Constitutive IP ₃ signaling underlies the sensitivity of B-cell cancers to the Bcl-2/IP ₃ receptor disruptor BIRD-2

Abstract

Anti-apoptotic Bcl-2 proteins are upregulated in different cancers, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), enabling survival by inhibiting pro-apoptotic Bcl-2-family members and inositol 1,4,5-trisphosphate (IP ₃) receptor (IP ₃R)-mediated Ca ²⁺-signaling. A peptide tool (Bcl-2/IP ₃R Disruptor-2; BIRD-2) was developed to abrogate the interaction of Bcl-2 with IP ₃Rs by targeting Bcl-2′s BH4 domain. BIRD-2 triggers cell death in primary CLL cells and in DLBCL cell lines. Particularly, DLBCL cells with high levels of IP ₃R2 were sensitive to BIRD-2. Here, we report that BIRD-2-induced cell death in DLBCL cells does not only depend on high IP ₃R2-expression levels, but also on constitutive IP ₃ signaling, downstream of the tonically active B-cell receptor. The basal Ca ²⁺ level in SU-DHL-4 DLBCL cells was significantly elevated due to the constitutive IP ₃ production. This constitutive IP ₃ signaling fulfilled a pro-survival role, since inhibition of phospholipase C (PLC) using U73122 (2.5 µM) caused cell death in SU-DHL-4 cells. Milder inhibition of IP ₃ signaling using a lower U73122 concentration (1 µM) or expression of an IP ₃ sponge suppressed both BIRD-2-induced Ca ²⁺ elevation and apoptosis in SU-DHL-4 cells. Basal PLC/IP ₃ signaling also fulfilled a pro-survival role in other DLBCL cell lines, including Karpas 422, RI-1 and SU-DHL-6 cells, whereas PLC inhibition protected these cells against BIRD-2-evoked apoptosis. Finally, U73122 treatment also suppressed BIRD-2-induced cell death in primary CLL, both in unsupported systems and in co-cultures with CD40L-expressing fibroblasts. Thus, constitutive IP ₃ signaling in lymphoma and leukemia cells is not only important for cancer cell survival, but also represents a vulnerability, rendering cancer cells dependent on Bcl-2 to limit IP ₃R activity. BIRD-2 seems to switch constitutive IP ₃ signaling from pro-survival into pro-death, presenting a plausible therapeutic strategy.