Haidar Akl

Selective regulation of IP3-receptor-mediated Ca2+ signaling and apoptosis by the BH4 domain of Bcl-2 versus Bcl-Xl.

Antiapoptotic B-cell lymphoma 2 (Bcl-2) targets the inositol 1,4,5-trisphosphate receptor (IP3R) via its BH4 domain, thereby suppressing IP3R Ca2+-flux properties and protecting against Ca2+-dependent apoptosis. Here, we directly compared IP3R inhibition by BH4-Bcl-2 and BH4-Bcl-Xl. In contrast to BH4-Bcl-2, BH4-Bcl-Xl neither bound the modulatory domain of IP3R nor inhibited IP3-induced Ca2+ release (IICR) in permeabilized and intact cells. We identified a critical residue in BH4-Bcl-2 (Lys17) not conserved in BH4-Bcl-Xl (Asp11). Changing Lys17 into Asp in BH4-Bcl-2 completely abolished its IP3R-binding and -inhibitory properties, whereas changing Asp11 into Lys in BH4-Bcl-Xl induced IP3R binding and inhibition. This difference in IP3R regulation between BH4-Bcl-2 and BH4-Bcl-Xl controls their antiapoptotic action. Although both BH4-Bcl-2 and BH4-Bcl-Xl had antiapoptotic activity, BH4-Bcl-2 was more potent than BH4-Bcl-Xl. The effect of BH4-Bcl-2, but not of BH4-Bcl-Xl, depended on its binding to IP3Rs. In agreement with the IP3R-binding properties, the antiapoptotic activity of BH4-Bcl-2 and BH4-Bcl-Xl was modulated by the Lys/Asp substitutions. Changing Lys17 into Asp in full-length Bcl-2 significantly decreased its binding to the IP3R, its ability to inhibit IICR and its protection against apoptotic stimuli. A single amino-acid difference between BH4-Bcl-2 and BH4-Bcl-Xl therefore underlies differential regulation of IP3Rs and Ca2+-driven apoptosis by these functional domains. Mutating this residue affects the function of Bcl-2 in Ca2+ signaling and apoptosis.

A dual role for the anti-apoptotic Bcl-2 protein in cancer: mitochondria versus endoplasmic reticulum.

Anti-apoptotic Bcl-2 contributes to cancer formation and progression by promoting the survival of altered cells. Hence, it is a prime target for novel specific anti-cancer therapeutics. In addition to its canonical anti-apoptotic role, Bcl-2 has an inhibitory effect on cell-cycle progression. Bcl-2 acts at two different intracellular compartments, the mitochondria and the endoplasmic reticulum (ER). At the mitochondria, Bcl-2 via its hydrophobic cleft scaffolds the Bcl-2-homology (BH) domain 3 (BH3) of pro-apoptotic Bcl-2-family members. Small molecules (like BH3 mimetics) can disrupt this interaction, resulting in apoptotic cell death in cancer cells. At the ER, Bcl-2 modulates Ca(2+) signaling, thereby promoting proliferation while increasing resistance to apoptosis. Bcl-2 at the ER acts via its N-terminal BH4 domain, which directly binds and inhibits the inositol 1,4,5-trisphosphate receptor (IP3R), the main intracellular Ca(2+)-release channel. Tools targeting the BH4 domain of Bcl-2 reverse Bcl-2s inhibitory action on IP3Rs and trigger pro-apoptotic Ca(2+) signaling in cancer B-cells, including chronic lymphocytic leukemia (CLL) cells and diffuse large B-cell lymphoma (DLBCL) cells. The sensitivity of DLBCL cells to BH4-domain targeting tools strongly correlated with the expression levels of the IP3R2 channel, the IP3R isoform with the highest affinity for IP3. Interestingly, bio-informatic analysis of a database of primary CLL patient cells also revealed a transcriptional upregulation of IP3R2. Finally, this review proposes a model, in which cancer cell survival depends on Bcl-2 at the mitochondria and/or the ER. This dependence likely will have an impact on their responses to BH3-mimetic drugs and BH4-domain targeting tools. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

