Function Of Bcl-xL Research Articles

Bcl-xL is translocated to the nucleus via CtBP2 to epigenetically promote metastasis

Nuclear Bcl-xL is found to promote cancer metastasis independently of its mitochondria-based anti-apoptotic activity. How Bcl-xL is translocated into the nucleus and how nuclear Bcl-xL regulates histone H3 trimethyl Lys4 (H3K4me3) modification have yet to be understood. Here, we report that C-terminal Binding Protein 2 (CtBP2) binds to Bcl-xL via its N-terminus and translocates Bcl-xL into the nucleus. Knockdown of CtBP2 by shRNA decreases the nuclear portion of Bcl-xL and reverses Bcl-xL-induced invasion and metastasis in mouse models. Furthermore, knockout of CtBP2 not only reduces the nuclear portion of Bcl-xL but also suppresses Bcl-xL transcription. The binding between Bcl-xL and CtBP2 is required for their interaction with MLL1, a histone H3K4 methyltransferase. Pharmacologic inhibition of the MLL1 enzymatic activity reverses Bcl-xL-induced H3K4me3 and TGFβ mRNA upregulation, as well as invasion. Moreover, the cleavage under targets and release using nuclease (CUT&RUN) assay coupled with next-generation sequencing reveals that H3K4me3 modifications are particularly enriched in the promotor regions of genes encoding TGFβ and its signaling pathway members in cancer cells overexpressing Bcl-xL. Altogether, the metastatic function of Bcl-xL is mediated by its interaction with CtBP2 and MLL1 and this study offers new therapeutic strategies to treat Bcl-xL-overexpressing cancer.

The endoplasmic reticulum pool of Bcl-xL prevents cell death through IP3R-dependent calcium release

Apoptosis plays a role in cell homeostasis in both normal development and disease. Bcl-xL, a member of the Bcl-2 family of proteins, regulates the intrinsic mitochondrial pathway of apoptosis. It is overexpressed in several cancers. Bcl-xL has a dual subcellular localisation and is found at the mitochondria as well as the endoplasmic reticulum (ER). However, the biological significance of its ER localisation is unclear. In order to decipher the functional contributions of the mitochondrial and reticular pools of Bcl-xL, we generated genetically modified mice expressing exclusively Bcl-xL at the ER, referred to as ER-xL, or the mitochondria, referred to as Mt-xL. By performing cell death assays, we demonstrated that ER-xL MEFs show increased vulnerability to apoptotic stimuli but are more resistant to ER stress. Furthermore, ER-xL MEFs displayed reduced 1,4,5-inositol trisphosphate receptor (IP3R)-mediated ER calcium release downstream of Phospholipase C activation. Collectively, our data indicate that upon ER stress, Bcl-xL negatively regulates IP3R-mediated calcium flux from the ER, which prevents ER calcium depletion and maintains the UPR and subsequent cell death in check. This work reveals a moonlighting function of Bcl-xL at the level of the ER, in addition to its well-known role in regulating apoptosis through the mitochondria.

Bclxl Prevents Progressive Exhaustion in BCMA CAR T Cells with Maintenance of High TCF1 Expressing T Cells

