Pro-apoptotic Bcl-2 Family Proteins Research Articles

A Novel Regimen for Treating Melanoma: MCL1 Inhibitors and Azacitidine.

Although treatment options for melanoma patients have expanded in recent years with the approval of immunotherapy and targeted therapy, there is still an unmet need for new treatment options for patients that are ineligible for, or resistant to these therapies. BH3 mimetics, drugs that mimic the activity of pro-apoptotic BCL2 family proteins, have recently achieved remarkable success in the clinical setting. The combination of BH3 mimetic ABT-199 (venetoclax) plus azacitidine has shown substantial benefit in treating acute myelogenous leukemia. We evaluated the efficacy of various combinations of BH3 mimetic + azacitidine in fourteen human melanoma cell lines from cutaneous, mucosal, acral and uveal subtypes. Using a combination of cell viability assay, BCL2 family knockdown cell lines, live cell imaging, and sphere formation assay, we found that combining inhibition of MCL1, an anti-apoptotic BCL2 protein, with azacitidine had substantial pro-apoptotic effects in multiple melanoma cell lines. Specifically, this combination reduced cell viability, proliferation, sphere formation, and induced apoptosis. In addition, this combination is highly effective at reducing cell viability in rare mucosal and uveal subtypes. Overall, our data suggest this combination as a promising therapeutic option for some patients with melanoma and should be further explored in clinical trials.

Autophagic degradation of NOXA underlies stromal cell-mediated resistance to proteasome inhibitors in mantle cell lymphoma

Mantle cell lymphoma (MCL) is usually resistant to the current standard-of-care regimens and also to novel agents such as the proteasome inhibitor bortezomib. A better prognosis of leukemic variants of MCL suggests that MCL cells acquire drug resistance in nodal and/or bone marrow microenvironments via interaction with supporting cells. Bortezomib exerts cytotoxic action in MCL cells via stabilization of the pro-apoptotic BCL-2 family protein NOXA. Here we show that autophagic degradation of NOXA is a mechanism of bortezomib resistance in MCL cells in a tumor microenvironment. First, we demonstrated that interaction with bone marrow-derived or nodal stromal cells conferred bortezomib resistance to MCL cells in vitro and in a murine model. Co-culture of MCL cells with stromal cells enhanced bortezomib-induced ubiquitination and subsequent binding of NOXA to the p62 adaptor, which escorted NOXA to the lysosome for autophagic degradation. Finally, we found that not only direct contact with stromal cells but also stroma-derived humoral factors, especially interleukin-6, promoted selective autophagy and NOXA degradation in MCL cells. Targeting protective autophagy, for example, using the lysosome inhibitor chloroquine, might increase the efficacy of bortezomib-containing regimens in MCL.

BCL-2 Antagonist ABT-199 Combined with Complex I Inhibitor IACS-010759 Blocks Mitochondrial Respiration and Facilitates Anti-Leukemia Efficacy in Pre-Clinical AML Models