Altered Ca2 + signaling in cancer cells: Proto-oncogenes and tumor suppressors targeting IP3 receptors

Proto-oncogenes and tumor suppressors critically control cell-fate decisions like cell survival, adaptation and death. These processes are regulated by Ca(2+) signals arising from the endoplasmic reticulum, which at distinct sites is in close proximity to the mitochondria. These organelles are linked by different mechanisms, including Ca(2+)-transport mechanisms involving the inositol 1,4,5-trisphosphate receptor (IP3R) and the voltage-dependent anion channel (VDAC). The amount of Ca(2+) transfer from the endoplasmic reticulum to mitochondria determines the susceptibility of cells to apoptotic stimuli. Suppressing the transfer of Ca(2+) from the endoplasmic reticulum to the mitochondria increases the apoptotic resistance of cells and may decrease the cellular responsiveness to apoptotic signaling in response to cellular damage or alterations. This can result in the survival, growth and proliferation of cells with oncogenic features. Clearly, proper maintenance of endoplasmic reticulum Ca(2+) homeostasis and dynamics including its links with the mitochondrial network is essential to detect and eliminate altered cells with oncogenic features through the apoptotic pathway. Proto-oncogenes and tumor suppressors exploit the central role of Ca(2+) signaling by targeting the IP3R. There are an increasing number of reports showing that activation of proto-oncogenes or inactivation of tumor suppressors directly affects IP3R function and endoplasmic reticulum Ca(2+) homeostasis, thereby decreasing mitochondrial Ca(2+) uptake and mitochondrial outer membrane permeabilization. In this review, we provide an overview of the current knowledge on the proto-oncogenes and tumor suppressors identified as IP3R-regulatory proteins and how they affect endoplasmic reticulum Ca(2+) homeostasis and dynamics.

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

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.

The selective BH4-domain biology of Bcl-2-family members: IP3Rs and beyond.

Anti-apoptotic Bcl-2-family members not only neutralize pro-apoptotic proteins but also directly regulate intracellular Ca2+ signaling from the endoplasmic reticulum (ER), critically controlling cellular health, survival, and death initiation. Furthermore, distinct Bcl-2-family members may selectively regulate inositol 1,4,5-trisphosphate receptor (IP3R): Bcl-2 likely acts as an endogenous inhibitor of the IP3R, preventing pro-apoptotic Ca2+ transients, while Bcl-XL likely acts as an endogenous IP3R-sensitizing protein promoting pro-survival Ca2+ oscillations. Furthermore, distinct functional domains in Bcl-2 and Bcl-XL may underlie the divergence in IP3R regulation. The Bcl-2 homology (BH) 4 domain, which targets the central modulatory domain of the IP3R, is likely to be Bcl-2’s determining factor. In contrast, the hydrophobic cleft targets the C-terminal Ca2+-channel tail and might be more crucial for Bcl-XL’s function. Furthermore, one amino acid critically different in the sequence of Bcl-2’s and Bcl-XL’s BH4 domains underpins their selective effect on Ca2+ signaling and distinct biological properties of Bcl-2 versus Bcl-XL. This difference is evolutionary conserved across five classes of vertebrates and may represent a fundamental divergence in their biological function. Moreover, these insights open novel avenues to selectively suppress malignant Bcl-2 function in cancer cells by targeting its BH4 domain, while maintaining essential Bcl-XL functions in normal cells. Thus, IP3R-derived molecules that mimic the BH4 domain’s binding site on the IP3R may function synergistically with BH3-mimetic molecules selectivity suppressing Bcl-2’s proto-oncogenic activity. Finally, a more general role for the BH4 domain on IP3Rs, rather than solely anti-apoptotic, may not be excluded as part of a complex network of molecular interactions.

HA14-1, but not the BH3 mimetic ABT-737, causes Ca2+ dysregulation in platelets and human cell lines.

