Clinical, biochemical, and genetic spectrum of MADD in a South African cohort: an ICGNMD study.

BACKGROUND: Multiple acyl-CoA dehydrogenase deficiency (MADD) is an autosomal recessive disorder resulting from pathogenic variants in three distinct genes, with most of the variants occurring in the electron transfer flavoprotein-ubiquinone oxidoreductase gene (ETFDH). Recent evidence of potential founder variants for MADD in the South African (SA) population, initiated this extensive investigation. As part of the International Centre for Genomic Medicine in Neuromuscular Diseases study, we recruited a cohort of patients diagnosed with MADD from academic medical centres across SA over a three-year period. The aim was to extensively profile the clinical, biochemical, and genomic characteristics of MADD in this understudied population. METHODS: Clinical evaluations and whole exome sequencing were conducted on each patient. Metabolic profiling was performed before and after treatment, where possible. The recessive inheritance and phase of the variants were established via segregation analyses using Sanger sequencing. Lastly, the haplotype and allele frequencies were determined for the two main variants in the four largest SA populations. RESULTS: Twelve unrelated families (ten of White SA and two of mixed ethnicity) with clinically heterogeneous presentations in 14 affected individuals were observed, and five pathogenic ETFDH variants were identified. Based on disease severity and treatment response, three distinct groups emerged. The most severe and fatal presentations were associated with the homozygous c.[1067G > A];c.[1067G > A] and compound heterozygous c.[976G > C];c.[1067G > A] genotypes, causing MADD types I and I/II, respectively. These, along with three less severe compound heterozygous genotypes (c.[1067G > A];c.[1448C > T], c.[740G > T];c.[1448C > T], and c.[287dupA*];c.[1448C > T]), resulting in MADD types II/III, presented before the age of five years, depending on the time and maintenance of intervention. By contrast, the homozygous c.[1448C > T];c.[1448C > T] genotype, which causes MADD type III, presented later in life. Except for the type I, I/II and II cases, urinary metabolic markers for MADD improved/normalised following treatment with riboflavin and L-carnitine. Furthermore, genetic analyses of the most frequent variants (c.[1067G > A] and c.[1448C > T]) revealed a shared haplotype in the region of ETFDH, with SA population-specific allele frequencies of < 0.00067-0.00084%. CONCLUSIONS: This study reveals the first extensive genotype-phenotype profile of a MADD patient cohort from the diverse and understudied SA population. The pathogenic variants and associated variable phenotypes were characterised, which will enable early screening, genetic counselling, and patient-specific treatment of MADD in this population.

Total recall: the role of PIDDosome components in neurodegeneration.

The PIDDosome is a multiprotein complex that includes p53-induced protein with a death domain 1 (PIDD1), receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD), and caspase-2, the activation of which is driven by PIDDosome assembly. In addition to the key role of the PIDDosome in the regulation of cell differentiation, tissue homeostasis, and organogenesis and regeneration, caspase-2, RAIDD and PIDD1 engagement in neuronal development was shown. Here, we focus on the involvement of PIDDosome components in neurodegenerative disorders, including retinal neuropathies, different types of brain damage, and Alzheimer's disease (AD), Huntington's disease (HD), and Lewy body disease. We also discuss pathogenic variants of PIDD1, RAIDD, and caspase-2 that are associated with intellectual, behavioral, and psychological abnormalities, together with prospective PIDDosome inhibition strategies and their potential clinical application.

Molecular basis of neurodevelopmental disorders caused by pathogenic variants of PIDD.

PIDDosome formation followed by caspase-2 activation is critical for genotoxic stress-induced apoptotic cell death. Failure of proper caspase-2 activation causes a neurodevelopmental disorder and intellectual disability. R815W, R862W, and Q863stop mutations in p53-induced protein with a death domain (PIDD), a component of the PIDDosome, also lead to this disorder. However, the molecular mechanisms underlying this pathogenesis remain elusive. In this study, we analyzed the molecular mechanisms underlying the pathogenesis of the PIDD DD pathogenic variants R815W, R862W, and Q863stop. We determined that these mutations prevented the interaction between PIDD and RIP-associated Ich-1/Ced-3 homologous protein with a death domain (RAIDD), a molecule that mediates PIDDosome formation. The disruption of this interaction affects PIDDosome formation and caspase-2 activation.

