Bindya Jacob

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.

Runx family genes, niche, and stem cell quiescence.

In multicellular organisms, terminally differentiated cells of most tissues are short-lived and therefore require constant replenishment from rapidly dividing stem cells for homeostasis and tissue repair. For the stem cells to last throughout the lifetime of the organism, however, a small subset of stem cells, which are maintained in a hibernation-like state known as stem cell quiescence, is required. Such dormant stem cells reside in the niche and are activated into proliferation only when necessary. A multitude of factors are required for the maintenance of stem cell quiescence and niche. In particular, the Runx family genes have been implicated in stem cell quiescence in various organisms and tissues. In this review, we discuss the maintenance of stem cell quiescence in various tissues, mainly in the context of the Runx family genes, and with special focus on the hematopoietic system.

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.

Stem cell exhaustion and leukemogenesis

Cancer is the result of a combination of genetic alterations, which aid transformation of cells. However, oncogenic alterations also simultaneously induce some detrimental effects on the cells such as apoptosis, senescence, and differentiation. Such negative effects caused by certain oncogenic events are overcome by other cooperating genetic hits. We propose stem cell exhaustion as a novel detrimental effect that is caused by a wide variety of oncogenic alterations. Interestingly, in most cases, the stem cell exhaustion due to oncogenic alterations is preceded by an abnormal expansion of stem/progenitor cells. This preceding stem/progenitor cell expansion may be a key feature that still promotes cancer development, along with cooperating hits that rescue stem cell exhaustion. This review summarizes current knowledge about hematopoietic stem cell exhaustion and the mechanisms to overcome stem cell exhaustion in cancer development. J. Cell. Biochem. 107: 393–399, 2009.

Mice Carrying a Hypomorphic Evi1 Allele Are Embryonic Viable but Exhibit Severe Congenital Heart Defects

The ecotropic viral integration site 1 (Evi1) oncogenic transcription factor is one of a number of alternative transcripts encoded by the Mds1 and Evi1 complex locus (Mecom). Overexpression of Evi1 has been observed in a number of myeloid disorders and is associated with poor patient survival. It is also amplified and/or overexpressed in many epithelial cancers including nasopharyngeal carcinoma, ovarian carcinoma, ependymomas, and lung and colorectal cancers. Two murine knockout models have also demonstrated Evi1s critical role in the maintenance of hematopoietic stem cell renewal with its absence resulting in the death of mutant embryos due to hematopoietic failure. Here we characterize a novel mouse model (designated Evi1fl3) in which Evi1 exon 3, which carries the ATG start, is flanked by loxP sites. Unexpectedly, we found that germline deletion of exon3 produces a hypomorphic allele due to the use of an alternative ATG start site located in exon 4, resulting in a minor Evi1 N-terminal truncation and a block in expression of the Mds1-Evi1 fusion transcript. Evi1δex3/δex3 mutant embryos showed only a mild non-lethal hematopoietic phenotype and bone marrow failure was only observed in adult Vav-iCre/+, Evi1fl3/fl3 mice in which exon 3 was specifically deleted in the hematopoietic system. Evi1δex3/δex3 knockout pups are born in normal numbers but die during the perinatal period from congenital heart defects. Database searches identified 143 genes with similar mutant heart phenotypes as those observed in Evi1δex3/δex3 mutant pups. Interestingly, 42 of these congenital heart defect genes contain known Evi1-binding sites, and expression of 18 of these genes are also effected by Evi1 siRNA knockdown. These results show a potential functional involvement of Evi1 target genes in heart development and indicate that Evi1 is part of a transcriptional program that regulates cardiac development in addition to the development of blood.