Publications by authors named "Peter van Galen"

25 Publications

  • Page 1 of 1

Single-Cell RNA Sequencing to Disentangle the Blood System.

Arterioscler Thromb Vasc Biol 2021 03 14;41(3):1012-1018. Epub 2021 Jan 14.

Division of Hematology, Brigham and Women's Hospital, Boston, MA. Department of Medicine, Harvard Medical School, Boston, MA. Broad Institute of MIT and Harvard, Cambridge, MA.

The blood system is often represented as a tree-like structure with stem cells that give rise to mature blood cell types through a series of demarcated steps. Although this representation has served as a model of hierarchical tissue organization for decades, single-cell technologies are shedding new light on the abundance of cell type intermediates and the molecular mechanisms that ensure balanced replenishment of differentiated cells. In this Brief Review, we exemplify new insights into blood cell differentiation generated by single-cell RNA sequencing, summarize considerations for the application of this technology, and highlight innovations that are leading the way to understand hematopoiesis at the resolution of single cells. Graphic Abstract: A graphic abstract is available for this article.
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http://dx.doi.org/10.1161/ATVBAHA.120.314654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901535PMC
March 2021

Single-cell lineage analysis reveals genetic and epigenetic interplay in glioblastoma drug resistance.

Genome Biol 2020 07 15;21(1):174. Epub 2020 Jul 15.

Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Background: Tumors can evolve and adapt to therapeutic pressure by acquiring genetic and epigenetic alterations that may be transient or stable. A precise understanding of how such events contribute to intratumoral heterogeneity, dynamic subpopulations, and overall tumor fitness will require experimental approaches to prospectively label, track, and characterize resistant or otherwise adaptive populations at the single-cell level. In glioblastoma, poor efficacy of receptor tyrosine kinase (RTK) therapies has been alternatively ascribed to genetic heterogeneity or to epigenetic transitions that circumvent signaling blockade.

Results: We combine cell lineage barcoding and single-cell transcriptomics to trace the emergence of drug resistance in stem-like glioblastoma cells treated with RTK inhibitors. Whereas a broad variety of barcoded lineages adopt a Notch-dependent persister phenotype that sustains them through early drug exposure, rare subclones acquire genetic changes that enable their rapid outgrowth over time. Single-cell analyses reveal that these genetic subclones gain copy number amplifications of the insulin receptor substrate-1 and substrate-2 (IRS1 or IRS2) loci, which activate insulin and AKT signaling programs. Persister-like cells and genomic amplifications of IRS2 and other loci are evident in primary glioblastomas and may underlie the inefficacy of targeted therapies in this disease.

Conclusions: A method for combined lineage tracing and scRNA-seq reveals the interplay between complementary genetic and epigenetic mechanisms of resistance in a heterogeneous glioblastoma tumor model.
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http://dx.doi.org/10.1186/s13059-020-02085-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7364565PMC
July 2020

Decoding the Noncoding Cancer Genome.

Authors:
Peter van Galen

Cancer Discov 2020 May;10(5):646-647

Division of Hematology, Brigham and Women's Hospital, Boston, Massachusetts. Department of Medicine, Harvard Medical School, Boston, Massachusetts. Broad Institute of MIT and Harvard, Cambridge, Massachusetts.

In this issue of , Li and colleagues provide a blueprint for the identification and functional validation of cancer-associated mutations in noncoding regions of the genome. Integration of whole-genome sequencing and high-throughput epigenome editing screens is starting to reveal the extent to which noncoding genetic lesions contribute to cancer..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0285DOI Listing
May 2020

Chromatin accessibility promotes hematopoietic and leukemia stem cell activity.

Nat Commun 2020 03 16;11(1):1406. Epub 2020 Mar 16.

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

Chromatin organization is a highly orchestrated process that influences gene expression, in part by modulating access of regulatory factors to DNA and nucleosomes. Here, we report that the chromatin accessibility regulator HMGN1, a target of recurrent DNA copy gains in leukemia, controls myeloid differentiation. HMGN1 amplification is associated with increased accessibility, expression, and histone H3K27 acetylation of loci important for hematopoietic stem cells (HSCs) and leukemia, such as HoxA cluster genes. In vivo, HMGN1 overexpression is linked to decreased quiescence and increased HSC activity in bone marrow transplantation. HMGN1 overexpression also cooperates with the AML-ETO9a fusion oncoprotein to impair myeloid differentiation and enhance leukemia stem cell (LSC) activity. Inhibition of histone acetyltransferases CBP/p300 relieves the HMGN1-associated differentiation block. These data nominate factors that modulate chromatin accessibility as regulators of HSCs and LSCs, and suggest that targeting HMGN1 or its downstream effects on histone acetylation could be therapeutically active in AML.
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http://dx.doi.org/10.1038/s41467-020-15221-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076002PMC
March 2020

Single-Cell RNA-Seq Reveals AML Hierarchies Relevant to Disease Progression and Immunity.