Nimbin analog N2 alleviates high testosterone induced oxidative stress in CHO cells and alters the expression of Tox3 and Dennd1a signal transduction pathway involved in the PCOS zebrafish.

Polycystic ovarian syndrome (PCOS) is a hormonal disorder that causes enlargement of ovaries and follicular maturation arrest, which lacks efficient treatment. N2, a semi-natural triterpenoid from the neem family, was already reported to have antioxidant and antiinflammatory properties in our previous report. This study investigated the anti-androgenic property of N2 on testosterone-induced oxidative stress in Chinese Hamster Ovarian cells (CHO) and PCOS zebrafish model. The testosterone exposure disrupted the antioxidant enzymes and ROS level and enhanced the apoptosis in both CHO cells and PCOS zebrafish. However, N2 significantly protected the CHO cells from ROS and apoptosis. N2 improved the Gonado somatic index (GSI) and upregulated the expression of the SOD enzyme in zebrafish ovaries. Moreover, the testosterone-induced follicular maturation arrest was normalized by N2 treatment in histopathology studies. In addition, the gene expression studies of Tox3 and Denndla in zebrafish demonstrated that N2 could impair PCOS condition. Furthermore, to confirm the N2 activity, the in-silico studies were performed against PCOS susceptible genes Tox3 and Dennd1a using molecular docking and molecular dynamic simulations. The results suggested that N2 alleviated the oxidative stress and apoptosis in-vitro and in-vivo and altered the expression of PCOS key genes.

Generation of a human induced pluripotent stem cell line (LZUSHi002-A) from a MADD patient with ETFDH mutation.

Multiple acyl-coenzyme A dehydrogenase deficiency (MADD) is an inborn metabolic disorder that affects fatty acid oxidation and the catabolism of branched-chain amino acids, vitamins B and energy metabolism. In this study, the induced pluripotent stem cell (iPSC) line LZUSHi002-A from PBMCs of a 10-year-old male patient with ETFDH mutations using the episomal plasmids was established, which is an ideal in vitro model to understand the exact pathogenesis of MADD.

PIDD1 in cell cycle control, sterile inflammation and cell death.

The death fold domain-containing protein PIDD1 has recently attracted renewed attention as a regulator of the orphan cell death-related protease, Caspase-2. Caspase-2 can activate p53 to promote cell cycle arrest in response to centrosome aberrations, and its activation requires formation of the PIDDosome multi-protein complex containing multimers of PIDD1 and the adapter RAIDD/CRADD at its core. However, PIDD1 appears to be able to engage with multiple client proteins to promote an even broader range of biological responses, such as NF-κB activation, translesion DNA synthesis or cell death. PIDD1 shows features of inteins, a class of self-cleaving proteins, to create different polypeptides from a common precursor protein that allow it to serve these diverse functions. This review summarizes structural information and molecular features as well as recent experimental advances that highlight the potential pathophysiological roles of this unique death fold protein to highlight its drug-target potential.

Autophagy induction regulates aquaporin 3-mediated skin fibroblast ageing.

