BH3-only Bcl-2 Family Member Research Articles

Involvement of a BH3-only apoptosis sensitizer gene Blm-s in hippocampus-mediated mood control.

Mood disorders are an important public health issue and recent advances in genomic studies have indicated that molecules involved in neurodevelopment are causally related to mood disorders. BLM-s (BCL-2-like molecule, small transcript isoform), a BH3-only proapoptotic BCL-2 family member, mediates apoptosis of postmitotic immature neurons during embryonic cortical development, but its role in the adult brain is unknown. To better understand the physiological role of Blm-s gene in vivo, we generated a Blm-s-knockout (Blm-s−/−) mouse. The Blm-s−/− mice breed normally and exhibit grossly normal development. However, global depletion of Blm-s is highly associated with depression- and anxiety-related behaviors in adult mutant mice with intact learning and memory capacity. Functional magnetic resonance imaging of adult Blm-s−/− mice reveals reduced connectivity mainly in the ventral dentate gyrus (vDG) of the hippocampus with no alteration in the dorsal DG connectivity and in total hippocampal volume. At the cellular level, BLM-s is expressed in DG granule cells (GCs), and Blm-s−/− mice show reduced dendritic complexity and decreased spine density in mature GCs. Electrophysiology study uncovers that mature vGCs in adult Blm-s−/− DG are intrinsically more excitable. Interestingly, certain genetic variants of the human Blm homologue gene (VPS50) are significantly associated with depression traits from publicly resourced UK Biobank data. Taken together, BLM-s is required for the hippocampal mood control function. Loss of BLM-s causes abnormality in the electrophysiology and morphology of GCs and a disrupted vDG neural network, which could underlie Blm-s-null-associated anxiety and depression.

Abstract 3691: Dynamic BH3 profiling identifies novel combinations in malignant pleural mesothelioma with in vivo efficacy

Abstract Currently, malignant pleural mesothelioma (MPM) is mainly treated with conventional therapies such as surgery, chemotherapy and radiation, and patients often suffer from the lack of second line therapy options. In this study, we aimed to identify efficacious drug combinations in primary MPM patient samples and novel MPM patient-derived xenograft (PDX) models to provide better therapeutic options for MPM patients. BH3 profiling is a functional assay that measures mitochondrial priming of the cell. It uses BH3 peptides derived from the BH3 domain of pro-apoptotic BH3-only Bcl-2 family members to provoke a response from viable mitochondria. When tumor cells are treated with drugs prior to BH3 profiling, early changes in their apoptosis signaling can be captured before frank apoptosis occurs. This method is termed dynamic BH3 profiling (DBP), which has been applied in multiple cancer types to identify efficacious drugs. Here, we used high throughput dynamic BH3 profiling (HTDBP) to assess hundreds of drug treatments simultaneously to identify drug combinations that increased apoptotic priming in primary MPM cells ex vivo. Next, we created novel MPM PDX models and tested these drug combinations to determine the ones that primed MPM PDX cells. The HTDBP result from MPM PDX cells recapitulated the chemical vulnerabilities of the primary MPM cells. Moreover, one of the top combination candidates that comprised of navitoclax (BCL-xL, BCL-2 and BCL-w antagonist) and AZD8055 (mTORC1/2 inhibitor) was shown to be safe and efficacious in vivo in our CPDM_011x MPM PDX model. Finally, on the molecular level, we revealed that treatment with navitoclax drove the anti-apoptotic dependence of MPM cell lines towards MCL-1, whose level was downregulated by AZD8055 treatment. In this study, we identified a novel drug combination for MPM with HTDBP that consists of navitoclax and AZD8055. In addition, our results further corroborated that DBP, as a functional assay, can be used to rationally construct novel drug combinations to improve treatment outcomes for cancer patients. Citation Format: Danielle S. Potter, Ruochen Du, Kin-Hoe Chow, Keith L. Ligon, Raphael Bueno, Anthony Letai. Dynamic BH3 profiling identifies novel combinations in malignant pleural mesothelioma with in vivo efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3691.

MAGE-A3 Promotes Chemotherapy Resistance and Proliferation in Multiple Myeloma through Regulation of BIM and p21Cip1

