BCL-XL exerts a protective role against anemia caused by radiation-induced kidney damage.

Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combination with DNA damage-inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage-inducing anti-cancer therapy plus a BCL-XL inhibitor could be tolerated in mice, at least when applied sequentially.

Osteosarcoma cells depend on MCL-1 for survival, and osteosarcoma metastases respond to MCL-1 antagonism plus regorafenib in vivo.

Osteosarcoma is the most common form of primary bone cancer, which primarily afflicts children and adolescents. Chemotherapy, consisting of doxorubicin, cisplatin and methotrexate (MAP) increased the 5-year osteosarcoma survival rate from 20% to approximately 60% by the 1980s. However, osteosarcoma survival rates have remained stagnant for several decades. Patients whose disease fails to respond to MAP receive second-line treatments such as etoposide and, in more recent years, the kinase inhibitor regorafenib. BCL-2 and its close relatives enforce cellular survival and have been implicated in the development and progression of various cancer types. BH3-mimetics antagonize pro-survival members of the BCL-2 family to directly stimulate apoptosis. These drugs have been proven to be efficacious in other cancer types, but their use in osteosarcoma has been relatively unexplored to date. We investigated the potential efficacy of BH3-mimetics against osteosarcoma cells in vitro and examined their cooperation with regorafenib in vivo. We demonstrated that osteosarcoma cell lines could be killed through inhibition of MCL-1 combined with BCL-2 or BCL-xL antagonism. Inhibition of MCL-1 also sensitized osteosarcoma cells to killing by second-line osteosarcoma treatments, particularly regorafenib. Importantly, we found that inhibition of MCL-1 with the BH3-mimetic S63845 combined with regorafenib significantly prolonged the survival of mice bearing pulmonary osteosarcoma metastases. Together, our results highlight the importance of MCL-1 in osteosarcoma cell survival and present a potential therapeutic avenue that may improve metastatic osteosarcoma patient outcomes.

BCL-2 protein family: attractive targets for cancer therapy.

Acquired resistance to cell death is a hallmark of cancer. The BCL-2 protein family members play important roles in controlling apoptotic cell death. Abnormal over-expression of pro-survival BCL-2 family members or abnormal reduction of pro-apoptotic BCL-2 family proteins, both resulting in the inhibition of apoptosis, are frequently detected in diverse malignancies. The critical role of the pro-survival and pro-apoptotic BCL-2 family proteins in the regulation of apoptosis makes them attractive targets for the development of agents for the treatment of cancer. This review describes the roles of the various pro-survival and pro-apoptotic members of the BCL-2 protein family in normal development and organismal function and how defects in the control of apoptosis promote the development and therapy resistance of cancer. Finally, we discuss the development of inhibitors of pro-survival BCL-2 proteins, termed BH3-mimetic drugs, as novel agents for cancer therapy.

Constitutive BAK activation as a determinant of drug sensitivity in malignant lymphohematopoietic cells.

Mitochondrial outer membrane permeabilization (MOMP), a key step in the intrinsic apoptotic pathway, is incompletely understood. Current models emphasize the role of BH3-only BCL2 family members in BAX and BAK activation. Here we demonstrate concentration-dependent BAK autoactivation under cell-free conditions and provide evidence that this autoactivation plays a key role in regulating the intrinsic apoptotic pathway in intact cells. In particular, we show that up to 80% of BAK (but not BAX) in lymphohematopoietic cell lines is oligomerized and bound to anti-apoptotic BCL2 family members in the absence of exogenous death stimuli. The extent of this constitutive BAK oligomerization is diminished by BAK knockdown and unaffected by BIM or PUMA down-regulation. Further analysis indicates that sensitivity of cells to BH3 mimetics reflects the identity of the anti-apoptotic proteins to which BAK is constitutively bound, with extensive BCLXL•BAK complexes predicting navitoclax sensitivity, and extensive MCL1•BAK complexes predicting A1210477 sensitivity. Moreover, high BAK expression correlates with sensitivity of clinical acute myelogenous leukemia to chemotherapy, whereas low BAK levels correlate with resistance and relapse. Collectively, these results inform current understanding of MOMP and provide new insight into the ability of BH3 mimetics to induce apoptosis without directly activating BAX or BAK.

T-Cell Prolymphocytic Leukemia: Diagnosis, Pathogenesis, and Treatment.

