REL overexpression and sustained NF-κB signaling associated with 2p gain induce resistance to BTK inhibitors in Chronic Lymphocytic Leukemia

Abstract

The gain of chromosome 2p (2p⁺) is a recurrent abnormality in chronic lymphocytic leukemia (CLL), frequently observed in advanced or relapsed disease, associated with poor prognosis and reduced response to Bruton’s tyrosine kinase inhibitors (BTKi). To investigate the mechanisms of 2p⁺-mediated resistance, we performed single-cell RNA sequencing, revealing NF-κB pathway enrichment and REL overexpression in 2p⁺ B cells. In vitro analyses confirmed increased REL expression and DNA-binding activity in a large cohort of 2p⁺ primary CLL samples. Functionally, 2p⁺ CLL cells showed reduced sensitivity to both covalent and non-covalent BTKi. Moreover, upon ibrutinib treatment, REL DNA-binding activity decreased in 2p^WT CLL cells but remained sustained in 2p⁺ CLL cells following BCR stimulation, suggesting that persistent NF-κB activation contributes to resistance. Consistently, CRISPR/Cas9-mediated inactivation of REL in a 2p⁺ B-lymphoid cell line led to downregulation of canonical NF-κB signaling and restored BTKi sensitivity. Clinically, patients with 2p⁺ CLL treated with BTKi had a shorter time-to-next-treatment than 2p^WT patients. Altogether, our study identifies REL overexpression as a novel 2p⁺-driven mechanism of BTKi resistance in CLL, complementing the well described BTK and PLCG2 mutations. These findings support the clinical relevance of detecting 2p gain to guide treatment strategies and improve outcomes in CLL.