Transducin Β-like Protein Research Articles

Design, Synthesis, and Biological Evaluation of Selective TBL1X Degraders.

Transducin β-like protein 1 X-linked (TBL1X) is an essential scaffold protein involved in multiple signaling pathways, such as the Wnt/β-catenin pathway, where it protects β-catenin from ubiquitination and proteasomal degradation. Recent studies, however, suggest that TBL1X might modulate Wnt-regulated genes independently of β-catenin in diffuse large B-cell lymphoma (DLBCL). Here, we developed selective TBL1X degraders against DLBCL using the Proteolysis Targeting Chimeras (PROTACs) strategy as a proof-of-concept. Eight PROTACs showed strong cytotoxic activity. Interestingly, N-linked PROTACs exhibited minimal TBL1X degradation, while most O-linked PROTACs significantly reduced TBL1X levels, suggesting the crucial role of the linker attachment site in successful TBL1X degradation. Our mechanistic study revealed that TBL1X degradation induced by TD11 relied on the formation of the ternary complex and was dependent on the proteasome. The TBL1X degraders developed in this study could be a valuable chemical tool for investigating TBL1X-related pathways.

Abstract 5920: Inhibition of TBL1 and β-catenin interaction sensitizes acute lymphoblastic leukemia to chemotherapy

Abstract BACKGROUND: Relapse of T-cell acute lymphoblastic leukemia (T-ALL) and B-cell acute lymphoblastic leukemia (B-ALL) post-chemotherapy or post-immunotherapy remains a therapeutic problem warranting novel therapeutic approaches. Aberrant Wnt pathway activation is a common feature in ALL as Wnt activation through β-catenin is involved in self- renewal of drug resistant leukemia cells. A therapeutical approach to target Wnt signaling in T-ALL and B-ALL remains elusive. Here, we focus on the effect of transducin β-like protein 1 (TBL1) inhibitor tegavivint in combination with chemotherapy in T-ALL and B-ALL. We hypothesize that interrupting the binding of TBL1 to β-catenin abrogates leukemia cell survival and test if tegavivint sensitizes these cells to chemotherapeutic agents such as VDL (Vincristine, Dexamethasone, L-asparaginase) as part of preclinical evaluation of tegavivint in T- and B-cell ALL. METHODS: Four T-ALL cell lines one patient-derived T-ALL case, B-ALL cell lines and eight patient-derived B-ALL cases were used in vitro. We performed cytotoxicity studies using Annexin V/7AAD stainings and flow cytometry or Trypanblue counts to exclude dead cells, western blots, and co-immunoprecipitation assays to investigate effects of TBL-1 inhibition using tegavivint on β-catenin-mediated signaling. Tegavivint was injected intraperitoneally along with chemotherapeutic drugs. RESULTS: β-catenin and TBL1 were uniformly expressed in all T-ALL and B-ALL cells as determined by western blotting, albeit at different levels. Tegavivint decreases β-catenin protein expression as seen through western blot as well as decreases the binding of TBL1 to β-catenin as seen through co-immunoprecipitation (co-IP) in both the T-ALL and B-ALL samples. We also determined that tegavivint as a monotherapy induced apoptosis in both cell lines and patient samples at as little of a dose of 5nM after 24 hours of incubation using Annexin-V/DAPI staining by flow cytometry. We observed that tegavivint can sensitize all cases to chemotherapy (V: Vincristine, D: Dexamethasone, L:L-Asparaginase or Peg-Asparaginase) within 24 hours of incubation. Lastly, in vivo evaluation of tegavivint showed that tegavivint alone and in combination with VD chemotherapy significantly extends survival of NSG mice engrafted with patient-derived T-ALL and B-ALL compared to control treated mice. CONCLUSION: Our preliminary data shows that T-ALL and B-ALL are sensitive to TBL1-β-catenin inhibition using tegavivint as a monotherapy. Tegavivint can also sensitize T-ALL and B-ALL to chemotherapy (VDL). As our data supports our hypothesis that TBL-1-β-catenin inhibition is a new target in ALL therapy, further studies are in progress to preclinically evaluate this approach for clinical care. Citation Format: Samantha Hurwitz, Rachel Friedmann, Tatiana Fourfouris, Ki Jun Lee, Zesheng Wan, Chintan Parekh, Fariba Navid, Deepa Bhojwani, Kimberly Holloway, Yong-Mi Kim. Inhibition of TBL1 and β-catenin interaction sensitizes acute lymphoblastic leukemia to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5920.

