Sustained NF-Kappa B Activity In Chronic Lymphocytic Leukemia Is Independent of Genetic and Epigenetic Modifications of the Tumor Suppressor Genes CYLD and A20
Authors: Frenzel Lukas P. , Schwamb Janine , Claus Rainer , Claasen Julia , Brinker Reinhild , Pallasch Christian P. , Plass Christoph , Wendtner Clemens-Martin 
Published: October 2019

Abstract
Abstract 3597

Introduction:
The microenvironment and especially the antigenic stimulation of the B-cell receptor on the surface of the malignant cell play a crucial role in the pathogenesis of chronic lymphocytic leukemia (CLL). Aberrant Nuclear Factor kappa B (NFκB) activity is another major hallmark of B-cell malignancies as well as of CLL. NFκB-dependent genes are involved in anti-apoptotic regulation, cell proliferation and metastasis and are responsible for survival and proliferation of tumors. However, the mechanisms of NFκB over-expression in CLL still remain to be elucidated. Prior studies revealed that cylindromatosis (CYLD) function might be of special interest in CLL since it inhibits signaling via TRAF2 and c-IAP1/2, which are known to be over-expressed in CLL. CYLD inactivation might therefore result in sustained NFκB signaling. The enzyme CYLD, a tumor suppressor that functions as a deubiquitinase, plays a role in other physiological aspects such as cell cycle response, inflammatory and immune processes. Moreover, it could be shown that impaired CYLD activity leads to increased NFκB activity in multiple myeloma cells demonstrating the negative regulatory function of CYLD regarding NFκB. Aside from CYLD, which is constitutively active preventing uncontrolled transcription factor activation, the enzyme A20, a key player in negative feedback loop regulation of NFκB, operates via induction, supposing that both enzymes might proceed at different phases of NFκB signaling. A20, also known as tumor necrosis factor alpha-induced protein 3 (TNFAIP3), acts as an ubiquitin-editing enzyme. Its inactivation is involved in immunopathologies (e.g. Crohn's disease, rheumatoid arthritis, systemic lupus erythematodes, psoriasis and type 1 diabetes mellitus) and in tumorigenesis. Frequent mutations in the A20 locus – leading to sustained NFκB activity – could be shown to play a dominant role in development of different B-cell malignancies. Experimental design and results: Based on genome-wide gene expression profiling analysis of CLL samples (n=8) compared to healthy donor B-cells (n=5), CYLD is expressed and its expression was reduced following B-cell receptor cross-linking (24 hours) (p=0,0036) contrary to A20 that could be induced after receptor stimulation (p=0,044). These results underline the role of B-cell receptor signaling in survival regulation of CLL cells and also its in-direct influence on NFκB activity. Recently, our report revealed by methylation analysis and additional sequence analysis that the A20 region neither contains any methylation (64 CLL patients versus 10 healthy donors) nor mutation (55 CLL patients with sequence analysis of exons 2–9 of the tnfaip3 gene) contrary to reports from other B-cell malignancies. Moreover, A20 expression could be confirmed by immunoblotting showing comparable results to healthy B-cells. In order to check if such alterations in the enzyme CYLD might occur in CLL leading to sustained activity of NFκB similar to other B-cell entities, we performed analysis of the methylation status of the promoter region of CYLD in 64 CLL patients compared to 10 DNAs of CD19-selected B-cells from healthy donors. Epigenetic alterations of the CYLD promoter could not be identified.

Conclusions:
Here we present the first report of epigenetic and mRNA expression analysis concerning the deubiquitinase CYLD in CLL. We identified that CYLD as well as A20 are regulated by B-cell receptor signaling. The opposed expression of CYLD and A20 after stimulation of the receptor might contribute to an almost balanced and well-adjusted NFκB activity. Our results of lacking epigenetic alteration in both proteins (A20 and CYLD) and absence of mutations in A20 indicate that malignant development in CLL differs from most of other B-cell malignancies, which show frequent inactivation of either CYLD or A20.

Disclosures:
No relevant conflicts of interest to declare.