The type I Melanoma Antigen Gene (MAGE) A3 is a promising functional target in multiple myeloma (MM). MAGE-A3 expression in primary MM specimens is associated with proliferation and progression of disease. Previous laboratory studies showed that MAGE-A3 inhibits p53-dependent and independent apoptosis and cell cycle regulation in human myeloma cell lines (HMCL) and primary cells. We investigated the mechanisms of these oncogenic activities in laboratory models and through analysis of gene expression and clinical outcome data. Gene expression profiling (GEP) by RNA sequencing (RNAseq) of p53+/+ HMCL MM.1r and H929 after MAGE-A knockdown identified a set of 201 differentially expressed genes (DEG, p<0.05). Of the top DEGs, eight were BH3-only Bcl-2 family members (BAX, BCL2L11/BIM, BBC3/PUMA, BMF, BCL2L14/BCLG) or apoptotic pathway genes (TNFRSF10B/TRAILR2, CASP3, TRIAP1). Four of the top DEGs were cell cycle regulation genes (TRAF4, CDKN1A/p21CIP1, TRIM5, MAP2K6/MEK6) and three were DNA binding/damage repair genes (APOBEC3B, DDB2, ARID3A). We interrogated protein expression after MAGE knockdown and demonstrated significantly increased levels of pro-apoptotic BIM, but other Bcl-2 proteins were either not altered or not detected. BIM protein stability is regulated by serine phosphorylation, which leads to subsequent ubiquitinylation and degradation. MAGE knockdown resulted in higher levels of ser69- and ser77-phosphorylated BIM, indicating that MAGE does not affect phosphorylation but likely mediates further post-translational modification and degradation. We also detected increased levels of the endogenous cyclin-dependent kinase (CDK) inhibitor p21Cip1, which correlated with the GEP results. Depletion of MAGE-A in HMCL increased the sensitivity to induction of apoptosis by the alkylating agent melphalan but not to proteasome inhibition, suggesting that inhibition of BIM expression may contribute to chemotherapy resistance. To assess the clinical significance of these mechanisms, we analyzed RNAseq data from the iA9 release of the Multiple Myeloma Research Foundation CoMMpass database of more than 650 newly diagnosed MM patients based on high or low MAGEA3expression, which revealed a set of significantly DEGs (p<0.05) that included several MAGE family members and related X-linked genes. Gene set enrichment analysis demonstrated associations with cell cycle and DNA replication pathways, similar to that observed in HMCL. We ranked subjects based on MAGEA3 mRNA expression levels and correlated the highest (219 subjects) and lowest quartiles (212 subjects) with clinical outcome, which showed that the MAGEA3high group had worse overall survival (Hazard Ratio = 2.5, p<0.01, data not shown). This survival difference was even more striking in the subgroup of subjects that had undergone high dose melphalan chemotherapy and autologous stem cell transplantation (ASCT, fig.). MAGEA3 high subjects (39 subs) had worse progression-free (p<0.1) and overall survival (p<0.01) compared to the lowest (37 subs). These results demonstrate that MAGE-A3 negatively regulates BIM at both the transcriptional and post-translational levels, which favor survival and resistance to chemotherapy. MAGE-A3 also inhibits p21Cip1transcription and protein expression, likely through ubiquitinylation and degradation of p53 protein, which promotes passage through the early G1 checkpoint and proliferation. These mechanisms are a biochemical basis for MAGE-A3-mediated resistance to chemotherapy-induced apoptosis and the associations with progression of disease and tumor proliferation. These results provide a rationale to investigate methods to inhibit MAGE-A3 activity in MM. [Display omitted] DisclosuresCho:Janssen: Consultancy; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agenus Inc.: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; J & J: Consultancy; Genentech Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Chari:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; The Binding Site: Consultancy; Bristol Myers Squibb: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Array Biopharma: Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jagannath:Medicom: Speakers Bureau; Merck: Consultancy; Multiple Myeloma Research Foundation: Speakers Bureau; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; Celgene: Consultancy.

Abstract 303: Novel role of nuclear BH3-only protein BNIP3 in regulation of cellular proliferation

Abstract The objective of this study is to investigate the role of Bcl-2 nineteen kilodalton interacting protein 3 (BNIP3) in regulating cellular proliferation and identifying the proliferation pathways mediated by BNIP3. BNIP3 is a BH3-only proapoptotic member of Bcl-2 family of cell death regulating proteins. The function of BNIP3 is dependent on its localization and expression levels. Under normal conditions, low levels of BNIP3 are present in the nucleus of cells. Low oxygen or hypoxic conditions lead to increased expression of BNIP3, and high levels of BNIP3 are found in the cytoplasm. Cytoplasmic BNIP3 is associated with caspase-independent apoptotic cell death through mitochondria. If, however, BNIP3 levels are increased in the nucleus, it promotes survival of cells by transcriptionally inhibiting several apoptosis-inducing factors. High levels of BNIP3 in nucleus are seen in majority of glioblastoma tumors, where hypoxic environment of tumor core causes an increase in expression of BNIP3 but high amounts of BNIP3 localize to the nucleus, possibly aiding in the survival of the tumor cells. In this study, we found that BNIP3 knockout mice brains have increased cellularity compared to wild-type brains. We also found that both mouse primary astrocytes and embryonic fibroblasts (MEFs) lacking BNIP3 expression have an increased capacity for proliferation compared to wild-type MEFs. Overexpressing BNIP3 in the nucleus of HEK 293 cells also resulted in reduced proliferation. In contrast, both astrocytes and MEFs lacking BNIP3 failed to show differences in cell death compared to wild-type cells. This reveals a novel function for BNIP3 in regulating cellular proliferation that may lead to new targets for cancer therapy. In future studies, we will identity genes regulated by nuclear BNIP3 that may lead to suppression of cellular proliferation in cancer cells. Citation Format: Amandeep Singh, Meghan Azad, Spencer Gibson. Novel role of nuclear BH3-only protein BNIP3 in regulation of cellular proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 303.