ABT-199 (Venetoclax) is a small-molecule BH3 mimetic that activates pro-apoptotic BCL-2 family proteins causing cell death. BCL-2 family regulates ADP/ATP exchange across the mitochondrial membrane by voltage-dependent anion channel (VDAC) to prevent the loss of coupled mitochondrial respiration during the apoptosis (Manfredi, J Bio Chem 2003). We and other have shown that AML is BCL-2 dependent and leukemia stem cell (LSC) rely on BCL-2 to regulate the oxidative phosphorylation (OxPhos) activity for survival (Pan, Cancer Discovery 2014; Lagadinou, Cell Stem Cell, 2013). IACS-010759, a novel complex I inhibitor, effectively inhibits cell respiration and exhibits potent anti-leukemia effect in AML (Molina, 2016 AACR abstract #335) . We hypothesized that BCL-2 facilitates OxPhos activity, and that BCL-2 inhibition with ABT-199 will enhance anti-leukemia efficacy of IACS-010759 in AML.Priming of AML cell lines with ABT-199 (0-33 nM) for 24 hrs, followed by IACS-010759 (0-33 nM) for 72 hrs drastically inhibited viable cell numbers as determined by flow cytometry. Analysis of cellular respiration using Seahorse showed that both ABT-199 at 20nM and IACS-010759 at 11nM partially (<15%) reduced oxygen consumption rate (OCR), while combination treatment fully (60%) inhibited basal and maximal OCR as early as 2 hrs. This translated into inhibition of ATP production at 6 hrs and loss of the mitochondrial membrane potential (MMP) measured by TMRE/MTG or JC-1 flow cytometry. Further, ABT-199 followed by IACS-010759 induced cleavage of caspase-3 and DNA damage shown by g-H2AX immunoblotting. Anti-BCL-2 immunoprecipitation showed decreased BCL-2/VDAC binding in ABT-199- and IACS-010759-treated cells (Fig 1A). Sequential treatment with ABT-199 followed by IACS-010759 (10-100 nM) in primary AML cells (n=3) caused additive decrease in viable cell numbers.To explore the role of BCL-2 in mitochondrial metabolism, we utilized HL-60 cells overexpressing BCL-2. The HL-60/BCL-2 cells (a kind gift by Dr. Kapil N. Bhalla) have increased OxPhos activity as measured by basal and maximal OCR by Seahorse (Fig.1B); and increased basal and mitochondrial ROS production measured by H2DCFDA and MitoSOX Red flow cytometry. BCL-2 inhibition by ABT-199 at 100 nM after 2 hrs treatment induced ROS production in control but not in HL-60/BCL-2 cells. Further, HL-60/BCL-2 cells were less sensitive to IACS-010759 compared to control cells. These data suggest that BCL-2 facilities OxPhos maintenance and reduces efficacy of mitochondrial inhibitors.We next tested the combined anti-AML efficacy of BCL-2 and OxPhos inhibitors in an in vivo leukemia model, by injecting NSG mice with AML PDX cells harboring FLT3-ITD. Upon engraftment, leukemia-bearing mice were randomized to receive vehicle; single agent ABT-199 at 100 mg/kg for 14 days; IACS-010759 at 7.5 mg/kg for 21 days; or sequential treatment with ABT-199 for 14 days followed by IACS-010759 for 21 days. Consistent with in vitro findings, mice in the IACS-010759 and IACS-010759/ABT-199 combination groups had lower circulating tumor burden compared to other groups as measured by hCD45% flow cytometry (Fig. 1C). Survival analysis in all groups is ongoing. Tumor cells collected from 2 mice of ABT-199, IACS-010759, or the combination treatment group at the end of drug dosing showed decreased OCR. The frequency of CD34+CD38-CD123+ AML stem/progenitor cells was decreased in ABT-199, IACS-010759 and combination groups, with notable induction of differentiation in combination group (CD11b and CD11c), measured by CyTOF mass cytometry (Fig. 1D).In summary, our data indicate that in addition to its known anti-apoptotic function, BCL-2 facilitates mitochondrial respiration and OxPhos in AML cells, possibly through direct binding of mitochondrial pore protein VDAC. Inhibition of BCL-2 with ABT-199 disrupts BCL-2/VDAC interactions, reduces OCR and induces ROS production. Combined blockade of BCL-2 with ABT-199 and of mitochondrial complex I with IACS-010759 produces a greater anti-AML efficacy in AML cells in vitro and in vivo . [Display omitted] DisclosuresBattula:United Therapeutics Corporation: Research Funding.

Everolimus Shows Synergistic Anti-Myeloma Effects with Bortezomib Via the AKT/mTOR Pathway

Introduction: Multiple myeloma (MM) is characterized by the proliferation of malignant plasma cells and a subsequent overabundance of monoclonal paraprotein (M protein). Everolimus ,as an oral mammalian target of rapamycin (mTOR) inhibitor, plays an important role in multiple myeloma. In the phase I trial explored the use of everolimus in relapsed and/or refractory multiple myeloma patients ,the median time to progression was 90 days (range, 13 to 278 days),and no dose-limiting toxicity was observed at daily dose of 10 mg. At the same time, the observed responses are promising in some testing combination strategies. The pre-clinical activity of pan-histone deacetylase inhibitor LBH589 in combination with everolimus caused potent G0/G1 arrest and induced pronounced apoptosis, the combination led to down regulation of pStat3, cyclins, CDKs and XIAP and up regulation of pro-apoptotic Bcl-2 family proteins. Everolimus enhances the cytotoxicity of bendamustine in multiple myeloma cells through a network of pro-apoptotic and cell-cycle-progression regulatory proteins.Down regulation of vascular endothelial cell growth factor and everolimus synergistic antitumor activity in multiple myeloma cells. Furthermore, In a phase I study of everolimus in combination with lenalidomide in relapsed or refractory multiple myeloma patients, the overall response rate was 65% and median progression-free survival was 5.5 months. Bortezomib was the first therapeutic proteasome inhibitor to be tested in humans with MM. However, the combination of bortezomib and everolimus for the treatment of MM is still less reported.Methods: To evaluate the in vitro and in vivo anti-tumor activity of the combination of everolimus with bortezomib.CCK-8 was used to explore the proliferation of MM cells. Flow cytometry was used to determine the effect on apoptosis of cells.Western blot and Immunohistochemical staining was used to determine the epression of proteins.Results: In our study, the experimental results demonstrated that everolimus combined with bortezomib suppressed cell proliferation. Treatment with 20 nM everolimus in combination with 10 nM bortezomib for 72h achieved the lowest cell survival rate in both U266 and LP-1 MM cell lines. The combination of everolimus and bortezomib was synergistic in both cell lines indicated by combination index (CI) values of <0.8.Flow cytometry was used to determine the effect on apoptosis of bortezomib in combination with everolimus. everolimus alone and bortezomib alone induced approximately 22.5% and 17.2% apoptosis, combination treatment induced28.8% apoptosis in U266 cells, and the results were 15.6%,10.0% and 16.7% in LP-1 cells. Western blot was used to explore the mechanisms. The expression of AKT1, mTOR, p70S6K1, 4EBP1 and NF-kB was significantly down regulated while the expression of Caspase-3was significantly up regulated in combination group than other alone group. Furthermore, Synergistic antitumor effect of everolimus and bortezomib was observed in the animal model carrying the myeloma xenograft on the 25th day after tumor implantation . The results of immunohistochemical staining further demonstrated that the expression of AKT1, mTOR, p70S6K1, 4EBP1 and NF-kB was significantly down regulated and the expression of Caspase-3 was significantly up regulated in combination group.Conclusion:Our results shows synergistic antitumor effects of the combination therapy of bortezomib and everolimus induced myeloma cell apoptosis in vitro and in vivo xenograft mouse model ,which mainly through the inhibition of the AKT/mTOR pathway. Our results provided provide a rationale application for the combinatorial treatment of MM with proteasome inhibitor agents and mTOR inhibitors in future clinical practice. DisclosuresNo relevant conflicts of interest to declare.

