The impact of phosphorylated PTEN at threonine 366 on cortical connectivity and behaviour.

The lipid phosphatase PTEN (phosphatase and tensin homologue on chromosome 10) is a key tumour suppressor gene and an important regulator of neuronal signalling. PTEN mutations have been identified in patients with autism spectrum disorders, characterized by macrocephaly, impaired social interactions and communication, repetitive behaviour, intellectual disability, and epilepsy. PTEN enzymatic activity is regulated by a cluster of phosphorylation sites at the C-terminus of the protein. Here, we focused on the role of PTEN T366 phosphorylation and generated a knock-in mouse line in which Pten T366 was substituted with alanine (PtenT366A/T366A). We identify that phosphorylation of PTEN at T366 controls neuron size and connectivity of brain circuits involved in sensory processing. We show in behavioural tests that PtenT366/T366A mice exhibit cognitive deficits and selective sensory impairments, with significant differences in male individuals. We identify restricted cellular overgrowth of cortical neurons in PtenT366A/T366A brains, linked to increases in both dendritic arborization and soma size. In a combinatorial approach of anterograde and retrograde monosynaptic tracing using rabies virus, we characterize differences in connectivity to the primary somatosensory cortex of PtenT366A/T366A brains, with imbalances in long-range cortico-cortical input to neurons. We conclude that phosphorylation of PTEN at T366 controls neuron size and connectivity of brain circuits involved in sensory processing and propose that PTEN T366 signalling may account for a subset of autism-related functions of PTEN.

PTEN deficiency leads to proteasome addiction: a novel vulnerability in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor in adults with a median survival of approximately 15 months; therefore, more effective treatment options for GBM are required. To identify new drugs targeting GBMs, we performed a high-throughput drug screen using patient-derived neurospheres cultured to preferentially retain their glioblastoma stem cell (GSC) phenotype. METHODS: High-throughput drug screening was performed on GSCs followed by a dose-response assay of the 5 identified original "hits." A PI3K/mTOR dependency to a proteasome inhibitor (carfilzomib), was confirmed by genetic and pharmacologic experiments. Proteasome Inhibition Response Signatures were derived from proteomic and bioinformatic analysis. Molecular mechanism of action was determined using three-dimensional (3D) GBM-organoids and preclinical orthotopic models. RESULTS: We found that GSCs were highly sensitive to proteasome inhibition due to an underlying dependency on an increased protein synthesis rate, and loss of autophagy, associated with PTEN loss and activation of the PI3K/mTOR pathway. In contrast, combinatory inhibition of autophagy and the proteasome resulted in enhanced cytotoxicity specifically in GSCs that did express PTEN. Finally, proteasome inhibition specifically increased cell death markers in 3D GBM-organoids, suppressed tumor growth, and increased survival of mice orthotopically engrafted with GSCs. As perturbations of the PI3K/mTOR pathway occur in nearly 50% of GBMs, these findings suggest that a significant fraction of these tumors could be vulnerable to proteasome inhibition. CONCLUSIONS: Proteasome inhibition is a potential synthetic lethal therapeutic strategy for GBM with proteasome addiction due to a high protein synthesis rate and autophagy deficiency.

Author Correction: Longitudinal assessment of tumor development using cancer avatars derived from genetically engineered pluripotent stem cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Longitudinal assessment of tumor development using cancer avatars derived from genetically engineered pluripotent stem cells.

Many cellular models aimed at elucidating cancer biology do not recapitulate pathobiology including tumor heterogeneity, an inherent feature of cancer that underlies treatment resistance. Here we introduce a cancer modeling paradigm using genetically engineered human pluripotent stem cells (hiPSCs) that captures authentic cancer pathobiology. Orthotopic engraftment of the neural progenitor cells derived from hiPSCs that have been genome-edited to contain tumor-associated genetic driver mutations revealed by The Cancer Genome Atlas project for glioblastoma (GBM) results in formation of high-grade gliomas. Similar to patient-derived GBM, these models harbor inter-tumor heterogeneity resembling different GBM molecular subtypes, intra-tumor heterogeneity, and extrachromosomal DNA amplification. Re-engraftment of these primary tumor neurospheres generates secondary tumors with features characteristic of patient samples and present mutation-dependent patterns of tumor evolution. These cancer avatar models provide a platform for comprehensive longitudinal assessment of human tumor development as governed by molecular subtype mutations and lineage-restricted differentiation.

