Qiu-Hua Huang

Arsenic Trioxide Controls the Fate of the PML-RARα Oncoprotein by Directly Binding PML

Arsenic on the Fingers Arsenic, an ancient drug used in traditional Chinese medicine, has attracted wide interest because it has therapeutic activity in patients with acute promyelocytic leukemia (APL). The drug acts by promoting degradation of an oncogenic protein, PML-RARα, a fusion protein containing sequences from the PML zinc finger protein and retinoic acid receptor α, which is found specifically in APL cells and helps drive their growth. Zhang et al. (p. 240; see the Perspective by Kogan) now explain how arsenic initiates the molecular events leading to PML-RARα degradation. Arsenic was found to bind directly to cysteine residues within zinc finger domains of PML. Arsenic binding then induced oligomerization of PML, which in turn enhanced its association with an enzyme that helps catalyze SUMOylation, a posttranslational modification that can target proteins for degradation. Arsenic, a drug used clinically for leukemia, binds directly to an oncogenic protein, thereby promoting its degradation. Arsenic, an ancient drug used in traditional Chinese medicine, has attracted worldwide interest because it shows substantial anticancer activity in patients with acute promyelocytic leukemia (APL). Arsenic trioxide (As2O3) exerts its therapeutic effect by promoting degradation of an oncogenic protein that drives the growth of APL cells, PML-RARα (a fusion protein containing sequences from the PML zinc finger protein and retinoic acid receptor alpha). PML and PML-RARα degradation is triggered by their SUMOylation, but the mechanism by which As2O3 induces this posttranslational modification is unclear. Here we show that arsenic binds directly to cysteine residues in zinc fingers located within the RBCC domain of PML-RARα and PML. Arsenic binding induces PML oligomerization, which increases its interaction with the small ubiquitin-like protein modifier (SUMO)–conjugating enzyme UBC9, resulting in enhanced SUMOylation and degradation. The identification of PML as a direct target of As2O3 provides new insights into the drug’s mechanism of action and its specificity for APL.

Insight into hepatocellular carcinogenesis at transcriptome level by comparing gene expression profiles of hepatocellular carcinoma with those of corresponding noncancerous liver

Human hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. In this work, we report on a comprehensive characterization of gene expression profiles of hepatitis B virus-positive HCC through the generation of a large set of 5′-read expressed sequence tag (EST) clusters (11,065 in total) from HCC and noncancerous liver samples, which then were applied to a cDNA microarray system containing 12,393 genes/ESTs and to comparison with a public database. The commercial cDNA microarray, which contains 1,176 known genes related to oncogenesis, was used also for profiling gene expression. Integrated data from the above approaches identified 2,253 genes/ESTs as candidates with differential expression. A number of genes related to oncogenesis and hepatic function/differentiation were selected for further semiquantitative reverse transcriptase–PCR analysis in 29 paired HCC/noncancerous liver samples. Many genes involved in cell cycle regulation such as cyclins, cyclin-dependent kinases, and cell cycle negative regulators were deregulated in most patients with HCC. Aberrant expression of the Wnt-β-catenin pathway and enzymes for DNA replication also could contribute to the pathogenesis of HCC. The alteration of transcription levels was noted in a large number of genes implicated in metabolism, whereas a profile change of others might represent a status of dedifferentiation of the malignant hepatocytes, both considered as potential markers of diagnostic value. Notably, the altered transcriptome profiles in HCC could be correlated to a number of chromosome regions with amplification or loss of heterozygosity, providing one of the underlying causes of the transcription anomaly of HCC.

Gain-of-function mutation of GATA-2 in acute myeloid transformation of chronic myeloid leukemia

Acquisition of additional genetic and/or epigenetic abnormalities other than the BCR/ABL fusion gene is believed to cause disease progression in chronic myeloid leukemia (CML) from chronic phase to blast crisis (BC). To gain insights into the underlying mechanisms of progression to BC, we screened DNA samples from CML patients during blast transformation for mutations in a number of transcription factor genes that are critical for myeloid–lymphoid development. In 85 cases of CML blast transformation, we identified two new mutations in the coding region of GATA-2, a negative regulator of hematopoietic stem/progenitor cell differentiation. A L359V substitution within zinc finger domain (ZF) 2 of GATA-2 was found in eight cases with myelomonoblastic features, whereas an in-frame deletion of 6 aa (Δ341–346) spanning the C-terminal border of ZF1 was detected in one patient at myeloid BC with eosinophilia. Further studies indicated that L359V not only increased transactivation activity of GATA-2 but also enhanced its inhibitory effects on the activity of PU.1, a major regulator of myelopoiesis. Consistent with the myelomonoblastic features of CML transformation with the GATA-2 L359V mutant, transduction of the GATA-2 L359V mutant into HL-60 cells or BCR/ABL-harboring murine cells disturbed myelomonocytic differentiation/proliferation in vitro and in vivo, respectively. These data strongly suggest that GATA-2 mutations may play a role in acute myeloid transformation in a subset of CML patients.

