Identification of drugs including a dopamine receptor antagonist that selectively target cancer stem cells.

Selective targeting of cancer stem cells (CSCs) offers promise for a new generation of therapeutics. However, assays for both human CSCs and normal stem cells that are amenable to robust biological screens are limited. Using a discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells (hPSC), we identify small molecules from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal. Surprisingly, thioridazine, an antipsychotic drug, selectively targets the neoplastic cells, and impairs human somatic CSCs capable of in vivo leukemic disease initiation while having no effect on normal blood SCs. The drug antagonizes dopamine receptors that are expressed on CSCs and on breast cancer cells as well. These results suggest that dopamine receptors may serve as a biomarker for diverse malignancies, demonstrate the utility of using neoplastic hPSCs for identifying CSC-targeting drugs, and provide support for the use of differentiation as a therapeutic strategy.

Evolution-guided discovery of antibiotics that inhibit peptidoglycan remodelling.

Addressing the ongoing antibiotic crisis requires the discovery of compounds with novel mechanisms of action that are capable of treating drug-resistant infections1. Many antibiotics are sourced from specialized metabolites produced by bacteria, particularly those of the Actinomycetes family2. Although actinomycete extracts have traditionally been screened using activity-based platforms, this approach has become unfavourable owing to the frequent rediscovery of known compounds. Genome sequencing of actinomycetes reveals an untapped reservoir of biosynthetic gene clusters, but prioritization is required to predict which gene clusters may yield promising new chemical matter2. Here we make use of the phylogeny of biosynthetic genes along with the lack of known resistance determinants to predict divergent members of the glycopeptide family of antibiotics that are likely to possess new biological activities. Using these predictions, we uncovered two members of a new functional class of glycopeptide antibiotics-the known glycopeptide antibiotic complestatin and a newly discovered compound we call corbomycin-that have a novel mode of action. We show that by binding to peptidoglycan, complestatin and corbomycin block the action of autolysins-essential peptidoglycan hydrolases that are required for remodelling of the cell wall during growth. Corbomycin and complestatin have low levels of resistance development and are effective in reducing bacterial burden in a mouse model of skin MRSA infection.

Author Correction: Direct conversion of human fibroblasts to multilineage blood progenitors.

In this Article, there were duplicated empty lanes in Supplementary Figs. 2e and 3b. The corrected figures are presented in the Supplementary Information to the accompanying Amendment. The original Article has not been corrected.

Distinct Molecular Features of NleG Type 3 Secreted Effectors Allow for Different Roles during Citrobacter rodentium Infection in Mice.

The NleGs are the largest family of type 3 secreted effectors in attaching and effacing (A/E) pathogens, such as enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and Citrobacter rodentium. NleG effectors contain a conserved C-terminal U-box domain acting as a ubiquitin protein ligase and target host proteins via a variable N-terminal portion. The specific roles of these effectors during infection remain uncertain. Here, we demonstrate that the three NleG effectors-NleG1Cr, NleG7Cr, and NleG8Cr-encoded by C. rodentium DBS100 play distinct roles during infection in mice. Using individual nleGCr knockout strains, we show that NleG7Cr contributes to bacterial survival during enteric infection while NleG1Cr promotes the expression of diarrheal symptoms and NleG8Cr contributes to accelerated lethality in susceptible mice. Furthermore, the NleG8Cr effector contains a C-terminal PDZ domain binding motif that enables interaction with the host protein GOPC. Both the PDZ domain binding motif and the ability to engage with host ubiquitination machinery via the intact U-box domain proved to be necessary for NleG8Cr function, contributing to the observed phenotype during infection. We also establish that the PTZ binding motif in the EHEC NleG8 (NleG8Ec) effector, which shares 60% identity with NleG8Cr, is engaged in interactions with human GOPC. The crystal structure of the NleG8Ec C-terminal peptide in complex with the GOPC PDZ domain, determined to 1.85 Å, revealed a conserved interaction mode similar to that observed between GOPC and eukaryotic PDZ domain binding motifs. Despite these common features, nleG8Ec does not complement the ΔnleG8Cr phenotype during infection, revealing functional diversification between these NleG effectors.

Low dietary fiber promotes enteric expansion of a Crohn's disease-associated pathobiont independent of obesity.

