The cell cycle regulatory DREAM complex is disrupted by high expression of oncogenic B-Myb.

Overexpression of the oncogene MYBL2 (B-Myb) is associated with increased cell proliferation and serves as a marker of poor prognosis in cancer. However, the mechanism by which B-Myb alters the cell cycle is not fully understood. In proliferating cells, B-Myb interacts with the MuvB core complex including LIN9, LIN37, LIN52, RBBP4, and LIN54, forming the MMB (Myb-MuvB) complex, and promotes transcription of genes required for mitosis. Alternatively, the MuvB core interacts with Rb-like protein p130 and E2F4-DP1 to form the DREAM complex that mediates global repression of cell cycle genes in G0/G1, including a subset of MMB target genes. Here, we show that overexpression of B-Myb disrupts the DREAM complex in human cells, and this activity depends on the intact MuvB-binding domain in B-Myb. Furthermore, we found that B-Myb regulates the protein expression levels of the MuvB core subunit LIN52, a key adapter for assembly of both the DREAM and MMB complexes, by a mechanism that requires S28 phosphorylation site in LIN52. Given that high expression of B-Myb correlates with global loss of repression of DREAM target genes in breast and ovarian cancer, our findings offer mechanistic insights for aggressiveness of cancers with MYBL2 amplification, and establish the rationale for targeting B-Myb to restore cell cycle control

Transcriptional activation of the miR-17-92 cluster is involved in the growth-promoting effects of MYB in human Ph-positive leukemia cells.

MicroRNAs, non-coding regulators of gene expression, are likely to function as important downstream effectors of many transcription factors including MYB. Optimal levels of MYB are required for transformation/maintenance of BCR-ABL-expressing cells. We investigated whether MYB silencing modulates microRNA expression in Philadelphia-positive (Ph+) leukemia cells and if MYB-regulated microRNAs are important for the MYB addiction of these cells. Thirty-five microRNAs were modulated by MYB silencing in lymphoid and erythromyeloid chronic myeloid leukemia-blast crisis BV173 and K562 cells; 15 of these were concordantly modulated in both lines. We focused on the miR-17-92 cluster because of its oncogenic role in tumors and found that: i) it is a direct MYB target; ii) it partially rescued the impaired proliferation and enhanced apoptosis of MYB-silenced BV173 cells. Moreover, we identified FRZB, a Wnt/β-catenin pathway inhibitor, as a novel target of the miR-17-92 cluster. High expression of MYB in blast cells from 2 Ph+leukemia patients correlated positively with the miR-17-92 cluster and inversely with FRZB. This expression pattern was also observed in a microarray dataset of 122 Ph+acute lymphoblastic leukemias. In vivo experiments in NOD scid gamma mice injected with BV173 cells confirmed that FRZB functions as a Wnt/β-catenin inhibitor even as they failed to demonstrate that this pathway is important for BV173-dependent leukemogenesis. These studies illustrate the global effects of MYB expression on the microRNAs profile of Ph+cells and supports the concept that the MYB addiction of these cells is, in part, caused by modulation of microRNA-regulated pathways affecting cell proliferation and survival. Copyright© 2019 Ferrata Storti Foundation