Abstract 1666: Novel peptides target the β-catenin/TBL1 complex to competitively inhibit dysregulated Wnt signaling in cancer

Abstract Purpose: The canonical Wnt pathway is a stem cell pathway essential during embryogenesis, and functions to direct stem cell fate, renewal, and proliferation. Mutations in this pathway induce overexpression of Wnt genes resulting in a variety of human cancers including breast, colorectal, and ovarian. Canonical Wnt signaling is β-catenin-dependent and when active, β-catenin forms a complex with transducin β-like protein 1 (TBL1) and transducin β-like related protein 1 (TBLR1) to promote gene TCF/LEF-dependent expression downstream the Wnt pathway. Previously we developed a small-molecule inhibitor that disrupts this protein complex to inhibit Wnt signaling and promote a cytotoxic effect in tumor cells. While this approach is effective, our current efforts are focused to develop a peptide-based therapy that targets the β-catenin/TBL1 complex which will result in increased target specificity and less overall toxicity compared to small-molecule counterparts. We have generated a library of peptides corresponding to the binding interface of β-catenin and TBL1 with the aim of developing a specific drug that disrupts the complex. Methods: Selected peptides were tested for their ability to affect the aberrant Wnt pathway on the Wnt-activated colon cancer cell line SW480. Displacement of β-catenin from TBL1 was assessed using a heterologous competition ELISA screen in cell lysate. Hit peptides were identified then tested for specific binding to recombinant TBL1 using an additional ELISA. Further validation to determine associated levels of β-catenin with and without peptide treatment was performed with a co-immunoprecipitation. In situ detection of β-catenin/TBL1 complex dissociation in response to peptide treatment was investigated with a proximity ligation assay (PLA). A ubiquitin enrichment kit was used to measure induced β-catenin degradation. Hit peptides were tested with a viability assay for efficacy in their ability to promote a cytotoxic effect on tumor cells. Transcriptional TCF/LEF activation, which is promoted by β-catenin, was evaluated with TOPFlash in the Wnt activated TCF/LEF reporter HEK293 cell line. Results: We identified a series of peptides that selectively bind TBL1 and displace β-catenin from the complex in Wnt-driven colon cancer cell line SW480 in a dose-dependent manner. Further, we found that treatment with 5 μM peptides in TCF/LEF reporter cells significantly blocks the activity of TCF/LEF, inhibiting downstream transcription of Wnt genes. The disruption of the β-catenin/TBL1 complex results in the ubiquitination of β-catenin. Our findings confirmed this data showing that peptide treatment depletes ubiquitinated β-catenin over time. When these peptides are used to treat Wnt-driven tumor cells, they display strong on-target cytotoxic effects indicating a potential clinical application for the use of these specific peptides as a novel therapeutic. Citation Format: Shelby Rheinschmidt, Trason Thode, Samuel Sampson, Alexis Weston, Tithi Ghosh Halder, Serina Ng, Sydney Adamson, Kate Gutowsky, Mohan Kaadige, Raffaella Soldi, Sunil Sharma. Novel peptides target the β-catenin/TBL1 complex to competitively inhibit dysregulated Wnt signaling in cancer [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 1666.