Obesity is associated with metabolic, immunological, and infectious disease comorbidities, including an increased risk of enteric infection and inflammatory bowel disease such as Crohn's disease (CD). Expansion of intestinal pathobionts such as adherent-invasive Escherichia coli (AIEC) is a common dysbiotic feature of CD, which is amplified by prior use of oral antibiotics. Although high-fat, high-sugar diets are associated with dysbiotic expansion of E. coli, it is unknown if the content of fat or another dietary component in obesogenic diets is sufficient to promote AIEC expansion. Here, we found that administration of an antibiotic combined with feeding mice an obesogenic low-fiber, high-sucrose, high-fat diet (HFD) that is typically used in rodent-obesity studies promoted AIEC intestinal expansion. Even a short-term (i.e., 1 day) pulse of HFD feeding before infection was sufficient to promote AIEC expansion, indicating that the magnitude of obesity was not the main driver of AIEC expansion. Controlled-diet experiments demonstrated that neither dietary fat nor sugar were the key determinants of AIEC colonization, but that lowering dietary fiber from approximately 13% to 5%-6% was sufficient to promote the intestinal expansion of AIEC when combined with antibiotics in mice. When combined with antibiotics, lowering fiber promoted AIEC intestinal expansion to a similar extent as widely used HFDs in mice. However, lowering dietary fiber was sufficient to promote AIEC intestinal expansion without affecting body mass. Our results show that low dietary fiber combined with oral antibiotics are environmental factors that promote the expansion of Crohn's disease-associated pathobionts in the gut.NEW & NOTEWORTHY It is commonly thought that obesity or a high-fat diet alters pathogenic bacteria and promotes inflammatory gut diseases. We found that lower dietary fiber is a key factor that expands a gut pathobiont linked to Crohn's disease, independent of obesity status in mice.

Cellular Reprogramming Allows Generation of Autologous Hematopoietic Progenitors From AML Patients That Are Devoid of Patient-Specific Genomic Aberrations.

Current treatments that use hematopoietic progenitor cell (HPC) transplantation in acute myeloid leukemia (AML) patients substantially reduce the risk of relapse, but are limited by the availability of immune compatible healthy HPCs. Although cellular reprogramming has the potential to provide a novel autologous source of HPCs for transplantation, the applicability of this technology toward the derivation of healthy autologous hematopoietic cells devoid of patient-specific leukemic aberrations from AML patients must first be evaluated. Here, we report the generation of human AML patient-specific hematopoietic progenitors that are capable of normal in vitro differentiation to myeloid lineages and are devoid of leukemia-associated aberration found in matched patient bone marrow. Skin fibroblasts were obtained from AML patients whose leukemic cells possessed a distinct, leukemia-associated aberration, and used to create AML patient-specific induced pluripotent stem cells (iPSCs). Through hematopoietic differentiation of AML patient iPSCs, coupled with cytogenetic interrogation, we reveal that AML patient-specific HPCs possess normal progenitor capacity and are devoid of leukemia-associated mutations. Importantly, in rare patient skin samples that give rise to mosaic fibroblast cultures that continue to carry leukemia-associated mutations; healthy hematopoietic progenitors can also be generated via reprogramming selection. Our findings provide the proof of principle that cellular reprogramming can be applied on a personalized basis to generate healthy HPCs from AML patients, and should further motivate advances toward creating transplantable hematopoietic stem cells for autologous AML therapy.

Direct conversion of human fibroblasts to multilineage blood progenitors.

As is the case for embryo-derived stem cells, application of reprogrammed human induced pluripotent stem cells is limited by our understanding of lineage specification. Here we demonstrate the ability to generate progenitors and mature cells of the haematopoietic fate directly from human dermal fibroblasts without establishing pluripotency. Ectopic expression of OCT4 (also called POU5F1)-activated haematopoietic transcription factors, together with specific cytokine treatment, allowed generation of cells expressing the pan-leukocyte marker CD45. These unique fibroblast-derived cells gave rise to granulocytic, monocytic, megakaryocytic and erythroid lineages, and demonstrated in vivo engraftment capacity. We note that adult haematopoietic programs are activated, consistent with bypassing the pluripotent state to generate blood fate: this is distinct from haematopoiesis involving pluripotent stem cells, where embryonic programs are activated. These findings demonstrate restoration of multipotency from human fibroblasts, and suggest an alternative approach to cellular reprogramming for autologous cell-replacement therapies that avoids complications associated with the use of human pluripotent stem cells.