Histone Deacetylase Inhibitor Depsipeptide (FK228) Induces Apoptosis in Leukemic Cells by Facilitating Mitochondrial Translocation of Bax, Which Is Enhanced by Proteasome Inhibitor Bortezomib.

Histone deacetylase (HDAC) inhibitors are a promising candidate for molecular-targeted therapy for leukemia. In this study, we investigated the mechanisms of cytotoxic effects of depsipeptide (FK228), one of the most effective HDAC inhibitors against leukemia, using human myeloid leukemic cell lines HL-60 and K562. We found that FK228 activated caspase-9 and a subsequent caspase cascade by perturbing the mitochondrial membrane to release cytochrome c, which was almost completely blocked by overexpression of Bcl-2. The mitochondrial damage was caused by the translocation of Bax but not other proapoptotic Bcl-2 family proteins to the mitochondria. FK228 did not affect the interaction between Bax and Bax-adaptor proteins such as 14–3–3 theta and Ku70. FK228-induced apoptosis and mitochondrial translocation of Bax were markedly enhanced by the proteasome inhibitor bortezomib. The synergistic action of FK228 and Bortezomib was at least partly mediated through conformational changes of Bax, which facilitate its translocation to the mitochondria. These results suggest that the combination of HDAC inhibitors and proteasome inhibitors is useful for the treatment of leukemia especially in the context of molecular-targeted therapy. The status of Bcl-2 and Bax may influence the sensitivity of tumors to this combination, and thus, can be a target of further therapeutic intervention.

Modeling hypoxia facilitates cancer cell survival through downregulation of p53 expression

A hypoxic environment of rapidly growing tumor cells makes them resistant to antitumor drugs. Mimicking hypoxia with iron chelator deferoxamine, suppressed cell death induced by widely used anticancer drugs doxorubicin or cisplatin. Deferoxamine decreased the number of dead (detached) cells, the size of SubG1 population, the release of cytochrome c, and the processing of caspase-3 in HCT116 colon carcinoma cells treated with cisplatin or doxorubicin. Deferoxamine-mediated suppression of apoptosis correlated with the level of pro-apoptotic Bcl-2 family proteins Bax, Bid, and Puma, which stimulate mitochondrial apoptotic pathway through permeabilization of the outer mitochondrial membrane and cytochrome c release. Here we show that one of the reasons for apoptosis suppression is downregulation of p53 expression under hypoxic conditions, and, as a result, attenuation of the expression of pro-apoptotic Bcl-2 family proteins. Indeed, p53 knock-out did not affect the stabilization of hypoxia-inducible factor but made undetectable the expression of pro-apoptotic proteins.

Evaluating the inhibitory priority of Bcl-xL to Bad, tBid and Bax by using live-cell imaging assay.

Molecular regulatory network among the B cell leukemia-2 (Bcl-2) family proteins is a research hotspot on apoptosis. The inhibitory priority of anti-apoptotic Bcl-2 family proteins (such as Bcl-xL) to pro-apoptotic Bcl-2 family proteins (such as Bad, tBid and Bax) determines the outcome of their interactions. Based on over-expression model system, we here evaluate the inhibitory priority of Bcl-xL to Bad, tBid and Bax by using live-cell imaging assay on cell viability. Fluorescence images of living cells co-expressing CFP-Bcl-xL and YFP-Bad or YFP-tBid or YFP-Bax showed that Bcl-xL markedly inhibited Bad/tBid/Bax-mediated apoptosis, revealing that Bcl-xL inhibits the proapoptotic function of Bad, tBid and Bax. In the case of equimolar co-expression of Bad and CFP-Bcl-xL, the inhibition of Bcl-xL on tBid/Bax mediate-apoptosis was completely relieved. Moreover, co-expression of tBid-P2A-CFP-Bcl-xL significantly relieved the inhibition of Bcl-xL on the pro-apoptotic ability Bax, suggesting that Bcl-xL preferentially inhibits the pro-apoptotic ability of Bad over tBid, subsequently to Bax.