Cell 2019 03 28;176(6):1265-1281.e24. Epub 2019 Feb 28.

Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA; Ludwig Center at Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA. Electronic address:

Acute myeloid leukemia (AML) is a heterogeneous disease that resides within a complex microenvironment, complicating efforts to understand how different cell types contribute to disease progression. We combined single-cell RNA sequencing and genotyping to profile 38,410 cells from 40 bone marrow aspirates, including 16 AML patients and five healthy donors. We then applied a machine learning classifier to distinguish a spectrum of malignant cell types whose abundances varied between patients and between subclones in the same tumor. Cell type compositions correlated with prototypic genetic lesions, including an association of FLT3-ITD with abundant progenitor-like cells. Primitive AML cells exhibited dysregulated transcriptional programs with co-expression of stemness and myeloid priming genes and had prognostic significance. Differentiated monocyte-like AML cells expressed diverse immunomodulatory genes and suppressed T cell activity in vitro. In conclusion, we provide single-cell technologies and an atlas of AML cell states, regulators, and markers with implications for precision medicine and immune therapies. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2019.01.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6515904PMC
March 2019

A stemness screen reveals as a promoter of human leukemia stem cell latency.

Blood 2019 05 22;133(20):2198-2211. Epub 2019 Feb 22.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.

There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene , in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34 cells, accumulation of cells in G, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, -OE resulted in the expansion of self-renewing LSCs. In contrast, silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that regulates G exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.
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http://dx.doi.org/10.1182/blood-2018-10-881441DOI Listing
May 2019

A novel method for detecting the cellular stemness state in normal and leukemic human hematopoietic cells can predict disease outcome and drug sensitivity.

Leukemia 2019 08 31;33(8):2061-2077. Epub 2019 Jan 31.

Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv-Yafo, Israel.

Acute leukemia is an aggressive blood malignancy with low survival rates. A high expression of stem-like programs in leukemias predicts poor prognosis and is assumed to act in an aberrant fashion in the phenotypically heterogeneous leukemia stem cell (LSC) population. A lack of suitable genome engineering tools that can isolate LSCs based on their stemness precludes their comprehensive examination and full characterization. We hypothesized that tagging endogenous stemness-regulatory regions could generate a genome reporter for the putative leukemia stemness-state. Our analysis revealed that the ERG  + 85 enhancer region can serve as a marker for stemness-state and a fluorescent lentiviral reporter was developed that can accurately recapitulate the endogenous activity. Using our novel reporter, we revealed cellular heterogeneity in several leukemia cell lines and patient-derived samples. Alterations in reporter activity were associated with transcriptomic and functional changes that were closely related to the hematopoietic stem cell (HSC) identity. Notably, the differentiation potential was skewed towards the erythro-megakaryocytic lineage. Moreover, an ERG  + 85 fraction of AML cells could regenerate the original cellular heterogeneity and was enriched for LSCs. RNA-seq analysis coupled with in silico drug-screen analysis identified 4HPR as an effective inhibitor of ERG  + 85 leukemia growth. We propose that further utilization of our novel molecular tool will identify crucial determinants of LSCs, thus providing a rationale for their therapeutic targeting.
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http://dx.doi.org/10.1038/s41375-019-0386-zDOI Listing
August 2019

Integrated Stress Response Activity Marks Stem Cells in Normal Hematopoiesis and Leukemia.

Cell Rep 2018 10;25(5):1109-1117.e5

Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 1L7, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Lifelong maintenance of the blood system requires equilibrium between clearance of damaged hematopoietic stem cells (HSCs) and long-term survival of the HSC pool. Severe perturbations of cellular homeostasis result in rapid HSC loss to maintain clonal purity. However, normal homeostatic processes can also generate lower-level stress; how HSCs survive these conditions remains unknown. Here we show that the integrated stress response (ISR) is uniquely active in HSCs and facilitates their persistence. Activating transcription factor 4 (ATF4) mediates the ISR and is highly expressed in HSCs due to scarcity of the eIF2 translation initiation complex. Amino acid deprivation results in eIF2α phosphorylation-dependent upregulation of ATF4, promoting HSC survival. Primitive acute myeloid leukemia (AML) cells also display eIF2 scarcity and ISR activity marks leukemia stem cells (LSCs) in primary AML samples. These findings identify a link between the ISR and stem cell survival in the normal and leukemic contexts.
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http://dx.doi.org/10.1016/j.celrep.2018.10.021DOI Listing
October 2018

Developmental and oncogenic programs in H3K27M gliomas dissected by single-cell RNA-seq.

