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1.
Mol Cell ; 81(16): 3246-3261.e11, 2021 08 19.
Article in English | MEDLINE | ID: mdl-34352208

ABSTRACT

The Wnt/ß-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by ß-catenin's phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein ß-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human ß-catenin destruction complex from purified components, recapitulating complex assembly, ß-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote ß-catenin capture, phosphorylation, and ubiquitylation. APC facilitates ß-catenin's flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCFß-TrCP E3 ligase complex in a ß-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes ß-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/ß-catenin signaling.


Subject(s)
Adenomatous Polyposis Coli Protein/genetics , Axin Protein/genetics , beta Catenin/genetics , Adenomatous Polyposis Coli Protein/ultrastructure , Axin Protein/chemistry , Axin Protein/ultrastructure , Humans , Multiprotein Complexes/genetics , Multiprotein Complexes/ultrastructure , Phosphorylation/genetics , Protein Multimerization/genetics , Proteolysis , Ubiquitination/genetics , Wnt Signaling Pathway
2.
J Immunother Cancer ; 7(1): 101, 2019 04 15.
Article in English | MEDLINE | ID: mdl-30982469

ABSTRACT

BACKGROUND: The T cell bispecific antibody cibisatamab (CEA-TCB) binds Carcino-Embryonic Antigen (CEA) on cancer cells and CD3 on T cells, which triggers T cell killing of cancer cell lines expressing moderate to high levels of CEA at the cell surface. Patient derived colorectal cancer organoids (PDOs) may more accurately represent patient tumors than established cell lines which potentially enables more detailed insights into mechanisms of cibisatamab resistance and sensitivity. METHODS: We established PDOs from multidrug-resistant metastatic CRCs. CEA expression of PDOs was determined by FACS and sensitivity to cibisatamab immunotherapy was assessed by co-culture of PDOs and allogeneic CD8 T cells. RESULTS: PDOs could be categorized into 3 groups based on CEA cell-surface expression: CEAhi (n = 3), CEAlo (n = 1) and CEAmixed PDOs (n = 4), that stably maintained populations of CEAhi and CEAlo cells, which has not previously been described in CRC cell lines. CEAhi PDOs were sensitive whereas CEAlo PDOs showed resistance to cibisatamab. PDOs with mixed expression showed low sensitivity to cibisatamab, suggesting that CEAlo cells maintain cancer cell growth. Culture of FACS-sorted CEAhi and CEAlo cells from PDOs with mixed CEA expression demonstrated high plasticity of CEA expression, contributing to resistance acquisition through CEA antigen loss. RNA-sequencing revealed increased WNT/ß-catenin pathway activity in CEAlo cells. Cell surface CEA expression was up-regulated by inhibitors of the WNT/ß-catenin pathway. CONCLUSIONS: Based on these preclinical findings, heterogeneity and plasticity of CEA expression appear to confer low cibisatamab sensitivity in PDOs, supporting further clinical evaluation of their predictive effect in CRC. Pharmacological inhibition of the WNT/ß-catenin pathway may be a rational combination to sensitize CRCs to cibisatamab. Our novel PDO and T cell co-culture immunotherapy models enable pre-clinical discovery of candidate biomarkers and combination therapies that may inform and accelerate the development of immuno-oncology agents in the clinic.


Subject(s)
Antibodies, Bispecific/pharmacology , Antineoplastic Agents, Immunological/pharmacology , Carcinoembryonic Antigen/genetics , Colorectal Neoplasms/drug therapy , Drug Resistance, Neoplasm/genetics , Antibodies, Bispecific/therapeutic use , Antineoplastic Agents, Immunological/therapeutic use , CD8-Positive T-Lymphocytes , Coculture Techniques , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Drug Screening Assays, Antitumor , GPI-Linked Proteins/antagonists & inhibitors , GPI-Linked Proteins/genetics , Gene Expression Regulation, Neoplastic , Genetic Heterogeneity , Humans , Tissue Culture Techniques
3.
Nat Commun ; 9(1): 1849, 2018 05 10.
Article in English | MEDLINE | ID: mdl-29748565

ABSTRACT

Although PARP inhibitors (PARPi) target homologous recombination defective tumours, drug resistance frequently emerges, often via poorly understood mechanisms. Here, using genome-wide and high-density CRISPR-Cas9 "tag-mutate-enrich" mutagenesis screens, we identify close to full-length mutant forms of PARP1 that cause in vitro and in vivo PARPi resistance. Mutations both within and outside of the PARP1 DNA-binding zinc-finger domains cause PARPi resistance and alter PARP1 trapping, as does a PARP1 mutation found in a clinical case of PARPi resistance. This reinforces the importance of trapped PARP1 as a cytotoxic DNA lesion and suggests that PARP1 intramolecular interactions might influence PARPi-mediated cytotoxicity. PARP1 mutations are also tolerated in cells with a pathogenic BRCA1 mutation where they result in distinct sensitivities to chemotherapeutic drugs compared to other mechanisms of PARPi resistance (BRCA1 reversion, 53BP1, REV7 (MAD2L2) mutation), suggesting that the underlying mechanism of PARPi resistance that emerges could influence the success of subsequent therapies.


