PF520 C‐REL NF‐KAPPA B SUBUNIT ACTIVITY REFINES A DISTINCT GERMINAL CENTER DIFFUSE LARGE B‐CELL LYMPHOMA SUBGROUP WITH FAVORABLE OUTCOME

Abstract

Background:Germinal Center (GC) B‐cell diffuse large B‐cell lymphomas (DLBCL) are associated with amplification of the REL locus that encodes the c‐Rel NF‐κB subunit. But, constitutive activation of NF‐κB is a feature of activated B‐cell (ABC) DLBCLs at gene expression profile signature, mutational and functional levels. Thus, functional relationship between REL amplification and NF‐κB activation in GC‐DLBCLs remains unclear.Aims:The aim of this study was to explore c‐Rel activation in DLBCLs and compare it with the gene expression profile of the tumors, survival of patients, the mutatome, REL imbalances and to functionally explore the c‐Rel oncogenic potential.Methods:From tumor biopsies, electro mobility shift assay (EMSA) with c‐Rel supershift was performed on a series of 40 ABC and 23 GC‐DLBCLs. Affymetrix transcriptomes were issued from series published by S Dubois et al (1) (GHEDI cohort) and by G Lenz et al (2). Exploring mutations of 34 oncogenes recurrently altered in DLBCLs was done by highthrouput‐targeted sequencing. REL imbalances were explored by CGH‐array. An in vitro cell model consisted in human EREB2–5 B‐cells (which cell proliferation is arrested by estradiol withdrawal) that were transfected with a doxycycline inducible vector for c‐Rel or IκBε molecule known to target c‐Rel.Results:By EMSA, a strong c‐Rel DNA binding activity was found in 13/63 (21%) cases, among them 10 (77%) and 3 (23%) were GC and ABC‐DLBCLs, respectively. Cases with increased c‐Rel DNA‐binding activity expressed higher levels of c‐Rel mRNA. Comparing transcriptome of c‐Rel and non c‐Rel EMSA cases led to identify a c‐Rel signature of 101 probesets with a positive fold change greater or equal than two. None of them belonged to the ABC signature while 42 (42%) overlapped with the GC signature. The c‐Rel signature was markedly discriminant for GC‐DLBCLs since it allowed to co‐clusterize 78% and 92% of GC‐DLBCLs of the GHEDI or the Lenz's DLBCL cohort respectively. Consistently, DLBCL cases of both series with the c‐Rel signature exhibited a better overall survival (logrank test, p = 0.03 and 0.001 respectively). Expression of c‐Rel on its own tended to refine a subgroup of GC‐DLBCLs with a better prognosis (logrank test, p = 0.056 and 0.014 respectively). BCL2, CREBBP, and EZH2 mutations were strongly associated with the c‐Rel signature while mutations of PIM1, MYD88 and TNFAIP3 were mainly found in non c‐Rel DLBCL cases. DNA‐binding activity, mRNA overexpression and transcriptomic signature of c‐Rel were markedly associated with REL amplification. Probesets of the c‐Rel signature were functionally related to metabolism, cell proliferation and various human cancers. Induction of c‐Rel in cell cycle arrested EREB2–5 cells led to both constitutive nuclear c‐Rel DNA binding activity and protection against apoptosis while IkBε expression increased cell death.Summary/Conclusion:These results demonstrate for the first time that most GC‐DLBCL exhibited a c‐Rel transcriptomic signature that was different from the ABC/GC signature. In tumors, DNA‐binding activity, overexpression and transcriptomic signature of c‐Rel were associated with REL amplification and refines a distinct GC‐DLBCL subgroup with a favorable outcome. Functionally, c‐Rel over‐expression led to constitutive c‐Rel DNA binding activity and was associated with decreased apoptosis of non‐proliferating EREB2–5 B‐cells.