Novel techniques for improved indoor positioning and localization using HF RFID

University of Technology, Sydney. Faculty of Engineering and Information Technology.This thesis investigates High Frequency Radio Frequency Identification (HF RFID) based positioning using a novel concept of multi-loop bridge reader antenna to localise moving objects such as autonomous wheelchairs in indoor environments. Typical HF RFIDs operate at 13.56 MHz and employ passive tags which are excited by the magnetic field radiated by the reader antenna. Positioning of moving objects using HF RFID systems derive location information by averaging the coordinates of detected passive floor tags by a portable reader antenna which are then recorded in the reader’s memory and database. To successfully detect floor tags, the reader’s antenna usually installed at the base of a moving object needs to be parallel to the floor. The magnetic field radiated by the HF RFID antenna is confined within its near field zone i.e., it is confined to a very close proximity of the antenna. This property of HF RFID helps to minimise interference to other appliances that may be present within the localisation area. Thus, HF RFID based positioning offers great potential benefit in providing location assistance in environments such as nursing homes, health care facilities, hospitals etc. However, despite the significant developments that have occurred in this field, there still exist problems with positioning accuracies obtainable mainly due to the uncertainty of the reader recognition area (RRA) of the reader antenna, which has not been fully addressed in literature. This thesis aims to address this problem by proposing the concept of multi-loop bridge reader antenna so that the reader recognition area is divided into multiple sub zones and an error signal (bridge signal) in terms of the position of the tag will be generated that helps to reduce the position uncertainty. The thesis starts with an investigation of the methods for creating multiple zones of RRA and the concept of bridge loop antenna from point of view of near magnetic fields. Different types of loop antennas for employing at the reader are electromagnetically analysed using both closed form solutions and numerical computations. The formation of reader recognition area (RRA) from different arrangements of loop reader antennas is also studied. To ensure that proposed bridge antennas can perform in realistic, non-ideal indoor environments where they are affected by proximity of metallic objects etc, we proposed methods of improvement. Equivalent circuits that reduce the computational complexity but can provide a broader understanding of the behaviour of bridge antennas have been formulated. This has lead to investigation of methods to minimise and/or eliminate the effect of metallic objects on the bridge signals. Next, we investigate the applicability of the proposed bridge loop antenna for the localisation and positioning of an autonomous wheel chair resulting in a realistic implementation of HF RFID based positioning system. The system is then tested to localise an autonomous wheelchair in an indoor environment using a grid of passive floor tags. Novel algorithms are proposed to estimate the position and orientation of the moving object using bridge signals generated by the bridge antenna coupled with the available dynamic information of the wheelchair. A comparison of our experimental results with the published results in the literature revealed significant improvements achieved by our proposed methods over existing techniques for estimating both, the orientation and position. Further, we demonstrate that the proposed technique obtains accurate position and estimation using much lesser number of floor tags (increased sparcity) than any of the currently published method, thus, contributing to simplified and easily expandable tag infrastructure deployment. We further extend the use of bridge loop antenna for situation when multiple tags are detected using the method of load modulation of the tags. When multiple tags present within the RRA of the bridge loop antenna, the resulting bridge signals incorporate information from all of the detected tags thus making it difficult to locate individual tags. To overcome this, we utilise states of the tag’s load modulation to separate these bridge signals, which then allow us to utilise them to estimate instantaneous position and orientation of the moving object. We performed analysis using equivalent circuits, as well as computational electromagnetic modelling of realistic antennas, which are then compared with experimental measurements carried on prototype systems. The comparison showed good agreement which validate our proposed method. Thus, the thesis incorporate contributions on various aspects of bridge loop reader antenna for HF RFID based positioning system. All full wave electromagnetic computations and simulations were carried by using a well known antenna design package “FEKO”. All the key analyses, equivalent circuits, antenna models and computational results for the proposed antennas and algorithms have been verified using extensive experimental campaigns to demonstrate the practical usefulness of the proposed methods. It is hoped that the findings in this thesis will result in newer efficient positioning systems in future

In vitro human embryonic stem cell hematopoiesis mimics MYB-independent yolk sac hematopoiesis

Although hematopoietic precursor activity can be generated in vitro from human embryonic stem cells, there is no solid evidence for the appearance of multipotent, self-renewing and transplantable hematopoietic stem cells. This could be due to short half-life of hematopoietic stem cells in culture or, alternatively, human embryonic stem cellinitiated hematopoiesis may be hematopoietic stem cell-independent, similar to yolk sac hematopoiesis, generating multipotent progenitors with limited expansion capacity. Since a MYB was reported to be an excellent marker for hematopoietic stem cell-dependent hematopoiesis, we generated a MYB-eGFP reporter human embryonic stem cell line to study formation of hematopoietic progenitor cells in vitro. We found CD34(+) hemogenic endothelial cells rounding up and developing into CD43(+) hematopoietic cells without expression of MYB-eGFP. MYB-eGFP+ cells appeared relatively late in embryoid body cultures as CD34(+) CD43(+) CD45(-/lo) cells. These MYB-eGFP(+) cells were CD33 positive, proliferated in IL-3 containing media and hematopoietic differentiation was restricted to the granulocytic lineage. In agreement with data obtained on murine Myb(-/-) embryonic stem cells, bright eGFP expression was observed in a subpopulation of cells, during directed myeloid differentiation, which again belonged to the granulocytic lineage. In contrast, CD14(+) macrophage cells were consistently eGFP-and were derived from eGFPprecursors only. In summary, no evidence was obtained for in vitro generation of MYB+ hematopoietic stem cells during embryoid body cultures. The observed MYB expression appeared late in culture and was confined to the granulocytic lineage

