UTX maintains the functional integrity of the murine hematopoietic system by globally regulating aging-associated genes.

Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. In this study, UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of COMPASS-like and SWI/SNF complexes, played an essential role in the hematopoietic system by globally regulating aging-associated genes. Utx-deficient (UtxΔ/Δ) mice exhibited myeloid skewing with dysplasia, extramedullary hematopoiesis, impaired hematopoietic reconstituting ability, and increased susceptibility to leukemia, which are the hallmarks of hematopoietic aging. RNA-sequencing (RNA-seq) analysis revealed that Utx deficiency converted the gene expression profiles of young hematopoietic stem-progenitor cells (HSPCs) to those of aged HSPCs. Utx expression in hematopoietic stem cells declined with age, and UtxΔ/Δ HSPCs exhibited increased expression of an aging-associated marker, accumulation of reactive oxygen species, and impaired repair of DNA double-strand breaks. Pathway and chromatin immunoprecipitation analyses coupled with RNA-seq data indicated that UTX contributed to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging via demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged induced neurons showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.

[A Case of Advanced Esophagogastric Junction Adenocarcinoma with Enlarged Lymph Node Metastases of the Middle Mediastinum and Intraperitoneal That Exhibited pCR by Preoperative SOX Therapy].

A 79-year-old man was diagnosed with esophagogastric junction adenocarcinoma, cT3N3M0, cStage Ⅲ, including enlarged lymph node metastases(Bulky N)in the middle mediastinum and intraperitoneal. A total of 2 cycles of S-1 plus oxaliplatin(SOX)was administered. After neoadjuvant chemotherapy, the primary tumor and enlarged lymph nodes had greatly decreased in size. Subsequently, thoracoscopic subtotal esophagectomy and reconstruction with a gastric tube were performed. Histopathological examinations showed no residual cancer cells in the primary lesion and dissected lymph nodes (pathological complete response). Preoperative chemotherapy containing SOX could be a useful treatment strategy for patients with esophagogastric junction adenocarcinoma with enlarged lymph node metastasis.

[Primary Carcinoid Tumor of the Urinary Bladder : A Case Report].

An 81-year-old male was referred to our department with a tumor in the left wall of the urinary bladder, which was detected by contrast-enhanced abdominal computed tomographic scan (CT), incidentally. Cystoscopy revealed a smooth non-papillary tumor. The patient underwent transurethral resection (TUR) of tumor. An immunohistochemical study showed the tumor cells positively stained for chromogranin A, synaptophysin, CD56, and Ki67. The Ki67 index of the tumor was ＞0.5%, which confirmed the diagnosis of a pure carcinoid tumor. There was no recurrence of bladder tumor and no metastasis after the primary treatment.

EMT-inducing transcription factor ZEB1-associated resistance to the BCL-2/BCL-XL inhibitor is overcome by BIM upregulation in ovarian clear cell carcinoma cells.

Ovarian clear cell carcinoma (OCCC) is an aggressive subtype of epithelial ovarian cancer, which generally exhibits chemoresistance. Effective therapy for OCCC is currently unavailable, requiring the development of new therapeutic strategies. ABT-263 (navitoclax), an inhibitor of the anti-apoptotic BCL-2/BCL-XL, has a potent ability of inducing death in cancer cells; however, the therapeutic effect of ABT-263 in OCCC remains unclear. Epithelial cells undergo epithelial-mesenchymal transition (EMT) to acquire a mesenchymal phenotype, which is known to contribute to the development of resistance against therapeutic agents. In this study, we revealed that the sensitivity of OCCC cells to ABT-263 was associated with the epithelial/mesenchymal status of the cells. While the OCCC cells with an epithelial phenotype were ABT-263-sensitive, those with a mesenchymal phenotype were ABT-263-resistant, which was accompanied by an insufficient expression of the pro-apoptotic BH3 protein BIM. Mechanistically, the EMT-inducing transcription factor, ZEB1 down-regulated BIM transcription by binding to BIM promoter, resulting in resistance to ABT-263. It is noteworthy that ZEB1-associated ABT-263 resistance was overcome by an HDAC inhibitor, FK228 (romidepsin), through the up-regulation of BIM. In summary, our study provides evidence for a mechanism for ABT-263 resistance in OCCC cells as well as a potential therapeutic strategy to overcome it.

