Developmental-stage-dependent transcriptional response to leukaemic oncogene expression

10.1038/ncomms8203Nature Communications6720

RUNX1B Expression is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis

The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny. RUNX1 also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter mouse model we demonstrate that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage- Sca1high cKithigh (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid (Ery) specification. Accordingly the PreMegE population can be prospectively separated into "pro-erythroid" and "pro-megakaryocyte" populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2+ and P2- populations indicated that levels of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will enable the further investigation of molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Having characterized the extensive activity of P1, we utilized a P1-GFP homozygous mouse model to analyze the impact of the complete absence of Runx1 P1 expression in adult mice and observed strong defects in the T cell lineage. Finally, we investigated how the leukemic fusion protein AML1-ETO9a might influence Runx1 promoter usage. Short-term AML1-ETO9a induction in BM resulted in preferential P2 upregulation, suggesting its expression may be important to establish a pre-leukemic environment

<i>Runx1 P1</i> and <i>P2</i> expression in mature hematopoietic lineages.

<p>(A, C, E, G, I, K) Contour plots of <i>Runx1 P1-GFP</i> and <i>P2-hCD4</i> expression in BM erythroid (A), granulocytic/macrophage (C) and B lymphocyte populations (E), spleen B (G) or T (K) lymphocytes and thymocytes (I), as defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005814#pgen.1005814.s001" target="_blank">S1 Fig</a>. (B, D, F, H, J, L) Numbers of <i>Runx1 P1</i>^<i>-</i><i>P2</i>^<i>-</i>, <i>P1</i>^<i>+</i><i>P2</i>^<i>-</i> and <i>P1</i>^<i>+</i><i>P2</i>^<i>+</i> cells as a proportion of defined BM erythroid (B), granulocytic/macrophage (D) and B lymphocyte populations (F), spleen B (H) or T (L) lymphocytes and thymocytes (J). Representative data of three independent experiments are shown.</p

<i>Runx1 P2</i> expression in GM-restricted progenitors enriches for monocyte/macrophage specification.

<p>(A) Contour plots of adult BM Lin^- Sca1^- cKit⁺ (LK) GM progenitors. The GMP and PreGM progenitors can be distinguished on the basis of CD41, CD16/32, SLAMF1 (CD150) and Endoglin expression. (B) Representative FACS plots of P1-GFP/P2-hCD4 expression in PreGM and GMP cells. (C–E) CFU-C activity of <i>WT</i>, <i>P1</i>^<i>+</i><i>P2</i>^<i>-</i> and <i>P1</i>^<i>+</i><i>P2</i>^<i>+</i> PreGMs and GMPs following culture in pro-myeloid semi-solid methylcellulose-based medium. (C) Total CFU-C numbers (%). (D-E) Granulocyte (CFU-G), macrophage (CFU-M) and granulocyte/macrophage (CFU-GM) colony forming unit numbers per 100 plated PreGMs (D) or 500 plated GMPs (E). n = 4 independent experiments.</p

Joint ABS-UKCGG-CanGene-CanVar consensus regarding the use of CanRisk in clinical practice.

Acknowledgements: Thank you to Rosie Way and Bethany Torr who helped with organisation of the meeting and preparation of the manuscript.Funder: National Institute for Health and Care Research Exeter Biomedical Research Centre (NIHR203320)Funder: NIHR Cambridge Biomedical Research Centre (NIHR203312)BACKGROUND: The CanRisk tool, which operationalises the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) is used by Clinical Geneticists, Genetic Counsellors, Breast Oncologists, Surgeons and Family History Nurses for breast cancer risk assessments both nationally and internationally. There are currently no guidelines with respect to the day-to-day clinical application of CanRisk and differing inputs to the model can result in different recommendations for practice. METHODS: To address this gap, the UK Cancer Genetics Group in collaboration with the Association of Breast Surgery and the CanGene-CanVar programme held a workshop on 16th of May 2023, with the aim of establishing best practice guidelines. RESULTS: Using a pre-workshop survey followed by structured discussion and in-meeting polling, we achieved consensus for UK best practice in use of CanRisk in making recommendations for breast cancer surveillance, eligibility for genetic testing and the input of available information to undertake an individualised risk assessment. CONCLUSIONS: Whilst consensus recommendations were achieved, the meeting highlighted some of the barriers limiting the use of CanRisk in clinical practice and identified areas that require further work and collaboration with relevant national bodies and policy makers to incorporate wider use of CanRisk into routine breast cancer risk assessments

Global gene expression analysis of <i>Runx1 P2-hCD4+</i> and <i>P2-hCD4-</i> PreMegEs enables identification of WT equivalents.

<p>(A) Heat map depiction of genes at least 2-fold differentially expressed between P1⁺ P2^- (P2^-) and P1⁺ P2⁺ (P2⁺) PreMegE samples, as determined by RNA Seq. Genes in red are upregulated and genes in blue are downregulated. (B) GSEA showing significantly enriched signaling pathways in the gene set upregulated in P2⁺ PreMegEs relative to P2^- PreMegEs. (C) Reads per kilobase per million mapped reads (RPKM) values of selected Mk/Ery- associated genes. (n = 3). (D) Quantitative PCR (qPCR) validation of expression of genes depicted in D. (n = 5). (E) Representative FACS plots of CD34 expression in P2⁺ and P2^- PreMegEs (top) and unsorted, purified CD34⁺ and purified CD34- WT PreMegEs (bottom). (<i>P1-GFP</i>::<i>P2-hCD4</i> mice n = 3; <i>WT</i> mice n = 5). (F) CFU-C activity of CD34⁺ and CD34^- PreMegEs following culture in pro-myeloid semi-solid methylcellulose-based medium. (n = 5). (G) Contour plots of OP9 co-cultured CD34⁺ and CD34^- PreMegE cells isolated on day 7 and stained with CD41 and Ter119 antibodies. (H) Quantification of CD41⁺ megakaryocytes and Ter119⁺ erythrocytes in the PreMegE/OP9 co-culture assays (n = 4).</p

Distinct megakaryocytic and erythroid progenitors can be isolated on the basis of <i>Runx1 P2-hCD4</i> expression.

