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

ABT-199 (Venetoclax), a BH3-mimetic Bcl-2 inhibitor, does not cause Ca^{2+}-signalling dysregulation or toxicity in pancreatic acinar cells

Background and Purpose: Many cancer cells depend on anti‐apoptotic B‐cell lymphoma 2 (Bcl‐2) proteins for their survival. Bcl‐2 antagonism through Bcl‐2 homology 3 (BH3) mimetics has emerged as a novel anti‐cancer therapy. ABT‐199 (Venetoclax), a recently developed BH3 mimetic that selectively inhibits Bcl‐2, was introduced into the clinic for treatment of relapsed chronic lymphocytic leukaemia. Early generations of Bcl‐2 inhibitors evoked sustained Ca2+ responses in pancreatic acinar cells (PACs) inducing cell death. Therefore, BH3 mimetics could potentially be toxic for the pancreas when used to treat cancer. Although ABT‐199 was shown to kill Bcl‐2‐dependent cancer cells without affecting intracellular Ca2+ signalling, its effects on PACs have not yet been determined. Hence, it is essential and timely to assess whether this recently approved anti‐leukaemic drug might potentially have pancreatotoxic effects. Experimental Approach: Single‐cell Ca2+ measurements and cell death analysis were performed on isolated mouse PACs. Key Results: Inhibition of Bcl‐2 via ABT‐199 did not elicit intracellular Ca2+ signalling on its own or potentiate Ca2+ signalling induced by physiological/pathophysiological stimuli in PACs. Although ABT‐199 did not affect cell death in PACs, under conditions that killed ABT‐199‐sensitive cancer cells, cytosolic Ca2+ extrusion was slightly enhanced in the presence of ABT‐199. In contrast, inhibition of Bcl‐xL potentiated pathophysiological Ca2+ responses in PACs, without exacerbating cell death. Conclusion and Implications: Our results demonstrate that apart from having a modest effect on cytosolic Ca2+ extrusion, ABT‐199 does not substantially alter intracellular Ca2+ homeostasis in normal PACs and should be safe for the pancreas during cancer treatment

Insights In Bcl-2 Dependence In Diffuse Large B-Cell Lymphomas -Two Sides Of The Same Anti-Apoptotic Coin

We aim to get a better insight in the pathology of diffuse large B-cell lymphoma (DLBCL) on the cellular and molecular level. To do this, we studied two different aspects of the anti-apoptotic role of the protein B-cell lymphoma-2 (Bcl-2), a protein involved in cell fate decisions. Typically, Bcl-2 prevents cell death by sequestering pro-apoptotic proteins via a hydrophobic groove on its surface. This working mechanism of Bcl-2 can be blocked by BH3 mimetics, small molecules that bind the hydrophobic groove and prevent capturing of pro-apoptotic proteins by Bcl-2. Venetoclax is a Bcl-2-specific BH3 mimetic and is able to kill cancer cells that rely on Bcl-2 for their survival. It is used in the clinic to treat relapsed patients suffering from chronic lymphocytic leukaemia. Despite its success, resistance against venetoclax has been reported in the clinic. Thus, a first aspect we wanted to study was venetoclax resistance in DLBCL cell lines, comparing a naïve cell line with a cell line previously exposed and rendered resistant to venetoclax. We report that venetoclax resistance in this specific instance occurred through upregulation of another anti-apoptotic protein from the Bcl-2-protein family: Bcl-XL. Apart from its canonical role, Bcl-2 exerts many "moonlighting" functions. One of these is its control over apoptosis by inhibiting Ca2+ release from the endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor (IP3R). As opposed to its classic anti-apoptotic function, the hydrophobic groove does not play a role in the Bcl-2-IP3R interaction. It is rather the BH4 domain of Bcl-2 that prevents IP3R-mediated Ca2+ release. Bcl-2-IP3R disruptor-2 (BIRD-2) is a decoy peptide that can break the Bcl-2-IP3R interaction, thereby killing cancer cells. However, its downstream signalling effects were not well understood. We observed that BIRD-2 triggers mitochondrial Ca2+ uptake and mitochondrial permeability transition pore opening, causing caspase activation and cell death in DLBCL cell lines. In this thesis, we studied both resistance towards venetoclax and downstream signalling triggered by peptide-mediated disruption of the IP3R-Bcl-2 interaction in DLBCL. We gained insight in the importance of both targeting the canonical role of Bcl-2 and targeting the non-canonical Bcl-2-IP3R interaction. We hope these results contribute to further exploitation of Bcl-2-centered therapies in DLBCL.status: publishe

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

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Emerging molecular mechanisms in chemotherapy: Ca2+signaling at the mitochondria-associated endoplasmic reticulum membranes