Abstract 3110: Design, synthesis, and biological evaluation of transducin β-like protein 1 (TBL1) degraders

Abstract Transducin β-like protein 1 (TBL1) is an essential scaffold protein that participates in multiple critical signaling pathways, such as the Wnt/β-catenin pathway, where it protects β-catenin from ubiquitination and proteasomal degradation. Only one compound, BC-2059 (tegavivint, Iterion Therapeutics), has been reported to promote apoptosis by disrupting the TBL1/β-catenin interactions and show promising therapeutic effects in Wnt-driven cancers, such as colorectal cancer, breast cancer, and leukemia. However, recent studies showed that TBL1 modulates Wnt-regulated genes in a β-catenin-independent manner in diffuse large B-cell lymphoma (DLBCL). Although the involvement of β-catenin is still under debate, TBL1’s engagement in the antitumor effects of BC-2059 is certain and critical. In this study, we aimed to develop proof-of-concept BC-2059-based TBL1 selective degraders against DLBCL using the Proteolysis Targeting Chimeras (PROTACs) strategy and use them as chemical probes to investigate TBL1-related pathways in DLBCL. Two series (N- and O-linked) of BC-2059-based PROTACs were synthesized, and their cytotoxicity and protein degradation profiles were tested in cellular assays. Several compounds showed low micromolar to nanomolar cytotoxic activity. The TBL1 degradation of these PROTACs is currently underway. In the meantime, we modeled the ternary complex formation by developing a workflow where two binary complexes and a library of PROTACs having varied linker lengths/chemical compositions were modeled by PRosettaC. The protein-protein interactions of the generated ternary complexes were analyzed by Rosetta and the interactions between the linkers and the protein were calculated by Autodock 4. The results suggested that short linkers (8-14 heavy atoms) are more suitable for N-linked series degraders in terms of PROTAC-induced ternary complex formation, while long linkers (>18 heavy atoms) are preferable for O-linked series. Citation Format: Rui Yang, Lapo Alinari, Pui-kai (Tom) Li, Xiaolin Cheng. Design, synthesis, and biological evaluation of transducin β-like protein 1 (TBL1) degraders [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 3110.

TBL1 Inhibitor, Tegavivint, Supresses Cell Survival and Promotes Sensitivity to Chemotherapy in ALL and AML

