Publications by authors named "Daniel G Tenen"

202 Publications

Myeloid lncRNA LOUP Mediates Opposing Regulatory Effects of RUNX1 and RUNX1-ETO in t(8;21) AML.

Blood 2021 05 10. Epub 2021 May 10.

Harvard Medical School Initiative for RNA Medicine, Harvard Medical School, Boston, Massachusetts, United States.

The mechanism underlying cell type-specific gene induction conferred by ubiquitous transcription factors as well as disruptions caused by their chimeric derivatives in leukemia is not well understood. Here we investigate whether RNAs coordinate with transcription factors to drive myeloid gene transcription. In an integrated genome-wide approach surveying for gene loci exhibiting concurrent RNA- and DNA-interactions with the broadly expressed transcription factor RUNX1, we identified the long noncoding RNA LOUP. This myeloid-specific and polyadenylated lncRNA induces myeloid differentiation and inhibits cell growth, acting as a transcriptional inducer of the myeloid master regulator PU.1. Mechanistically, LOUP recruits RUNX1 to both the PU.1 enhancer and the promoter, leading to the formation of an active chromatin loop. In t(8;21) acute myeloid leukemia, wherein RUNX1 is fused to ETO, the resulting oncogenic fusion protein RUNX1-ETO limits chromatin accessibility at the LOUP locus, causing inhibition of LOUP and PU.1 expression. These findings highlight the important role of the interplay between cell type-specific RNAs and transcription factors as well as their oncogenic derivatives in modulating lineage-gene activation and raise the possibility that RNA regulators of transcription factors represent alternative targets for therapeutic development.
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http://dx.doi.org/10.1182/blood.2020007920DOI Listing
May 2021

Chronic interleukin-1 exposure triggers selection for Cebpa-knockout multipotent hematopoietic progenitors.

J Exp Med 2021 Jun;218(6)

Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO.

The early events that drive myeloid oncogenesis are not well understood. Most studies focus on the cell-intrinsic genetic changes and how they impact cell fate decisions. We consider how chronic exposure to the proinflammatory cytokine, interleukin-1β (IL-1β), impacts Cebpa-knockout hematopoietic stem and progenitor cells (HSPCs) in competitive settings. Surprisingly, we found that Cebpa loss did not confer a hematopoietic cell-intrinsic competitive advantage; rather chronic IL-1β exposure engendered potent selection for Cebpa loss. Chronic IL-1β augments myeloid lineage output by activating differentiation and repressing stem cell gene expression programs in a Cebpa-dependent manner. As a result, Cebpa-knockout HSPCs are resistant to the prodifferentiative effects of chronic IL-1β, and competitively expand. We further show that ectopic CEBPA expression reduces the fitness of established human acute myeloid leukemias, coinciding with increased differentiation. These findings have important implications for the earliest events that drive hematologic disorders, suggesting that chronic inflammation could be an important driver of leukemogenesis and a potential target for intervention.
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http://dx.doi.org/10.1084/jem.20200560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8094119PMC
June 2021

The Interplay between Transcription Factor SALL4 and Histone Modifiers in Hematopoietic Stem and Progenitor Cells.

J Cell Immunol 2021 ;3(1):26-30

Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.

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http://dx.doi.org/10.33696/immunology.3.073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8057709PMC
January 2021

Identification of a targetable KRAS-mutant epithelial population in non-small cell lung cancer.

Commun Biol 2021 Apr 14;4(1):370. Epub 2021 Apr 14.

NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Pisa, Italy.

Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencing in aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer. We previously reported a key role for the oncogene BMI-1 in adenocarcinomas. We therefore investigated the effects of in vivo PTC596 treatment, which affects BMI-1 activity, in our murine model. Post-treatment, MRI analysis showed decreased tumor size, while single cell transcriptomics concomitantly detected near complete ablation of the mutant-KRAS-associated subpopulation, signifying the presence of a pharmacologically targetable, tumor-associated subpopulation. Our findings therefore hold promise for the development of a targeted therapy for KRAS-mutant adenocarcinomas.
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http://dx.doi.org/10.1038/s42003-021-01897-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046784PMC
April 2021

Emerging therapies for inv(16) AML.

Blood 2021 Apr 1. Epub 2021 Apr 1.

Medical College of Wisconsin, United States.

The core-binding factors (CBFs), composed of CBFβ and RUNX subunits, play critical roles in most hematopoietic lineages, and are deregulated in Acute myeloid leukemia (AML). The fusion oncogene CBFβ-SMMHC expressed in AML with the chromosome inversion inv(16)(p13q22) acts as a driver oncogene in hematopoietic stem cells and induces AML. This review focuses on novel insights on the molecular mechanisms involving CBFβ-SMMHC driven leukemogenesis and recent advances in therapeutic approaches to target CBFβ-SMMHC in inv(16) AML.
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http://dx.doi.org/10.1182/blood.2020009933DOI Listing
April 2021

Metabolic alterations and vulnerabilities in hepatocellular carcinoma.

Gastroenterol Rep (Oxf) 2021 Jan 18;9(1):1-13. Epub 2020 Nov 18.