Dissecting the genome-wide evolution and function of R2R3-MYB transcription factor family in Rosa chinensis

Rosa chinensis, an important ancestor species of Rosa hybrida, the most popular ornamental plant species worldwide, produces flowers with diverse colors and fragrances. The R2R3-MYB transcription factor family controls a wide variety of plant-specific metabolic processes, especially phenylpropanoid metabolism. Despite their importance for the ornamental value of flowers, the evolution of R2R3-MYB genes in plants has not been comprehensively characterized. In this study, 121 predicted R2R3-MYB gene sequences were identified in the rose genome. Additionally, a phylogenomic synteny network (synnet) was applied for the R2R3-MYB gene families in 35 complete plant genomes. We also analyzed the R2R3-MYB genes regarding their genomic locations, Ka/Ks ratio, encoded conserved motifs, and spatiotemporal expression. Our results indicated that R2R3-MYBs have multiple synteny clusters. The RcMYB114a gene was included in the Rosaceae-specific Cluster 54, with independent evolutionary patterns. On the basis of these results and an analysis of RcMYB114a-overexpressing tobacco leaf samples, we predicted that RcMYB114a functions in the phenylpropanoid pathway. We clarified the relationship between R2R3-MYB gene evolution and function from a new perspective. Our study data may be relevant for elucidating the regulation of floral metabolism in roses at the transcript level

Cellular senescence and aging: the role of B-MYB

Cellular senescence is a stable cell cycle arrest, caused by insults, such as: telomere erosion, oncogene activation, irradiation, DNA damage, oxidative stress, and viral infection. Extrinsic stimuli such as cell culture stress can also trigger this growth arrest. Senescence is thought to have evolved as an example of antagonistic pleiotropy, as it acts as a tumor suppressor mechanism during the reproductive age, but can promote organismal aging by disrupting tissue renewal, repair, and regeneration later in life. The mechanisms underlying the senescence growth arrest are broadly considered to involve p16(INK4A) -pRB and p53-p21(CIP1/WAF1/SDI1) tumor suppressor pathways; but it is not known what makes the senescence arrest stable and what the critical downstream targets are, as they are likely to be key to the establishment and maintenance of the senescent state. MYB-related protein B (B-MYB/MYBL2), a member of the myeloblastosis family of transcription factors, has recently emerged as a potential candidate for regulating entry into senescence. Here, we review the evidence which indicates that loss of B-MYB expression has an important role in causing senescence growth arrest. We discuss how B-MYB acts, as the gatekeeper, to coordinate transit through the cell cycle, in conjunction with the multivulval class B (MuvB) complex and FOXM1 transcription factors. We also evaluate the evidence connecting B-MYB to the mTOR nutrient signaling pathway and suggest that inhibition of this pathway leading to an extension of healthspan may involve activation of B-MYB

SpMyb functions as an intramodular repressor to regulate spatial expression of CyIIIa in sea urchin embryos

The CyIIIa actin gene of Strongylocentrotus purpuratus is transcribed exclusively in the embryonic aboral ectoderm, under the control of 2.3 kb cis-regulatory domain that contains a proximal module that controls expression in early embryogenesis, and a middle module that controls expression in later embryogenesis. Previous studies demonstrated that the SpRunt-1 target site within the middle module is required for the sharp increase in CyIIIa transcription which accompanies differentiation of the aboral ectoderm, and that a negative regulatory region near the SpRunt-1 target site is required to prevent ectopic transcription in the oral ectoderm and skeletogenic mesenchyme. This negative regulatory region contains a consensus binding site for the myb family of transcription factors. In vitro DNA-binding experiments reveal that a protein in blastula-stage nuclei interacts specifically with the myb target site. Gene transfer experiments utilizing CyIIIa reporter constructs containing oligonucleotide substitutions indicate that this site is both necessary and sufficient to prevent ectopic expression of CyIIIa. Synthetic oligonucleotides containing the myb target site were used to purify a protein from sea urchin embryo nuclear extracts by affinity chromatography. This protein is immunoprecipitated by antibodies specific to the evolutionarily conserved myb domain, and amino acid sequences obtained from the purified protein were found to be identical to sequences within the myb domain. Sequence information was used to obtain cDNA clones of SpMyb, the S. purpuratus member of the myb family of transcription factors. Through interactions within the middle module, SpMyb functions to repress activation of CyIIIa in the oral ectoderm and skeletogenic mesenchyme