Although BCMA targeted CAR T cell therapies are highly effective in the treatment of multiple myeloma (MM), patients with high disease burden have not fully benefited from this therapy with shorter disease remissions despite initial responses. It is postulated that high disease burden results in chronic antigenic stimulation of CAR T cells with the induction of exhaustion-associated gene signatures and CAR T cells contraction, limiting their in vivo potency. We have previously reported that BCL2L1 (gene encoding for BCLxL) armoring of BCMA CAR T cells enhanced CAR T cells functional fitness and improved proliferation and cytotoxicity after chronic antigenic stimulation in vitro, as well as superior tumor clearance in xenograft models of MM. In the current study we aimed to define the molecular mechanisms that mediates BCL2L1-armored CAR T cell and examine the effect of BCLxL variants on CAR T cells fitness. In in vivo studies, NSG mice were systemically injected with OPM2 cells (stably transduced with firefly luciferase) and led to develop large MM disease burden, treatment with BCLxL armored (BCMA_BCL2L1) vs unarmored CARs (BCMA_CAR) or control T cells completely eliminated the MM disease and significantly improved animal survival. In animals treated with armored CARs (BCMA_BCL2L1) no disease recurrence was noted 180 days post treatment (in contrast to unarmored CARs where disease recurrence was noted within 60 days). BCLxL also improved CARs persistence, with armored CAR T cells being detectable in the marrow of these mice 7 and 14 days post treatment. Of note, BCLxL armored CAR T cells did not demonstrate uncontrolled proliferation as they were no longer detectable on day 120 post-infusion. Furthermore, CITEseq profiling on harvested human T cells 7 days post CAR T cells infusion to diseased mice revealed a differential T cell repertoires and phenotypes between BCMA_CAR and BCMA_BCL2L1 CARs. While unarmored CARs were significantly enriched for exhausted T cells (CD8_Tex), BCMA_BCL2L1 CAR T were enriched with early activated non-exhausted CD8 T cells, precursor exhausted T cells (CD8_Tpex) and naïve CD4 and CD8 T cells. Importantly, single cell genes set enrichment analysis (ssGSEA) revealed a differential metabolic signature with higher oxidative phosphorylation in BCMA_BCL2L1 CAR T cells compared to unarmored CARs. These findings suggested that armoring CAR T cells with BCL2L1 maintain CAR T cells in an activated and precursor exhausted T cell states with improved mitochondrial and metabolic fitness, preventing terminal T cells exhaustion. To further evaluate the impact of BCLxL in suppressing BCMA CAR T cells' exhaustion, we engineered BCMA CAR T cells with armored expression of two BCLxL mutants (BCLxL Δ26-83 or BCLxL Δ46-83) harboring large or small deletions involving a BCLxL regulatory loop. These mutants were previously reported to enhance BCLxL function (Chang et al The EMBO Journal 1997,16:968-977). Under chronic antigenic stimulation (CAS) or hypoxic conditions, wild type or mutants BClxL overexpression in BCMA CAR T cells resulted in higher proliferation rates compared to unarmored CARs. Compared to wild type BClxL, BCLxL Δ26-83 and BCLxL Δ46-83 mutants demonstrated higher proliferation capacity post CARs or under hypoxic conditions. Intriguingly, majority of BCLxL-overexpressing BCMA CAR-T cells, particularly BCMA CAR-T BCLxL_Δ26-83 cells, maintained a central memory (T CM) phenotype following a long co-culture with MM tumor cells with lower levels of exhaustion markers. Importantly BCLxL-overexpressing BCMA CAR-T cells had substantially higher level of TCF1 with lower TOX/TOX2 expression compared to unarmored BCMA CAR T cells. TCF1 high population was also enriched in unstimulated BCLxL-overexpressing BCMA CAR T cells suggesting a positive regulatory impact of BCL2L1 on TCF1 expression, either through direct or indirect pathways. Expectedly, TCF1 high BCMA CAR-T cells overexpressing a BCLxL were mainly associated with the naive/T CM phenotype. In summary, we have identified a role for BCLxL and two mutant variants lacking a loop regulatory domain (BCLxL Δ26-83 or BCLxL Δ46-83), in maintenance of BCMA CAR T cells in central memory and precursor exhausted states with retained high TCF1 expression, even under hypoxic and chronic antigenic stimulation conditions. These studies further support the clinical development of a BCL2L1 armored CAR T cells for the treatment of MM.

Ser14 phosphorylation of Bcl-xL mediates compensatory cardiac hypertrophy in male mice

The anti-apoptotic function of Bcl-xL in the heart during ischemia/reperfusion is diminished by K-Ras-Mst1-mediated phosphorylation of Ser14, which allows dissociation of Bcl-xL from Bax and promotes cardiomyocyte death. Here we show that Ser14 phosphorylation of Bcl-xL is also promoted by hemodynamic stress in the heart, through the H-Ras-ERK pathway. Bcl-xL Ser14 phosphorylation-resistant knock-in male mice develop less cardiac hypertrophy and exhibit contractile dysfunction and increased mortality during acute pressure overload. Bcl-xL Ser14 phosphorylation enhances the Ca2+ transient by blocking the inhibitory interaction between Bcl-xL and IP3Rs, thereby promoting Ca2+ release and activation of the calcineurin-NFAT pathway, a Ca2+-dependent mechanism that promotes cardiac hypertrophy. These results suggest that phosphorylation of Bcl-xL at Ser14 in response to acute pressure overload plays an essential role in mediating compensatory hypertrophy by inducing the release of Bcl-xL from IP3Rs, alleviating the negative constraint of Bcl-xL upon the IP3R-NFAT pathway.

Exploring association of melanoma-specific Bcl-xL with tumor immune microenvironment.