Cancer Science Institute of Singapore, National University of Singapore, Singapore.

Liver cancer is a serious disease. It is ranked as the cancer with the second highest number of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC), which arises from transformed hepatocytes, is the major subtype of liver cancer. It accounts for 85% of total liver-cancer cases. An important aspect of HCC that has been actively studied is its metabolism. With the liver as the primary site of numerous metabolic processes in the body, it has been shown that the metabolism of HCC cells is highly dysregulated compared to that of normal hepatocytes. It is therefore crucial to understand the metabolic alterations caused by HCC and the underlying mechanisms for these alterations. This deeper understanding will allow diagnostic and therapeutic advancements in the treatment of HCC. In this review, we will summarize the current literature in HCC metabolic alterations, induced vulnerabilities, and potential therapeutic interventions.
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http://dx.doi.org/10.1093/gastro/goaa066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962738PMC
January 2021

Dual DNA and protein tagging of open chromatin unveils dynamics of epigenomic landscapes in leukemia.

Nat Methods 2021 03 1;18(3):293-302. Epub 2021 Mar 1.

Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

The architecture of chromatin regulates eukaryotic cell states by controlling transcription factor access to sites of gene regulation. Here we describe a dual transposase-peroxidase approach, integrative DNA and protein tagging (iDAPT), which detects both DNA (iDAPT-seq) and protein (iDAPT-MS) associated with accessible regions of chromatin. In addition to direct identification of bound transcription factors, iDAPT enables the inference of their gene regulatory networks, protein interactors and regulation of chromatin accessibility. We applied iDAPT to profile the epigenomic consequences of granulocytic differentiation of acute promyelocytic leukemia, yielding previously undescribed mechanistic insights. Our findings demonstrate the power of iDAPT as a platform for studying the dynamic epigenomic landscapes and their transcription factor components associated with biological phenomena and disease.
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http://dx.doi.org/10.1038/s41592-021-01077-8DOI Listing
March 2021

E-cadherin is regulated by GATA-2 and marks the early commitment of mouse hematopoietic progenitors to the basophil and mast cell fates.

Sci Immunol 2021 02;6(56)

Division of Hematology/Oncology, O'Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

E-cadherin is a calcium-dependent cell-cell adhesion molecule extensively studied for its involvement in tissue formation, epithelial cell behavior, and suppression of cancer. However, E-cadherin expression in the hematopoietic system has not been fully elucidated. Combining single-cell RNA-sequencing analyses and immunophenotyping, we revealed that progenitors expressing high levels of E-cadherin and contained within the granulocyte-monocyte progenitors (GMPs) fraction have an enriched capacity to differentiate into basophils and mast cells. We detected E-cadherin expression on committed progenitors before the expression of other reported markers of these lineages. We named such progenitors pro-BMPs (pro-basophil and mast cell progenitors). Using RNA sequencing, we observed transcriptional priming of pro-BMPs to the basophil and mast cell lineages. We also showed that GATA-2 directly regulates E-cadherin expression in the basophil and mast cell lineages, thus providing a mechanistic connection between the expression of this cell surface marker and the basophil and mast cell fate specification.
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http://dx.doi.org/10.1126/sciimmunol.aba0178DOI Listing
February 2021

Super-enhancers for RUNX3 are required for cell proliferation in EBV-infected B cell lines.

Gene 2021 Mar 12;774:145421. Epub 2021 Jan 12.

Cancer Science Institute of Singapore, National University of Singapore, Singapore; Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; International Research Center for Medical Sciences, Kumamoto University, Japan. Electronic address:

Epstein-Barr virus nuclear antigens 2 (EBNA2) mediated super-enhancers, defined by in silico data, localize near genes associated with B cell transcription factors including RUNX3. However, the biological function of super-enhancer for RUNX3 gene (seR3) remains unclear. Here, we show that two seR3s, tandemly-located at 59- and 70-kb upstream of RUNX3 transcription start site, named seR3 -59h and seR3 -70h, are required for RUNX3 expression and cell proliferation in Epstein-Barr virus (EBV)-positive malignant B cells. A BET bromodomain inhibitor, JQ1, potently suppressed EBV-positive B cell growth through the reduction of RUNX3 and MYC expression. Excision of either or both seR3s by employing CRISPR/Cas9 system resulted in the decrease in RUNX3 expression and the subsequent suppression of cell proliferation and colony forming capability. The expression of MYC was also reduced when seR3s were deleted, probably due to the loss of trans effect of seR3s on the super-enhancers for MYC. These findings suggest that seR3s play a pivotal role in expression and biological function of both RUNX3 and MYC. seR3s would serve as a potential therapeutic target in EBV-related widespread tumors.
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http://dx.doi.org/10.1016/j.gene.2021.145421DOI Listing
March 2021

Zinc Finger Protein SALL4 Functions through an AT-Rich Motif to Regulate Gene Expression.

