Publications by authors named "Daniel E Bauer"

87 Publications

Author Correction: Transcription factor competition at the γ-globin promoters controls hemoglobin switching.

Nat Genet 2021 Apr;53(4):586

Cancer and Blood Disorders Center, Dana-Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

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http://dx.doi.org/10.1038/s41588-021-00834-xDOI Listing
April 2021

Editing GWAS: experimental approaches to dissect and exploit disease-associated genetic variation.

Genome Med 2021 Mar 10;13(1):41. Epub 2021 Mar 10.

Division of Hematology/Oncology, Boston Children's Hospital; Department of Pediatric Oncology, Dana-Farber Cancer Institute; Harvard Stem Cell Institute; Broad Institute; Department of Pediatrics, Harvard Medical School, Boston, MA, USA.

Genome-wide association studies (GWAS) have uncovered thousands of genetic variants that influence risk for human diseases and traits. Yet understanding the mechanisms by which these genetic variants, mainly noncoding, have an impact on associated diseases and traits remains a significant hurdle. In this review, we discuss emerging experimental approaches that are being applied for functional studies of causal variants and translational advances from GWAS findings to disease prevention and treatment. We highlight the use of genome editing technologies in GWAS functional studies to modify genomic sequences, with proof-of-principle examples. We discuss the challenges in interrogating causal variants, points for consideration in experimental design and interpretation of GWAS locus mechanisms, and the potential for novel therapeutic opportunities. With the accumulation of knowledge of functional genetics, therapeutic genome editing based on GWAS discoveries will become increasingly feasible.
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http://dx.doi.org/10.1186/s13073-021-00857-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948363PMC
March 2021

Transcription factor competition at the γ-globin promoters controls hemoglobin switching.

Nat Genet 2021 04 1;53(4):511-520. Epub 2021 Mar 1.

Cancer and Blood Disorders Center, Dana-Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

BCL11A, the major regulator of fetal hemoglobin (HbF, αγ) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult hemoglobin (HbA, αβ). To uncover how BCL11A initiates repression, we used CRISPR-Cas9, dCas9, dCas9-KRAB and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A-binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established following BCL11A depletion, and precedes γ-globin derepression and locus control region (LCR)-globin loop formation. Our findings reveal that the switch from fetal to adult globin gene expression within the >50-kb β-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters.
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http://dx.doi.org/10.1038/s41588-021-00798-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038971PMC
April 2021

Motif-Raptor: A Cell Type-Specific and Transcription Factor Centric Approach for Post-GWAS Prioritization of Causal Regulators.

Bioinformatics 2021 Feb 3. Epub 2021 Feb 3.

Department of Pathology, Massachusetts General Hospital, Charlestown, MA, USA.

Motivation: Genome-wide association studies (GWAS) have identified thousands of common trait-associated genetic variants but interpretation of their function remains challenging. These genetic variants can overlap the binding sites of transcription factors (TFs) and therefore could alter gene expression. However, we currently lack a systematic understanding on how this mechanism contributes to phenotype.

Results: We present Motif-Raptor, a TF-centric computational tool that integrates sequence-based predictive models, chromatin accessibility, gene expression datasets and GWAS summary statistics to systematically investigate how TF function is affected by genetic variants. Given trait associated non-coding variants, Motif-Raptor can recover relevant cell types and critical TFs to drive hypotheses regarding their mechanism of action. We tested Motif-Raptor on complex traits such as rheumatoid arthritis and red blood cell count and demonstrated its ability to prioritize relevant cell types, potential regulatory TFs and non-coding SNPs which have been previously characterized and validated.

Availability: Motif-Raptor is freely available as a Python package at: https://github.com/pinellolab/MotifRaptor.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btab072DOI Listing
February 2021

Dissecting ELANE neutropenia pathogenicity by human HSC gene editing.

Cell Stem Cell 2021 Jan 25. Epub 2021 Jan 25.