Macrophages take center stage in the tumor microenvironment, a niche composed of extracellular matrix and a heterogeneous group of cells, including immune ones. They can evolve during tumor progression and acquire Tumor-Associated Macrophage (TAMs) phenotype. The release of cytokines by tumor and stromal cells, influence the secretion of cytokines by TAMs, which can guarantee tumor progression and influence the response to therapy. Among all factors able to recruit and polarize macrophages, we focused our attention on Bcl-xL, a multifaceted member of the Bcl-2 family, whose expression is deregulated in melanoma. It acts not only as a canonical pro-survival and anti-apoptotic protein, but also as a promoter of tumor progression. Human melanoma cells silencing or overexpressing Bcl-xL protein, THP-1 monocytic cells and monocyte-derived macrophages were used in this study. Protein array and specific neutralizing antibodies were used to analyze cytokines and chemokines secreted by melanoma cells. qRT-PCR, ELISA and Western Blot analyses were used to evaluate macrophage polarization markers and protein expression levels. Transwell chambers were used to evaluate migration of THP-1 and monocyte-derived macrophages. Mouse and zebrafish models were used to evaluate the ability of melanoma cells to recruit and polarize macrophages in vivo. We demonstrated that melanoma cells overexpressing Bcl-xL recruit macrophages at the tumor site and induce a M2 phenotype. In addition, we identified that interleukin-8 and interleukin-1β cytokines are involved in macrophage polarization, and the chemokine CCL5/RANTES in the macrophages recruitment at the tumor site. We also found that all these Bcl-xL-induced factors are regulated in a NF-kB dependent manner in human and zebrafish melanoma models. Our findings confirmed the pro-tumoral function of Bcl-xL in melanoma through its effects on macrophage phenotype.

The Bcl-2/Bcl-xL Inhibitor ABT-263 Attenuates Retinal Degeneration by Selectively Inducing Apoptosis in Senescent Retinal Pigment Epithelial Cells.

Age-related macular degeneration (AMD) is one of the leading causes of blindness in elderly individuals. However, the currently used intravitreal injections of anti-vascular endothelial growth factor are invasive, and repetitive injections are also accompanied by a risk of intraocular infection. The pathogenic mechanism of AMD is still not completely understood, but a multifactorial mechanism that combines genetic predisposition and environmental factors, including cellular senescence, has been suggested. Cellular senescence refers to the accumulation of cells that stop dividing due to the presence of free radicals and DNA damage. Characteristics of senescent cells include nuclear hypertrophy, increased levels of cell cycle inhibitors such as p16 and p21, and resistance to apoptosis. Senolytic drugs remove senescent cells by targeting the main characteristics of these cells. One of the senolytic drugs, ABT-263, which inhibits the antiapoptotic functions of Bcl-2 and Bcl-xL, may be a new treatment for AMD patients because it targets senescent retinal pigment epithelium (RPE) cells. We proved that it selectively kills doxorubicin (Dox)-induced senescent ARPE-19 cells by activating apoptosis. By removing senescent cells, the expression of inflammatory cytokines was reduced, and the proliferation of the remaining cells was increased. When ABT-263 was orally administered to the mouse model of senescent RPE cells induced by Dox, we confirmed that senescent RPE cells were selectively removed and retinal degeneration was alleviated. Therefore, we suggest that ABT-263, which removes senescent RPE cells through its senolytic effect, has the potential to be the first orally administered senolytic drug for the treatment of AMD.

Structural transitions in TCTP tumor protein upon binding to the anti-apoptotic protein family member Mcl-1

Translationally Controlled Tumor Protein (TCTP) serves as a pro-survival factor in tumor cells, inhibiting the mitochondrial apoptosis pathway by enhancing the function of anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL. TCTP specifically binds to Bcl-xL, preventing Bax-dependent Bcl-xL-induced cytochrome c release, and it reduces Mcl-1 turnover by inhibiting its ubiquitination, thereby decreasing Mcl-1-mediated apoptosis. TCTP harbors a BH3-like motif that forms a β-strand buried in the globular domain of the protein. In contrast, the crystal structure of the TCTP BH3-like peptide in complex with the Bcl-2 family member Bcl-xL reveals an α-helical conformation for the BH3-like motif, suggesting significant structural changes upon complex formation. Employing biochemical and biophysical methods, including limited proteolysis, circular dichroism, NMR, and SAXS, we describe the TCTP complex with the Bcl-2 homolog Mcl-1. Our findings demonstrate that full-length TCTP binds to the BH3 binding groove of Mcl-1 via its BH3-like motif, experiencing conformational exchange at the interface on a micro- to milli-second timescale. Concurrently, the TCTP globular domain becomes destabilized, transitioning into a molten-globule state. Furthermore, we establish that the non-canonical residue D16 within the TCTP BH3-like motif reduces stability while enhancing the dynamics of the intermolecular interface. In conclusion, we detail the structural plasticity of TCTP and discuss its implications for partner interactions and future anticancer drug design strategies aimed at targeting TCTP complexes.