Abstract 267: Nonsteroidal anti-inflammatory drugs induce ER stress- and BID-dependent immunogenic cell death to suppress colorectal tumorigenesis

Abstract Prevention or early detection represents a key approach for reducing the mortality and morbidity of CRC. Use of non-steroidal anti-inflammatory drugs (NSAIDs) has been associated with decreased risk of CRC in numerous epidemiological studies, clinical trials, and animal model studies. However, the mechanism by which NSAIDs suppress colorectal tumorigenesis has remained unclear. We previously demonstrated that the chemopreventive effect of NSAIDs is mediated by a synthetic lethal interaction between death receptor signaling and loss of the gatekeeper APC tumor suppressor, which is dependent on the BH3-only Bcl-2 family member BID. In this study, we found a critical role of endoplasmic reticulum (ER) stress upstream of death receptor signaling and BID activation. Elevated levels of ER stress and cell death markers were detected in advanced adenomas from patients taking NSAIDs including aspirin. Inhibiting ER stress using a pharmacological approach abolished the apoptotic effect of NSAIDs in CRC cells and normal colonic epithelial cells with APC loss and also suppressed the chemopreventive activity of the NSAID sulindac in APCMin/+ mice. Importantly, our results unveiled a critical role of ER stress and BID-dependent cell death in triggering immune-mediated elimination of nascent cells that lose APC. Markers of immunogenic cell death (ICD), including plasma membrane translocation of calreticulin and phagocytosis by dendritic cells, were suppressed in cells with BID knockout or ER stress inhibition. BID knockout or ER stress inhibition also reduced tumor infiltrating lymphocytes (TILs) in sulindac-treated APCMin/+ mice. Collectively, our results suggest that NSAIDs suppress colorectal tumorigenesis by enhancing immunosurveillance through ER stress and BID-dependent ICD. These results provide novel insight into the chemopreventive mechanism of NSAIDs, which may help design more effective chemopreventive strategies and agents. Citation Format: Rochelle Elayne Fletcher, Brian Leibowitz, Yijun Wang, Fernando Concha-Benavente, Robert Ferris, Robert Schoen, Jian Yu, Lin Zhang. Nonsteroidal anti-inflammatory drugs induce ER stress- and BID-dependent immunogenic cell death to suppress colorectal tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 267.

PUMA amplifies necroptosis signaling by activating cytosolic DNA sensors

Necroptosis, a form of regulated necrotic cell death, is governed by RIP1/RIP3-mediated activation of MLKL. However, the signaling process leading to necroptotic death remains to be elucidated. In this study, we found that PUMA, a proapoptotic BH3-only Bcl-2 family member, is transcriptionally activated in an RIP3/MLKL-dependent manner following induction of necroptosis. The induction of PUMA, which is mediated by autocrine TNF-α and enhanced NF-κB activity, contributes to necroptotic death in RIP3-expressing cells with caspases inhibited. On induction, PUMA promotes the cytosolic release of mitochondrial DNA and activation of the DNA sensors DAI/Zbp1 and STING, leading to enhanced RIP3 and MLKL phosphorylation in a positive feedback loop. Furthermore, deletion of PUMA partially rescues necroptosis-mediated developmental defects in FADD-deficient embryos. Collectively, our results reveal a signal amplification mechanism mediated by PUMA and cytosolic DNA sensors that is involved in TNF-driven necroptotic death in vitro and in vivo.

Bid-deficient fish delay grass carp reovirus (GCRV) replication and attenuate GCRV-triggered apoptosis.

Bid, BH3-interacting domain death agonist, is a pro-apoptotic BH3-only member of Bcl-2 family, playing an important role in apoptosis. In the study, Bid genes from grass carp (Ctenopharyngodon idellus) and rare minnow (Gobiocypris rarus), named CiBid and GrBid, were cloned and analyzed. Bid was constitutively expressed in all examined tissues of grass carp, but the expression level varied in different tissues. Following grass carp reovirus (GCRV) stimulation in vivo, Bid and apoptosis related genes Caspase-9 and Caspase-3 was up-regulated significantly at the late stage of infection. Moreover, we generated a Bid-deficient rare minnow (Bid-/-) to investigate the possible role of Bid in GCRV-triggered apoptosis. We found that the survival time of Bid-/- rare minnow after GCRV infection was extended when compared with wild-type fish, the relative copy number of GCRV in Bid-/- rare minnow was lower than that in wild-type fish, and the expression level of Caspase-9 and Caspase-3 in Bid-/- rare minnow were significantly lower than that in the wild-type fish. Collectively, the current data revealed the important role of Bid during virus-induced apoptosis in teleost fish. Our study would provide new insight into understanding the GCRV induced apoptosis and may provide a target gene for virus-resistant breeding in grass carp.