BACKGROUND Abnormal wnt signaling has been linked to different forms of cancer and has been described to be critical in acute myeloblastic leukemia (AML) and acute lymphoblastic leukemia (ALL). AML is a subtype of leukemia with a poor prognosis as long-term disease-free survival is only 30~40%. While B-ALL has a better prognosis, relapse of B-ALL still has a poor overall survival rate. Aberrant Wnt pathway activation is a common feature in AML and B-ALL as Wnt/catenin activation through β-catenin is involved in self- renewal of leukemia initiating cells. In the canonical Wnt pathway, the signaling of wnt inhibits β-catenin from being degraded. β-catenin regulates transcription of several genes by its translocation into the nucleus. Recently, it has been shown that transducin β-like protein 1 (TBL1) and its highly related family member TBLR1 are required for Wnt-β-catenin-mediated transcription. Overexpression of TBL1 or TBLR1 can lead to significantly enhanced β-catenin-mediated transcription. Conversely, depletion of TBL1 or TBLR1 abolishes the activation of β-catenin. It has been shown that Tegavivint, a novel small molecule inhibitor of Wnt-ß-catenin signaling, binds to transducin ß-like protein 1 (TBL1) preventing the binding of TBL1 to ß-catenin, which results in nuclear degradation of ß-catenin in MV4-11 cells. Here we focus on the effect of Tegavivint in combination with chemotherapy: We hypothesize that interrupting the binding of TBL1 to beta-catenin abrogates not only AML, but also B-ALL cell survival and tests if Tegavivint sensitizes AML cells to AraC and B-ALL cells to chemotherapeutic agents including Vincristine, Dexamethasone or the tyrosine kinase inhibitor Nilotinib as part of preclinical evaluation in acute leukemias. RESULTS As AML is a genetically heterogenous disease, we have tested Tegavivint in vitro in six AML cell lines (THP1, KG1, MOLM14, MOLM13m U937 and MV4-11). These six AML cell lines express β-catenin and TBL1 as determined by Western Blotting. Tegavivint decreases nuclear β-catenin without affecting cytoplasmic β-catenin and decreases non-phospho β-catenin, the active form of β-catenin. We also determined that the IC50 of Tegavivint after 24 hours of incubation ranged between 5-16 nM in these 6 cell lines using AnnexinV/ DAPI staining and flow cytometry. We observed that Tegavivint can sensitize all six AML cell lines to chemotherapy (Ara-C) within 24 hours of incubation. We have also tested Tegavivint on 5 ALL cell lines with different karyotypes (RCH-ACV, NALM6, REH, SUPB15, and RS4;11). We determined that the IC50 of Tegavivint ranged between 5-6.5nM in these 5 cell lines. We also found that Tegavivint can sensitize four ALL cells to chemotherapy using Vincristine and Dexamethasone and one B-ALL cell line (SUBP-B15: BCR-ABL+) to the tyrosine kinase inhibitor Nilotinib within 24 hours of incubation. Further in vitro evaluation of patient-derived B-ALL cells and in vivo evaluation of the sensitization of B-ALL and AML to chemotherapy are in progress. CONCLUSION Our preliminary data shows that AML and B-ALL are sensitive to TBL1- β-catenin inhibition using Tegavivint as a monotherapy and can be sensitized to chemotherapy when AraC or Vincristine, dexamethasone or Nilotinib treatment is combined with Tegavivint. As our data supports our hypothesis that TBL-1- β-catenin inhibition is a new target in B-ALL and AML therapy, further studies are in progress to preclinically evaluate this approach for clinical care.

Abstract 2567: Targeting TBL1/β-catenin complex using peptides designed to competitively inhibit aberrant Wnt signaling in cancer

Abstract Purpose: Canonical Wnt signaling is a key cascade in regulating development and stemness, however it can become dysregulated and tightly associated with oncogenesis. In human cancers, Wnt/β-catenin signaling is highly activated due to mutations of members associated with the pathway such as adenomatous polyposis coli (APC) and β-catenin. Transducin β-like protein 1 (TBL1) and highly related TBLR1, form a complex with β-catenin facilitating the translocation of β-catenin to the nucleus. The formation of this complex protects β-catenin from degradation by SIAH-1 ubiquitinase and aids β-catenin binding to TCF/LEF transcription factors to transcribe downstream Wnt target genes. We recently reported that the destruction of the TBL1/β-catenin complex using a small molecule results in ubiquitination of β-catenin and inhibition of downstream signaling. As an alternative approach, we designed a series of novel peptides to interfere with binding of β-catenin with TBL1. Methods: Homologous competitive ELISA and thermal shift assays were performed to identify potential peptides that were able to displace β-catenin from TBL1. Additionally, we evaluated the ability of the peptides to disrupt the TBL1/β-catenin complex by performing pull-down assays and monitored the levels of β-catenin ubiquitination in response to peptide treatment in colon cell lines. To assess the levels of TCF/LEF transcriptional activation downstream the Wnt/β-catenin pathway, we performed TOPFlash assays in Wnt-activated cells transfected with plasmids for peptide expression. Results: Our preliminary results identified a set of peptides that efficiently displaces β-catenin from TBL1. Homologous competitive ELISA showed that the peptides were able to displace β-catenin from TBL1 in a dose dependent manner. The top five peptide hits were then tested for their ability to inhibit Wnt signaling using the TOPFlash assay. Among these, one peptide in particular showed high efficiency in inhibiting TCF/LEF transcriptional activation downstream the Wnt/β-catenin pathway. Our study suggests that β-catenin therapeutic peptides may represent a new and exciting approach for Wnt driven cancer therapy. Citation Format: Shelby Rheinschmidt, Trason Thode, Samuel Sampson, Alexis Weston, Tithi Ghosh Halder, Serina Ng, Ryan Rodriguez del Villar, Mohan Kaadige, Anton Zernov, Marcelle Machluf, Raffaella Soldi, Sunil Sharma. Targeting TBL1/β-catenin complex using peptides designed to competitively inhibit aberrant Wnt signaling in cancer [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 2567.

