2006 — 2010 |
Hsieh, James J-D |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Integrated Analyses of Taspase 1 (Mll Cleaving Protease)
[unreadable] DESCRIPTION (provided by applicant): Chromosome 11q23 translocations disrupting human Mixed Lineage Leukemia (MLL) gene are found in 80% of infantile leukemia and almost all cases of treatment induced secondary acute myeloid leukemia (AML). The MLL gene is required for proper HOX gene expression. Our prior studies demonstrated that full-length 500kD MLL protein undergoes proteolysis to generate N-terminal 320kD (MLLN320) and C-terminal 180kD (MLLC180) fragments. Processed MLL fragments form a complex to regulate the stability and availability of MLLN320 for downstream gene regulation. We subsequently purified and cloned the responsible protease and entitled it Taspase1 (Threonine Aspartase 1). The discovery of Taspase1 initiates a new class of proteases utilizing their N- terminal Threonine of mature ? subunit to cleave polypeptide substrates after P1 aspartate. Preliminary studies in HeLa cells indicated the importance of Taspase1-mediated MLL cleavage in HOX gene expression. Recently, we also identified a basal transcription factor, TFIIA, as a bona fide Taspase1 substrate. To investigate the physiological functions of Taspase1 in vivo, we generated Taspase1 knockout mice. Initial studies on Taspase1 deficient animals indicate the essential role of Taspase1 in body patterning, nervous system development, and cell cycle progression. With these unique reagents, we will further interrogate Taspase1 functions via the following specific aims: Specific Aim 1: We will characterize the role of Taspase1 in mouse embryonic development. It entails the creation of straight and conditional Taspase1 knockout mice to determine whether Taspase1 deficiency in mice results in embryonic lethality, homeotic transformations and/or other developmental abnormalities. We will dissect the mechanisms by which Taspase1 regulates Hox gene expression. Specific Aim 2: We will investigate the requirement of Taspase1 in normal cell cycle progression. We will start with studying cell cycle progression defects in Taspase1 deficient animals and cells, followed by dissecting the mechanisms by which Taspase1 regulates cell cycle progression and perform genetic reconstitutions of processed MLL family proteins into Taspase1 deficient cells to determine whether MLL proteolysis regulates cell proliferation. Specific Aim 3: We will perform studies to identify additional Taspase1 substrates and will validate their importance in vitro and in vivo. We will utilize 2-D difference in-gel electrophoresis in conjunction with mass spectrometry for the initial discovery, followed by phenotypic analyses of individual non-cleavable substrates. Since deregulation of HOX genes and cell cycle genes contribute to tumorigenesis, this combined genetic, biochemical, and proteomic approach to investigate Taspase1 functions will provide further insights regarding MLL leukemia and may lay the foundation for future development of Taspase1 inhibitors as anti-cancer therapeutics. [unreadable] [unreadable] [unreadable]
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1 |
2007 |
Hsieh, James J-D |
K01Activity Code Description: For support of a scientist, committed to research, in need of both advanced research training and additional experience. |
Genetic and Biochemical Analyses of Mixed Lineage Leukemia Cleavage
DESCRIPTION (provided by applicant): The mixed-lineage leukemia gene (MLL, ALL1, HRX) encodes a 3,969 amino acid nuclear protein homologous to Drosophila trithorax and is required to maintain proper Hox gene expression. Deregulation of Hox and perhaps other gene expression causes transformation of segmental identities and contributes to human malignancy. Chromosome translocations in human leukemia disrupt MLL (11q23), generating chimeric proteins between the N-terminus of MLL and multiple translocation partners. More than 20 MLL translocation partners have been identified. They vary widely from nuclear factors to cytoplasmic structural proteins and there are no common characteristics identified among them. However, mouse models demonstrated an indispensable role played by the various fusion partners in MLL leukemias. Gene expression profiles of human leukemia bearing an MLL translocation identified a pattern of upregulated genes. Among these genes were some of the well-recognized targets of wild type MLL. This argues that the common MLL N-terminus is sufficient to confer at least some target gene specificity to MLL-fusion proteins. However, the mechanism by which MLL regulates downstream gene expression is still unclear. In preliminary studies, I demonstrate that MLL is normally cleaved at two conserved sites (D/GADD and D/GVDD) and that mutation of these sites abolishes the proteolysis. The cleavage site sequences are highly conserved from flies to mammals. MLL cleavage generates N-terminal p320 (N320) and C-terminal p180 (C180) fragments, which then interact to form a stable complex that localizes to a subnuclear compartment. Disruption of the interaction between N320 and C180 leads to a marked decrease in the level of N320 and a redistribution of C180 to a diffuse nuclear pattern. Based on these data, I propose a model in which a dynamic post-cleavage association confers stability to N320 and directs correct nuclear sublocalization of the complex, thereby controlling the availability of N320 for target gene regulation. This model predicts that MLL-fusion proteins of leukemia lose the ability to complex with C180 and instead have their stability conferred by the fusion partners, thus providing one mechanism for the altered target gene expression observed in MLL leukemic cells. Further characterization of MLL cleavage will help elucidate how MLL regulates target genes, which is crucial for both development and leukemogenesis. In this regard, I propose the following specific aims: (1) Characterize the MLL cleavage and determine its role in protein stability and nuclear sublocalization; (2) Generate knock-in mice with a noncleavable MLL; (3) Identify and characterize the protease responsible for MLL cleavage.