BACKGROUND: Long- and short-term ultraviolet (UV) exposure have distinct biological effects on human fibroblasts. OBJECTIVES: To elucidate the underlying mechanisms of the biological effects of UV exposure on human skin fibroblasts. METHODS: We subjected human skin fibroblast cells with or without aquaporin 3 (AQP3), death effector domain-containing protein (DEDD) or Beclin1 manipulation to UVA treatment and evaluated autophagy and senescence in them. RESULTS: Short-term UVA irradiation induced autophagy and upregulated AQP3 but not senescence, whereas long-term UVA irradiation inhibited autophagy, AQP3 and trigger senescence in vitro and in vivo. Silencing AQP3 abolished short-term UVA irradiation-induced autophagy and led to cellular senescence, whereas AQP3 overexpression partially rescued the senescence and autophagy inhibition induced by long-term UVA exposure in vitro. Mechanistically, the transcription factor Jun was found to bind to the AQP3 promoter to activate its transcription following short-term UVA exposure. Subsequently, AQP3 interacted with DEDD to induce its ubiquitination-mediated degradation and promote autophagy, and bound to Beclin1 to directly activate autophagy. Finally, autophagy induced by AQP3 overexpression robustly prevented UVA-induced senescence in vitro and in vivo. CONCLUSIONS: Our study indicates that AQP3 controls skin fibroblast photoageing by regulating autophagy and represents a potential target for future interventions against skin ageing.

CircNFIC Balances Inflammation and Apoptosis by Sponging miR-30e-3p and Regulating DENND1B Expression.

Disordered inflammation and apoptosis are closely related to diseases, and inflammation can also promote cell apoptosis, where growing evidence has shown that circular RNAs (circRNAs) play important roles. Lipopolysaccharide (LPS) is the main component of the cytoderm of gram-negative bacterium, which can cause inflammatory responses in macrophages. We constructed an inflammatory model by exposing chicken macrophage cell lines (also known as HD11) to LPS for in vitro experiments. In this study, we validated a novel circRNA-circNFIC-which was dramatically up-regulated in tissues infected by coccidia and cells exposed to LPS. Besides, circNFIC could significantly promote the expression levels of pro-inflammation factors, including (IL-1ß, TNFα, and IFNγ) and pro-apoptosis maker genes (caspase 3 and caspase 8) in HD11 exposed to LPS or not. In terms of mechanism, circNFIC exerted notable effects on DENND1B to regulate cell inflammation and apoptosis by sponging miR-30e-3p. The molecular functions played by miR-30e-3p and DENND1B have been explored, respectively. In addition, the effects of circNFIC knockdown suppressing the expression of pro-inflammatory and pro-apoptosis functions could be reversed by a miR-30e-3p inhibitor. On the whole, circNFIC promoted cell inflammation and apoptosis via the miR-30e-3p/DENND1B axis.

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex.

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1ß-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

FANCI functions as a repair/apoptosis switch in response to DNA crosslinks.

Cells counter DNA damage through repair or apoptosis, yet a direct mechanism for this choice has remained elusive. When facing interstrand crosslinks (ICLs), the ICL-repair protein FANCI heterodimerizes with FANCD2 to initiate ICL excision. We found that FANCI alternatively interacts with a pro-apoptotic factor, PIDD1, to enable PIDDosome (PIDD1-RAIDD-caspase-2) formation and apoptotic death. FANCI switches from FANCD2/repair to PIDD1/apoptosis signaling in the event of ICL-repair failure. Specifically, removing key endonucleases downstream of FANCI/FANCD2, increasing ICL levels, or allowing damaged cells into mitosis (when repair is suppressed) all suffice for switching. Reciprocally, apoptosis-committed FANCI reverts from PIDD1 to FANCD2 after a failed attempt to assemble the PIDDosome. Monoubiquitination and deubiquitination at FANCI K523 impact interactor selection. These data unveil a repair-or-apoptosis switch in eukaryotes. Beyond ensuring the removal of unrepaired genomes, the switch's bidirectionality reveals that damaged cells can offset apoptotic defects via de novo attempts at lesion repair.

Mechanistic insights into TNFR1/MADD death domains in Alzheimer's disease through conformational molecular dynamic analysis.