Division of Hematology/Oncology, Boston Children's Hospital, Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Broad Institute, Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Severe congenital neutropenia (SCN) is a life-threatening disorder most often caused by dominant mutations of ELANE that interfere with neutrophil maturation. We conducted a pooled CRISPR screen in human hematopoietic stem and progenitor cells (HSPCs) that correlated ELANE mutations with neutrophil maturation potential. Highly efficient gene editing of early exons elicited nonsense-mediated decay (NMD), overcame neutrophil maturation arrest in HSPCs from ELANE-mutant SCN patients, and produced normal hematopoietic engraftment function. Conversely, terminal exon frameshift alleles that mimic SCN-associated mutations escaped NMD, recapitulated neutrophil maturation arrest, and established an animal model of ELANE-mutant SCN. Surprisingly, only -1 frame insertions or deletions (indels) impeded neutrophil maturation, whereas -2 frame late exon indels repressed translation and supported neutrophil maturation. Gene editing of primary HSPCs allowed faithful identification of variant pathogenicity to clarify molecular mechanisms of disease and encourage a universal therapeutic approach to ELANE-mutant neutropenia, returning normal neutrophil production and preserving HSPC function.
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http://dx.doi.org/10.1016/j.stem.2020.12.015DOI Listing
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.

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

BCL11A enhancer-edited hematopoietic stem cells persist in rhesus monkeys without toxicity.

J Clin Invest 2020 12;130(12):6677-6687

Cellular and Molecular Therapeutics Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, Bethesda, Maryland, USA.

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from patients with sickle cell disease (SCD) induces fetal hemoglobin (HbF) without detectable toxicity, as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer-edited HSPCs in 4 nonhuman primates. We used a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer- or control locus-targeted (AAVS1-targeted) cells. Biallelic BCL11A enhancer editing resulted in robust γ-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Nonhomologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we found that edited HSCs can persist for at least 101 weeks after transplant and biallelic-edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach.
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http://dx.doi.org/10.1172/JCI140189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685754PMC
December 2020

Author Correction: Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.

Nat Biotechnol 2020 Jul;38(7):901

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41587-020-0562-8DOI Listing
July 2020

Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.

Nat Biotechnol 2020 07 16;38(7):883-891. Epub 2020 Mar 16.

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Applications of adenine base editors (ABEs) have been constrained by the limited compatibility of the deoxyadenosine deaminase component with Cas homologs other than SpCas9. We evolved the deaminase component of ABE7.10 using phage-assisted non-continuous and continuous evolution (PANCE and PACE), which resulted in ABE8e. ABE8e contains eight additional mutations that increase activity (k) 590-fold compared with that of ABE7.10. ABE8e offers substantially improved editing efficiencies when paired with a variety of Cas9 or Cas12 homologs. ABE8e is more processive than ABE7.10, which could benefit screening, disruption of regulatory regions and multiplex base editing applications. A modest increase in Cas9-dependent and -independent DNA off-target editing, and in transcriptome-wide RNA off-target editing can be ameliorated by the introduction of an additional mutation in the TadA-8e domain. Finally, we show that ABE8e can efficiently install natural mutations that upregulate fetal hemoglobin expression in the BCL11A enhancer or in the the HBG promoter in human cells, targets that were poorly edited with ABE7.10. ABE8e augments the effectiveness and applicability of adenine base editing.
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http://dx.doi.org/10.1038/s41587-020-0453-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357821PMC
July 2020

Small-Molecule PAPD5 Inhibitors Restore Telomerase Activity in Patient Stem Cells.

Cell Stem Cell 2020 06 21;26(6):896-909.e8. Epub 2020 Apr 21.

Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Harvard Stem Cell Institute, Boston, MA, USA; Department of Pediatrics, Harvard Medical School, Boston, MA, USA; Stem Cell Program, Boston Children's Hospital, Boston, MA, USA; Manton Center for Orphan Disease Research, Boston Children's Hospital, Boston, MA, USA; Harvard Initiative in RNA Medicine, Boston, MA, USA. Electronic address:

Genetic lesions that reduce telomerase activity inhibit stem cell replication and cause a range of incurable diseases, including dyskeratosis congenita (DC) and pulmonary fibrosis (PF). Modalities to restore telomerase in stem cells throughout the body remain unclear. Here, we describe small-molecule PAPD5 inhibitors that demonstrate telomere restoration in vitro, in stem cell models, and in vivo. PAPD5 is a non-canonical polymerase that oligoadenylates and destabilizes telomerase RNA component (TERC). We identified BCH001, a specific PAPD5 inhibitor that restored telomerase activity and telomere length in DC patient induced pluripotent stem cells. When human blood stem cells engineered to carry DC-causing PARN mutations were xenotransplanted into immunodeficient mice, oral treatment with a repurposed PAPD5 inhibitor, the dihydroquinolizinone RG7834, rescued TERC 3' end maturation and telomere length. These findings pave the way for developing systemic telomere therapeutics to counteract stem cell exhaustion in DC, PF, and possibly other aging-related diseases.
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http://dx.doi.org/10.1016/j.stem.2020.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275922PMC
June 2020

Therapeutic base editing of human hematopoietic stem cells.