Cell Rep 2021 Jan;34(1):108574

Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Boston, MA 02115, USA. Electronic address:

The zinc finger transcription factor SALL4 is highly expressed in embryonic stem cells, downregulated in most adult tissues, but reactivated in many aggressive cancers. This unique expression pattern makes SALL4 an attractive therapeutic target. However, whether SALL4 binds DNA directly to regulate gene expression is unclear, and many of its targets in cancer cells remain elusive. Here, through an unbiased screen of protein binding microarray (PBM) and cleavage under targets and release using nuclease (CUT&RUN) experiments, we identify and validate the DNA binding domain of SALL4 and its consensus binding sequence. Combined with RNA sequencing (RNA-seq) analyses after SALL4 knockdown, we discover hundreds of new SALL4 target genes that it directly regulates in aggressive liver cancer cells, including genes encoding a family of histone 3 lysine 9-specific demethylases (KDMs). Taken together, these results elucidate the mechanism of SALL4 DNA binding and reveal pathways and molecules to target in SALL4-dependent tumors.
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http://dx.doi.org/10.1016/j.celrep.2020.108574DOI Listing
January 2021

ZNF143 mediates CTCF-bound promoter-enhancer loops required for murine hematopoietic stem and progenitor cell function.

Nat Commun 2021 01 4;12(1):43. Epub 2021 Jan 4.

Cancer Science Institute of Singapore, National University of Singapore, 117599, Singapore, Singapore.

CCCTC binding factor (CTCF) is an important factor in the maintenance of chromatin-chromatin interactions, yet the mechanism regulating its binding to chromatin is unknown. We demonstrate that zinc finger protein 143 (ZNF143) is a key regulator for CTCF-bound promoter-enhancer loops. In the murine genome, a large percentage of CTCF and ZNF143 DNA binding motifs are distributed 37 bp apart in the convergent orientation. Furthermore, deletion of ZNF143 leads to loss of CTCF binding on promoter and enhancer regions associated with gene expression changes. CTCF-bound promoter-enhancer loops are also disrupted after excision of ZNF143. ZNF143-CTCF-bound promoter-enhancer loops regulate gene expression patterns essential for maintenance of murine hematopoietic stem and progenitor cell integrity. Our data suggest a common feature of gene regulation is that ZNF143 is a critical factor for CTCF-bound promoter-enhancer loops.
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http://dx.doi.org/10.1038/s41467-020-20282-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782510PMC
January 2021

Scavenging of Labile Heme by Hemopexin Is a Key Checkpoint in Cancer Growth and Metastases.

Cell Rep 2020 09;32(12):108181

Department of Surgery, Division of Surgical Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02214, USA; Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02214, USA. Electronic address:

Hemopexin (Hx) is a scavenger of labile heme. Herein, we present data defining the role of tumor stroma-expressed Hx in suppressing cancer progression. Labile heme and Hx levels are inversely correlated in the plasma of patients with prostate cancer (PCa). Further, low expression of Hx in PCa biopsies characterizes poorly differentiated tumors and correlates with earlier time to relapse. Significantly, heme promotes tumor growth and metastases in an orthotopic murine model of PCa, with the most aggressive phenotype detected in mice lacking Hx. Mechanistically, labile heme accumulates in the nucleus and modulates specific gene expression via interacting with guanine quadruplex (G4) DNA structures to promote PCa growth. We identify c-MYC as a heme:G4-regulated gene and a major player in heme-driven cancer progression. Collectively, these results reveal that sequestration of labile heme by Hx may block heme-driven tumor growth and metastases, suggesting a potential strategy to prevent and/or arrest cancer dissemination.
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http://dx.doi.org/10.1016/j.celrep.2020.108181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7551404PMC
September 2020

β-Catenin-TCF/LEF signaling promotes steady-state and emergency granulopoiesis via G-CSF receptor upregulation.

Blood 2020 11;136(22):2574-2587

Department of Hemato-oncology, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.

The canonical Wnt signaling pathway is mediated by interaction of β-catenin with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and subsequent transcription activation of Wnt-target genes. In the hematopoietic system, the function of the pathway has been mainly investigated by rather unspecific genetic manipulations of β-catenin that yielded contradictory results. Here, we used a mouse expressing a truncated dominant negative form of the human TCF4 transcription factor (dnTCF4) that specifically abrogates β-catenin-TCF/LEF interaction. Disruption of the β-catenin-TCF/LEF interaction resulted in the accumulation of immature cells and reduced granulocytic differentiation. Mechanistically, dnTCF4 progenitors exhibited downregulation of the Csf3r gene, reduced granulocyte colony-stimulating factor (G-CSF) receptor levels, attenuation of downstream Stat3 phosphorylation after G-CSF treatment, and impaired G-CSF-mediated differentiation. Chromatin immunoprecipitation assays confirmed direct binding of TCF/LEF factors to the promoter and putative enhancer regions of CSF3R. Inhibition of β-catenin signaling compromised activation of the emergency granulopoiesis program, which requires maintenance and expansion of myeloid progenitors. Consequently, dnTCF4 mice were more susceptible to Candida albicans infection and more sensitive to 5-fluorouracil-induced granulocytic regeneration. Importantly, genetic and chemical inhibition of β-catenin-TCF/LEF signaling in human CD34+ cells reduced granulocytic differentiation, whereas its activation enhanced myelopoiesis. Altogether, our data indicate that the β-catenin-TCF/LEF complex directly regulates G-CSF receptor levels, and consequently controls proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Our results uncover a role for the β-catenin signaling pathway in fine tuning the granulocytic production, opening venues for clinical intervention that require enhanced or reduced production of neutrophils.
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http://dx.doi.org/10.1182/blood.2019004664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714095PMC
November 2020

Lessons learned from early compassionate use of convalescent plasma on critically ill patients with Covid-19.

