IL-16 production is a mechanism of resistance to BTK inhibitors and R-CHOP in lymphomas

Resistance to Bruton tyrosine kinase (BTK) inhibitors remains a major clinical challenge in B-cell lymphomas and often occurs in the absence of BTK or PLCG2 mutations. Here, we investigated nongenetic mechanisms of ibrutinib resistance in marginal zone lymphoma (MZL) and their broader therapeutic implications. Continuous 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 the activation of phosphatidylinositol 3-kinase (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+ 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 patients with ibrutinib-refractory CLL without BTK/PLCG2 mutations. Mechanistically, IL-16 engaged CD9-enriched membrane microdomains to activate PI3Kδ, thereby sustaining AKT and extracellular signal-regulated kinase 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 (rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine [Oncovin], and prednisone) 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.