Detailed Functional Characterization of Splenic Marginal Zone Lymphoma: Uncovering Links between the Epigenetic and the Signaling Machinery
Authors: Ntoufa Stavroula , Gounari Maria , Papakonstantinou Nikos , Binou Despoina , Tyritidis Ioannis , Stivarou Theodora , Sarrigeorgiou Ioannis , Iatrou Anastasia , Stavroyianni Niki , Anagnostopoulos Achilles , Lymberi Peggy , Stamatopoulos Kostas 
Published: November 2019

Splenic marginal-zone lymphoma (SMZL) ontogeny and evolution is a complex process, involving, amongst others, (super)antigenic stimulation, molecular deregulation of genes involved in the physiological differentiation of splenic marginal zone B cells (e.g. NOTCH2 and KLF2), and epigenetic alterations leading to silencing of different tumor suppressor genes and overexpression of oncogenes, including the PRC2-complex (EZH2, EED, and SUZ12). However, despite important recent progress, advances in the fundamental understanding of SMZL have not yet been translated in improved patient management, highlighting the need for further molecular investigations. In this context, here we aimed at detailed characterization of: (i) epigenetic modifiers, particularly the histone methyltransferase EZH2, a marker of clinical aggressiveness and druggable target in lymphoma; (ii) immune signaling capacity of SMZL B cells; and, (iii) antigen reactivity profile of the SMZL clonotypic B Cell Receptor immunoglobulin (BcR IG).
Quantification of EZH2 mRNA levels using quantitative Real-time PCR (n=26 SMZL patients) and protein levels using western blotting (WB) (n=14) revealed that EZH2 was expressed in all cases yet in a heterogeneous manner (2^-ΔCt range: 0.079-1.38; ΕΖΗ2+ cells: 3.11%-44.8%). Interestingly, higher proliferation rates (Ki67+ cells) were observed in cases with high EZH2 protein levels (n=4) compared to cases with low EZH2 (n=6) (FD=2.6; p&lt;0.05). Stimulation of SMZL CD19+ B cells (n=8) through i) the BcR with anti-IgM ii) Toll-Like receptor 9 (TLR9) with CpG and iii) BcR/TLR for 60 minutes affected pBTK, pERK and pNF-kB levels as revealed by WB and flow cytometry (FCM). Although, great heterogeneity was observed in the responses between different cases, categorization of SMZL cases based on EZH2 levels showed that, at basal level, EZH2high cases expressed higher pBTK, pERK and pNF-κB compared to EZH2low cases (FC=2.4, FC=6,1, FC=12.7, respectively; p&lt;0.05 for all comparisons). BcR and TLR9 activation and, especially, co-stimulation with anti-IgM/CpG induced the expression of pBTK, pERK and pNF-κB in EZH2low cases, while the opposite was observed in EZH2high cases. Moreover, stimulation through both the BcR and BcR/TLR9 for 24 hours could also induce EZH2 expression compared to the unstimulated control cells (FC=1.6; p&lt;0.05 for both comparisons).
To gain insight into the type of antigenic stimuli that could activate BcR signaling through the BcR we investigated the antigen binding profile of 10 SMZL clonotypic BcR IGs expressed as recombinant human IgM monoclonal antibodies (mAbs). Cases were representative of the most common IGHV genes in SMZL (IGHV1-2*04, IGHV4-34 and IGHV3-23). Reactivity (at 15 μg/ml) against various autoantigens was evaluated by ELISA. Five of 9 (56%), 4/7 (57%), 5/10 (50%), 2/9(22%), 2/9 (22%), 3/9 (33%), 3/8 (38%), 4/8 (50%) and 4 of 9 (44%) SMZL IgM mAbs displayed reactivity against native DNA, nuclear Hep-2 cell extract, actin, myosin, thyroglobulin, β-amyloid, carbonic anhydrase, F(ab')2 and the hapten trinitrophenyl respectively, with optical density (OD) values greater than the cut-off point. Moreover, 5/10 (50%) SMZL IgM mAbs recognized more than 2 antigens and were characterized as polyreactive. Each of these SMZL mAbs exhibited a distinct antigen polyreactivity profile, while heterogeneity was observed even amongst cases expressing the same gene or cases with similar IGHV gene mutational load.
Recognition of epitopes on the surface of viable MEC1 B and HEK293 cells was assessed by FCΜ. Two of 3 and 5/11 mAbs bound to viable MEC1 B cells and HEK293 cells, respectively: binding to HEK293 cells was more pronounced for minimally mutated/unmutated versus mutated mAbs (p&lt;0.05). Importantly, stimulation of SMZL CD19+ B cells with the SMZL BcR cognate antigens actin and myosin resulted in regulation of pERK/pPLCγ2 in 3/6 and 1/2 cases, respectively, as revealed by WB.
Overall, SMZL cells can be activated by microenvironmental signals albeit in a different manner depending on EZH2 levels, highlighting links between the epigenetic and the signaling machinery with potential therapeutic implications. Moreover, the recognition of a wide range of autoantigens by the SMZL mAbs indicates that SMZL B cell clones may arise from polyreactive B cells, likely resident in the normal splenic marginal zone.

Disclosures
Stamatopoulos: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.