IL16 PRODUCTION IS A MECHANISM OF RESISTANCE TO BTK INHIBITORS AND TO R‐CHOP

Introduction: Covalent and non-covalent BTK inhibitors (BTKi) are used or being explored for the treatment of patients with B-cell malignancies, such as marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) of the activated B-cell type (ABC). The identification of mechanisms of resistance can lead to improvements in the current therapeutic approaches. We had reported IL16 production in a MZL model of secondary resistance to ibrutinib (Arribas ENA2019). Here, we expand the initial observation to additional lymphoma types and treatments modalities, performing therapeutic interventions to block it and providing clinical validation data on its expression and role in patients. Methods: VL51 (marginal zone lymphoma, MZL) parental (PAR) and Ibrutinib-resistant (RES) cells underwent RNA (mRNA & miRNA by RNA-Seq), DNA (promoter methylation, WES) and secretome (Cytokine array) profiling. RES cells exhibited resistance to covalent and non-covalent BTK inhibitors. No DNA copy number variations or mutations were detected by WES in RES compared to PAR cells, including those affecting BTK or PLCG2 genes. Results: Multi-omics (DNA, mRNA, miRNA, secretome) characterization of RES and PAR cells identified hypomethylation, up-regulation and secretion of IL16 in RES but not in PAR cells. Elevated levels of p-AKT and p-ERK was observed in RES compared to PAR, suggesting a signaling activation in RES cells. Stimulation with recombinant IL16 reduced sensitivity to BTKi not only in VL51 PAR cells, but also in models derived from MCL (REC1) and CLL (MEC1). Conversely, the use of IL16 blocking antibody recovered sensitivity to BTKi in RES cells, along with other lines with high expression of IL16 and low sensitivity to ibrutinib derived from MCL (MAVER1) and MZL (SSK41). IL16 is part of the ABC-DLBCL signature. We observed that high IL16 expression was associated with shorter overall survival in ABC but not in germinal center like DLBCL patients (n = 233, log-rank test p = 0.013, multivariate Cox: HR (95% CI) 0.43 (0.22–0.85), p = 0.016). IL16 stimulation also decreased sensitivity to RCHOP in ABC DBCL cells (OCILy10, HBL1). Finally, secreted levels of IL16 in the serum of CLL patients (n = 17) with resistance to ibrutinib (in absence of BTK or PLCG2 mutations) were significantly higher compared to patients responding to the drug and paired for clinical features. Conclusions: Starting from a MZL model of secondary resistance to ibrutinib, we showed that IL16 can sustain resistance to BTK inhibitors also in CLL and MCL. Furthermore, increased expression of IL16 associated with shorter outcome in ABC DLBCL patients and the interleukin induced in vitro resistance to RCHOP. The demonstration that IL16 blocking therapies recovered sensitivity to therapies can lead to novel therapeutic approaches to overcome resistance in lymphoma patients. The research was funded by: Swiss National Science Foundation (SNSF 31003A_163232/1) Keyword: molecular targeted therapies Conflicts of interests pertinent to the abstract A. Arribas Educational grants: Astra Zeneca