A systems biology understanding of the synergistic effects of arsenic sulfide and Imatinib in BCR/ABL-associated leukemia

In this study, we show that combined use of Imatinib (IM) and arsenic sulfide [As4S4 (AS)] exerts more profound therapeutic effects in a BCR/ABL-positive mouse model of chronic myeloid leukemia (CML) than either drug as a single agent. A systematic analysis of dynamic changes of the proteome, phosphoproteome, and transcriptome in K562 cells after AS and/or IM treatment was performed to address the mechanisms underlying this synergy. Our data indicate that AS promotes the activities of the unfolded protein reaction (UPR) and ubiquitination pathway, which could form the biochemical basis for the pharmacological effects of this compound. In this CML model, AS targets BCR/ABL through the ubiquitination of key lysine residues, leading to its proteasomal degradation, whereas IM inhibits the PI3K/AKT/mTOR pathway. Combination of the 2 agents synergistically arrests the cell cycle, decreases activity of BCR/ABL, and leads to activation of intrinsic and extrinsic apoptosis pathways through complex modifications to both transcription and protein levels. Thus, these results suggest potential clinical benefits of IM/AS combination therapy for human CML.

Mir-144 selectively regulates embryonic α-hemoglobin synthesis during primitive erythropoiesis

Precise transcriptional control of developmental stage-specific expression and switching of alpha- and beta-globin genes is significantly important to understand the general principles controlling gene expression and the pathogenesis of thalassemia. Although transcription factors regulating beta-globin genes have been identified, little is known about the microRNAs and trans-acting mechanism controlling alpha-globin genes transcription. Here, we show that an erythroid lineage-specific microRNA gene, miR-144, expressed at specific developmental stages during zebrafish embryogenesis, negatively regulates the embryonic alpha-globin, but not embryonic beta-globin, gene expression, through physiologically targeting klfd, an erythroid-specific Krüppel-like transcription factor. Klfd selectively binds to the CACCC boxes in the promoters of both alpha-globin and miR-144 genes to activate their transcriptions, thus forming a negative feedback circuitry to fine-tune the expression of embryonic alpha-globin gene. The selective effect of the miR-144-Klfd pathway on globin gene regulation may thereby constitute a novel therapeutic target for improving the clinical outcome of patients with thalassemia.

Genome-wide survey and developmental expression mapping of zebrafish SET domain-containing genes.

SET domain-containing proteins represent an evolutionarily conserved family of epigenetic regulators, which are responsible for most histone lysine methylation. Since some of these genes have been revealed to be essential for embryonic development, we propose that the zebrafish, a vertebrate model organism possessing many advantages for developmental studies, can be utilized to study the biological functions of these genes and the related epigenetic mechanisms during early development. To this end, we have performed a genome-wide survey of zebrafish SET domain genes. 58 genes total have been identified. Although gene duplication events give rise to several lineage-specific paralogs, clear reciprocal orthologous relationship reveals high conservation between zebrafish and human SET domain genes. These data were further subject to an evolutionary analysis ranging from yeast to human, leading to the identification of putative clusters of orthologous groups (COGs) of this gene family. By means of whole-mount mRNA in situ hybridization strategy, we have also carried out a developmental expression mapping of these genes. A group of maternal SET domain genes, which are implicated in the programming of histone modification states in early development, have been identified and predicted to be responsible for all known sites of SET domain-mediated histone methylation. Furthermore, some genes show specific expression patterns in certain tissues at certain stages, suggesting the involvement of epigenetic mechanisms in the development of these systems. These results provide a global view of zebrafish SET domain histone methyltransferases in evolutionary and developmental dimensions and pave the way for using zebrafish to systematically study the roles of these genes during development.

Disruption of palladin results in neural tube closure defects in mice.