Acquired expression of CblQ367P in mice induces dysplastic myelopoiesis mimicking chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a hematological malignancy characterized by uncontrolled proliferation of dysplastic myelomonocytes and frequent progression to acute myeloid leukemia (AML). We identified mutations in the Cbl gene, which encodes a negative regulator of cytokine signaling, in a subset of CMML patients. To investigate the contribution of mutant Cbl in CMML pathogenesis, we generated conditional knockin mice for Cbl that express wild-type Cbl in a steady state and inducibly express CblQ367P , a CMML-associated Cbl mutant. CblQ367P mice exhibited sustained proliferation of myelomonocytes, multilineage dysplasia, and splenomegaly, which are the hallmarks of CMML. The phosphatidylinositol 3-kinase (PI3K)-AKT and JAK-STAT pathways were constitutively activated in CblQ367P hematopoietic stem cells, which promoted cell cycle progression and enhanced chemokine-chemokine receptor activity. Gem, a gene encoding a GTPase that is upregulated by CblQ367P , enhanced hematopoietic stem cell activity and induced myeloid cell proliferation. In addition, Evi1, a gene encoding a transcription factor, was found to cooperate with CblQ367P and progress CMML to AML. Furthermore, targeted inhibition for the PI3K-AKT and JAK-STAT pathways efficiently suppressed the proliferative activity of CblQ367P -bearing CMML cells. Our findings provide insights into the molecular mechanisms underlying mutant Cbl-induced CMML and propose a possible molecular targeting therapy for mutant Cbl-carrying CMML patients.

Propagation of trimethylated H3K27 regulated by polycomb protein EED is required for embryogenesis, hematopoietic maintenance, and tumor suppression.

Polycomb repressive complex 2 (PRC2) catalyzes the monomethylation, dimethylation, and trimethylation of histone H3 Lys27 (H3K27) and acts as a central epigenetic regulator that marks the repressive chromatin domain. Embryonic ectoderm development (EED), an essential component of PRC2, interacts with trimethylated H3K27 (H3K27me3) through the aromatic cage structure composed of its three aromatic amino acids, Phe97, Trp364, and Tyr365. This interaction allosterically activates the histone methyltransferase activity of PRC2 and thereby propagates repressive histone marks. In this study, we report the analysis of knock-in mice harboring the myeloid disorder-associated EED Ile363Met (I363M) mutation, analogous to the EED aromatic cage mutants. The I363M homozygotes displayed a remarkable and preferential reduction of H3K27me3 and died at midgestation. The heterozygotes increased the clonogenic capacity and bone marrow repopulating activity of hematopoietic stem/progenitor cells (HSPCs) and were susceptible to leukemia. Lgals3, a PRC2 target gene encoding a multifunctional galactose-binding lectin, was derepressed in I363M heterozygotes, which enhanced the stemness of HSPCs. Thus, our work provides in vivo evidence that the structural integrity of EED to H3K27me3 propagation is critical, especially for embryonic development and hematopoietic homeostasis, and that its perturbation increases the predisposition to hematologic malignancies.

Eicosapentaenoic acid attenuates obesity-related hepatocellular carcinogenesis.

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of obesity, is an emerging risk factor for hepatocellular carcinoma (HCC). Accumulating evidence has shown that chronic inflammation represents a plausible link between obesity and HCC and that the pro-inflammatory cytokine interleukin (IL)-6 contributes to the development of obesity-related HCC. In the present study, we aimed to examine the therapeutic potential of the omega-3 polyunsaturated fatty acid, eicosapentaenoic acid (EPA), which exerts anti-inflammatory effects. The results showed that the development of carcinogen-induced HCC was significantly less in mice fed a high-fat diet (HFD) supplemented with EPA than in those fed HFD only, suggesting that EPA attenuates the development of obesity-related HCC. Although EPA did not appear to affect obesity-linked inflammation, it suppressed the activation of the pro-tumorigenic IL-6 effector STAT3, contributing to the inhibition of tumor growth. These findings suggest a clinical implication of EPA as a treatment for obesity-related HCC.

Fbxl10 overexpression in murine hematopoietic stem cells induces leukemia involving metabolic activation and upregulation of Nsg2.