T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive neoplasm of mature T-cells. Most patients with T-PLL present with lymphocytosis, anemia, thrombocytopenia, and hepatosplenomegaly. Correct identification of T-PLL is essential because treatment for this disease is distinct from that of other T-cell neoplasms. In 2019, the T-PLL International Study Group (TPLL-ISG) established criteria for the diagnosis, staging, and assessment of response to treatment of T-PLL with the goal of harmonizing research efforts and supporting clinical decision-making. T-PLL pathogenesis is commonly driven by T-cell leukemia 1 (TCL1) overexpression and ATM loss, genetic alterations that are incorporated into the TPLL-ISG diagnostic criteria. The cooperativity between TCL1 family members and ATM is seemingly unique to T-PLL across the spectrum of T-cell neoplasms. The role of the T-cell receptor, its downstream kinases, and JAK/STAT signaling are also emerging themes in disease pathogenesis and have obvious therapeutic implications. Despite improved understanding of disease pathogenesis, alemtuzumab remains the frontline therapy in the treatment of naïve patients with indications for treatment given its high response rate. Unfortunately, the responses achieved are rarely durable, and the majority of patients are not candidates for consolidation with hematopoietic stem cell transplantation. Improved understanding of T-PLL pathogenesis has unveiled novel therapeutic vulnerabilities that may change the natural history of this lymphoproliferative neoplasm and will be the focus of this concise review.

MCL1 Inhibition Overcomes the Aggressiveness Features of Triple-Negative Breast Cancer MDA-MB-231 Cells.

Triple-Negative Breast Cancer (TNBC) is a particularly aggressive subtype among breast cancers (BCs), characterized by anoikis resistance, high invasiveness, and metastatic potential as well as Epithelial-Mesenchymal Transition (EMT) and stemness features. In the last few years, our research focused on the function of MCL1, an antiapoptotic protein frequently deregulated in TNBC. Here, we demonstrate that MCL1 inhibition by A-1210477, a specific BH3-mimetic, promotes anoikis/apoptosis in the MDA-MB-231 cell line, as shown via an increase in proapoptotic markers and caspase activation. Our evidence also shows A-1210477 effects on Focal Adhesions (FAs) impairing the integrin trim and survival signaling pathways, such as FAK, AKT, ERK, NF-κB, and GSK3ß-inducing anoikis, thus suggesting a putative role of MCL1 in regulation of FA dynamics. Interestingly, in accordance with these results, we observed a reduction in migratory and invasiveness capabilities as confirmed by a decrease in metalloproteinases (MMPs) levels following A-1210477 treatment. Moreover, MCL1 inhibition promotes a reduction in EMT characteristics as demonstrated by the downregulation of Vimentin, MUC1, DNMT1, and a surprising re-expression of E-Cadherin, suggesting a possible mesenchymal-like phenotype reversion. In addition, we also observed the downregulation of stemness makers such as OCT3/4, SOX2, NANOG, as well as CD133, EpCAM, and CD49f. Our findings support the idea that MCL1 inhibition in MDA-MB-231 could be crucial to reduce anoikis resistance, aggressiveness, and metastatic potential and to minimize EMT and stemness features that distinguish TNBC.

Fibroblast growth factor receptor inhibitor erdafitinib promotes Mcl-1 degradation and synergistically induces apoptosis with Bcl-xL/Bcl-2 inhibitor in urothelial cancer cells.

Metastatic urothelial cancer is a lethal disease. Although recent advances in immunotherapies and targeted therapy against fibroblast growth factor receptor (FGFR)2/3 mutation (erdafitinib) have improved patient survival, there is still a critical need for novel therapeutic strategies for patients who do not benefit from these treatments. Evasion of apoptosis through amplifying anti-apoptotic Bcl-2 family proteins (Mcl-1, Bcl-xL, Bcl-2) is one mechanism responsible for treatment resistance in urothelial cancers, suggesting that targeting anti-apoptotic proteins may be a possible therapeutic strategy for urothelial cancers. Here, we showed that erdafitinib increased Mcl-1 degradation mainly through previously unknown mechanisms and synergized with a BH3 mimetic drug targeting Bcl-xL/Bcl-2 to induce apoptosis in FGFR wild-type urothelial cancer cells. Strikingly, clinical sequencing data showed amplification of MCL1 or BCL2L1 (encoding Bcl-xL) in subsets of FGFR mutation-negative bladder cancer tissues. In conclusion, these findings suggest that exploiting apoptosis pathways may be a promising treatment strategy for patients with FGFR wild-type metastatic urothelial cancer with Mcl-1 or Bcl-xL overexpression.