Transfusion 2020 10 8;60(10):2210-2216. Epub 2020 Aug 8.

Department of Emergency, The Central Hospital of Wuhan, Tongji Medical College, Hua Zhong University of Science and Technology, Wuhan, China.

Background: The management of critically ill patients with coronavirus disease 2019 (COVID-19), caused by a new human virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is challenging. Recently, there have been several reports with inconsistent results after treatment with convalescent plasma (CP) on critically ill patients with COVID-19, which was produced with a neutralizing antibody titer and tested in a P3 or P4 laboratory. However, due to the limitation of the conditions on mass production of plasma, most producers hardly had the capability to isolate the neutralizing antibody. Here, we report the clinical courses of three critically ill patients with COVID-19 receiving CP treatments by total immunoglobulin G (IgG) titer collection.

Methods: Three patients with COVID-19 in this study were laboratory confirmed to be positive for SARS-CoV-2, with radiographic and clinical features of pneumonia. CP was collected by total IgG titer of 160 (range, 200-225 mL), and patients were transfused between 20 and 30 days after disease onset at the critical illness stage as a trial in addition to standard care. The clinical courses of these patients, including laboratory results and pulmonary functional and image studies after receiving convalescent plasma infusions, were reviewed.

Results: No therapeutic effect of CP was observed in any of the patients; instead, all three patients deteriorated and required extracorporeal membrane oxygenation treatment. A potential cytokine storm 4 hours after infusion of CP in Patient 2 was observed. No more patients were put on the trial of CP transfusion.

Conclusions: We recommend extreme caution in using CP in critically ill patients more than 2 weeks after the onset of COVID-19 pneumonia.
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http://dx.doi.org/10.1111/trf.15975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7436937PMC
October 2020

Lysine acetyltransferase Tip60 is required for hematopoietic stem cell maintenance.

Blood 2020 10;136(15):1735-1747

Cancer Science Institute of Singapore, National University of Singapore, Singapore.

Hematopoietic stem cells (HSCs) have the potential to replenish the blood system for the lifetime of the organism. Their 2 defining properties, self-renewal and differentiation, are tightly regulated by the epigenetic machineries. Using conditional gene-knockout models, we demonstrated a critical requirement of lysine acetyltransferase 5 (Kat5, also known as Tip60) for murine HSC maintenance in both the embryonic and adult stages, which depends on its acetyltransferase activity. Genome-wide chromatin and transcriptome profiling in murine hematopoietic stem and progenitor cells revealed that Tip60 colocalizes with c-Myc and that Tip60 deletion suppress the expression of Myc target genes, which are associated with critical biological processes for HSC maintenance, cell cycling, and DNA repair. Notably, acetylated H2A.Z (acH2A.Z) was enriched at the Tip60-bound active chromatin, and Tip60 deletion induced a robust reduction in the acH2A.Z/H2A.Z ratio. These results uncover a critical epigenetic regulatory layer for HSC maintenance, at least in part through Tip60-dependent H2A.Z acetylation to activate Myc target genes.
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http://dx.doi.org/10.1182/blood.2019001279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544546PMC
October 2020

Patients with Cancer Appear More Vulnerable to SARS-CoV-2: A Multicenter Study during the COVID-19 Outbreak.

Cancer Discov 2020 06 28;10(6):783-791. Epub 2020 Apr 28.

Department of Gynecological Oncology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei, China.

The novel COVID-19 outbreak has affected more than 200 countries and territories as of March 2020. Given that patients with cancer are generally more vulnerable to infections, systematic analysis of diverse cohorts of patients with cancer affected by COVID-19 is needed. We performed a multicenter study including 105 patients with cancer and 536 age-matched noncancer patients confirmed with COVID-19. Our results showed COVID-19 patients with cancer had higher risks in all severe outcomes. Patients with hematologic cancer, lung cancer, or with metastatic cancer (stage IV) had the highest frequency of severe events. Patients with nonmetastatic cancer experienced similar frequencies of severe conditions to those observed in patients without cancer. Patients who received surgery had higher risks of having severe events, whereas patients who underwent only radiotherapy did not demonstrate significant differences in severe events when compared with patients without cancer. These findings indicate that patients with cancer appear more vulnerable to SARS-CoV-2 outbreak. SIGNIFICANCE: Because this is the first large cohort study on this topic, our report will provide much-needed information that will benefit patients with cancer globally. As such, we believe it is extremely important that our study be disseminated widely to alert clinicians and patients..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309152PMC
June 2020

Targeting microtubules sensitizes drug resistant lung cancer cells to lysosomal pathway inhibitors.

