Chronic spindle assembly checkpoint activation causes myelosuppression and gastrointestinal atrophy.

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor, CDC20, preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However, the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression, we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for, gastrointestinal atrophy is detrimental. Remarkably, deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome, while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect, identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast, only overexpression of anti-apoptotic BCL2, but none of the BH3-only protein deficiencies mentioned above, can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo.

Apoptotic mitochondrial poration by a growing list of pore-forming BCL-2 family proteins.

The pore-forming BCL-2 family proteins are effectors of mitochondrial poration in apoptosis initiation. Two atypical effectors-BOK and truncated BID (tBID)-join the canonical effectors BAK and BAX. Gene knockout revealed developmental phenotypes in the absence the effectors, supporting their roles in vivo. During apoptosis effectors are activated and change shape from dormant monomers to dynamic oligomers that associate with and permeabilize mitochondria. BID is activated by proteolysis, BOK accumulates on inhibition of its degradation by the E3 ligase gp78, while BAK and BAX undergo direct activation by BH3-only initiators, autoactivation, and crossactivation. Except tBID, effector oligomers on the mitochondria appear as arcs and rings in super-resolution microscopy images. The BH3-in-groove dimers of BAK and BAX, the tBID monomers, and uncharacterized BOK species are the putative building blocks of apoptotic pores. Effectors interact with lipids and bilayers but the mechanism of membrane poration remains elusive. I discuss effector-mediated mitochondrial poration.

Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death receptor-mediated extrinsic apoptosis, specifically in cancer cells, and Bid (BH3-interacting domain death agonist) plays an important role in TRAIL-induced apoptosis. Ferroptosis is a newly defined form of regulated cell death known to be distinct from other forms of cell death. However, our previous studies have shown that ferroptosis shares common pathways with other types of programmed cell death such as apoptosis. In this study, we investigated the role of Bid in the crosstalk between the ferroptotic agent-induced endoplasmic reticulum (ER) stress response and TRAIL-induced apoptosis. When human colorectal carcinoma HCT116 cells were treated with the ferroptosis-inducing agents artesunate and erastin in combination with TRAIL, TRAIL-induced activation of caspase-8 was enhanced, and subsequently, the truncation of Bid was increased. Similar results were observed when ovarian adenocarcinoma OVCAR-3 cells were treated with the ferroptotic agents in combination with TRAIL. Results from studies with Bid mutants reveal that the truncation of Bid and the presence of intact BH3 domains are critical for synergistic apoptosis. Nonfunctional Bid mutants were not able to activate the mitochondria-dependent apoptosis pathway, which is required for the conversion of p19 to p17, the active form of caspase-3. These results indicate that Bid plays a critical role in the crosstalk between the ferroptotic agent-induced ER stress response and TRAIL-induced apoptosis.

The BCL-2 family member BID plays a role during embryonic development in addition to its BH3-only protein function by acting in parallel to BAX, BAK and BOK.

The intrinsic apoptosis pathway, regulated by the BCL-2 protein family, is essential for embryonic development. Using mice lacking all known apoptosis effectors, BAX, BAK and BOK, we have previously defined the processes during development that require apoptosis. Rare Bok-/- Bax-/- Bak-/- triple knockout (TKO) mice developed to adulthood and several tissues that were thought to require apoptosis during development appeared normal. This raises the question if all apoptosis had been abolished in the TKO mice or if other BCL-2 family members could act as effectors of apoptosis. Here, we investigated the role of BID, generally considered to link the extrinsic and intrinsic apoptosis pathways, acting as a BH3-only protein initiating apoptosis upstream of BAX and BAK. We found that Bok-/- Bax-/- Bak-/- Bid-/- quadruple knockout (QKO) mice have additional developmental anomalies compared to TKO mice, consistent with a role of BID, not only upstream but also in parallel to BAX, BAK and BOK. Mitochondrial experiments identified a small cytochrome c-releasing activity of full-length BID. Collectively, these findings suggest a new effector role for BID in the intrinsic apoptosis pathway.

Structural basis of BAK activation in mitochondrial apoptosis initiation.

BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric "BH3-in-groove" triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.

BCL-2-family protein tBID can act as a BAX-like effector of apoptosis.