Palladin is a newly identified actin-associated protein which was proposed to be involved in actin cytoskeleton organization and nervous system development. Here, we show that inactivation of palladin leads to embryonic lethality due to severe defects of cranial neural tube closure and herniation of liver and intestine. It was found that palladin(-/-) embryos died around E15.5 and developed cranial neural tube closure defects (NTDs) with 100% penetrance. Whole mount in situ hybridization revealed that expression of palladin in early wild type embryos (E8.5) was specifically restricted in the elevating cranial neural folds where the neural tube closure is initiated. Palladin expression closely mirrors the phenotypic defects observed in palladin(-/-) mutants. While in E 9.5 and E10.5 embryos palladin was ubiquitously expressed. In vitro study revealed that formation of stress fibers in cytoplasm, cell adherent ability to extra-cellular matrix protein fibronectin and cell migration were dramatically disturbed in palladin(-/-) murine embryonic fibroblast cells (MEFs). Our findings suggest that palladin plays important roles in actin stress fiber formation, cell adhesion and migration. We propose that palladin is required for the initiation of neural tube closure and provides an important new candidate that may be implicated in the etiology of human NTDs.

Histone H3 lysine 36 methyltransferase Hypb/Setd2 is required for embryonic vascular remodeling

HYPB is a human histone H3 lysine 36 (H3K36)–specific methyltransferase and acts as the ortholog of yeast Set2. This study explored the physiological function of mammalian HYPB using knockout mice. Homozygous disruption of Hypb impaired H3K36 trimethylation but not mono- or dimethylation, and resulted in embryonic lethality at E10.5-E11.5. Severe vascular defects were observed in the Hypb −/− embryo, yolk sac, and placenta. The abnormally dilated capillaries in mutant embryos and yolk sacs could not be remodeled into large blood vessels or intricate networks, and the aberrantly rounded mesodermal cells exhibited weakened interaction with endothelial cells. The embryonic vessels failed to invade the labyrinthine layer of placenta, which impaired the embryonic–maternal vascular connection. These defects could not be rescued by wild-type tetraploid blastocysts, excluding the possibility that they were caused by the extraembryonic tissues. Consistent with these phenotypes, gene expression profiling in wild-type and Hypb −/− yolk sacs revealed that the Hypb disruption altered the expression of some genes involved in vascular remodeling. At the cellular level, Hypb −/− embryonic stem cell–derived embryonic bodies, as well as in vitro–cultured human endothelial cells with siRNA-mediated suppression of HYPB, showed obvious defects in cell migration and invasion during vessel formation, suggesting an intrinsic role of Hypb in vascular development. Taken together, these results indicate that Hypb is required for embryonic vascular remodeling and provide a tool to study the function of H3K36 methylation in vasculogenesis/angiogenesis.

As4S4 targets RING-type E3 ligase c-CBL to induce degradation of BCR-ABL in chronic myelogenous leukemia

Arsenic, a curative agent for acute promyelocytic leukemia, induces cell apoptosis and degradation of BCR-ABL in chronic myelogenous leukemia (CML). We demonstrated that ubiquitination and degradation of BCR-ABL was mediated by c-CBL, a RING-type E3 ligase that was also shown to be involved in ubiquitination for many other receptor/protein tyrosine kinases. Our data showed that c-CBL protein was considerably up-regulated by arsenic sulfide (As4S4). Interestingly, arsenic directly bound the RING finger domain of c-CBL to inhibit its self-ubiquitination/degradation without interfering with the enhancement of ubiquitination and subsequent proteolysis of its substrate BCR-ABL. Degradation of BCR-ABL due to c-CBL induction as a result of arsenic treatment was also observed in vivo in CML mice. These findings provide insight into the molecular mechanisms of arsenic and further support its therapeutic applications in CML in combination with tyrosine kinase inhibitors and potentially also in other malignancies involving aberrant receptor/protein tyrosine kinase signaling.

RIG-I plays a critical role in negatively regulating granulocytic proliferation

RIG-I has been implicated in innate immunity by sensing intracellular viral RNAs and inducing type I IFN production. However, we have found a significant RIG-I induction in a biological setting without active viral infection—namely, during RA-induced terminal granulocytic differentiation of acute myeloid leukemias. Here, we present evidence that a significant Rig-I induction also occurs during normal myelopoiesis and that the disruption of the Rig-I gene in mice leads to the development of a progressive myeloproliferative disorder. The initiation of progressive myeloproliferative disorder is mainly due to an intrinsic defect of Rig-I−/− myeloid cells, which are characterized by a reduced expression of IFN consensus sequence binding protein, a major regulator of myeloid differentiation. Thus, our study reveals a critical regulatory role of Rig-I in modulating the generation and differentiation of granulocytes.