Recent insights into the SWI/SNF complex and the molecular mechanism of hSNF5 deficiency in rhabdoid tumors.

Genetic information encoded by DNA is packaged in the nucleus using the chromatin structure. The accessibility of transcriptional elements in DNA is controlled by the dynamic structural changes of chromatin for the appropriate regulation of gene transcription. Chromatin structure is regulated by two general mechanisms, one is histone modification and the other is chromatin remodeling in an ATP-dependent manner. Switch/sucrose nonfermentable (SWI/SNF) complexes utilize the energy from ATP hydrolysis to mobilize nucleosomes and remodel the chromatin structure, contributing to conformational changes in chromatin. Recently, the inactivation of encoding genes for subunits of the SWI/SNF complexes has been documented in a series of human cancers, accounting for up to almost 20% of all human cancers. For example, human SNF5 (hSNF5), the gene that encodes a subunit of the SWI/SNF complexes, is the sole mutation target that drives malignant rhabdoid tumors (MRT). Despite remarkably simple genomes, the MRT has highly malignant characteristics. As a key to understanding MRT tumorigenesis, it is necessary to fully examine the mechanism of chromatin remodeling by the SWI/SNF complexes. Herein, we review the current understanding of chromatin remodeling by focusing on SWI/SNF complexes. In addition, we describe the molecular mechanisms and influences of hSNF5 deficiency in rhabdoid tumors and the prospects for developing new therapeutic targets to overcome the epigenetic drive of cancer that is caused by abnormal chromatin remodeling.

Dual targeting of EZH1 and EZH2 for the treatment of malignant rhabdoid tumors.

Malignant rhabdoid tumors (MRTs) are rare and highly aggressive pediatric cancers with no standard of care. MRTs are characterized by loss of SMARCB1, which results in upregulated expression of enhancer of zeste homolog 2 (EZH2), which is responsible for the methylation of lysine 27 of histone H3 (H3K27me3), leading to the repression of gene expression. Although previous reports suggest EZH2 as an effective therapeutic target, the functions of EZH1, the other homolog of EZH, in MRT remain unknown. Here, we show that EZH1, as well as EZH2, contributes to MRT cell growth and H3K27 methylation. Depletion or selective inhibition of EZH2 led to a compensatory increase in EZH1 expression, and depletion of EZH1 enhanced the effect of EZH2 inhibition. EZH1/2 dual inhibitors suppressed MRT cell growth markedly, reflecting the reduction of H3K27me3 accumulation at one of the EZH1/2 targets, the CDKN2A locus. Dual inhibition of EZH1/2 in vivo suppressed tumor growth completely, with no significant adverse effects. These findings indicate that both EZH1 and EZH2 are potential targets for MRT therapy, and that EZH1/2 dual inhibitors may be promising therapeutic strategies for MRT.

Tumor necrosis factor­related apoptosis­inducing ligand is a novel transcriptional target of runt­related transcription factor 1.

Runt­related transcription factor 1 (RUNX1), which is also known as acute myeloid leukemia 1 (AML1), has been frequently found with genomic aberrations in human leukemia. RUNX1 encodes a transcription factor that can regulate the expression of hematopoietic genes. In addition, tumor necrosis factor­related apoptosis­inducing ligand (TRAIL) performs an important function for malignant tumors in immune surveillance. However, the regulatory mechanism of TRAIL expression remain to be fully elucidated. In the present study, tetradecanoylphorbol 13­acetate­treated megakaryocytic differentiated K562 cells was used to examine the effect of RUNX1 on TRAIL expression. Luciferase assay series of TRAIL promoters for the cells co­transfected with RUNX1 and core­binding factor ß (CBFß) expression vectors were performed to evaluate the nature of TRAIL transcriptional regulation. Electrophoresis mobility shift assay of the RUNX1 consensus sequence of the TRAIL promoter with recombinant RUNX1 and CBFß proteins was also performed. BloodSpot database analysis for TRAIL expression in patients with acute myeloid leukemia were performed. The expression of TRAIL, its receptor Death receptor 4 and 5 and RUNX1 in K562 cells transfected with the RUNX1 expression vector and RUNX1 siRNA were evaluated by reverse transcription­quantitative PCR (RT­qPCR). TRAIL and RUNX1­ETO expression was also measured in Kasumi­1 cells transfected with RUNX1­ETO siRNA and in KG­1 cells transfected with RUNX1­ETO expression plasmid, both by RT­qPCR. Cell counting, lactate dehydrogenase assay and cell cycle analysis by flow cytometry were performed on Kasumi­1, KG­1, SKNO­1 and K562 cells treated with TRAIL and HDAC inhibitors sodium butyrate or valproic acid. The present study demonstrated that RUNX1 is a transcriptional regulator of TRAIL. It was initially found that the induction of TRAIL expression following the megakaryocytic differentiation of human leukemia cells was RUNX1­dependent. Subsequently, overexpression of RUNX1 was found to increase TRAIL mRNA expression by activating its promoter activity. Additional analyses revealed that RUNX1 regulated the expression of TRAIL in an indirect manner, because RUNX1 retained its ability to activate this promoter following the mutation of all possible RUNX1 consensus sites. Furthermore, TRAIL expression was reduced in leukemia cells carrying the t(8;21) translocation, where the RUNX1­ETO chimeric protein interfere with normal RUNX1 function. Exogenous treatment of recombinant TRAIL proteins was found to induce leukemia cell death. To conclude, the present study provided a novel mechanism, whereby TRAIL is a target gene of RUNX1 and TRAIL expression was inhibited by RUNX1­ETO. These results suggest that TRAIL is a promising agent for the clinical treatment of t(8;21) AML.

