Patient-derived models recapitulate heterogeneity of molecular signatures and drug response in pediatric high-grade glioma.

Pediatric high-grade glioma (pHGG) is a major contributor to cancer-related death in children. In vitro and in vivo disease models reflecting the intimate connection between developmental context and pathogenesis of pHGG are essential to advance understanding and identify therapeutic vulnerabilities. Here we report establishment of 21 patient-derived pHGG orthotopic xenograft (PDOX) models and eight matched cell lines from diverse groups of pHGG. These models recapitulate histopathology, DNA methylation signatures, mutations and gene expression patterns of the patient tumors from which they were derived, and include rare subgroups not well-represented by existing models. We deploy 16 new and existing cell lines for high-throughput screening (HTS). In vitro HTS results predict variable in vivo response to PI3K/mTOR and MEK pathway inhibitors. These unique new models and an online interactive data portal for exploration of associated detailed molecular characterization and HTS chemical sensitivity data provide a rich resource for pediatric brain tumor research.

Ehrlichia chaffeensis TRP120 nucleomodulin binds DNA with disordered tandem repeat domain.

Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis, secretes several effector proteins that bind host DNA to modulate host gene expression. The tandem repeat protein 120 (TRP120), one of the largest effector proteins, has four nearly identical tandem repeat (TR) regions that each consists of 80 amino acids. In addition to playing a role in ehrlichial binding and internalization, TRP120 translocates to the host nucleus where it is thought to function as a transcription factor that modulates gene expression. However, sequence analysis of TRP120 does not identify the presence of DNA-binding or trans-activation domains typical of classical eukaryotic transcription factors. Thus, the mechanism by which TRP120 binds DNA and modulates gene expression remains elusive. Herein, we expressed the TR regions of the TRP120 protein, and characterized its solution structure and ability to bind DNA. TRP120, expressed as either a one or two TR repeat, is a monomer in solution, and is mostly disordered as determined by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Using NMR spectroscopy, we further show that the 1 TR construct selectively binds GC-rich DNA. Although low pH was required for TRP120 TR-DNA interaction, acidic pH alone does not induce any significant structural changes in the TR region. This suggests that TRP120 folds into an ordered structure upon forming a protein-DNA complex, and thus folding of TRP120 TR is coupled with DNA binding.

Ehrlichia chaffeensis TRP120 Effector Targets and Recruits Host Polycomb Group Proteins for Degradation To Promote Intracellular Infection.

Ehrlichia chaffeensis has a group of well-characterized type I secreted tandem repeat protein (TRP) effectors that have moonlighting capabilities. TRPs modulate various cellular processes, reprogram host gene transcription as nucleomodulins, function as ubiquitin ligases, and directly activate conserved host cell signaling pathways to promote E. chaffeensis infection. One TRP-interacting host target is polycomb group ring finger protein 5 (PCGF5), a member of the polycomb group (PcG) protein family and a component of the polycomb repressive complex 1 (PRC1). The current study demonstrates that during early infection, PCGF5 strongly colocalizes with TRP120 in the nucleus and later dramatically redistributes to the ehrlichial vacuole along with other PCGF isoforms. Ectopic expression and immunoprecipitation of TRP120 confirmed the interaction of TRP120 with multiple different PCGF isoforms. At 48 h postinfection, a dramatic redistribution of PCGF isoforms from the nucleus to the ehrlichial vacuole was observed, which also temporally coincided with proteasomal degradation of PCGF isoforms and TRP120 expression on the vacuole. A decrease in PRC1-mediated repressive chromatin mark and an altered transcriptional activity in PRC1-associated Hox genes primarily from HOXB and HOXC clusters were observed along with the degradation of PCGF isoforms, suggesting disruption of the PRC1 in E. chaffeensis-infected cells. Notably, small interfering RNA (siRNA)-mediated knockdown of PCGF isoforms resulted in significantly increased E. chaffeensis infection. This study demonstrates a novel strategy in which E. chaffeensis manipulates PRC complexes through interactions between TRP120 and PCGF isoforms to promote infection.

Ehrlichia chaffeensis Tandem Repeat Effector Targets Differentially Influence Infection.

Ehrlichia chaffeensis infects mononuclear phagocytes and survives intracellularly by exploiting host cell processes to evade host defenses. The mechanisms involved are not fully defined, but appear to rely largely on a subset of tandem repeat proteins (TRP) effectors. E. chaffeensis TRPs are type 1 secreted effectors that interact with a functionally diverse group of host cell targets associated with various biological processes. In this study, we investigated the influence of TRP host target proteins on ehrlichial infection by RNA interference. In total, 138 TRP-interacting host proteins identified by yeast two-hybrid were targeted by siRNA and the infection level determined by real-time qPCR. Knockdown of 124 (89%) TRP target proteins had significant influence on infection either by inhibiting (85%) or promoting (15%) ehrlichial infection. Notably, knockdown of 18 host proteins which interacted with TRP120 promoted the infection, suggesting that these targets may be degraded to promote infection. Host proteins that interact with TRPs are involved in cellular processes, including cell signaling, vesicle trafficking and intracellular transport, transcriptional regulation, metabolism, protein posttranslational modification, and apoptosis. Selected host targets were examined by immunofluorescent microscopy during infection and were found to localize with the morulae, or in the host cell cytoplasm adjacent to morulae. This study confirms that the majority of host proteins known to interact with TRP effectors influence infection and further extends the current knowledge that E. chaffeensis TRPs participate in a complex array of host protein interactions in order to reprogram the host cell and promote intracellular survival.

