IP3R2 levels dictate the apoptotic sensitivity of diffuse large B-cell lymphoma cells to an IP3R-derived peptide targeting the BH4 domain of Bcl-2

Abstract

Disrupting 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-IDPS)) 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-IDPS 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-IDPS with SU-DHL-4 being most sensitive and OCI-LY-1 being most resistant. This sensitivity strongly correlated with the ability of TAT-IDPS 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-IDPS-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-IDPS-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.

HA14-1 potentiates apoptosis in B-cell cancer cells sensitive to a peptide disrupting IP3 receptor / Bcl-2 complexes

“…in IP 3 R2 expression levels (Akl et al, 2013a). In this study, we explored the possibility to use HA14-1, which inhibits Bcl-2 by targeting its hydrophobic cleft (Wang et al, 2000) and dysregulates Ca 2+ homeostasis by inhibiting SERCA (Akl et al, 2013b), to enhance the sensitivity of B-cell cancer cells to BIRD-2.…”

“…Bcl-2 binding to IP 3 Rs dampens the pro-apoptotic Ca 2+ flux from the ER into the mitochondria (Rong et al, 2009). A cell-permeable peptide, corresponding to the Bcl-2-binding site located in the modulatory domain of IP 3 R1, was developed and able to disrupt IP 3 R/Bcl-2 complexes in a variety of cell models, including B-cell cancer cells (Zhong et al, 2011;Akl et al, 2013a). The stabilized version of this peptide (with a mutated aspartate cleavage site) and previously referred to as TAT-IDP DD / AA (Zhong et al, 2011) or TAT-IDP S (Akl et al, 2013a;Akl et al, 2014), will for convenience be referred to as BIRD-2 (Bcl-2-IP 3 R Disrupter-2) to distinguish it from unrelated IP 3 R sequence-derived peptides now in use in our laboratories.…”

“…A cell-permeable peptide, corresponding to the Bcl-2-binding site located in the modulatory domain of IP 3 R1, was developed and able to disrupt IP 3 R/Bcl-2 complexes in a variety of cell models, including B-cell cancer cells (Zhong et al, 2011;Akl et al, 2013a). The stabilized version of this peptide (with a mutated aspartate cleavage site) and previously referred to as TAT-IDP DD / AA (Zhong et al, 2011) or TAT-IDP S (Akl et al, 2013a;Akl et al, 2014), will for convenience be referred to as BIRD-2 (Bcl-2-IP 3 R Disrupter-2) to distinguish it from unrelated IP 3 R sequence-derived peptides now in use in our laboratories. BIRD-2 triggered IP 3 R-mediated Ca 2+ signaling and subsequent apoptotic cell death in primary CLL cells (Zhong et al, 2011) and in DLBCL cell lines (Akl et al, 2013a), while normal peripheral mononuclear blood cells were resistant.…”

“…The stabilized version of this peptide (with a mutated aspartate cleavage site) and previously referred to as TAT-IDP DD / AA (Zhong et al, 2011) or TAT-IDP S (Akl et al, 2013a;Akl et al, 2014), will for convenience be referred to as BIRD-2 (Bcl-2-IP 3 R Disrupter-2) to distinguish it from unrelated IP 3 R sequence-derived peptides now in use in our laboratories. BIRD-2 triggered IP 3 R-mediated Ca 2+ signaling and subsequent apoptotic cell death in primary CLL cells (Zhong et al, 2011) and in DLBCL cell lines (Akl et al, 2013a), while normal peripheral mononuclear blood cells were resistant. DLBCL cancer cells displayed a large heterogeneity in their apoptotic responses to BIRD-2 with the SU-DHL-4 cells being most sensitive and the OCI-LY-1 cells being most resistant, a behavior linked to differences …”

Feedback regulation mediated by Bcl-2 and DARPP-32 regulates inositol 1,4,5-trisphosphate receptor phosphorylation and promotes cell survival

“…A 20-amino acid peptide (InsP 3 Rderived peptide, or IDP) corresponding to the Bcl-2 binding site on the InsP 3 R inhibits Bcl-2-InsP 3 R interaction, thus eliminating Bcl-2's control over InsP 3 R-mediated Ca 2+ elevation (6,7). This peptide induces marked Ca 2+ elevation and Ca 2+ -mediated apoptosis in primary chronic lymphocytic leukemia (CLL) cells and in B-cell lymphoma lines, indicating that Bcl-2-InsP 3 R interaction contributes to the apoptosis-resistance characteristic of these lymphoid malignancies (13,14).…”