Publications by authors named "Leonard I Zon"

334 Publications

Dissecting melanocytes to predict melanoma.

Nat Cell Biol 2021 Sep;23(9):930-931

Stem Cell Program and Division of Hematology/Oncology, Children's Hospital Boston, Howard Hughes Medical Institute, Boston, MA, USA.

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http://dx.doi.org/10.1038/s41556-021-00748-0DOI Listing
September 2021

Telomerase RNA recruits RNA polymerase II to target gene promoters to enhance myelopoiesis.

Proc Natl Acad Sci U S A 2021 Aug;118(32)

Grupo de Telomerasa, Cáncer y Envejecimiento, Hospital Clínico Universitario Virgen de la Arrixaca, 30120 Murcia, Spain;

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure and cancer predisposition syndrome caused by mutations in telomerase or telomeric proteins. Here, we report that zebrafish telomerase RNA () binds to specific DNA sequences of master myeloid genes and controls their expression by recruiting RNA Polymerase II (Pol II). Zebrafish harboring the CR4-CR5 domain mutation found in DC patients hardly interacted with Pol II and failed to regulate myeloid gene expression in vivo and to increase their transcription rates in vitro. Similarly, regulated myeloid gene expression and Pol II promoter occupancy in human myeloid progenitor cells. Strikingly, induced pluripotent stem cells derived from DC patients with a mutation in the CR4-CR5 domain showed impaired myelopoiesis, while those with mutated telomerase catalytic subunit differentiated normally. Our findings show that acts as a transcription factor, revealing a target for therapeutic intervention in DC patients.
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http://dx.doi.org/10.1073/pnas.2015528118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8364177PMC
August 2021

Synergistic melanoma cell death mediated by inhibition of both MCL1 and BCL2 in high-risk tumors driven by NF1/PTEN loss.

Oncogene 2021 Jul 30. Epub 2021 Jul 30.

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

Melanomas driven by loss of the NF1 tumor suppressor have a high risk of treatment failure and effective therapies have not been developed. Here we show that loss-of-function mutations of nf1 and pten result in aggressive melanomas in zebrafish, representing the first animal model of NF1-mutant melanomas harboring PTEN loss. MEK or PI3K inhibitors show little activity when given alone due to cross-talk between the pathways, and high toxicity when given together. The mTOR inhibitors, sirolimus, everolimus, and temsirolimus, were the most active single agents tested, potently induced tumor-suppressive autophagy, but not apoptosis. Because addition of the BCL2 inhibitor venetoclax resulted in compensatory upregulation of MCL1, we established a three-drug combination composed of sirolimus, venetoclax, and the MCL1 inhibitor S63845. This well-tolerated drug combination potently and synergistically induces apoptosis in both zebrafish and human NF1/PTEN-deficient melanoma cells, providing preclinical evidence justifying an early-stage clinical trial in patients with NF1/PTEN-deficient melanoma.
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http://dx.doi.org/10.1038/s41388-021-01926-yDOI Listing
July 2021

Nucleotide stress responses in neural crest cell fate and melanoma.

Cell Cycle 2021 Aug 19;20(15):1455-1467. Epub 2021 Jul 19.

Harvard Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.

Melanoma is the deadliest form of skin cancer. While clinical developments have significantly improved patient prognosis, effective treatment is often obstructed by limited response rates, intrinsic or acquired resistance to therapy, and adverse events. Melanoma initiation and progression are associated with transcriptional reprogramming of melanocytes to a cell state that resembles the lineage from which the cells are specified during development, that is the neural crest. Convergence to a neural crest cell (NCC)-like state revealed the therapeutic potential of targeting developmental pathways for the treatment of melanoma. Neural crest cells have a unique sensitivity to metabolic dysregulation, especially nucleotide depletion. Mutations in the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) particularly affect neural crest-derived tissues and cause Miller syndrome, a genetic disorder characterized by craniofacial malformations in patients. The developmental susceptibility of the neural crest to nucleotide deficiency is conserved in melanoma and provides a metabolic vulnerability that can be exploited for therapeutic purposes. We review the current knowledge on nucleotide stress responses in neural crest and melanoma and discuss how the recent scientific advances that have improved our understanding of transcriptional regulation during nucleotide depletion can impact melanoma treatment.
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http://dx.doi.org/10.1080/15384101.2021.1947567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8354612PMC
August 2021

NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism.