Transducin β-like protein 1 controls multiple oncogenic networks in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common non- Hodgkin lymphoma and is characterized by a remarkable heterogeneity with diverse variants that can be identified histologically and molecularly. Large-scale gene expression profiling studies have identified the germinal center B-cell (GCB-) and activated B-cell (ABC-) subtypes. Standard chemo-immunotherapy remains standard front-line therapy, curing approximately two thirds of patients. Patients with refractory disease or those who relapse after salvage treatment have an overall poor prognosis highlighting the need for novel therapeutic strategies. Transducin b-like protein 1 (TBL1) is an exchange adaptor protein encoded by the TBL1X gene and known to function as a master regulator of the Wnt signaling pathway by binding to β-CATENIN and promoting its downstream transcriptional program. Here, we show that, unlike normal B cells, DLBCL cells express abundant levels of TBL1 and its overexpression correlates with poor clinical outcome regardless of DLBCL molecular subtype. Genetic deletion of TBL1 and pharmacological approach using tegavivint, a first-in-class small molecule targeting TBL1 (Iterion Therapeutics), promotes DLBCL cell death in vitro and in vivo. Through an integrated genomic, biochemical, and pharmacologic analyses, we characterized a novel, β-CATENIN independent, post-transcriptional oncogenic function of TBL1 in DLBCL where TBL1 modulates the stability of key oncogenic proteins such as PLK1, MYC, and the autophagy regulatory protein BECLIN-1 through its interaction with a SKP1-CUL1-F-box (SCF) protein supercomplex. Collectively, our data provide the rationale for targeting TBL1 as a novel therapeutic strategy in DLBCL.

PPARα and NCOR/SMRT corepressor network in liver metabolic regulation.

Peroxisome proliferator-activated receptor alpha (PPARα, NR1C1) belongs to a large family of ligand-dependent nuclear receptors (NRs). It is one of the best studied NRs which controls the lipid metabolism (mainly fatty acid oxidation) and inflammation, and has been a promising target for treating metabolic disorders such as fatty liver and cardiometabolic diseases. The function of PPARα relies on its interaction with various coregulators upon different stimulating contexts, and, thereby, activates or represses its transcription targets in a gene-selective manner. Understanding the transcription factor and coregulator network underlying the PPARα regulation is prerequisite to decipher its gene- and context-selectivity for designing better therapeutic ligands. In this review, we will summarize current knowledge of PPARα coregulator network, with major focus on a relatively well-studied corepressor complex containing core subunits of nuclear receptor corepressor (NCOR or NCOR1), silencing mediator of retinoic acid and thyroid hormone receptor (SMRT or NCOR2), G-protein suppressor 2 (GPS2), transducin β-like protein 1 (TBL1 or TBL1X), TBL-related 1 (TBLR1 or TBL1XR1), and the catalytic core of histone deacetylase 3 (HDAC3). We will mainly review the molecular events of the complex and sub-complexes in controlling the liver metabolism. We will also discuss the potential perturbation of the subunit expression in human livers during liver metabolic disorder progression which potentially defines the patient disease susceptibility and drug responses.

Mutations in TBL1X Are Associated With Central Hypothyroidism.