Nat Med 2020 04 16;26(4):535-541. Epub 2020 Mar 16.

Division of Hematology/Oncology, Boston Children's Hospital, Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Broad Institute, Department of Pediatrics, Harvard Medical School, Boston, MA, USA.

Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34 hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A erythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11A enhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and β-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11A erythroid enhancer and correction of the HBB -28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self-renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification.
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http://dx.doi.org/10.1038/s41591-020-0790-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869435PMC
April 2020

End points for sickle cell disease clinical trials: renal and cardiopulmonary, cure, and low-resource settings.

Blood Adv 2019 12;3(23):4002-4020

Division of Pediatric Hematology Oncology and Stem Cell Transplant, Department of Pediatrics, Washington University School of Medicine, St. Louis, MO.

To address the global burden of sickle cell disease and the need for novel therapies, the American Society of Hematology partnered with the US Food and Drug Administration to engage the work of 7 panels of clinicians, investigators, and patients to develop consensus recommendations for clinical trial end points. The panels conducted their work through literature reviews, assessment of available evidence, and expert judgment focusing on end points related to patient-reported outcome, pain (non-patient-reported outcomes), the brain, end-organ considerations, biomarkers, measurement of cure, and low-resource settings. This article presents the findings and recommendations of the end-organ considerations, measurement of cure, and low-resource settings panels as well as relevant findings and recommendations from the biomarkers panel.
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http://dx.doi.org/10.1182/bloodadvances.2019000883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963248PMC
December 2019

Genome editing of HBG1 and HBG2 to induce fetal hemoglobin.

Blood Adv 2019 11;3(21):3379-3392

Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN.

Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress γ-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and β-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34+ hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2rγ-/-/KitW41/W41 immunodeficient mice. On-target editing did not impair CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies.
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http://dx.doi.org/10.1182/bloodadvances.2019000820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855127PMC
November 2019

Production of foetal globin in adult monkeys.

Authors:
Daniel E Bauer

Nat Biomed Eng 2019 11;3(11):857-859

Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA.

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http://dx.doi.org/10.1038/s41551-019-0475-3DOI Listing
November 2019

Single-cell cloning of human T-cell lines reveals clonal variation in cell death responses to chemotherapeutics.

Cancer Genet 2019 09 12;237:69-77. Epub 2019 Jun 12.

Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, United States; Department of Pediatrics, Harvard Medical School, Boston, MA, United States. Electronic address:

Genetic modification of human leukemic cell lines using CRISPR-Cas9 has become a staple of gene-function studies. Single-cell cloning of modified cells is frequently used to facilitate studies of gene function. Inherent in this approach is an assumption that the genetic drift, amplified in some cell lines by mutations in DNA replication and repair machinery, as well as non-genetic factors will not introduce significant levels of experimental cellular heterogeneity in clones derived from parental populations. In this study, we characterize the variation in cell death of fifty clonal cell lines generated from human Jurkat and MOLT-4 T-cells edited by CRISPR-Cas9. We demonstrate a wide distribution of sensitivity to chemotherapeutics between non-edited clonal human leukemia T-cell lines, and also following CRISPR-Cas9 editing at the NLRP1 locus, or following transfection with non-targeting sgRNA controls. The cell death sensitivity profile of clonal cell lines was consistent across experiments and failed to revert to the non-clonal parental phenotype. Whole genome sequencing of two clonal cell lines edited by CRISPR-Cas9 revealed unique and shared genetic variants, which had minimal read support in the non-clonal parental population and were not suspected CRISPR-Cas9 off-target effects. These variants included genes related to cell death and drug metabolism. The variation in cell death phenotype of clonal populations of human T-cell lines may be a consequence of T-cell line genetic instability, and to a lesser extent clonal heterogeneity in the parental population or CRISPR-Cas9 off-target effects not predicted by current models. This work highlights the importance of genetic variation between clonal T-cell lines in the design, conduct, and analysis of experiments to investigate gene function after single-cell cloning.
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http://dx.doi.org/10.1016/j.cancergen.2019.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6711394PMC
September 2019

Rational targeting of a NuRD subcomplex guided by comprehensive in situ mutagenesis.