Science 2018 04;360(6386):331-335

Department of Pathology and Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.

Gliomas with histone H3 lysine27-to-methionine mutations (H3K27M-glioma) arise primarily in the midline of the central nervous system of young children, suggesting a cooperation between genetics and cellular context in tumorigenesis. Although the genetics of H3K27M-glioma are well characterized, their cellular architecture remains uncharted. We performed single-cell RNA sequencing in 3321 cells from six primary H3K27M-glioma and matched models. We found that H3K27M-glioma primarily contain cells that resemble oligodendrocyte precursor cells (OPC-like), whereas more differentiated malignant cells are a minority. OPC-like cells exhibit greater proliferation and tumor-propagating potential than their more differentiated counterparts and are at least in part sustained by signaling. Our study characterizes oncogenic and developmental programs in H3K27M-glioma at single-cell resolution and across genetic subclones, suggesting potential therapeutic targets in this disease.
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http://dx.doi.org/10.1126/science.aao4750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5949869PMC
April 2018

Dual Targeting of Oncogenic Activation and Inflammatory Signaling Increases Therapeutic Efficacy in Myeloproliferative Neoplasms.

Cancer Cell 2018 01 14;33(1):29-43.e7. Epub 2017 Dec 14.

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box 20, New York, NY 10065, USA; Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Center for Hematologic Malignancies, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Leukemia Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Electronic address:

Genetic and functional studies underscore the central role of JAK/STAT signaling in myeloproliferative neoplasms (MPNs). However, the mechanisms that mediate transformation in MPNs are not fully delineated, and clinically utilized JAK inhibitors have limited ability to reduce disease burden or reverse myelofibrosis. Here we show that MPN progenitor cells are characterized by marked alterations in gene regulation through differential enhancer utilization, and identify nuclear factor κB (NF-κB) signaling as a key pathway activated in malignant and non-malignant cells in MPN. Inhibition of BET bromodomain proteins attenuated NF-κB signaling and reduced cytokine production in vivo. Most importantly, combined JAK/BET inhibition resulted in a marked reduction in the serum levels of inflammatory cytokines, reduced disease burden, and reversed bone marrow fibrosis in vivo.
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http://dx.doi.org/10.1016/j.ccell.2017.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760343PMC
January 2018

Adaptive Chromatin Remodeling Drives Glioblastoma Stem Cell Plasticity and Drug Tolerance.

Cell Stem Cell 2017 02 15;20(2):233-246.e7. Epub 2016 Dec 15.

Department of Pathology and Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA; Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. Electronic address:

Glioblastoma, the most common and aggressive malignant brain tumor, is propagated by stem-like cancer cells refractory to existing therapies. Understanding the molecular mechanisms that control glioblastoma stem cell (GSC) proliferation and drug resistance may reveal opportunities for therapeutic interventions. Here we show that GSCs can reversibly transition to a slow-cycling, persistent state in response to targeted kinase inhibitors. In this state, GSCs upregulate primitive developmental programs and are dependent upon Notch signaling. This transition is accompanied by widespread redistribution of repressive histone methylation. Accordingly, persister GSCs upregulate, and are dependent on, the histone demethylases KDM6A/B. Slow-cycling cells with high Notch activity and histone demethylase expression are present in primary glioblastomas before treatment, potentially contributing to relapse. Our findings illustrate how cancer cells may hijack aspects of native developmental programs for deranged proliferation, adaptation, and tolerance. They also suggest strategies for eliminating refractory tumor cells by targeting epigenetic and developmental pathways.
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http://dx.doi.org/10.1016/j.stem.2016.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291795PMC
February 2017

miR-126 Regulates Distinct Self-Renewal Outcomes in Normal and Malignant Hematopoietic Stem Cells.

Cancer Cell 2016 Feb 28;29(2):214-28. Epub 2016 Jan 28.

Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, ON M5G 1L7, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5G 1L7, Canada; Princess Margaret Cancer Research Tower, Room 8-301, 101 College Street, Toronto M5G 1L7, Canada. Electronic address:

To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance.
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http://dx.doi.org/10.1016/j.ccell.2015.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4749543PMC
February 2016

A Multiplexed System for Quantitative Comparisons of Chromatin Landscapes.

Mol Cell 2016 Jan 10;61(1):170-80. Epub 2015 Dec 10.

Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Center for Cancer Research, Massachusetts General Hospital, Boston, MA 02114, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Electronic address:

Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here, we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of p300, EZH2, or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions, and drug treatments.
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http://dx.doi.org/10.1016/j.molcel.2015.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4707994PMC
January 2016

EHMT1 and EHMT2 inhibition induces fetal hemoglobin expression.

Blood 2015 Oct 28;126(16):1930-9. Epub 2015 Aug 28.

Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Boston, MA; Harvard Medical School, Boston, MA; Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA;

Fetal hemoglobin (HbF, α2γ2) induction is a well-validated strategy for sickle cell disease (SCD) treatment. Using a small-molecule screen, we found that UNC0638, a selective inhibitor of EHMT1 and EHMT2 histone methyltransferases, induces γ-globin expression. EHMT1/2 catalyze mono- and dimethylation of lysine 9 on histone 3 (H3K9), raising the possibility that H3K9Me2, a repressive chromatin mark, plays a role in silencing γ-globin expression. In primary human adult erythroid cells, UNC0638 and EHMT1 or EHMT2 short hairpin RNA-mediated knockdown significantly increased γ-globin expression, HbF synthesis, and the percentage of cells expressing HbF. At effective concentrations, UNC0638 did not alter cell morphology, proliferation, or erythroid differentiation of primary human CD34(+) hematopoietic stem and progenitor cells in culture ex vivo. In murine erythroleukemia cells, UNC0638 and Ehmt2 CRISPR/Cas9-mediated knockout both led to a marked increase in expression of embryonic β-globin genes Hbb-εy and Hbb-βh1. In primary human adult erythroblasts, chromatin immunoprecipitation followed by sequencing analysis revealed that UNC0638 treatment leads to genome-wide depletion in H3K9Me2 and a concomitant increase in the activating mark H3K9Ac, which was especially pronounced at the γ-globin gene region. In RNA-sequencing analysis of erythroblasts, γ-globin genes were among the most significantly upregulated genes by UNC0638. Further increase in γ-globin expression in primary human adult erythroid cells was achieved by combining EHMT1/2 inhibition with the histone deacetylase inhibitor entinostat or hypomethylating agent decitabine. Our data provide genetic and pharmacologic evidence that EHMT1 and EHMT2 are epigenetic regulators involved in γ-globin repression and represent a novel therapeutic target for SCD.
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http://dx.doi.org/10.1182/blood-2015-06-649087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608240PMC
October 2015

Two new routes to make blood: Hematopoietic specification from pluripotent cell lines versus reprogramming of somatic cells.

Exp Hematol 2015 Sep 2;43(9):756-9. Epub 2015 Jul 2.

Stem Cell Transplantation Program, Division of Hematology and Oncology, Boston Children's Hospital, Harvard Medical School, Harvard Stem Cell Institute, Cambridge, MA, USA.

Transplantation of hematopoietic stem cells (HSCs) to treat hematologic disorders is routinely used in the clinic. However, HSC therapy is hindered by the requirements of finding human leukocyte antigen (HLA)-matched donors and attaining sufficient numbers of long-term HSCs in the graft. Therefore, ex vivo expansion of transplantable HSCs remains one of the "holy grails" of hematology. Without the ability to maintain and expand human HSCs in vitro, two complementary approaches involving cellular reprogramming to generate transplantable HSCs have emerged. Reprogrammed HSCs represent a potentially inexhaustible supply of autologous tissue. On March 18th, 2015, Dr. George Q. Daley and Dr. Derrick J. Rossi, two pioneers in the field, presented and discussed their most recent research on these topics in a webinar organized by the International Society for Experimental Hematology (ISEH). Here, we summarize these seminars and discuss the possibilities and challenges in the field of hematopoietic specification.
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http://dx.doi.org/10.1016/j.exphem.2015.05.007DOI Listing
September 2015

Isolation and identification of 4-α-rhamnosyloxy benzyl glucosinolate in Noccaea caerulescens showing intraspecific variation.