C11orf21, a novel RUNX1 target gene, is down-regulated by RUNX1-ETO.

The fusion protein RUNX1-ETO is an oncogenic transcription factor generated by t(8;21) chromosome translocation, which is found in FAB-M2-type acute myeloid leukemia (AML). RUNX1-ETO is known to dysregulate the normal RUNX1 transcriptional network, which should involve essential factors for the onset of AML with t(8;21). In this study, we screened for possible transcriptional targets of RUNX1 by reanalysis of public data in silico, and identified C11orf21 as a novel RUNX1 target gene because its expression was down-regulated in the presence of RUNX1-ETO. The expression level of C11orf21 was low in AML patient samples with t(8;21) and in Kasumi-1 cells, which carry RUNX1-ETO. Knockdown of RUNX1-ETO in Kasumi-1 cells restored C11orf21 expression, whereas overexpression of RUNX1 up-regulated C11orf21 expression. In addition, knockdown of RUNX1 in other human leukemia cells without RUNX-ETO, such as K562, led to a decrease in C11orf21 expression. Of note, the C11orf21 promoter sequence contains a consensus sequence for RUNX1 binding and it was activated by exogenously expressed RUNX1 based on our luciferase reporter assay. This luciferase signal was trans-dominantly suppressed by RUNX1-ETO and site-directed mutagenesis of the consensus site abrogated the reporter activity. This study demonstrated that C11orf21 is a novel transcriptional target of RUNX1 and RUNX1-ETO suppressed C11orf21 transcription in t(8;21) AML. Thus, through this in silico approach, we identified a novel transcriptional target of RUNX1, and the depletion of C11orf21, the target gene, may be associated with the onset of t(8;21) AML.

Novel Two MRT Cell Lines Established from Multiple Sites of a Synchronous MRT Patient.

BACKGROUND/AIM: Malignant rhabdoid tumor (MRT) is a rare, aggressive neoplasm found in young children, caused by inactivation of a single gene, SNF5 (INI1, SMARCB1). MRT cases with multifocal tumors at diagnosis are categorized as synchronous MRT, often with a germline mutation of SNF5. The aim of this study was to establish new models useful in clarifying the biological basis of synchronous MRT. MATERIALS AND METHODS: We established two novel MRT cell lines, designated as KP-MRT-KS and KP-MRT-KSa, derived from different lesions and at a different time from a synchronous multifocal 7-month-old female MRT patient. RESULTS: Both cells showed typical morphology of MRT, with a compound genomic mutation in exons 2 and 5 of the SNF5 gene. The exon 2 mutation was found in the germline. CONCLUSION: These cell lines could serve as powerful tools for unveiling the molecular mechanism of refractory synchronous MRT.

Hematopoietic stem cells acquire survival advantage by loss of RUNX1 methylation identified in familial leukemia.

