Search

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, due to lack of experimental models that mimic different disease stages. We describe a novel androgen-dependent PCa patient-derived xenograft (PDX) model from treatment-naïve, soft tissue metastasis (PNPCa). RNA and whole-exome sequencing of the PDX tissue and organoids confirmed transcriptomic and genomic similarity to primary tumor. PNPCa harboursBRCA2 and CHD1somatic mutations, shows anSPOP/FOXA1-like transcriptomic signature and microsatellite instability, which occurs in 3% of advanced PCa and has never been modelledin vivo. Comparison of the treatment-naïve PNPCa with additional metastatic PDXs (BM18, LAPC9), in a medium-throughput organoid screen of FDA-approved compounds, revealed differential drug sensitivities. Multikinase inhibitors (ponatinib, sunitinib, sorafenib) were broadly effective on all PDX- and patient-derived organoids from advanced cases with acquired resistance to standard-of-care compounds. This proof-of-principle study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified, repurposed compounds.

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, due to lack of experimental models that mimic different disease stages. We describe an androgen-dependent PCa patient-derived xenograft (PDX) model from treatment-naïve, soft tissue metastasis (PNPCa). RNA and whole-exome sequencing of the PDX tissue and organoids confirmed transcriptomic and genomic similarity to primary tumor. PNPCa harbors BRCA2 and CHD1 somatic mutations, shows an SPOP/FOXA1-like transcriptomic signature and microsatellite instability, which occurs in 3% of advanced PCa and has never been modeled in vivo. Comparison of the treatment-naïve PNPCa with additional metastatic PDXs (BM18, LAPC9), in a medium-throughput organoid screen of FDA-approved compounds, revealed differential drug sensitivities. Multikinase inhibitors (ponatinib, sunitinib, sorafenib) were broadly effective on all PDX- and patient-derived organoids from advanced cases with acquired resistance to standard-of-care compounds. This proof-of-principle study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified, repurposed compounds., To date, patients still succumb to cancer, due to tumors not responding to therapy or ultimately acquiring resistance. Here the authors show that by exploiting patient derived organoids and a treatment-naïve patient derived xenograft, patient therapy can be personalized.

Transcription factors (TFs) are thought to function with partners to achieve specificity and precise quantitative outputs. In the developing heart, heterotypic TF interactions, such as between the T-box TF TBX5 and the homeodomain TF NKX2-5, have been proposed as a mechanism for human congenital heart defects. We report extensive and complex interdependent genomic occupancy of TBX5, NKX2-5, and the zinc finger TF GATA4, coordinately controlling cardiac gene expression, differentiation, and morphogenesis. Interdependent binding serves not only to co-regulate gene expression, but also to prevent TFs from distributing to ectopic loci and activate lineage-inappropriate genes. We define preferential motif arrangements for TBX5 and NKX2-5 cooperative binding sites, supported at the atomic level by their co-crystal structure bound to DNA, revealing direct interaction between the two factors, and induced DNA bending. Complex interdependent binding mechanisms reveal tightly regulated TF genomic distribution and define a combinatorial logic for heterotypic TF regulation of differentiation.

