The type 2 inositol 1,4,5-trisphosphate receptor, emerging functions for an intriguing Ca2+-release channel

AbstractThe inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) type 2 (IP3R2) is an intracellular Ca2+-release channel located on the endoplasmic reticulum (ER). IP3R2 is characterized by a high sensitivity to both IP3 and ATP and is biphasically regulated by Ca2+. Furthermore, IP3R2 is modulated by various protein kinases. In addition to its regulation by protein kinase A, IP3R2 forms a complex with adenylate cyclase 6 and is directly regulated by cAMP. Finally, in the ER, IP3R2 is less mobile than the other IP3R isoforms, while its functional properties appear dominant in heterotetramers. These properties make the IP3R2 a Ca2+ channel with exquisite properties for setting up intracellular Ca2+ signals with unique characteristics. IP3R2 plays a crucial role in the function of secretory cell types (e.g. pancreatic acinar cells, hepatocytes, salivary gland, eccrine sweat gland). In cardiac myocytes, the role of IP3R2 appears more complex, because, together with IP3R1, it is needed for normal cardiogenesis, while its aberrant activity is implicated in cardiac hypertrophy and arrhythmias. Most importantly, its high sensitivity to IP3 makes IP3R2 a target for anti-apoptotic proteins (e.g. Bcl-2) in B-cell cancers. Disrupting IP3R/Bcl-2 interaction therefore leads in those cells to increased Ca2+ release and apoptosis. Intriguingly, IP3R2 is not only implicated in apoptosis but also in the induction of senescence, another tumour-suppressive mechanism. These results were the first to unravel the physiological and pathophysiological role of IP3R2 and we anticipate that further progress will soon be made in understanding the function of IP3R2 in various tissues and organs

DLBCL cells with acquired resistance to venetoclax are not sensitized to BIRD-2 but can be resensitized to venetoclax through Bcl-XL inhibition

Anti-apoptotic Bcl-2-family members are frequently dysregulated in both blood and solid cancers, contributing to their survival despite ongoing oncogenic stress. Yet, such cancer cells often are highly dependent on Bcl-2 for their survival, a feature that is exploited by so-called BH3-mimetic drugs. Venetoclax (ABT-199) is a selective BH3-mimetic Bcl-2 antagonist that is currently used in the clinic for treatment of chronic lymphocytic leukemia patients. Unfortunately, venetoclax resistance has already emerged in patients, limiting the therapeutic success. Here, we examined strategies to overcome venetoclax resistance. Therefore, we used two diffuse large B-cell lymphoma (DLBCL) cell lines, Riva WT and venetoclax-resistant Riva (VR). The latter was obtained by prolonged culturing in the presence of venetoclax. We report that Riva VR cells did not become more sensitive to BIRD-2, a peptide targeting the Bcl-2 BH4 domain, and established cross-resistance towards BDA-366, a putative BH4-domain antagonist of Bcl-2. However, we found that Bcl-XL, another Bcl-2-family protein, is upregulated in Riva VR, while Mcl-1 expression levels are not different in comparison with Riva WT, hinting towards an increased dependence of Riva VR cells to Bcl-XL. Indeed, Riva VR cells could be resensitized to venetoclax by A-1155463, a selective BH3 mimetic Bcl-XL inhibitor. This is underpinned by siRNA experiments, demonstrating that lowering Bcl-XL-expression levels also augmented the sensitivity of Riva VR cells to venetoclax. Overall, this work demonstrates that Bcl-XL upregulation contributes to acquired resistance of DLBCL cancer cells towards venetoclax and that antagonizing Bcl-XL can resensitize such cells towards venetoclax

Antagonizing Bcl-2's BH4 domain in cancer

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Recent advances in uncovering the mechanisms contributing to BIRD-2-induced cell death in B-cell cancer cells

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The type 2 inositol 1,4,5-trisphosphate receptor, emerging functions for an intriguing Ca2+-release channel

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) type 2 (IP3R2) is an intracellular Ca²⁺-release channel located on the endoplasmic reticulum (ER). IP3R2 is characterized by a high sensitivity to both IP3 and ATP and is biphasically regulated by Ca²⁺. Furthermore, IP3R2 is modulated by various protein kinases. In addition to its regulation by protein kinase A, IP3R2 forms a complex with adenylate cyclase 6 and is directly regulated by cAMP. Finally, in the ER, IP3R2 is less mobile than the other IP3R isoforms, while its functional properties appear dominant in heterotetramers. These properties make the IP3R2 a Ca²⁺ channel with exquisite properties for setting up intracellular Ca²⁺ signals with unique characteristics. IP3R2 plays a crucial role in the function of secretory cell types (e.g. pancreatic acinar cells, hepatocytes, salivary gland, eccrine sweat gland). In cardiac myocytes, the role of IP3R2 appears more complex, because, together with IP3R1, it is needed for normal cardiogenesis, while its aberrant activity is implicated in cardiac hypertrophy and arrhythmias. Most importantly, its high sensitivity to IP3 makes IP3R2 a target for anti-apoptotic proteins (e.g. Bcl-2) in B-cell cancers. Disrupting IP3R/Bcl-2 interaction therefore leads in those cells to increased Ca²⁺ release and apoptosis. Intriguingly, IP3R2 is not only implicated in apoptosis but also in the induction of senescence, another tumour-suppressive mechanism. These results were the first to unravel the physiological and pathophysiological role of IP3R2 and we anticipate that further progress will soon be made in understanding the function of IP3R2 in various tissues and organs.publisher: Elsevier articletitle: The type 2 inositol 1,4,5-trisphosphate receptor, emerging functions for an intriguing Ca2+-release channel journaltitle: Biochimica et Biophysica Acta (BBA) - Molecular Cell Research articlelink: http://dx.doi.org/10.1016/j.bbamcr.2014.12.006 content_type: article copyright: Copyright © 2014 Elsevier B.V. All rights reserved.status: publishe