During apoptosis, the BCL-2-family protein tBID promotes mitochondrial permeabilization by activating BAX and BAK and by blocking anti-apoptotic BCL-2 members. Here, we report that tBID can also mediate mitochondrial permeabilization by itself, resulting in release of cytochrome c and mitochondrial DNA, caspase activation and apoptosis even in absence of BAX and BAK. This previously unrecognized activity of tBID depends on helix 6, homologous to the pore-forming regions of BAX and BAK, and can be blocked by pro-survival BCL-2 proteins. Importantly, tBID-mediated mitochondrial permeabilization independent of BAX and BAK is physiologically relevant for SMAC release in the immune response against Shigella infection. Furthermore, it can be exploited to kill leukaemia cells with acquired venetoclax resistance due to lack of active BAX and BAK. Our findings define tBID as an effector of mitochondrial permeabilization in apoptosis and provide a new paradigm for BCL-2 proteins, with implications for anti-bacterial immunity and cancer therapy.

CstF64-Induced Shortening of the BID 3'UTR Promotes Esophageal Squamous Cell Carcinoma Progression by Disrupting ceRNA Cross-talk with ZFP36L2.

The majority of human genes have multiple polyadenylation sites, which are differentially used through the process of alternative polyadenylation (APA). Dysregulation of APA contributes to numerous diseases, including cancer. However, specific genes subject to APA that impact oncogenesis have not been well characterized, and many cancer APA landscapes remain underexplored. Here, we used dynamic analyses of APA from RNA-seq (DaPars) to define both the 3'UTR APA profile in esophageal squamous cell carcinoma (ESCC) and to identify 3'UTR shortening events that may drive tumor progression. In four distinct squamous cell carcinoma datasets, BID 3'UTRs were recurrently shortened and BID mRNA levels were significantly upregulated. Moreover, system correlation analysis revealed that CstF64 is a candidate upstream regulator of BID 3'UTR length. Mechanistically, a shortened BID 3'UTR promoted proliferation of ESCC cells by disrupting competing endogenous RNA (ceRNA) cross-talk, resulting in downregulation of the tumor suppressor gene ZFP36L2. These in vitro and in vivo results were supported by human patient data whereby 3'UTR shortening of BID and low expression of ZFP36L2 are prognostic factors of survival in ESCC. Collectively, these findings demonstrate that a key ceRNA network is disrupted through APA and promotes ESCC tumor progression.Significance: High-throughput analysis of alternative polyadenylation in esophageal squamous cell carcinoma identifies recurrent shortening of the BID 3'UTR as a driver of disease progression.

Methylation of Promoters of Apoptosis-Related Genes in Blood Lymphocytes of Workers Exposed to Occupational External Irradiation.

We studied the effect of technogenic radiation on the degree of promoter methylation in genes involved in apoptosis in blood lymphocytes of workers exposed to long-term γ-radiation during their professional activities. Blood samples for the analysis were obtained from 11 conventionally healthy men aged from 54 to 71 years (mean 66 years), workers of the Siberian Group of Chemical Enterprises working experience from 27 to 40 years (mean 30 years); the external exposure dose was 175.88 mSv (158.20-207.81 mSv). In all examined subjects, the degree of methylation of the promoters of apoptosis-related genes ranged from 0.22 to 50.00%. A correlation was found between the degree of methylation of BCLAF1 promoters (p=0.035) with the age of workers, BAX promoters (p=0.0289) with high content of aberrant cells, and APAF1 promoters (p=0.0152) with increased number of dicentric chromosomes. A relationship was found between the dose of external irradiation and the degree of methylation of gene promoters of BAD (p=0.0388), BID (р=0.0426), and HRK (р=0.0101) genes.

miR-1283 Contributes to Endoplasmic Reticulum Stress in the Development of Hypertension Through the Activating Transcription Factor-4 (ATF4)/C/EBP-Homologous Protein (CHOP) Signaling Pathway.

