Synergistic disruption of BTK and BCL-2 causes apoptosis while inducing ferroptosis in double-hit lymphoma

Double-hit lymphoma (DHL) is an aggressive subset of Diffuse Large B-cell Lymphoma (DLBCL) with poor outcomes and without satisfying treatment options. BTK inhibitor monotherapy is ineffective to suppress aggressive lymphoma. Hence, combination with other potential agents is warranted. Here, we demonstrated the second generation of BTK inhibitor, zanubrutinib, and a BCL-2 inhibitor, navitoclax, worked in synergistic manner to suppress DHL. Comprehensive in silico approach by interrogating single-cell to bulk-level profiling was employed along with in vitro and in vivo validation in DHL cell lines. Ablation of BTK enhanced sensitivity to navitoclax and suppressed proliferation of DHL cells. Combination of second generation of BTK inhibitor with navitoclax synergistically suppressed DLBCL cells with higher synergy score in DHL subset. The drug combi-nation triggered apoptosis and ferroptosis, with the latter being characterized by reactive oxygen species (ROS) accumulation, extensive lipid peroxidation, and depletion of reduced glutathione. Moreover, ablation of BTK sensitized DHL cells to ferroptosis. Mechanistically, disruption of BTK and BCL-2 triggered ferroptosis by downregulating NRF2 and HMOX1, while deactivating GPX4. Combination of zanubrutinib and navitoclax effectively suppressed tumor growth in vivo. Our data suggest that zanubrutinib and navitoclax synergistically suppressed DHL by inducing apoptosis and ferroptosis.

Automated summary

In this study, the synergistic effects of the second generation BTK inhibitor zanubrutinib and the BCL-2 inhibitor navitoclax in suppressing Double-hit lymphoma (DHL) were demonstrated. Comprehensive in silico analysis from single-cell to bulk-level profiling validated the combination's ability to induce apoptosis and ferroptosis, effectively inhibiting DHL cell proliferation. Mechanistically, the combination disrupted NRF2 and HMOX1 while deactivating GPX4, triggering ferroptosis. In vivo studies showed that the combination of zanubrutinib and navitoclax effectively suppressed tumor growth.