Many Bcl-2-dependent cancer cells are primed to death due to their upregulation of BH3-only proteins in response to ongoing oncogenic stress. BH3-mimetic drugs like ABT-737 compete with Bim for binding to Bcl-2, releasing Bim and triggering Bax/Bak-mediated apoptosis.1 While ABT-737 causes regression of established tumors,2 it also limits platelet survival.3 The mechanisms underlying the observed thrombocytopenia have been the subject of debate. Since Bcl-Xl is essential for platelet life span,4 it was proposed that ABT-737, which does not discriminate between Bcl-2 and Bcl-Xl,5 kills platelets by antagonizing Bcl-Xl.3 However, these devastating effects of ABT-737 on platelet function were also associated with disturbed intracellular Ca2+ homeostasis and dynamics,3 but this remains poorly understood and controversial.6,7 We, therefore, explored the effect of ABT-737 on apoptosis in platelets and on intracellular Ca2+ signaling in platelets and human cell lines. We compared its effects on Ca2+ homeostasis with another Bcl-2 antagonist, the HA14-1 compound, which was reported to exert inhibitory properties on sarco/endoplasmic reticulum Ca2+-ATPases (SERCA).8 Washed platelets were prepared as described.9 For this, venous blood was collected from healthy donors. Permission was given by the Ethical Committee of the Leuven University Hospital to use blood from healthy individuals for further analysis. For apoptosis measurements, platelets were incubated with Annexin-V-FITC and analyzed with an Attune® Acoustic Focusing Flow Cytometer (Applied Biosystems). For the Ca2+ measurements in intact cells, platelets were seeded the day of measurement in poly-L-lysine-coated 96-well plates (Greiner) at a density of approximately 3 × 108 platelets/mL. Ca2+ measurements in intact Hela cells and unidirectional 45Ca2+-flux experiments in permeabilized cells were basically performed as previously described.10 SERCA2b ATPase activity was determined by colorimetric monitoring11 in a Ca2+-buffered solution containing microsomes (10 μg of protein) from HEK-293T cells ectopically expressing SERCA2b. Results are expressed as average ± standard deviation (SD). Significance was determined using two-tailed paired Students t-test. P<0.05 was considered significant. We confirmed that both HA14-1 (3 and 10 μM; 2 h) and ABT-737 (0.03, 0.1 and 0.3 μM; 2 h) provoked apoptosis in blood platelets (Figure 1A and B). However, only HA14-1, but not ABT-737, triggered a slow and steady increase in the cytosolic [Ca2+] originating from the intracellular Ca2+ stores in Fura2-loaded platelets exposed to extracellular EGTA (Figure 1C). Moreover, pre-treatment (10 μM; 30 min) of HA14-1, but not of ABT-737, reduced the total Ca2+ released from the ER in response to thapsigargin, an irreversible SERCA inhibitor, while not affecting store-operated Ca2+ influx (Figure 1D). To confirm our result in another cell model, we used the human cell line HeLa. Again, HA14-1, but not ABT-737, affected intracellular Ca2+ homeostasis in these cells in a concentration-dependent manner (Figure 2A). To investigate the underlying mechanism, we applied a highly quantitative 45Ca2+-flux assay10 in permeabilized HeLa cells to specifically assess ER 45Ca2+-uptake activity in the absence of plasmalemmal and mitochondrial Ca2+ fluxes and of IP3R-mediated Ca2+-release. Application of HA14-1 during the ER 45Ca2+-loading phase caused a strong decrease in the steady-state 45Ca2+ loading levels, while ABT-737 was much less effective (Figure 2B). The steady-state ER Ca2+ levels are determined by the balance between the ER Ca2+-uptake and ER Ca2+-leak activities. Importantly, application of either ABT-737 or HA14-1 (up to 30 μM) during the efflux phase did not affect the ER Ca2+-leak rate (Figure 2C), suggesting an inhibition of the ER Ca2+-uptake activity. Next, we directly assessed the effect of ABT-737 and HA14-1 on SERCA2b activity, the housekeeping isoform in human platelets,12 which was ectopically expressed in HEK-293T cells (Figure 2D). HA14-1 effectively inhibited SERCA2b Ca2+ ATPase activity (IC50 = 14 μM) while ABT-737 (up to 30 μM) had no effect. Finally, we assessed whether ABT-737 or HA14-1 directly affected IP3R-mediated Ca2+ release (e.g. which occurs in response to thrombin exposure of platelets). We used the 45Ca2+-flux assay to accurately measure IP3-induced Ca2+ release in permeabilized HeLa cells. We found that 30 μM HA14-1, but not ABT-737, inhibited the IP3R (Figure 2E). Performing a dose-response curve, we found that ABT-737 up to 100 μM did not inhibit IP3Rs. In contrast, HA14-1 potently inhibited IP3Rs at concentrations higher than 10 μM with an IC50 of approximately 50 mM and with an IP3R inhibition of approximately 80% at 100 μM (Figure 2F). Figure 1. The effect of HA14-1 and ABT-737 on apoptosis and intracellular Ca2+ signaling in platelets. (A-B) Flow-cytometry analysis of HA14-1 and ABT-737-induced apoptosis in platelets. (A) Histogram overlay representation of Annexin V-FITC staining of platelets ... Figure 2. The effect of HA14-1 and ABT-737 on ER Ca2+-uptake and –release mechanisms. (A) Fluorimetric analysis of the HA14-1 and ABT-737-induced Ca2+ responses in HeLa cells. The ratio of emitted fluorescence of Fura2 (excitation wavelength 340 nm/380 ... Our experiments clearly indicate that disrupted intracellular Ca2+ homeostasis is not a proximal event in ABT-737-induced thrombocytopenia. Thus, earlier studies indicating depleted intracellular Ca2+ homeostasis in platelets exposed for 10 μM ABT-737 for prolonged periods (e.g. 2 h) may reflect a late event that is the consequence of Bcl-Xl inhibition and ongoing cell death in platelets.6 We conclude that the dysregulation of intracellular Ca2+ signaling in platelets and human cell lines by HA14-1 is an off-target effect on SERCA2b and on IP3Rs, since on-target inhibition of Bcl-2/Bcl-Xl by ABT-737 does not disrupt intracellular Ca2+ signaling. Thus, ABT-737 has a safe Ca2+-signaling profile, since ABT-737, at therapeutically relevant concentrations (i.e. below 1 μM), does not affect intracellular Ca2+-transport mechanisms essential for cellular homeostasis.