Cell 2021 Aug 6;184(16):4268-4283.e20. Epub 2021 Jul 6.

Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.
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http://dx.doi.org/10.1016/j.cell.2021.06.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8349839PMC
August 2021

Single-cell ATAC-seq reveals GATA2-dependent priming defect in myeloid and a maturation bottleneck in lymphoid lineages.

Blood Adv 2021 07;5(13):2673-2686

Boston Children's Hospital, Boston, MA.

Germline heterozygous mutations in GATA2 are associated with a syndrome characterized by cytopenias, atypical infections, and increased risk of hematologic malignancies. Here, we generated a zebrafish mutant of gata2b that recapitulated the myelomonocytopenia and B-cell lymphopenia of GATA2 deficiency syndrome. Using single-cell assay for transposase accessible chromatin with sequencing of marrow cells, we showed that loss of gata2b led to contrasting alterations in chromosome accessibility in early myeloid and lymphoid progenitors, associated with defects in gene expression. Within the myeloid lineage in gata2b mutant zebrafish, we identified an attenuated myeloid differentiation with reduced transcriptional priming and skewing away from the monocytic program. In contrast, in early lymphoid progenitors, gata2b loss led to accumulation of B-lymphoid transcription factor accessibility coupled with increased expression of the B-cell lineage-specification program. However, gata2b mutant zebrafish had incomplete B-cell lymphopoiesis with loss of lineage-specific transcription factor accessibility in differentiating B cells, in the context of aberrantly reduced oxidative metabolic pathways. Our results establish that transcriptional events in early progenitors driven by Gata2 are required to complete normal differentiation.
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http://dx.doi.org/10.1182/bloodadvances.2020002992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8221101PMC
July 2021

Zebrafish disease models in drug discovery: from preclinical modelling to clinical trials.

Nat Rev Drug Discov 2021 08 11;20(8):611-628. Epub 2021 Jun 11.

Department of Pathology, Massachusetts General Research Institute, Boston, MA, USA.

Numerous drug treatments that have recently entered the clinic or clinical trials have their genesis in zebrafish. Zebrafish are well established for their contribution to developmental biology and have now emerged as a powerful preclinical model for human disease, as their disease characteristics, aetiology and progression, and molecular mechanisms are clinically relevant and highly conserved. Zebrafish respond to small molecules and drug treatments at physiologically relevant dose ranges and, when combined with cell-specific or tissue-specific reporters and gene editing technologies, drug activity can be studied at single-cell resolution within the complexity of a whole animal, across tissues and over an extended timescale. These features enable high-throughput and high-content phenotypic drug screening, repurposing of available drugs for personalized and compassionate use, and even the development of new drug classes. Often, drugs and drug leads explored in zebrafish have an inter-organ mechanism of action and would otherwise not be identified through targeted screening approaches. Here, we discuss how zebrafish is an important model for drug discovery, the process of how these discoveries emerge and future opportunities for maximizing zebrafish potential in medical discoveries.
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http://dx.doi.org/10.1038/s41573-021-00210-8DOI Listing
August 2021

From development toward therapeutics, a collaborative effort on blood progenitors.

Stem Cell Reports 2021 Jul 10;16(7):1674-1685. Epub 2021 Jun 10.

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Stem Cell Program and Division of Hematology/Oncology, Children's Hospital Boston, Howard Hughes Medical Institute, Boston, MA 02115, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Harvard Stem Cell Institute, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115, USA. Electronic address:

The National Heart, Lung, and Blood Institute Progenitor Cell Translational Consortium Blood Progenitor Meeting was hosted virtually on November 5, 2020, with 93 attendees across 20 research groups. The purpose of this meeting was to exchange recent findings, discuss current efforts, and identify prospective opportunities in the field of hematopoietic stem and progenitor cell research and therapeutic discovery.
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http://dx.doi.org/10.1016/j.stemcr.2021.05.010DOI Listing
July 2021

Mitochondrial function in development and disease.