BAD-Mediated Neuronal Apoptosis and Neuroinflammation Contribute to Alzheimer's Disease Pathology

Alzheimer's disease is the most common progressive neurodegenerative disease. However, the underlying molecular mechanism is incompletely understood. Here we report the identification of the pro-apoptotic BCL-2 family protein BAD as a key regulator for neuronal apoptosis, neuroinflammation and β-amyloid (Aβ) clearance in Alzheimer's disease. The pro-apoptotic activity of BAD is significantly increased in neurons of patients with Alzheimer's disease and 5XFAD mice. Conversely, genetic disruption of Bad alleles restores spatial learning and memory deficits in 5XFAD mice. Mechanistically, phosphorylation and inactivation of BAD by neurotropic factor-activated Akt is abrogated in cortical neurons in patients with Alzheimer's disease and 5XFAD mice, thereby triggering mitochondrial translocation of hypo-phosphorylated BAD to promote neuronal apoptosis and neurodegeneration. Through reactive oxygen species (ROS)-oxidized mitochondrial DNA (mtDNA) axis, BAD also promotes activation of microglial NLRP3 inflammasome, thereby skewing microglia toward neuroinflammatory microglia to inhibit microglial phagocytosis of Aβ plaques in 5XFAD mice. AAV-mediated Bad deletion in hippocampal neurons prevents neuronal apoptosis and rescues memory deficits in 5XFAD mice. In another Alzheimer's disease murine model, APP/PS1 mice, Bad loss also inhibits activation of microglial NLRP3 inflammasome and Aβ deposition, thereby restoring memory deficits. Our results support a model in which BAD contributes to memory deficits and neuropathology in Alzheimer's disease by driving neuronal apoptosis, neuroinflammation but suppressing microglial phagocytosis of Aβ plaques, suggesting that BAD is a potential therapeutic target for Alzheimer's disease.

Membrane-dependent amyloid aggregation of human BAX α9 (173-192).

Mitochondrial outer membrane permeabilization, which is a critical step in apoptosis, is initiated upon transmembrane insertion of the C-terminal α-helix (α9) of the proapoptotic Bcl-2 family protein BAX. The isolated α9 fragment (residues 173-192) is also competent to disrupt model membranes, and the structures of its membrane-associated oligomers are of interest in understanding the potential roles of this sequence in apoptosis. Here, we used ultrafast two-dimensional infrared (2D IR) spectroscopy, thioflavin T binding, and transmission electron microscopy to show that the synthetic BAX α9 peptide (α9p) forms amyloid aggregates in aqueous environments and on the surfaces of anionic small unilamellar vesicles. Its inherent amyloidogenicity was predicted by sequence analysis, and 2D IR spectra reveal that vesicles modulate the β-sheet structures of insoluble aggregates, motivating further examination of the formation or suppression of BAX amyloids in apoptosis.

Non-steroidal anti-inflammatory drugs induce immunogenic cell death in suppressing colorectal tumorigenesis.

Use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with reduced risk of colorectal cancer (CRC). However, the mechanism by which NSAIDs suppress colorectal tumorigenesis remains unclear. We previously showed that NSAIDs selectively kill emerging tumor cells via death receptor (DR) signaling and a synthetic lethal interaction mediated by the proapoptotic Bcl-2 family protein BID. In this study, we found NSAIDs induce endoplasmic reticulum (ER) stress to activate DR signaling and BID in tumor suppression. Importantly, our results unveiled an ER stress- and BID-dependent immunogenic effect of NSAIDs, which may be critical for tumor suppression. NSAID treatment induced hallmarks of immunogenic cell death (ICD) in CRC cells and colonic epithelial cells upon loss of APC tumor suppressor, and elevated tumor-infiltrating lymphocytes (TILs) in the polyps of APCMin/+ mice. ER stress inhibition or BID deletion abrogated the antitumor and immunogenic effects of NSAIDs. Furthermore, increased ER stress and TILs were detected in human advanced adenomas from NSAID-treated patients. Together, our results suggest that NSAIDs induce ER stress- and BID-mediated ICD to restore immunosurveillance and suppress colorectal tumor formation.

BAD inactivation exacerbates rheumatoid arthritis pathology by promoting survival of sublining macrophages.

The resistance of synovial sublining macrophages to apoptosis has a crucial role in joint inflammation and destruction in rheumatoid arthritis (RA). However, the underlying mechanism is incompletely understood. Here we report that inactivation of the pro-apoptotic BCL-2 family protein BAD is essential for survival of synovial sublining macrophage in RA. Genetic disruption of Bad leads to more severe joint inflammation and cartilage and bone damage with reduced apoptosis of synovial sublining macrophages in collagen-induced arthritis (CIA) and TNFα transgenic (TNF-Tg) mouse models. Conversely, Bad3SA/3SA mice, in which BAD can no longer be inactivated by phosphorylation, are protected from collagen-induced arthritis. Mechanistically, phosphorylation-mediated inactivation of BAD specifically protects synovial sublining macrophages from apoptosis in highly inflammatory environment of arthritic joints in CIA and TNF-Tg mice, and in patients with RA, thereby contributing to RA pathology. Our findings put forward a model in which inactivation of BAD confers the apoptosis resistance on synovial sublining macrophages, thereby contributing to the development of arthritis, suggesting that BAD may be a potential therapeutic target for RA.

