Namiko Yamashita

Causal relationship between the loss of RUNX3 expression and gastric cancer.

Runx3/Pebp2alphaC null mouse gastric mucosa exhibits hyperplasias due to stimulated proliferation and suppressed apoptosis in epithelial cells, and the cells are resistant to growth-inhibitory and apoptosis-inducing action of TGF-beta, indicating that Runx3 is a major growth regulator of gastric epithelial cells. Between 45% and 60% of human gastric cancer cells do not significantly express RUNX3 due to hemizygous deletion and hypermethylation of the RUNX3 promoter region. Tumorigenicity of human gastric cancer cell lines in nude mice was inversely related to their level of RUNX3 expression, and a mutation (R122C) occurring within the conserved Runt domain abolished the tumor-suppressive effect of RUNX3, suggesting that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer.

Runx3 controls the axonal projection of proprioceptive dorsal root ganglion neurons

Dorsal root ganglion (DRG) neurons specifically project axons to central and peripheral targets according to their sensory modality. The Runt-related genes Runx1 and Runx3 are expressed in DRG neuronal subpopulations, suggesting that they may regulate the trajectories of specific axons. Here we report that Runx3-deficient (Runx3−/−) mice displayed severe motor discoordination and that few DRG neurons synthesized the proprioceptive neuronal marker parvalbumin. Proprioceptive afferent axons failed to project to their targets in the spinal cord as well as those in the muscle. NT-3-responsive Runx3−/− DRG neurons showed less neurite outgrowth in vitro. However, we found no changes in the fate specification of Runx3−/− DRG neurons or in the number of DRG neurons that expressed trkC. Our data demonstrate that Runx3 is critical in regulating the axonal projections of a specific subpopulation of DRG neurons.

Runx1 Protects Hematopoietic Stem/Progenitor Cells from Oncogenic Insult

The RUNX1/AML1 gene encodes a transcription factor essential for the generation of hematopoietic stem cells and is frequently targeted in human leukemia. In human RUNX1‐related leukemias, the RAS pathway is often concurrently mutated, but the mechanism of the synergism remains elusive. Here, we found that inactivation of Runx1 in mouse bone marrow cells results in an increase in the stem/progenitor cell fraction due to suppression of apoptosis and elevated expression of the polycomb gene Bmi‐1, which is important for stem cell self‐renewal. Introduction of oncogenic N‐RAS into wild‐type cells, in contrast, reduced the stem/progenitor cell fraction because of senescence, apoptosis, and differentiation. Such detrimental events presumably occurred because of the cellular fail‐safe program, although hyperproliferation was initially induced by an oncogenic stimulus. Runx1 insufficiency appears to impair such a fail‐safe mechanism, particularly in the stem/progenitor cells, thereby supporting the clonal maintenance of leukemia‐initiating cells expressing an activated oncogene.

A Runx1 Intronic Enhancer Marks Hemogenic Endothelial Cells and Hematopoietic Stem Cells

Runx1 is essential for the generation of hematopoietic stem cells (HSCs) and is frequently mutated in human leukemias. However, the cis‐regulatory mechanisms modulating the Runx1 gene expression remain to be elucidated. Herewith, we report the identification of an intronic Runx1 enhancer, Runx1 +24 mouse conserved noncoding element (mCNE), using a combinatorial in silico approach involving comparative genomics and retroviral integration sites mapping. The Runx1 +24 mCNE was found to possess hematopoietic‐specific enhancer activity in both zebrafish and mouse models. Significantly, this enhancer is active specifically in hemogenic endothelial cells (ECs) at sites where the de novo generation of HSCs occurs. The activity of this enhancer is also strictly restricted to HSCs within the hematopoietic compartment of the adult bone marrow. We anticipate that Runx1 +24 mCNE HSC enhancer will serve as a molecular handle for tracing and/or manipulating hemogenic ECs/HSCs behavior in vivo, and consequently become an invaluable tool for research on stem cell and cancer biology. STEM CELLS 2010;28:1869–1881

Haploinsufficiency of Runx1/AML1 promotes myeloid features and leukaemogenesis in BXH2 mice

Haploinsufficiency of RUNX1/AML1 is associated with familial platelet disorder with a predisposition to acute myeloid leukaemia (FPD/AML), but the causal relationship remains to be addressed experimentally. Mice heterozygous for the Runx1 null mutation, Runx1+/−, are considered to be genetically comparable with human FPD/AML patients but do not develop spontaneous leukaemia. To induce additional genetic alterations, retroviral insertional mutagenesis was employed with the use of BXH2 mice, which develop myeloid leukaemia because of the random integration of retrovirus present in the mouse. Heterozygous disruption of Runx1 in BXH2 mice resulted in a shortening of the latency period of leukaemia. In addition, BXH2‐Runx1+/− mice exhibited more marked myeloid features than control mice. Moreover, the c‐Kit gene, mutated in human RUNX leukaemias, was recurrently activated in BXH2‐Runx1+/− mice, and a colony‐forming assay revealed synergism between the Runx1+/− status and c‐KIT overexpression. In conclusion, the BXH2‐Runx1+/− system is a promising mouse model to investigate the mechanism of leukaemogenesis in FPD/AML.