Dis Model Mech 2021 Jun 11;14(6). Epub 2021 Jun 11.

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 01238, USA.

Mitochondria are organelles with vital functions in almost all eukaryotic cells. Often described as the cellular 'powerhouses' due to their essential role in aerobic oxidative phosphorylation, mitochondria perform many other essential functions beyond energy production. As signaling organelles, mitochondria communicate with the nucleus and other organelles to help maintain cellular homeostasis, allow cellular adaptation to diverse stresses, and help steer cell fate decisions during development. Mitochondria have taken center stage in the research of normal and pathological processes, including normal tissue homeostasis and metabolism, neurodegeneration, immunity and infectious diseases. The central role that mitochondria assume within cells is evidenced by the broad impact of mitochondrial diseases, caused by defects in either mitochondrial or nuclear genes encoding for mitochondrial proteins, on different organ systems. In this Review, we will provide the reader with a foundation of the mitochondrial 'hardware', the mitochondrion itself, with its specific dynamics, quality control mechanisms and cross-organelle communication, including its roles as a driver of an innate immune response, all with a focus on development, disease and aging. We will further discuss how mitochondrial DNA is inherited, how its mutation affects cell and organismal fitness, and current therapeutic approaches for mitochondrial diseases in both model organisms and humans.
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http://dx.doi.org/10.1242/dmm.048912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214736PMC
June 2021

A phase II trial of all-trans retinoic acid (ATRA) in advanced adenoid cystic carcinoma.

Oral Oncol 2021 Aug 3;119:105366. Epub 2021 Jun 3.

Department of Medical Oncology, Center for Head & Neck Oncology, Center for Salivary and Rare Head and Neck Cancers, Dana-Farber Cancer Institute, Boston, USA.

Background: Effective therapies are lacking for recurrent, metastatic adenoid cystic carcinoma (R/M ACC) and preclinical models suggest retinoic acid agonists inhibit ACC growth. This phase II trial evaluated all-trans retinoic acid (ATRA) as a novel therapy for ACC.

Methods: Patients with R/M ACC (any site) with clinical and/or radiographic progression ≤12 months prior to study entry were eligible. Cohort 1 (CH1) received ATRA 45 mg/m split oral daily dosing on days 1-14 of a 28-day cycle; Cohort 2 (CH2) received the same dosing continuously. Primary endpoint was best overall response rate (CR + PR) (RECIST v1.1). Secondary endpoints: safety and progression-free survival (PFS). Exploratory analyses: ATRA impact on MYB expression and genomic predictors of response.

Results: Eighteen patients enrolled. There were no responses, but 61% (11/18) had stable disease (SD) and 28% (5/18) progression as best response; 11% (2/18) unevaluable. Median duration of stability: 3.7 months (95%CI, 1.9-3.9). One patient (CH1) remains on drug with SD approaching 1 year. Half of those who received prior VEGFR therapy achieved SD (4/8). At median follow up of 7.9 months, median PFS was 3.2 months (95%CI, 1.8-3.9). N = 1 required dose adjustment; N = 1 came off drug for toxicity. There were no grade 3-4 adverse events. NOTCH1 and PI3K pathway alterations were most frequent. Low MYB protein expression was associated with longer duration of stability on ATRA (P < 0.01).

Conclusion(s): While the trial did not meet its prespecified response endpoint, ATRA alone or in combination may be a low toxicity treatment for disease growth stabilization in R/M ACC.
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http://dx.doi.org/10.1016/j.oraloncology.2021.105366DOI Listing
August 2021

Cell-specific transcriptional control of mitochondrial metabolism by TIF1γ drives erythropoiesis.

Science 2021 05;372(6543):716-721

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 01238, USA.