PALVEN: phase Ib trial of palbociclib, letrozole and venetoclax in estrogen receptor- and BCL2-positive advanced breast cancer.

The addition of a CDK4/6 inhibitor to endocrine therapy improves progression-free and overall survival in women with metastatic estrogen receptor-positive breast cancer. In that setting, CDK4/6 inhibitors induce a potent cell-cycle arrest (which may be accompanied by tumor senescence) but fail to induce apoptotic cell death. Venetoclax is a potent inhibitor of BCL2, a pro-survival protein overexpressed in the majority of estrogen receptor-positive cancers. Pre-clinical findings indicate that venetoclax augments tumor response to the CDK4/6 inhibitor palbociclib by triggering apoptosis, including in senescent cells. The PALVEN phase Ib trial will further examine this finding. The primary objective is to identify the maximum tolerated dose and determine the recommended phase II dose for palbociclib, letrozole and venetoclax combination therapy. Clinical Trial Registration: NCT03900884 (ClinicalTrials.gov).

The current 'gold standard' treatment for estrogen receptor-positive, HER2-negative metastatic breast cancer is endocrine therapy with a CDK4/6 inhibitor. This combination improves tumor response and patient outcomes, primarily by reducing tumor cell growth. Paradoxically, less killing of tumor cells is observed in the presence of a CDK4/6 inhibitor. The authors hypothesize that co-treatment with venetoclax, an inhibitor of the BCL2 survival protein, will help trigger tumor death, thereby further improving tumor responses and patient outcomes. As a first step, combination therapy comprising letrozole, palbociclib and venetoclax will be tested in a phase I trial to identify the recommended doses for subsequent studies.

Induction of multiple subroutines of regulated necrosis in murine macrophages by natural BH3-mimetic gossypol.

Macrophages are critical sentinel cells armed with multiple regulated necrosis pathways, including pyroptosis, apoptosis followed by secondary necrosis, and necroptosis, and are poised to undergo distinct form(s) of necrosis for tackling dangers of pathogenic infection or toxic exposure. The natural BH3-mimetic gossypol is a toxic phytochemical that can induce apoptosis and/or pyroptotic-like cell death, but what exact forms of regulated necrosis are induced remains largely unknown. Here we demonstrated that gossypol induces pyroptotic-like cell death in both unprimed and lipopolysaccharide-primed mouse bone marrow-derived macrophages (BMDMs), as evidenced by membrane swelling and ballooning accompanied by propidium iodide incorporation and lactic acid dehydrogenase release. Notably, gossypol simultaneously induces the activation of both pyroptotic and apoptotic (followed by secondary necrosis) pathways but only weakly activates the necroptosis pathway. Unexpectedly, gossypol-induced necrosis is independent of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, as neither inhibitor for the NLRP3 pathway nor NLRP3 deficiency protects the macrophages from the necrosis. Furthermore, necrotic inhibitors or even pan-caspase inhibitor alone does not or only partly inhibit such necrosis. Instead, a combination of inhibitors composed of pan-caspase inhibitor IDN-6556, RIPK3 inhibitor GSK'872 and NADPH oxidase inhibitor GKT137831 not only markedly inhibits the necrosis, with all apoptotic and pyroptotic pathways being blocked, but also attenuates gossypol-induced peritonitis in mice. Lastly, the activation of the NLRP3 pathway and apoptotic caspase-3 appears to be independent of each other. Collectively, gossypol simultaneously induces the activation of multiple subroutines of regulated necrosis in macrophages depending on both apoptotic and inflammatory caspases.

Comparison of putative BH3 mimetics AT-101, HA14-1, sabutoclax and TW-37 with ABT-737 in platelets.

Platelet lifespan is regulated by intrinsic apoptosis. Platelet apoptosis can be triggered by BH3 mimetics that inhibit the pro-survival Bcl-2 family protein, Bcl-xL. Here, we investigated several small molecules that are reported to act as BH3 mimetics and compared their effects to the well-established BH3 mimetic, ABT-737. Platelet phosphatidylserine (PS) exposure was determined by flow cytometry. Changes in cytosolic Ca2+ signaling were detected using Cal-520. Plasma membrane integrity was determined by calcein leakage. The roles of caspases and calpain in these processes were determined using Q-VD-OPh and calpeptin, respectively. As previously reported, ABT-737 triggered PS exposure in a caspase-dependent manner and calcein loss in a caspase and calpain-dependent manner. In contrast, AT-101 and sabutoclax triggered PS exposure independently of caspases. HA14-1 also triggered PS exposure in a caspase-independent but calpain-dependent manner. There were also significant differences in the pattern and protease-dependency of cytosolic Ca2+ signaling in response to these drugs compared to ABT-737. Since there are clear differences between the action of ABT-737 and the other putative BH3 mimetics investigated here, AT-101, HA14-1 and sabutoclax cannot be considered as acting as BH3 mimetics in platelets. Furthermore, the platelet death caused by these drugs is likely to be distinct from apoptosis.

