AbstractDisrupting inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R)/B-cell lymphoma 2 (Bcl-2) complexes using a cell-permeable peptide (stabilized TAT-fused IP₃R-derived peptide (TAT-IDP^S)) that selectively targets the BH4 domain of Bcl-2 but not that of B-cell lymphoma 2-extra large (Bcl-Xl) potentiated pro-apoptotic Ca²⁺ signaling in chronic lymphocytic leukemia cells. However, the molecular mechanisms rendering cancer cells but not normal cells particularly sensitive to disrupting IP₃R/Bcl-2 complexes are poorly understood. Therefore, we studied the effect of TAT-IDP^S in a more heterogeneous Bcl-2-dependent cancer model using a set of ‘primed to death’ diffuse large B-cell lymphoma (DL-BCL) cell lines containing elevated Bcl-2 levels. We discovered a large heterogeneity in the apoptotic responses of these cells to TAT-IDP^S with SU-DHL-4 being most sensitive and OCI-LY-1 being most resistant. This sensitivity strongly correlated with the ability of TAT-IDP^S to promote IP₃R-mediated Ca²⁺ release. Although total IP₃R-expression levels were very similar among SU-DHL-4 and OCI-LY-1, we discovered that the IP₃R2-protein level was the highest for SU-DHL-4 and the lowest for OCI-LY-1. Strikingly, TAT-IDP^S-induced Ca²⁺ rise and apoptosis in the different DL-BCL cell lines strongly correlated with their IP₃R2-protein level, but not with IP₃R1-, IP₃R3- or total IP₃R-expression levels. Inhibiting or knocking down IP₃R2 activity in SU-DHL-4-reduced TAT-IDP^S-induced apoptosis, which is compatible with its ability to dissociate Bcl-2 from IP₃R2 and to promote IP₃-induced pro-apoptotic Ca²⁺ signaling. Thus, certain chronically activated B-cell lymphoma cells are addicted to high Bcl-2 levels for their survival not only to neutralize pro-apoptotic Bcl-2-family members but also to suppress IP₃R hyperactivity. In particular, cancer cells expressing high levels of IP₃R2 are addicted to IP₃R/Bcl-2 complex formation and disruption of these complexes using peptide tools results in pro-apoptotic Ca²⁺ signaling and cell death.