Theranostics 2020 3;10(6):2727-2743. Epub 2020 Feb 3.

Cancer Science Institute, National University of Singapore, 14 Medical Drive, Singapore 117599.

Oncogene-addicted cancers are predominantly driven by specific oncogenic pathways and display initial exquisite sensitivity to designer therapies, but eventually become refractory to treatments. Clear understanding of lung tumorigenic mechanisms is essential for improved therapies. : Lysosomes were analyzed in EGFR-WT and mutant cells and corresponding patient samples using immunofluorescence or immunohistochemistry and immunoblotting. Microtubule organization and dynamics were studied using immunofluorescence analyses. Also, we have validated our findings in a transgenic mouse model that contain EGFR-TKI resistant mutations. : We herein describe a novel mechanism that a mutated kinase disrupts the microtubule organization and results in a defective endosomal/lysosomal pathway. This prevents the efficient degradation of phosphorylated proteins that become trapped within the endosomes and continue to signal, therefore amplifying downstream proliferative and survival pathways. Phenotypically, a distinctive subcellular appearance of LAMP1 secondary to microtubule dysfunction in cells expressing EGFR kinase mutants is seen, and this may have potential diagnostic applications for the detection of such mutants. We demonstrate that lysosomal-inhibitors re-sensitize resistant cells to EGFR tyrosine-kinase inhibitors (TKIs). Identifying the endosome-lysosome pathway and microtubule dysfunction as a mechanism of resistance allows to pharmacologically intervene on this pathway. : We find that the combination of microtubule stabilizing agent and lysosome inhibitor could reduce the tumor progression in EGFR TKI resistant mouse models of lung cancer.
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http://dx.doi.org/10.7150/thno.38729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052910PMC
February 2020

NanoVar: accurate characterization of patients' genomic structural variants using low-depth nanopore sequencing.

Genome Biol 2020 03 3;21(1):56. Epub 2020 Mar 3.

Cancer Science Institute of Singapore, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive, #12-01, Singapore, 117599, Singapore.

The recent advent of third-generation sequencing technologies brings promise for better characterization of genomic structural variants by virtue of having longer reads. However, long-read applications are still constrained by their high sequencing error rates and low sequencing throughput. Here, we present NanoVar, an optimized structural variant caller utilizing low-depth (8X) whole-genome sequencing data generated by Oxford Nanopore Technologies. NanoVar exhibits higher structural variant calling accuracy when benchmarked against current tools using low-depth simulated datasets. In patient samples, we successfully validate structural variants characterized by NanoVar and uncover normal alternative sequences or alleles which are present in healthy individuals.
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http://dx.doi.org/10.1186/s13059-020-01968-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055087PMC
March 2020

High-speed automatic characterization of rare events in flow cytometric data.

PLoS One 2020 11;15(2):e0228651. Epub 2020 Feb 11.

Public Health Dynamics Laboratory, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, United States of America.

A new computational framework for FLow cytometric Analysis of Rare Events (FLARE) has been developed specifically for fast and automatic identification of rare cell populations in very large samples generated by platforms like multi-parametric flow cytometry. Using a hierarchical Bayesian model and information-sharing via parallel computation, FLARE rapidly explores the high-dimensional marker-space to detect highly rare populations that are consistent across multiple samples. Further it can focus within specified regions of interest in marker-space to detect subpopulations with desired precision.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228651PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012421PMC
April 2020

CAV1 - GLUT3 signaling is important for cellular energy and can be targeted by Atorvastatin in Non-Small Cell Lung Cancer.

Theranostics 2019 14;9(21):6157-6174. Epub 2019 Aug 14.

Cancer Science Institute Singapore, National University of Singapore, MD6, #12-01, 14 Medical Drive, Singapore 117599.

: The development of molecular targeted therapies, such as EGFR-TKIs, has positively impacted the management of EGFR mutated NSCLC. However, patients with innate and acquired resistance to EGFR-TKIs still face limited effective therapeutic options. Statins are the most frequently prescribed anti-cholesterol agents and have been reported to inhibit the progression of various malignancies, including in lung. However, the mechanism by which statin exerts its anti-cancer effects is unclear. This study is designed to investigate the anti-proliferative effects and identify the mechanism-of-action of statins in NSCLC. : In this study, the anti-tumoral properties of Atorvastatin were investigated in NSCLC utilizing cell culture system and models. : We demonstrate a link between elevated cellular cholesterol and TKI-resistance in NSCLC, which is independent of EGFR mutation status. Atorvastatin suppresses growth by inhibiting Cav1 expression in tumors in cell culture system and in models. Subsequent interrogations demonstrate an oncogenic physical interaction between Cav1 and GLUT3, and glucose uptake found distinctly in TKI-resistant NSCLC and this may be due to changes in the physical properties of Cav1 favoring GLUT3 binding in which significantly stronger Cav1 and GLUT3 physical interactions were observed in TKI-resistant than in TKI-sensitive NSCLC cells. Further, the differential effects of atorvastatin observed between EGFR-TKI resistant and sensitive cells suggest that EGFR mutation status may influence its actions. : This study reveals the inhibition of oncogenic role of Cav1 in GLUT3-mediated glucose uptake by statins and highlights its potential impact to overcome NSCLC with EGFR-TKI resistance.
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http://dx.doi.org/10.7150/thno.35805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735519PMC
September 2020

New High-Throughput Screening Identifies Compounds That Reduce Viability Specifically in Liver Cancer Cells That Express High Levels of SALL4 by Inhibiting Oxidative Phosphorylation.

