Molecular and Functional Profiling of Microenvironmental Interactions in Splenic Marginal Zone Lymphoma

Microenvironmental interactions play a critical role in the natural history of splenic marginal-zone lymphoma (SMZL) as evidenced by the pronounced skewing of the immunoglobulin gene repertoire and the enrichment for recurrent aberrations in genes of critical B cell signaling pathways. That notwithstanding, the precise impact of microenvironmental triggering in SMZL has been much less investigated. In this study, we aimed to (i) uncover molecular signatures that arise after microenvironmental stimulation in SMZL and obtain functional evidence regarding the implicated pathways and processes; and, (ii) characterize B cell receptor (BcR)-antigen interactions at the molecular level. Regarding the former aim, first we performed transcriptome analysis (RNA-Seq) in circulating malignant B cells from 8 SMZL cases both at basal level and after triggering both the BcR and TLR9, the latter known to activate SMZL cells. Overall, 1037 genes were differentially expressed (p≤0.05) in stimulated vs untreated cells with 507 and 530 being up- or down-regulated in the former, respectively. Pathway enrichment and gene ontology analysis of the differentially expressed genes revealed significant enrichment in genes related to signal transduction (e.g. MAPK, PI3K-AKT signaling pathways), immune system, regulation of metabolic processes and gene transcription (e.g. E2F transcription family). Focusing on signaling pathways, flow cytometric analysis showed that BcR/TLR9-stimulated cells exhibited increased pERK levels (FD=1.5, p<0.001, n=15 cases) and pNF-κB levels (FD=1.7, p=0.1, n=22 cases), hence validating the RNA-Seq results. Next, we explored whether microenvironmental triggering can modulate the transcriptional program of SMZL cells. The E2F transcription factor family, found enriched by RNA-Seq analysis, is known to cooperate with the histone methyltransferase EZH2 to stimulate gene transcription. On these grounds, we investigated the expression of EZH2 and H3k27me3, the main target of EZH2, and found that: (i) EZH2 was expressed in all SMZL cases, albeit variably; (ii) cases with high (n=9) vs low (n=16) EZH2 protein levels showed higher cell proliferation, assessed by Ki67 expression (FD=3.4, p<0.01); (iii) H3k27me3 levels were increased in all studied cases (28-99.7%, n=20), indicating that EZH2 is active in SMZL; and, (iv) BcR/TLR9 triggering resulted in significant upregulation of EZH2 expression (FD=4.3, p<0.0001, n=14), overall supporting transcriptional rewiring induced by immune stimulation in SMZL. Moving to our second aim of gaining insight into BcR-antigen interactions in SMZL, we investigated the antigen binding profile of 41 SMZL clonotypic BcR IGs expressed as recombinant human IgM monoclonal antibodies (rmAbs); comparisons were performed against a series of CLL rmAbs. ELISA documented significantly (p<0.05) stronger reactivity of SMZL mAbs (n=30) vs CLL mAbs (n=35) against various human (auto)antigens, including native DNA, skeletal muscle actin, myosin, thyroglobulin, β-amyloid peptide, carbonic anhydrase, the F(ab')2 fragment of serum IgG, and the non-self haptentrinitrophenyl (coupled to a carrier molecule). IGHV1-2*04 is the predominant gene in SMZL (~30% of all cases), which is noteworthy since the nucleotide sequence of allele *04 encodes for a tryptophan (W) residue at position VH FR3-75 instead of the arginine (R) residue that is encoded by all remaining alleles of the IGHV1-2 gene and almost all other human IGHV genes and alleles. To assess the effect of the W residue on IGHV1-2*04, we used PCR-based site-directed mutagenesis for modifying W-75 to R (the corresponding modified rmAbs were called IGHV1-2*04 W75R). Comprehensive characterization of the antigen reactivity profile of the authentic IGHV1-2*04 rmAbs vs the respective IGHV1-2*04 W75R mAbs through ELISA, flow cytometry, immunohistochemistry and protein microarrays revealed significant (p<0.05) differences (Figure), supporting functional selection as the cause for the biased usage of IGHV1-2*04 in SMZL. In conclusion, we show that microenvironmental interactions result in transcriptional rewiring of SMZL cells through affecting key regulatory molecules such as EZH2. Our findings also suggest that relevant microenvironmental triggers can be provided by a wide range of autoantigens, supporting the derivation of SMZL from polyreactive B cells of the splenic marginal zone.

Ghia:MSD: Consultancy, Honoraria; Lilly/Loxo: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Chatzidimitriou:AbbVie: Research Funding; Janssen: Research Funding. Stamatopoulos:AbbVie: Research Funding; Janssen: Research Funding.