John Brown

Clinical outcomes of newborn screening for cystic fibrosis.

AIM To determine how early diagnosis of cystic fibrosis, using neonatal screening, affects long term clinical outcome. METHODS Fifty seven children with cystic fibrosis born before neonatal screening was introduced (1978 to mid 1981) and a further 60 children born during the first three years of the programme (mid 1981 to 1984), were followed up to the age of 10. The cohorts were compared on measures of clinical outcome, including height, weight, lung function tests, chest x-ray picture and Shwachman score. RESULTS Age and sex adjusted standard deviation scores (SDS) for height and weight were consistently higher in children screened for cystic fibrosis than in those born before screening. At 10 years of age, average differences in SDS between groups were 0.4 (95% CI −0.1, 0.8) for weight and 0.3 (95% CI −0.1, 0.7) for height. This translates to an average difference of about 2.7 cm in height and 1.7 kg in weight. Mean FEV1 and FVC (as percentage predicted) were significantly higher in the screened cohort at 5 and 10 years of age, with an average difference of 9.4% FEV1(95% CI 0.8, 17.9) and 8.4% FVC (95% CI 1.8, 15.0) at 10 years. Chest x-ray scores were not different between the groups at any age, but by 10 years screened patients scored an average 5.3 (95% CI 1.2, 9.4) points higher on the Shwachman score. CONCLUSION Although not a randomised trial, this long term observational study indicates that early treatment made possible by neonatal screening may be important in determining subsequent clinical outcomes for children with cystic fibrosis. For countries contemplating the introduction of neonatal screening for cystic fibrosis, its introduction to some areas in a cluster randomised design will permit validation of studies performed to date.

Dynamic Analysis of Gene Expression and Genome-wide Transcription Factor Binding during Lineage Specification of Multipotent Progenitors.

Summary We used the paradigmatic GATA-PU.1 axis to explore, at the systems level, dynamic relationships between transcription factor (TF) binding and global gene expression programs as multipotent cells differentiate. We combined global ChIP-seq of GATA1, GATA2, and PU.1 with expression profiling during differentiation to erythroid and neutrophil lineages. Our analysis reveals (1) differential complexity of sequence motifs bound by GATA1, GATA2, and PU.1; (2) the scope and interplay of GATA1 and GATA2 programs within, and during transitions between, different cell compartments, and the extent of their hard-wiring by DNA motifs; (3) the potential to predict gene expression trajectories based on global associations between TF-binding data and target gene expression; and (4) how dynamic modeling of DNA-binding and gene expression data can be used to infer regulatory logic of TF circuitry. This rubric exemplifies the utility of this cross-platform resource for deconvoluting the complexity of transcriptional programs controlling stem/progenitor cell fate in hematopoiesis.

Single-Cell Network Analysis Identifies DDIT3 as a Nodal Lineage Regulator in Hematopoiesis

Summary We explore cell heterogeneity during spontaneous and transcription-factor-driven commitment for network inference in hematopoiesis. Since individual genes display discrete OFF states or a distribution of ON levels, we compute and combine pairwise gene associations from binary and continuous components of gene expression in single cells. Ddit3 emerges as a regulatory node with positive linkage to erythroid regulators and negative association with myeloid determinants. Ddit3 loss impairs erythroid colony output from multipotent cells, while forcing Ddit3 in granulo-monocytic progenitors (GMPs) enhances self-renewal and impedes differentiation. Network analysis of Ddit3-transduced GMPs reveals uncoupling of myeloid networks and strengthening of erythroid linkages. RNA sequencing suggests that Ddit3 acts through development or stabilization of a precursor upstream of GMPs with inherent Meg-E potential. The enrichment of Gata2 target genes in Ddit3-dependent transcriptional responses suggests that Ddit3 functions in an erythroid transcriptional network nucleated by Gata2.

Diagnosis of cystic fibrosis.

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A Human IPS Model Implicates Embryonic B-Myeloid Fate Restriction as Developmental Susceptibility to B Acute Lymphoblastic Leukemia-Associated ETV6-RUNX1.