Gastroenterology 2019 12 22;157(6):1615-1629.e17. Epub 2019 Aug 22.

Cancer Science Institute of Singapore, National University of Singapore, Singapore; Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

Background & Aims: Some oncogenes encode transcription factors, but few drugs have been successfully developed to block their activity specifically in cancer cells. The transcription factor SALL4 is aberrantly expressed in solid tumor and leukemia cells. We developed a screen to identify compounds that reduce the viability of liver cancer cells that express high levels of SALL4, and we investigated their mechanisms.

Methods: We developed a stringent high-throughput screening platform comprising unmodified SNU-387 and SNU-398 liver cancer cell lines and SNU-387 cell lines engineered to express low and high levels of SALL4. We screened 1597 pharmacologically active small molecules and 21,575 natural product extracts from plant, bacteria, and fungal sources for those that selectively reduce the viability of cells with high levels of SALL4 (SALL4 cells). We compared gene expression patterns of SALL4 cells vs SALL4-knockdown cells using RNA sequencing and real-time polymerase chain reaction analyses. Xenograft tumors were grown in NOD/SCID gamma mice from SALL4 SNU-398 or HCC26.1 cells or from SALL4 patient-derived xenograft (PDX) cells; mice were given injections of identified compounds or sorafenib, and the effects on tumor growth were measured.

Results: Our screening identified 1 small molecule (PI-103) and 4 natural compound analogues (oligomycin, efrapeptin, antimycin, and leucinostatin) that selectively reduced viability of SALL4 cells. We performed validation studies, and 4 of these compounds were found to inhibit oxidative phosphorylation. The adenosine triphosphate (ATP) synthase inhibitor oligomycin reduced the viability of SALL4 hepatocellular carcinoma and non-small-cell lung cancer cell lines with minimal effects on SALL4 cells. Oligomycin also reduced the growth of xenograft tumors grown from SALL4 SNU-398 or HCC26.1 cells to a greater extent than sorafenib, but oligomycin had little effect on tumors grown from SALL4 PDX cells. Oligomycin was not toxic to mice. Analyses of chromatin immunoprecipitation sequencing data showed that SALL4 binds approximately 50% of mitochondrial genes, including many oxidative phosphorylation genes, to activate their transcription. In comparing SALL4 and SALL4-knockdown cells, we found SALL4 to increase oxidative phosphorylation, oxygen consumption rate, mitochondrial membrane potential, and use of oxidative phosphorylation-related metabolites to generate ATP.

Conclusions: In a screening for compounds that reduce the viability of cells that express high levels of the transcription factor SALL4, we identified inhibitors of oxidative phosphorylation, which slowed the growth of xenograft tumors from SALL4 cells in mice. SALL4 activates the transcription of genes that regulate oxidative phosphorylation to increase oxygen consumption, mitochondrial membrane potential, and ATP generation in cancer cells. Inhibitors of oxidative phosphorylation might be used for the treatment of liver tumors with high levels of SALL4.
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http://dx.doi.org/10.1053/j.gastro.2019.08.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309153PMC
December 2019

Mapping Distinct Bone Marrow Niche Populations and Their Differentiation Paths.

Cell Rep 2019 07;28(2):302-311.e5

Division of Hematology/Oncology, University of Alabama at Birmingham, Birmingham, AL, USA. Electronic address:

The bone marrow microenvironment is composed of heterogeneous cell populations of non-hematopoietic cells with complex phenotypes and undefined trajectories of maturation. Among them, mesenchymal cells maintain the production of stromal, bone, fat, and cartilage cells. Resolving these unique cellular subsets within the bone marrow remains challenging. Here, we used single-cell RNA sequencing of non-hematopoietic bone marrow cells to define specific subpopulations. Furthermore, by combining computational prediction of the cell state hierarchy with the known expression of key transcription factors, we mapped differentiation paths to the osteocyte, chondrocyte, and adipocyte lineages. Finally, we validated our findings using lineage-specific reporter strains and targeted knockdowns. Our analysis reveals differentiation hierarchies for maturing stromal cells, determines key transcription factors along these trajectories, and provides an understanding of the complexity of the bone marrow microenvironment.
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http://dx.doi.org/10.1016/j.celrep.2019.06.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684313PMC
July 2019

Styryl quinazolinones and its ethynyl derivatives induce myeloid differentiation.

Bioorg Med Chem Lett 2019 08 20;29(16):2286-2289. Epub 2019 Jun 20.

Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA; Division of Translational Genomics, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Kashiwa, Chiba 277-8577, Japan. Electronic address:

The tumor suppressor transcription factor CCAAT enhancer-binding protein α (C/EBPα) expression is downregulated in myeloid leukemias and enhancement of C/EBPα expression induces granulocytic differentiation in leukemic cells. Previously we reported that Styryl quinazolinones induce myeloid differentiation in HL-60 cells by upregulating C/EBPα expression. To identify more potent molecule that can induce leukemic cell differentiation we synthesized and evaluated new series of styryl quinazolinones, ethynyl styryl quinazolinones, styryl quinolinones and thienopyrimidinones. Thienopyrimidinones were found toxic and styryl quinolinones were found inactive. Ethynyl styryl quinazolinone 39 and styryl quinazolinone 5 were found active on par with the earlier reported analogues 1 and 2 suggesting that the 5-nitro furan-2-yl styryl quinazolinones find a real promise in leukemic cell differentiation. The improved potency of 5 suggested that further modifications in the 5-nitro furan-2-yl styryl quinazolinones can be at the phenyl substitution at the 3-position of the quinazolinone ring apart from the 5-position of the heteroaryl ring.
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http://dx.doi.org/10.1016/j.bmcl.2019.06.024DOI Listing
August 2019

Maintenance and enhancement of human peripheral blood mobilized stem/progenitor cell engraftment after ex vivo culture via an HDACi/SALL4 axis (3465).

Exp Hematol 2019 07 28;75:53-63.e11. Epub 2019 Jun 28.

Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts. Electronic address:

Currently, there is a growing need for culturing hematopoietic stem/progenitor cells (HSPCs) in vitro for various clinical applications including gene therapy. Compared with cord blood (CB) CD34 HSPCs, it is more challenging to maintain or expand CD34 peripheral blood mobilized stem/progenitor cells (PBSCs) ex vivo. To fill this knowledge gap, we have systematically surveyed 466 small-molecule drug compounds for their potential in cytokine-dependent expansion of human CD34CD90 HSPCs. We found that epigenetic modifiers, especially histone deacetylase inhibitors (HDACis), could preferentially maintain and expand these cells. In particular, treatment of CD34 PBSCs with a single dose of HDACi trichostatin A (TSA) at a concentration of 50 nmol/L ex vivo yielded the greatest expansion (11.7-fold) of CD34CD90 cells when compared with the control (dimethyl sulfoxide [DMSO] plus cytokines) group. Additionally, TSA-treated PBSC CD34 cells had a statistically significant higher engraftment rate than the control-treated group in xenotransplantation experiments. Mechanistically, TSA treatment was associated with increased expression of HSPC-related genes such as GATA2 and SALL4. Furthermore, TSA-mediated CD34CD90 expansion was reduced by downregulation of SALL4 but not GATA2. Overall, we have developed a robust, short-term (5-day), PBSC ex vivo maintenance/expansion culture technique and found that the HDACi-TSA/SALL4 axis is important for the biological process.
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http://dx.doi.org/10.1016/j.exphem.2019.06.473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719293PMC
July 2019

DNMT3B shapes the mCA landscape and regulates mCG for promoter bivalency in human embryonic stem cells.

Nucleic Acids Res 2019 08;47(14):7460-7475

Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, #12-01, Singapore 117599.

DNMT3B is known as a de novo DNA methyltransferase. However, its preferential target sites for DNA methylation are largely unknown. Our analysis on ChIP-seq experiment in human embryonic stem cells (hESC) revealed that DNMT3B, mCA and H3K36me3 share the same genomic distribution profile. Deletion of DNMT3B or its histone-interacting domain (PWWP) demolished mCA in hESCs, suggesting that PWWP domain of DNMT3B directs the formation of mCA landscape. In contrast to the common presumption that PWWP guides DNMT3B-mediated mCG deposition, we found that deleting PWWP does not affect the mCG landscape. Nonetheless, DNMT3B knockout led to the formation of 2985 de novo hypomethylated regions at annotated promoter sites. Upon knockout, most of these promoters gain the bivalent marks, H3K4me3 and H3K27me3. We call them spurious bivalent promoters. Gene ontology analysis associated spurious bivalent promoters with development and cell differentiation. Overall, we found the importance of DNMT3B for shaping the mCA landscape and for maintaining the fidelity of the bivalent promoters in hESCs.
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http://dx.doi.org/10.1093/nar/gkz520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698669PMC
August 2019

Hlf marks the developmental pathway for hematopoietic stem cells but not for erythro-myeloid progenitors.

J Exp Med 2019 07 10;216(7):1599-1614. Epub 2019 May 10.

Department of Hematology, Juntendo University Graduate School of Medicine, Tokyo, Japan.

