Reversal of MYB-dependent suppression of MAFB expression overrides leukaemia phenotype in MLL-rearranged AML.

The transcription factor MYB plays a pivotal role in haematopoietic homoeostasis and its aberrant expression is involved in the genesis and maintenance of acute myeloid leukaemia (AML). We have previously demonstrated that not all AML subtypes display the same dependency on MYB expression and that such variability is dictated by the nature of the driver mutation. However, whether this difference in MYB dependency is a general trend in AML remains to be further elucidated. Here, we investigate the role of MYB in human leukaemia by performing siRNA-mediated knock-down in cell line models of AML with different driver lesions. We show that the characteristic reduction in proliferation and the concomitant induction of myeloid differentiation that is observed in MLL-rearranged and t(8;21) leukaemias upon MYB suppression is not seen in AML cells with a complex karyotype. Transcriptome analyses revealed that MYB ablation produces consensual increase of MAFB expression in MYB-dependent cells and, interestingly, the ectopic expression of MAFB could phenocopy the effect of MYB suppression. Accordingly, in silico stratification analyses of molecular data from AML patients revealed a reciprocal relationship between MYB and MAFB expression, highlighting a novel biological interconnection between these two factors in AML and supporting new rationales of MAFB targeting in MLL-rearranged leukaemias.

Expression dynamics of metalloproteinases during mandibular bone formation: association with Myb transcription factor.

As the dentition forms and becomes functional, the alveolar bone is remodelled. Metalloproteinases are known to contribute to this process, but new regulators are emerging and their contextualization is challenging. This applies to Myb, a transcription factor recently reported to be involved in bone development and regeneration. The regulatory effect of Myb on Mmps expression has mostly been investigated in tumorigenesis, where Myb impacted the expression of Mmp1, Mmp2, Mmp7, and Mmp9. The aim of this investigation was to evaluate the regulatory influence of the Myb on Mmps gene expression, impacting osteogenesis and mandibular bone formation. For that purpose, knock-out mouse model was used. Gene expression of bone-related Mmps and the key osteoblastic transcription factors Runx2 and Sp7 was analysed in Myb knock-out mice mandibles at the survival limit. Out of the metalloproteinases under study, Mmp13 was significantly downregulated. The impact of Myb on the expression of Mmp13 was confirmed by the overexpression of Myb in calvarial-derived cells causing upregulation of Mmp13. Expression of Mmp13 in the context of other Mmps during mandibular/alveolar bone development was followed in vivo along with Myb, Sp7 and Runx2. The most significant changes were observed in the expression of Mmp9 and Mmp13. These MMPs and MYB were further localized in situ by immunohistochemistry and were identified in pre/osteoblastic cells as well as in pre/osteocytes. In conclusion, these results provide a comprehensive insight into the expression dynamics of bone related Mmps during mandibular/alveolar bone formation and point to Myb as another potential regulator of Mmp13.

Author Correction: High WBP5 expression correlates with elevation of HOX genes levels and is associated with inferior survival in patients with acute myeloid leukaemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

High WBP5 expression correlates with elevation of HOX genes levels and is associated with inferior survival in patients with acute myeloid leukaemia.

WW domain binding protein 5 (WBP5), also known as Transcriptional Elongation Factor A like 9 (TCEAL9) has been proposed as a candidate oncogene for human colorectal cancers with microsatellite instability and as a predictive indicator of small cell lung cancers. Furthermore, several independent studies have proposed WBP5, and its association with Wilms Tumor-1 (WT1) expression, as part of a gene expression-based risk score for predicting survival and clinical outcome in patients with Acute Myeloid Leukaemia (AML). To date, the prognostic significance of the sole WBP5 expression and its impact on the survival outcome in AML patients remains largely understudied. In the present study, we have made use of publicly available patient expression arrays and have developed an unbiased approach to classify AML patients into low versus high WBP5 expressers and to balance them for known mutations and cytogenetic findings. Interestingly, we found that patients characterized by high WBP5 expression displayed inferior overall and event-free survival rates. Notably, gene expression profiling showed that patients with high WBP5 had elevated expression of several HOX cluster genes, such as HOXA5, HOXA7, HOXA9 and HOXA10, and several of their partner proteins, such as MEIS1 and FOXC1, which have been demonstrated to be causative for AML. Taken together, our data suggest that WBP5 expression level could serve as an indicator for prognosis and survival outcome in patients with AML.

Author Correction: Yolk sac macrophage progenitors traffic to the embryo during defined stages of development.