Resistance to anti-EGFR targeted therapy mediated by oncogenetic mutations in colorectal cancer: Revision of the dogma?

Traditionally, systemic treatment for high stage colorectal carcinoma (CRC) is mainly fluorouracil-based chemotherapy [1]. The anti-epidermal growth factor receptor (EGFR) monoclonal antibodies, by acting on specific molecular pathways in tumor growth or modulating immune response towards tumor cells, provide a more targeted response, a better side effect profile and greater impact on patient survival compared with conventional molecules. This monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor, is known to be effective only in a subset of KRAS wild-type colorectal cancers. Patients with mutations in either KRAS or NRAS gene are not eligible for anti-EGFR monoclonal antibody therapy [3]. This is due to downstream activation of the Ras/Raf/MAPK pathway by mutated RAS protein, leading to cell proliferation which cannot be sufficiently inhibited by anti-EGFR receptor monoclonal antibodies [4]. With the increasing choices of targeted agents, more and more biomarkers are tested. Currently, the standard recommended biomarker panel for colorectal carcinoma would include KRAS, NRAS, BRAF gene hotspot mutation detection and microsatellite instability test [5]. With the advances in genomic profiling and sequencing and the understanding of the resistance mechanisms, the contraindication of anti-EGFR therapy in mutant KRAS patients may be revised. Based on the fact that the KRAS mutation in CRC suppresses the phosphorylation of the AMP-activated protein kinase (AMPK) known to be toxic for the tumor cells, Hua et al obtained a satisfactory response to for the anti-EGFR antibodies in mutant KRAS CRC xenograft models after reactivation of the AMPK [6]. Knickelbein et al demonstrated that the anti-EGFR antibodies induce the death of CRC cells via a p73- dependent transcriptional activation of the pro-apoptotic Bcl-2 family protein (PUMA). This action is abolished in case of KRAS mutation. These authors admitted that the restoration of this pathway by inhibiting aurora kinases preferentially kills mutant KRAS CRC cells and overcomes KRAS-mediated resistance to anti-EGFR antibodies [7]. In fifty-one CRC patient-derived xenografts study, Lee et al showed that KRAS mutants expressed remarkably elevated autocrine levels of high-affinity EGFR ligands compared with wild-type KRAS. The use of an anti-EGFR IgG1 antibody that displays potent inhibitory effects on highaffinity EGFR ligand enhanced CRC KRAS mutant cells cytotoxicity [8]. Dealing with the resistance to targeted therapies in CRC patient looks feasible. It could allow to broaden the indications of anti-EGFR therapy and provide a better survival for a larger group of CRC patients. In this era of precision and personalized medicine, a complete case specific tumor profiling and the comprehensive study of the tumorogenesis mechanisms should allow to overcome the intrinsic and acquired resistance to these targeted high effective therapies.

Combination of CS2164 and Venetoclax Shows Synergistic Antitumor Effect in High-Grade B-Cell Lymphomas with ConcomitantMYCandBCL2Rearrangements

High-grade B-cell lymphoma with concurrentMYCandBCL2rearrangements (HGBCL-DHL) is a rare and aggressive B-cell disorder with a high likelihood of nonresponsiveness to the front-line immunochemotherapy. Patients with HGBCL-DHL who develop a recurrent or progressive disease have limited effective therapeutics and show very poor clinical outcomes. Therefore, it calls for the development of novel targeted therapies for this specific patient populations. In this study, we showed that combination of BCL2 inhibitor venetoclax and CS2164, a novel orally active multitarget inhibitor, is a potent therapeutic strategy against HGBCL-DHL in the preclinical setting. BCL2rearrangement, a hallmark feature of HGBCL-DHL, often results in increased BCL2 protein that counteracts the proapoptotic proteins of BCL2 family, and thus blocks cell death. BCL2 blockage with venetoclax shows promising clinical responses in various human malignancies. However, resistance to venetoclax takes place following its continuous administration, suggesting that venetoclax should combine with other agents to prevent disease progression. CS2164 is originally developed by China and designed to perturb three key characteristics of neoplasms: tumor angiogenesis, cell mitosis and chronic inflammation. Our previous study demonstrated that CS2164 exerted potently antilymphoma activities in MYC-driven lymphomas, providing evidence that CS2164 could potentiate the effect of venetoclax for MYC/BCL2 driven HGBCL-DHL. In the present work, we first observed that both venetoclax and CS2164 as single agent reduced the capability of cell proliferation in HGBCL-DHL cell lines. Next, we found that CS2164 synergized with venetoclax to suppress cell proliferation and trigger cell apoptosis in HGBCL-DHLin vitro. More importantly, when compared with each single drug groups, coadministration of venetoclax and CS2164 resulted in superior suppression of HGBCL-DHL cell growth and remarkably abrogated tumor burden in a HGBCL-DHL-xenografted mouse model. The synergistic lethality of venetoclax and CS2164 towards HGBCL-DHL cells was associated with the modulation of multiple molecular mechanisms. The underlying mechanisms for the synergy of the two drugs included the blockade of Rad51 recombinase-dependent DNA repair, the perturbation of the delicate balance of BCL2 family proteins that induced mitochondrial membrane depolarization and subsequently led to the proapoptotic effect, as well as the inhibition of PI3K/AKT/mTOR pathway and MYC expression. In summary, these findings suggest that the regimen of CS2164 and venetoclax combination is highly effective to eliminate HGBCL-DHL cellsin vitroandin vivoand thus provide a rational treatment paradigm to strip HGBCL-DHL of its protection fromMYCandBCL2rearrangements. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: CS2164 originally developed by China is a novel orally active multitarget inhibitor that is evaluating in clinical trials against multiple solid tumors.