Inter-organellar communication often takes the form of Ca2+signals. These Ca2+signals originate from the endoplasmic reticulum (ER) and regulate different cellular processes like metabolism, fertilization, migration, and cell fate. A prime target for Ca2+signals are the mitochondria. ER-mitochondrial Ca2+transfer is possible through the existence of mitochondria-associated ER membranes (MAMs), ER structures that are in the proximity of the mitochondria. This creates a micro-domain in which the Ca2+concentrations are manifold higher than in the cytosol, allowing for rapid mitochondrial Ca2+uptake. In the mitochondria, the Ca2+signal is decoded differentially depending on its spatiotemporal characteristics. While Ca2+oscillations stimulate metabolism and constitute pro-survival signaling, mitochondrial Ca2+overload results in apoptosis. Many chemotherapeutics depend on efficient ER-mitochondrial Ca2+signaling to exert their function. However, several oncogenes and tumor suppressors present in the MAMs can alter Ca2+signaling in cancer cells, rendering chemotherapeutics ineffective. In this review, we will discuss recent studies that connect ER-mitochondrial Ca2+transfer, tumor suppressors and oncogenes at the MAMs, and chemotherapy

Endoplasmic Reticulum–Mitochondrial Ca2+ Fluxes Underlying Cancer Cell Survival

Calcium ions (Ca2+) are crucial, ubiquitous, intracellular second messengers required for functional mitochondrial metabolism during uncontrolled proliferation of cancer cells. The mitochondria and the endoplasmic reticulum (ER) are connected via “mitochondria-associated ER membranes” (MAMs) where ER–mitochondria Ca2+ transfer occurs, impacting the mitochondrial biology related to several aspects of cellular survival, autophagy, metabolism, cell death sensitivity, and metastasis, all cancer hallmarks. Cancer cells appear addicted to these constitutive ER–mitochondrial Ca2+ fluxes for their survival, since they drive the tricarboxylic acid cycle and the production of mitochondrial substrates needed for nucleoside synthesis and proper cell cycle progression. In addition to this, the mitochondrial Ca2+ uniporter and mitochondrial Ca2+ have been linked to hypoxia-inducible factor 1α signaling, enabling metastasis and invasion processes, but they can also contribute to cellular senescence induced by oncogenes and replication. Finally, proper ER–mitochondrial Ca2+ transfer seems to be a key event in the cell death response of cancer cells exposed to chemotherapeutics. In this review, we discuss the emerging role of ER–mitochondrial Ca2+ fluxes underlying these cancer-related features

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

Pathophysiological consequences of isoform-specific IP3 receptor mutations

Ca2+ signaling governs a diverse range of cellular processes and, as such, is subject to tight regulation. A main component of the complex intracellular Ca2+-signaling network is the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), a tetrameric channel that mediates Ca2+ release from the endoplasmic reticulum (ER) in response to IP3. IP3R function is controlled by a myriad of factors, such as Ca2+, ATP, kinases and phosphatases and a plethora of accessory and regulatory proteins. Further complexity in IP3R-mediated Ca2+ signaling is the result of the existence of three main isoforms (IP3R1, IP3R2 and IP3R3) that display distinct functional characteristics and properties. Despite their abundant and overlapping expression profiles, IP3R1 is highly expressed in neurons, IP3R2 in cardiomyocytes and hepatocytes and IP3R3 in rapidly proliferating cells as e.g. epithelial cells. As a consequence, dysfunction and/or dysregulation of IP3R isoforms will have distinct pathophysiological outcomes, ranging from neurological disorders for IP3R1 to dysfunctional exocrine tissues and autoimmune diseases for IP3R2 and -3. Over the past years, several IP3R mutations have surfaced in the sequence analysis of patient-derived samples. Here, we aimed to provide an integrative overview of the clinically most relevant mutations for each IP3R isoform and the subsequent molecular mechanisms underlying the etiology of the disease.status: publishe

Emerging molecular mechanisms in chemotherapy: Ca2+ signaling at the mitochondria-associated endoplasmic reticulum membranes

Inter-organellar communication often takes the form of Ca2+ signals. These Ca2+ signals originate from the endoplasmic reticulum (ER) and regulate different cellular processes like metabolism, fertilization, migration, and cell fate. A prime target for Ca2+ signals are the mitochondria. ER-mitochondrial Ca2+ transfer is possible through the existence of mitochondria-associated ER membranes (MAMs), ER structures that are in the proximity of the mitochondria. This creates a micro-domain in which the Ca2+ concentrations are manifold higher than in the cytosol, allowing for rapid mitochondrial Ca2+ uptake. In the mitochondria, the Ca2+ signal is decoded differentially depending on its spatiotemporal characteristics. While Ca2+ oscillations stimulate metabolism and constitute pro-survival signaling, mitochondrial Ca2+ overload results in apoptosis. Many chemotherapeutics depend on efficient ER-mitochondrial Ca2+ signaling to exert their function. However, several oncogenes and tumor suppressors present in the MAMs can alter Ca2+ signaling in cancer cells, rendering chemotherapeutics ineffective. In this review, we will discuss recent studies that connect ER-mitochondrial Ca2+ transfer, tumor suppressors and oncogenes at the MAMs, and chemotherapy.status: publishe