Transcription and metabolism both influence cell function, but dedicated transcriptional control of metabolic pathways that regulate cell fate has rarely been defined. We discovered, using a chemical suppressor screen, that inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) rescues erythroid differentiation in bloodless zebrafish mutant embryos defective for transcriptional intermediary factor 1 gamma (). This rescue depends on the functional link of DHODH to mitochondrial respiration. The transcription elongation factor TIF1γ directly controls coenzyme Q (CoQ) synthesis gene expression. Upon loss, CoQ levels are reduced, and a high succinate/α-ketoglutarate ratio leads to increased histone methylation. A CoQ analog rescues 's bloodless phenotype. These results demonstrate that mitochondrial metabolism is a key output of a lineage transcription factor that drives cell fate decisions in the early blood lineage.
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http://dx.doi.org/10.1126/science.aaz2740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8177078PMC
May 2021

A uniform format for manuscript submission.

Cell 2021 Apr;184(7):1654-1656

Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA; Stem Cell and Regenerative Biology Department, Harvard University, Cambridge, MA, USA.

Many scientists spend unnecessary time reformatting papers to submit them to different journals. We propose a uniform submission format that we hope journals will include in their options for submission. Widespread adoption of this uniform submission format could shorten the submission and publishing process, freeing up time for research.
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http://dx.doi.org/10.1016/j.cell.2021.01.030DOI Listing
April 2021

'Enhancing' red cell fate through epigenetic mechanisms.

Curr Opin Hematol 2021 05;28(3):129-137

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge.

Purpose Of Review: Transcription of erythroid-specific genes is regulated by the three-dimensional (3D) structure and composition of chromatin, which dynamically changes during erythroid differentiation. Chromatin organization and dynamics are regulated by several epigenetic mechanisms involving DNA (de-)methylation, posttranslational modifications (PTMs) of histones, chromatin-associated structural proteins, and higher-order structural changes and interactions. This review addresses examples of recent developments in several areas delineating the interface of chromatin regulation and erythroid-specific lineage transcription.

Recent Findings: We survey and discuss recent studies that focus on the erythroid chromatin landscape, erythroid enhancer-promotor interactions, super-enhancer functionality, the role of chromatin modifiers and epigenetic crosstalk, as well as the progress in mapping red blood cell (RBC) trait-associated genetic variants within cis-regulatory elements (CREs) identified in genome-wide association study (GWAS) efforts as a step toward determining their impact on erythroid-specific gene expression.

Summary: As one of the best characterized and accessible cell differentiation systems, erythropoiesis has been at the forefront of studies aiming to conceptualize how chromatin dynamics regulate transcription. New emerging technologies that bring a significantly enhanced spatial and temporal resolution of chromatin structure, and allow investigation of small cell numbers, have advanced our understanding of chromatin dynamics during erythroid differentiation in vivo.
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http://dx.doi.org/10.1097/MOH.0000000000000654DOI Listing
May 2021

Melanoma models for the next generation of therapies.

Cancer Cell 2021 May 4;39(5):610-631. Epub 2021 Feb 4.

Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, and Department of Oncology, Sidney Kimmel Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.

There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.
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http://dx.doi.org/10.1016/j.ccell.2021.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8378471PMC
May 2021

SATB2 induction of a neural crest mesenchyme-like program drives melanoma invasion and drug resistance.

Elife 2021 Feb 2;10. Epub 2021 Feb 2.

Howard Hughes Medical Institute, Stem Cell Program and the Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Harvard Medical School, Boston, United States.

Recent genomic and scRNA-seq analyses of melanoma demonstrated a lack of recurrent genetic drivers of metastasis, while identifying common transcriptional states correlating with invasion or drug resistance. To test whether transcriptional adaptation can drive melanoma progression, we made use of a zebrafish mitfa:; model, in which malignant progression is characterized by minimal genetic evolution. We undertook an overexpression-screen of 80 epigenetic/transcriptional regulators and found neural crest-mesenchyme developmental regulator SATB2 to accelerate aggressive melanoma development. Its overexpression induces invadopodia formation and invasion in zebrafish tumors and human melanoma cell lines. SATB2 binds and activates neural crest-regulators, including and . The transcriptional program induced by SATB2 overlaps with known MITFAXL and AQP1NGFR1 drug-resistant states and functionally drives enhanced tumor propagation and resistance to Vemurafenib in vivo. In summary, we show that melanoma transcriptional rewiring by SATB2 to a neural crest mesenchyme-like program can drive invasion and drug resistance in autochthonous tumors.
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http://dx.doi.org/10.7554/eLife.64370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880683PMC
February 2021

SPRED1 deletion confers resistance to MAPK inhibition in melanoma.