This article contains errors in Figs. 5 and 6, for which we apologize. In Fig. 5f, the image 'E12.5 tail' was inadvertently replaced with a duplicate of the image 'E12.5 trunk' from the same panel. In Figure 6d, the image 'E9.5/OH-TAM E8.5, embryo' was inadvertently replaced with a duplicate of the image 'E10.5/ OH-TAM E8.5, embryo' from Fig. 6b. The corrected versions of these figures appear in the Author Correction associated with this Article.

Inducible disruption of the c-myb gene allows allogeneic bone marrow transplantation without irradiation.

Allogeneic bone marrow (BM) transplantation enables the in vivo functional assessment of hematopoietic cells. As pre-conditioning, ionizing radiation is commonly applied to induce BM depletion, however, it exerts adverse effects on the animal and can limit experimental outcome. Here, we provide an alternative method that harnesses conditional gene deletion to ablate c-myb and thereby deplete BM cells, hence allowing BM substitution without other pre-conditioning. The protocol results in a high level of blood chimerism after allogeneic BM transplantation, whereas immune cells in peripheral tissues such as resident macrophages are not replaced. Further, mice featuring a low chimerism after initial transplantation can undergo a second induction cycle for efficient deletion of residual BM cells without the necessity to re-apply donor cells. In summary, we present an effective c-myb-dependent genetic technique to generate BM chimeras in the absence of irradiation or other methods for pre-conditioning.

Yolk sac macrophage progenitors traffic to the embryo during defined stages of development.

Tissue macrophages in many adult organs originate from yolk sac (YS) progenitors, which invade the developing embryo and persist by means of local self-renewal. However, the route and characteristics of YS macrophage trafficking during embryogenesis are incompletely understood. Here we show the early migration dynamics of YS-derived macrophage progenitors in vivo using fate mapping and intravital microscopy. From embryonic day 8.5 (E8.5) CX3CR1+ pre-macrophages are present in the mouse YS where they rapidly proliferate and gain access to the bloodstream to migrate towards the embryo. Trafficking of pre-macrophages and their progenitors from the YS to tissues peaks around E10.5, dramatically decreases towards E12.5 and is no longer evident from E14.5 onwards. Thus, YS progenitors use the vascular system during a restricted time window of embryogenesis to invade the growing fetus. These findings close an important gap in our understanding of the development of the innate immune system.

Templated Assembly of Collagen Fibers Directs Cell Growth in 2D and 3D.

Collagen is widely used in tissue engineering and regenerative medicine, with many examples of collagen-based biomaterials emerging in recent years. While there are numerous methods available for forming collagen scaffolds from isolated collagen, existing biomaterial processing techniques are unable to efficiently align collagen at the microstructural level, which is important for providing appropriate cell recognition and mechanical properties. Although some attention has shifted to development of fiber-based collagen biomaterials, existing techniques for producing and aligning collagen fibers are not appropriate for large-scale fiber manufacturing. Here, we report a novel biomaterial fabrication approach capable of efficiently generating collagen fibers of appropriate sizes using a viscous solution of dextran as a dissolvable template. We demonstrate that myoblasts readily attach and align along 2D collagen fiber networks created by this process. Furthermore, encapsulation of collagen fibers with myoblasts into non-cell-adherent hydrogels promotes aligned growth of cells and supports their differentiation. The ease-of-production and versatility of this technique will support future development of advanced in vitro tissue models and materials for regenerative medicine.

Transcriptional regulation of SPROUTY2 by MYB influences myeloid cell proliferation and stem cell properties by enhancing responsiveness to IL-3.

Myeloproliferative neoplasms (MPN), which overproduce blood cells in the bone marrow, have recently been linked with a genetically determined decrease in expression of the MYB transcription factor. Here, we use a mouse MYB knockdown model with an MPN-like phenotype to show how lower levels of MYB lead to stem cell characteristics in myeloid progenitors. The altered progenitor properties feature elevated cytokine responsiveness, especially to interleukin-3, which results from increased receptor expression and increased MAPK activity leading to enhanced phosphorylation of a key regulator of protein synthesis, ribosomal protein S6. MYB acts on MAPK signaling by directly regulating transcription of the gene encoding the negative modulator SPRY2. This mechanistic insight points to pathways that might be targeted therapeutically in MPN.

Comparison of interactive voice response, patient mailing, and mailed registry to encourage screening for osteoporosis: a randomized controlled trial.