CCR2 Expression Signature Can Classify and Predict Outcome in a Subpopulation of Chronic Lymphocytic Leukemia (CLL) Patients

Despite the recent advances in chronic lymphocytic leukemia (CLL) treatment with the development of novel targeted agents such as Bruton's tyrosine kinase (BTK) inhibitor ibrutinib and the BCL2 antagonist Venetoclax, the disease remains incurable for most patients, and resistance mechanisms to these novel agents have already been identified. Therefore, identifying new therapeutic vulnerabilities to treat and prevent resistant tumors is of great importance. Stromal cells provide CLL cells a protective niche which increases resistance to drug-induced apoptosis. Therefore, a current challenge is to develop new strategies by targeting not only CLL cells, but also the microenvironment. We found that soluble factors can recapitulate much of the protective function of stromal cells. To understand the role of microenvironmental factors on CLL cells, we took advantage of the conditioned media (CM) collected from the stromal cell line NK-tert, which protects CLL cells from spontaneous cell death. First, we identified three major cytokines (IL6, IL8, CCL2) from the conditioned media using Proteome Profiler Human XL Cytokine Array. Next, we evaluated the effect of each cytokine on peripheral blood (PB) CLL survival (n=5, treatment naïve, unselected regarding genetic background). CLL cells were cultured ex-vivo in CM and treated with neutralizing antibody against either Il6, IL8 or CCL2 for 48hrs and found a significant decrease in cell viability in presence of anti-CCL2 antibody (median % decrease in cell viability over isotype control is 50%, p&amp;lt;0.0001). Similarly, we also found an increase in cell viability when CLL cells were cultured in RPMI in presence of recombinant-CCL2 protein than those are cultured in RPMI only, suggesting that CCL2 has an important role in CLL survival. BH3 profiling quantifies apoptotic signaling based on cytochrome c loss as a measure of mitochondrial outer membrane permeabilization (MOMP) by flow cytometry in response to BH3-only synthetic peptides that mimic pro-apoptotic BCL-2 family proteins, applied to digitonin permeabilized cells. We performed BH3 profile on PB derived -CLL samples (n=5, treatment naive) cultured in CM and exposed to either isotype control or anti-CCL2 antibody. Anti-CCL2 treated CLL samples showed an increase in MOMP in response to BIM, PUMA, MS1 and HRK peptides compared to CLL samples exposed to isotype control. This suggests that CCL2 suppresses apoptotic signaling in CLL cells. Furthermore, Dynamic BH3 profiling (DBP), a measure of drug-induced apoptotic signaling as previously described (Montero et al., Cell, 2015) showed that a combination of anti-CCL2 and venetoclax increased dependence on BCL-2 (BAD peptide)MCL-1(MS1 peptide) as well as BCL-xL (HRK peptide) in CLL cells. To our surprise, we also found a subgroup of treatment naive PB-CLL samples (CM independent) that undergo minimal spontaneous cell death in the absence of stroma support. In addition to this, IL-6, IL8, CCL2 neutralizing antibodies showed no effect on their survival. These CM independent CLL samples(n=5) are less sensitive to venetoclax treatment compared to the CLL samples that need stroma support (CM dependent). Moreover, baseline BH3 profile showed that CM independent CLL cells are less primed for apoptosis compared to CM dependent CLL cells, and exposure to venetoclax and anti-CCL2 antibody did not increase any drug-mediated apoptotic priming. Next, we checked mRNA expression level of the CCL2 receptor, CCR2 from CM dependent and CM independent CLL samples and found that CM dependent CLL cells have higher expression of CCR2, suggesting a distinct signature discriminating CM dependent and independent patients. In summary, our study shows that CCL2 may serve as a novel biomarker to target in CLL, especially in relapsed/refractory patients. Disclosures Davids: Verastem: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Surface Oncology: Research Funding; MEI Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Gilead Sciences: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Research Funding; Janssen: Consultancy; Genentech: Consultancy, Research Funding; Eli Lilly: Consultancy; Celgene: Consultancy; AstraZeneca: Consultancy, Research Funding; BeiGene: Consultancy; Ascentage Pharma: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; AbbVie: Consultancy; Zentalis: Consultancy; Sunesis: Consultancy; Syros Pharmaceuticals: Consultancy; Research to Practice: Honoraria. Letai:Chugai: Other: Lecture Fees; Novartis: Research Funding; AbbVie: Consultancy; AstraZeneca: Consultancy; Zentalis: Membership on an entity's Board of Directors or advisory committees; Flash Therapeutics: Membership on an entity's Board of Directors or advisory committees; Dialectic: Membership on an entity's Board of Directors or advisory committees.