We previously reported that deficiency for Samd9L, which was cloned as a candidate gene for -7/7q- syndrome, accelerated leukemia cooperatively with enhanced expression of a histone demethylase: F-box and leucine-rich repeat protein 10 (Fbxl10, also known as Jhdm1b, Kdm2b, and Ndy1). To further investigate the role of Fbxl10 in leukemogenesis, we generated transgenic (Tg) mice that overexpress Fbxl10 in hematopoietic stem cells (HSCs). Interestingly, Fbxl10 Tg mice developed myeloid or B-lymphoid leukemia with complete penetrance. HSCs from the Tg mice exhibited an accelerated G0/G1-to-S transition with a normal G0 to G1 entry, resulting in pleiotropic progenitor cell expansion. Fbxl10 Tg HSCs displayed enhanced expression of neuron-specific gene family member 2 (Nsg2), and forced expression of Nsg2 in primary bone marrow cells resulted in expansion of immature cells. In addition, the genes involved in mitochondrial oxidative phosphorylation were markedly enriched in Fbxl10 Tg HSCs, coupled with increased cellular adenosine 5'-triphosphate levels. Moreover, chromatin immunoprecipitation followed by sequencing analysis demonstrated that Fbxl10 directly binds to the regulatory regions of Nsg2 and oxidative phosphorylation genes. These findings define Fbxl10 as a bona fide oncogene, whose deregulated expression contributes to the development of leukemia involving metabolic proliferative advantage and Nsg2-mediated impaired differentiation.

Identification of cooperative genes for E2A-PBX1 to develop acute lymphoblastic leukemia.

E2A-PBX1 is a chimeric gene product detected in t(1;19)-bearing acute lymphoblastic leukemia (ALL) with B-cell lineage. To investigate the leukemogenic process, we generated conditional knock-in (cKI) mice for E2A-PBX1, in which E2A-PBX1 is inducibly expressed under the control of the endogenous E2A promoter. Despite the induced expression of E2A-PBX1, no hematopoietic disease was observed, strongly suggesting that additional genetic alterations are required to develop leukemia. To address this possibility, retroviral insertional mutagenesis was used. Virus infection efficiently induced T-cell, B-cell, and biphenotypic ALL in E2A-PBX1 cKI mice. Inverse PCR identified eight retroviral common integration sites, in which enhanced expression was observed in the Gfi1, Mycn, and Pim1 genes. In addition, it is of note that viral integration and overexpression of the Zfp521 gene was detected in one tumor with B-cell lineage; we previously identified Zfp521 as a cooperative gene with E2A-HLF, another E2A-involving fusion gene with B-lineage ALL. The cooperative oncogenicity of E2A-PBX1 with overexpressed Zfp521 in B-cell tumorigenesis was indicated by the finding that E2A-PBX1 cKI, Zfp521 transgenic compound mice developed B-lineage ALL. Moreover, upregulation of ZNF521, the human counterpart of Zfp521, was found in several human leukemic cell lines bearing t(1;19). These results indicate that E2A-PBX1 cooperates with additional gene alterations to develop ALL. Among them, enhanced expression of ZNF521 may play a clinically relevant role in E2A fusion genes to develop B-lineage ALL.

Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms.

