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Biomedical subjects

Kathleen M Sakamoto

Publications and source records attributed to Kathleen M Sakamoto.

At least 19 recordsLinked to original sources
PubMed2007

ABT-869, a multitargeted receptor tyrosine kinase inhibitor: inhibition of FLT3 phosphorylation and signaling in acute myeloid leukemia.

In 15% to 30% of patients with acute myeloid leukemia (AML), aberrant proliferation is a consequence of a juxtamembrane mutation in the FLT3 gene (FMS-like tyrosine kinase 3-internal tandem duplication [FLT3-ITD]), causing constitutive kinase activity. ABT-869 (a multitargeted receptor tyrosine kinase inhibitor) inhibited the phosphorylation of FLT3, STAT5, and ERK, as well as Pim-1 expression in MV-4-11 and MOLM-13 cells (IC(50) approximately 1-10 nM) harboring the FLT3-ITD. ABT-869 inhibited the proliferation of these cells (IC(50) = 4 and 6 nM, respectively) through the induction of apoptosis (increased sub-G(0)/G(1) phase, caspase activation, and PARP cleavage), whereas cells harboring wild-type (wt)-FLT3 were less sensitive. In normal human blood spiked with AML cells, ABT-869 inhibited phosphorylation of FLT3 (IC(50) approximately 100 nM), STAT5, and ERK, and decreased Pim-1 expression. In methylcellulose-based colony-forming assays, ABT-869 had no significant effect up to 1000 nM on normal hematopoietic progenitor cells, whereas in AML patient samples harboring both FLT3-ITD and wt-FLT3, ABT-869 inhibited colony formation (IC(50) = 100 and 1000 nM, respectively). ABT-869 dose-dependently inhibited MV-4-11 and MOLM-13 flank tumor growth, prevented tumor formation, regressed established MV-4-11 xenografts, and increased survival by 20 weeks in an MV-4-11 engraftment model. In tumors, ABT-869 inhibited FLT3 phosphorylation, induced apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]) and decreased proliferation (Ki67). ABT-869 is under clinical development for AML.

Animals↗
PubMed2006

Calcium-dependent upregulation of E4BP4 expression correlates with glucocorticoid-evoked apoptosis of human leukemic CEM cells.

Glucocorticoid (GC)-evoked apoptosis of T-lymphoid cells is preceded by increases in the intracellular Ca2+ concentration ([Ca2+]i), which may contribute to apoptosis. This report demonstrates that GC-mediated upregulation of the bZIP transcriptional repressor gene, E4BP4, is dependent on [Ca2+]i levels, and correlates with GC-evoked apoptosis of GC-sensitive CEM-C7-14 cells. Calcium chelators EGTA and BAPTA reduced [Ca2+]i levels and protected CEM-C7-14 cells from Dex-evoked E4BP4 upregulation as well as apoptosis. In the GC-resistant sister clone, CEM-C1-15, Dex treatment did not induce [Ca2+]i levels, E4BP4 expression or apoptosis, however, the calcium ionophore A23187 restored Dex-evoked E4BP4 upregulation and apoptosis. CEM-C7-14 cells were more sensitive to GC-independent increases in [Ca2+]i levels by thapsigargin, and a corresponding increase in E4BP4 expression and cell death, compared to CEM-C1-15 cells, suggesting a direct correlation between [Ca2+]i levels, E4BP4 expression, and apoptosis.

Apoptosis↗
PubMed2006

FMS-like tyrosine kinase 3 in normal hematopoiesis and acute myeloid leukemia.

Ligand-mediated activation of the FMS-like tyrosine kinase 3 (FLT3) receptor is important for normal proliferation of primitive hematopoietic cells. However, activating mutations in FLT3 induce ligand-independent downstream signaling that promotes oncogenesis through pathways involved in proliferation, differentiation, and survival. FLT3 mutations are identified as the most frequent genetic abnormality in acute myeloid leukemia and are also observed in other leukemias. Multiple small-molecule inhibitors are under development to target aberrant FLT3 activity that confers a poor prognosis in patients.

Clinical Trials as Topic↗
PubMed2005

Role of cyclic AMP response element binding protein in human leukemias.