Nat Genet 2019 07 28;51(7):1149-1159. Epub 2019 Jun 28.

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

Developmental silencing of fetal globins serves as both a paradigm of spatiotemporal gene regulation and an opportunity for therapeutic intervention of β-hemoglobinopathy. The nucleosome remodeling and deacetylase (NuRD) chromatin complex participates in γ-globin repression. We used pooled CRISPR screening to disrupt NuRD protein coding sequences comprehensively in human adult erythroid precursors. Essential for fetal hemoglobin (HbF) control is a non-redundant subcomplex of NuRD protein family paralogs, whose composition we corroborated by affinity chromatography and proximity labeling mass spectrometry proteomics. Mapping top functional guide RNAs identified key protein interfaces where in-frame alleles resulted in loss-of-function due to destabilization or altered function of subunits. We ascertained mutations of CHD4 that dissociate its requirement for cell fitness from HbF repression in both primary human erythroid precursors and transgenic mice. Finally we demonstrated that sequestering CHD4 from NuRD phenocopied these mutations. These results indicate a generalizable approach to discover protein complex features amenable to rational biochemical targeting.
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http://dx.doi.org/10.1038/s41588-019-0453-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650275PMC
July 2019

Single-cell trajectories reconstruction, exploration and mapping of omics data with STREAM.

Nat Commun 2019 04 23;10(1):1903. Epub 2019 Apr 23.

Molecular Pathology Unit & Cancer Center, Massachusetts General Hospital Research Institute and Harvard Medical School, Boston, MA, 02114, USA.

Single-cell transcriptomic assays have enabled the de novo reconstruction of lineage differentiation trajectories, along with the characterization of cellular heterogeneity and state transitions. Several methods have been developed for reconstructing developmental trajectories from single-cell transcriptomic data, but efforts on analyzing single-cell epigenomic data and on trajectory visualization remain limited. Here we present STREAM, an interactive pipeline capable of disentangling and visualizing complex branching trajectories from both single-cell transcriptomic and epigenomic data. We have tested STREAM on several synthetic and real datasets generated with different single-cell technologies. We further demonstrate its utility for understanding myoblast differentiation and disentangling known heterogeneity in hematopoiesis for different organisms. STREAM is an open-source software package.
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http://dx.doi.org/10.1038/s41467-019-09670-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478907PMC
April 2019

CRISPR-suppressor scanning reveals a nonenzymatic role of LSD1 in AML.

Nat Chem Biol 2019 05 15;15(5):529-539. Epub 2019 Apr 15.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.

Understanding the mechanism of small molecules is a critical challenge in chemical biology and drug discovery. Medicinal chemistry is essential for elucidating drug mechanism, enabling variation of small molecule structure to gain structure-activity relationships (SARs). However, the development of complementary approaches that systematically vary target protein structure could provide equally informative SARs for investigating drug mechanism and protein function. Here we explore the ability of CRISPR-Cas9 mutagenesis to profile the interactions between lysine-specific histone demethylase 1 (LSD1) and chemical inhibitors in the context of acute myeloid leukemia (AML). Through this approach, termed CRISPR-suppressor scanning, we elucidate drug mechanism of action by showing that LSD1 enzyme activity is not required for AML survival and that LSD1 inhibitors instead function by disrupting interactions between LSD1 and the transcription factor GFI1B on chromatin. Our studies clarify how LSD1 inhibitors mechanistically operate in AML and demonstrate how CRISPR-suppressor scanning can uncover novel aspects of target biology.
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http://dx.doi.org/10.1038/s41589-019-0263-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679026PMC
May 2019

Synthetic Lethality of Wnt Pathway Activation and Asparaginase in Drug-Resistant Acute Leukemias.