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

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) is commonly upregulated in hematological cancers, including B-cell chronic lymphocytic leukemia (B-CLL) and diffuse large B-cell lymphoma (DLBCL), thereby protecting neoplastic cells from oncogenic-stress-induced apoptosis. Bcl-2 executes its anti-apoptotic function at two different sites in the cell. At the mitochondria, Bcl-2 via its hydrophobic cleft interacts with pro-apoptotic Bcl-2 family members to inhibit apoptosis. At the endoplasmic reticulum (ER), Bcl-2 via its Bcl-2 homology (BH)4 domain, prevents excessive Ca(2+) signals by interacting with the inositol 1,4,5-trisphosphate receptor (IP3R), an intracellular Ca(2+)-release channel. A peptide tool (BIRD-2) that targets the BH4 domain of Bcl-2 reverses Bcl-2s inhibitory action on IP3Rs and can trigger pro-apoptotic Ca(2+)signals in B-cell cancer cells. Here, we explored whether HA14-1, a Bcl-2 inhibitor that also inhibits sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCA), could potentiate BIRD-2-induced cell death. We measured apoptosis in Annexin V/7-AAD stained cells using flow cytometry and intracellular Ca(2+) signals in Fura2-AM-loaded cells using an automated fluorescent plate reader. HA14-1 potentiated BIRD-2-induced Ca(2+) release from the ER and apoptosis in both BIRD-2-sensitive DLBCL cell lines (SU-DHL-4) and in primary B-CLL cells. BIRD-2-resistant DLBCL cells (OCI-LY-1) were already very sensitive to HA14-1. Yet, although BIRD-2 moderately increased Ca(2+) levels in HA14-1-treated cells, apoptosis was not potentiated by BIRD-2 in these cells. These results further underpin the relevance of IP3R-mediated Ca(2+) signaling as a therapeutic target in the treatment of Bcl-2-dependent B-cell malignancies and the advantage of combination regimens with HA14-1 to enhance BIRD-2-induced cell death.