Dissecting the lineage specific roles of BCL-XL in breast cancer metastasis

Abstract B-cell lymphoma X-large (BCL-XL) has been shown to promote breast cancer metastasis independently of its anti-apoptotic function. We recently found that proteolysis-targeting chimeras (PROTACs) against BCL-XL kill cancer cells as well as tumor-induced regulatory T cells (TI-Tregs). BCL-XL-targeting PROTACs can be potentially used to treat metastatic breast cancer – by direct killing of circulating tumor cells at their vulnerable stages and/or by eliciting anti-cancer immunity via T-reg cell depletion. Our pilot experiments showed opposing results with one model showing reduced metastasis by BCL-XL PROTAC treatment and the other model showing increased metastasis. Genetic deletion of BCL-XL (KO) in those two cell lines reproduces the effect of PROTAC on metastasis. We hypothesize that BCL-XL may play different roles in breast cancer metastasis depending on cellular cues from different cancer cells. We generated 4 other syngeneic mouse breast cancer BCL-XL-KO lines and confirmed that most of the breast cancer lines depend on BCL-XL for metastasis, whereas only 1 model exhibits an opposite phenotype. Additionally, we treated 4T1-tumor bearing mice with BCL-XL-targeting PROTAC that nearly significantly reduces metastatic spread to the lung. We also found a systematic inhibition of TI-Tregs by PROTACs indicating a lineage-specific function of BCL-XL in breast cancer metastasis. We conclude that BCL-XL is a viable therapeutic target for treating breast cancer metastasis, but further investigation into lineage-specific BCL-XL targeting is needed. Our future efforts will focus on investigating why BCL-XL inhibits metastasis in some cancer types, which can be used as an exclusion criterion for BCL-XL-targeting therapy. Supported by grants from NIH/National Cancer Institute (R01 CA260239)

Abstract 2486: Dissecting the lineage specific roles of BCL-XL in breast cancer metastasis

Abstract B-cell lymphoma X-large (BCL-XL) is a member of the BCL-2 protein family and has been known as an anti-apoptotic protein. As a viable target for cancer therapy, we recently found that proteolysis-targeting chimeras (PROTAC, currently under phase I clinical investigation) against BCL-XL kill cancer cells as well as tumor-induced regulatory T cells (TI-Tregs). Interestingly, BCL-XL has been shown to promote breast cancer metastasis via a function independent of its anti-apoptotic function. We reason that BCL-XL-targeting PROTACs can be potentially used to treat metastatic breast cancer - by direct killing of circulating tumor cells at their vulnerable stages and/or by eliciting anti-cancer immunity via T-reg cell depletion. To our surprise, our pilot experiments using two syngeneic breast cancer models showed opposite results with one model showing reduced metastasis by BCL-XL PROTAC treatment and the other model showing increased metastasis. Genetic deletion of BCL-XL (KO) in those two cell lines reproduces the effect of PROTAC on metastasis, suggesting the on-targeting but contrasting function of BCL-XL in breast cancer metastasis. Our central hypothesis is that BCL-XL may play different roles in breast cancer metastasis depending on the genetics/proteomics of cellular cues from different cancer cells. To confirm the function of BCL-XL in different breast cancer cell lines, we generated BCL-XL KO lines from 4 other syngeneic mouse breast cancers. We confirmed that the majority of breast cancer cells depend on BCL-XL for metastasis, including 4T1, AT-3, E0771.lmb etc; whereas only the Py8119 model exhibits an opposite phenotype. For primary tumor growth, AT-3 and E0771.lmb cells grow bigger tumors in the WT group than in the BCL-XL KO group; and 4T1 and Py8119 cells exhibit similar tumor growth rate between WT and KO cells. For metastatic breast cancer therapy, we treated 4T1-tumor bearing mice with BCL-XL-targeting PROTAC that nearly significantly reduces metastatic spread in the lung without significant change in primary tumor growth. In addition, we found a systematic inhibition of TI-Tregs by PROTACs indicating a lineage-specific function of BCL-XL in breast cancer metastasis. We conclude that BCL-XL is a viable therapeutic target for treating breast cancer metastasis, but caution is required as BCL-XL inhibition may be detrimental for certain cancers. Our future effort will focus on the mechanisms explaining why BCL-XL inhibits metastasis in some cancer types, which can be used as an exclusion criterion for BCL-XL-targeting therapy. Citation Format: Madison E. Carelock, Mo Jiao, Weizhou Zhang. Dissecting the lineage specific roles of BCL-XL in breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2486.