Summary ETV6-RUNX1 is associated with childhood acute B-lymphoblastic leukemia (cALL) functioning as a first-hit mutation that initiates a clinically silent pre-leukemia in utero. Because lineage commitment hierarchies differ between embryo and adult, and the impact of oncogenes is cell-context dependent, we hypothesized that the childhood affiliation of ETV6-RUNX1 cALL reflects its origins in a progenitor unique to embryonic life. We characterize the first emerging B cells in first-trimester human embryos, identifying a developmentally restricted CD19−IL-7R+ progenitor compartment, which transitions from a myeloid to lymphoid program during ontogeny. This developmental series is recapitulated in differentiating human pluripotent stem cells (hPSCs), thereby providing a model for the initiation of cALL. Genome-engineered hPSCs expressing ETV6-RUNX1 from the endogenous ETV6 locus show expansion of the CD19−IL-7R+ compartment, show a partial block in B lineage commitment, and produce proB cells with aberrant myeloid gene expression signatures and potential: features (collectively) consistent with a pre-leukemic state.

A network including TGFβ/Smad4, Gata2, and p57 regulates proliferation of mouse hematopoietic progenitor cells

Transforming growth factor β (TGFβ) is a potent inhibitor of hematopoietic stem and progenitor cell proliferation. However, the precise mechanism for this effect is unknown. Here, we have identified the transcription factor Gata2, previously described as an important regulator of hematopoietic stem cell function, as an early and direct target gene for TGFβ-induced Smad signaling in hematopoietic progenitor cells. We also report that Gata2 is involved in mediating a significant part of the TGFβ response in primitive hematopoietic cells. Interestingly, the cell cycle regulator and TGFβ signaling effector molecule p57 was found to be upregulated as a secondary response to TGFβ. We observed Gata2 binding upstream of the p57 genomic locus, and importantly, loss of Gata2 abolished TGFβ-stimulated induction of p57 as well as the resulting growth arrest of hematopoietic progenitors. Our results connect key molecules involved in hematopoietic stem cell self-renewal and reveal a functionally relevant network, regulating proliferation of primitive hematopoietic cells.

Modelling the in-utero initation of ETV6-RUNX1 in childhood acute lymphoblastic leukaemia using human pluripotent stem cells

Abstract Background We hypothesise that the clinical differences between adult and childhood acute lymphoblastic leukaemia (cALL) arise partly through their origin in developmentally distinct target cells. Since cALL frequently initiates in utero we aimed to characterise the earliest stages of lymphoid development in human fetal liver. In parallel we used human pluripotent stem cells (hPSC) to recapitulate fetal liver lymphopoiesis and model the impact of the fusion oncogene ETV6-RUNX1 , the commonest genetic aberration in cALL. Methods H1 human embyronic stem cells and MIFF3 human induced hPSCs (provided by University of Sheffield Centre for Stem Cell Biology) were maintained in vitro with mTeSR1/Matrigel. Vectors were produced by recombineering an ETV6 bacterial artificial chromosome with a custom DNA cassette (GeneArt, ThermoFisher, Waltham MA, USA). Vectors were transfected (Nucleofector II, Lonza, Basle, Switzerland) and G418-selected clones were screened by Southern blot. hPSCs were differentiated by sequential OP9/MS5 coculture. Populations sorted by fluorescence-activated cell sorting were analysed by single cell real-time PCR (Biomark 48.48, Fluidigm, San Fransisco, CA, USA) and 200 cell RNA sequencing. Human fetal livers were donated under informed consent with approval of Lund University Ethical Review Board and the Swedish National Board of Health and Welfare. Findings B lymphopoiesis in the fetal liver was distinct from that in adult bone marrow or neonatal cord blood: the earliest fetal liver B cells did not fully express mature lymphoid effectors and the earliest identified lymphoid-capable progenitors coexpressed both lymphoid and myeloid gene expression programmes. Global and single cell gene expression profiling of B lymphopoietic progenitors generated by in vitro differentiation of hPSCs showed that they share the same transcriptional programme. hPSCs that were CRISPR-engineered to express ETV6-RUNX1 were partly arrested in B cell differentiation at the level of the lymphomyeloid progenitor. B cells that passed this block shared the global gene expression signature of the fetal lymphomyeloid progenitor, and single cell real-time PCR showed that they aberrantly coexpressed B and myeloid lineage genes. Interpretation Our results identify a B lymphoid progenitor in human fetal liver characterised by coexpression of lymphoid and myeloid gene expression programmes and suggest that this progenitors B myeloid signature provides a permissive transcriptional context for ETV6-RUNX1 to effect a partial differentiation arrest. The developmental specificity of the gene expression signature of this cell could offer unique therapeutic targets, and the hSPC model might provide a novel drug-screening platform. Funding Wellcome Trust Research Training Fellowship and NIHR Academic Clinical Fellowship (SR); Swedish Childhood Cancer Foundation (CB); Bloodwise, Cancer Research UK, Children with Cancer, and Great Ormond Street Hospital Childrens Charity (TE).

