BDA-366, a putative Bcl-2 BH4 domain antagonist, induces apoptosis independently of Bcl-2 in a variety of cancer cell models.

Several cancer cell types, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) upregulate antiapoptotic Bcl-2 to cope with oncogenic stress. BH3 mimetics targeting Bcl-2's hydrophobic cleft have been developed, including venetoclax as a promising anticancer precision medicine for treating CLL patients. Recently, BDA-366 was identified as a small molecule BH4-domain antagonist that could kill lung cancer and multiple myeloma cells. BDA-366 was proposed to switch Bcl-2 from an antiapoptotic into a proapoptotic protein, thereby activating Bax and inducing apoptosis. Here, we scrutinized the therapeutic potential and mechanism of action of BDA-366 in CLL and DLBCL. Although BDA-366 displayed selective toxicity against both cell types, the BDA-366-induced cell death did not correlate with Bcl-2-protein levels and also occurred in the absence of Bcl-2. Moreover, although BDA-366 provoked Bax activation, it did neither directly activate Bax nor switch Bcl-2 into a Bax-activating protein in in vitro Bax/liposome assays. Instead, in primary CLL cells and DLBCL cell lines, BDA-366 inhibited the activity of the PI3K/AKT pathway, resulted in Bcl-2 dephosphorylation and reduced Mcl-1-protein levels without affecting the levels of Bcl-2 or Bcl-xL. Hence, our work challenges the current view that BDA-366 is a BH4-domain antagonist of Bcl-2 that turns Bcl-2 into a pro-apoptotic protein. Rather, our results indicate that other mechanisms beyond switching Bcl-2 conformation underlie BDA-366's cell-death properties that may implicate Mcl-1 downregulation and/or Bcl-2 dephosphorylation.

APOL1 C-Terminal Variants May Trigger Kidney Disease through Interference with APOL3 Control of Actomyosin.

The C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate [PI(4)P] synthesis by the Golgi PI(4)-kinase IIIB (PI4KB). Both APOL1 and APOL3 can form K+ channels, but only APOL3 exhibits Ca2+-dependent binding of high affinity to neuronal calcium sensor-1 (NCS-1), promoting NCS-1-PI4KB interaction and stimulating PI4KB activity. Alteration of the APOL1 C-terminal helix triggers APOL1 unfolding and increased binding to APOL3, affecting APOL3-NCS-1 interaction. Since the podocytes of G1 and G2 patients exhibit an APOL1Δ or APOL3KO-like phenotype, APOL1 C-terminal variants may induce kidney disease by preventing APOL3 from activating PI4KB, with consecutive actomyosin reorganization of podocytes.

Constitutive IP3 signaling underlies the sensitivity of B-cell cancers to the Bcl-2/IP3 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.

Extracellular and ER-stored Ca2+ contribute to BIRD-2-induced cell death in diffuse large B-cell lymphoma cells.

The anti-apoptotic protein Bcl-2 is upregulated in several cancers, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL). In a subset of these cancer cells, Bcl-2 blocks Ca2+-mediated apoptosis by suppressing the function of inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) located at the endoplasmic reticulum (ER). A peptide tool, called Bcl-2/IP3 receptor disruptor-2 (BIRD-2), was developed to disrupt Bcl-2/IP3R complexes, triggering pro-apoptotic Ca2+ signals and killing Bcl-2-dependent cancer cells. In DLBCL cells, BIRD-2 sensitivity depended on the expression level of IP3R2 channels and constitutive IP3 signaling downstream of the B-cell receptor. However, other cellular pathways probably also contribute to BIRD-2-provoked cell death. Here, we examined whether BIRD-2-induced apoptosis depended on extracellular Ca2+ and more particularly on store-operated Ca2+ entry (SOCE), a Ca2+-influx pathway activated upon ER-store depletion. Excitingly, DPB162-AE, a SOCE inhibitor, suppressed BIRD-2-induced cell death in DLBCL cells. However, DPB162-AE not only inhibits SOCE but also depletes the ER Ca2+ store. Treatment of the cells with YM-58483 and GSK-7975A, two selective SOCE inhibitors, did not protect against BIRD-2-induced apoptosis. Similar data were obtained by knocking down STIM1 using small interfering RNA. Yet, extracellular Ca2+ contributed to BIRD-2 sensitivity in DLBCL, since the extracellular Ca2+ buffer ethylene glycol tetraacetic acid (EGTA) blunted BIRD-2-triggered apoptosis. The protective effects observed with DPB162-AE are likely due to ER Ca2+-store depletion, since a similar protective effect could be obtained using the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. Thus, both the ER Ca2+-store content and extracellular Ca2+, but not SOCE, are critical factors underlying BIRD-2-provoked cell death.

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.

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.

IP3 Receptor Properties and Function at Membrane Contact Sites.

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is a ubiquitously expressed Ca2+-release channel localized in the endoplasmic reticulum (ER). The intracellular Ca2+ signals originating from the activation of the IP3R regulate multiple cellular processes including the control of cell death versus cell survival via their action on apoptosis and autophagy. The exact role of the IP3Rs in these two processes does not only depend on their activity, which is modulated by the cytosolic composition (Ca2+, ATP, redox status, …) and by various types of regulatory proteins, including kinases and phosphatases as well as by a number of oncogenes and tumor suppressors, but also on their intracellular localization, especially at the ER-mitochondrial and ER-lysosomal interfaces. At these interfaces, Ca2+ microdomains are formed, in which the Ca2+ concentration is finely regulated by the different ER, mitochondrial and lysosomal Ca2+-transport systems and also depends on the functional and structural interactions existing between them. In this review, we therefore discuss the most recent insights in the role of Ca2+ signaling in general, and of the IP3R in particular, in the control of basal mitochondrial bioenergetics, apoptosis, and autophagy at the level of inter-organellar contact sites.

Intracellular Ca(2+) signaling and Ca(2+) microdomains in the control of cell survival, apoptosis and autophagy.

The endoplasmic reticulum (ER), mitochondria and lysosomes are physically and/or functionally linked, establishing close contact sites between these organelles. As a consequence, Ca(2+) release events from the ER, the major intracellular Ca(2+)-storage organelle, have an immediate effect on the physiological function of mitochondria and lysosomes. Also, the lysosomes can act as a Ca(2+) source for Ca(2+) release into the cytosol, thereby influencing ER-based Ca(2+) signaling. Given the important role for mitochondria and lysosomes in cell survival, cell death and cell adaptation processes, it has become increasingly clear that Ca(2+) signals from or towards these organelles impact these processes. In this review, we discuss the most recent insights in the emerging role of Ca(2+) signaling in cellular survival by controlling basal mitochondrial bioenergetics and by regulating apoptosis, a mitochondrial process, and autophagy, a lysosomal process, in response to cell damage and cell stress.

HA14-1 potentiates apoptosis in B-cell cancer cells sensitive to a peptide disrupting IP 3 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-2's 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.

The BH4 domain of anti-apoptotic Bcl-XL, but not that of the related Bcl-2, limits the voltage-dependent anion channel 1 (VDAC1)-mediated transfer of pro-apoptotic Ca2+ signals to mitochondria.

Excessive Ca(2+) fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting Ca(2+)-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP3Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca(2+)-dependent apoptosis by binding and inhibiting IP3Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP3Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca(2+) uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1(-/-) cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca(2+) uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca(2+) release in the cytosol or to prevent apoptosis, as done instead by an IP3R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca(2+) uptake into the mitochondria.