In vivo generation of neural tumors from neoplastic pluripotent stem cells models early human pediatric brain tumor formation.

Recent studies have identified gene signatures in malignant tumors that are associated with human embryonic stem cells, suggesting a molecular relationship between aggressive cancers and pluripotency. Here, we characterize neural precursors (NPs) derived from transformed human embryonic stem cells (N-t-hESCs) that exhibit neoplastic features of human brain tumors. NPs derived from t-hESCs have enhanced cell proliferation and an inability to mature toward the astrocytic lineage, compared with progeny derived from normal human embryonic stem cells (N-hESCs) independent of adherent or neurosphere outgrowth. Intracranial transplantation of NPs derived from N-t-hESCs and N-hESCs into NOD SCID mice revealed development of neuroectoderm tumors exclusively from the N-t-hESCs NPs and not from normal N-hESCs. These tumors infiltrated the ventricles and the cerebellum of recipient mice and displayed morphological, phenotypic, and molecular features associated with classic medulloblastoma including retention of a pluripotent signature. Importantly, N-t-hESCs did not exhibit cytogenetic changes associated with medulloblastoma, suggesting that aberrant cellular and molecular properties precede the acquisition of karyotypic changes thus underscoring the value of this model system of human medulloblastoma. Our study demonstrates that NPs from a starting population of neoplastic human pluripotent parent cells possess brain tumor-initiating cell capacity, thereby providing a model system to investigate initiation and progression of primitive human neural cancers that are difficult to assess using somatic sources.

Synthetic Biology Facilitates Semisynthetic Development of Type V Glycopeptide Antibiotics Targeting Vancomycin-Resistant Enterococcus.

The continued efficacy of glycopeptide antibiotics (GPAs) against Gram-positive bacteria is challenged by the emergence and spread of GPA-resistant pathogens, particularly vancomycin-resistant enterococci (VRE). The growing frequency of GPA resistance propels the need for innovative development of more effective antibiotics. Unlike canonical GPAs like vancomycin, Type V GPAs adopt a distinct mode of action by binding peptidoglycan and blocking the activity of autolysins essential for cell division, rendering them a promising class of antibiotics for further development. In this study, the Type V GPA, rimomycin A, was modified to generate 32 new analogues. Compound 17, derived from rimomycin A through N-terminal acylation and C-terminal amidation, exhibited improved anti-VRE activity and solubility. In a VRE-A neutropenic thigh infection mouse model, compound 17 significantly lowered the bacterial load by 3-4 orders of magnitude. This study sets the stage to develop next-generation GPAs in response to growing VRE infections.

Genetic and Chemical Screening in Human Blood Serum Reveals Unique Antibacterial Targets and Compounds against Klebsiella pneumoniae.

Antibiotics halt the growth of bacteria by targeting core, essential physiology that is required for life on standard microbiological media. Many more biochemical and virulence processes, however, are required for bacteria to cause infection in a host. Indeed, chemical inhibitors of the latter processes are overlooked using conventional antibiotic drug discovery approaches. Here, we use human blood serum as an alternative growth medium to explore new targets and compounds. High-throughput screening of genetic and chemical libraries identified compounds targeting biological activities required by Klebsiella pneumoniae to grow in serum, such as nucleobase biosynthesis and iron acquisition, and showed that serum can chemically transform compounds to reveal cryptic antibacterial activity. One of these compounds, ruthenium red, was effective in a rat bloodstream infection model. Our data demonstrate that human serum is an effective tool to find new chemical matter to address the current antibiotic resistance crisis.

Complete Genome Sequence of Citrobacter rodentium Strain DBS100.

Citrobacter rodentium strain DBS100 causes an infection of the intestines in mice. It provides an important model for human gastrointestinal pathogens, such as enteropathogenic and enterohemorrhagic Escherichia coli, which cause life-threatening infections. To identify the genetic determinants that are common across the enteropathogenic bacteria, we sequenced the DBS100 genome.

Antibiotics Potentiate Adherent-Invasive E. coli Infection and Expansion.