Abstract 602: High-Density Lipoprotein Protects Macrophages Against Apoptosis Through BH3-only Bcl-2 Family Member Bim

Macrophage apoptosis contributes to the formation of necrotic cores within atherosclerotic lesions, increasing the susceptibility of atherosclerotic plaques to rupture and the risk of cardiovascular events. In advanced atherosclerotic plaques, lipid-laden foam cells are exposed to a diverse number of apoptosis inducers, including prolonged endoplasmic reticulum (ER) stress. The pro-apoptotic BH3-only protein Bim has been reported to mediate apoptosis in response to ER stress induction in a variety of cells including macrophages. Treatment of macrophages with high-density lipoprotein (HDL) protects them against apoptosis induced by a variety of stressors. We hypothesize that HDL mediated protection against macrophage apoptosis occurs via pathways that regulate Bim activity. To test this, thioglycollate-elicted peritoneal macrophages were treated in lipoprotein deficient culture with different apoptosis inducers in the absence or presence of HDL. Cell apoptosis was assessed by cleaved caspase 3 staining and terminal deoxynucleotidal transferase dUTP nick end labeling. Treatment of mouse peritoneal macrophages in culture with 50μg/ml of human HDL protected against apoptosis induced by tunicamycin (P&lt;0.05) without affecting ER stress markers, Grp78, Grp94 and CHOP. Peritoneal macrophages from mice deficient in Bim were less susceptible to apoptosis induced by treatment with either tunicamycin or 7-ketocholesterol. Furthermore, treatment with HDL provided no additional protection to apoptosis in Bim KO macrophages. Our results suggest that protection of macrophage apoptosis by HDL is regulated through pathways involving the activity of BH3-only Bcl-2 family member Bim.

DR5 and caspase-8 are dispensable in ER stress-induced apoptosis.

The endoplasmic reticulum (ER) stress response constitutes cellular reactions triggered by a wide variety of stimuli that disturb folding of proteins, often leading to apoptosis. ER stress-induced apoptotic cell death is thought to be an important contributor to many human pathological conditions. The molecular mechanism of this apoptosis process has been highly controversial with both the receptor and the mitochondrial pathways being implicated. Using knockout mouse models and RNAi-mediated gene silencing in cell lines, our group and others had demonstrated the importance of the mitochondrial apoptotic pathway in ER stress-induced cell death, particularly the role of the pro-apoptotic BH3-only BCL-2 family members, BIM and PUMA. However, a recent report suggested a central role for the death receptor, DR5, activated in a ligand-independent manner, and the initiator caspase, caspase-8, in ER stress-induced cell death. This prompted us to re-visit our previous observations and attempt to reproduce the newly published findings. Here we report that the mitochondrial apoptotic pathway, activated by BH3-only proteins, is essential for ER stress-induced cell death and that, in contrast to the previous report, DR5 as well as caspase-8 are not required for this process.