BACKGROUND Hypertension-related microRNA(miR)-1283 and its target gene, activating transcription factor-4 (ATF4), can regulate vascular endothelial dysfunction. This study aimed to explore whether miR-1283 prevents hypertension through targeting ATF4. MATERIAL AND METHODS Transcriptome sequencing was performed after overexpression or inhibition of miR-1283 in human amniotic epithelial cells (HAECs). After miR-1283 was overexpressed or inhibited in HAECs, ATF4+/- and wild-type mice were induced with a high-salt diet. We detected the expression of ATF4, C/EBP-homologous protein (CHOP), BH3-interacting domain death agonist (BID), Bcl-2, Bcl-2-like protein 11 (BIM), Bcl-2-like protein 1 (BCL-X), and caspase-3 by PCR and western blotting. We detected the changes of vasoactive substances including nitric oxide (NO), endothelin 1 (ET-1), endothelial protein C receptor (EPCR), thrombin (TM), and von Willebrand factor (vWF) by ELISA. RESULTS Compared with that of the miR-1283- inhibited group, NO was higher in the miR-1283 overexpression group, while the expression of ET-1, EPCR, TM, and vWF were lower. Similarly, compared with that of the miR-1283 inhibited group, the expression of ATF4, CHOP, BID, BIM, and caspase-3 in the miR-1283 overexpression group was downregulated, while the expression of BCL-2 and BCL-X was upregulated (P<0.05). In vivo experiments showed the lack of ATF4 gene could prevent hypertension in mice induced by high-salt diet and protect endothelial function. CONCLUSIONS The mechanism of regulating blood pressure and endothelial function of the miR-1283/ATF4 axis was related to inhibiting endoplasmic reticulum stress and cell apoptosis through the ATF4/CHOP signaling pathway. Therefore, the miR-1283/ATF4 axis may be a target for the prevention and treatment of hypertension.

The effect of bee bread (Perga) with chemotherapy on MDA-MB-231 cells.

Bee bread (BB) is a bee product like propolis and honey. It is the main food for larvae and bees producing royal jelly in the hive. It also known as Perga. As with other bee products, it is increasingly popular due to its antioxidant properties. The aim of this study was to examine the effects of BB on MDA-MB-231 breast cancer cells and the effects on these cells when administered together with Doxorubicin (DOX) and Cisplatin (CDDP), used in cancer treatment. The proliferation of the cells was determined by applying 5 mg/mL BB together with different concentrations of DOX and CDDP. In addition to these studies, the effect of DOX+BB and CDDP+BB combinations on the migration of MDA-MB-231 cells was determined by the wound healing method. The expression levels of Bid and Bcl-2 were determined by RtqPCR. According to these studies, as expected, BB did not show a significant toxic effect on MDA-MB-231 cells at different concentrations. BB significantly suppressed the effect of DOX and CDDP on the proliferation of MDA-MB-231 cells. BB with DOX and CDDP suppressed the proapoptotic Bid gene while overexpressing the anti-apoptotic Bcl-2 gene, separately. Interestingly, BB blocked the migration of MDA-MB-231 cells by 50% even after 72 h. As a result, BB significantly reduced the toxicity of DOX and CDDP on MDA-MB-231 cells. The most interesting result of the study is that BB prevented the migration of cancer cells.

Bid is involved in apoptosis induced by Chinese giant salamander iridovirus and contributes to the viral replication in an amphibian cell line.

Bid is a pro-apoptotic BH3-only member of the Bcl-2 superfamily that functions to link the extrinsic apoptotic pathway and the mitochondrial amplification loop of the intrinsic pathway. In this study, the expression and functions of Chinese giant salamander (Andrias davidianus) Bid (AdBid) were investigated. The AdBid cDNA sequence contains an open reading frame (ORF) of 576 nucleotides, encoding a putative protein of 191 aa. AdBid possesses the conserved BH3 interacting domain and shared 34-52% sequence identities with other amphibian Bid. mRNA expression of AdBid was most abundant in muscle. The expression level of AdBid in Chinese giant salamander muscle, kidney and spleen significantly increased after Chinese giant salamander iridovirus (GSIV) infection. Additionally, a plasmid expressing AdBid was constructed and transfected into the Chinese giant salamander muscle cell line (GSM cells). The morphology and cytopathic effect (CPE) and apoptotic process in AdBid over-expressed GSM cells was significantly enhanced during GSIV infection compared with that in control cells. Moreover, a higher level of the virus major capsid protein (MCP) gene copies and protein synthesis was confirmed in the AdBid over-expressed cells. These results indicated that AdBid played a positive role in GSIV induced apoptosis and the viral replication. This study may contribute to the better understanding on the infection mechanism of iridovirus-induced apoptosis.

Plants expressing murine pro-apoptotic protein Bid do not have enhanced PCD.