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1 |
2011 — 2015 |
Hsieh, James J-D |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanisms of Impaired Dan Damage Response in Leukemia Pathogenesis @ Sloan-Kettering Inst Can Research
DESCRIPTION (provided by applicant): Cell cycle checkpoints are implemented to safeguard our genome and the deregulation of which contributes to the pathogenesis of human cancers. Hence, it is of paramount importance to discover and interrogate novel key constituents of the mammalian DNA damage response network. Among G1-, S-, G2- and M-phase checkpoints, genetic studies indicate the essence of an intact S-phase checkpoint in maintaining genome integrity. Although basic framework of the S-phase checkpoint in multi-cellular organisms has been outlined, the mechanistic details remain to be elucidated. Human chromosome band 11q23 translocation disrupting the MLL gene results in poor prognostic leukemias that carry pathognomonic MLL fusions. MLL is a transcription co-activator that is best known to maintain HOX gene expression. The importance of HOXA gene deregulation in MLL leukemogenesis has been intensively investigated. However, physiological murine MLL leukemia knockin models indicated that MLL fusion-induced HOXA gene aberration alone is insufficient to initiate MLL leukemia. Therefore, further dysregulation must exit and contribute to the ultimate leukemia phenotype. Our recent studies demonstrated a close relationship between MLL and the regulation of mammalian cell cycle. MLL not only assists in the G1/S and G2/M phase transition during a normal cell division cycle but also executes the S-phase checkpoint upon DNA damage. We found that (1) MLL functions as a key effector of ATR-mediated S-phase checkpoint response, (2) activated ATR phosphorylates and thus stabilizes MLL, (3) upon checkpoint activation MLL accumulates at the late replication origin, methylates histone H3K4, and thus delays DNA replication, (4) MLL deficient cells exhibit defects in the S-phase checkpoint response, and (5) MLL fusions work as dominant negative mutants that compromise the integrity of S-phase checkpoint. Here we will determine the mechanisms by which MLL executes the S phase checkpoint response and examine whether and to what extent an S-phase checkpoint dysfunction contributes to MLL leukemogenesis. Our proposal connects MLL/MLL fusions to the S-phase checkpoint response network, which not only provides novel insights into the mammalian cell cycle checkpoint control but also shed light on the pathogenesis of poor prognostic human leukemias. PUBLIC HEALTH RELEVANCE: Cancer is a public health issue that directly impacts millions of lives and costs billions of dollars in the United States each year. Through a better understanding of the molecular pathogenesis of cancer, targeted therapeutics may be developed and eventually benefit advanced- stage cancer patients (1-2). Mixed lineage leukemia, resulted from chromosome translocation of the MLL gene, portends poor prognosis (3-6). Consequently, novel treatment strategies for this dreadful illness are urgently needed. My laboratory over the years has helped elucidate novel regulations and functions of the MLL protein (7-14). Cell cycle checkpoints safeguard our genome and compromised checkpoints contribute to the evolution of human cancer (15-18). Hence, we are particularly excited about our recent studies that link MLL to the DNA damage response network (14). Based on these findings, we propose to further investigate how MLL regulates the S-phase checkpoint and determine whether checkpoint dysfunction contributes to the MLL leukemogenesis. Data obtained from this current grant expect to provide novel insights concerning the mammalian DNA damage response network and shed light on the molecular pathogenesis of MLL leukemias.
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0.909 |