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, mainly due to the lack of experimental models representing different disease stages. We aim to provide functional experimental and preclinical drug tools applicable for direct use with patient-derived material. Biopsies from metastatic PCa were used for the establishment of patient-derived xenografts (PDXs) by subcutaneous implantation. In vivo tumor growth kinetic in response to androgens was assessed by surgical castration and testosterone supplementation. RNA and whole-exome sequencing (WES) and organoid drug screens were used to characterize the genomic, transcriptomic, and functional properties. Organoid culture methodology was adapted in order to set up an automated medium-throughput organoid drug screen (Nexus Theragnostics automated platform) for screening standard-of-care compounds and candidates for drug repurposing. A novel case of PCa xenograft model derived from soft tissue metastasis (PNPCa) was established. RNAseq and WES confirmed transcriptomic and genomic similarity to the primary tumor. PNPCa harbors BRAC2 and CHD1 mutations and shows SPOP-like and FOXA1-like transcriptomic signature with microsatellite instability. PNPCa tumor growth is inhibited after androgen deprivation by castration, while androgen replacement leads to tumor reformation. No spontaneous tumor growth occurred after prolonged period of castration; however, scarce micrometastases were found in the bone marrow and lymph nodes. The treatment-naive and androgen-dependent properties of the PNPCa made it a candidate model for identification of potent drug compounds. PNPCa-derived organoid screens on standard-of-care and 74 FDA-approved compounds showed that mTOR pathway and multi-tyrosine kinase inhibitors, used for clinical treatment of other cancer types, have high impact on PCa organoid viability. Application of the organoid drug screen panel on two additional metastatic PDXs of advanced disease has allowed shortlisting of compounds for repurposing use in patient-derived organoid screens that can be routinely performed in a timeframe of few weeks, and thus, provide information on treatment decision. Treatment response of a treatment-naive, early metastatic PCa case (PNPCa PDX) highlights the potential of organoid screens to assess therapy response with extended applications for personalized screens. Citation Format: Sofia Karkampouna, Federico la Manna, Maria R. De Filippo, Mirjam Kiener, Marta De Menna, Eugenio Zoni, Joel Grosjean, Irena Klima, Andrea Garofoli, Marco Bolis, Jean-Philippe Theurillat, Vera Genitsch, David Keller, Booij Tijmen, Christian U. Stirnimann, Andrea Sboner, Charlotte K.Y. Ng, Salvatore Piscuoglio, Martin Spahn, Mark A. Rubin, George N. Thalmann, Marianna Kruithof-de Julio. Patient-derived xenograft and organoids models of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B18.

Summary The ability of circulating tumor cells (CTCs) to form clusters has been linked to increased metastatic potential. Yet biological features and vulnerabilities of CTC clusters remain largely unknown. Here, we profile the DNA methylation landscape of single CTCs and CTC clusters from breast cancer patients and mouse models on a genome-wide scale. We find that binding sites for stemness- and proliferation-associated transcription factors are specifically hypomethylated in CTC clusters, including binding sites for OCT4, NANOG, SOX2, and SIN3A, paralleling embryonic stem cell biology. Among 2,486 FDA-approved compounds, we identify Na+/K+ ATPase inhibitors that enable the dissociation of CTC clusters into single cells, leading to DNA methylation remodeling at critical sites and metastasis suppression. Thus, our results link CTC clustering to specific changes in DNA methylation that promote stemness and metastasis and point to cluster-targeting compounds to suppress the spread of cancer., Graphical Abstract, Highlights • Binding sites for OCT4, SOX2, NANOG, and SIN3A are hypomethylated in CTC clusters • CTC cluster hypomethylation profile correlates with a poor prognosis in breast cancer • Treatment with FDA-approved Na+/K+-ATPase inhibitors dissociates CTC clusters • Dissociation reverts the methylation profile of CTC clusters and suppresses metastasis, A comparative analysis of the methylation landscape of single and clusters of circulating tumor cells reveals patterns of similarity to embryonic stem cells and identifies pharmacological agents that can target clustering, suppress stemness, and blunt metastatic spreading.

Drosophila Nurf55 is a component of different chromatin-modifying complexes, including the PRC2 (Polycomb repressive complex 2). Based on the 1.75-Å crystal structure of Nurf55 bound to histone H4 helix 1, we analyzed interactions of Nurf55 (Nurf55 or p55 in fly and RbAp48/46 in human) with the N-terminal tail of histone H3, the first helix of histone H4, and an N-terminal fragment of the PRC2 subunit Su(z)12 using isothermal calorimetry and pulldown experiments. Site-directed mutagenesis identified the binding site of histone H3 at the top of the Nurf55 WD40 propeller. Unmodified or K9me3- or K27me3-containing H3 peptides were bound with similar affinities, whereas the affinity for K4me3-containing H3 peptides was reduced. Helix 1 of histone H4 and Su(z)12 bound to the edge of the β-propeller using overlapping binding sites. Our results show similarities in the recognition of histone H4 and Su(z)12 and identify Nurf55 as a versatile interactor that simultaneously contacts multiple partners.