Reciprocal sensitivity of diffuse large B-cell lymphoma cells to Bcl-2 inhibitors BIRD-2 versus venetoclax

Bcl-2 is often upregulated in cancers to neutralize the BH3-only protein Bim at the mitochondria. BH3 mimetics (e.g. ABT-199 (venetoclax)) kill cancer cells by targeting Bcl-2’s hydrophobic cleft and disrupting Bcl-2/Bim complexes. Some cancers with elevated Bcl-2 display poor responses towards BH3 mimetics, suggesting an additional function for anti-apoptotic Bcl-2 in these cancers. Indeed, Bcl-2 via its BH4 domain prevents cytotoxic Ca2+ release from the endoplasmic reticulum (ER) by directly inhibiting the inositol 1,4,5-trisphosphate receptor (IP3R). The cell-permeable Bcl-2/IP3R disruptor-2 (BIRD-2) peptide can kill these Bcl-2-dependent cancers by targeting Bcl-2’s BH4 domain, unleashing pro-apoptotic Ca2+-release events. We compared eight “primed to death” diffuse large B-cell lymphoma cell lines (DLBCL) for their apoptotic sensitivity towards BIRD-2 and venetoclax. By determining their IC50 using cytometric cell-death analysis, we discovered a reciprocal sensitivity towards venetoclax versus BIRD-2. Using immunoblotting, we quantified the expression levels of IP3R2 and Bim in DLBCL cell lysates, revealing that BIRD-2 sensitivity correlated with IP3R2 levels but not with Bim levels. Moreover, the requirement of intracellular Ca2+ for BIRD-2- versus venetoclax-induced cell death was different. Indeed, BAPTA-AM suppressed BIRD-2-induced cell death, but promoted venetoclax-induced cell death in DLBCL cells. Finally, compared to single-agent treatments, combining BIRD-2 with venetoclax synergistically enhanced cell-death induction, correlating with a Ca2+-dependent upregulation of Bim after BIRD-2 treatment. Our findings suggest that some cancer cells require Bcl-2 proteins at the mitochondria, preventing Bax activation via its hydrophobic cleft, while others require Bcl-2 proteins at the ER, preventing cytotoxic Ca2+-signaling events via its BH4 domain.

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

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 (IP3) receptor (IP3R)-mediated Ca2+-signaling. A peptide tool (Bcl-2/IP3R Disruptor-2; BIRD-2) was developed to abrogate the interaction of Bcl-2 with IP3Rs 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 IP3R2 were sensitive to BIRD-2. Here, we report that BIRD-2-induced cell death in DLBCL cells does not only depend on high IP3R2-expression levels, but also on constitutive IP3 signaling, downstream of the tonically active B-cell receptor. The basal Ca2+ level in SU-DHL-4 DLBCL cells was significantly elevated due to the constitutive IP3 production. This constitutive IP3 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 IP3 signaling using a lower U73122 concentration (1 µM) or expression of an IP3 sponge suppressed both BIRD-2-induced Ca2+ elevation and apoptosis in SU-DHL-4 cells. Basal PLC/IP3 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 IP3 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 IP3R activity. BIRD-2 seems to switch constitutive IP3 signaling from pro-survival into pro-death, presenting a plausible therapeutic strategy.

Abstract B42: The regulation of the ER-mitochondria-Ca2+ cross-talk by Bcl-2 and Bcl-XL: A new scenario for the development of selective tools in oncology?

