ABSTRACT: Resistance to Bruton tyrosine kinase (BTK) inhibitors remains a major clinical challenge in B-cell lymphomas and often occurs without BTK or PLCG2 mutations. Here, we investigated non-genetic mechanisms of ibrutinib resistance in marginal zone lymphoma (MZL) and their broader therapeutic implications. Chronic ibrutinib exposure generated a resistant MZL model that showed cross-resistance to BTK inhibitors and degraders, without evidence of multidrug resistance or genetic alterations. Integrated transcriptomic, epigenetic, and proteomic analyses revealed extensive reprogramming, including activation of PI3K/AKT, MAPK, and MYC pathways, repression of apoptosis and oxidative phosphorylation, and a prominent cytokine-secretory phenotype. Interleukin-16 (IL-16) emerged as a central mediator of resistance. IL-16 was transcriptionally upregulated, actively secreted, and sufficient to induce ibrutinib resistance across multiple CD9-positive models of MZL, mantle cell lymphoma, chronic lymphocytic leukemia (CLL), and activated B-cell-like diffuse large B-cell lymphoma. Serum IL-16 levels were elevated in ibrutinib-refractory CLL patients without BTK/PLCG2 mutations. Mechanistically, IL-16 engaged CD9-enriched membrane microdomains to activate PI3Kδ, thereby sustaining AKT and ERK signaling, stabilizing MYC, inducing NF-κB–dependent programs, and upregulating antiapoptotic effectors, including BFL1 (BCL2A1). Pharmacological or genetic disruption of the IL-16/CD9/PI3K axis restored sensitivity to BTK inhibitors and R-CHOP and abrogated IL-16-induced signaling in primary CLL samples. In conclusion, an IL-16/CD9-driven, epigenetically regulated survival pathway represents one possible mechanism of resistance to BTK inhibitors and chemoimmunotherapy, supporting therapeutic targeting of this axis in refractory B-cell lymphomas.