PI3Kδ Inhibition Elicits Anti-Leukemic Effects through Bim-Dependent Apoptosis

Introduction: Phosphatidylinositol-3-kinase δ (PI3Kδ)-signaling provides key maintenance, proliferation, and survival cues during both normal and malignant B-lymphocyte development. Consequently, isoform-selective PI3Kδ inhibitors (PI3Kδi) have generated huge interest as a potential treatment for lymphoid malignancies. In particular, PI3Kδi demonstrate impressive clinical efficacy in combination with anti-CD20 monoclonal antibodies (mAbs) for relapsed chronic lymphocytic leukemia (CLL). However, these combinations function primarily to delay disease progression, but are not curative. With the ever-rising number of new targeted therapeutics, the challenge is to identify combinations that will ultimately deliver curative regimes. In order to guide these selections, a detailed mechanistic understanding is required. To date, only limited data are available regarding the exact in vivo therapeutic mechanism of PI3Kδi. Currently, studies identify immunomodulation, inhibition of BCR-, chemokine/cytokine-signaling, and induction of apoptosis as putative therapeutic mechanisms. Here we characterize the molecular mechanisms responsible for PI3Kδi-induced cytotoxicity and determine the relative contribution toward in vivo therapeutic responses utilizing the Eµ-TCL1-Tg mouse model of CLL alongside human CLL samples.Methods: The molecular mechanisms of PI3Kδi alone or in combination with anti-CD20 mAbs were assessed using the Eµ-TCL1-Tg mouse model, an in vivo model system of CLL. To inhibit PI3Kδ, the δ isoform-selective inhibitor GS-9820 was chosen, as it is highly structurally related to idelalisib, and critically demonstrates improved pharmacokinetic properties in the mouse in comparison to idelalisib. In vitro GS-9820 IC50 are as follows: PI3Kδ 27 nM; PI3Kα 83,424 nM; PI3Kβ 14,899 nM; and PI3Kγ 15,606 nM. Assays to measure its effects on BCR-mediated kinase activation, chemokine signaling/chemotaxis, and cytokine- and cell-mediated support were performed. GS-9820 was administered in vivo at 10 mg/kg per os BID (formulated in 0.5% methylcellulose, 0.05% tween-80), once leukemias were detected, and maintained throughout the treatment period (GS-9820 Cmax 3114 nM, Ctrough 48.6 nM).Results: GS-9820 induced substantive in vitro cell death and disruptedBCR-mediated kinase activation, chemokine signaling/chemotaxis, and inhibited both cytokine- and cell-mediated support in murine (Eµ-TCL1) and human CLL cells. In vivo administration of GS-9820 imparted significant therapeutic responses in Eµ-TCL1-bearing animals, reducing leukemic burden by 75% and splenic tumor deposits by 66% 4 weeks posttreatment. GS-9820 appeared well tolerated in recipient animals with no obvious toxicity apparent (e.g. weight loss or behavioral symptoms). When in combination with anti-CD20 mAbs, GS-9820 extended leukemia depletion by several weeks. GS-9820 enhanced overall survival by 66% in comparison with vehicle control-recipient animals and enhanced the survival benefit of anti-CD20 mAb therapy. These therapeutic responses were associated with a 2-fold increase in expression of the pro-apoptotic BH3-only Bcl-2 family member Bim and a 3-fold increase in the extent of Bim/Bcl-2 interaction. Accordingly, Bim-/- Eµ-TCL1-Tg leukaemias exhibited profound resistance to PI3Kδi-induced cytotoxicity, were refractory to PI3Kδi in vivo, and failed to display combination efficacy with anti-CD20 mAbs. These findings informed the rational design of a GS-9820 + ABT199 (Venetoclax) complementary drug combination strategy. Combinations of GS-9820 and ABT199 were well tolerated with an absence of weight loss or altered behavioral symptoms. The GS-9820 + ABT199 combination effectively halted leukemia progression in vivo with increased efficacy compared to monotherapy regimes, resulting in a 90% reduction in leukemic burden at the end of the treatment period.Conclusions: Bim-dependent apoptosis represents the key in vivo effector mechanism for PI3Kδi in the Eµ-TCL1-Tg mouse model, both alone and in combination with anti-CD20 mAbs. As such, combinations of PI3Kδ and Bcl-2 inhibitors may represent an efficacious drug combination strategy. DisclosuresTannheimer:Gilead Sciences: Employment. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding. Cragg:Baxalta: Consultancy; Roche: Consultancy, Research Funding; Bioinvent International: Consultancy, Research Funding; Gilead Sciences: Research Funding; GSK: Research Funding.

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

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

Abstract 971: Pro-apoptotic Bid preserves hematopoietic stem cell function through restraint of necrosis: implications for myelodysplastic syndrome

Abstract Multicellular organisms remove damaged or superfluous cells through a highly regulated cellular process known as programmed cell death. There are two main forms of programmed cell death, apoptosis and necrosis. In apoptotic cell death, caspases digest the cell. In necroptotic cell death, increased Rip kinase signaling effects plasma membrane rupture, and activation of an inflammatory response. Death receptors, such as the TNF receptor, can activate either apoptotic or necroptotic death. The upstream activators and transducers including Caspase-8, Rip1, and Fadd, are common to both forms of cell death. Interestingly, Caspase-8 and c-FlipL, were recently shown to inhibit the necrotic pathway through the formation of a catalytically active complex. The BH3-only Bcl-2 family member, Bid is a substrate of Caspase-8, and is thus in position to mediate cell death fate. We developed a mouse model of unrestrained bone marrow necroptosis in which apoptosis is prevented by the deletion of the pro-apoptotic effectors Bax and Bak. We further deleted the upstream activator Bid (VavBaxBakBid TKO mice). These mice die of bone marrow failure with myeloid dysplasia between 3 and 12 months, and some develop leukemia, closely resembling MDS. TKO bone marrow displays necroptotic cells by electron microscopy, and markedly increased Rip1 expression by immunofluorescence. TKO SLAM-HSCs display increased BrdU incorporation, consistent with compensatory HSC proliferation in response to bone marrow cell death. TKO bone marrow outcompetes WT bone marrow in competitive reconstitution experiments, but the mice succumb to bone marrow failure at 5 months. Thus, increased necroptosis results in a cell autonomous stem cell defect. In addition, necroptotic bone marrow kills normal HSCs by feed-forward TNF signaling. Myeloid progenitor cell lines (MPCs) derived from the bone marrow of BaxBakBid TKO mice, display increased activation (phosphorylation) and increased levels of Rip1 in the pronecrotic complex (Complex II) following LPS treatment. This association is abrogated by reintroduction of Bid by retrovirus into TKO MPCs, demonstrating that Bid inhibits Rip1 association with complex II, to determine cell death fate. Finally, we found increased RIP1 expression, but not activated caspase 3 in bone marrow samples from patients with the RCMD, RAEB-1, and RAEB-2 subtypes of MDS. We thus demonstrate that increased necroptosis signaling can result in bone marrow failure with dysplasia, and that necroptotic cell death signaling is increased in bone marrow from MDS patients, highlighting the potential importance of this targetable signaling pathway in bone marrow failure disorders such as MDS. Citation Format: Sandra S. Zinkel. Pro-apoptotic Bid preserves hematopoietic stem cell function through restraint of necrosis: implications for myelodysplastic syndrome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 971. doi:10.1158/1538-7445.AM2015-971

Hepatocyte-specific Bid depletion reduces tumor development by suppressing inflammation-related compensatory proliferation.