RUNX1 is among the most frequently mutated genes in human leukemia, and the loss or dominant-negative suppression of RUNX1 function is found in myelodysplastic syndrome and acute myeloid leukemia (AML). How posttranslational modifications (PTMs) of RUNX1 affect its in vivo function, however, and whether PTM dysregulation of RUNX1 can cause leukemia are largely unknown. We performed targeted deep sequencing on a family with 3 occurrences of AML and identified a novel RUNX1 mutation, R237K. The mutated R237 residue is a methylation site by protein arginine methyltransferase 1, and loss of methylation reportedly impairs the transcriptional activity of RUNX1 in vitro. To explore the biologic significance of RUNX1 methylation in vivo, we used RUNX1 R233K/R237K double-mutant mice, in which 2 arginine-to-lysine mutations precluded RUNX1 methylation. Genetic ablation of RUNX1 methylation led to loss of quiescence and expansion of hematopoietic stem cells (HSCs), and it changed the genomic and epigenomic signatures of phenotypic HSCs to a poised progenitor state. Furthermore, loss of RUNX1 R233/R237 methylation suppressed endoplasmic reticulum stress-induced unfolded protein response genes, including Atf4, Ddit3, and Gadd34; the radiation-induced p53 downstream genes Bbc3, Pmaip1, and Cdkn1a; and subsequent apoptosis in HSCs. Mechanistically, activating transcription factor 4 was identified as a direct transcriptional target of RUNX1. Collectively, defects in RUNX1 methylation in HSCs confer resistance to apoptosis and survival advantage under stress conditions, a hallmark of a preleukemic clone that may predispose affected individuals to leukemia. Our study will lead to a better understanding of how dysregulation of PTMs can contribute to leukemogenesis.

Interaction of Cx43 with Hsc70 regulates G1/S transition through CDK inhibitor p27.

Connexin 43 (Cx43) functions as a cell growth suppressor. We have demonstrated that Cx43 interacts with heat shock cognate protein 70 (Hsc70) for regulating cell proliferation. Hsc70 interacts with CDK inhibitor p27, which regulates the assembly and subcellular localization of cyclin D1-CDK4-p27 complex. However, the involvement of p27 with Cx43-mediated cell cycle suppression is still poorly understood. Here, we report that nuclear accumulation of p27 is reduced by overexpression of Cx43, and that this reduction is restored by co-overexpression with Hsc70. We found that Cx43 competes with p27 for binding to Hsc70, and as a result, decreases the level of Hsc70 in cyclin D1-CDK4-p27 complex, leading to prevention of the nuclear translocation of the complex and the G1/S transition. Collectively, our findings suggest that, in Cx43 up-regulation, which is most likely an emergency measure, Cx43-Hsc70 interaction regulates cell cycle G1/S progression through a novel mechanism by which Cx43-Hsc70 interaction prevents the nuclear accumulation of p27 through controlling the nuclear translocation of cyclin D1-CDK4-p27 complex.

MAZR and Runx Factors Synergistically Repress ThPOK during CD8+ T Cell Lineage Development.

Th-inducing Pox virus and zinc finger/Krüppel-like factor (ThPOK) is a key commitment factor for CD4(+) lineage T cells and is essential for the maintenance of CD4 lineage integrity; thus, the expression of ThPOK has to be tightly controlled. In this article, we demonstrate that Myc-associated zinc finger-related factor (MAZR) and Runt-related transcription factor 1 (Runx1) together repressed ThPOK in preselection double-positive thymocytes, whereas MAZR acted in synergy with Runx3 in the repression of ThPOK in CD8(+) T cells. Furthermore, MAZR-Runx1 and MAZR-Runx3 double-mutant mice showed enhanced derepression of Cd4 in double-negative thymocytes and in CD8(+) T cells in comparison with Runx1 or Runx3 single-deficient mice, respectively, indicating that MAZR modulates Cd4 silencing. Thus, our data demonstrate developmental stage-specific synergistic activities between MAZR and Runx/core-binding factor ß (CBFß) complexes. Finally, retroviral Cre-mediated conditional deletion of MAZR in peripheral CD8(+) T cells led to the derepression of ThPOK, thus showing that MAZR is also part of the molecular machinery that maintains a repressed state of ThPOK in CD8(+) T cells.

Loss of RUNX1/AML1 arginine-methylation impairs peripheral T cell homeostasis.

RUNX1 (previously termed AML1) is a frequent target of human leukaemia-associated gene aberrations, and it encodes the DNA-binding subunit of the Core-Binding Factor transcription factor complex. RUNX1 expression is essential for the initiation of definitive haematopoiesis, for steady-state thrombopoiesis, and for normal lymphocytes development. Recent studies revealed that protein arginine methyltransferase 1 (PRMT1), which accounts for the majority of the type I PRMT activity in cells, methylates two arginine residues in RUNX1 (R206 and R210), and these modifications inhibit corepressor-binding to RUNX1 thereby enhancing its transcriptional activity. In order to elucidate the biological significance of these methylations, we established novel knock-in mouse lines with non-methylable, double arginine-to-lysine (RTAMR-to-KTAMK) mutations in RUNX1. Homozygous Runx1(KTAMK) (/) (KTAMK) mice are born alive and appear normal during adulthood. However, Runx1(KTAMK) (/) (KTAMK) mice showed a reduction in CD3(+) T lymphoid cells and a decrease in CD4(+) T cells in peripheral lymphoid organs, in comparison to their wild-type littermates, leading to a reduction in the CD4(+) to CD8(+) T-cell ratio. These findings suggest that arginine-methylation of RUNX1 in the RTAMR-motif is dispensable for the development of definitive haematopoiesis and for steady-state platelet production, however this modification affects the role of RUNX1 in the maintenance of the peripheral CD4(+) T-cell population.