TBX5, a member of the T-box transcription factor family, plays an important role in heart and limb development. More than 60 single point or deletion mutations of human TBX5 are associated with Holt-Oram syndrome that manifests itself as heart and limb malformations in 1 out of 100,000 live births. The majority of these mutations are located in the TBX5 T-box domain. We solved the crystal structures of the human TBX5 T-box domain in its DNA-unbound form and in complex with a natural DNA target site allowing for the first time the comparison between unbound and DNA-bound forms. Our analysis identifies a 3(10)-helix at the C-terminus of the T-box domain as an inducible recognition element, critically required for the interaction with DNA, as it only forms upon DNA binding and is unstructured in the DNA-unbound form. Using circular dichroism, we characterized the thermal stability of six TBX5 mutants containing single point mutations in the T-box domain (M74V, G80R, W121G, G169R, T223M, and R237W) and compared them with wild-type protein. Mutants G80R and W121G show drastically reduced thermal stability, while the other mutants only show a marginal stability decrease. For all TBX5 mutants, binding affinities to specific and nonspecific DNA sequences were determined using isothermal titration calorimetry. All TBX5 mutants show reduced binding affinities to a specific DNA target site, although to various degrees. Interestingly, all tested TBX5 mutants differ in their ability to bind unspecific DNA, indicating that both sequence-specific and unspecific binding might contribute to the misregulation of target gene expression.

Proteins of the thioredoxin (Trx) superfamily catalyze disulfide-bond formation, reduction and isomerization in substrate proteins both in prokaryotic and in eukaryotic cells. All members of the Trx family with thiol-disulfide oxidoreductase activity contain the characteristic Cys-X-X-Cys motif in their active site. Here, using Poisson-Boltzmann-based protonation-state calculations based on 100-ns molecular dynamics simulations, we investigate the catalytic mechanism of DsbL, the most oxidizing Trx-like protein known to date. We observed several correlated transitions in the protonation states of the buried active-site cysteine and a neighboring lysine coupled to the exposure of the active-site thiolate. These results support the view of an internal proton shuffling mechanism during oxidation crucial for the uptake of two electrons from the substrate protein. Intramolecular disulfide-bond formation is probably steered by the conformational switch facilitating interaction with the active-site thiolate. A consistent catalytic mechanism for DsbL, probably conferrable to other proteins of the same class, is presented. Our results suggest a functional role of hydration entropy of active-site groups.

The causative agent of Legionnaires' pneumonia, Legionella pneumophila, colonizes diverse environmental niches, including biofilms, plant material, and protozoa. In these habitats, myo-inositol hexakisphosphate (phytate) is prevalent and used as a phosphate storage compound or as a siderophore. L. pneumophila replicates in protozoa and mammalian phagocytes within a unique "Legionella-containing vacuole." The bacteria govern host cell interactions through the Icm/Dot type IV secretion system (T4SS) and ∼300 different "effector" proteins. Here we characterize a hitherto unrecognized Icm/Dot substrate, LppA, as a phytate phosphatase (phytase). Phytase activity of recombinant LppA required catalytically essential cysteine (Cys(231)) and arginine (Arg(237)) residues. The structure of LppA at 1.4 A resolution revealed a mainly α-helical globular protein stabilized by four antiparallel β-sheets that binds two phosphate moieties. The phosphates localize to a P-loop active site characteristic of dual specificity phosphatases or to a non-catalytic site, respectively. Phytate reversibly abolished growth of L. pneumophila in broth, and growth inhibition was relieved by overproduction of LppA or by metal ion titration. L. pneumophila lacking lppA replicated less efficiently in phytate-loaded Acanthamoeba castellanii or Dictyostelium discoideum, and the intracellular growth defect was complemented by the phytase gene. These findings identify the chelator phytate as an intracellular bacteriostatic component of cell-autonomous host immunity and reveal a T4SS-translocated L. pneumophila phytase that counteracts intracellular bacterial growth restriction by phytate. Thus, bacterial phytases might represent therapeutic targets to combat intracellular pathogens.

