技术资料

In human B-acute lymphoblastic leukemia (B-ALL),RAG1-induced genomic alterations are important for disease progression. However,given that biallelic loss of the RAG1 locus is observed in a subset of cases,RAG1's role in the development of B-ALL remains unclear. We chose a p19Arf(-/-)Rag1(-/-) mouse model to confirm the previously published results concerning the contribution of CDKN2A (p19ARF /INK4a) and RAG1 copy number alterations in precursor B cells to the initiation and/or progression to B-acute lymphoblastic leukemia (B-ALL). In this murine model,we identified a new,Rag1-independent leukemia-initiating mechanism originating from a Sca1(+)CD19(+) precursor cell population and showed that Notch1 expression accelerates the cells' self-renewal capacity in vitro. In human RAG1-deficient BM,a similar CD34(+)CD19(+) population expressed p19ARF. These findings suggest that combined loss of p19Arf and Rag1 results in B-cell precursor leukemia in mice and may contribute to the progression of precursor B-ALL in humans. View Publication

Complementary DNA clones encoding mouse cytokine synthesis inhibitory factor (CSIF; interleukin-10),which inhibits cytokine synthesis by TH1 helper T cells,were isolated and expressed. The predicted protein sequence shows extensive homology with an uncharacterized open reading frame,BCRFI,in the Epstein-Barr virus genome,suggesting the possibility that this herpes virus exploits the biological activity of a captured cytokine gene to enhance its survival in the host. View Publication

Estrogens regulate osteoblast differentiation and mineralization. We identified GATA4 as a transcription factor expressed in osteoblasts and directly regulated by 17β-estradiol in this cell type but not in breast cancer cells,another estrogen-responsive tissue. Chromatin immunoprecipitation sequencing (chromatin immunoprecipitation sequencing) reveals that estrogen receptor α (ERα) binds to chromatin near GATA4 at five different enhancers. GATA4 and ERα are both recruited to ERα binding sites near genes that are specifically expressed in osteoblasts and control osteoblast differentiation. Maximal binding of GATA4 precedes ERα binding,and GATA4 is necessary for histone 3 lysine 4 dimethylation at ERα binding sites,suggesting that GATA4 is a pioneer factor for ERα. As such,knockdown of GATA4 reduced recruitment of ERα to DNA. Our study illustrates that GATA4 is a pioneer factor for ERα recruitment to osteoblast-specific enhancers. View Publication

Xenotransplantation of acute myeloid leukemia (AML) into immunodeficient mice has been critical for understanding leukemogenesis in vivo and defining self-renewing leukemia-initiating cell subfractions (LICs). Although AML-engraftment capacity is considered an inherent property of LICs,substrains of NOD/SCID mice that possess additional deletions such as the IL2Rγc(null) (NSG) have been described as a more sensitive recipient to assay human LIC function. Using 23 AML-patient samples,39% demonstrated no detectable engraftment in NOD/SCID and were categorized as AMLs devoid of LICs. However,33% of AML patients lacking AML-LICs were capable of engrafting NSG recipients,but produced a monoclonal T-cell proliferative disorder similar to T-ALL. These grafts demonstrated self-renewal capacity as measured by in vivo serial passage and were restricted to CD34-positive fraction,and were defined as LICs. Molecular analysis for translocations in MLL genes indicated that these AML patient-derived LICs all expressed the MLL-AFX1 fusion product. Our results reveal that the in vivo human versus xenograft host microenvironment dictates the developmental capacity of human LICs residing in a small subset of patients diagnosed with AML harboring MLL mutations. These findings have implications both for the basic biology of CSC function,and for the use of in vivo models of the leukemogenic process in preclinical or diagnostic studies. View Publication

