P1225: CYLD, A NOVEL REGULATOR OF SPLENIC MARGINAL ZONE LYMPHOMA PATHOGENESIS

Topic: 20. Lymphoma Biology & Translational Research Background: Splenic Marginal zone lymphoma (SMZL) is a distinct clinical and pathologic entity among indolent B-cell lymphomas arising from marginal zone B-cells. Genetic and microenvironmental factors, leading to the aberrant activation of the NF-κB pathway, have been implicated in its pathogenesis. CYLD is a negative regulator of NF-κB and other signaling pathways, acting as a deubiquitinase of regulatory molecules and has been reported as a tumor suppressor in different types of neoplasias such as skin, colon and breast cancer but also in B-cell malignancies like multiple myeloma and CLL. Aims: We aimed at defining the potential role of CYLD dysregulation in the pathogenesis of SMZL, taking advantage of both primary samples and human cell lines and exploring at biochemical and molecular level the consequences of its aberrant expression. Methods: A bioinformatic analysis in FFPE biopsy samples of 35 SMZL patients was conducted to explore CYLD expression levels and the correlation with mutation status of significant genes that are frequently mutated in SMZL. Moreover, a “chronic” (SL-15) and an “aggressive” (SL-22) SMZL cell line generated from the same patient were genetically modified using CRISPR/Cas9 methodology, in order to establish CYLD-knockout (KO) SMZL cell lines. Possible aberrations in cell signaling, cell behavior and response to therapeutic agents such as the BTK inhibitor Ibrutinib and the BCL2 inhibitor Venetoclax were analyzed through biochemical assays in vitro. Furthermore, effect of CYLD ablation on cell migration and tumorigenicity was evaluated in vitro and in vivo through injection of wild-type and KO cell lines into RAG2-/-γc-/- immunodeficient mice. Results: A bioinformatic analysis of gene expression profiling data in 35 primary SMZL patient samples revealed lower CYLD expression levels and significant correlation with mutations in genes previously implicated in SMZL, including NOTCH2, TRAF3 and IKBKB. CYLD ablation in SMZL cell lines led to an increase in their proliferation and activation of NF-κB, Wnt and Notch signaling pathways. Interestingly, more prominent activation of the BCR signaling pathway was observed in CYLD KO SMZL cell lines. In addition, we observed that CYLD deletion increased not only basal but also CCL19 and CCL21-stimulated migration capacity of SMZL cell lines, in line with the known role played by CYLD in the regulation of the expression of chemokine receptors such as CCR7. Accordingly, CYLD KO SMZL cell lines showed higher dissemination in the spleen, liver, peripheral blood and peritoneal cavity after intravenous injection in RAG2-/-γc-/- immunodeficient mice. Finally, CYLD ablation was also associated to a significant resistance to target therapies such as BTK and BCL2 inhibitors. Summary/Conclusion: CYLD appears to be a key regulator in SMZL pathogenesis, dissemination and resistance to targeted agents and could be proposed as a novel target for diagnostic and therapeutic approaches. Keywords: Signaling, Splenic marginal zone lymphoma, Tumor suppressor, ibrutinib