J Exp Med 2021 03;218(3)

Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA.

Functional evaluation of genetic lesions can discover a role in cancer initiation and progression and help develop novel therapeutic strategies. We previously identified the negative MAPK regulator SPRED1 as a novel tumor suppressor in KIT-driven melanoma. Here, we show that SPRED1 is also frequently deleted in human melanoma driven by mutant BRAF. We found that SPRED1 inactivation in human melanoma cell lines and primary zebrafish melanoma conferred resistance to BRAFV600E inhibition in vitro and in vivo. Mechanistically, SPRED1 loss promoted melanoma cell proliferation under mutant BRAF inhibition by reactivating MAPK activity. Consistently, biallelic deletion of SPRED1 was observed in a patient whose melanoma acquired resistance to MAPK-targeted therapy. These studies combining work in human cells and in vivo modeling in zebrafish demonstrate a new mechanism of resistance to BRAFV600E inhibition in melanoma.
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http://dx.doi.org/10.1084/jem.20201097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927430PMC
March 2021

Blood in the water: recent uses of zebrafish to study myeloid biology.

Curr Opin Hematol 2021 01;28(1):43-49

Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Medical School, Boston.

Purpose Of Review: Myeloid cells contribute to immune response to infection and tissue regeneration after injury as well as to the developmental induction of the hematopoietic system overall. Here we review recent uses of zebrafish to advance the study of myeloid biology in development and disease.

Recent Findings: Recent studies have made use of advanced imaging and genetic strategies and have highlighted key concepts in myeloid cell behavior. These include immune-cell cross-talk and subpopulation response in infection and regeneration, and tightly regulated inflammatory and tissue remodeling behaviors in development.

Summary: These new findings will shape our understanding of the developmental origins of immune populations as well as their specific cellular behaviors at all stages of infection, regeneration, and myeloid neoplasms.
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http://dx.doi.org/10.1097/MOH.0000000000000627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737468PMC
January 2021

Common variants in signaling transcription-factor-binding sites drive phenotypic variability in red blood cell traits.

Nat Genet 2020 12 23;52(12):1333-1345. Epub 2020 Nov 23.

Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

Genome-wide association studies identify genomic variants associated with human traits and diseases. Most trait-associated variants are located within cell-type-specific enhancers, but the molecular mechanisms governing phenotypic variation are less well understood. Here, we show that many enhancer variants associated with red blood cell (RBC) traits map to enhancers that are co-bound by lineage-specific master transcription factors (MTFs) and signaling transcription factors (STFs) responsive to extracellular signals. The majority of enhancer variants reside on STF and not MTF motifs, perturbing DNA binding by various STFs (BMP/TGF-β-directed SMADs or WNT-induced TCFs) and affecting target gene expression. Analyses of engineered human blood cells and expression quantitative trait loci verify that disrupted STF binding leads to altered gene expression. Our results propose that the majority of the RBC-trait-associated variants that reside on transcription-factor-binding sequences fall in STF target sequences, suggesting that the phenotypic variation of RBC traits could stem from altered responsiveness to extracellular stimuli.
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http://dx.doi.org/10.1038/s41588-020-00738-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876911PMC
December 2020

Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia.

Sci Transl Med 2020 10;12(566)

Stem Cell Program, Boston Children's Hospital and Harvard Stem Cell Institute, Boston, MA 02115, USA.

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 () mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34 hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34 cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.
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http://dx.doi.org/10.1126/scitranslmed.abb5831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709865PMC
October 2020

Recurrent co-alteration of HDGF and SETDB1 on chromosome 1q drives cutaneous melanoma progression and poor prognosis.

Pigment Cell Melanoma Res 2021 05 17;34(3):641-647. Epub 2020 Nov 17.

