Abstract A127: Secretion of IL16 is associated with resistance to ibrutinib in pre-clinical models of lymphoma
Authors: Arribas Alberto J. , Napoli Sara , Gaudio Eugenio , Cascione Luciano , Veroli Alessandra Di , Tarantelli Chiara , Spriano Filipppo , Zucchetto Antonella , Rossi Francesca , Sartori Giulio , Rinaldi Andrea , Stathis Anastasios , Stussi Georg , Gattei Valter , Cruciani Gabriele , Zucca Emanuele , Rossi Davide , Bertoni Francesco 
Published: March 2020

Abstract
Background.The first-in-class BTK inhibitor ibrutinib has been approved by the U.S. Food and Drug Administration (FDA) for different indications including the treatment of patients with marginal zone lymphoma, who are in need of systemic therapy and have received at least one prior anti-CD20-based therapy. We have generated and characterized a model resistant to ibrutinib and derived from splenic marginal zone lymphoma. Materials and Methods.The splenic MZL VL51 cell line was kept under ibrutinib (IC90) until acquisition of resistance or with no drug (parental, PAR). Cell identity was confirmed by STR DNA fingerprinting. Resistance was determined by MTT assay as stable if present after 2-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out. Cells underwent transcriptome profiling (RNA-Seq), whole exome sequencing, lipidomic profiling, pharmacological screening (348 compounds), and immunophenotypic analysis (FACS). Secreted cytokines and growth factors were analyzed by ELISA. Results. We developed a stable ibrutinib resistance model derived from VL51 cell line (VL51-ibR) with 8-fold times higher IC50 than parental cells. Specific mutations associated with resistance were not detected, including those in BTKor PLCG2 genes. Conditioned media from VL51-ibR conferred resistance to ibrutinib in the parental cells, indicating the involvement of secreted factors in the mechanism of resistance. At transcriptome level, VL51-ibR exhibited overexpression of genes coding for secreted molecules (IL16, CXCL10), integrins (ITGAM, ITGA1), members of the NFKB (TNF, LTA) and RAS-RAF (RASGRP4, RASGRP2) signaling pathways. The pharmacologic screening identified acquired sensitivity to a RAS- inhibitor. Lipidomic profiling showed high levels of specific triacylglycerols, glycerophosphocholines and cardiolipins with a down-regulation of sphingomyelins. Also, in agreement with transcriptomic data, VL51-ibR had increased levels of p-PLCG2 and p-ERK, paired with the presence of IL6 and CXCL10 in the medium and double positive surface expression of CXCR5 and CD49d. We extended our findings to other models and to clinical specimens. Lastly, we investigated whether these results might be extrapolated into different in vitro models and splenic marginal zone lymphoma clinical cases. First, IL16 and CXCL10 expression levels were inversely correlated with sensitivity to ibrutinib in a panel of 13 B-cell lymphoma cell lines (Tarantelli et al, CCR 2018) (P&lt;0.05). Second, we determined the top 200 genes positively correlated genes with IL16 in a series of splenic marginal zone lymphoma clinical cases (Arribas et al, Mod Pathol 2013), and we observed that these genes were also more enriched in the VL51-ibR when compared to the parental VL51. Conclusions. We have developed and characterized a preclinical model, driven by secreted factors, of secondary resistance to the BTK-inhibitor ibrutinib in splenic marginal zone lymphoma. The current work provides new insights into the mechanisms of resistance to ibrutinib and can lead to novel therapeutic approaches to overcome the resistance.
Citation Format: Alberto J. Arribas, Sara Napoli, Eugenio Gaudio, Luciano Cascione, Alessandra Di Veroli, Chiara Tarantelli, Filipppo Spriano, Antonella Zucchetto, Francesca Rossi, Giulio Sartori, Andrea Rinaldi, Anastasios Stathis, Georg Stussi, Valter Gattei, Gabriele Cruciani, Emanuele Zucca, Davide Rossi, Francesco Bertoni. Secretion of IL16 is associated with resistance to ibrutinib in pre-clinical models of lymphoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A127. doi:10.1158/1535-7163.TARG-19-A127