We also identified novel targets of NFKBIZ including HCK and PD-L1, both of which have implications as therapeutic targets in this subset of DLBCLs. In addition, we found that these mutant lines were more resistant to some targeted lymphoma drugs.
 
 
 Scott: NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding. Steidl:Seattle Genetics: Consultancy; AbbVie: Consultancy; Bayer: Consultancy; Curis Inc: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy. Morin:Celgene: Consultancy.