Abstract 3709: Synergistic activity of ABT-263 and ONC201 against solid tumor cell lines is associated with suppression of BAG3, Mcl-1, pAkt, and upregulation of Noxa along with Bax cleavage during apoptosis

Abstract There is an urgent need for the development of novel therapeutic approaches that can address the complex nature of cancer, including resistance to therapy. A major underlying cause of the resistance is the evasion of cell death following anti-cancer drug treatment. A great deal of progress has been made in the discovery and early development of cancer therapeutics that target cell death via the intrinsic or extrinsic cell death pathways. Two such drugs are ABT-263 and ONC201. ABT-263 is a small molecule that targets the pro-apoptotic cell death of tumor cells by blocking the anti-apoptotic function of BCL-XL and BCL-2; ONC201/TIC10 is a first-in-class small molecule originally discovered as a TRAIL-inducing compound. Previous studies have found that a mechanism of resistance to ABT-263 involves upregulation of anti-apoptotic Mcl-1 in ABT-263-treated tumors, while the efficacy of ONC201 is limited by BCL-2 overexpression. Given that ONC201 is known to downregulate Mcl-1 expression and that BCL-2 is a target of ABT-263, the combination of the two drugs has great synergistic potential. ONC201 and ABT-263 been tested in glioblastoma; however, ABT-263 is not able to cross the blood-brain barrier, and the combination of ONC201 and ABT-263 has not been tested in other solid tumors. It remains a gap in the field to test this combination therapy in tissues to which ONC201 and ABT-263 are bioavailable. We explored the combination of TRAIL-inducing compound ONC201/TIC10 and BCL-xl/BCL-2 inhibitor ABT-263 to target the extrinsic and intrinsic apoptotic pathways, respectively, in solid tumor cell lines (N=13) derived from different tissues (colon, prostate, lung, breast, ovary, bladder). We found an IC50 range of 0.83-20.10 μM for ONC201 and 0.06-14.75 μM for ABT-263 among the 13 cell lines. We show that combination of ONC201 and ABT-263 produces a strong synergistic effect leading to tumor cell death across the 1cell line panel, with maximum synergy scores ranging from 13.56-52.23 (scores were calculated using SynergyFinder; scores 0-10 indicate additive effects, while scores >10 indicate synergism), and that the combination is not toxic to human fibroblast cells. We also found that the combination of these two agents resulted in caspase activation and PARP cleavage consistent with induction of apoptosis, and that these effects where over and above individual drug effects. Combination therapy-induced cell death correlated with decreased levels of Mcl-1, BAG3, pAkt, and upregulation of Noxa along with Bax cleavage during apoptosis. Our data provide evidence for synergy from the combination of ONC201 and ABT-263 against human solid tumor cell lines associated with alterations in cell death and pro-survival mediators. The combination of ONC201 and ABT-263 merits further exploration in vivo in a variety of solid malignancies. Citation Format: Francesca R. Di Cristofano, Mara Fong, Lanlan Zhou, Wafik S. El-Deiry. Synergistic activity of ABT-263 and ONC201 against solid tumor cell lines is associated with suppression of BAG3, Mcl-1, pAkt, and upregulation of Noxa along with Bax cleavage during apoptosis [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 3709.

Increased apoptotic sensitivity of glioblastoma enables therapeutic targeting by BH3-mimetics

Glioblastoma (GBM) is the most prevalent malignant primary brain tumour in adults. GBM typically has a poor prognosis, mainly due to a lack of effective treatment options leading to tumour persistence or recurrence. We investigated the therapeutic potential of targeting anti-apoptotic BCL-2 proteins in GBM. Levels of anti-apoptotic BCL-xL and MCL-1 were consistently increased in GBM compared with non-malignant cells and tissue. Moreover, we found that relative to their differentiated counterparts, patient-derived GBM stem-like cells also displayed higher expression of anti-apoptotic BCL-2 family members. High anti-apoptotic BCL-xL and MCL-1 expression correlated with heightened susceptibility of GBM to BCL-2 family protein-targeting BH3-mimetics. This is indicative of increased apoptotic priming. Indeed, GBM displayed an obligate requirement for MCL-1 expression in both tumour development and maintenance. Investigating this apoptotic sensitivity, we found that sequential inhibition of BCL-xL and MCL-1 led to robust anti-tumour responses in vivo, in the absence of overt toxicity. These data demonstrate that BCL-xL and MCL-1 pro-survival function is a fundamental prerequisite for GBM survival that can be therapeutically exploited by BH3-mimetics.