BACKGROUND: Crohn's disease (CD) is an inflammatory bowel disease with a complex etiology. Paradoxically, CD is associated with the use of antibiotics and with an increased abundance of an unusual phenotypic group of Escherichia coli known as adherent-invasive E. coli (AIEC). However, the impact of antibiotics on AIEC infection has not been well studied in controlled models of infection. METHODS: We infected mice with AIEC before or after treatment with a variety of different classes of antibiotics. We assessed levels of AIEC in the feces and tissues, AIEC localization by immunofluorescence microscopy, and tissue pathology. RESULTS: We found that a wide range of antibiotic classes strongly potentiated initial AIEC infection and expanded AIEC in chronically infected mice. We found that the ability of antibiotics to potentiate AIEC infection did not correlate with a stereotyped shift in the gut bacterial community but was correlated with a decrease in overall diversity and a divergence from the pre-antibiotic state. We found that antibiotic-induced inflammation provided a fitness advantage for AIEC expansion through their use of oxidized metabolites in the postantibiotic period. CONCLUSIONS: Our results show that antibiotics can render hosts more susceptible to initial AIEC infection and can worsen infection in previously colonized hosts. AIEC appears to exploit host inflammatory responses that arise in the postantibiotic period, highlighting a previously unknown interaction between CD risk factors.

Overcoming mcr-1 mediated colistin resistance with colistin in combination with other antibiotics.

Plasmid-borne colistin resistance mediated by mcr-1 may contribute to the dissemination of pan-resistant Gram-negative bacteria. Here, we show that mcr-1 confers resistance to colistin-induced lysis and bacterial cell death, but provides minimal protection from the ability of colistin to disrupt the Gram-negative outer membrane. Indeed, for colistin-resistant strains of Enterobacteriaceae expressing plasmid-borne mcr-1, clinically relevant concentrations of colistin potentiate the action of antibiotics that, by themselves, are not active against Gram-negative bacteria. The result is that several antibiotics, in combination with colistin, display growth-inhibition at levels below their corresponding clinical breakpoints. Furthermore, colistin and clarithromycin combination therapy displays efficacy against mcr-1-positive Klebsiella pneumoniae in murine thigh and bacteremia infection models at clinically relevant doses. Altogether, these data suggest that the use of colistin in combination with antibiotics that are typically active against Gram-positive bacteria poses a viable therapeutic alternative for highly drug-resistant Gram-negative pathogens expressing mcr-1.

Lineage-Specific Differentiation Is Influenced by State of Human Pluripotency.

Human pluripotent stem cells (hPSCs) have been reported in naive and primed states. However, the ability to generate mature cell types remains the imperative property for utility of hPSCs. Here, we reveal that the naive state enhances self-renewal while restricting lineage differentiation in vitro to neural default fate. Molecular analyses indicate expression of multiple lineage-associated transcripts in naive hPSCs that failed to predict biased functional differentiation capacity. Naive hPSCs can be converted to primed state over long-term serial passage that permits recovery of multi-germ layer differentiation. Suppression of OCT4 but not NANOG allows immediate recovery directly from naive state. To this end, we identified chemical inhibitors of OCT4 that restore naive hPSC differentiation. Our study reveals unique cell-fate restrictions in human pluripotent states and provides an approach to overcome these barriers that harness both efficient naive hPSC growth while maintaining in vitro differentiation essential for hPSC applications.

GSK3 Deficiencies in Hematopoietic Stem Cells Initiate Pre-neoplastic State that Is Predictive of Clinical Outcomes of Human Acute Leukemia.

Initial pathway alternations required for pathogenesis of human acute myeloid leukemia (AML) are poorly understood. Here we reveal that removal of glycogen synthase kinase-3α (GSK-3α) and GSK-3ß dependency leads to aggressive AML. Although GSK-3α deletion alone has no effect, GSK-3ß deletion in hematopoietic stem cells (HSCs) resulted in a pre-neoplastic state consistent with human myelodysplastic syndromes (MDSs). Transcriptome and functional studies reveal that each GSK-3ß and GSK-3α uniquely contributes to AML by affecting Wnt/Akt/mTOR signaling and metabolism, respectively. The molecular signature of HSCs deleted for GSK-3ß provided a prognostic tool for disease progression and survival of MDS patients. Our study reveals that GSK-3α- and GSK-3ß-regulated pathways can be responsible for stepwise transition to MDS and subsequent AML, thereby providing potential therapeutic targets of disease evolution.