A proteome-wide screen to identify transcription factors interacting with the Vibrio cholerae rpoS promoter.

We describe a proteomic approach to identify transcription factors binding to a target promoter. The method's usefulness was tested by identifying proteins binding to the Vibrio cholerae rpoS promoter in response to cell density. Proteins identified in this screen included the nucleoid-associated protein Fis and the quorum sensing regulator HapR.

Inhibition of Nuclear PTEN Tyrosine Phosphorylation Enhances Glioma Radiation Sensitivity through Attenuated DNA Repair.

Ionizing radiation (IR) and chemotherapy are standard-of-care treatments for glioblastoma (GBM) patients and both result in DNA damage, however, the clinical efficacy is limited due to therapeutic resistance. We identified a mechanism of such resistance mediated by phosphorylation of PTEN on tyrosine 240 (pY240-PTEN) by FGFR2. pY240-PTEN is rapidly elevated and bound to chromatin through interaction with Ki-67 in response to IR treatment and facilitates the recruitment of RAD51 to promote DNA repair. Blocking Y240 phosphorylation confers radiation sensitivity to tumors and extends survival in GBM preclinical models. Y240F-Pten knockin mice showed radiation sensitivity. These results suggest that FGFR-mediated pY240-PTEN is a key mechanism of radiation resistance and is an actionable target for improving radiotherapy efficacy.

Chromatin Immunoprecipitation.

Chromatin immunoprecipitation (ChIP) measures the physical association between a protein and DNA in the cell. In combination with next-generation sequencing, the technique enables the identification of DNA targets for the corresponding protein across an entire genome. Here we describe the immunoprecipitation of Vibrio cholerae DNA bound to the histone-like nucleoid structuring protein (H-NS) tagged with the Flag epitope. The quality of the DNA obtained in this protocol is suitable for next-generation sequencing. The procedure described herein can be readily adapted to other bacteria and DNA-binding proteins.

YAP and MRTF-A, transcriptional co-activators of RhoA-mediated gene expression, are critical for glioblastoma tumorigenicity.

The role of YAP (Yes-associated protein 1) and MRTF-A (myocardin-related transcription factor A), two transcriptional co-activators regulated downstream of GPCRs (G protein-coupled receptors) and RhoA, in the growth of glioblastoma cells and in vivo glioblastoma multiforme (GBM) tumor development was explored using human glioblastoma cell lines and tumor-initiating cells derived from patient-derived xenografts (PDX). Knockdown of these co-activators in GSC-23 PDX cells using short hairpin RNA significantly attenuated in vitro self-renewal capability assessed by limiting dilution, oncogene expression, and neurosphere formation. Orthotopic xenografts of the MRTF-A and YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild-type cells. In vitro studies used PDX and 1321N1 glioblastoma cells to examine functional responses to sphingosine 1-phosphate (S1P), a GPCR agonist that activates RhoA signaling, demonstrated that YAP signaling was required for cell migration and invasion, whereas MRTF-A was required for cell adhesion; both YAP and MRTF-A were required for proliferation. Gene expression analysis by RNA-sequencing of S1P-treated MRTF-A or YAP knockout cells identified 44 genes that were induced through RhoA and highly dependent on YAP, MRTF-A, or both. Knockdown of F3 (tissue factor (TF)), a target gene regulated selectively through YAP, blocked cell invasion and migration, whereas knockdown of HBEGF (heparin-binding epidermal growth factor-like growth factor), a gene selectively induced through MRTF-A, prevented cell adhesion in response to S1P. Proliferation was sensitive to knockdown of target genes regulated through either or both YAP and MRTF-A. Expression of TF and HBEGF was also selectively decreased in tumors from PDX cells lacking YAP or MRTF-A, indicating that these transcriptional pathways are regulated in preclinical GBM models and suggesting that their activation through GPCRs and RhoA contributes to growth and maintenance of human GBM.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Deletion of gene encoding the nucleoid-associated protein H-NS unmasks hidden regulatory connections in El Tor biotype Vibrio cholerae.