Context:Isolated congenital central hypothyroidism (CeH) can result from mutations in TRHR, TSHB, and IGSF1, but its etiology often remains unexplained. We identified a missense mutation in the transducin β-like protein 1, X-linked (TBL1X) gene in three relatives diagnosed with isolated CeH. TBL1X is part of the thyroid hormone receptor-corepressor complex.Objective:The objectives of the study were the identification of TBL1X mutations in patients with unexplained isolated CeH, Sanger sequencing of relatives of affected individuals, and clinical and biochemical characterization; in vitro investigation of functional consequences of mutations; and mRNA expression in, and immunostaining of, human hypothalami and pituitary glands.Design:This was an observational study.Setting:The study was conducted at university medical centers.Patients:Nineteen individuals with and seven without a mutation participated in the study.Main Outcome Measures:Outcome measures included sequencing results, clinical and biochemical characteristics of mutation carriers, and results of in vitro functional and expression studies.Results:Sanger sequencing yielded five additional mutations. All patients (n = 8; six males) were previously diagnosed with CeH (free T4 [FT4] concentration below the reference interval, normal thyrotropin). Eleven relatives (two males) also carried mutations. One female had CeH, whereas 10 others had low-normal FT4 concentrations. As a group, adult mutation carriers had 20%–25% lower FT4 concentrations than controls. Twelve of 19 evaluated carriers had hearing loss. Mutations are located in the highly conserved WD40-repeat domain of the protein, influencing its expression and thermal stability. TBL1X mRNA and protein are expressed in the human hypothalamus and pituitary.Conclusions:TBL1X mutations are associated with CeH and hearing loss. FT4 concentrations in mutation carriers vary from low-normal to values compatible with CeH.

SUMOylation of TBL1 and TBLR1 promotes androgen-independent prostate cancer cell growth.

Chronic inflammation is strongly associated with prostate cancer pathogenesis. Transducin β-like protein (TBL1) and Transducin β-like 1X-linked receptor 1 (TBLR1) have been identified recently as a coactivator for NF-κB-mediated transcription; however, the underlying mechanism by which TBL1 and TBLR1 activate NF-κB function during inflammation remains unknown. Here, we demonstrate that cytokine production is significantly elevated in androgen-independent PC-3 prostate cancer cells compared with androgen-dependent LNCaP prostate cancer cells. Elevated cytokine production positively correlates with the TBL1 and TBLR1 SUMOylation level in PC-3 cells. We show that both TBL1 and TBLR1 are SUMOylated in response to TNF-α treatment, and this increases formation of the TBL1-TBLR1-NF-κB complex, which leads to NF-κB-mediated transcriptional activation of cytokine gene expression. Conversely, SENP1-mediated deSUMOylation of TBL1 and TBLR1 inhibits NF-κB-target gene expression by dissociating TBL1 and TBLR1 from the nuclear hormone receptor corepressor (NCoR) complex. TBL1 knockdown substantially suppresses inflammatory signaling and PC-3 cell proliferation. Collectively, these results suggest that targeted SUMOylation of TBL1 and TBLR1 may be a useful strategy for therapeutic treatment of androgen-independent prostate cancer.

Theranostical nanosystem-mediated identification of an oncogene and highly effective therapy in hepatocellular carcinoma.

Because the primary surgical treatment options for hepatocellular carcinoma (HCC)-including hepatic resection and liver transplantation-often fail due to recurrence and metastasis, identifying early prognostic biomarkers and therapeutic targets for HCC is of great importance. This study shows that transducin β-like protein 1-related protein (TBLR1) is a key HCC oncogene that plays important roles in HCC proliferation, antiapoptosis, and angiogenesis by regulating the Wnt/β-catenin pathway. The folate-targeted theranostic small interfering RNA (siRNA) nanomedicine Fa-PEG-g-PEI-SPION/psiRNA-TBLR1 effectively silences the TBLR1 gene in different human HCC cell lines in vitro and in human HCC samples in vivo, resulting in the simultaneous suppression of HCC cell proliferation, antiapoptosis, and angiogenesis. Because of its multi-anticancer functions against HCC, intravenous injection of the folate-targeted siRNA nanomedicine into nude mice bearing intrahepatic or subcutaneous xenografts of human HCC has a significant therapeutic effect. Tumor growth in those animals was almost completely inhibited by treatment with Fa-PEG-g-PEI-SPION/psiRNA-TBLR1. Moreover, the SPION-encapsulated polyplexes possess high magnetic resonance imaging (MRI) detection sensitivity, which makes tumor-targeted siRNA delivery easily trackable using the clinical MRI technique. The theranostic siRNA nanomedicine examined here possesses great theranostic potential for combined gene therapy and MRI diagnosis of HCC.