Liver cancer is a major health-care concern and its oncogenic mechanisms are still largely unclear. Persistent hepatocyte cell death is a common feature among various chronic liver diseases, the blocking of which presents as logical treatment. Therefore, we aimed at investigating tumor development in mice with hepatocyte-specific Bid depletion--a BH3-only Bcl-2 family member that amplifies apoptotic death signals. Hepatocyte-specific conditional Bid-knockout mice (Bid(Δhep)) were injected with 25 mg/kg diethylnitrosamine (DEN) at 14 days of age, and liver tumorigenesis was investigated 9 months later. Additionally, different models of acute liver injury were used including: acute high-dose DEN challenge, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet and carbon tetrachloride (CCL4) injection. Bid(Δhep) mice developed significantly fewer tumors, showed smaller maximal and average tumor size and reduced tumor incidence. In the acute DEN model, 48 h post injection we observed a significant reduction in liver injury in Bid(Δhep) animals, assessed via serum transaminases and liver histopathology. Furthermore, TNF-α, IL-1ß, cJUN and IL-6 mRNA expression was reduced. These findings were accompanied by reduced compensatory hepatocyte proliferation in Bid(Δhep) mice when compared with controls by immunohistochemistry for Ki67 and proliferating cell nuclear antigen 48 h after DEN injection. In the acute CCL4 model, Bid(Δhep) mice displayed reductions in liver injury and inflammation when compared with controls. No differences in liver injury and serum bilirubin levels were detected in Bid(Δhep) and Bid(flo/flo) mice fed with DDC, which induces bile duct injury and a ductular reaction. Our study demonstrates that in DEN-induced hepatocellular carcinoma, the inhibition of hepatocyte death pathways through Bid deletion protects animals from tumorigenesis. These results suggest that reducing hepatocyte cell death, liver inflammation and compensatory proliferation has a stronger beneficial effect than the potential side effect of enhancing tumor cell survival.

Inhibition of AKT/FoxO3a signaling induced PUMA expression in response to p53-independent cytotoxic effects of H1: A derivative of tetrandrine

PUMA (p53 unregulated modulator of apoptosis), a BH3-only Bcl-2 family member, can be induced by p53-dependent and p53-independent manners. It plays an important role as regulator of cellular apoptosis. Herein, we evaluate the effects of H1 (a derivative of tetrandrine) on induction of PUMA and underlie its potential mechanism in p53-independent cytotoxic response. Anti-proliferative activity and evidently cytotoxic activity of H1 were observed in wild-type and p53 null cells. Further studies demonstrated that H1 resulted in an increase of cleaved PARP, decease of survivin and elevation of p-H2AX. What is more, H1 significantly induced PUMA expression in a concentration- and time-dependent manner and caused an increase of Bax/Bcl-2 ratio in p53 null cells. Of note, knockdown of PUMA attenuated cytotoxic activity of H1. Further studies demonstrated that inhibition of AKT/FoxO3a signaling contributed to H1-mediated PUMA induction. Targeted suppression of AKT/FoxO3a signaling by siRNA could overcome H1-mediated PUMA induction. In addition, H1 significantly suppressed NF-κB activity and caused an increase of early apoptotic and late apoptotic cells, and elevated caspase-3 activity. Taken together, we found that inhibition of AKT/FoxO3a signaling may contribute to H1-mediated PUMA induction, suggesting that inhibition of AKT/FoxO3a signaling result in PUMA expression in response to p53-independent cytotoxic effects of H1.