UNLABELLED: Guidelines recommend screening for osteoporosis with bone mineral density (BMD) testing in menopausal women, particularly those with additional risk factors for fracture. Many eligible women remain unscreened. This randomized study demonstrates that a single outreach interactive voice response phone call improves rates of BMD screening among high-risk women age 50-64. INTRODUCTION: Osteoporotic fractures are a major cause of disability and mortality. Guidelines recommend screening with BMD for menopausal women, particularly those with additional risk factors for fracture. However, many women remain unscreened. We examined whether telephonic interactive voice response (IVR) or patient mailing could increase rates of BMD testing in high risk, menopausal women. METHODS: We studied 4,685 women age 50-64 years within a not-for-profit health plan in the United States. All women had risk factors for developing osteoporosis and no prior BMD testing or treatment for osteoporosis. Patients were randomly allocated to usual care, usual care plus IVR, or usual care plus mailed educational materials. To avoid contamination, patients within a single primary care physician practice were randomized to receive the same intervention. The primary endpoint was BMD testing at 12 months. Secondary outcomes included BMD testing at 6 months and medication use at 12 months. RESULTS: Mean age was 57 years. Baseline demographic and clinical characteristics were similar across the three study groups. In adjusted analyses, the incidence of BMD screening was 24.6% in the IVR group compared with 18.6% in the usual care group (P < 0.001). There was no difference between the patient mailing group and the usual care group (P = 0.3). CONCLUSIONS: In this large community-based randomized trial of high risk, menopausal women age 50-64, IVR, but not patient mailing, improved rates of BMD screening. IVR remains a viable strategy to incorporate in population screening interventions.

C/EBPα and MYB regulate FLT3 expression in AML.

The interaction between the receptor FLT3 (FMS-like tyrosine kinase-3) and its ligand FL leads to crucial signalling during the early stages of the commitment of haematopoietic stem cells. Mutation or over-expression of the FLT3 gene, leading to constitutive signalling, enhances the survival and expansion of a variety of leukaemias and is associated with an unfavourable clinical outcome for acute myeloid leukaemia (AML) patients. In this study, we used a murine cellular model for AML and primary leukaemic cells from AML patients to investigate the molecular mechanisms underlying the regulation of FLT3 gene expression and identify its key cis- and trans-regulators. By assessing DNA accessibility and epigenetic markings, we defined regulatory domains in the FLT3 promoter and first intron. These elements permit in vivo binding of several AML-related transcription factors, including the proto-oncogene MYB and the CCAAT/enhancer binding protein C/EBPα, which are recruited to the FLT3 promoter and intronic module, respectively. Substantiating their relevance to the human disease, our analysis of gene expression profiling arrays from AML patients uncovered significant correlations between FLT3 expression level and that of MYB and CEBPA. The latter relationship permits discrimination between patients with CEBPA mono- and bi-allelic mutations, and thus connects two major prognostic factors for AML.

MYBL2 haploinsufficiency increases susceptibility to age-related haematopoietic neoplasia.

The haematopoietic system is prone to age-related disorders ranging from deficits in functional blood cells to the development of neoplastic states. Such neoplasms often involve recurrent cytogenetic abnormalities, among which a deletion in the long arm of chromosome 20 (del20q) is common in myeloid malignancies. The del20q minimum deleted region contains nine genes, including MYBL2, which encodes a key protein involved in the maintenance of genome integrity. Here, we show that mice expressing half the normal levels of Mybl2 (Mybl2(+/Δ)) develop a variety of myeloid disorders upon ageing. These include myeloproliferative neoplasms, myelodysplasia (MDS) and myeloid leukaemia, mirroring the human conditions associated with del20q. Moreover, analysis of gene expression profiles from patients with MDS demonstrated reduced levels of MYBL2, regardless of del20q status and demonstrated a strong correlation between low levels of MYBL2 RNA and reduced expression of a subset of genes related to DNA replication and checkpoint control pathways. Paralleling the human data, we found that these pathways are also disturbed in our Mybl2(+/Δ) mice. This novel mouse model, therefore, represents a valuable tool for studying the initiation and progression of haematological malignancies during ageing, and may provide a platform for preclinical testing of therapeutic approaches.

Distinct regulation of c-myb gene expression by HoxA9, Meis1 and Pbx proteins in normal hematopoietic progenitors and transformed myeloid cells.