Proteins are tiny players involved in the activation and deactivation of multiple signaling cascades through interactions in cells. The TNFR1 and MADD interact with each other and mediate downstream protein signaling pathways which cause neuronal cell death and Alzheimer's disease. In the current study, a molecular docking approach was employed to explore the interactive behavior of TNFR1 and MADD proteins and their role in the activation of downstream signaling pathways. The computational sequential and structural conformational results revealed that Asp400, Arg58, Arg59 were common residues of TNFR1 and MADD which are involved in the activation of downstream signaling pathways. Aspartic acid in negatively charged residues is involved in the biosynthesis of protein. However, arginine is a positively charged residue with the potential to interact with oppositely charged amino acids. Furthermore, our molecular dynamic simulation results also ensured the stability of the backbone of TNFR1 and MADD death domains (DDs) in binding interactions. This DDs interaction mediates some conformational changes in TNFR1 which leads to the activation of mediators proteins in the cellular signaling pathways. Taken together, a better understanding of TNFR1 and MADD receptors and their activated signaling cascade may help treat Alzheimer's disease. The death domains of TNFR1 and MADD could be used as a novel pharmacological target for the treatment of Alzheimer's disease by inhibiting the MAPK pathway.

The calcium sensing receptor (CaSR) promotes Rab27B expression and activity to control secretion in breast cancer cells.

Chemotactic and angiogenic factors secreted within the tumor microenvironment eventually facilitate the metastatic dissemination of cancer cells. Calcium-sensing receptor (CaSR) activates secretory pathways in breast cancer cells via a mechanism driven by vesicular trafficking of this receptor. However, it remains to be elucidated how endosomal proteins in secretory vesicles are controlled by CaSR. In the present study, we demonstrate that CaSR promotes expression of Rab27B and activates this secretory small GTPase via PI3K, PKA, mTOR and MADD, a guanine nucleotide exchange factor, also known as DENN/Rab3GEP. Active Rab27B leads secretion of various cytokines and chemokines, including IL-6, IL-1ß, IL-8, IP-10 and RANTES. Expression of Rab27B is stimulated by CaSR in MDA-MB-231 and MCF-7 breast epithelial cancer cells, but not in non-cancerous MCF-10A cells. This regulatory mechanism also occurs in HeLa and PC3 cells. Our findings provide insightful information regarding how CaSR activates a Rab27B-dependent mechanism to control secretion of factors known to intervene in paracrine communication circuits within the tumor microenvironment.

Homozygous variant in MADD, encoding a Rab guanine nucleotide exchange factor, results in pleiotropic effects and a multisystemic disorder.

Rab proteins coordinate inter-organellar vesicle-mediated transport, facilitating intracellular communication, protein recycling, and signaling processes. Dysfunction of Rab proteins or their direct interactors leads to a wide range of diseases with diverse manifestations. We describe seven individuals from four consanguineous Arab Muslim families with an infantile-lethal syndrome, including failure to thrive (FTT), chronic diarrhea, neonatal respiratory distress, variable pituitary dysfunction, and distal arthrogryposis. Exome sequencing analysis in the independent families, followed by an internal gene-matching process using a local exome database, identified a homozygous splice-site variant in MADD (c.2816 + 1 G > A) on a common haplotype. The variant segregated with the disease in all available family members. Determination of cDNA sequence verified single exon skipping, resulting in an out-of-frame deletion. MADD encodes a Rab guanine nucleotide exchange factor (GEF), which activates RAB3 and RAB27A/27B and is thus a crucial regulator of neuromuscular junctions and endocrine secretory granule release. Moreover, MADD protects cells from caspase-mediated TNF-α-induced apoptosis. The combined roles of MADD and its downstream effectors correlate with the phenotypic spectrum of disease, and call for additional studies to confirm the pathogenic mechanism and to investigate possible therapeutic avenues through modulation of TNF-α signaling.

Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate.

Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.

Mechanism of filament formation in UPA-promoted CARD8 and NLRP1 inflammasomes.

NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase-1 activation, cytokine maturation and/or pyroptotic cell death. NLRP1 and CARD8 use their C-terminal (CT) fragments containing a caspase recruitment domain (CARD) and the UPA (conserved in UNC5, PIDD, and ankyrins) subdomain for self-oligomerization, which in turn form the platform to recruit the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a CARD) or caspase-1, respectively. Here, we report cryo-EM structures of NLRP1-CT and CARD8-CT assemblies, in which the respective CARDs form central helical filaments that are promoted by oligomerized, but flexibly linked, UPAs surrounding the filaments. Through biochemical and cellular approaches, we demonstrate that the UPA itself reduces the threshold needed for NLRP1-CT and CARD8-CT filament formation and signalling. Structural analyses provide insights on the mode of ASC recruitment by NLRP1-CT and the contrasting direct recruitment of caspase-1 by CARD8-CT. We also discover that subunits in the central NLRP1CARD filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. Finally, we engineer and determine the structure of an ASCCARD-caspase-1CARD octamer, which suggests that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. Together these structures capture the architecture and specificity of the active NLRP1 and CARD8 inflammasomes in addition to key heteromeric CARD-CARD interactions governing inflammasome signalling.

Progesterone induces apoptosis by activation of caspase-8 and calcitriol via activation of caspase-9 pathways in ovarian and endometrial cancer cells in vitro.

Previously we have shown inhibition of endometrial cancer cell growth with progesterone and calcitriol. However, the mechanisms by which the two agents attenuate proliferation have not been well characterized yet. Herein, we investigated how progesterone and calcitriol induce apoptosis in cancer cells. DNA fragmentation was upregulated by progesterone and calcitriol in ovarian and endometrial cancer cells. Time-dependent treatment of ovarian cancer cells, ES-2, and TOV-21G with progesterone enhanced caspase -8 activity after 12 h, whereas OV-90, TOV-112D, HEC-1A, and HEC-59 cells showed increased activity after 24 h. Caspase 9 activity was increased in all cell lines after 24 h treatment with calcitriol. Pretreatment of cancer cells with a caspase-8 inhibitor (z-IETD-fmk) or caspase-9 inhibitor (Z-LEHD-fmk) significantly attenuated progesterone and calcitriol induced caspase-8 and caspase-9 expression, respectively. The expression of FasL, Fas, FAD, and pro-caspase-8, which constitute the death-inducing signaling complex (DISC), was upregulated in progesterone treated cancer cells. Knockdown of FAS or FADD with specific siRNAs significantly blocked progesterone-induced caspase-8. Cleavage of the BID was not affected by caspase-8 activation suggesting the absence of cross-talk between caspase-8 and caspase-9 pathways. Calcitriol treatment decreased mitochondrial membrane potential and increased the release of cancer cytochrome C. These findings indicate that progesterone induces apoptosis through activation of caspase-8 and calcitriol through caspase-9 activation in cancer cells. A combination of progesterone-calcitriol activates both extrinsic and intrinsic apoptotic pathways in cancer cells.

Centriolar distal appendages activate the centrosome-PIDDosome-p53 signalling axis via ANKRD26.

Centrosome amplification results into genetic instability and predisposes cells to neoplastic transformation. Supernumerary centrosomes trigger p53 stabilization dependent on the PIDDosome (a multiprotein complex composed by PIDD1, RAIDD and Caspase-2), whose activation results in cleavage of p53's key inhibitor, MDM2. Here, we demonstrate that PIDD1 is recruited to mature centrosomes by the centriolar distal appendage protein ANKRD26. PIDDosome-dependent Caspase-2 activation requires not only PIDD1 centrosomal localization, but also its autoproteolysis. Following cytokinesis failure, supernumerary centrosomes form clusters, which appear to be necessary for PIDDosome activation. In addition, in the context of DNA damage, activation of the complex results from a p53-dependent elevation of PIDD1 levels independently of centrosome amplification. We propose that PIDDosome activation can in both cases be promoted by an ANKRD26-dependent local increase in PIDD1 concentration close to the centrosome. Collectively, these findings provide a paradigm for how centrosomes can contribute to cell fate determination by igniting a signalling cascade.

ANKRD26 recruits PIDD1 to centriolar distal appendages to activate the PIDDosome following centrosome amplification.