The MAPK pathway is one of the most important pathways for novel anticancer drug development. We performed high-throughput screening for compounds that induce expression of p15INK4b,and identified JTP-74057 (GSK1120212),which is being evaluated in ongoing phase I,II and III clinical trials. We characterized its antitumor activities in vitro and in vivo. JTP-74057 strongly inhibited MEK1/2 kinase activities,but did not inhibit another 98 kinase activities. Treatment by JTP-74057 resulted in growth inhibition accompanied with upregulation of p15INK4b and/or p27KIP1 in most of the colorectal cancer cell lines tested. Daily oral administration of JTP-74057 for 14 days suppressed tumor growth of HT-29 and COLO205 xenografts in nude mice. Notably,tumor regression was observed only in COLO205 xenografts,and COLO205 was much more sensitive to JTP-74057-induced apoptosis than HT-29 in vitro. Treatment with an Akt inhibitor enhanced the JTP-74057-induced apoptosis in HT-29 cells. Finally,JTP-74057 exhibited an additive or a synergistic effect in combination with the standard-of-care agents,5-fluorouracil,oxaliplatin or SN-38. JTP-74057,a highly specific and potent MEK1/2 inhibitor,exerts favorable antitumor activities in vitro and in vivo. Sensitivity to JTP-74057-induced apoptosis may be an important factor for the estimation of in vivo efficacy,and sensitivity was enhanced by an Akt inhibitor. These results suggest the usefulness of JTP-74057 in therapeutic applications for colorectal cancer patients. View Publication

Bone morphogenetic protein (BMP) signaling regulates embryonic hematopoiesis via receptor-mediated activation of downstream SMAD proteins,including SMAD1. In previous work,we showed that Smad1 expression is sufficient to enhance commitment of mesoderm to hemangioblast fate. We also found indirect evidence to support a subsequent repressive function for Smad1 in hematopoiesis. To test this hypothesis directly,we developed a novel system allowing temporal control of Smad1 levels by conditional knockdown in embryonic stem cell derivatives. Depletion of Smad1 in embryoid body cultures before hemangioblast commitment limits hematopoietic potential because of a block in mesoderm development. Conversely,when Smad1 is depleted in FlK1(+) mesoderm,at a stage after hemangioblast commitment,the pool of hematopoietic progenitors is expanded. This involves enhanced expression levels for genes specific to hematopoiesis,including Gata1,Runx1 and Eklf,rather than factors required for earlier specification of the hemangioblast. The phenotype correlates with increased nuclear SMAD2 activity,indicating molecular cross-regulation between the BMP and TGF-β signaling pathways. Consistent with this mechanism,hematopoiesis was enhanced when Smad2 was directly expressed during this same developmental window. Therefore,this study reveals a temporally defined function for Smad1 in restricting the expansion of early hematopoietic progenitors. View Publication

Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) kinase (CaMKK) is a member of the CaMK cascade that mediates the response to intracellular Ca(2+) elevation. CaMKK phosphorylates and activates CaMKI and CaMKIV,which directly activate transcription factors. In this study,we determined the 2.4 Å crystal structure of the catalytic kinase domain of the human CaMKKβ isoform complexed with its selective inhibitor,STO-609. The structure revealed that CaMKKβ lacks the αD helix and that the equivalent region displays a hydrophobic molecular surface,which may reflect its unique substrate recognition and autoinhibition. Although CaMKKβ lacks the activation loop phosphorylation site,the activation loop is folded in an active-state conformation,which is stabilized by a number of interactions between amino acid residues conserved among the CaMKK isoforms. An in vitro analysis of the kinase activity confirmed the intrinsic activity of the CaMKKβ kinase domain. Structure and sequence analyses of the STO-609-binding site revealed amino acid replacements that may affect the inhibitor binding. Indeed,mutagenesis demonstrated that the CaMKKβ residue Pro(274),which replaces the conserved acidic residue of other protein kinases,is an important determinant for the selective inhibition by STO-609. Therefore,the present structure provides a molecular basis for clarifying the known biochemical properties of CaMKKβ and for designing novel inhibitors targeting CaMKKβ and the related protein kinases. View Publication