The selective Bcl-2 inhibitor venetoclax, a BH3 mimetic, does not dysregulate intracellular Ca2+ signaling

Anti-apoptotic B cell-lymphoma-2 (Bcl-2) proteins are emerging as therapeutic targets in a variety of cancers for precision medicines, like the BH3-mimetic drug venetoclax (ABT-199), which antagonizes the hydrophobic cleft of Bcl-2. However, the impact of venetoclax on intracellular Ca2+ homeostasis and dynamics in cell systems has not been characterized in detail. Here, we show that venetoclax did not affect Ca2+-transport systems from the endoplasmic reticulum (ER) in permeabilized cell systems. Venetoclax (1μM) did neither trigger Ca2+ release by itself nor affect agonist-induced Ca2+ release in a variety of intact cell models. Among the different cell types, we also studied two Bcl-2-dependent cancer cell models with a varying sensitivity towards venetoclax, namely SU-DHL-4 and OCI-LY-1, both diffuse large B-cell lymphoma cell lines. Acute application of venetoclax did also not dysregulate Ca2+ signaling in these Bcl-2-dependent cancer cells. Moreover, venetoclax-induced cell death was independent of intracellular Ca2+ overload, since Ca2+ buffering using BAPTA-AM did not suppress venetoclax-induced cell death. This study therefore shows that venetoclax does not dysregulate the intracellular Ca2+ homeostasis in a variety of cell types, which may underlie its limited toxicity in human patients. Furthermore, venetoclax-induced cell death in Bcl-2-dependent cancer cells is not mediated by intracellular Ca2+ overload. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.publisher: Elsevier articletitle: The selective Bcl-2 inhibitor venetoclax, a BH3 mimetic, does not dysregulate intracellular Ca2+ signaling journaltitle: Biochimica et Biophysica Acta (BBA) - Molecular Cell Research articlelink: http://dx.doi.org/10.1016/j.bbamcr.2016.11.024 content_type: article copyright: © 2016 Elsevier B.V. All rights reserved.status: publishe

The type 2 inositol 1,4,5-trisphosphate receptor, emerging functions for an intriguing Ca2+-release channel

AbstractThe inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) type 2 (IP3R2) is an intracellular Ca2+-release channel located on the endoplasmic reticulum (ER). IP3R2 is characterized by a high sensitivity to both IP3 and ATP and is biphasically regulated by Ca2+. Furthermore, IP3R2 is modulated by various protein kinases. In addition to its regulation by protein kinase A, IP3R2 forms a complex with adenylate cyclase 6 and is directly regulated by cAMP. Finally, in the ER, IP3R2 is less mobile than the other IP3R isoforms, while its functional properties appear dominant in heterotetramers. These properties make the IP3R2 a Ca2+ channel with exquisite properties for setting up intracellular Ca2+ signals with unique characteristics. IP3R2 plays a crucial role in the function of secretory cell types (e.g. pancreatic acinar cells, hepatocytes, salivary gland, eccrine sweat gland). In cardiac myocytes, the role of IP3R2 appears more complex, because, together with IP3R1, it is needed for normal cardiogenesis, while its aberrant activity is implicated in cardiac hypertrophy and arrhythmias. Most importantly, its high sensitivity to IP3 makes IP3R2 a target for anti-apoptotic proteins (e.g. Bcl-2) in B-cell cancers. Disrupting IP3R/Bcl-2 interaction therefore leads in those cells to increased Ca2+ release and apoptosis. Intriguingly, IP3R2 is not only implicated in apoptosis but also in the induction of senescence, another tumour-suppressive mechanism. These results were the first to unravel the physiological and pathophysiological role of IP3R2 and we anticipate that further progress will soon be made in understanding the function of IP3R2 in various tissues and organs

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.status: publishe

Bcl-2 inhibitors as anti-cancer therapeutics: the impact of and on calcium signaling

Bcl-2-protein family members are essential regulators of apoptosis. Anti-apoptotic Bcl-2 proteins ensure cell survival via different mechanisms, including via binding of pro-apoptotic Bcl-2-family members and the modulation of intracellular Ca2+-transport systems. Many cancer cells upregulate these proteins to overcome the consequences of ongoing oncogenic stress. Bcl-2 inhibition leading to cell death, therefore emerged as a novel cancer therapy. Different Bcl-2 inhibitors have already been developed including the hydrophobic cleft-targeting BH3 mimetics, which antagonize Bcl-2's ability to scaffold and neutralize pro-apoptotic Bcl-2-family members. As such, the BH3 mimetics have progressed into clinical studies as precision medicines. Furthermore, new inhibitors that target Bcl-2's BH4 domain have been developed as promising anti-cancer tools. Given Bcl-2's role in Ca2+ signaling, these drugs and tools can impact Ca2+ signaling. In addition to this, some Bcl-2 inhibitors may have "off-target" effects that cause Ca2+-signaling dysregulation not only in cancer cells but also in healthy cells, resulting in adverse effects. In this review, we aim to provide an up-to-date overview of the involvement of intracellular Ca2+ signaling in the working mechanism and "off-target" effects of the different Bcl-2-antagonizing small molecules and peptides.status: publishe