Identification of additional genetic alterations in RUNX1 related leukemias

Ph.DDOCTOR OF PHILOSOPH

Starting Day Care in Espoo from the Viewpoint of Chinese Parents

The thesis is a small-scale evaluative research. The day care start folder, which provided practical and detailed information about starting Finnish day care to support multicultural parents in Otaniemi day-care centre and Servin-Maija day-care centre, was assessed among nine Chinese parents of children in five municipal and outsourced day-care centres in the City of Espoo. The purposes of the current research were to find out how Chinese parents perceive starting day care in the City of Espoo and their difficulties and suggestions concerning starting day care in the City of Espoo; to evaluate how the day care start folder is experienced as a working method in opinion of Chinese parents living in the City of Espoo; and to further improve the day care start folder as a working method for multicultural families and personnel in Otaniemi day-care centre and possibly for other day-care centres in the similar situation. Two types of interview, namely semi-structured individual interview and focus group interview, were utilised as the primary qualitative data-collecting methods in the current research. In addition, questionnaire as an assistant quantitative data-collecting method was employed as well to ensure verification of the consistency of the data, and supplement the abundance of the narrative data collected from interviews. The qualitative data gathered from the six interviews was coded, categorised and analysed in accordance with the method of content analysis. The results showed that the participating Chinese parents basically concerned about the information on starting day care; their child’s language learning; their communication and cooperation with day-care staff; cultures, religions and festivals; playing, learning and friends; child protection and legislation and so forth. Furthermore, these Chinese parents also put forward some important problems and suggestions on Finnish day care, such as problems caused by cultural differences, different opinions on learning by playing and Finnish as a second language-teaching, difficulties to obtain enough English information about Finnish day care, and the lack of mediums for foreign families to receive help concerning Finnish day care. In addition, these Chinese parents perceived the day care start folder as useful and adequate. The results based on the questionnaires supported and verified the results generated from the qualitative data analysis

Disruption of Runx1 and Runx3 Leads to Bone Marrow Failure and Leukemia Predisposition due to Transcriptional and DNA Repair Defects

The RUNX genes encode transcription factors involved in development and human disease. RUNX1 and RUNX3 are frequently associated with leukemias, yet the basis for their involvement in leukemogenesis is not fully understood. Here, we show that Runx1;Runx3 double-knockout (DKO) mice exhibited lethal phenotypes due to bone marrow failure and myeloproliferative disorder. These contradictory clinical manifestations are reminiscent of human inherited bone marrow failure syndromes such as Fanconi anemia (FA), caused by defective DNA repair. Indeed, Runx1;Runx3 DKO cells showed mitomycin C hypersensitivity, due to impairment of monoubiquitinated-FANCD2 recruitment to DNA damage foci, although FANCD2 monoubiquitination in the FA pathway was unaffected. RUNX1 and RUNX3 interact with FANCD2 independently of CBFβ, suggesting a nontranscriptional role for RUNX in DNA repair. These findings suggest that RUNX dysfunction causes DNA repair defect, besides transcriptional misregulation, and promotes the development of leukemias and other cancers

Stem cell exhaustion due to Runx1 deficiency is prevented by Evi5 activation in leukemogenesis

The RUNX1/AML1 gene is the most frequently mutated gene in human leukemia. Conditional deletion of Runx1 in adult mice results in an increase of hematopoietic stem cells (HSCs), which serve as target cells for leukemia; however, Runx1−/− mice do not develop spontaneous leukemia. Here we show that maintenance of Runx1−/− HSCs is compromised, progressively resulting in HSC exhaustion. In leukemia development, the stem cell exhaustion was rescued by additional genetic changes. Retroviral insertional mutagenesis revealed Evi5 activation as a cooperating genetic alteration and EVI5 overexpression indeed prevented Runx1−/− HSC exhaustion in mice. Moreover, EVI5 was frequently overexpressed in human RUNX1-related leukemias. These results provide insights into the mechanism for maintenance of pre-leukemic stem cells and may provide a novel direction for therapeutic applications

Evi1 regulates the expression of other CHD genes during embryonic heart development.

<p>(A) The number of CHD genes represented in Evi1 ChIP-Seq data (Evi1 bound genes) or in the list of genes regulated by Mecom. An enriched number of CHD genes were found bound or regulated by Mecom (50 out of 143 genes), p = 0.0453 and p = 0.0276, respectively. These genes represent potential Mecom target genes in heart development. (B) Mecom regulates the expression of 23 CHD genes, which contain Evi1-binding sites specifically in heart. Heart and head (neural crest) tissues were harvested from WT and Evi1^δex3/δex3 embryos of somite number 9 to 18. RT-qPCR assays were performed. Genes considered to be mis-regulated in Evi1^δex3/δex3 hearts were increased or decreased in expression by at least three fold in average for all samples of the same time-point. These graphs are representative of two to five independent experiments.</p

Cardiac malformations and failure in Evi1^δex3/δex3 mice.