The proto-oncogenic protein c-Myb is an essential regulator of hematopoiesis and is frequently deregulated in hematological diseases such as lymphoma and leukemia. To gain insight into the mechanisms underlying the aberrant expression of c-Myb in myeloid leukemia, we analyzed and compared c-myb gene transcriptional regulation using two cell lines modeling normal hematopoietic progenitor cells (HPCs) and transformed myelomonocytic blasts. We report that the transcription factors HoxA9, Meis1, Pbx1 and Pbx2 bind in vivo to the c-myb locus and maintain its expression through different mechanisms in HPCs and leukemic cells. Our analysis also points to a critical role for Pbx2 in deregulating c-myb expression in murine myeloid cells cotransformed by the cooperative activity of HoxA9 and Meis1. This effect is associated with an intronic positioning of epigenetic marks and RNA polymerase II binding in the orthologous region of a previously described alternative promoter for c-myb. Taken together, our results could provide a first hint to explain the abnormal expression of c-myb in leukemic cells.

c-Myb and GATA-1 alternate dominant roles during megakaryocyte differentiation.

BACKGROUND: Transcription factors are essential for blood cell formation. Mice expressing low levels of c-Myb (c-Myb(low)) have an increased number of bone marrow megakaryocytes (MKs) and corresponding thrombocytosis. In contrast, mice engineered to express low levels of GATA-1 (GATA-1(low)) in the megakaryocytic lineage exhibit aberrant megakaryocytopoiesis with hyperproliferation of progenitors and defective terminal differentiation leading to thrombocytopenia. These seemingly opposite roles may affect platelet turnover and thus be of clinical relevance. OBJECTIVE: To determine how these two transcription factors act together to control megakaryocytopoiesis and platelet formation. METHODS: We used a combination of cellular and molecular in vitro assays to examine the ability of bone marrow cells from mice expressing low levels of both c-Myb and GATA-1 (referred to as double(low)) to produce MKs and platelets. RESULTS: Double(low) cells, or those with low GATA-1 levels in which c-Myb is conditionally deleted, lack the hyperproliferative capacity of GATA-1(low) cells, allowing the cells to proceed towards more committed MKs that are, however, impaired in their capacity to produce fully differentiated cells, as confirmed by the abundance of morphologically aberrant cells that lack the ability to form proplatelets. CONCLUSION: c-Myb and GATA-1 act in concert to achieve correct megakaryocytic differentiation. GATA-1 regulates both the proliferation of megakaryocytic progenitors and their terminal maturation. c-Myb also acts at the level of the progenitor by influencing its commitment to differentiation, but in contrast to GATA-1 it does not have any effect on the process of terminal differentiation.

EMMPRIN (CD147/basigin) mediates platelet-monocyte interactions in vivo and augments monocyte recruitment to the vascular wall.

BACKGROUND: Platelets play a central role in hemostasis, in inflammatory diseases such as atherosclerosis, and during thrombus formation following vascular injury. Thereby, platelets interact intensively with monocytes and enhance their recruitment to the vascular wall. OBJECTIVES: To investigate the role of the extracellular matrix metalloproteinase inducer (EMMPRIN) in platelet-monocyte interactions. METHODS AND RESULTS: Isolated human monocytes were perfused in vitro over firmly adherent platelets to allow investigation of the role of EMMPRIN in platelet-monocyte interactions under flow conditions. Monocytes readily bound to surface-adherent platelets. Both antibody blockade and gene silencing of monocyte EMMPRIN substantially attenuated firm adhesion of monocytes to platelets at arterial and venous shear rates. In vivo, platelet interactions with the murine monocyte cell line ANA-1 were significantly decreased when ANA-1 cells were pretreated with EMMPRIN-silencing small interfering RNA prior to injection into wild-type mice. Using intravital microscopy, we showed that recruitment of EMMPRIN-silenced ANA-1 to the injured carotid artery was significantly reduced as compared with control cells. Further silencing of EMMPRIN resulted in significantly fewer ANA-1-platelet aggregates in the mouse circulation as determined by flow cytometry. Finally, we identified glycoprotein (GP)VI as a critical corresponding receptor on platelets that mediates interaction with monocyte EMMPRIN. Thus, blocking of GPVI inhibited the effect of EMMPRIN on firm monocyte adhesion to platelets under arterial flow conditions in vitro, and abrogated EMMPRIN-mediated platelet-monocyte aggregate formation in vivo. CONCLUSIONS: EMMPRIN supports platelet-monocyte interactions and promotes monocyte recruitment to the arterial wall. Therefore, EMMPRIN might represent a novel target to reduce vascular inflammation and atherosclerotic lesion development.

Fabrication and optimization of alginate hydrogel constructs for use in 3D neural cell culture.