Brain-Targeted Codelivery of Bcl-2/Bcl-xl and Mcl-1 Inhibitors by Biomimetic Nanoparticles for Orthotopic Glioblastoma Therapy.

Glioblastoma (GBM) is among the most treatment-resistant solid tumors and often recurrs after resection. One of the mechanisms through which GBM escapes various treatment modalities is the overexpression of anti-apoptotic Bcl-2 family proteins (e.g., Bcl-2, Bcl-xl, and Mcl-1) in tumor cells. Small-molecule inhibitors such as ABT-263 (ABT), which can promote mitochondrial-mediated cell apoptosis by selectively inhibiting the function of Bcl-2 and Bcl-xl, have been proven to be promising anticancer agents in clinical trials. However, the therapeutic prospects of ABT for GBM treatment are hampered by its limited blood-brain barrier (BBB) penetration, dose-dependent thrombocytopenia, and the drug resistance driven by Mcl-1, which is overexpressed in GBM cells and further upregulated upon treatment with ABT. Herein, we reported that the Mcl-1-specific inhibitor A-1210477 (A12) can act synergistically with ABT to induce potent cell apoptosis in U87 MG cells, drug-resistant U251 cells, and patient-derived GBM cancer stem cells. We further designed a biomimetic nanomedicine, based on the apolipoprotein E (ApoE) peptide-decorated red blood cell membrane and pH-sensitive dextran nanoparticles, for the brain-targeted delivery of ABT and A12. The synergistic anti-GBM effect was retained after encapsulation in the nanomedicine. Additionally, the obtained nanomedicine possessed good biocompatibility, exhibited efficient BBB penetration, and could effectively suppress tumor growth and prolong the survival time of mice bearing orthotopic GBM xenografts without inducing detectable adverse effects.

Impact of Deamidation on the Structure and Function of Antiapoptotic Bcl-xL.

Bcl-xL is an antiapoptotic mitochondrial trans-membrane protein, which is known to play a crucial role in the survival of tumor cells. The deamidation of Bcl-xL is a pivotal switch that regulates its biological function. The potential impact of deamidation on the structure and dynamics of Bcl-xL is directly linked to the intrinsically disordered region (IDR), which is the main site for post-translational modifications (PTMs). In this study, we explored deamidation-induced conformational changes in Bcl-xL to gain insight into its loss of function by performing microsecond-long molecular dynamics (MD) simulations. MD simulation outcomes showed that the IDR motion and interaction patterns have changed notably upon deamidation. Principal component analysis (PCA) demonstrates significant differences between wild-type and deamidated Bcl-xL and suggests that deamidation affects the structure and dynamics of Bcl-xL. The combination of clustering analysis, H-bond analysis, and PCA revealed changes in conformation, interaction, and dynamics upon deamidation. Differences in contact patterns and essential dynamics that lead to a narrowing in the binding groove (BG) are clear indications of deamidation-induced allosteric effects. In line with previous studies, we show that the IDR plays a very important role in the loss of apoptotic functions of Bcl-xL while providing a unique perspective on the underlying mechanism of Bcl-xL deamidation-induced cell death.

Acetogenins Exhibit Potential BCL-XL Inhibitor for the Induction of Apoptosis in the Molecular Docking Study.

Anti-apoptotic protein BCL-XL plays a vital role in tumorigenesis and cancer chemotherapy resistance, resulting in a good target for cancer treatment. Understanding the function of BCL-XL has driven the progression of a new class of cancer drugs that can mimic its natural inhibitors, BH3-only proteins, to trigger apoptosis. This mimicking is initiated through acetogenins due to their excellent biological properties. Acetogenins, which can be isolated from Annonaceae plants, have a unique structure along with several oxygenated functionalities. Based on their biological capability, various acetogenins were studied in the present study and compared alongside ABT-737 on molecular docking. The docking simulation of acetogenins was performed using AutoDock Vina software. Our findings have shown eleven acetogenins-BCL-XL protein complex, namely, muricin B (2), muricin F (4), muricin H (6), muricin I (7), xylomaticin (9), annomontacin (12), annonacin (14), squamocin (15), squamostatin A (16), bullatacin (20) and annoreticulin (21) exhibited strong binding affinities lower than - 10.4 kcalmol-1 as compared to ABT-373-BCL-XL complex. Six hydrogen bonds along with hydrophobic interaction were detected on the complex of BCL-XL with muricin B (2), muricin G (5), corossolone (11), and isoannonacin-10-one A (18). These findings indicated that some acetogenins could represent a new potential BCLXL inhibitor that could mimic the BH3-only protein for the induction of apoptosis in cancer chemotherapy.