Proapoptotic Bid Preserves HSC Function through Restraint of Necroptosis

Multicellular organisms remove damaged or superfluous cells through a highly regulated cellular process known as programmed cell death. There are two main forms of programmed cell death, apoptosis and necrosis. Necrosis (necroptosis) previously thought to be an unregulated death pathway was recently found to be highly regulated. The manner by which a cell dies has important implications. In apoptotic cell death, caspases digest the cell to cause implosion in an immunologically silent process. In necroptotic cell death, increased Rip kinase signaling effects rupture of the plasma membrane, cellular explosion, and the activation of an inflammatory response.Death receptors, such as the TNFα receptor, can activate either apoptotic or necroptotic death. The upstream activators and transducers including Caspase-8, Rip1, and Fadd, are common to both forms of cell death. Interestingly, Caspase-8 and c-FlipL, a caspase homolog, were recently shown to inhibit the necrotic pathway during embryonic development through the formation of a catalytically active complex. The BH3-only Bcl-2 family member, Bid is one of the strongest substrates of Caspase-8, placing it at the interface of the apoptotic and necroptotic pathways, and in position to mediate cell death fate.The role of apoptosis in hematopoietic homeostasis has been well characterized. We developed a mouse model of unrestrained necroptosis in order to determine how unrestrained necroptosis impacts hematopoietic homeostasis and bone marrow function. To do this we generated a mouse model in which apoptosis is prevented by the deletion of the pro-apoptotic effectors Bax and Bak. We further deleted the upstream activator Bid (VavBaxBakBid TKO mice). Surprisingly, these mice die of bone marrow failure due to unrestrained necroptotic cell death. TKO bone marrow displays necroptotic cells by electron microscopy, and markedly increased Rip1 expression by immunofluorescence. TKO mice die of bone marrow failure with marked myeloid dysplasia between the age of 3 and 12 months, and a small number develop leukemia, a phenotype that closely resembles MDS. Further analysis revealed expansion and increased BrdU incorporation of the SLAM-HSC population, consistent with increased HSC proliferation in response to death of more mature cells. To assess function of these HSCs, we performed competitive reconstitution assays. TKO bone marrow initially outcompetes WT bone marrow, but the mice eventually succumb to bone marrow failure beginning at 5 months post–transplantation, despite the presence of ~10-15% wild type bone marrow. These results demonstrate that increased necroptotic signaling results in a cell autonomous stem cell defect. In addition, the presence of necroptotic bone marrow also kills normal HSCs in a non cell-autonomous manner, due to a feed-forward inflammatory process.To further characterize how necroptotic cell death is regulated, we developed myeloid progenitor cell lines (MPCs) from the bone marrow of WT, Bid KO, BaxBak DKO, and BaxBakBid TKO mice to facilitate biochemical and mechanistic studies. Our studies demonstrated increased activation (phosphorylation) and markedly increased levels of Rip1 in the pronecrotic complex (Complex II) with Rip3, Caspase-8, and Fadd in our TKO MPCs following LPS treatment. This association of Rip1 with Complex II is abrogated by reintroduction of Bid by retrovirus into TKO MPCs, demonstrating that Bid inhibits Rip1 association with complex II, suggesting that Bid is a key factor that determines cell death fate.Increased bone marrow cell death is well documented in MDS. To determine if necroptosis plays a role in this bone marrow cell death, we evaluated RIP1 and Caspase 3 expression in 17 human MDS samples. Remarkably, we found increased RIP1 expression, but not activated caspase 3 in bone marrow samples from patients with the RCMD, RAEB-1, and RAEB-2 subtype of MDS, but not in 4 control bone marrow samples (normal lymphoma staging marrows). Our study thus demonstrates that increased necroptosis signaling can result in bone marrow failure with dysplasia, and that necroptotic cell death signaling is increased in bone marrow from MDS patients, highlighting the potential importance of this targetable signaling pathway in bone marrow failure disorders such as MDS. DisclosuresNo relevant conflicts of interest to declare.

Assessing the Mechanism of Cytarabine-Induced Killing in Acute Leukemia

The nucleoside analogue cytarabine is part of standard therapy for acute myelogenous leukemia (AML) and is also widely used in various acute lymphocytic leukemia (ALL) regimens. During a 7-day infusion at 100 mg/m2/d, which is part of the “7 + 3” AML induction regimen, steady-state cytarabine concentrations in the 100-200 nM range are achieved. Previous literature suggesting the mechanism of cytarabine-induced killing in AML examined cytarabine concentrations in the micromolar range, which might be less pertinent to routinely achieved drug exposure. To better understand the mechanism of cytarabine-induced killing, AML and ALL cell lines were exposed to varying cytarabine concentrations (25-200 nM) for 24-48 hours and assayed for cell death using a variety of assays. Dose- and time-dependent induction of apoptosis was observed in both AML and ALL cell lines, as detected by phosphatidylserine externalization (Annexin V staining), DNA fragmentation (propidium iodide staining for subdiploid cells), nuclear morphological changes, and cleavage of caspase substrates. Jurkat T cell ALL variants lacking critical components of the death receptor pathway (FADD or procaspase 8) underwent apoptosis with kinetics indistinguishable from parental cells, arguing against involvement of the death receptor pathway. Immunoblotting for Bcl-2 family members in AML and ALL cell lines revealed upregulation of the BH3-only proteins Bim, Puma and Noxa across multiple lines. In further experiments, small interfering RNA (siRNA)-mediated knockdown of Bim, Puma, and Noxa or the mitochondrial proteins Bax and Bak markedly diminished the apoptotic response to cytarabine. These results suggest that cytarabine at nanomolar concentrations kills cells through the mitochondrial apoptotic pathway but, in contrast to more targeted agents, does so by upregulating multiple BH3-only Bcl-2 family members simultaneously. Additional studies to determine the mechanisms of Bim, Puma, and Noxa upregulation are underway. (Supported in part by R01 CA166741 and predoctoral fellowships from the Mayo Foundation for Education and Research.) DisclosuresNo relevant conflicts of interest to declare.