Iterative optimization yields Mcl-1-targeting stapled peptides with selective cytotoxicity to Mcl-1-dependent cancer cells.

Bcl-2 family proteins regulate apoptosis, and aberrant interactions of overexpressed antiapoptotic family members such as Mcl-1 promote cell transformation, cancer survival, and resistance to chemotherapy. Discovering potent and selective Mcl-1 inhibitors that can relieve apoptotic blockades is thus a high priority for cancer research. An attractive strategy for disabling Mcl-1 involves using designer peptides to competitively engage its binding groove, mimicking the structural mechanism of action of native sensitizer BH3-only proteins. We transformed Mcl-1-binding peptides into α-helical, cell-penetrating constructs that are selectively cytotoxic to Mcl-1-dependent cancer cells. Critical to the design of effective inhibitors was our introduction of an all-hydrocarbon cross-link or "staple" that stabilizes α-helical structure, increases target binding affinity, and independently confers binding specificity for Mcl-1 over related Bcl-2 family paralogs. Two crystal structures of complexes at 1.4 Å and 1.9 Å resolution demonstrate how the hydrophobic staple induces an unanticipated structural rearrangement in Mcl-1 upon binding. Systematic sampling of staple location and iterative optimization of peptide sequence in accordance with established design principles provided peptides that target intracellular Mcl-1. This work provides proof of concept for the development of potent, selective, and cell-permeable stapled peptides for therapeutic targeting of Mcl-1 in cancer, applying a design and validation workflow applicable to a host of challenging biomedical targets.

Co-Operativity between MYC and BCL-2 Pro-Survival Proteins in Cancer.

B-Cell Lymphoma 2 (BCL-2), c-MYC and related proteins are arguably amongst the most widely studied in all of biology. Every year there are thousands of papers reporting on different aspects of their biochemistry, cellular and physiological mechanisms and functions. This plethora of literature can be attributed to both proteins playing essential roles in the normal functioning of a cell, and by extension a whole organism, but also due to their central role in disease, most notably, cancer. Many cancers arise due to genetic lesions resulting in deregulation of both proteins, and indeed the development and survival of tumours is often dependent on co-operativity between these protein families. In this review we will discuss the individual roles of both proteins in cancer, describe cancers where co-operativity between them has been well-characterised and finally, some strategies to target these proteins therapeutically.

Dual targeting of bromodomain-containing 4 by AZD5153 and BCL2 by AZD4320 against B-cell lymphomas concomitantly overexpressing c-MYC and BCL2.

Despite the recent therapeutic progress, the prognoses of diffuse large B-cell lymphomas (DLBCLs) that concomitantly overexpress c-MYC and BCL2, i.e., double hit lymphoma (DHL) and double expressing lymphoma (DEL), remain poor. This study examined triple targeting of c-MYC, BCL2 and the B-cell receptor (BCR) signaling pathway for DHL and DEL. We first used AZD5153, a novel bivalent inhibitor for bromodomain-containing 4 (BRD4), in DHL- and DEL-derived cell lines, because BRD4 regulates disease type-oriented key molecules for oncogenesis. AZD5153 was more effective than conventional monovalent BRD4 inhibitors, JQ1 and I-BET151, in inhibiting cell proliferation of a DHL-derived cell line and two DEL-derived cell lines, with at least 10-fold lower half growth inhibitory concentrations. AZD5153 caused G1/S cell cycle blockade, while the apoptosis-inducing effect was relatively modest. At the molecular level, AZD5153 was potent in downregulating various molecules for oncogenesis, such as c-MYC, AKT2 and MAP3K; those involved in the BCR signaling pathway, such as CD19, BLNK and CD79B; and those associated with B-cell development, such as IKZF1, IKZF3, PAX5, POU2AF1 and EBF1. In contrast, AZD5153 did not decrease anti-apoptotic BCL2 proteins, and did not activate pro-apoptotic BH3-only proteins, except BAD. To augment cell death induction, we added a novel BH3-mimicking BCL2 inhibitor AZD4320 to AZD5153, and found that these two agents had a mostly synergistic antitumor effect by increasing cells undergoing apoptosis in all three cell lines. These results provide a rationale for dual targeting of BRD4 and BCL2 using AZD5153 and AZD4320 as a therapeutic strategy against DHL and DEL.