Apoptotic Ablation of Platelets Reduces Atherosclerosis in Mice With Diabetes.

People with diabetes are at a significantly higher risk of cardiovascular disease, in part, due to accelerated atherosclerosis. Diabetic subjects have increased number of platelets that are activated, more reactive, and respond suboptimally to antiplatelet therapies. We hypothesized that reducing platelet numbers by inducing their premature apoptotic death would decrease atherosclerosis. Approach and Results: This was achieved by targeting the antiapoptotic protein Bcl-xL (B-cell lymphoma-extra large; which is essential for platelet viability) via distinct genetic and pharmacological approaches. In the former, we transplanted bone marrow from mice carrying the Tyr15 to Cys loss of function allele of Bcl-x (known as Bcl-xPlt20) or wild-type littermate controls into atherosclerotic-prone Ldlr+/- mice made diabetic with streptozotocin and fed a Western diet. Reduced Bcl-xL function in hematopoietic cells significantly decreased platelet numbers, exclusive of other hematologic changes. This led to a significant reduction in atherosclerotic lesion formation in Bcl-xPlt20 bone marrow transplanted Ldlr+/- mice. To assess the potential therapeutic relevance of reducing platelets in atherosclerosis, we next targeted Bcl-xL with a pharmacological strategy. This was achieved by low-dose administration of the BH3 (B-cell lymphoma-2 homology domain 3) mimetic, ABT-737 triweekly, in diabetic Apoe-/- mice for the final 6 weeks of a 12-week study. ABT-737 normalized platelet numbers along with platelet and leukocyte activation to that of nondiabetic controls, significantly reducing atherosclerosis while promoting a more stable plaque phenotype. These studies suggest that selectively reducing circulating platelets, by targeting Bcl-xL to promote platelet apoptosis, can reduce atherosclerosis and lower cardiovascular disease risk in diabetes. Graphic Abstract: A graphic abstract is available for this article.

Bcl-xL inhibits tBid and Bax via distinct mechanisms.

The proteins of the Bcl-2 family are key regulators of apoptosis. They form a complex interaction network in the cytosol and in cellular membranes, whose outcome determines mitochondrial permeabilization and commitment to death. However, we still do not understand how the action of the different family members is orchestrated to regulate apoptosis. Here, we combined quantitative analysis of the interactions and the localization dynamics of the family representatives Bcl-xL, Bax and tBid, in living cells. We discovered that Bax and tBid are able to constitutively shuttle between cytosol and mitochondria in the absence of other Bcl-2 proteins. Bcl-xL clearly stabilized tBid at mitochondria, where they formed tight complexes. In contrast, Bcl-xL promoted Bax retrotranslocation to the cytosol without affecting its shuttling rate, but by forming weak inhibitory mitochondrial complexes. Furthermore, analysis of phospho-mimetics of Bcl-xL suggested that phosphorylation regulates the function of Bcl-xL via multiple mechanisms. Altogether, our findings support a model in which the Bcl-2 network not only modulates protein/protein interactions among the family members, but also their respective intracellular localization dynamics, to regulate apoptosis.

Functional impact of cancer patient-associated Bcl-xL mutations.

Bcl‐xL, an antiapoptotic protein, is frequently overexpressed in cancer to promote survival of tumor cells. However, we have previously shown that Bcl‐xL promotes migration, invasion, and metastasis independent of its antiapoptotic function in mitochondria. The pro‐metastatic function of Bcl‐xL may require its translocation into the nucleus. Besides overexpression, patient‐associated mutations of Bcl‐xL have been identified in large‐scale cancer genomics projects. Understanding the functions of these mutations will guide the development of precision medicine. Here, we selected four patient‐associated Bcl‐xL mutations, R132W, N136K, R165W, and A201T, to investigate their impacts on antiapoptosis, migration, and nuclear translocation. We found that all four mutation proteins could be detected in both the nucleus and cytosol. Although all four mutations disrupted the antiapoptosis function, one of these mutants, N136K, significantly improved the ability to promote cell migration. These data suggest the importance of developing novel Bcl‐xL inhibitors to ablate both antiapoptotic and pro‐metastatic functions of Bcl‐xL in cancer.