Stem Cell Program and the Division of Pediatric Hematology/Oncology, Howard Hughes Medical Institute, Boston Children's Hospital and Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.

A progressive increase in copy number variation (CNV) characterizes the natural history of cutaneous melanoma progression toward later disease stages, but our understanding of genetic drivers underlying chromosomal arm-level CNVs remains limited. To identify candidate progression drivers, we mined the TCGA SKCM dataset and identified HDGF as a recurrently amplified gene whose high mRNA expression correlates with poor patient survival. Using melanocyte-specific overexpression in the zebrafish BRAF -driven MiniCoopR melanoma model, we show that HDGF accelerates melanoma development in vivo. Transcriptional analysis of HDGF compared to control EGFP tumors showed the activation of endothelial/angiogenic pathways. We validated this observation using an endothelial kdrl:mCherry reporter line which showed HDGF to increases tumor vasculature. HDGF is frequently co-altered with the established melanoma driver SETDB1. Both genes are located on chromosome 1q, and their co-amplification is observed in up to 13% of metastatic melanoma. TCGA patients with both genes amplified and/or overexpressed have a worse melanoma specific survival. We tested co-expression of HDGF and SETDB1 in the MiniCoopR model, which resulted in faster and more aggressive melanoma development than either gene individually. Our work identifies the co-amplification of HDGF and SETDB1 as a functional driver of melanoma progression and poor patient prognosis.
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http://dx.doi.org/10.1111/pcmr.12937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050117PMC
May 2021

CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation.

Proc Natl Acad Sci U S A 2020 09 3;117(38):23626-23635. Epub 2020 Sep 3.

Division of Oncology and Center for Childhood Cancer Research, Department of Biomedical and Health Informatics, Children's Hospital of Philadelphia, Philadelphia, PA 19104.

Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation.
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http://dx.doi.org/10.1073/pnas.2003228117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519295PMC
September 2020

A Transgenic System for Rapid Magnetic Enrichment of Rare Embryonic Cells.

Zebrafish 2020 10 29;17(5):354-357. Epub 2020 Jul 29.

Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA.

Collecting large numbers of rare cells for high-throughput molecular analysis remains a technical challenge, primarily due to limitations in existing technologies. In developmental biology this has impeded single-cell analysis of primordial organs, which derive from few cells. In this study, we share novel transgenic lines for rapid cell enrichment from zebrafish embryos using human surface antigens for immunological binding and magnetic sorting. As proof of principle, we tagged, enriched, and performed single-cell RNA sequencing on nascent hematopoietic stem/progenitor cells and endothelial cells from early embryos. Our method is a quick, efficient, and cost-effective approach to a previously intractable problem.
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http://dx.doi.org/10.1089/zeb.2020.1904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578180PMC
October 2020

PRL3-DDX21 Transcriptional Control of Endolysosomal Genes Restricts Melanocyte Stem Cell Differentiation.

Dev Cell 2020 08 10;54(3):317-332.e9. Epub 2020 Jul 10.

MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh EH4 2XU, UK; Cancer Research UK Edinburgh Centre, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK. Electronic address:

Melanocytes, replenished throughout life by melanocyte stem cells (MSCs), play a critical role in pigmentation and melanoma. Here, we reveal a function for the metastasis-associated phosphatase of regenerating liver 3 (PRL3) in MSC regeneration. We show that PRL3 binds to the RNA helicase DDX21, thereby restricting productive transcription by RNAPII at master transcription factor (MITF)-regulated endolysosomal vesicle genes. In zebrafish, this mechanism controls premature melanoblast expansion and differentiation from MSCs. In melanoma patients, restricted transcription of this endolysosomal vesicle pathway is a hallmark of PRL3-high melanomas. Our work presents the conceptual advance that PRL3-mediated control of transcriptional elongation is a differentiation checkpoint mechanism for activated MSCs and has clinical relevance for the activity of PRL3 in regenerating tissue and cancer.
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http://dx.doi.org/10.1016/j.devcel.2020.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435699PMC
August 2020

Long-Range Optogenetic Control of Axon Guidance Overcomes Developmental Boundaries and Defects.

