BIRD-2, a BH4-domain-targeting peptide of Bcl-2, provokes Bax/Bak-independent cell death in B-cell cancers through mitochondrial Ca²⁺-dependent mPTP opening

Anti-apoptotic Bcl-2 critically controls cell death by neutralizing pro-apoptotic Bcl-2-family members at the mitochondria. Bcl-2 proteins also act at the endoplasmic reticulum, the main intracellular Ca²⁺-storage organelle, where they inhibit IP₃ receptors (IP₃R) and prevent pro-apoptotic Ca²⁺-signaling events. IP₃R channels are targeted by the BH4 domain of Bcl-2. Some cancer types rely on the IP₃R-Bcl-2 interaction for survival. We previously developed a cell-permeable, BH4-domain-targeting peptide that can abrogate Bcl-2′s inhibitory action on IP₃Rs, named Bcl-2 IP₃ receptor disrupter-2 (BIRD-2). This peptide kills several Bcl-2-dependent cancer cell types, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukaemia (CLL) cells, by eliciting intracellular Ca²⁺ signalling. However, the exact mechanisms by which these excessive Ca²⁺ signals triggered by BIRD-2 provoke cancer cell death remain elusive. Here, we demonstrate in DLBCL that although BIRD-2 activates caspase 3/7 and provokes cell death in a caspase-dependent manner, the cell death is independent of pro-apoptotic Bcl-2-family members, Bim, Bax and Bak. Instead, BIRD-2 provokes mitochondrial Ca²⁺ overload that is rapidly followed by opening of the mitochondrial permeability transition pore (mPTP). Inhibiting mitochondrial Ca²⁺ overload using Ru265, an inhibitor of the mitochondrial Ca²⁺ uniporter complex counteracts BIRD-2-induced cancer cell death. Finally, we validated our findings in primary CLL patient samples where BIRD-2 provoked mitochondrial Ca²⁺ overload and Ru265 counteracted BIRD-2-induced cell death. Overall, this work reveals the mechanisms by which BIRD-2 provokes cell death, which occurs via mitochondrial Ca²⁺ overload but acts independently of pro-apoptotic Bcl-2-family members.