Microfluidic technology has opened new possibilities for the crystallization of biological macromolecules during the past decade. Microfluidic systems offer numerous advantages over conventional crystal growth methods. They enable easy handling of nanovolumes of solutions, extreme miniaturization, and parallelization of crystallization assays, especially for high-throughput screening applications. Our goal was to design a versatile, low cost, and easy-to-use crystallization chip based on counter-diffusion that is compatible with on-chip crystallographic characterization. The ChipX is a microfluidic chip made of cyclic olefin copolymer. It was used to grow crystals of biomolecules and perform complete X-ray diffraction analyses on synchrotron sources. Our results demonstrate that accurate crystallographic data can be collected at room temperature directly from ChipX microfluidic devices for both experimental single-wavelength anomalous dispersion phasing and structure refinement.

Coiled coils are well suited to drive subunit oligomerization and are widely used in applications ranging from basic research to medicine. The optimization of these applications requires a detailed understanding of the molecular determinants that control of coiled-coil formation. Although many of these determinants have been identified and characterized in great detail, a puzzling observation is that their presence does not necessarily correlate with the oligomerization state of a given coiled-coil structure. Thus, other determinants must play a key role. To address this issue, we recently investigated the unrelated coiled-coil domains from GCN4, ATF1 and cortexillin-1 as model systems. We found that well-known trimer-specific oligomerization-state determinants, such as the distribution of isoleucine residues at heptad-repeat core positions or the trimerization motif Arg-h-x-x-h-Glu (where h = hydrophobic amino acid; x = any amino acid), switch the peptide’s topology from a dimer to a trimer only when inserted into the trigger sequence, a site indispensable for coiled-coil formation. Because high-resolution structural information could not be obtained for the full-length, three-stranded cortexillin-1 coiled coil, we here report the detailed biophysical and structural characterization of a shorter variant spanning the trigger sequence using circular dichroism, anatytical ultracentrifugation and x-ray crystallography. We show that the peptide forms a stable α-helical trimer in solution. We further determined the crystal structure of an optimised variant at a resolution of 1.65 Å, revealing that the peptide folds into a parallel, three-stranded coiled coil. The two complemented R-IxxIE trimerization motifs and the additional hydrophobic core isoleucine residue adopt the conformations seen in other extensively characterized parallel, three-stranded coiled coils. These findings not only confirm the structural basis for the switch in oligomerization state from a dimer to a trimer observed for the full-length cortexillin-1 coiled-coil domain, but also provide further evidence for a general link between oligomerization-state specificity and trigger-sequence function. ISSN:1932-6203

Receptor agonism remains poorly understood at the molecular and mechanistic level. In this study, we identified a fully human anti-Fas antibody that could efficiently trigger apoptosis and therefore function as a potent agonist. Protein engineering and crystallography were used to mechanistically understand the agonistic activity of the antibody. The crystal structure of the complex was determined at 1.9 A resolution and provided insights into epitope recognition and comparisons with the natural ligand FasL (Fas ligand). When we affinity-matured the agonist antibody, we observed that, surprisingly, the higher-affinity antibodies demonstrated a significant reduction, rather than an increase, in agonist activity at the Fas receptor. We propose and experimentally demonstrate a model to explain this non-intuitive impact of affinity on agonist antibody signalling and explore the implications for the discovery of therapeutic agonists in general.

Asparagine-linked glycosylation is a common posttranslational modification of diverse secretory and membrane proteins in eukaryotes, where it is catalyzed by the multiprotein complex oligosaccharyltransferase. The functions of the protein subunits of oligoasccharyltransferase, apart from the catalytic Stt3p, are ill defined. Here we describe functional and structural investigations of the Ost3/6p components of the yeast enzyme. Genetic, biochemical and structural analyses of the lumenal domain of Ost6p revealed oxidoreductase activity mediated by a thioredoxin-like fold with a distinctive active-site loop that changed conformation with redox state. We found that mutation of the active-site cysteine residues of Ost6p and its paralogue Ost3p affected the glycosylation efficiency of a subset of glycosylation sites. Our results show that eukaryotic oligosaccharyltransferase is a multifunctional enzyme that acts at the crossroads of protein modification and protein folding.