Acquired uniparental disomy (aUPD) is a common feature of cancer genomes, leading to loss of heterozygosity. aUPD is associated not only with loss-of-function mutations of tumour suppressor genes, but also with gain-of-function mutations of proto-oncogenes. Here we show unique gain-of-function mutations of the C-CBL (also known as CBL) tumour suppressor that are tightly associated with aUPD of the 11q arm in myeloid neoplasms showing myeloproliferative features. The C-CBL proto-oncogene, a cellular homologue of v-Cbl, encodes an E3 ubiquitin ligase and negatively regulates signal transduction of tyrosine kinases. Homozygous C-CBL mutations were found in most 11q-aUPD-positive myeloid malignancies. Although the C-CBL mutations were oncogenic in NIH3T3 cells, c-Cbl was shown to functionally and genetically act as a tumour suppressor. C-CBL mutants did not have E3 ubiquitin ligase activity, but inhibited that of wild-type C-CBL and CBL-B (also known as CBLB), leading to prolonged activation of tyrosine kinases after cytokine stimulation. c-Cbl(-/-) haematopoietic stem/progenitor cells (HSPCs) showed enhanced sensitivity to a variety of cytokines compared to c-Cbl(+/+) HSPCs, and transduction of C-CBL mutants into c-Cbl(-/-) HSPCs further augmented their sensitivities to a broader spectrum of cytokines, including stem-cell factor (SCF, also known as KITLG), thrombopoietin (TPO, also known as THPO), IL3 and FLT3 ligand (FLT3LG), indicating the presence of a gain-of-function that could not be attributed to a simple loss-of-function. The gain-of-function effects of C-CBL mutants on cytokine sensitivity of HSPCs largely disappeared in a c-Cbl(+/+) background or by co-transduction of wild-type C-CBL, which suggests the pathogenic importance of loss of wild-type C-CBL alleles found in most cases of C-CBL-mutated myeloid neoplasms. Our findings provide a new insight into a role of gain-of-function mutations of a tumour suppressor associated with aUPD in the pathogenesis of some myeloid cancer subsets.

Dok adaptors play anti-inflammatory roles in pulmonary homeostasis.

Asthma is a chronic inflammatory disease of the lung with airflow obstruction and bronchospasm, characterized by pulmonary eosinophilia, airway remodeling, increased airway hyperresponsiveness to environmental stimuli, and excessive Th2-type cytokine production. Recent studies indicate that crosstalk between the innate and adaptive immune systems is crucial for this disease. We and others have showed that the Dok (downstream of tyrosine kinases) family adaptors, Dok-1, Dok-2, and Dok-3, play essential roles in negative regulation of a wide variety of signaling pathways in both innate and adaptive immunities. Here, histopathology and bronchoalveolar lavage fluid (BALF) cellularity showed spontaneous pulmonary inflammation in Dok-1-/- Dok-2-/- Dok-3-/- (TKO) mice, but not in Dok-1-/- Dok-2-/- or Dok-3-/- mice, with hallmarks of asthma, including eosinophilia, goblet cell hyperplasia, and subepithelial fibrosis. Consistently, TKO mice, but not the other mutants, showed increased airway hyperresponsiveness to methacholine inhalation. In addition, Th2-type cytokine concentrations in BALF were increased in TKO mice. These findings provide strong evidence that Dok-1, Dok-2, and Dok-3 cooperatively play critical anti-inflammatory roles in lung homeostasis.

Hemp, an mbt domain-containing protein, plays essential roles in hematopoietic stem cell function and skeletal formation.

To clarify the molecular pathways governing hematopoietic stem cell (HSC) development, we screened a fetal liver (FL) HSC cDNA library and identified a unique gene, hematopoietic expressed mammalian polycomb (hemp), encoding a protein with a zinc-finger domain and four malignant brain tumor (mbt) repeats. To investigate its biological role, we generated mice lacking Hemp (hemp(-/-)). Hemp(-/-) mice exhibited a variety of skeletal malformations and died soon after birth. In the FL, hemp was preferentially expressed in the HSC and early progenitor cell fractions, and analyses of fetal hematopoiesis revealed that the number of FL mononuclear cells, including HSCs, was reduced markedly in hemp(-/-) embryos, especially during early development. In addition, colony-forming and competitive repopulation assays demonstrated that the proliferative and reconstitution abilities of hemp(-/-) FL HSCs were significantly impaired. Microarray analysis revealed alterations in the expression levels of several genes implicated in hematopoietic development and differentiation in hemp(-/-) FL HSCs. These results demonstrate that Hemp, an mbt-containing protein, plays essential roles in HSC function and skeletal formation. It is also hypothesized that Hemp might be involved in certain congenital diseases, such as Klippel-Feil anomaly.

Experience with chemotherapy for postoperative metastases of adenosquamous carcinoma of the esophagogastric junction and pathological study of its development.