Derivation of human induced pluripotent stem cells through continued exposure of OCT4-induced plastic human fibroblasts to reprogramming media.

The combination of OCT4 expression and short-term exposure to reprogramming media induces a state of transcriptional plasticity in human fibroblasts, capable of responding to changes in the extracellular environment. Here we provide characterization of iPSCs established through continued culture of OCT4-induced plastic human fibroblasts in pluripotent-supportive reprogramming media. Human iPSC(OCT4) are morphologically indistinguishable from conventionally derived iPSCs and express core proteins involved in maintenance of pluripotency. iPSC(OCT4) display bona fide functional pluripotency as measured by in vivo teratoma formation consisting of the three germ layers.

Acquisition of pluripotency through continued environmental influence on OCT4-induced plastic human fibroblasts.

The combination of OCT4 expression and short-term exposure to reprogramming media induces a state of transcriptional plasticity in human fibroblasts, capable of responding to changes in the extracellular environment that facilitate direct cell fate conversion toward lineage specific progenitors. Here we reveal that continued exposure of OCT4-induced plastic human fibroblasts to reprogramming media (RM) is sufficient to induce pluripotency. OCT4-derived induced pluripotent stem cell (iPSC(OCT4)) colonies emerged after prolonged culture in RM, and formed independently of lineage specific progenitors. Human iPSC(OCT4) are morphologically indistinguishable from conventionally derived iPSCs and express core proteins involved in maintenance of pluripotency. iPSC(OCT4) display in vivo functional pluripotency as measured by teratoma formation consisting of the three germ layers, and are capable of targeted in vitro differentiation. Our study indicates that acquisition of pluripotency is one of multiple cell fate choices that can be facilitated through environmental stimulation of OCT4-induced plasticity, and suggests the role of other reprogramming factors to induce pluripotency can be substituted by prolonged culture of plastic fibroblasts.

Single Transcription Factor Conversion of Human Blood Fate to NPCs with CNS and PNS Developmental Capacity.

The clinical applicability of direct cell fate conversion depends on obtaining tissue from patients that is easy to harvest, store, and manipulate for reprogramming. Here, we generate induced neural progenitor cells (iNPCs) from neonatal and adult peripheral blood using single-factor OCT4 reprogramming. Unlike fibroblasts that share molecular hallmarks of neural crest, OCT4 reprogramming of blood was facilitated by SMAD+GSK-3 inhibition to overcome restrictions on neural fate conversion. Blood-derived (BD) iNPCs differentiate in vivo and respond to guided differentiation in vitro, producing glia (astrocytes and oligodendrocytes) and multiple neuronal subtypes, including dopaminergic (CNS related) and nociceptive neurons (peripheral nervous system [PNS]). Furthermore, nociceptive neurons phenocopy chemotherapy-induced neurotoxicity in a system suitable for high-throughput drug screening. Our findings provide an easily accessible approach for generating human NPCs that harbor extensive developmental potential, enabling the study of clinically relevant neural diseases directly from patient cohorts.

Somatic transcriptome priming gates lineage-specific differentiation potential of human-induced pluripotent stem cell states.

Human-induced pluripotent stem cells (hiPSCs) provide an invaluable source for regenerative medicine, but are limited by proficient lineage-specific differentiation. Here we reveal that hiPSCs derived from human fibroblasts (Fibs) versus human cord blood (CB) exhibit indistinguishable pluripotency, but harbour biased propensities for differentiation. Genes associated with germ layer specification were identical in Fib- or CB-derived iPSCs, whereas lineage-specific marks emerge upon differentiation induction of hiPSCs that were correlated to the cell of origin. Differentiation propensities come at the expense of other lineages and cannot be overcome with stimuli for alternative cell fates. Although incomplete DNA methylation and distinct histone modifications of lineage-specific loci correlate to lineage-specific transcriptome priming, transitioning hiPSCs into naive state of pluripotency removes iPSC-memorized transcriptome. Upon re-entry to the primed state, transcriptome memory is restored, indicating a human-specific phenomenon whereby lineage gated developmental potential is not permanently erased, but can be modulated by the pluripotent state.