Dev Cell 2020 06;53(5):577-588.e7

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02138, USA. Electronic address:

Axons connect neurons together, establishing the wiring architecture of neuronal networks. Axonal connectivity is largely built during embryonic development through highly constrained processes of axon guidance, which have been extensively studied. However, the inability to control axon guidance, and thus neuronal network architecture, has limited investigation of how axonal connections influence subsequent development and function of neuronal networks. Here, we use zebrafish motor neurons expressing a photoactivatable Rac1 to co-opt endogenous growth cone guidance machinery to precisely and non-invasively direct axon growth using light. Axons can be guided over large distances, within complex environments of living organisms, overriding competing endogenous signals and redirecting axons across potent repulsive barriers to construct novel circuitry. Notably, genetic axon guidance defects can be rescued, restoring functional connectivity. These data demonstrate that intrinsic growth cone guidance machinery can be co-opted to non-invasively build new connectivity, allowing investigation of neural network dynamics in intact living organisms.
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http://dx.doi.org/10.1016/j.devcel.2020.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375170PMC
June 2020

Massively parallel reporter assays of melanoma risk variants identify MX2 as a gene promoting melanoma.

Nat Commun 2020 06 1;11(1):2718. Epub 2020 Jun 1.

Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, 20892, USA.

Genome-wide association studies (GWAS) have identified ~20 melanoma susceptibility loci, most of which are not functionally characterized. Here we report an approach integrating massively-parallel reporter assays (MPRA) with cell-type-specific epigenome and expression quantitative trait loci (eQTL) to identify susceptibility genes/variants from multiple GWAS loci. From 832 high-LD variants, we identify 39 candidate functional variants from 14 loci displaying allelic transcriptional activity, a subset of which corroborates four colocalizing melanocyte cis-eQTL genes. Among these, we further characterize the locus encompassing the HIV-1 restriction gene, MX2 (Chr21q22.3), and validate a functional intronic variant, rs398206. rs398206 mediates the binding of the transcription factor, YY1, to increase MX2 levels, consistent with the cis-eQTL of MX2 in primary human melanocytes. Melanocyte-specific expression of human MX2 in a zebrafish model demonstrates accelerated melanoma formation in a BRAF background. Our integrative approach streamlines GWAS follow-up studies and highlights a pleiotropic function of MX2 in melanoma susceptibility.
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http://dx.doi.org/10.1038/s41467-020-16590-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264232PMC
June 2020

An evolutionarily ancient mechanism for regulation of hemoglobin expression in vertebrate red cells.

Blood 2020 07;136(3):269-278

Department of Cell Biology, Erasmus University Medical Center (Erasmus MC), Rotterdam, The Netherlands.

The oxygen transport function of hemoglobin (HB) is thought to have arisen ∼500 million years ago, roughly coinciding with the divergence between jawless (Agnatha) and jawed (Gnathostomata) vertebrates. Intriguingly, extant HBs of jawless and jawed vertebrates were shown to have evolved twice, and independently, from different ancestral globin proteins. This raises the question of whether erythroid-specific expression of HB also evolved twice independently. In all jawed vertebrates studied to date, one of the HB gene clusters is linked to the widely expressed NPRL3 gene. Here we show that the nprl3-linked hb locus of a jawless vertebrate, the river lamprey (Lampetra fluviatilis), shares a range of structural and functional properties with the equivalent jawed vertebrate HB locus. Functional analysis demonstrates that an erythroid-specific enhancer is located in intron 7 of lamprey nprl3, which corresponds to the NPRL3 intron 7 MCS-R1 enhancer of jawed vertebrates. Collectively, our findings signify the presence of an nprl3-linked multiglobin gene locus, which contains a remote enhancer that drives globin expression in erythroid cells, before the divergence of jawless and jawed vertebrates. Different globin genes from this ancestral cluster evolved in the current NPRL3-linked HB genes in jawless and jawed vertebrates. This provides an explanation of the enigma of how, in different species, globin genes linked to the same adjacent gene could undergo convergent evolution.
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http://dx.doi.org/10.1182/blood.2020004826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531998PMC
July 2020

Transcriptome Dynamics of Hematopoietic Stem Cell Formation Revealed Using a Combinatorial Runx1 and Ly6a Reporter System.