We report here a case of postoperative recurrent adenosquamous carcinoma (ASC) of the esophagogastric junction (EGJ) treated with S-1 therapy. A 79-year-old woman was diagnosed with carcinoma of the EGJ. Thoracoscopic subtotal esophagectomy was performed, and pathological examination revealed advanced ASC with lymph node metastasis. Five months after surgery, multiple lung metastases and multiple lymph node metastases were observed, and the patient was treated with S-1 monotherapy, which showed partial response and may be effective for advanced ASC of the EGJ. On the other hand, immunohistological analysis of the tumors showed a relatively wide range of areas that could differentiate into both adenocarcinoma and squamous cell carcinoma, suggesting that tumor cells with multidifferentiation potential, or at least the ability to differentiate into both adeno-epithelial and squamous epithelial cells, were the likely source of the tumors.

Advanced glycation end products suppress lysyl oxidase and induce bone collagen degradation in a rat model of renal osteodystrophy.

Renal osteodystrophy (ROD) is a major problem in patients with renal insufficiency. The present study was designed to elucidate the role of bone collagen changes and osteoblast differentiation in a rat model of ROD pathogenesis induced by adenine. Typical characteristics of renal failure, including increased serum urea nitrogen, creatinine, inorganic phosphorus, and intact parathyroid hormone levels, and decreased serum calcium and 1,25(OH)2D3 levels, were observed in adenine-induced rats. Micro-computed tomography analysis of the femur in adenine-induced rats showed decreased bone mineral density and osteoporotic changes, confirmed by the three-point bending test. The cancellous bone histomorphometric parameters of the tibia showed increased osteoblast number, decreased osteoclast surface with peritrabecular fibrosis, and increased osteoid tissue, indicating a severe mineralization disorder similar to clinical ROD. Scanning and transmission electron microscopy revealed irregular alignment and increased diameter of bone collagen fibrils in adenine-induced rats. Protein expression analysis showed greater accumulation of advanced glycation end products (AGEs) in peritrabecular osteoblasts of adenine-induced rats than in the controls. In contrast, suppressed expression of runt-related transcription factor 2, alkaline phosphatase, secreted phosphoprotein 1 (Spp1), and lysyl oxidase (Lox) mRNA levels, particularly the amount of active LOX protein, were observed. In in-vitro experiments, mineralizing MC3T3-E1 osteoblastic cells stimulated with AGE-modified bovine serum albumin had attenuated the expression of Spp1 mRNA levels and active LOX protein, with a decrease in extracellular nodules of mineralization. These observations provide clues to ROD pathogenesis, as they indicate that the suppression of osteoblast differentiation and decreased active LOX protein associated with accumulation of AGEs in osteoblasts caused structural abnormalities of bone collagen fibrils and a severe mineralization disorder, leading to bone fragility.

Depletion of mitochondrial fission factor DRP1 causes increased apoptosis in human colon cancer cells.

Mitochondria play a critical role in regulation of apoptosis, a form of programmed cell death, by releasing apoptogenic factors including cytochrome c. Growing evidence suggests that dynamic changes in mitochondrial morphology are involved in cellular apoptotic response. However, whether DRP1-mediated mitochondrial fission is required for induction of apoptosis remains speculative. Here, we show that siRNA-mediated DRP1 knockdown promoted accumulation of elongated mitochondria in HCT116 and SW480 human colon cancer cells. Surprisingly, DRP1 down-regulation led to decreased proliferation and increased apoptosis of these cells. A higher rate of cytochrome c release and reductions in mitochondrial membrane potential were also revealed in DRP1-depleted cells. Taken together, our present findings suggest that mitochondrial fission factor DRP1 inhibits colon cancer cell apoptosis through the regulation of cytochrome c release and mitochondrial membrane integrity.

The role of the basic helix-loop-helix transcription factor Dec1 in the regulatory T cells.

Naturally occurring regulatory T (Treg) cells play a central role in the maintenance of immune homeostasis and in restraining the development of spontaneous inflammatory responses. However, the underlying mechanisms of Treg homeostasis remain incompletely understood. Of particular note, the IL-2Rα (CD25) is crucial for the homeostasis of Treg cells and the prevention of lymphoproliferative autoimmune disease. In this paper, we report that the basic helix-loop-helix transcription factor Dec1 is involved in the homeostasis of Treg cells and plays a role in their survival or expansion after adoptive transfer to lymphopenic recipients. Hence, it is crucial for the suppression of effector T cell-mediated inflammatory responses. Enforced expression of Dec1 upregulates CD25 expression during thymocyte development and increases the number of Treg cells in the periphery. Dec1 binds the transcription factor Runx1 and colocalizes with Runx1 in Treg cells. Specifically, we demonstrate that in Treg cells the Dec1/Runx1 complex binds to regulatory elements present in the Il-2rα locus. Collectively, these data show how Dec1 mechanistically acts in Treg cells.