Hypervirulent atypical El Tor biotype Vibrio cholerae O1 isolates harbour mutations in the DNA-binding domain of the nucleoid-associated protein H-NS and the receiver domain of the response regulator VieA. Here, we provide two examples in which inactivation of H-NS in El Tor biotype vibrios unmasks hidden regulatory connections. First, deletion of the helix-turn-helix domain of VieA in an hns mutant background diminished biofilm formation and exopolysaccharide gene expression, a function that phenotypically opposes its phosphodiesterase activity. Second, deletion of vieA in an hns mutant diminished the expression of σE, a virulence determinant that mediates the envelope stress response. hns mutants were highly sensitive to envelope stressors compared to wild-type. However, deletion of vieA in the hns mutant restored or exceeded wild-type resistance. These findings suggest an evolutionary path for the emergence of hypervirulent strains starting from nucleotide sequence diversification affecting the interaction of H-NS with DNA.

Publisher Correction: PTEN regulates glioblastoma oncogenesis through chromatin-associated complexes of DAXX and histone H3.3.

This corrects the article DOI: 10.1038/ncomms15223.

A simple cell-based high throughput screening (HTS) assay for inhibitors of Salmonella enterica RNA polymerase containing the general stress response regulator RpoS (σS).

RNA polymerase containing the stress response regulator σS subunit (RpoS) plays a key role in bacterial survival in hostile environments in nature and during infection. Here we devise and validate a simple cell-based high throughput luminescence assay for this holoenzyme suitable for screening large chemical libraries in a robotic platform.

Molecular basis for the differential expression of the global regulator VieA in Vibrio cholerae biotypes directed by H-NS, LeuO and quorum sensing.

VieA is a cyclic diguanylate phosphodiesterase that modulates biofilm development and motility in Vibrio cholerae O1 of the classical biotype. vieA is part of an operon encoding the VieSAB signal transduction pathway that is nearly silent in V. cholerae of the El Tor biotype. A DNA pull-down assay for proteins interacting with the vieSAB promoter identified the LysR-type regulator LeuO. We show that in classical biotype V. cholerae, LeuO cooperates with the nucleoid-associated protein H-NS to repress vieSAB transcription. LeuO and H-NS interacted with the vieSAB promoter of both biotypes with similar affinities and protected overlapping DNA sequences. H-NS was expressed at similar levels in both cholera biotypes. In contrast, El Tor biotype strains expressed negligible LeuO under identical conditions. In El Tor biotype vibrios, transcription of vieSAB is repressed by the quorum sensing regulator HapR, which is absent in classical biotype strains. Restoring HapR expression in classical biotype V. cholerae repressed vieSAB transcription by binding to its promoter. We propose that double locking of the vieSAB promoter by H-NS and HapR in the El Tor biotype prior to the cessation of exponential growth results in a more pronounced decline in VieA specific activity compared to the classical biotype.

Flagellar motility, extracellular proteases and Vibrio cholerae detachment from abiotic and biotic surfaces.

Vibrio cholerae of serogroups O1 and O139, the causative agent of Asiatic cholera, continues to be a major global health threat. This pathogen utilizes substratum-specific pili to attach to distinct surfaces in the aquatic environment and the human small intestine and detaches when conditions become unfavorable. Both attachment and detachment are critical to bacterial environmental survival, pathogenesis and disease transmission. However, the factors that promote detachment are less understood. In this study, we examine the role of flagellar motility and hemagglutinin/protease (HapA) in vibrio detachment from a non-degradable abiotic surface and from the suckling mouse intestine. Flagellar motility facilitated V. cholerae detachment from abiotic surfaces. HapA had no effect on the stability of biofilms formed on abiotic surfaces despite representing >50% of the proteolytic activity present in the extracellular matrix. We developed a balanced lethal plasmid system to increase the bacterial cyclic diguanylate (c-di-GMP) pool late in infection, a condition that represses motility and HapA expression. Increasing the c-di-GMP pool enhanced V. cholerae colonization of the suckling mouse intestine. The c-di-GMP effect was fully abolished in hapA isogenic mutants. These results suggest that motility facilitates detachment in a substratum-independent manner. Instead, HapA appears to function as a substratum-specific detachment factor.