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.

AML1/RUNX1 works as a negative regulator of c-Mpl in hematopoietic stem cells.

In this study, we analyzed the roles for AML1/RUNX1 in the regulation of the c-mpl promoter. Wild-type AML1 activated the c-mpl promoter through the proximal AML-binding site in luciferase assays using 293T and HeLa cells. In accord with this result, electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrated that AML1 bound to this site. Next, we analyzed the function of AML1 using a mutant of AML1 lacking the C terminus (AML1dC), which was originally found in a patient with myelodysplastic syndromes. AML1dC dominant-negatively suppressed transcriptional activity of wild-type AML1. However, unexpectedly, AML1dC-transduced murine c-Kit(+)Sca1(+)Lineage(-) cells expressed c-mpl mRNA and c-Mpl protein more abundantly than mock-transduced cells, which led to the enhanced thrombopoietin-mediated proliferation. Moreover, when AML1dC was induced to express during the development of hematopoietic cells from embryonic stem (ES) cells, AML1dC augmented the c-Mpl expression on hematopoietic stem/progenitor cells. Furthermore, we found that early hematopoietic cells that derived from AML1(+/-) ES cells expressed c-Mpl more intensely than those that developed from wild-type ES cells. In contrast, AML1dC hardly affected c-Mpl expression and maturation of megakaryocytes. As for the mechanism of the different roles of AML1 in the regulation of the c-mpl promoter, we found that AML1 forms a complex with a transcription repressor mSin3A on the c-mpl promoter in hematopoietic stem/progenitor cells, although it forms a complex with a transcription activator p300 on the same promoter in megakaryocytic cells. Together, these data indicate that AML1 can regulate the c-mpl promoter both positively and negatively by changing the binding partner according to cell types.

Repression of the transcription factor Th-POK by Runx complexes in cytotoxic T cell development.

Mouse CD4+CD8+ double-positive (DP) thymocytes differentiate into CD4+ helper-lineage cells upon expression of the transcription factor Th-POK but commit to the CD8+ cytotoxic lineage in its absence. We report the redirected differentiation of class I-restricted thymocytes into CD4+CD8- helper-like T cells upon loss of Runx transcription factor complexes. A Runx-binding sequence within the Th-POK locus acts as a transcriptional silencer that is essential for Th-POK repression and for development of CD8+ T cells. Thus, Th-POK expression and genetic programming for T helper cell development are actively inhibited by Runx-dependent silencer activity, allowing for cytotoxic T cell differentiation. Identification of the transcription factors network in CD4 and CD8 lineage choice provides insight into how distinct T cell subsets are developed for regulating the adaptive immune system.

Mechanisms of compartmentalized expression of Mrg class G-protein-coupled sensory receptors.

Mrg class G-protein-coupled receptors (GPCRs) are expressed exclusively in sensory neurons in the trigeminal and dorsal root ganglia. Pharmacological activation of Mrg proteins is capable of modulating sensory neuron activities and elicits nociceptive effects. In this study, we illustrate a control mechanism that allows the Runx1 runt domain transcription factor to generate compartmentalized expression of these sensory GPCRs. Expression of MrgA, MrgB, and MrgC subclasses is confined to an "A/B/C" neuronal compartment that expresses Runx1 transiently (or does not express Runx1), whereas MrgD expression is restricted to a "D" compartment with persistent Runx1 expression. Runx1 is initially required for the expression of all Mrg genes. However, during late development Runx1 becomes a repressor for MrgA/B/C genes. As a result, MrgA/B/C expression persists only in the Runx1- "A/B/C" compartment. In delta446 mice, in which Runx1 lacks the C-terminal repression domain, expression of MrgA/B/C genes is dramatically expanded into the Runx1+ "D" compartment. MrgD expression, however, is resistant to Runx1-mediated repression in the "D" compartment. Therefore, the creation of Runx1+ and Runx1- compartments, in conjunction with different responses of Mrg genes to Runx1-mediated repression, results in the compartmentalized expression of MrgA/B/C versus MrgD genes. Within the MrgA/B/C compartment, MrgB4-expressing neurons innervate exclusively the hairy skin. Here we found that Smad4, a downstream component of bone morphological protein-mediated signaling, is required selectively for the expression of MrgB4. Our study suggests a new line of evidence that specification of sensory subtypes is established progressively during perinatal and postnatal development.