Subject(s)
Drug Resistance, Neoplasm/genetics , Neoplasms/drug therapy , Poly (ADP-Ribose) Polymerase-1/genetics , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Aged , Animals , BRCA1 Protein/genetics , CRISPR-Cas Systems , Cell Line, Tumor , DNA Mutational Analysis/methods , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Mouse Embryonic Stem Cells , Mutagenesis , Neoplasms/genetics , Neoplasms/pathology , Phthalazines/pharmacology , Phthalazines/therapeutic use , Point Mutation , Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Precision Medicine/methods , Whole Genome Sequencing/methods , Xenograft Model Antitumor Assays , Zinc Fingers/genetics
4.
Methods Mol Biol ; 1608: 445-473, 2017.
Article in English | MEDLINE | ID: mdl-28695526

ABSTRACT

The poly(ADP-ribose)polymerase (PARP) enzyme tankyrase (TNKS/ARTD5, TNKS2/ARTD6) uses its ankyrin repeat clusters (ARCs) to recognize degenerate peptide motifs in a wide range of proteins, thereby recruiting such proteins and their complexes for scaffolding and/or poly(ADP-ribosyl)ation. Here, we provide guidance for predicting putative tankyrase-binding motifs, based on the previously delineated peptide sequence rules and existing structural information. We present a general method for the expression and purification of tankyrase ARCs from Escherichia coli and outline a fluorescence polarization assay to quantitatively assess direct ARC-TBM peptide interactions. We provide a basic protocol for evaluating binding and poly(ADP-ribosyl)ation of full-length candidate interacting proteins by full-length tankyrase in mammalian cells.


Subject(s)
Poly ADP Ribosylation/physiology , Tankyrases/chemistry , Tankyrases/metabolism , Animals , Binding Sites , Humans , Poly ADP Ribosylation/genetics , Protein Binding/genetics , Protein Binding/physiology , Telomere/genetics , Telomere/metabolism
5.
Mol Cell ; 63(3): 498-513, 2016 08 04.
Article in English | MEDLINE | ID: mdl-27494558

ABSTRACT

The poly(ADP-ribose) polymerase (PARP) Tankyrase (TNKS and TNKS2) is paramount to Wnt-ß-catenin signaling and a promising therapeutic target in Wnt-dependent cancers. The pool of active ß-catenin is normally limited by destruction complexes, whose assembly depends on the polymeric master scaffolding protein AXIN. Tankyrase, which poly(ADP-ribosyl)ates and thereby destabilizes AXIN, also can polymerize, but the relevance of these polymers has remained unclear. We report crystal structures of the polymerizing TNKS and TNKS2 sterile alpha motif (SAM) domains, revealing versatile head-to-tail interactions. Biochemical studies informed by these structures demonstrate that polymerization is required for Tankyrase to drive ß-catenin-dependent transcription. We show that the polymeric state supports PARP activity and allows Tankyrase to effectively access destruction complexes through enabling avidity-dependent AXIN binding. This study provides an example for regulated signal transduction in non-membrane-enclosed compartments (signalosomes), and it points to novel potential strategies to inhibit Tankyrase function in oncogenic Wnt signaling.


Subject(s)
Sterile Alpha Motif , Tankyrases/metabolism , Wnt Signaling Pathway , Axin Protein/metabolism , Binding Sites , Caspase Activation and Recruitment Domain , Catalysis , Crystallography , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Models, Molecular , Mutation , Poly(ADP-ribose) Polymerases/metabolism , Protein Binding , Protein Conformation , Protein Multimerization , Structure-Activity Relationship , Tankyrases/chemistry , Tankyrases/genetics , Transfection
6.
Nucleic Acids Res ; 44(11): 5246-55, 2016 06 20.
Article in English | MEDLINE | ID: mdl-27060134

ABSTRACT

Cockayne syndrome B (CSB), best known for its role in transcription-coupled nucleotide excision repair (TC-NER), contains a ubiquitin-binding domain (UBD), but the functional connection between protein ubiquitylation and this UBD remains unclear. Here, we show that CSB is regulated via site-specific ubiquitylation. Mass spectrometry analysis of CSB identified lysine (K) 991 as a ubiquitylation site. Intriguingly, mutation of this residue (K991R) does not affect CSB's catalytic activity or protein stability, but greatly affects genome stability, even in the absence of induced DNA damage. Moreover, cells expressing CSB K991R are sensitive to oxidative DNA damage, but proficient for TC-NER. K991 becomes ubiquitylated upon oxidative DNA damage, and while CSB K991R is recruited normally to such damage, it fails to dissociate in a timely manner, suggesting a requirement for K991 ubiquitylation in CSB activation. Interestingly, deletion of CSB's UBD gives rise to oxidative damage sensitivity as well, while CSB ΔUBD and CSB K991R affects expression of overlapping groups of genes, further indicating a functional connection. Together, these results shed new light on the regulation of CSB, with K991R representing an important separation-of-function-mutation in this multi-functional protein.