Stem Cell Reports 2020 05 16;14(5):956-971. Epub 2020 Apr 16.

Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Stem Cell Program, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Boston, MA, USA. Electronic address:

Studies of hematopoietic stem cell (HSC) development from pre-HSC-producing hemogenic endothelial cells (HECs) are hampered by the rarity of these cells and the presence of other cell types with overlapping marker expression profiles. We generated a Tg(Runx1-mKO2; Ly6a-GFP) dual reporter mouse to visualize hematopoietic commitment and study pre-HSC emergence and maturation. Runx1-mKO2 marked all intra-arterial HECs and hematopoietic cluster cells (HCCs), including pre-HSCs, myeloid- and lymphoid progenitors, and HSCs themselves. However, HSC and lymphoid potential were almost exclusively found in reporter double-positive (DP) cells. Robust HSC activity was first detected in DP cells of the placenta, reflecting the importance of this niche for (pre-)HSC maturation and expansion before the fetal liver stage. A time course analysis by single-cell RNA sequencing revealed that as pre-HSCs mature into fetal liver stage HSCs, they show signs of interferon exposure, exhibit signatures of multi-lineage differentiation gene expression, and develop a prolonged cell cycle reminiscent of quiescent adult HSCs.
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http://dx.doi.org/10.1016/j.stemcr.2020.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7220988PMC
May 2020

Gain-of-Function Genetic Alterations of G9a Drive Oncogenesis.

Cancer Discov 2020 07 8;10(7):980-997. Epub 2020 Apr 8.

Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts.

Epigenetic regulators, when genomically altered, may become driver oncogenes that mediate otherwise unexplained pro-oncogenic changes lacking a clear genetic stimulus, such as activation of the WNT/β-catenin pathway in melanoma. This study identifies previously unrecognized recurrent activating mutations in the G9a histone methyltransferase gene, as well as G9a genomic copy gains in approximately 26% of human melanomas, which collectively drive tumor growth and an immunologically sterile microenvironment beyond melanoma. Furthermore, the WNT pathway is identified as a key tumorigenic target of G9a gain-of-function, via suppression of the WNT antagonist DKK1. Importantly, genetic or pharmacologic suppression of mutated or amplified G9a using multiple and models demonstrates that G9a is a druggable target for therapeutic intervention in melanoma and other cancers harboring G9a genomic aberrations. SIGNIFICANCE: Oncogenic G9a abnormalities drive tumorigenesis and the "cold" immune microenvironment by activating WNT signaling through DKK1 repression. These results reveal a key druggable mechanism for tumor development and identify strategies to restore "hot" tumor immune microenvironments..
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http://dx.doi.org/10.1158/2159-8290.CD-19-0532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334057PMC
July 2020

Zebrafish patient avatars in cancer biology and precision cancer therapy.

Nat Rev Cancer 2020 05 6;20(5):263-273. Epub 2020 Apr 6.

Stem Cell Program and Division of Hematology/Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA.

In precision oncology, two major strategies are being pursued for predicting clinically relevant tumour behaviours, such as treatment response and emergence of drug resistance: inference based on genomic, transcriptomic, epigenomic and/or proteomic analysis of patient samples, and phenotypic assays in personalized cancer avatars. The latter approach has historically relied on in vivo mouse xenografts and in vitro organoids or 2D cell cultures. Recent progress in rapid combinatorial genetic modelling, the development of a genetically immunocompromised strain for xenotransplantation of human patient samples in adult zebrafish and the first clinical trial using xenotransplantation in zebrafish larvae for phenotypic testing of drug response bring this tiny vertebrate to the forefront of the precision medicine arena. In this Review, we discuss advances in transgenic and transplantation-based zebrafish cancer avatars, and how these models compare with and complement mouse xenografts and human organoids. We also outline the unique opportunities that these different models present for prediction studies and current challenges they face for future clinical deployment.
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http://dx.doi.org/10.1038/s41568-020-0252-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011456PMC
May 2020
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