Quantitative Measurement of BIM Protein in Lung Cancer: Diagnosis/Staging

A proportion of EGFR-mutant and ALK-rearranged lung cancers do not respond to tyrosine kinase inhibition for as-yet unknown reasons. The BH3-only Bcl-2 family member BIM has been implicated as a potential biomarker of clinical benefit from kinase inhibitors. Quantifying BIM expression in tumor samples of patients with lung cancer may help to predict outcome, and potentially to guide therapy.

Impact of conditional deletion of the pro-apoptotic BCL-2 family member BIM in mice.

The pro-apoptotic BH3-only BCL-2 family member BIM is a critical determinant of hematopoietic cell development and homeostasis. It has been argued that the striking hematopoietic abnormalities of BIM-deficient mice (accumulation of lymphocytes and granulocytes) may be the result of the loss of the protein throughout the whole animal rather than a consequence intrinsic to the loss of BIM in hematopoietic cells. To address this issue and allow the deletion of BIM in specific cell types in future studies, we have developed a mouse strain with a conditional Bim allele as well as a new Cre transgenic strain, Vav-CreER, in which the tamoxifen-inducible CreER recombinase (fusion protein) is predominantly expressed in the hematopoietic system. We show that acute loss of BIM in the adult mouse rapidly results in the hematopoietic phenotypes previously observed in mice lacking BIM in all tissues. This includes changes in thymocyte subpopulations, increased white blood cell counts and resistance of lymphocytes to BIM-dependent apoptotic stimuli, such as cytokine deprivation. We have validated this novel conditional Bim knockout mouse model using established and newly developed CreER strains (Rosa26-CreER and Vav-CreER) and will make these exciting new tools for studies on cell death and cancer available.

Overexpression of CREB protein protects from tunicamycin-induced apoptosis in various rat cell types.

Endoplasmic reticulum (ER) stress plays an essential role in unfolded protein response induced apoptosis contributing to several pathological conditions. Glycogen synthase kinase-3β (GSK-3β) plays a central role in several apoptotic signaling, including ER stress, as the active form of GSK-3β induces apoptosis. The phosphorylation of cAMP responsive element (CRE) binding protein (CREB) Ser-133 (S133) residue is the end-point of various signaling pathways, like growth factor signaling, while the Ser-129 (S129) residue is phosphorylated by GSK-3β. The significance of the ubiquitously expressed transcription factor CREB is demonstrated in prolonged, tunicamycin (TM)-induced ER stress in this study. In the experiments wild-type (wt) CREB, S129Ala, S133Ala or S129Ala-S133Ala mutant CREB expressing PC12 rat pheochromocytoma cell lines showed increased survival under TM-evoked prolonged ER stress compared to wtPC12 cells. After TM treatment ER stress was activated in all PC12 cell types. Lithium and SB-216763, the selective, well-known inhibitors of GSK-3β, decreased TM-induced apoptosis and promoted cell survival. The proapoptotic BH3-only Bcl-2 family member Bcl-2-interacting mediator of cell death (Bim) level was decreased in the different CREB overexpressing PC12 cells as a result of TM treatment. CREB overexpression also inhibited the sequestration of Bim protein from tubulin molecules, as it was demonstrated in wtPC12 cells. Transient expression of wtCREB diminished TM-induced apoptosis in wtPC12, Rat-1 and primary rat vascular smooth muscle cells. These findings demonstrate a novel role of CREB in different cell types as a potent protector against ER stress.

Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival

Bcl-2 family proteins act as essential regulators and mediators of intrinsic apoptosis. Several lines of evidence suggest that the anti-apoptotic members of the family, including Bcl-2, Bcl-xL and Mcl-1, exhibit functional redundancy. However, the current evidence is largely indirect, and based mainly on pharmacological data using small-molecule inhibitors. In order to study compensation and redundancy of anti-apoptotic Bcl-2 proteins at the molecular level, we used a combined knockdown/overexpression strategy to essentially replace the function of one member with another. The results show that HeLa cells are strictly dependent on Mcl-1 for survival and correspondingly refractory to the Bcl-2/Bcl-xL inhibitor ABT-263, and remain resistant to ABT-263 in the context of Bcl-xL overexpression because endogenous Mcl-1 continues to provide the primary guardian role. However, if Mcl-1 is knocked down in the context of Bcl-xL overexpression, the cells become Bcl-xL-dependent and sensitive to ABT-263. We also show that Bcl-xL compensates for loss of Mcl-1 by sequestration of two key pro-apoptotic Bcl-2 family members, Bak and Bim, normally bound to Mcl-1, and that Bim is essential for cell death induced by Mcl-1 knockdown. To our knowledge, this is the first example where cell death induced by loss of Mcl-1 was rescued by the silencing of a single BH3-only Bcl-2 family member. In colon carcinoma cell lines, Bcl-xL and Mcl-1 also play compensatory roles, and Mcl-1 knockdown sensitizes cells to ABT-263. The results, obtained employing a novel strategy of combining knockdown and overexpression, provide unique molecular insight into the mechanisms of compensation by pro-survival Bcl-2 family proteins.