OBJECTIVES: The purpose of this study was to explore whether plant programmed cell death (PCD) cascade can sense the presence of the animal-only BH3 protein Bid, a BCL-2 family protein known to play a regulatory role in the signaling cascade of animal apoptosis. RESULTS: We have expressed the mouse pro-apoptotic protein Bid in Arabidopsis thaliana and in Nicotiana tabacum. We did not obtain any transformed plant constitutively expressing the truncated protein (tBid-i.e. the caspase-activated form) whereas ectopic expression of the full-length protein (flBid) does not interfere with growth and development of the transformed plants. To verify whether the presence of this animal pro-apoptotic protein modified stress responses and PCD execution, both N. tabacum and A. thaliana plants constitutively expressing flBid have been studied under different stress conditions triggering cell death activation. The results show that the presence of flBid in transgenic plants did not significantly change the responses to abiotic stress (H2O2 or NO) and biotic stress treatments. Moreover, the finding that no Bid active form was present in treated tobacco plants suggests an absence of a proper activation of Bid.

BCL-2 family deregulation in colorectal cancer: potential for BH3 mimetics in therapy.

Apoptosis is a form of programmed cell death that is essential for tissue homeostasis. De-regulation of the balance between proliferation and apoptosis contributes to tumor initiation. Particularly in the colon where apoptosis is a crucial process in intestinal turnover, inhibition of apoptosis facilitates transformation and tumor progression. The BCL-2 family of proteins are key regulators of apoptosis and have been implicated in colorectal cancer (CRC) initiation, progression and resistance to therapy. In this review we outline the current knowledge on the BCL-2 family-regulated intrinsic apoptosis pathway and mechanisms by which it is de-regulated in CRC. We further review BH3 mimetics as a therapeutic opportunity to target this pathway and evaluate their potential for CRC treatment.

Caspase-1 cleaves Bid to release mitochondrial SMAC and drive secondary necrosis in the absence of GSDMD.

Caspase-1 drives a lytic inflammatory cell death named pyroptosis by cleaving the pore-forming cell death executor gasdermin-D (GSDMD). Gsdmd deficiency, however, only delays cell lysis, indicating that caspase-1 controls alternative cell death pathways. Here, we show that in the absence of GSDMD, caspase-1 activates apoptotic initiator and executioner caspases and triggers a rapid progression into secondary necrosis. GSDMD-independent cell death required direct caspase-1-driven truncation of Bid and generation of caspase-3 p19/p12 by either caspase-8 or caspase-9. tBid-induced mitochondrial outer membrane permeabilization was also required to drive SMAC release and relieve inhibitor of apoptosis protein inhibition of caspase-3, thereby allowing caspase-3 auto-processing to the fully active p17/p12 form. Our data reveal that cell lysis in inflammasome-activated Gsdmd-deficient cells is caused by a synergistic effect of rapid caspase-1-driven activation of initiator caspases-8/-9 and Bid cleavage, resulting in an unusually fast activation of caspase-3 and immediate transition into secondary necrosis. This pathway might be advantageous for the host in counteracting pathogen-induced inhibition of GSDMD but also has implications for the use of GSDMD inhibitors in immune therapies for caspase-1-dependent inflammatory disease.

Truncated Bid Regulates Cisplatin Response via Activation of Mitochondrial Apoptosis Pathway in Ovarian Cancer.

Refractoriness to conventional chemotherapy is a major challenge in the treatment of advanced ovarian cancer (OC). There is increasing evidence that mitochondrial priming correlates with cisplatin response in various cancers. Notably, Bim and Bid, two of the proapoptotic BH3-only proteins, are recognized as the most effective inducers of mitochondrial priming in OC. In this study, we constructed two tumor-specific oncolytic adenoviruses (Ads) coding for Bim (Ad-Bim) or truncated Bid (Ad-tBid), respectively, and performed gain-of-function assays in nine OC cell lines. Ad-tBid exhibited significant antitumor efficacy than the controls. On addition of Ad-tBid pretreatment, mito-primed cells displayed more sensitivity to cisplatin both in vitro and ex vivo. We also found that Ad-tBid induced mitochondrial apoptosis in a Bak-dependent manner. Furthermore, a combined cisplatin plus Ad-tBid therapy markedly inhibited tumor growth in a subcutaneous xenotransplanted tumor model. In mice bearing peritoneal disseminated OC, intraperitoneal administration of Ad-tBid potentiated the antitumor effect of cisplatin. Our findings suggest that Ad-tBid enhances cisplatin response in OC cells, establishing the potential treatment of advanced OC via a combination of cisplatin and Ad-tBid.

Alterations of the interactome of Bcl-2 proteins in breast cancer at the transcriptional, mutational and structural level.