Shared and distinct roles mediated through C-terminal subdomains of acute myeloid leukemia/Runt-related transcription factor molecules in murine development.

AML1/Runx1 is a frequent target of human leukemia-associated gene aberration and encodes a transcription factor with nonredundant biologic functions in initial development of definitive hematopoiesis, T-cell development, and steady-state platelet production. AML1/Runx1 and 2 closely related family genes, AML2/Runx3 and AML3/Runx2/Cbfa1, present in mammals, comprise the Runt-domain transcription factor family. Although they have similar structural and biochemical properties, gene-targeting experiments have identified distinct biologic roles. To directly determine the presence of functional overlap among runt-related transcription factor (Runx) family molecules, we replaced the C-terminal portion of acute myeloid leukemia 1 (AML1) with that derived from its family members, which are variable in contrast to conserved Runt domain, using the gene knock-in method. We found that C-terminal portions of either AML2 or AML3 could functionally replace that of AML1 for myeloid development in culture and within the entire mouse. However, while AML2 substituted for AML1 could effectively rescue lymphoid lineages, AML3 could not, resulting in a smaller thymus and lymphoid deficiency in peripheral blood. Substitution by the C-terminal portion of AML3 also led to high infantile mortality and growth retardation, suggesting that AML1 has as yet unidentified effects on these phenotypes. Thus, the C-terminal portions of Runx family members have both similar and distinct biologic functions.

Novel loss-of-function mutations of the haematopoiesis-related transcription factor, acute myeloid leukaemia 1/runt-related transcription factor 1, detected in acute myeloblastic leukaemia and myelodysplastic syndrome.

AML1/RUNX1, which encodes a transcription factor essential for definitive haematopoiesis, is a frequent target of leukaemia-associated chromosome translocations. Point mutations of this gene have also recently been associated with leukaemia and myelodysplastic syndrome (MDS). To further define the frequency and biological characteristics of AML1 mutations, we have examined 170 cases of such diseases. Mutations within the runt-domain were identified in five cases: one of de novo acute myeloid leukaemia (AML) and four of MDS. Where multiple time point samples were available, mutations were detected in the earliest samples, which persisted throughout the disease course. Of the five mutations, one was a silent mutation, two were apparent loss-of-function mutations caused by N-terminal truncation, and two were insertions, I150ins and K168ins, which preserved most of the AML1 DNA-binding domain. Both AML1 molecules with insertion mutations were non-functional in that they were unable to rescue haematological defects in AML1-deficient mouse embryonic stem cells. In addition, activating mutations of N-ras, deletion of chromosome 12p, or inactivation of TP53 accompanied some of the AML1 mutations. Together, these observations strongly suggest that one-allele inactivation of AML1 serves as an initial or early event that plays an important role in the eventual development of overt diseases with additional genetic alterations.

VWRPY motif-dependent and -independent roles of AML1/Runx1 transcription factor in murine hematopoietic development.

AML1/Runx1 is a frequent target of leukemia-associated gene aberration, and it encodes a transcription factor essential for definitive hematopoiesis. We previously reported that the AML1 molecules with trans-activation subdomains retained can rescue in vitro hematopoietic defects of AML1-deficient mouse embryonic stem (ES) cells when expressed by using a knock-in approach. Extending this notion to in vivo conditions, we found that the knock-in ES cell clones with AML1 mutants, which retain trans-activation subdomains but lack C-terminal repression subdomains including the conserved VWRPY motif, contribute to hematopoietic tissues in chimera mice. We also found that germline mice homozygous for the mutated AML1 allele, which lacks the VWRPY motif, exhibit a minimal effect on hematopoietic development, as was observed in control knock-in mice with full-length AML1. On the other hand, reduced cell numbers and deviant CD4 expression were observed during early T-lymphoid ontogeny in the VWRPY-deficient mice, whereas the contribution to the thymus by the corresponding ES cell clones was inadequate. These findings demonstrate that AML1 with its trans-activating subdomains is essential and sufficient for hematopoietic development in the context of the entire mouse. In addition, its trans-repression activity, depending on the C-terminal VWRPY motif, plays a role in early thymocyte development.