Computational investigation of 4,5-diphenyl-1H-pyrrole-3-carboxylic acid derivatives as B-cell lymphoma-extra large (Bcl-xL) inhibitors by using 3D-QSAR, molecular docking, and molecular dynamics simulations

B-cell lymphoma-extra large (Bcl-xL) can inhibit apoptosis via heterodimerization with pro-apoptotic Bcl-2 family proteins, and is over-expressed in many different types of human tumors and has been regarded as a novel cancer therapeutic strategy. Due to the fact that current Bcl-xl inhibitors lack sustained effectiveness and the occurrence of some unpredictable side effects, the development of new inhibitors is necessary. In this study, computational study was applied to a series of Bcl-xL inhibitors to reveal the relationship between structure and activities through applying molecular docking, three-dimensional qualitative structure-activity relationship (3D-QSAR), and molecular dynamic (MD) simulations. A molecular docking study was performed to explore possible modes of action between inhibitors and Bcl-xL protein. Subsequently, 3D-QSAR models were generated with comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). For the best CoMFA model, the Q2 and R2 values were computed as 0.927 and 0.999, while those were computed as 0.943 and 0.998 for the best CoMSIA model. Twenty new Bcl-xL inhibitors were designed, and all their predictive activities were improved than molecules in the dataset based on the contour maps. In addition, MD simulations were conducted to evaluate the stability of the complexes conformed by two inhibitors and Bcl-xL, and the results were consistent with those of the molecular docking and 3D-QSAR studies. Finally, binding free energy was computed through molecular mechanics performed by surface area approach (MM-GBSA), and the result congruent with the activities which indicated van der Waals as well as lipophilic energy contributing the most during the molecular with Bcl-xL protein binding. In brief, our research provided valuable information for further development of Bcl-xL inhibitors.

Kill one or kill the many: interplay between mitophagy and apoptosis.

Mitochondria are key players of cellular metabolism, Ca2+ homeostasis, and apoptosis. The functionality of mitochondria is tightly regulated, and dysfunctional mitochondria are removed via mitophagy, a specialized form of autophagy that is compromised in hereditary forms of Parkinson's disease. Through mitophagy, cells are able to cope with mitochondrial stress until the damage becomes too great, which leads to the activation of pro-apoptotic BCL-2 family proteins located on the outer mitochondrial membrane. Active pro-apoptotic BCL-2 proteins facilitate the release of cytochrome c from the mitochondrial intermembrane space (IMS) into the cytosol, committing the cell to apoptosis by activating a cascade of cysteinyl-aspartate specific proteases (caspases). We are only beginning to understand how the choice between mitophagy and the activation of caspases is determined on the mitochondrial surface. Intriguingly in neurons, caspase activation also plays a non-apoptotic role in synaptic plasticity. Here we review the current knowledge on the interplay between mitophagy and caspase activation with a special focus on the central nervous system.

The chaperone Hsp70 is a BH3 receptor activated by the pro-apoptotic Bim to stabilize anti-apoptotic clients

The chaperone heat shock protein 70 (Hsp70) is crucial for avoiding protein misfolding under stress, but is also up-regulated in many kinds of cancers, where its ability to buffer cellular stress prevents apoptosis. Previous research has suggested Hsp70 interacts with pro-apoptotic Bcl-2 family proteins, including Bim and Bax. However, a definitive demonstration of this interaction awaits, and insights into the structural basis and molecular mechanism remain unclear. Earlier studies have identified a Bcl-2 homology 3 (BH3) domain present in Bcl-2 family members that engages receptors to stimulate apoptosis. We now show that Hsp70 physically interacts with pro-apoptotic multidomain and BH3-only proteins via a BH3 domain, thereby serving as a novel BH3 receptor, using in vitro fluorescent polarization (FP), isothermal titration calorimetry (ITC), and cell-based co-immunoprecipitation (co-IP) experiments, 1H-15N-transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, ATPase activity, and denatured rhodanese aggregation measurements further demonstrated that BimBH3 binds to a novel allosteric site in the nucleotide-binding domain (NBD) of Hsp70, by which Bim acts as a positive co-chaperone to promote the ATPase activity and chaperone functions. A dual role of Hsp70's anti-apoptotic function was revealed that when it keeps Bim in check to inhibit apoptosis, it simultaneously stabilizes oncogenic clients including AKT and Raf-1 with the aid of Bim. Two faces of Bim in cell fate regulation were revealed that in opposite to its well-established pro-apoptotic activator role, Bim could help the folding of oncogenic proteins.