Apoptosis is an essential defensive mechanism against tumorigenesis. Proteins of the B-cell lymphoma-2 (Bcl-2) family regulate programmed cell death by the mitochondrial apoptosis pathway. In response to intracellular stress, the apoptotic balance is governed by interactions of three distinct subgroups of proteins; the activator/sensitizer BH3 (Bcl-2 homology 3)-only proteins, the pro-survival, and the pro-apoptotic executioner proteins. Changes in expression levels, stability, and functional impairment of pro-survival proteins can lead to an imbalance in tissue homeostasis. Their overexpression or hyperactivation can result in oncogenic effects. Pro-survival Bcl-2 family members carry out their function by binding the BH3 short linear motif of pro-apoptotic proteins in a modular way, creating a complex network of protein-protein interactions. Their dysfunction enables cancer cells to evade cell death. The critical role of Bcl-2 proteins in homeostasis and tumorigenesis, coupled with mounting insight in their structural properties, make them therapeutic targets of interest. A better understanding of gene expression, mutational profile, and molecular mechanisms of pro-survival Bcl-2 proteins in different cancer types, could help to clarify their role in cancer development and may guide advancement in drug discovery. Here, we shed light on the pro-survival Bcl-2 proteins in breast cancer using different bioinformatic approaches, linking -omics with structural data. We analyzed the changes in the expression of the Bcl-2 proteins and their BH3-containing interactors in breast cancer samples. We then studied, at the structural level, a selection of interactions, accounting for effects induced by mutations found in the breast cancer samples. We find two complexes between the up-regulated Bcl2A1 and two down-regulated BH3-only candidates (i.e., Hrk and Nr4a1) as targets associated with reduced apoptosis in breast cancer samples for future experimental validation. Furthermore, we predict L99R, M75R as damaging mutations altering protein stability, and Y120C as a possible allosteric mutation from an exposed surface to the BH3-binding site.

Characterization of the Anti-Cancer Activity of the Probiotic Bacterium Lactobacillus fermentum Using 2D vs. 3D Culture in Colorectal Cancer Cells.

The aim of this study was to investigate the potential anti-cancer effects of probiotic cell-free supernatant (CFS) treatment using Lactobacillusfermentum for colorectal cancer (CRC) in 3D culture systems. Cell viability was assessed using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assays, whereas apoptosis was monitored through RT-qPCR analysis of Bax, Bak, Noxa, and Bid mRNA expressions in addition to flow cytometry analysis of Lactobacillus cell-free supernatant (LCFS) treatment. Our results showed that the anti-cancer effect of LCFS on cell viability was pronouncedly enhanced in 3D-cultured HCT-116 cells, which was linked to the increased level of cleaved caspase 3. Additionally, upregulation of apoptotic marker gene mRNA transcription was dramatically increased in 3D cultured cells compared to 2D systems. In conclusion, this study suggests that LCFS enhances the activation of intrinsic apoptosis in HCT-116 cells and the potential anti-cancer effects of Lactobacilli mixtures in 3D culture systems. All in all, our study highlights the benefits of 3D culture models over 2D culture modeling in studying the anti-cancer effects of probiotics.

Liver-specific Bid silencing inhibits APAP-induced cell death in mice.

Acetaminophen (APAP)-induced acute liver failure (ALF) is a life-threatening disease with only a few treatment options available. Though extensive research has been conducted for more than 40 years, the underlying pathomechanisms are not completely understood. Here, we studied as to whether APAP-induced ALF can be prevented in mice by silencing the BH3-interacting domain death agonist (Bid) as a potential key player in APAP pathology. For silencing Bid expression in mice, siRNABid was formulated with the liver-specific siRNA delivery system DBTC and administered 48 h prior to APAP exposure. Mice which were pre-treated with HEPES (vehicleHEPES) and siRNALuci served as siRNA controls. Hepatic pathology was assessed by in vivo fluorescence microscopy, molecular biology, histology and laboratory analysis 6 h after APAP or PBS exposure. Application of siRNABid caused a significant decrease of mRNA and protein expression of Bid in APAP-exposed mice. Off-targets, such as cytochrome P450 2E1 and glutathione, which are known to be consumed under APAP intoxication, were comparably reduced in all APAP-exposed mice, underlining the specificity of Bid silencing. In APAP-exposed mice non-sterile inflammation with leukocyte infiltration and perfusion failure remained almost unaffected by Bid silencing. However, the Bid silencing reduced hepatocellular damage, evident by a remarkable decrease of DNA fragmented cells in APAP-exposed mice. In these mice, the expression of the pro-apoptotic protein Bax, which recently gained importance in the cell death pathway of regulated necrosis, was also significantly reduced, in line with a decrease in both, necrotic liver tissue and plasma transaminase activities. In addition, plasma levels of HMGB1, a marker of sterile inflammation, were significantly diminished. In conclusion, the liver-specific silencing of Bid expression did not protect APAP-exposed mice from microcirculatory dysfunction, but markedly protected the liver from necrotic cell death and in consequence from sterile inflammation. The study contributes to the understanding of the molecular mechanism of the APAP-induced pathogenic pathway by strengthening the importance of Bid and Bid silencing associated effects.