Cancer Cell 2019 04;35(4):664-676.e7

Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02445, USA; Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142, USA. Electronic address:

Resistance to asparaginase, an antileukemic enzyme that depletes asparagine, is a common clinical problem. Using a genome-wide CRISPR/Cas9 screen, we found a synthetic lethal interaction between Wnt pathway activation and asparaginase in acute leukemias resistant to this enzyme. Wnt pathway activation induced asparaginase sensitivity in distinct treatment-resistant subtypes of acute leukemia, but not in normal hematopoietic progenitors. Sensitization to asparaginase was mediated by Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits glycogen synthase kinase 3 (GSK3)-dependent protein ubiquitination and proteasomal degradation, a catabolic source of asparagine. Inhibiting the alpha isoform of GSK3 phenocopied this effect, and pharmacologic GSK3α inhibition profoundly sensitized drug-resistant leukemias to asparaginase. Our findings provide a molecular rationale for activation of Wnt/STOP signaling to improve the therapeutic index of asparaginase.
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http://dx.doi.org/10.1016/j.ccell.2019.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541931PMC
April 2019

Highly efficient therapeutic gene editing of human hematopoietic stem cells.

Nat Med 2019 05 25;25(5):776-783. Epub 2019 Mar 25.

Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.

Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal hemoglobin (HbF, αγ). Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.
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http://dx.doi.org/10.1038/s41591-019-0401-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6512986PMC
May 2019

Editing aberrant splice sites efficiently restores β-globin expression in β-thalassemia.

Blood 2019 05 31;133(21):2255-2262. Epub 2019 Jan 31.

Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA.

The thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here, we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34 hematopoietic stem and progenitor cells (HSPCs) from β-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of β-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent β-thalassemia genotypes with currently available gene-editing technology.
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http://dx.doi.org/10.1182/blood-2019-01-895094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533605PMC
May 2019

Getting Past HSC Security: Cyclosporine H Gives Lentiviruses an Entry Pass.

Cell Stem Cell 2018 12;23(6):775-776

Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Harvard Medical School, Boston, MA, USA. Electronic address:

In this issue of Cell Stem Cell, Petrillo et al. (2018) improve lentiviral transduction of hematopoietic stem cells (HSCs) by using cyclosporine H to relieve viral entry restriction by interferon-induced transmembrane protein 3 (IFITM3). This finding promises to enhance the efficiency of ex vivo therapeutic gene transfer and gene editing of HSCs.
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http://dx.doi.org/10.1016/j.stem.2018.11.008DOI Listing
December 2018

DrugThatGene: integrative analysis to streamline the identification of druggable genes, pathways and protein complexes from CRISPR screens.

Bioinformatics 2019 06;35(11):1981-1984

Molecular Pathology Unit, Center for Computational and Integrative Biology, Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA.

Motivation: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) nuclease system has allowed for high-throughput, large scale pooled screens for functional genomic studies. To aid in the translation of functional genomics to therapeutics, we developed DrugThatGene (DTG) as a web-based application that streamlines analysis of potential therapeutic targets identified from functional genetic screens.

Results: Starting from a gene list as input, DTG offers automated identification of small molecules along with supporting information from human genetic and other relevant databases. Furthermore, DTG aids in the identification of common biological pathways and protein complexes in conjunction with associated small molecule inhibitors. Taken together, DTG aims to expedite the identification of small molecules from the abundance of functional genetic data generated from CRISPR screens.

Availability And Implementation: DTG is an open-source and free software available as a website at http://drugthatgene.pinellolab.org. Source code is available at: https://github.com/pinellolab/DrugThatGene, which can be downloaded in order to run DTG locally.
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http://dx.doi.org/10.1093/bioinformatics/bty913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546128PMC
June 2019

FAM210B is an erythropoietin target and regulates erythroid heme synthesis by controlling mitochondrial iron import and ferrochelatase activity.

J Biol Chem 2018 12 26;293(51):19797-19811. Epub 2018 Oct 26.

the Division of Hematology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.

Erythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates iron status are not well-understood. To this end, here we profiled gene expression in EPO-treated 32D pro-B cells and developing fetal liver erythroid cells to identify additional iron regulatory genes. We determined that FAM210B, a mitochondrial inner-membrane protein, is essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation. deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron-sulfur cluster formation. These defects were corrected with a lipid-soluble, small-molecule iron transporter, hinokitiol, in -deficient murine erythroid cells and zebrafish morphants. Genetic complementation experiments revealed that FAM210B is not a mitochondrial iron transporter but is required for adequate mitochondrial iron import to sustain heme synthesis and iron-sulfur cluster formation during erythroid differentiation. FAM210B was also required for maximal ferrochelatase activity in differentiating erythroid cells. We propose that FAM210B functions as an adaptor protein that facilitates the formation of an oligomeric mitochondrial iron transport complex, required for the increase in iron acquisition for heme synthesis during terminal erythropoiesis. Collectively, our results reveal a critical mechanism by which EPO signaling regulates terminal erythropoiesis and iron metabolism.
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http://dx.doi.org/10.1074/jbc.RA118.002742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314115PMC
December 2018

Emerging Genetic Therapy for Sickle Cell Disease.

Annu Rev Med 2019 01 24;70:257-271. Epub 2018 Oct 24.

Dana Farber/Boston Children's Cancer and Blood Disorders Center, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA; email:

The genetic basis of sickle cell disease (SCD) was elucidated >60 years ago, yet current therapy does not rely on this knowledge. Recent advances raise prospects for improved, and perhaps curative, treatment. First, transcription factors, BCL11A and LRF/ZBTB7A, that mediate silencing of the β-like fetal (γ-) globin gene after birth have been identified and demonstrated to act at the γ-globin promoters, precisely at recognition sequences disrupted in rare individuals with hereditary persistence of fetal hemoglobin. Second, transformative advances in gene editing and progress in lentiviral gene therapy provide diverse opportunities for genetic strategies to cure SCD. Approaches include hematopoietic gene therapy by globin gene addition, gene editing to correct the SCD mutation, and genetic manipulations to enhance fetal hemoglobin production, a potent modifier of the clinical phenotype. Clinical trials may soon identify efficacious and safe genetic approaches to the ultimate goal of cure for SCD.
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http://dx.doi.org/10.1146/annurev-med-041817-125507DOI Listing
January 2019

CRISPRO: identification of functional protein coding sequences based on genome editing dense mutagenesis.

Genome Biol 2018 10 19;19(1):169. Epub 2018 Oct 19.

Division of Hematology/Oncology, Boston Children's Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Broad Institute, Harvard Medical School, Boston, MA, 02115, USA.

CRISPR/Cas9 pooled screening permits parallel evaluation of comprehensive guide RNA libraries to systematically perturb protein coding sequences in situ and correlate with functional readouts. For the analysis and visualization of the resulting datasets, we develop CRISPRO, a computational pipeline that maps functional scores associated with guide RNAs to genomes, transcripts, and protein coordinates and structures. No currently available tool has similar functionality. The ensuing genotype-phenotype linear and three-dimensional maps raise hypotheses about structure-function relationships at discrete protein regions. Machine learning based on CRISPRO features improves prediction of guide RNA efficacy. The CRISPRO tool is freely available at gitlab.com/bauerlab/crispro .
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http://dx.doi.org/10.1186/s13059-018-1563-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195731PMC
October 2018

An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities.

Nat Biotechnol 2018 11 30;36(10):977-982. Epub 2018 Jul 30.

Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

Base editor technology, which uses CRISPR-Cas9 to direct cytidine deaminase enzymatic activity to specific genomic loci, enables the highly efficient introduction of precise cytidine-to-thymidine DNA alterations. However, existing base editors create unwanted C-to-T alterations when more than one C is present in the enzyme's five-base-pair editing window. Here we describe a strategy for reducing bystander mutations using an engineered human APOBEC3A (eA3A) domain, which preferentially deaminates cytidines in specific motifs according to a TCR>TCY>VCN hierarchy. In direct comparisons with the widely used base editor 3 (BE3) fusion in human cells, our eA3A-BE3 fusion exhibits similar activities on cytidines in TC motifs but greatly reduced editing on cytidines in other sequence contexts. eA3A-BE3 corrects a human β-thalassemia promoter mutation with much higher (>40-fold) precision than BE3. We also demonstrate that eA3A-BE3 shows reduced mutation frequencies on known off-target sites of BE3, even when targeting promiscuous homopolymeric sites.
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http://dx.doi.org/10.1038/nbt.4199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6181770PMC
November 2018