Glioblastoma cellular cross-talk converges on NF-κB to attenuate EGFR inhibitor sensitivity.

In glioblastoma (GBM), heterogeneous expression of amplified and mutated epidermal growth factor receptor (EGFR) presents a substantial challenge for the effective use of EGFR-directed therapeutics. Here we demonstrate that heterogeneous expression of the wild-type receptor and its constitutively active mutant form, EGFRvIII, limits sensitivity to these therapies through an interclonal communication mechanism mediated by interleukin-6 (IL-6) cytokine secreted from EGFRvIII-positive tumor cells. IL-6 activates a NF-κB signaling axis in a paracrine and autocrine manner, leading to bromodomain protein 4 (BRD4)-dependent expression of the prosurvival protein survivin (BIRC5) and attenuation of sensitivity to EGFR tyrosine kinase inhibitors (TKIs). NF-κB and survivin are coordinately up-regulated in GBM patient tumors, and functional inhibition of either protein or BRD4 in in vitro and in vivo models restores sensitivity to EGFR TKIs. These results provide a rationale for improving anti-EGFR therapeutic efficacy through pharmacological uncoupling of a convergence point of NF-κB-mediated survival that is leveraged by an interclonal circuitry mechanism established by intratumoral mutational heterogeneity.

PTEN regulates glioblastoma oncogenesis through chromatin-associated complexes of DAXX and histone H3.3.

Glioblastoma (GBM) is the most lethal type of human brain cancer, where deletions and mutations in the tumour suppressor gene PTEN (phosphatase and tensin homolog) are frequent events and are associated with therapeutic resistance. Herein, we report a novel chromatin-associated function of PTEN in complex with the histone chaperone DAXX and the histone variant H3.3. We show that PTEN interacts with DAXX and, in turn PTEN directly regulates oncogene expression by modulating DAXX-H3.3 association on the chromatin, independently of PTEN enzymatic activity. Furthermore, DAXX inhibition specifically suppresses tumour growth and improves the survival of orthotopically engrafted mice implanted with human PTEN-deficient glioma samples, associated with global H3.3 genomic distribution changes leading to upregulation of tumour suppressor genes and downregulation of oncogenes. Moreover, DAXX expression anti-correlates with PTEN expression in GBM patient samples. Since loss of chromosome 10 and PTEN are common events in cancer, this synthetic growth defect mediated by DAXX suppression represents a therapeutic opportunity to inhibit tumorigenesis specifically in the context of PTEN deletion.

3-Amino 1,8-naphthalimide, a structural analog of the anti-cholera drug virstatin inhibits chemically-biased swimming and swarming motility in vibrios.

A screen for inhibitors of Vibrio cholerae motility identified the compound 3-amino 1,8-naphthalimide (3-A18NI), a structural analog of the cholera drug virstatin. Similar to virstatin, 3-A18NI diminished cholera toxin production. In contrast, 3-A18NI impeded swimming and/or swarming motility of V. cholerae and V. parahemolyticus suggesting that it could target the chemotaxis pathway shared by the polar and lateral flagellar system of vibrios. 3-A18NI did not inhibit the expression of V. cholerae major flagellin FlaA or the assembly of its polar flagellum. Finally, 3-A18NI enhanced V. cholerae colonization mimicking the phenotype of chemotaxis mutants that exhibit counterclockwise-biased flagellum rotation.

H-NS: an overarching regulator of the Vibrio cholerae life cycle.

Vibrio cholerae has become a model organism for studies connecting virulence, pathogen evolution and infectious disease ecology. The coordinate expression of motility, virulence and biofilm enhances its pathogenicity, environmental fitness and fecal-oral transmission. The histone-like nucleoid structuring protein negatively regulates gene expression at multiple phases of the V. cholerae life cycle. Here we discuss: (i) the regulatory and structural implications of H-NS chromatin-binding in the two-chromosome cholera bacterium; (ii) the factors that counteract H-NS repression; and (iii) a model for the regulation of the V. cholerae life cycle that integrates H-NS repression, cyclic diguanylic acid signaling and the general stress response.