Generation of incednine derivatives by mutasynthesis.

The macrolactam antibiotic incednine, isolated from Streptomyces sp. ML694-90F3, contains a (S)-3-aminobutyric acid moiety in its polyketide aglycon. In this study, we performed mutasynthesis to generate incednine derivatives. We successfully obtained 28-methylincednine by feeding 3-aminopentanoic acid into culture of a strain in which the glutamate 2,3-aminomutase gene idnL4, whose product is responsible for supplying 3-aminobutyric acid, was disrupted. 28-Methylincednine showed similar suppressive activity of the antiapoptotic function of oncoprotein Bcl-xL to that of incednine. Thus, this study highlights the applicability of the mutasynthesis approach in generation of novel β-amino acid-containing macrolactam polyketide derivatives.

MiR-124-3p.1 Sensitizes Ovarian Cancer Cells to Mitochondrial Apoptosis Induced by Carboplatin.

BackgroundCarboplatin is a platinum-based chemotherapeutic drug that is commonly used as a treatment for ovarian cancer. However, high doses and repeated use of carboplatin usually reduce the sensitivity of cancer cells to the drug. There is an urgent need to develop strategies to increase the sensitivity of ovarian cancer cells to carboplatin.Materials and MethodsQuantitative reverse-transcriptase real-time PCR was used to detect miR-124-3p.1 levels in ovarian cancer tissues and cell lines. Transfection with miR-124-3p.1 and caveolin-1 (CAV1) was used for gain-of-function experiments. Western blot and immunoprecipitation assays were performed to evaluate the expression and function of CAV1, AKT, Bad, and Bcl-xl. Flow cytometry analysis was used to measure the apoptosis rates of SKOV3 and A2780 cells.ResultsExpression levels of miR-124-3p.1 were decreased in ovarian cancer tissues and cell lines. Furthermore, overexpression of miR-124-3p.1 enhanced carboplatin-induced apoptotic cell death of ovarian cancer cell lines. Regarding the mechanism of this effect, we showed that CAV1 was the target of miR-124-3p.1 in ovarian cancer. Overexpression of miR-124-3p.1 suppressed the expression of CAV1, thereby reducing the activation of AKT and phosphorylation of Bad. As a result, the function of Bcl-xl was inhibited and carboplatin-induced mitochondrial apoptosis was enhanced.ConclusionmiR-124-3p.1 sensitizes carboplatin-induced mitochondrial apoptosis through suppression of CAV1 in ovarian cancer. Increasing miR-124-3p.1 expression may represent a novel strategy to improve carboplatin sensitivity in ovarian cancer.

326-LB: BCL-xL/BCL2L1 Is a Critical Anti-Apoptotic Protein that Suppresses BAK to Promote Pancreatic Specification from Human Pluripotent Stem Cells

The differentiation of human pluripotent stem cells (hPSCs) into desired cell types such as the pancreatic cells involve cellular proliferation and apoptosis during cell fate transitions. Although different rates of cell growth and death are typically observed during differentiation, its implications for pancreatic tissue formation in humans remain unclear. Here, we profiled the expression of BCL-2 family of proteins during pancreatic specification from hPSCs and observed an upregulation of BCL- XL, downregulation of BAK and corresponding downregulation of cleaved CASP3 representative of apoptosis. The inhibition or downregulation of BCL-xL reciprocally increased apoptosis and resulted in a decreased gene expression of pancreatic markers despite a compensatory increase in companion anti-apoptotic protein BCL2. RNA-Seq analyses revealed a downregulation of multiple metabolic genes upon inhibition of BCL-xL function. Bioenergetics assays then revealed a broad downregulation of both glycolysis and oxidative phosphorylation when BCL-xL function was inhibited. In conclusion, we have identified anti-apoptotic protein BCL-xL to be necessary for suppressing the expression of pro-apoptotic protein BAK to facilitate proper pancreas formation in human cells. This is the first report that links a member of the BCL-2 family with pancreatic specification. Therefore, modulation of these two BCL-2 family of proteins can potentially increase the survival and robustness of pancreatic progenitors that ultimately defines human pancreatic beta cell mass and function. Disclosure L. Loo: None. A. Teo: None. S. Ghosh: None. A. Soetedjo: None. L. Nguyen: None. V.G. Krishnan: None. S. Hoon: None. Funding Agency for Science, Technology and Research; Institute of Molecular and Cell Biology