Ehrlichia chaffeensis TRP32 is a Nucleomodulin that Directly Regulates Expression of Host Genes Governing Differentiation and Proliferation.

Ehrlichia chaffeensis is an obligately intracellular bacterium that reprograms the mononuclear phagocyte through diverse effector-host interactions to modulate numerous host cell processes, including transcription. In a previous study, we reported that E. chaffeensis TRP32, a type 1 secreted effector, interacts with multiple host nucleus-associated proteins and also auto-activates reporter gene expression in yeast. In this study, we demonstrate that TRP32 is a nucleomodulin that binds host DNA and alters host gene transcription. TRP32 enters the host cell nucleus via a noncanonical translocation mechanism that involves phosphorylation of Y179 located in a C-terminal tri-tyrosine motif. Both genistein and mutation of Y179 inhibited TRP32 nuclear entry. An electromobility shift assay (EMSA) demonstrated TRP32 host DNA binding via its tandem repeat domain. TRP32 DNA binding and motif preference were further confirmed by supershift assays, as well as competition and mutant probe analyses. Using ChIP-Seq, we determined that TRP32 binds a G-rich motif primarily within ±500 bp of the gene transcription start site. An ontology analysis identified genes involved in processes such as immune cell differentiation, chromatin remodeling, and RNA transcription and processing, as primary TRP32 targets. TRP32 bound genes (n=1223) were distributed on all chromosomes and included several global regulators of proliferation and inflammation such as FOS and JUN, AKT3 and NRAS, and non-coding RNA genes, miRNA 21 and miRNA 142. TRP32 target genes were differentially regulated during infection, the majority of which were repressed, and direct repression/activation of these genes by TRP32 was confirmed in vitro with a cellular luciferase reporter assay.

Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival.

UNLABELLED: Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival. IMPORTANCE: E. chaffeensis is an obligately intracellular bacterium and the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection and avoids innate host defenses are not understood, but functionally relevant host-pathogen interactions with type 1 secreted TRP effectors are essential for the ehrlichial cellular reprogramming strategy. This study provides further insight into the molecular strategies used by obligately intracellular pathogens such as E. chaffeensis, which have small genomes and a limited number of effector proteins and exploit evolutionarily conserved host cell programs such as Notch signaling to promote infection and intracellular survival.

Ehrlichia chaffeensis Exploits Canonical and Noncanonical Host Wnt Signaling Pathways To Stimulate Phagocytosis and Promote Intracellular Survival.

Ehrlichia chaffeensis invades and survives in phagocytes by modulating host cell processes and evading innate defenses, but the mechanisms are not fully defined. Recently we have determined that E. chaffeensis tandem repeat proteins (TRPs) are type 1 secreted effectors involved in functionally diverse interactions with host targets, including components of the evolutionarily conserved Wnt signaling pathways. In this study, we demonstrated that induction of host canonical and noncanonical Wnt pathways by E. chaffeensis TRP effectors stimulates phagocytosis and promotes intracellular survival. After E. chaffeensis infection, canonical and noncanonical Wnt signalings were significantly stimulated during early stages of infection (1 to 3 h) which coincided with dephosphorylation and nuclear translocation of ß-catenin, a major canonical Wnt signal transducer, and NFATC1, a noncanonical Wnt transcription factor. In total, the expression of ∼44% of Wnt signaling target genes was altered during infection. Knockdown of TRP120-interacting Wnt pathway components/regulators and other critical components, such as Wnt5a ligand, Frizzled 5 receptor, ß-catenin, nuclear factor of activated T cells (NFAT), and major signaling molecules, resulted in significant reductions in the ehrlichial load. Moreover, small-molecule inhibitors specific for components of canonical and noncanonical (Ca(2+) and planar cell polarity [PCP]) Wnt pathways, including IWP-2, which blocks Wnt secretion, significantly decreased ehrlichial infection. TRPs directly activated Wnt signaling, as TRP-coated microspheres triggered phagocytosis which was blocked by Wnt pathway inhibitors, demonstrating a key role of TRP activation of Wnt pathways to induce ehrlichial phagocytosis. These novel findings reveal that E. chaffeensis exploits canonical and noncanonical Wnt pathways through TRP effectors to facilitate host cell entry and promote intracellular survival.