Phytochemistry 2015 Feb 4;110:166-71. Epub 2014 Dec 4.

Molecular Interaction Ecology, Institute of Water and Wetland Research (IWWR), Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands; German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e, 04103 Leipzig, Germany; Friedrich Schiller University Jena, Institute of Ecology, Dornburger-Str. 159, 07743 Jena, Germany.

Glucosinolates are secondary plant compounds typically found in members of the Brassicaceae and a few other plant families. Usually each plant species contains a specific subset of the ∼ 130 different glucosinolates identified to date. However, intraspecific variation in glucosinolate profiles is commonly found. Sinalbin (4-hydroxybenzyl glucosinolate) so far has been identified as the main glucosinolate of the heavy metal accumulating plant species Noccaea caerulescens (Brassicaceae). However, a screening of 13 N. caerulescens populations revealed that in 10 populations a structurally related glucosinolate was found as the major component. Based on nuclear magnetic resonance (NMR) and mass spectrometry analyses of the intact glucosinolate as well as of the products formed after enzymatic conversion by sulfatase or myrosinase, this compound was identified as 4-α-rhamnosyloxy benzyl glucosinolate (glucomoringin). So far, glucomoringin had only been reported as the main glucosinolate of Moringa spp. (Moringaceae) which are tropical tree species. There was no apparent relation between the level of soil pollution at the location of origin, and the presence of glucomoringin. The isothiocyanate that is formed after conversion of glucomoringin is a potent antimicrobial and antitumor agent. It has yet to be established whether glucomoringin or its breakdown product have an added benefit to the plant in its natural habitat.
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http://dx.doi.org/10.1016/j.phytochem.2014.11.016DOI Listing
February 2015

The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.

Nature 2014 Jun 28;510(7504):268-72. Epub 2014 Apr 28.

1] Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario M5G 2M9, Canada [2] Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage. Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis. Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis. Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the co-chaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells.
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http://dx.doi.org/10.1038/nature13228DOI Listing
June 2014

Reduced lymphoid lineage priming promotes human hematopoietic stem cell expansion.

Cell Stem Cell 2014 Jan;14(1):94-106

Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 1L7, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5G 1L7, Canada. Electronic address:

The hematopoietic system sustains regeneration throughout life by balancing self-renewal and differentiation. To stay poised for mature blood production, hematopoietic stem cells (HSCs) maintain low-level expression of lineage-associated genes, a process termed lineage priming. Here, we modulated expression levels of Inhibitor of DNA binding (ID) proteins to ask whether lineage priming affects self-renewal of human HSCs. We found that lentiviral overexpression of ID proteins in cord blood HSCs biases myeloerythroid commitment at the expense of lymphoid differentiation. Conversely, reducing ID2 expression levels increases lymphoid potential. Mechanistically, ID2 inhibits the transcription factor E47 to attenuate B-lymphoid priming in HSCs and progenitors. Strikingly, ID2 overexpression also results in a 10-fold expansion of HSCs in serial limiting dilution assays, indicating that early lymphoid transcription factors antagonize human HSC self-renewal. The relationship between lineage priming and self-renewal can be exploited to increase expansion of transplantable human HSCs and points to broader implications for other stem cell populations.
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http://dx.doi.org/10.1016/j.stem.2013.11.021DOI Listing
January 2014

Self-renewal as a therapeutic target in human colorectal cancer.

Nat Med 2014 Jan 1;20(1):29-36. Epub 2013 Dec 1.

1] Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada. [2] Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. [3] Department of Surgery, Toronto General Hospital, Toronto, Ontario, Canada. [4].

Tumor recurrence following treatment remains a major clinical challenge. Evidence from xenograft models and human trials indicates selective enrichment of cancer-initiating cells (CICs) in tumors that survive therapy. Together with recent reports showing that CIC gene signatures influence patient survival, these studies predict that targeting self-renewal, the key 'stemness' property unique to CICs, may represent a new paradigm in cancer therapy. Here we demonstrate that tumor formation and, more specifically, human colorectal CIC function are dependent on the canonical self-renewal regulator BMI-1. Downregulation of BMI-1 inhibits the ability of colorectal CICs to self-renew, resulting in the abrogation of their tumorigenic potential. Treatment of primary colorectal cancer xenografts with a small-molecule BMI-1 inhibitor resulted in colorectal CIC loss with long-term and irreversible impairment of tumor growth. Targeting the BMI-1-related self-renewal machinery provides the basis for a new therapeutic approach in the treatment of colorectal cancer.
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http://dx.doi.org/10.1038/nm.3418DOI Listing
January 2014

Combination of pantothenamides with vanin inhibitors as a novel antibiotic strategy against gram-positive bacteria.

