Session: 622. Lymphoma Biology—Non-Genetic Studies: Mechanisms of Lymphomagenesis, Progression, and Response
Hematology Disease Topics & Pathways:
Diseases, Non-Hodgkin Lymphoma, DLBCL, Biological Processes, Lymphoid Malignancies, genomics, integrative -omics, pathways
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.
Disclosures: Scott: Janssen: Consultancy, Research Funding; AstraZeneca: Consultancy; Abbvie: Consultancy; Celgene: Consultancy; Roche/Genentech: Research Funding; NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; 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.
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