Two-dimensional (2D) culture systems provide useful information about many biological processes. However, some applications including tissue engineering, drug transport studies, and analysis of cell growth and dynamics are better studied using three-dimensional (3D) culture systems. 3D culture systems can potentially offer higher degrees of organization and control of cell growth environments, more physiologically relevant diffusion characteristics, and permit the formation of more extensive 3D networks of cell-cell interactions. A 3D culture system has been developed using alginate as a cell scaffold, capable of maintaining the viability and function of a variety of neural cell types. Alginate was functionalized by the covalent attachment of a variety of whole proteins and peptide epitopes selected to provide sites for cell attachment. Alginate constructs were used to entrap a variety of neural cell types including astroglioma cells, astrocytes, microglia and neurons. Neural cells displayed process outgrowth over time in culture. Cell-seeded scaffolds were characterized in terms of their biochemical and biomechanical properties, effects on seeded neural cells, and suitability for use as 3D neural cell culture models.

Haematopoietic stem cell recruitment to injured murine liver sinusoids depends on (alpha)4(beta)1 integrin/VCAM-1 interactions.

OBJECTIVE: Evidence suggests haematopoietic stem cells (HSCs) can migrate to injured liver and influence tissue repair. However, mechanisms governing HSC recruitment to injured hepatic microcirculation are poorly understood. These were investigated in vivo following hepatic ischaemia-reperfusion (IR) injury and in vitro using flow-based adhesion assays. DESIGN: Partial IR was induced in anaesthetised WT or PECAM-1(-/-) mice for 90 min. Recruitment of systemically administered HSCs was monitored and effects of function blocking antibodies against alpha(4)beta(1) integrin, CD18, CD44, PECAM-1 or VCAM-1 investigated. The kinetics and molecular events governing adhesion to murine cardiac endothelial cells in vitro were also determined. Effects of conditioned media from IR injured liver on HSC adhesion molecule expression was determined by FACS. RESULTS: Administered HSCs homed predominantly to lungs rather than liver, highlighting a potential therapeutic hurdle. Hepatic HSC recruitment following IR injury was inhibited by anti-alpha(4)beta(1) and anti-VCAM-1 antibodies. A role for alpha(4)beta(1) was also confirmed using flow-based adhesion assays. Incubating HSCs with conditioned media from IR injured liver increased alpha(4)beta(1) expression. CD18, CD44 and PECAM-1 were not involved in recruitment. CONCLUSIONS: This novel study demonstrates that the alpha(4)beta(1)/VCAM-1 pathway mediates HSC recruitment to injured liver. Manipulating this pathway may enhance delivery of HSCs to the liver.

Three-dimensional hydrogel cultures for modeling changes in tissue impedance around microfabricated neural probes.

One limitation to the use of neuroprosthestic devices for chronic application, in the treatment of disease, is the reactive cell responses that occur surrounding the device after insertion. These cell and tissue responses result in increases in device impedance and failure of the device to interact with target populations of neurons. However, few tools are available to assess which components of the reactive response contribute most to changes in tissue impedance. An in vitro culture system has been developed that is capable of assessing individual components of the reactive response. The system utilizes alginate cell encapsulation to construct three-dimensional architectures that approach the cell densities found in rat cortex. The system was constructed around neuroNexus acute probes with on-board circuitry capable of monitoring the electrical properties of the surrounding tissue. This study demonstrates the utility of the system by demonstrating that differences in cell density within the three-dimensional alginate constructs result in differences in resistance and capacitance as measured by electrochemical impedance spectroscopy. We propose that this system can be used to model components of the reactive responses in brain tissue, and that the measurements recorded in vitro are comparable to measurements recorded in vivo.

Targeting an E2F site in the mouse genome prevents promoter silencing in quiescent and post-mitotic cells.

Previous studies have shown that the cell cycle-regulated B-myb promoter contains a conserved E2F binding site that is critical for repressing transcription in quiescent cells. To investigate its significance for permanent promoter silencing, we have inactivated this binding site in the mouse genome. Mice homozygous for the mutant B-mybmE2F allele were fully viable, however, B-myb transcription was derepressed during quiescence in mouse embryo fibroblasts (MEFs) derived from mutant animals. Moreover, it was found that mutation of the E2F site resulted in abnormal maintenance of B-myb expression in senescent MEFs and in differentiated brain tissue. These findings therefore reveal a direct and primary role for repressive E2F complexes in silencing gene expression in post-mitotic cells. Analysis of histone modifications at the promoter showed that histone H3 lysine 9 was constitutively acetylated throughout the cell cycle in homozygous mutant MEFs. This mouse system is the first description of an E2F site mutation in situ and will facilitate the study of E2F function in vivo.