NF-κB Subunit c-Rel Cooperates with Myc and Mutated p53 to Confer Significantly Worse Survival in Patients with Diffuse Large B-Cell Lymphoma: A Report from the International DLBCL Rituximab-CHOP Consortium Program
Authors: Li Ling , Xu-Monette Zijun Yidan , Ok Chi Young , Tzanko Alexander , Visco Carlo , Gisin Nora , Montes-Moreno Santiago , Dybkær Karen , Chiu April , Orazi Attilio , Zu Youli , Bhagat Govind , Richards Kristy L. , Hsi Eric D. , Choi William W. L. , Van Krieken Joannes H.J.M. , Huh Jooryung , Ponzoni Maurilio , Ferreri Andres J.M. , Møller Michael B. , Go Ronald S. , Winter Jane N. , Piris Miguel A. , Medeiros L. Jeffrey , Young Ken H. 
Published: October 2019

Abstract
Objective :To evaluate the prognostic significance of RELamplification, expression and c-Rel activation in DLBCL patients and to identify potential mechanisms for impact of c-Rel activation on patient survival.
Patients and Design : The study cohort consists 460 de novo DLBCL patients (median follow-up, 46.8 moths) treated with R-CHOP. We assessed the nuclear expression/activation of c-Rel and other NF-κB subunits by immunohistochemistry, REL gene amplification by fluorescence in situ hybridization, and gene expression profiling using Affymetrix GeneChips array. Correlations between expression of nuclear c-Rel, REL mRNA, and expression of TP53, MDM2, MDM4, MYC, BCL2, AKT1, NFKB1, RELA, NFKB2, and RELB, both at the mRNA and protein levels were analyzed using t-tests. The prognostic significance of c-Rel activation, REL mRNA expression, and REL amplification was evaluated in the overall cohort, and different subgroups stratified by COO, status of TP53 mutation (wide type/WT, or mutated/MUT) and expression, Myc, Bcl-2 overexpression, and nuclear expression of other NF-κB subunits.
Results :Nuclear c-Rel expression was observed in 29.6% of DLBCL patients and did not correlate with REL mRNA levels (P=0.95) and COO (P=0.77). In contrast, REL mRNA was significantly higher in germinal center B-like (GCB) subtype (P<0.0001). In GCB-DLBCL, nuclear c-Rel expression was associated with significantly lower Myc, p53, MDM4, and pAKT protein levels but not at the transcriptional level. In contrast, in ABC-DLBCL, c-Rel activation was associated with significantly higher MUT-TP53mRNA level and reduced pAKT expression.
Correspondingly to the lack of associations with reduced Myc, pAKT, and p53 in ABC-DLBCL, nuclear c-Rel expression showed prognostic impact only in ABC- but not in GCB- DLBCL, especially when it was concurrent with Myc overexpression (P<0.0001 for OS and P=0.0012 for PFS). Importantly, patients with c-Rel activation and p53 mutations had significantly worse survival (for OS, hazard ratio, 3.56; P=0.0011; median survival, 16.2 vs 87.3 months. For PFS, hazard ratio, 3.976; P=0.0004; median survival, 10.4 vs 55.5 months) compared with other MUT-p53 DLBCL patients. The additive impact of c-Rel activation to TP53 mutations was more apparent in the ABC-DLBCL subtype, in which c-Rel activation was associated with significantly upregulated MUT-TP53 transcription (P=0.0087). Conversely, MUT-p53 expression was associated significantly with upregulated REL mRNA expression (P=0.0021), predominantly in the ABC-DLBCL subtype (P=0.0047). Only in ABC-DLBCL patients, TP53 mutations were associated with elevated nuclear c-Rel levels with a borderline P value (P=0.05).
In addition to the association of nuclear p65 in GCB-DLBCL (P=0.003), and p50, NFKB1 and RELA mRNA in ABC-DLBCL (P=0.0023), the prognostic significance of c-Rel appears to depend on p50 (P=0.08) and p65 expression (P=0.12). Also, supporting that c-Rel transactivates anti-apoptotic BCL2L1/BCLXL by previous studies, there was no significant difference in survival of ABC-DLBCL patients with isolated BCL2 overexpression and with nuclear c-Rel expression. Comprehensive gene-expression profiling analysis and cell line study are undergoing in order to identify pathways to activate c-Rel and oncogenic mechanisms for c-Rel–mediated chemoresistance.
REL amplification was predominantly observed in GCB-DLBCL (frequency, 7%) and correlated with significantly higher mRNA level (P<0.0001). However, REL amplification did not correlation with nuclear c-Rel expression or patient survival, illustrating the importance of posttranslational regulations in c-Rel activation and function. Consistent with the adverse impact of c-Rel activation at the protein level, a strong trend toward poor survival was observed for elevated RELmRNA in ABC- but not in GCB-DLBCL.
Conclusions : Nuclear c-Rel activation was associated with reduced level of proteins whose degradation involves with ubiquitin-proteasome in GCB-DLBCL, and upregulated TP53 transcription in ABC-DLBCL. Reciprocal regulation of c-Rel and MUT-p53 at the transcriptional level may underlie the synergetic adverse effect of c-Rel activation and TP53 mutations. c-Rel cooperated with Myc and conferred significantly worse survival in ABC-DLBCL patients. These subsets of c-Rel+ DLBCL patients likely will benefit from c-Rel targeted therapies.

Disclosures
No relevant conflicts of interest to declare.