Abstract 2692: ETV6-RUNX1 targets a developmentally restricted embryonic human B-myeloid progenitor

Childhood acute lymphoblastic leukemia (cALL) is distinct from that in adults with higher incidence, better prognosis and a distinct mutational spectrum. One hypothesis for this difference is that cALL arises in transient cells unique to early human development. We explored this in ETV6-RUNX1 cALL where evidence from twins and neonatal heel prick testing has shown that this mutation arises in utero and is an initiating event. We characterized B cell development in first trimester human fetal liver (FL) to identify compartments vulnerable to ETV6-RUNX1. Using CD19 as a marker of B lineage commitment we found the first CD19+ B cells emerge in the human FL at Carnegie Stage (CS) 17 and were distinct from adult in that the majority expressed surface IL7 receptor. We used IL7R to identify a CD19-IL7R+ B progenitor compartment that produced B cells in vitro, possessed DJH recombination, but also had monocytic potential. Single cell analysis of CS20 IL7R+ progenitors revealed co-expression of lymphoid and myeloid programmes, whereas at CS17 they were strongly myeloid primed indicating that IL7R+ progenitors acquire lymphoid potential in this developmental window. Some co-expression of lymphoid and myeloid programmes also persisted in CS20 FL B cells. We tested whether FL B cell development could be modeled using human pluripotent stem cells (hPSCs). In vitro B cell differentiation of hPSCs produced IL7R expressing pro and preB cells as well as an IL7R+ progenitor that switched from myeloid to B-myeloid priming during culture. At the global transcriptional level the hPSC lymphoid hierarchy mapped closely with FL, with both separating from adult suggesting that hPSCs provide a developmentally relevant model of early FL B lymphopoiesis. We next used CRIPSR-directed homologous recombination to engineer the expression of ETV6-RUNX1 under the endogenous ETV6 promoter. ETV6-RUNX1 hPSCs displayed a partial block in B cell differentiation at the level of the IL7R+ progenitor. ETV6-RUNX1 expressing B cells co-expressed an abnormal B-myeloid gene expression signature akin to that seen in the IL7R+ progenitor. Both the transcriptional and differentiation phenotypes were dependent on ETV6-RUNX1 as demonstrated by their reversion upon cre-mediated excision of the knock-in cassette. Our data support a model where expression of ETV6-RUNX1 inhibits lymphoid specification in an early FL IL7R+ lymphomyeloid progenitor, arresting B lineage differentiation and resulting in the production of myeloid-primed B cells. This may explain the relatively high levels of myeloid antigen expression lineage promiscuity seen in cALL. ETV6-RUNX1 hPSCs will afford the systematic evaluation of the contribution of additional mutations seen in cALL and may offer a tractable platform for drug screening. In conclusion we propose that a novel IL7R+ lymphomyeloid progenitor in the human FL is a candidate target cell for in utero pre-leukemic initiation in cALL. Citation Format: Simon E. Richardson, Charlotta Boiers, Alya Zriwil, Virginia Turati, John Brown, Dapeng Wang, Javier Herrero, Stefan Karlsson, Andrew J. H. Smith, Sten Erik Jacobsen, Tariq Enver. ETV6-RUNX1 targets a developmentally restricted embryonic human B-myeloid progenitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2692.