Centriole copy number is tightly maintained by the once-per-cycle duplication of these organelles. Centrioles constitute the core of centrosomes, which organize the microtubule cytoskeleton and form the poles of the mitotic spindle. Centrosome amplification is frequently observed in tumors, where it promotes aneuploidy and contributes to invasive phenotypes. In non-transformed cells, centrosome amplification triggers PIDDosome activation as a protective response to inhibit cell proliferation, but how extra centrosomes activate the PIDDosome remains unclear. Using a genome-wide screen, we identify centriole distal appendages as critical for PIDDosome activation in cells with extra centrosomes. The distal appendage protein ANKRD26 is found to interact with and recruit the PIDDosome component PIDD1 to centriole distal appendages, and this interaction is required for PIDDosome activation following centrosome amplification. Furthermore, a recurrent ANKRD26 mutation found in human tumors disrupts PIDD1 localization and PIDDosome activation in cells with extra centrosomes. Our data support a model in which ANKRD26 initiates a centriole-derived signal to limit cell proliferation in response to centrosome amplification.

TRAIL Triggers CRAC-Dependent Calcium Influx and Apoptosis through the Recruitment of Autophagy Proteins to Death-Inducing Signaling Complex.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills various cancer cell types, but also leads to the activation of signaling pathways that favor resistance to cell death. Here, we investigated the as yet unknown roles of calcium signaling and autophagy regulatory proteins during TRAIL-induced cell death in leukemia cells. Taking advantage of the Gene Expression Profiling Interactive Analysis (GEPIA) project, we first found that leukemia patients present a unique TRAIL receptor gene expression pattern that may reflect their resistance to TRAIL. The exposure of NB4 acute promyelocytic leukemia cells to TRAIL induces intracellular Ca2+ influx through a calcium release-activated channel (CRAC)-dependent mechanism, leading to an anti-apoptotic response. Mechanistically, we showed that upon TRAIL treatment, two autophagy proteins, ATG7 and p62/SQSTM1, are recruited to the death-inducing signaling complex (DISC) and are essential for TRAIL-induced Ca2+ influx and cell death. Importantly, the treatment of NB4 cells with all-trans retinoic acid (ATRA) led to the upregulation of p62/SQSTM1 and caspase-8 and, when added prior to TRAIL stimulation, significantly enhanced DISC formation and the apoptosis induced by TRAIL. In addition to uncovering new pleiotropic roles for autophagy proteins in controlling the calcium response and apoptosis triggered by TRAIL, our results point to novel therapeutic strategies for sensitizing leukemia cells to TRAIL.

Pharmacological targeting of c-FLIPL and Bcl-2 family members promotes apoptosis in CD95L-resistant cells.

The development of efficient combinatorial treatments is one of the key tasks in modern anti-cancer therapies. An apoptotic signal can either be induced by activation of death receptors (DR) (extrinsic pathway) or via the mitochondria (intrinsic pathway). Cancer cells are characterized by deregulation of both pathways. Procaspase-8 activation in extrinsic apoptosis is controlled by c-FLIP proteins. We have recently reported the small molecules FLIPinB/FLIPinBγ targeting c-FLIPL in the caspase-8/c-FLIPL heterodimer. These small molecules enhanced caspase-8 activity in the death-inducing signaling complex (DISC), CD95L/TRAIL-induced caspase-3/7 activation and subsequent apoptosis. In this study to increase the pro-apoptotic effects of FLIPinB/FLIPinBγ and enhance its therapeutic potential we investigated costimulatory effects of FLIPinB/FLIPinBγ in combination with the pharmacological inhibitors of the anti-apoptotic Bcl-2 family members such as ABT-263 and S63845. The combination of these inhibitors together with FLIPinB/FLIPinBγ increased CD95L-induced cell viability loss, caspase activation and apoptosis. Taken together, our study suggests new approaches for the development of combinatorial anti-cancer therapies specifically targeting both intrinsic and extrinsic apoptosis pathways.