Subject(s)
Cockayne Syndrome/genetics , Cockayne Syndrome/metabolism , DNA Damage , DNA Repair , Oxidative Stress , Transcription, Genetic , Amino Acid Sequence , Cell Cycle , Cell Line , Cell Survival , Cluster Analysis , DNA Damage/radiation effects , Gene Expression , Gene Expression Profiling , Genomic Instability , Humans , Mutation , Recombinant Fusion Proteins , Ubiquitination
7.
Proc Natl Acad Sci U S A ; 111(40): 14454-9, 2014 Oct 07.
Article in English | MEDLINE | ID: mdl-25249633

ABSTRACT

Cockayne syndrome (CS) is a multisystem disorder with severe neurological symptoms. The majority of CS patients carry mutations in Cockayne syndrome group B (CSB), best known for its role in transcription-coupled nucleotide excision repair. Indeed, because various repair pathways are compromised in patient cells, CS is widely considered a genome instability syndrome. Here, we investigate the connection between the neuropathology of CS and dysregulation of gene expression. Transcriptome analysis of human fibroblasts revealed that even in the absence of DNA damage, CSB affects the expression of thousands of genes, many of which are neuronal genes. CSB is present in a significant subset of these genes, suggesting that regulation is direct, at the level of transcription. Importantly, reprogramming of CS fibroblasts to neuron-like cells is defective unless an exogenous CSB gene is introduced. Moreover, neuroblastoma cells from which CSB is depleted show defects in gene expression programs required for neuronal differentiation, and fail to differentiate and extend neurites. Likewise, neuron-like cells cannot be maintained without CSB. Finally, a number of disease symptoms may be explained by marked gene expression changes in the brain of patients with CS. Together, these data point to dysregulation of gene regulatory networks as a cause of the neurological symptoms in CS.


Subject(s)
Cockayne Syndrome/genetics , DNA Helicases/genetics , DNA Repair Enzymes/genetics , Gene Expression Profiling , Gene Expression Regulation , Animals , Blotting, Western , Cell Line , Cell Line, Tumor , Cell Transdifferentiation/genetics , Cells, Cultured , Cockayne Syndrome/metabolism , Cockayne Syndrome/pathology , DNA Helicases/metabolism , DNA Repair Enzymes/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Ontology , Gene Regulatory Networks , HEK293 Cells , Humans , Mice , Microscopy, Fluorescence , Neurons/cytology , Neurons/metabolism , Oligonucleotide Array Sequence Analysis , Poly-ADP-Ribose Binding Proteins , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction
8.
Mol Cell Biol ; 31(4): 674-85, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21149575

ABSTRACT

Formation of a ribonucleoprotein particle (mRNP) competent for export requires the coupling of transcription with mRNA processing and RNA export. A key link between these processes is provided by the THO complex. To progress in our understanding of this coupling, we have performed a search for suppressors of the transcription defect caused by the hpr1Δ mutation. This has permitted us to identify mutations in the genes for the RNA polymerase II mediator component Med10, the Sch9 protein kinase, and the Ypr045c protein. We report a role in transcription elongation for Ypr045c (Thp3) and the Csn12 component of the COP9 signalosome. Thp3 and Csn12 form a complex that is recruited to transcribed genes. Their mutations suppress the gene expression defects of THO complex mutants involved in mRNP biogenesis and export and show defects in mRNA accumulation. Transcription elongation impairment of thp3Δ mutants is shown by in vivo transcript run-on analysis performed in G-less systems. Thp3-Csn12 establishes a novel link between transcription and mRNA processing that opens new perspectives on our understanding of gene expression and reveals novel functions for a component of the COP9 signalosome. Thp3-Csn12 also copurifies with ribosomal proteins, which opens the possibility that it has other functions in addition to transcription.


Subject(s)
Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Base Sequence , COP9 Signalosome Complex , DNA, Fungal/genetics , Genes, Fungal , Genome, Fungal , Mediator Complex/genetics , Mediator Complex/metabolism , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Open Reading Frames , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA Processing, Post-Transcriptional , Suppression, Genetic , Transcription, Genetic
9.
Biochim Biophys Acta ; 1769(3): 153-71, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17395283

ABSTRACT

ATP-dependent chromatin remodeling is performed by multi-subunit protein complexes. Over the last years, the identity of these factors has been unveiled in yeast and many parallels have been drawn with animal and plant systems, indicating that sophisticated chromatin transactions evolved prior to their divergence. Here we review current knowledge pertaining to the molecular mode of action of ATP-dependent chromatin remodeling, from single molecule studies to genome-wide genetic and proteomic studies. We focus on the budding yeast versions of SWI/SNF, RSC, DDM1, ISWI, CHD1, INO80 and SWR1.


Subject(s)
Adenosine Triphosphate/pharmacology , Chromatin Assembly and Disassembly , Nuclear Proteins/metabolism , Proteome , Saccharomycetales/metabolism , Genome, Fungal , Saccharomycetales/genetics , Saccharomycetales/growth & development
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