Acquired hemophilia A in a patient with essential thrombocythemia.

A 69-year-old woman with essential thrombocythemia (ET) developed giant ecchymosis, and she was admitted to hospital. Marked anemia (Hb 8.1 g/dl) accompanied by a prolonged activated partial thromboplastin time (89.6 s) was observed, and she received red blood cells (RBC) and fresh frozen plasma (FFP). On day 2 after admission, consciousness disturbance suddenly occurred, whereas computed tomography of the brain showed no evidence of bleeding. As the ecchymosis progressed, she developed shock. Although RBC and FFP transfusions were administered, she developed multi-organ failure and died 48 h after admission. Low factor VIII activity (<1%) accompanied by factor VIII inhibitor (17 Bethesda units) was found after her death. An autopsy revealed cerebral infarction without cerebral herniation. To date, acquired hemophilia A accompanying ET has been described in only one other patient. Although acquired factor VIII inhibitor is a rare disease, it should be tested for in ET patients with marked hemorrhagic tendency.

An autopsy case of clinically un-diagnosed autosomal recessive polycystic kidney disease in 77-year-old male.

Autosomal recessive polycystic kidney disease (ARPKD) is caused by genetic mutations of the gene encoding fibrocystin, and is characterized by the collecting duct cysts and congenital hepatic fibrosis. We report an autopsy-proven case of ARPKD in a 77-year-old male who presented with rapidly progressive renal and liver dysfunction. He had refused hemodialysis, and died 4 months later. At autopsy, both kidneys were enlarged with numerous small cysts throughout the cortex, which were revealed immunohistochemically to be the collecting ducts. Liver involvement was characterized by ductal plate malformation accompanied with portal fibrosis. The morphological appearances were compatible with ARPKD and the negative immunostaining for fibrocystin in the collecting ducts and bile ducts confirmed the diagnosis. ARPKD is known to occur in the neonatal period or in infancy with a high mortality rate. Although some patients who survive infancy are expected to live longer into young adulthood, most patients with ARPKD die of renal and hepatic failure in their childhood. The present case is extremely exceptional, in that no clinical symptoms suggestive of ARPKD were noticed until old age, and suggests that the disease spectrum of ARPKD is variable, and that a slowly progressive form of ARPKD may not be diagnosed until old age.

A 5' untranslated region containing the IRES element in the Runx1 gene is required for angiogenesis, hematopoiesis and leukemogenesis in a knock-in mouse model.

Although internal ribosome entry site (IRES)-mediated translation is considered important for proper cellular function, its precise biological role is not fully understood. Runx1 gene, which encodes a transcription factor implicated in hematopoiesis, angiogenesis, and leukemogenesis, contains IRES sequences in the 5' untranslated region. To clarify the roles of the IRES element in Runx1 function, we generated knock-in mice for either wild-type Runx1 or Runx1/Evi1, a Runx1 fusion protein identified in human leukemia. In both cases, native promoter-dependent transcription was retained, whereas IRES-mediated translation was eliminated. Interestingly, homozygotes expressing wild-type Runx1 deleted for the IRES element (Runx1(Delta IRES/Delta IRES)) died in utero with prominent dilatation of peripheral blood vessels due to impaired pericyte development. In addition, hematopoietic cells in the Runx1(Delta IRES/Delta IRES) fetal liver were significantly decreased, and exhibited an altered differentiation pattern, a reduced proliferative activity, and an impaired reconstitution ability. On the other hand, heterozygotes expressing Runx1/Evi1 deleted for the IRES element (Runx1(+/RE Delta IRES)) were born normally and did not show any hematological abnormalities, in contrast that conventional Runx1/Evi1 heterozygotes die in utero with central nervous system hemorrhage and Runx1/Evi1 chimeric mice develop acute leukemia. The findings reported here demonstrate the essential roles of the IRES element in Runx1 function under physiological and pathological conditions.