Carbonic anhydrase IV inhibits colon cancer development by inhibiting the Wnt signalling pathway through targeting the WTAP–WT1–TBL1 axis

ObjectiveWe found that carbonic anhydrase IV (CA4), a member of the carbonic anhydrases, is silenced in colorectal cancer (CRC). We analysed its epigenetic inactivation, biological effects and prognostic significance in...

Ebi/AP-1 Suppresses Pro-Apoptotic Genes Expression and Permits Long-Term Survival of Drosophila Sensory Neurons

Sensory organs are constantly exposed to physical and chemical stresses that collectively threaten the survival of sensory neurons. Failure to protect stressed neurons leads to age-related loss of neurons and sensory dysfunction in organs in which the supply of new sensory neurons is limited, such as the human auditory system. Transducin β-like protein 1 (TBL1) is a candidate gene for ocular albinism with late-onset sensorineural deafness, a form of X-linked age-related hearing loss. TBL1 encodes an evolutionarily conserved F-box–like and WD40 repeats–containing subunit of the nuclear receptor co-repressor/silencing mediator for retinoid and thyroid hormone receptor and other transcriptional co-repressor complexes. Here we report that a Drosophila homologue of TBL1, Ebi, is required for maintenance of photoreceptor neurons. Loss of ebi function caused late-onset neuronal apoptosis in the retina and increased sensitivity to oxidative stress. Ebi formed a complex with activator protein 1 (AP-1) and was required for repression of Drosophila pro-apoptotic and anti-apoptotic genes expression. These results suggest that Ebi/AP-1 suppresses basal transcription levels of apoptotic genes and thereby protects sensory neurons from degeneration.

Abstract 3103: Transducin β-like protein 1 promotes cancer cell invasion by recruiting nuclear factor-κB to target gene promoter for transcriptional activation

Abstract Background and Purpose: Nuclear factor-kappaB (NF-κB) signaling controls a wide range of cellular functions such as tumor progression and invasion by inducing gene expression. Although significant progress has been made in understanding NF-κB-mediated gene transactivation, little is known about how NF-κB is recruited to its target gene promoters. Therefore the purpose of the study is to determine the role of transducin β-like protein 1 (TBL1) in the recruitment of NF-κB to target gene promoter. Methods:TBL1 knock down breast and squamous cell carcinoma cells were used to asses the significance of TBL1 depletion on NF-κB recruitment to target gene promoter and its consequence on the invasion of cancer cells. Results: Our results revealed that transducin β-like protein 1 (TBL1) controlled the expression of NF-κB target genes by directly binding with NF-κB and facilitating its recruitment to target gene promoters. TNF-α stimulation triggered the formation of NF-κB and TBL1 complex and subsequent target gene promoter binding. Knock-down of TBL1 impaired the recruitments of NF-κB to its target gene promotes. Interestingly, the mRNA microarray studies available from Oncomine revealed that TBL1 expression levels were higher in invasive breast cancer tissues than in breast adenocarcinoma. Consistently, TBL1 knockdown significantly reduced the invasive potential of breast cancer cells by inhibiting NF-κB. Conclusions: Our results unravel a new mechanism for the regulation of NF-κB activation, with important implications in the development of novel strategies for cancer therapy by targeting NF-κB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3103. doi:10.1158/1538-7445.AM2011-3103