The process of “programmed cell suicide”, referred to as apoptosis is classically controlled by the B-cell lymphoma-2 (Bcl-2) family members, a group of intracellular proteins with a pro-death (e.g. Bax and Bak) or anti-death (e.g. Bcl-2, Bcl-Xl) role. Thus, the excessive expression of the protective Bcl-2 members is a typical trademark associated with the survival advantage of many hematological as well as solid cancers [1-2]. The recently identified interaction of the anti-apoptotic Bcl-2 protein with the inositol 1,4,5-trisphosphate receptor (IP 3 R), a ubiquitous Ca 2+ -release channel in the endoplasmic reticulum (ER), represents one of the stratagems by which Bcl-2 extends the life-span of cancer cells [3]. Bcl-2 can thereby inhibit the IP 3 R-mediated Ca 2+ firing and reduce the lethal Ca 2+ transfer from the ER to the mitochondria. The only Bcl-2 molecular component essential and sufficient for this inhibitory activity on the IP 3 R is the homology domain 4 (Bcl-2-BH4) [4]. In our study, we first revealed that although Bcl-2 shares high structural and biochemical similarity with Bcl-Xl, its closest protein relative, the latter could not curb the pro-apoptotic ER-Ca 2+ discharge. This differential action of Bcl-2 versus Bcl-Xl on the IP3R could be attributable to one critical amino acid difference in their BH4 domains [5]. Furthermore, we found that BH4-Bcl-Xl, which is also able to protect against Ca 2+ -mediated apoptosis, acted downstream of IP 3 R signaling, by physically interacting with the voltage-dependent anion channel 1 (VDAC1), an outer mitochondrial Ca 2+ gateway. Our findings opened novel avenues to selectively suppress malignant Bcl-2 or Bcl-Xl functions in cancer cells by targeting their BH4 domain biology. Accordingly, we screened for apoptosis induction a set of diffuse large-B-cell lymphoma cells (DL-BCL) treated with a cell-permeable version of a peptide that selectively antagonized Bcl-2-BH49s binding to the IP 3 R (IP 3 R-derived peptide: IDP). As a result, we observed that the most responsive subclasses of DL-BCL are also the ones that express the highest levels of the type 2 IP 3 R (IP 3 R2), which is the most sensitive isoform to IP3 signaling [6]. This is compatible with the higher metabolic needs of the DL-BCL, which in the responsive DL-BCL, was reflected in: 1) a general chronic activation of the B-cell receptor (BCR) signaling [7]; 2) an up-regulation of IP 3 R2 and 3) an addiction to Bcl-2 in order to suppress the potentially lethal IP 3 R hyperactivity. Collectively, our work provided a better understanding of the molecular and functional conversation between the Ca 2+ channels at the ER/mitochondria interface and the Bcl-2/Bcl-Xl proteins. These new findings could lead to the development of more selective and safer peptidomimetics targeting the adaptive Ca 2+ -signaling dysregulation of cancer cells. [1] (http://www.ncbi.nlm.nih.gov/pubmed/18362212) [2] (http://www.ncbi.nlm.nih.gov/pubmed/14996506) [3] (http://www.ncbi.nlm.nih.gov/pubmed/15263017) [4] (http://www.ncbi.nlm.nih.gov/pubmed/19706527) [5] (http://www.ncbi.nlm.nih.gov/pubmed/21818117) [6] (http://www.ncbi.nlm.nih.gov/pubmed/23681227) [7] (http://www.ncbi.nlm.nih.gov/pubmed/23449308) Citation Format: Haidar Akl, Giovanni Monaco, Elke Decrock, Rita La Rovere, Kirsten Welkenhuyzen, Tomas Luyten, Santeri Kiviluoto, Tim Vervliet, Jordi Molgo, Ludwig Missiaen, Katsuhiko Mikoshiba, Luc Leybaert, Jan B. Parys, Humbert De Smedt, Clark W. Distelhorst, Geert Bultynck. The regulation of the ER-mitochondria-Ca2+ cross-talk by Bcl-2 and Bcl-XL: A new scenario for the development of selective tools in oncology? [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B42.