Involvement of Upregulated P53-Induced Death Domain Protein in Retinal Ganglion Cells Apoptosis After Optic Nerve Crush.

PURPOSE: Retinal ganglion cells (RGCs) apoptosis is a common characteristic of optic neuropathies. p53-induced protein with a death domain (PIDD) is a well-known regulator of genotoxic stress-induced apoptosis, which is constitutively cleaved into three main fragments: PIDD-N, PIDD-C and PIDD-CC. Thus, we aim to determine the physiological relevance of PIDD in RGCs apoptosis in an optic nerve crush (ONC) model. METHODS: All animals were evenly randomized into four groups: sham-control group, con-siRNA group, ONC group, and PIDD-siRNA group (ONC +PIDD-siRNA). Expressions of PIDD, caspase-2, Brn3a and tBid in ONC model were analyzed by Western blot and immunofluorescence. Mean densities of RGCs/mm2 were calculated with Fluoro-Gold (FG). Moreover, we tested the effect of PIDD-siRNA on ONC-induced RGCs apoptosis using TUNEL staining. RESULTS: The level of full-length PIDD was weakly present and showed no significant differences at any time points. PIDD-CC and PIDD-C were significantly up-regulated in the retina at 3 days after ONC. Meanwhile, the expression of PIDD was significantly increased in Brn3a (a marker of RGCs) positive cells, indicating that the localization of PIDD appeared to be confined to RGCs. Furthermore, inhibition of PIDD prevented RGCs apoptosis by inhibiting caspase-2 and tBid activation. CONCLUSION: Taken together, PIDD may play a crucial role in RGCs apoptosis after ONC, and this process may be relevant to caspase-2 and tBid.

Prediction of venetoclax activity in precursor B-ALL by functional assessment of apoptosis signaling.

Deregulated cell death pathways contribute to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Intrinsic apoptosis signaling is regulated by different proapoptotic and antiapoptotic molecules: proapoptotic BCL-2 homology domain 3 (BH3) proteins activate prodeath molecules leading to cellular death, while antiapoptotic molecules including B-cell lymphoma 2 (BCL-2) prevent activation of prodeath proteins and counter-regulate apoptosis induction. Inhibition of these antiapoptotic regulators has become a promising strategy for anticancer treatment, but variable anticancer activities in different malignancies indicate the need for upfront identification of responsive patients. Here, we investigated the activity of the BCL-2 inhibitor venetoclax (VEN, ABT-199) in B-cell precursor acute lymphoblastic leukemia and found heterogeneous sensitivities in BCP-ALL cell lines and in a series of patient-derived primografts. To identify parameters of sensitivity and resistance, we evaluated genetic aberrations, gene-expression profiles, expression levels of apoptosis regulators, and functional apoptosis parameters analyzed by mitochondrial profiling using recombinant BH3-like peptides. Importantly, ex vivo VEN sensitivity was most accurately associated with functional BCL-2 dependence detected by BH3 profiling. Modeling clinical application of VEN in a preclinical trial in a set of individual ALL primografts, we identified that leukemia-free survival of VEN treated mice was precisely determined by functional BCL-2 dependence. Moreover, the predictive value of ex vivo measured functional BCL-2 dependence for preclinical in vivo VEN response was confirmed in an independent set of primograft ALL including T- and high risk-ALL. Thus, integrative analysis of the apoptosis signaling indicating mitochondrial addiction to BCL-2 accurately predicts antileukemia activity of VEN, robustly identifies VEN-responsive patients, and provides information for stratification and clinical guidance in future clinical applications of VEN in patients with ALL.