Antimicrob Agents Chemother 2013 Oct 22;57(10):4794-800. Epub 2013 Jul 22.

Department of Dermatology and Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

The emergence of resistance against current antibiotics calls for the development of new compounds to treat infectious diseases. Synthetic pantothenamides are pantothenate analogs that possess broad-spectrum antibacterial activity in vitro in minimal media. Pantothenamides were shown to be substrates of the bacterial coenzyme A (CoA) biosynthetic pathway, causing cellular CoA depletion and interference with fatty acid synthesis. In spite of their potential use and selectivity for bacterial metabolic routes, these compounds have never made it to the clinic. In the present study, we show that pantothenamides are not active as antibiotics in the presence of serum, and we found that they were hydrolyzed by ubiquitous pantetheinases of the vanin family. To address this further, we synthesized a series of pantetheinase inhibitors based on a pantothenate scaffold that inhibited serum pantetheinase activity in the nanomolar range. Mass spectrometric analysis showed that addition of these pantetheinase inhibitors prevented hydrolysis of pantothenamides by serum. We found that combinations of these novel pantetheinase inhibitors and prototypic pantothenamides like N5-Pan and N7-Pan exerted antimicrobial activity in vitro, particularly against Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Streptococcus pyogenes) even in the presence of serum. These results indicate that pantothenamides, when protected against degradation by host pantetheinases, are potentially useful antimicrobial agents.
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http://dx.doi.org/10.1128/AAC.00603-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811400PMC
October 2013

Variable clonal repopulation dynamics influence chemotherapy response in colorectal cancer.

Science 2013 Feb 13;339(6119):543-8. Epub 2012 Dec 13.

Campbell Family Institute, Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

Intratumoral heterogeneity arises through the evolution of genetically diverse subclones during tumor progression. However, it remains unknown whether cells within single genetic clones are functionally equivalent. By combining DNA copy number alteration (CNA) profiling, sequencing, and lentiviral lineage tracking, we followed the repopulation dynamics of 150 single lentivirus-marked lineages from 10 human colorectal cancers through serial xenograft passages in mice. CNA and mutational analysis distinguished individual clones and showed that clones remained stable upon serial transplantation. Despite this stability, the proliferation, persistence, and chemotherapy tolerance of lentivirally marked lineages were variable within each clone. Chemotherapy promoted the dominance of previously minor or dormant lineages. Thus, apart from genetic diversity, tumor cells display inherent functional variability in tumor propagation potential, which contributes to both cancer growth and therapy tolerance.
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http://dx.doi.org/10.1126/science.1227670DOI Listing
February 2013

Attenuation of miR-126 activity expands HSC in vivo without exhaustion.

Cell Stem Cell 2012 Dec 8;11(6):799-811. Epub 2012 Nov 8.

Campbell Family Institute, Ontario Cancer Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 1L7, Canada.

Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks, making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function.
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http://dx.doi.org/10.1016/j.stem.2012.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517970PMC
December 2012

Stem cell gene expression programs influence clinical outcome in human leukemia.

Nat Med 2011 Aug 28;17(9):1086-93. Epub 2011 Aug 28.

Division of Stem Cell and Developmental Biology, Campbell Family Institute for Cancer Research, Ontario Cancer Institute, University Health Network and Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

Xenograft studies indicate that some solid tumors and leukemias are organized as cellular hierarchies sustained by cancer stem cells (CSCs). Despite the promise of the CSC model, its relevance in humans remains uncertain. Here we show that acute myeloid leukemia (AML) follows a CSC model on the basis of sorting multiple populations from each of 16 primary human AML samples and identifying which contain leukemia stem cells (LSCs) using a sensitive xenograft assay. Analysis of gene expression from all functionally validated populations yielded an LSC-specific signature. Similarly, a hematopoietic stem cell (HSC) gene signature was established. Bioinformatic analysis identified a core transcriptional program shared by LSCs and HSCs, revealing the molecular machinery underlying 'stemness' properties. Both stem cell programs were highly significant independent predictors of patient survival and were found in existing prognostic signatures. Thus, determinants of stemness influence the clinical outcome of AML, establishing that LSCs are clinically relevant and not artifacts of xenotransplantation.
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http://dx.doi.org/10.1038/nm.2415DOI Listing
August 2011