Transducin β-Like Protein 1 Recruits Nuclear Factor κB to the Target Gene Promoter for Transcriptional Activation

Nuclear factor κB (NF-κB) signaling controls a wide range of cellular functions such as tumor progression and invasion by inducing gene expression. Upon stimulation, NF-κB is translocated to the nucleus and binds to its target gene promoters to activate transcription by recruiting transcription coactivators. Although significant progress has been made in understanding NF-κB-mediated transactivation, little is known about how NF-κB is recruited to its target gene promoters. Here, we report that transducin β-like protein 1 (TBL1) controls the expression of NF-κB target genes by directly binding with NF-κB and facilitating its recruitment to target gene promoters. Tumor necrosis factor alpha stimulation triggered the formation of an NF-κB and TBL1 complex and subsequent target gene promoter binding. Knockdown of TBL1 impaired the recruitment of NF-κB to its target gene promoters. Interestingly, analysis of the Oncomine database revealed that TBL1 mRNA levels were significantly higher in invasive breast cancer tissues than in breast adenocarcinoma tissue. Consistently, TBL1 knockdown significantly reduced the invasive potential of breast cancer cells by inhibiting NF-κB. Our results reveal a new mechanism for the regulation of NF-κB activation, with important implications for the development of novel strategies for cancer therapy by targeting NF-κB.

Negative Regulation of TSHα Target Gene by Thyroid Hormone Involves Histone Acetylation and Corepressor Complex Dissociation

Currently, little is known about histone modifications and molecular mechanisms of negatively regulated transcription. In pituitary cells, thyroid hormone (T(3)) decreased transcription, and surprisingly increased histone acetylation, of TSHalpha promoter. This increase was mediated directly by thyroid hormone receptor. Histone acetylation of H3K9 and H3K18 sites, two modifications usually associated with transcriptional activation, occur in negative regulation of TSHalpha promoter. T(3) also caused release of a corepressor complex composed of histone deacetylase 3 (HDAC3), transducin beta-like protein 1, and nuclear receptor coprepressor (NCoR)/ silencing mediator for retinoic and thyroid hormone receptor from TSHalpha promoter in chromatin immunoprecipitation assays. NCoR and HDAC3 overexpression selectively increased ligand-independent basal transcription. Two histone acetyltransferase inhibitors increased overall transcription but did not abrogate negative regulation or NCoR/HDAC3 complex release by T(3). Chromatin immunoprecipitation analyses of an endogenous positively regulated target gene showed increased histone acetylation and corepressor complex release with T(3) treatment. Finally, microarray analyses suggested there is a subset of negatively regulated genes with increased histone acetylation. These findings demonstrate the critical role of NCoR/HDAC3 complex in negative regulation of TSHalpha gene expression and show that similar complexes and overlapping epigenetic modifications can participate in both negative and positive transcriptional regulation.

A core SMRT corepressor complex containing HDAC3 and TBL1, a WD40-repeat protein linked to deafness

The corepressor SMRT mediates repression by thyroid hormone receptor (TR) as well as other nuclear hormone receptors and transcription factors. Here we report the isolation of a novel SMRT-containing complex from HeLa cells. This complex contains transducin beta-like protein 1 (TBL1), whose gene is mutated in human sensorineural deafness. It also contains HDAC3, a histone deacetylase not previously thought to interact with SMRT. TBL1 displays structural and functional similarities to Tup1 and Groucho corepressors, sharing their ability to interact with histone H3. In vivo, TBL1 is bridged to HDAC3 through SMRT and can potentiate repression by TR. Intriguingly, loss-of-function TRbeta mutations cause deafness in mice and humans. These results define a new TR corepressor complex with a physical link to histone structure and a potential biological link to deafness.