Targeting CDK6 and BCL2 Exploits the MYB Addiction of Ph+ Acute Lymphoblastic Leukemia

Philadelphia chromosome–positive acute lymphoblastic leukemia (Phþ ALL) is currently treated with BCR-ABL1 tyrosine kinase inhibitors (TKI) in combination with chemotherapy. However, most patients develop resistance to TKI through BCR-ABL1–dependent and –independent mechanisms. Newly developed TKI can target Phþ ALL cells with BCR-ABL1–dependent resistance; however, overcoming BCR-ABL1–independent mechanisms of resistance remains challenging because transcription factors, which are difficult to inhibit, are often involved. We show here that (i) the growth of Phþ ALL cell lines and primary cells is highly dependent on MYB-mediated transcriptional upregulation of CDK6, cyclin D3, and BCL2, and (ii) restoring their expression in MYB-silenced Phþ ALL cells rescues their impaired proliferation and survival. Levels of MYB and CDK6 were highly correlated in adult Phþ ALL (P ¼ 0.00008). Moreover, Phþ ALL cells exhibited a specific requirement for CDK6 but not CDK4 expression, most likely because, in these cells, CDK6 was predominantly localized in the nucleus, whereas CDK4 was almost exclusively cytoplasmic. Consistent with their essential role in Phþ ALL, pharmacologic inhibition of CDK6 and BCL2 markedly suppressed proliferation, colony formation, and survival of Phþ ALL cells ex vivo and in mice. In summary, these findings provide a proof-of-principle, rational strategy to target the MYB addiction of Phþ ALL. © 2017 American Association for Cancer Research

Adenoid cystic carcinoma: emerging role of translocations and gene fusions.

Adenoid cystic carcinoma (ACC), the second most common salivary gland malignancy, is notorious for poor prognosis, which reflects the propensity of ACC to progress to clinically advanced metastatic disease. Due to high long-term mortality and lack of effective systemic treatment, the slow-growing but aggressive ACC poses a particular challenge in head and neck oncology. Despite the advancements in cancer genomics, up until recently relatively few genetic alterations critical to the ACC development have been recognized. Although the specific chromosomal translocations resulting in MYB-NFIB fusions provide insight into the ACC pathogenesis and represent attractive diagnostic and therapeutic targets, their clinical significance is unclear, and a substantial subset of ACCs do not harbor the MYB-NFIB translocation. Strategies based on detection of newly described genetic events (such as MYB activating super-enhancer translocations and alterations affecting another member of MYB transcription factor family-MYBL1) offer new hope for improved risk assessment, therapeutic intervention and tumor surveillance. However, the impact of these approaches is still limited by an incomplete understanding of the ACC biology, and the manner by which these alterations initiate and drive ACC remains to be delineated. This manuscript summarizes the current status of gene fusions and other driver genetic alterations in ACC pathogenesis and discusses new therapeutic strategies stemming from the current research

Radial or bilateral? The molecular basis of floral symmetry.

In the plant kingdom, the flower is one of the most relevant evolutionary novelties. Floral symmetry has evolved multiple times from the ancestral condition of radial to bilateral symmetry. During evolution, several transcription factors have been recruited by the different developmental pathways in relation to the increase of plant complexity. The MYB proteins are among the most ancient plant transcription factor families and are implicated in different metabolic and developmental processes. In the model plant Antirrhinum majus, three MYB transcription factors (DIVARICATA, DRIF, and RADIALIS) have a pivotal function in the establishment of floral dorsoventral asymmetry. Here, we present an updated report of the role of the DIV, DRIF, and RAD transcription factors in both eudicots and monocots, pointing out their functional changes during plant evolution. In addition, we discuss the molecular models of the establishment of flower symmetry in different flowering plants