Increased dosage of Runx1/AML1 acts as a positive modulator of myeloid leukemogenesis in BXH2 mice

The RUNX1/AML1 gene on chromosome 21 is most frequently inactivated in human leukemias. In addition, an increased dose of RUNX1 is suggested as a basis for several kinds of leukemias. Amplifications of chromosome 21 or the RUNX1 gene are shown to be associated with leukemias with lymphoid lineage, whereas its involvement in myeloid lineage remains unclear. In this study, we generated GATA-1 promoter-driven Runx1 transgenic (Tg) mice, which showed a transient mild increase of megakaryocyte marker-positive myeloid cells but no spontaneous leukemia. These mice were then crossed with BXH2 mice, which have a replication-competent retrovirus in the mouse and develop myeloid leukemia due to insertional mutagenesis by random integration of the virus. Overexpressed Runx1 transgene in BXH2 mice resulted in shortening of the latency of leukemia with increased frequency of megakaryoblastic leukemia, suggesting that increased Runx1 dosage is leukemogenic in myeloid lineage. Identifications of retroviral integration sites revealed the genetic alterations that may cooperate with Runx1 overdose in myeloid leukemogenesis. This mouse model may be useful for analysing the pathogenesis of myeloid leukemias with RUNX1 overdose, especially to examine whether an extra-copy of RUNX1 by trisomy 21 is causally related to Downs syndrome-related acute megakaryoblastic leukemia (DS-AMKL).

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.

Runx3 is essential for the target-specific axon pathfinding of trkc-expressing dorsal root ganglion neurons

Dorsal root ganglion (DRG) neurons project their axons to specific target layers in the gray matter of the spinal cord, according to their sensory modality (Neuron 30 (2001), 707; Cell 101 (2000), 485; Neuron 31 (2001), 59; J. Comp. Neurol. 380 (1997), 215; Sensory Neurons, Oxford Univ. Press, New York, 1992, p. 131). Expression of runt-related Runx/AML genes (Mech. Dev. 109 (2001), 413) on subtypes of DRG neurons suggests their involvement in lamina-specific afferent differentiation and maturation. Here we show that Runx3-/- mice display severe limb ataxia and abnormal posture and that most of them die shortly after birth. They show that proprioceptive afferent axons fail to reach the ventral horn and have a smaller dorsal funiculus in their spinal cords. Despite the strong resemblance of this phenotype to that of knockout mice deficient in neurotrophin-3 (NT-3) (Cell 77 (1994), 503; Nature 369 (1994), 658) and its receptor, trkC, (Nature 368 (1994), 249), which show proprioceptive afferent loss through selective neuronal cell death, Runx3-/- mice maintain normal number of TrkC/trkC positive DRG neurons throughout development. Our results suggest that Runx3 controls the target-specific axon pathfinding of trkC-expressing DRG neurons in the spinal cord.

Glutamine repeat variants in human RUNX2 associated with decreased femoral neck BMD, broadband ultrasound attenuation and target gene transactivation.

RUNX2 is an essential transcription factor required for skeletal development and cartilage formation. Haploinsufficiency of RUNX2 leads to cleidocranial displaysia (CCD) a skeletal disorder characterised by gross dysgenesis of bones particularly those derived from intramembranous bone formation. A notable feature of the RUNX2 protein is the polyglutamine and polyalanine (23Q/17A) domain coded by a repeat sequence. Since none of the known mutations causing CCD characterised to date map in the glutamine repeat region, we hypothesised that Q-repeat mutations may be related to a more subtle bone phenotype. We screened subjects derived from four normal populations for Q-repeat variants. A total of 22 subjects were identified who were heterozygous for a wild type allele and a Q-repeat variant allele: (15Q, 16Q, 18Q and 30Q). Although not every subject had data for all measures, Q-repeat variants had a significant deficit in BMD with an average decrease of 0.7SD measured over 12 BMD-related parameters (p = 0.005). Femoral neck BMD was measured in all subjects (−0.6SD, p = 0.0007). The transactivation function of RUNX2 was determined for 16Q and 30Q alleles using a reporter gene assay. 16Q and 30Q alleles displayed significantly lower transactivation function compared to wild type (23Q). Our analysis has identified novel Q-repeat mutations that occur at a collective frequency of about 0.4%. These mutations significantly alter BMD and display impaired transactivation function, introducing a new class of functionally relevant RUNX2 mutants.