PURPOSE: The purpose of this study was to determine whether histone deacetylase (HDAC) inhibitors (HDACI) such as vorinostat or entinostat (SNDX-275) could increase the lethality of the dual Bcr/Abl-Aurora kinase inhibitor KW-2449 in various Bcr/Abl(+) human leukemia cells,including those resistant to imatinib mesylate (IM). EXPERIMENTAL DESIGN: Bcr/Abl(+) chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) cells,including those resistant to IM (T315I,E255K),were exposed to KW-2449 in the presence or absence of vorinostat or SNDX-275,after which apoptosis and effects on signaling pathways were examined. In vivo studies combining HDACIs and KW2449 were carried out by using a systemic IM-resistant ALL xenograft model. RESULTS: Coadministration of HDACIs synergistically increased KW-2449 lethality in vitro in multiple CML and Ph(+) ALL cell types including human IM resistant cells (e.g.,BV-173/E255K and Adult/T315I). Combined treatment resulted in inactivation of Bcr/Abl and downstream targets (e.g.,STAT5 and CRKL),as well as increased reactive oxygen species (ROS) generation and DNA damage (γH2A.X). The latter events and cell death were significantly attenuated by free radical scavengers (TBAP). Increased lethality was also observed in primary CD34(+) cells from patients with CML,but not in normal CD34(+) cells. Finally,minimally active vorinostat or SNDX275 doses markedly increased KW2449 antitumor effects and significantly prolonged the survival of murine xenografts bearing IM-resistant ALL cells (BV173/E255K). CONCLUSIONS: HDACIs increase KW-2449 lethality in Bcr/Abl(+) cells in association with inhibition of Bcr/Abl,generation of ROS,and induction of DNA damage. This strategy preferentially targets primary Bcr/Abl(+) hematopoietic cells and exhibits enhanced in vivo activity. Combining KW-2449 with HDACIs warrants attention in IM-resistant Bcr/Abl(+) leukemias. View Publication

α-Lipoic acid (LA) is a thiol with antioxidant properties that protects against oxidative stress-induced apoptosis. LA is absorbed from the diet,taken up by cells and tissues,and subsequently reduced to dihydrolipoic acid (DHLA). Recently,DHLA has been used as the hydrophilic nanomaterial preparations,and therefore,determination of its bio-safety profile is essential. In this article,we show that DHLA (50-100 μM) induces apoptotic processes in mouse embryonic stem cells (ESC-B5),but exerts no injury effects at treatment dosages below 50 μM. Higher concentrations of DHLA (50-100 μM) directly increased the reactive oxygen species (ROS) content in ESC-B5 cells,along with a significant increase in cytoplasmic free calcium and nitric oxide (NO) levels,loss of mitochondrial membrane potential (MMP),activation of caspases-9 and -3,and cell death. Pretreatment with NO scavengers suppressed the apoptotic biochemical changes induced by 100 μM DHLA and promoted the gene expression levels of p53 and p21 involved in apoptotic signaling. Our results collectively indicate that DHLA at concentrations of 50-100 μM triggers apoptosis of ESC-B5 cells,which involves both ROS and NO. Importantly,at doses of less than 50 μM (0-25 μM),DHLA does not exert hazardous effects on ESC-B5 cell properties,including viability,development and differentiation. These results provide important information in terms of dosage safety and biocompatibility of DHLA to facilitate its further use as a precursor for biomaterial preparation. View Publication

A number of human malignancies exhibit sustained stimulation,mutation,or gene amplification of the receptor tyrosine kinase human mesenchymal-epithelial transition factor (c-Met). ARQ 197 is a clinically advanced,selective,orally bioavailable,and well tolerated c-Met inhibitor,currently in Phase 3 clinical testing in non-small cell lung cancer patients. Herein,we describe the molecular and structural basis by which ARQ 197 selectively targets c-Met. Through our analysis we reveal a previously undisclosed,novel inhibitory mechanism that utilizes distinct regulatory elements of the c-Met kinase. The structure of ARQ 197 in complex with the c-Met kinase domain shows that the inhibitor binds a conformation that is distinct from published kinase structures. ARQ 197 inhibits c-Met autophosphorylation and is highly selective for the inactive or unphosphorylated form of c-Met. Through our analysis of the interplay between the regulatory and catalytic residues of c-Met,and by comparison between the autoinhibited canonical conformation of c-Met bound by ARQ 197 to previously described kinase domains of type III receptor tyrosine kinases,we believe this to be the basis of a powerful new in silico approach for the design of similar inhibitors for other protein kinases of therapeutic interest. View Publication