The deubiquitinating enzyme OTUD1 antagonizes BH3-mimetic inhibitor induced cell death through regulating the stability of the MCL1 protein.

BACKGROUND: Myeloid cell leukaemia 1 (MCL1) is a pro-survival Bcl-2 family protein that plays important roles in cell survival, proliferation, differentiation and tumourigenesis. MCL1 is a fast-turnover protein that is degraded via an ubiquitination/proteasome-dependent mechanism. Although several E3 ligases have been discovered to promote the ubiquitination of MCL1, the deubiquitinating enzyme (DUB) that regulates its stability requires further investigation. METHODS: The immunoprecipitation was used to determine the interaction between OTUD1 and MCL1. The ubiquitination assays was performed to determine the regulation of MCL1 by OTUD1. The cell viability was used to determine the regulation of BH3-mimetic inhibitor induced cell death by OTUD1. The survival analysis was used to determine the relationship between OTUD1 expression levels and the survival rate of cancer patients. RESULTS: By screening a DUB expression library, we determined that the deubiquitinating enzyme OTUD1 regulates MCL1 protein stability in an enzymatic-activity dependent manner. OTUD1 interacts with MCL1 and promotes its deubiquitination. Knockdown of OTUD1 increases the sensitivity of tumour cells to the BH3-mimetic inhibitor ABT-263, while overexpression of OTUD1 increases tumour cell tolerance of ABT-263. Furthermore, bioinformatics analysis data reveal that OTUD1 is a negative prognostic factor for liver cancer, ovarian cancer and specific subtypes of breast and cervical cancer. CONCLUSIONS: The deubiquitinating enzyme OTUD1 antagonizes BH3-mimetic inhibitor induced cell death through regulating the stability of the MCL1 protein. Thus, OTUD1 could be considered as a therapeutic target for curing these cancers.

Allosteric modulation of sigma receptors by BH3 mimetics ABT-737, ABT-263 (Navitoclax) and ABT-199 (Venetoclax).

ABT-737, ABT-263 (Navitoclax) and ABT-199 (Venetoclax) are under intensive preclinical and clinical investigation as treatments for hematologic and other malignancies. These small molecules mimic pro-death B-cell lymphoma-2 (Bcl-2) Homology 3 (BH3) domain-only proteins. They also bear a structural resemblance to certain sigma (σ) receptor ligands. Moreover, the Bcl-2 and σ receptor protein families are both located primarily at the endoplasmic reticulum, mediate cell death and survival through protein-protein interactions, and physically associate. Accordingly, we examined the ability of the ABT series of BH3 mimetics to interact with σ receptors using radioligand-binding techniques. Negative allosteric modulation of [3H](+)-pentazocine, an agonist, binding to σ1 receptors in guinea pig brain membranes was observed for ABT-737, ABT-263 and ABT-199. Findings included reduction of specific binding to distinct plateaus in concentration-dependent fashion, significant slowing of radioligand dissociation kinetics, and decreases in radioligand affinity with no or modest changes in maximal receptor densities. Using a ternary complex model, dissociation constants (KX) for modulator binding to the σ1 receptor ranged from 1 to 2.5 µM, while negative cooperativity factors (α), representing the changes in affinity of ligand and modulator when bound as a ternary complex with the receptor, ranged from 0.15 to 0.42. These observations were extended and reinforced by studies using intact small cell (NCI-H69) and non-small cell (NCI-H23) lung cancer cells, and by using an antagonist σ1 receptor radioligand, E-N-1-(3'-[125I]iodoallyl)-N'-4-(3″,4″-dimethoxyphenethyl)piperazine, in mouse brain membranes. By contrast, exploratory studies indicate marked enhancement of the σ2 receptor binding of [3H]1,3-di-(o-tolyl)guanidine/(+)-pentazocine in NCI-H23 cells and guinea pig brain membranes. These findings raise intriguing questions regarding mechanism and potential functional outcomes.

Discovery of novel biaryl sulfonamide based Mcl-1 inhibitors.