Rapid and quantitative in vitro analysis of mitochondrial fusion and its interplay with apoptosis

Mitochondrial fusion is essential to maintain genomic stability and physiological functions of mitochondria. Since mitochondrial fusion and fission work in concert to regulate mitochondrial morphology and functions, it has been challenging to quantitatively measure the direct roles of mitochondrial fusion in apoptosis and cancer progression. Here, we report the development of a high-throughput in vitro method to quantify mitochondrial fusion through single mitochondria analysis by a laboratory-built nano-flow cytometer (nFCM). Isolated mitochondria expressing green fluorescent protein (GFP-mito) or discosoma red fluorescent protein (DsRed-mito) were mixed together, induced to fuse, and analyzed by nFCM. A particle exhibiting both green and red fluorescence was identified as an event of heterotypic fusion, and the efficiency of heterotypic fusion was used as a surrogate of overall fusion efficiency. The as-developed method was applied to reveal the interplay between mitochondrial fusion and apoptosis without the interference of fission. We show that cytosolic components promoted mitochondrial fusion, and this upregulation was diminished during apoptosis. Combined with the translocation of Bid and Bax from cytosol to mitochondria, these findings suggest that cytosolic pro-apoptotic Bcl-2 family proteins could be the positive mediators of mitochondrial fusion. On the other hand, fusion also renders mitochondria more resistant to membrane potential collapse upon apoptosis induction. Our data suggest that disruption of mitochondrial fusion could be a potent strategy for cancer therapy. Furthermore, the as-developed method offers an effective approach to identify fusion inhibitors, including betulinic acid and antimycin A, giving reasons for their powerful utility in cancer treatment.

EGFR/FOXO3a/BIM signaling pathway determines chemosensitivity of BMP4-differentiated glioma stem cells to temozolomide

Accumulating evidence suggests that glioma stem cells (GSCs), which are rare cells characterized by pluripotency and self-renewal ability, are responsible for glioblastoma (GBM) propagation, recurrence and resistance to therapies. Bone morphogenic proteins (BMPs) induce GSC differentiation, which leads to elimination of GSCs and sensitization of glioma to chemotherapeutics. Alterations in the epidermal growth factor receptor (EGFR) gene are detected in more than half of GBMs; however, the role of EGFR in the chemoresistance of GSCs remains unknown. Here, we examined whether EGFR signaling affects BMP4-induced differentiation of GSCs and their response to the alkylating drug temozolomide (TMZ). We show that BMP4 triggers the SMAD signaling cascade in GSCs independent of the EGFR level. BMP4 downregulated the levels of pluripotency markers (SOX2 and OLIG2) with a concomitant induction of an astrocytic marker (GFAP) and a neuronal marker (β-Tubulin III). However, GSCs with different EGFR levels responded differently to treatments. BMP4-induced differentiation did not enhance sensitivity to TMZ in EGFRlow GSCs, in contrast to EGFRhigh GSCs, which underwent apoptosis. We then identified differences in cell cycle regulation. In EGFRlow cells, BMP4-triggered G1 cell cycle arrest which was not detected in EGFRhigh cells. RNA-seq profiles further highlighted transcriptomic alterations and distinct processes characterizing EGFR-dependent responses in the course of BMP4-induced differentiation. We found that the control of BIM (the pro-apoptotic BCL-2 family protein) by the AKT/FOXO3a axis only operated in BMP4-differentiated EGFRhigh cells upon TMZ treatment.

Bax and Bak jointly control survival and dampen the early unfolded protein response in pancreatic \u03b2-cells under glucolipotoxic stress

ER stress and apoptosis contribute to the loss of pancreatic β-cells under pro-diabetic conditions of glucolipotoxicity. Although activation of canonical intrinsic apoptosis is known to require pro-apoptotic Bcl-2 family proteins Bax and Bak, their individual and combined involvement in glucolipotoxic β-cell death are not known. It has also remained an open question if Bax and Bak in β-cells have non-apoptotic roles in mitochondrial function and ER stress signaling, as suggested in other cell types. Using mice with individual or combined β-cell deletion of Bax and Bak, we demonstrated that glucolipotoxic β-cell death in vitro occurs by both non-apoptotic and apoptotic mechanisms, and the apoptosis could be triggered by either Bax or Bak alone. In contrast, they had non-redundant roles in mediating staurosporine-induced apoptosis. We further established that Bax and Bak do not affect normal glucose-stimulated β-cell Ca2+ responses, insulin secretion, or in vivo glucose tolerance. Finally, our experiments revealed that combined deletion of Bax and Bak amplified the unfolded protein response in islets during the early stages of chemical- or glucolipotoxicity-induced ER stress. These findings shed new light on roles of the core apoptosis machinery in β-cell survival and stress signals of importance for the pathobiology of diabetes.