Acute myeloid leukemia (AML) in adults has a 20% 5-year disease-free survival despite treatment with aggressive cytotoxic chemotherapy. Previous work from our laboratory demonstrated that the majority of patients with acute lymphoid and myeloid leukemia overexpress CREB in the bone marrow. CREB overexpression is associated with poor initial outcome of clinical disease in AML patients. CREB is a transcription factor that functions in glucose homeostasis, growth-factor-dependent cell survival, and memory. Signaling by hematopoietic growth factors, such as GM-CSF, results in activation of CREB and up-regulation of CREB target genes. To study its role in hematopoiesis, we overexpressed CREB in leukemia cell lines and in mice. CREB overexpression resulted in increased survival and proliferation of myeloid cells and blast-transformation of bone marrow progenitor cells from transgenic mice expressing CREB in the myeloid lineage. CREB transgenic mice also develop myeloproliferative disease after 1 year. Thus, CREB acts as a protooncogene to regulate hematopoiesis and contributes to the leukemia phenotype. Our results suggest that CREB-dependent pathways may serve as targets for directed therapies in leukemia in the future.

Acute Disease↗
PubMed2005

The role of CREB as a proto-oncogene in hematopoiesis.

Cyclic-AMP response element binding protein (CREB) is a transcription factor that functions in glucose homeostasis, growth-factor- dependent cell survival, proliferation and memory. Signaling by hematopoietic growth factors, such as GM-CSF, results in activation of CREB and upregulation of CREB target genes. Data from our laboratory shows that a majority of patients with acute lymphoid and myeloid leukemia overexpress CREB in the bone marrow. CREB overexpression is associated with poor initial outcome of clinical disease in AML patients. To study its role in hematopoiesis, we overexpressed CREB in leukemia cell lines and in mice. CREB overexpression resulted in increased survival and proliferation of myeloid cells and blast-transformation of bone marrow progenitor cells from transgenic mice expressing CREB in the myeloid lineage. CREB transgenic mice also develop myeloproliferative disease after one year. Thus, CREB acts as a proto-oncogene to regulate hematopoiesis and contributes to the leukemia phenotype. Our results suggest that CREB-dependent pathways may serve as targets for directed therapies in leukemia in the future.

Animals↗
PubMed2005

The role of CREB as a proto-oncogene in hematopoiesis and in acute myeloid leukemia.

CREB is a transcription factor that functions in glucose homeostasis, growth factor-dependent cell survival, and memory. In this study, we describe a role of CREB in human cancer. CREB overexpression is associated with increased risk of relapse and decreased event-free survival. CREB levels are elevated in blast cells from patients with acute myeloid leukemia. To understand the role of CREB in leukemogenesis, we studied the biological consequences of CREB overexpression in primary human leukemia cells, leukemia cell lines, and transgenic mice. Our results demonstrate that CREB promotes abnormal proliferation and survival of myeloid cells in vitro and in vivo through upregulation of specific target genes. Thus, we report that CREB is implicated in myeloid cell transformation.

ATP-Binding Cassette Transporters↗
PubMed2005

Chimeric molecules to target proteins for ubiquitination and degradation.

Protein degradation is one of the tactics used by the cell for irreversibly inactivating proteins. In eukaryotes, ATP-dependent protein degradation in the cytoplasm and nucleus is carried out by the 26S proteasome. Most proteins are targeted to the 26S proteasome by covalent attachment of a multiubiquitin chain. A key component of the enzyme cascade that results in attachment of the multiubiquitin chain to the target or labile protein is the ubiquitin ligase that controls the specificity of the ubiquitination reaction. Defects in ubiquitin-dependent proteolysis have been shown to result in a variety of human diseases, including cancer, neurodegenerative diseases, and metabolic disorders. The SCF (Skp1-Cullin-F-box-Hrt1) complex is a heteromeric ubiquitin ligase that multiubiquitinates proteins important for signal transduction and cell cycle progression. A technology was developed known as Protac (Proteolysis Targeting Chimeric Molecule) that acts as a bridge, bringing together the SCF ubiquitin ligase with a protein target, resulting in its ubiquitination and degradation. The Protac contains an SCF-binding peptide moiety at one end that is recognized by SCF that is chemically linked to the binding partner or ligand of the target protein. The first demonstration of the efficacy of Protac technology was the successful recruitment, ubiquitination, and degradation of the protein methionine aminopeptidase-2 (MetAP-2) through a covalent interaction between MetAP-2 and Protac. Subsequently, we demonstrated that Protacs could effectively ubiquitinate and degrade cancer-promoting proteins (estrogen and androgen receptors) through noncovalent interactions in vitro and in cells. Finally, cell-permeable Protacs can also promote the degradation of proteins in cells. This chapter includes experiments to test the ability of Protacs to target proteins in vitro and in cells.