<p>(A) Contour plots of adult BM LK Mk/Ery progenitors. The PreMegE, MkP, PreCFUe and CFUe populations can by distinguished on the basis of cKit, CD41, SLAMF1 (CD150), CD16/32 and Endoglin cell surface expression. (B) Representative FACS plots of P1-GFP/P2-hCD4 expression in PreMegE, MkP, PreCFUe and CFUe subsets. (n = 8). (C) P1-GFP/P2-hCD4 expression in BM MkP-derived cultured CD41⁺ megakaryocytes. (n = 4). (D–H) CFU-C activity of <i>WT</i>, <i>P1</i>^<i>+</i><i>P2</i>^<i>-</i> and <i>P1</i>^<i>+</i><i>P2</i>^<i>+</i> PreMegEs. Cells were cultured either in pro-myeloid semi-solid methylcellulose-based medium (D) or in pro-megakaryocytic collagen-based MegaCultTM medium (F). Photographs of representative PreMegE-derived methylcellulose (E) and MegaCultTM (G) colonies. (n = 5) (H) Numbers of megakaryocytes per MegaCultTM CFU-Mk colony from 3 independent experiments (mean ± SD, Mann-Whitney <i>U</i> test). (I) Contour plots of OP9 co-cultured PreMegE cells isolated on day 7 and stained with CD41 and Ter119 antibodies. (J) Quantification of CD41⁺ megakaryocytes and Ter119⁺ erythrocytes in the PreMegE/OP9 co-culture assays. (n = 5). (K) Representative FACS plots of PreMegE cells following short-term (12 hours) culture in pro-myeloid liquid medium. Top: CD150/CD41 expression of LK CD16/32^- progenitor cells. Middle: Endoglin/CD150 expression of LK CD41 negative CD16/32 negative progenitors. Bottom: <i>P1-GFP</i>/<i>P2-hCD4</i> expression of immunophenotypic PreMegE (LK CD41- CD16/32- Endoglin- CD150⁺) cells (n = 3).</p

Effect of short-term induction of <i>AML1-ETO9a</i> expression on <i>Runx1</i> expression in BM HSPCs.

<p>(A) Top: Schematic representation of the <i>Rosa26</i> and <i>Hprt</i> loci in the Doxycycline-inducible AML1-ETO9a GFP mouse model. The reverse tetracycline-controlled transcriptional activator (rtTA) is constitutively expressed under the control of the <i>Rosa</i> promoter. Upon binding to Doxycycline, the rtTA is capable of binding to and activating a tetracycline responsive element (TRE) located in the ubiquitously expressed <i>Hprt</i> locus, resulting in expression of the hemagglutinin (HA)-tagged AML1-ETO9a::IRES-GFP construct also incorporated into this locus. Bottom: Schematic diagram of the experimental design to induce short-term expression of <i>AML1-ETO9a</i> in adult mice by administering Doxycycline in food for 8 days. Bone marrow cells were the harvested and GFP⁺ and GFP^- HSPC populations were isolated by FACS sorting for RNA extraction and gene expression analysis. (B-E) Gene expression analysis by qPCR of <i>AML1-ETO9a</i> (B), total <i>Runx1</i> (C), <i>Runx1 P1</i> (D) and <i>Runx1 P2</i> (E). (F) Ratio of <i>Runx1 P1</i>:<i>P2</i> expression (n = 3).</p

Impact of the absence of <i>P1</i>-directed <i>Runx1</i> expression on adult hematopoiesis.

<p>(A) Top: Schematic diagrams of the <i>Runx1 WT</i> (top) and <i>P1-GFP</i> (bottom) alleles. Expression of GFP is directed by <i>Runx1</i> promoter <i>P1</i> but expression of <i>Runx1</i> from the <i>P2</i> promoter remains intact. Bottom: Schematic diagram of the experimental design for the investigation of the impact of <i>Runx1 P1</i> deletion on adult hematopoiesis. Peripheral blood, BM, thymus and spleen samples were collected from adult WT, <i>P1-GFP</i> heterozygous (<i>P1-GFP/+</i>) and homozygous (<i>P1-GFP/GFP</i>) adult mice. All samples were analyzed for mature blood cell surface marker expression. In addition, blood samples were subjected to automated cell counts (Sysmex) and CFU-C assays were performed on unfractionated BM. (B) Peripheral blood cell counts of WT, <i>P1-GFP/+</i> and <i>P1-GFP/GFP</i> mice as determined by Sysmex automated cell counting. (C) Numbers of CD3e+ T cells, CD11b+ Gr1+ GM cells and B220+ CD19+ B cells as a proportion of total ACK-lysed blood cells from WT, <i>P1-GFP/+</i> and <i>P1-GFP/GFP</i> mice. (D) CFU-C activity of WT, <i>P1-GFP/+</i> and <i>P1-GFP/GFP</i> unfractionated ACK-lysed BM following culture in pro-myeloid semi-solid methylcellulose-based medium. (n = 4.) (E) Numbers of erythroid lineage (ProE, EryA, EryB and EryC) cells as a proportion of live unfractionated BM cells. (F) Numbers of T cell lineage populations as a proportion of live unfractionated thymus cells. (G) Numbers of CD4 SP and CD8 SP T cells as a proportion of live unfractionated spleen cells. (H) Ratio of splenic CD4 SP T cells to splenic CD8 SP T cells (n = 4).</p