BACKGROUND: Chronic Lymphocytic Leukemia (CLL) is a B-cell malignancy driven by a hyperactive B-cell receptor pathway (BCR-P). BCR signalling includes Bruton’s tyrosine kinase (BTK) activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2)-mediated proliferation via phospholipase C gamma 2 (PLCγ2), and phosphatidylinositol-3-Kinaseδ-Protein Kinase B (PI3Kδ-AKT) signalling promoting cell survival. The BTK inhibitor ibrutinib revolutionized CLL treatment; unfortunately, resistance is now on the rise, creating the need for a better understanding of the effects of long-term ibrutinib use. This project aims to establish and characterize a cell line model of ibrutinib resistance in order to identify cell signalling events and other changes induced by long-term ibrutinib use. METHODS: The BJAB cell line known to partly mimic CLL cell responses in vitro, was used for all investigations. Ibrutinib-resistant BJAB (IBR102) cells were created via a chronic dose-escalation approach. BJAB and IBR102 cells were treated with the BCR-P inhibitors ibrutinib (BTK), idelalisib (PI3Kδ), copanlisib (PI3Kα/β/δ/γ) and ravoxertinib (ERK1/2) alone and in combinations for 72 hours. Combinations were assessed for synergistic/additive/antagonistic cell death via flow cytometry and western blot was used for protein expression analyses. RESULTS: IBR102 cells were resistant to all BCR-P inhibitors used. Synergism in cell viability reduction was observed in ibrutinib-idelalisib/copanlisib combinations. Ravoxertinib displayed antagonism in both BJAB and IBR102 cells (to a lesser extent) when combined with all inhibitors used. Protein analysis showed PI3K-AKT-mediated ERK activation and 45% less baseline activity of ERK in IBR102 relative to BJABs; BTK activation was abolished in IBR102 cells. Ibrutinib-idelalisib combination resulted in a statistically significant synergistic reduction in ERK activation in both cell lines, but also AKT activation in BJAB cells. Notably, ibrutinib-idelalisib was shown to be synergistic in patient-derived CLL cells, and transcriptomic analysis showed that IBR102 cells expressed reduced levels of BCR, PI3K-AKT and MAPK pathway targets. CONCLUSION: Our work on IBR102 cells shows a new mechanism of ibrutinib resistance characterized by BCR pathway rewiring. We also provide evidence for an additional benefit on combining ibrutinib and a PI3K inhibitor in primary CLL cells. In summary, we propose a novel mechanism of ibrutinib resistance and define combination therapies against this resistance.