Before the emergence of hematopoietic stem cells (HSCs), lineage-restricted progenitors, such as erythro-myeloid progenitors (EMPs), are detected in the embryo or in pluripotent stem cell cultures in vitro. Although both HSCs and EMPs are derived from hemogenic endothelium, it remains unclear how and when these two developmental programs are segregated during ontogeny. Here, we show that hepatic leukemia factor (Hlf) expression specifically marks a developmental continuum between HSC precursors and HSCs. Using the -tdTomato reporter mouse, we found that is expressed in intra-aortic hematopoietic clusters and fetal liver HSCs. In contrast, EMPs and yolk sac hematopoietic clusters before embryonic day 9.5 do not express HSC specification, regulated by the Evi-1/Hlf axis, is activated only within Hlf nascent hematopoietic clusters. These results strongly suggest that HSCs and EMPs are generated from distinct cohorts of hemogenic endothelium. Selective induction of the Hlf lineage pathway may lead to the in vitro generation of HSCs from pluripotent stem cells.
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http://dx.doi.org/10.1084/jem.20181399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605751PMC
July 2019

The enhancer RNA activates the oncogenic transcriptional program in T-cell acute lymphoblastic leukemia.

Blood 2019 07 10;134(3):239-251. Epub 2019 May 10.

Cancer Science Institute of Singapore, National University of Singapore, Singapore.

The oncogenic transcription factor TAL1 regulates the transcriptional program in T-ALL. ARID5B is one of the critical downstream targets of TAL1, which further activates the oncogenic regulatory circuit in T-ALL cells. Here, we elucidated the molecular functions of the noncoding RNA, ARID5B-inducing enhancer associated long noncoding RNA (), in T-ALL pathogenesis. We demonstrated that is specifically activated in T-ALL cases, and its expression is associated with enhancer activity. recruits mediator proteins to the enhancer, promotes enhancer-promoter interactions, and activates the expression of , thereby positively regulating the TAL1-induced transcriptional program and the oncogene. The TAL1 complex coordinately regulates the expression of Knockdown of inhibits cell growth and survival of T-ALL cells in culture and blocks disease progression in a murine xenograft model. Our results indicate that plays an oncogenic role as an enhancer RNA in T-ALL.
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http://dx.doi.org/10.1182/blood.2018874503DOI Listing
July 2019

Single-Cell Transcriptomics of Human and Mouse Lung Cancers Reveals Conserved Myeloid Populations across Individuals and Species.

Immunity 2019 05 9;50(5):1317-1334.e10. Epub 2019 Apr 9.

Department of Systems Biology, Harvard Medical School, Boston, MA, USA. Electronic address:

Tumor-infiltrating myeloid cells (TIMs) comprise monocytes, macrophages, dendritic cells, and neutrophils, and have emerged as key regulators of cancer growth. These cells can diversify into a spectrum of states, which might promote or limit tumor outgrowth but remain poorly understood. Here, we used single-cell RNA sequencing (scRNA-seq) to map TIMs in non-small-cell lung cancer patients. We uncovered 25 TIM states, most of which were reproducibly found across patients. To facilitate translational research of these populations, we also profiled TIMs in mice. In comparing TIMs across species, we identified a near-complete congruence of population structures among dendritic cells and monocytes; conserved neutrophil subsets; and species differences among macrophages. By contrast, myeloid cell population structures in patients' blood showed limited overlap with those of TIMs. This study determines the lung TIM landscape and sets the stage for future investigations into the potential of TIMs as immunotherapy targets.
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http://dx.doi.org/10.1016/j.immuni.2019.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620049PMC
May 2019

Monitoring structural modulation of redox-sensitive proteins in cells with MS-CETSA.

Redox Biol 2019 06 14;24:101168. Epub 2019 Mar 14.

School of Biological Sciences, Nanyang Technological University, 637551, Singapore; Institute of Molecular and Cell Biology, A*STAR, 138673, Singapore; Department of Oncology and Pathology, Karolinska Institutet, Stockholm, 17177, Sweden. Electronic address:

Reactive oxygen species (ROS) induce different cellular stress responses but can also mediate cellular signaling. Augmented levels of ROS are associated with aging, cancer as well as various metabolic and neurological disorders. ROS can also affect the efficacy and adverse effects of drugs. Although proteins are key mediators of most ROS effects, direct studies of ROS-modulated-protein function in the cellular context are very challenging. Therefore the understanding of specific roles of different proteins in cellular ROS responses is still relatively rudimentary. In the present work we show that Mass Spectrometry-Cellular Thermal Shift Assay (MS-CETSA) can directly monitor ROS and redox modulations of protein structure at the proteome level. By altering ROS levels in cultured human hepatocellular carcinoma cell lysates and intact cells, we detected CETSA responses in many proteins known to be redox sensitive, and also revealed novel candidate ROS sensitive proteins. Studies in intact cells treated with hydrogen peroxide and sulfasalazine, a ROS modulating drug, identified not only proteins that are directly modified, but also proteins reporting on downstream cellular effects. Comprehensive changes are seen on rate-limiting proteins regulating key cellular processes, including known redox control systems, protein degradation, epigenetic control and protein translational processes. Interestingly, concerted shifts on ATP-binding proteins revealed redox-induced modulation of ATP levels, which likely control many cellular processes. Collectively, these studies establish CETSA as a novel method for cellular studies of redox modulations of proteins, which implicated in a wide range of processes and for the discovery of CETSA-based biomarkers reporting on the efficacy as well as adverse effects of drugs.
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http://dx.doi.org/10.1016/j.redox.2019.101168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6439307PMC
June 2019