<p>(A) Transverse sections and (B) 3D reconstruction (left-ventral oblique view) of hearts from Evi1^δex3/δex3 or wild type littermate (+/+) E15.5 embryos analyzed by magnetic resonance imaging (MRI). The aorta (Ao), right ventricle (RV), left ventricle (LV), ventricular septum (VS), trachea (Tr), aortic arch (AoA) and ductus arteriosus (DA) are indicated. Ventricular septal defect (VSD), interrupted aortic arch (IAA) and common arterial trunk (CAT) were observed in Evi1^δex3/δex3 hearts. (C) List of the congenital heart defects identified in fifteen E15.5 embryos of various different genotypes by MRI and 3D reconstruction. (D) Hematoxylin and eosin staining of 5 µm sections of a sick Evi1^δex3/δex3 pup. Subcutaneous and other tissue edema (white spaces) was present, consistent with heart failure.</p

Expression of Mecom mRNA in cardiac structures of wild type embryos.

<p>(A–D) Whole mount mRNA <i>in situ</i> hybridization to show Mecom expression. A–C) Expression during subsequent stages of heart tube formation E8.5 (black brackets). D) At E9.5 Evi1 is expressed in the endothelial cells and in the endocardium of the heart and in the mesenchyme of the aortic arches. Expression also includes a population of migrating neural crest cells (white arrowhead). E–J) E10.5 Sagittal sections (from right to left) showing Evi1 in the aortic arches (a), mesenchyme of the secondary heart field (black arrowheads), outflow and atrio-ventricular canal endocardium including the cushions.</p

Overview of Major Reported Expression Domains.

<p><b>Key</b></p><p>AA – Aortic Arch and Aortic Arch Arteries.</p><p>CC/HT - Cardiac Crescent/Heart Tube.</p><p>End – Endocardium (+Csn – including Cushions).</p><p>Myo – Myocardium.</p><p>NC –Neural Crest (Cardiac).</p><p>OFT – Outflow Tract.</p><p>SHF – Secondary Heart Field.</p

Spontaneous lethal bone marrow depletion in mice harboring an Evi1 exon3 deletion in the hematopoietic system.

<p>(A) Histology was performed on sick Vav-iCre; Evi1^fl3/fl3 and littermate control mice. Bone marrow depletion was observed in the mutant mice. Adipose tissue replaced the hematopoietic cells in the bone marrow. (B) Increased erythropoiesis in the spleen of Vav-iCre; Evi1^fl3/fl3 mice. No visible border was found between the red pulp and white pulp. Erythroid cells are shown by the arrows. Excess erythropoiesis in spleen likely happens to compensate for bone marrow loss. (C) H&E stained sections of the brain of a dying Vav-iCre; Evi1^fl3/fl3 mouse. Hemorrhages (red areas) were visible at several locations (also see Fig. S3E in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089397#pone.0089397.s001" target="_blank">File S1</a>). (D) Histological sections of tissues from dying Vav-iCre; Evi1^fl3/fl3 animals showing bacteremia. Red arrows indicate the presence of bacteria in alveolar capillaries. Giemsa stains reveal the presence of cocci or small rods within glomerular capillaries. No sign of immune system defense (inflammatory cells) was observed despite the infection.</p

Deletion of Evi1 exon3 generates a hypomorphic allele.

<p>(A) Sequenced products obtained after 5′RACE from wild type or Evi1^δex3/δex3 mutant embryos. (B) Table showing the fraction of embryos of each genotype detected at different stages of embryonic development. The Mendelian ratios were not affected by the Evi1 exon3 deletion. (C) Pictures of 28 hr-old littermates highlight the poor health of dying Evi1^δex3/δex3 pups. (D) Kaplan-Meyer curves for wild type, Evi1^δex3/+ and Evi1^δex3/δex3 progeny indicate lethality of all Evi1^δex3/δex3 pups by three days after birth (n = 5 to 16 per genotype). Log rank test, Chi square p value <0.0001. (E) RT-qPCR from cDNA of E14.5 embryos. The primers used amplified the regions between Evi1 exons 2 and 3, 3 and 4 or 13 and 14. Mean of three different samples per condition. The standard deviation is shown. (F) Expression of Evi1 and γ-tubulin protein products in E14.5 wild type or E17.5 Evi1^δex3/+ and Evi1^δex3/δex3 mutant embryos (100 µg protein/lane). (G) Nucleotide sequence of Evi1 cDNA in the exon 3 and 4 genomic region. Two ATG sites are present in exon 3 and one in exon 4. All ATGs are conserved in higher vertebrates.</p