Mcl-1 is an anti-apoptotic protein overexpressed in hematological malignancies and several human solid tumors. Small molecule inhibition of Mcl-1 would offer an effective therapy to Mcl-1 mediated resistance. Subsequently, it has been the target of extensive research in the pharmaceutical industry. The discovery of a novel class of Mcl-1 small molecule inhibitors is described beginning with a simple biaryl sulfonamide hit derived from a high through put screen. A medicinal chemistry effort aided by SBDD generated compounds capable of disrupting the Mcl-1/Bid protein-protein interaction in vitro. The crystal structure of the Mcl-1 bound ligand represents a unique binding mode to the BH3 binding pocket where binding affinity is achieved, in part, through a sulfonamide oxygen/Arg263 interaction. The work highlights the some of the key challenges in designing effective protein-protein inhibitors for the Bcl-2 class of proteins.

The Structural Biology of Bcl-xL.

Interactions between the pro-survival and pro-apoptotic members of the Bcl-2 family of proteins dictate whether a cell lives or dies. Much of our knowledge of the molecular details of these interactions has come from biochemical and structural studies on the pro-survival protein Bcl-xL. The first high-resolution structure of any Bcl-2 family member was of Bcl-xL, which revealed the conserved topology amongst all family members. Subsequent structures of Bcl-xL complexes with pro-apoptotic ligands demonstrated the general features of all pro-survival:pro-apoptotic complexes. Structural studies involving Bcl-xL were also the basis for the discovery of the first small-molecule pro-survival protein inhibitors, leading ultimately to the development of a new class of drugs now successfully used for cancer treatment in the clinic. This article will review our current knowledge of the structural biology of Bcl-xL and how this has impacted our understanding of the molecular details of the intrinsic apoptotic pathway.

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.

Hypertonicity primes malignant melanoma cells for apoptosis.

The tumor environment critically influences responsiveness of cancer cells to chemotherapies, most of which activate the mitochondria-regulated (intrinsic) apoptotic cascade to kill malignant cells. Especially skin tumors encounter an environment with remarkable biophysical properties. Cutaneous accumulation of Na+ locally establishes osmotic pressure gradients in vivo (hypertonicity or hyperosmotic stress), but whether cutaneous hypertonicity is a factor that modulates the responsiveness of skin cancers to therapeutic apoptosis-induction has thus far not been investigated. Here, we show that hyperosmotic stress lowers the threshold for apoptosis induction in malignant melanoma, the deadliest form of skin cancer. Hypertonic conditions enforce addiction to BCL-2-like proteins to prevent initiation of the mitochondria-regulated (intrinsic) apoptotic pathway. Essentially, hyperosmotic stress primes mitochondria for death. Our work identifies osmotic pressure in the tumor microenvironment as a cell extrinsic factor that modulates responsiveness of malignant melanoma cells to therapy.

MEK1/2 inhibition by binimetinib is effective as a single agent and potentiates the actions of Venetoclax and ABT-737 under conditions that mimic the chronic lymphocytic leukaemia (CLL) tumour microenvironment.

The survival and proliferation of chronic lymphocytic leukaemia (CLL) cells is driven by multiple signalling pathways, including those mediated by the B cell, Toll-like and chemokine receptors. Many of these pathways converge on the same signalling molecules, including those involved in the Raf-1/MEK/Erk1/2-MAPK pathway. We investigated the effects of the MEK1/2 (also termed MAP2K1/2) inhibitor, binimetinib, against CLL cells cultured under conditions that mimic aspects of the tumour microenvironment. Binimetinib blocked CLL cell survival induced by stroma-conditioned media and phorbol myristylate (PMA). Binimetinib was also significantly more toxic towards CLL cells cultured in the presence of either anti-IgM antibody or stroma-derived factor-1α (SDF-1α) and reduced CLL cell cycle progression and proliferation. Furthermore, binimetinib significantly increased the sensitivity of CLL cells co-cultured with CD40 ligand (CD40L)-expressing fibroblasts to the BH3-mimetics ABT-737 and Venetoclax (ABT-199) via a mechanism involving down-regulation of Mcl-1 (MCL1) activity and Bim (BCL2L11) and Bcl-xL (BCL2L1) expression. Collectively, these data suggest that binimetinib may have both cytotoxic and cytostatic effects on CLL cells by blocking microenvironment-derived signals known to drive survival and proliferation. The combination of binimetinib with a BH3 mimetic may be an effective treatment strategy for CLL, particularly against the proliferative fraction of the disease within the tumour microenvironment.