Animals↗
PubMed2004

G-CSF induces stabilization of ETS protein Fli-1 during myeloid cell development.

Granulocyte colony-stimulating factor (G-CSF) is a growth factor that regulates the production and function of neutrophils. G-CSF has been used to treat neutropenia in neonates, pediatric cancer patients, and patients undergoing stem cell transplantation. The regulation of transcription factors mediating G-CSF activity has not been well characterized. The goal of this study was to examine the regulation of the ETS binding protein, Friend leukemia integration site 1 (Fli-1), in myeloid cells treated with G-CSF. Fli-1 has oncogenic properties in humans and mice, and plays a role in vascular and hematopoietic cell development. We previously reported that Fli-1 and the serum response factor bind at adjacent sites within the serum response element-1 of the early growth response gene-1 promoter in the murine myeloid leukemic cell line, NFS60. We also identified that Fli-1 DNA binding increased in G-CSF-treated cells compared with untreated cells. To determine whether the change in binding activity is due to increased Fli-1 transcription or protein stability, we examined endogenous Fli-1 expression in G-CSF-treated or -untreated NFS60 cells. Our results demonstrated that levels of Fli-1 protein, but not RNA, were higher in extracts from cells treated with G-CSF. The increase in Fli-1 protein was also dependent on protein synthesis. Finally, we showed that the half-life of Fli-1 is prolonged in G-CSF-treated cells compared with control-treated cells. These results suggest that G-CSF induces stabilization of Fli-1 protein in myeloid cells, thus proposing a novel mechanism by which hematopoietic growth factors regulate transcription factors.

Animals↗
PubMed2004

The emerging role of RNA interference in the design of novel therapeutics in oncology.

RNA interference (RNAi) has emerged as the tool of choice for studying gene function. Dubbed the "breakthrough of the year" in 2002 by the journal Science, RNAi is a naturally occurring host defense mechanism that mediates the sequence-specific degradation of target mRNA transcripts and their protein products. The specificity of RNAi makes it an ideal tool for targeted therapeutics against unique fusion oncogene sequences. RNAi may also be effective against viral-mediated oncogenesis and has the potential to enhance tumor sensitivities to existing chemotherapy. The current interest in the success of RNAi-based therapies will depend on the delivery systems that protect the silencing apparatus from endogenous nucleases, sustain tissue-specific expression of the small-interfering RNAs, and prevent the activation of a destructive nonspecific host immune response.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2004

Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain.

To identify previously unknown small molecules that inhibit cell cycle machinery, we performed a chemical genetic screen in Xenopus extracts. One class of inhibitors, termed ubistatins, blocked cell cycle progression by inhibiting cyclin B proteolysis and inhibited degradation of ubiquitinated Sic1 by purified proteasomes. Ubistatins blocked the binding of ubiquitinated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of Lys(48)-linked chains. The same interface is recognized by ubiquitin-chain receptors of the proteasome, indicating that ubistatins act by disrupting a critical protein-protein interaction in the ubiquitin-proteasome system.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2004

The role of cyclic-AMP binding protein (CREB) in leukemia cell proliferation and acute leukemias.

Leukemia is a result of accumulating genetic alterations. The collaboration of mutations that offer survival and proliferative signals, together with mutations that result in lack of differentiation, is thought to cause a leukemic phenotype. The cyclic-AMP Response Element Binding Protein (CREB) is a transcription factor that is known to be a downstream component of the GM-CSF and IL-3 signaling pathways. We previously showed that CREB is overexpressed in blast cells from patients with acute leukemias. In this paper, we review the role of CREB in hematopoiesis, cell proliferation and acute leukemias.

Animals↗