Disulfide bond formation in the Escherichia coli periplasm requires the transfer of electrons from substrate proteins to DsbA, which is recycled as an oxidant by the membrane protein DsbB. The highly virulent, uropathogenic E. coli strain CFT073 contains a second, homologous pair of proteins, DsbL and DsbI, which are encoded in a tri-cistronic operon together with a periplasmic, uropathogen-specific arylsulfate sulfotransferase (ASST). We show that DsbL and DsbI form a functional redox pair, and that ASST is a substrate of DsbL/DsbI in vivo. DsbL is the most reactive oxidizing thioredoxin-like protein known to date. In contrast to DsbA, however, DsbL oxidizes reduced RNaseA with a much lower rate and prevents unspecific aggregation of reduced insulin. The 1.55 A resolution crystal structure of reduced DsbL provides insight into the reduced state of thioredoxin-like dithiol oxidases at high resolution, and reveals an unusual cluster of basic residues stabilizing the thiolate anion of the nucleophilic active-site cysteine. We propose that the DsbL/DsbI pair of uropathogenic E. coli was acquired as an additional, specific redox couple that guarantees biological activity of ASST.

Cellular and Molecular Life Sciences, 63 (14), ISSN:1420-682X, ISSN:1420-9071

Escherichia coli DsbD transports electrons from cytoplasmic thioredoxin to periplasmic target proteins. DsbD is composed of an N-terminal (nDsbD) and a C-terminal (cDsbD) periplasmic domain, connected by a central transmembrane domain. Each domain possesses two cysteine residues essential for electron transport. The transport proceeds via disulfide exchange reactions from cytoplasmic thioredoxin to the central transmembrane domain and via cDsbD to nDsbD, which then reduces the periplasmic target proteins. We determined four high-resolution structures of cDsbD: oxidized (1.65 A resolution), chemically reduced (1.3 A), photo-reduced (1.1 A) and chemically reduced at pH increased from 4.6 to 7. The latter structure was refined at 0.99 A resolution, the highest achieved so far for a thioredoxin superfamily member. The data reveal unprecedented structural details of cDsbD, demonstrating that the domain is very rigid and undergoes hardly any conformational change upon disulfide reduction or interaction with nDsbD. In full agreement with the crystallographic results, guanidinium chloride-induced unfolding and refolding experiments indicate that oxidized and reduced cDsbD are equally stable. We confirmed the structural rigidity of cDsbD by molecular dynamics simulations. A remarkable feature of cDsbD is the p K a of 9.3 for the active site Cys461: this value, determined using two different experimental methods, surprisingly was around 2.5 units higher than expected on the basis of the redox potential. Additionally, taking advantage of the very high quality of the cDsbD structures, we carried out p K a calculations, which gave results in agreement with the experimental findings. In conclusion, our wide-scope analysis of cDsbD, encompassing atomic-resolution crystallography, computational chemistry and biophysical measurements, highlighted two so far unrecognized key aspects of this domain: its unusual redox properties and extreme rigidity. Both are likely to be correlated to the role of cDsbD as a covalently linked electron shuttle between the membrane domain and the N-terminal periplasmic domain of DsbD.

Summary DsbD from Escherichia coli transports two electrons from cytoplasmic thioredoxin to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of an N-terminal periplasmic domain (nDsbD), a C-terminal periplasmic domain, and a central transmembrane domain. Each domain possesses two cysteines required for electron transport. Herein, we demonstrate fast (3.9 × 10 5 M −1 s −1 ) and direct disulfide exchange between nDsbD and CcmG, a highly specific disulfide reductase essential for cytochrome c maturation. We determined the crystal structure of the disulfide-linked complex between nDsbD and the soluble part of CcmG at 1.94 A resolution. In contrast to the other two known complexes of nDsbD with target proteins, the N-terminal segment of nDsbD contributes to specific recognition of CcmG. This and other features, like the possibility of using an additional interaction surface, constitute the structural basis for the adaptability of nDsbD to different protein substrates.