Diamond Blackfan anemia is mediated by hyperactive Nemo-like kinase.

Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome associated with ribosomal gene mutations that lead to ribosomal insufficiency. DBA is characterized by anemia, congenital anomalies, and cancer predisposition. Treatment for DBA is associated with significant morbidity. Here, we report the identification of Nemo-like kinase (NLK) as a potential target for DBA therapy. To identify new DBA targets, we screen for small molecules that increase erythroid expansion in mouse models of DBA. This screen identified a compound that inhibits NLK. Chemical and genetic inhibition of NLK increases erythroid expansion in mouse and human progenitors, including bone marrow cells from DBA patients. In DBA models and patient samples, aberrant NLK activation is initiated at the Megakaryocyte/Erythroid Progenitor (MEP) stage of differentiation and is not observed in non-erythroid hematopoietic lineages or healthy erythroblasts. We propose that NLK mediates aberrant erythropoiesis in DBA and is a potential target for therapy.

Granulocyte-macrophage colony-stimulating factor and interleukin-3 signaling pathways converge on the CREB-binding site in the human egr-1 promoter.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates myeloid progenitor cell proliferation and enhances the function of terminally differentiated effector cells. Interleukin-3 (IL-3) stimulation results in the proliferation and maturation of early bone marrow progenitor cells. These activities are mediated by non-tyrosine kinase-containing receptors which consist of ligand-specific alpha subunits that complex with a common beta subunit required for signal transduction. Both GM-CSF and IL-3 rapidly and transiently induce expression of early growth response gene 1 (egr-1) in the human factor-dependent cell line TF-1. To define the mechanism of early response gene induction by GM-CSF and IL-3, growth factor- and serum-starved TF-1 cells transfected with recombinant constructs containing sequences of the human egr-1 promoter were stimulated with GM-CSF or IL-3. A 116-nucleotide (nt) region of the egr-1 promoter which contains sequences inducible by GM-CSF and IL-3 was defined. DNase I footprint analysis identified a 20-nt region, including nt -57 to -76, which contains a potential cyclic AMP (cAMP) response element (CRE). Electrophoretic mobility shift assays performed with CREB antibody confirmed the presence of CREB in the DNA-binding complex. Mutational analysis of the cytokine-responsive region of the egr-1 promoter revealed that both the cAMP response and serum response elements are required for induction by GM-CSF and IL-3. Nuclear extracts from GM-CSF- or IL-3-stimulated but not unstimulated TF-1 cells contain factors which specifically bind to the Egr-1-binding site in the nt -600 to -480 region of the promoter. Electrophoretic mobility shift assays were performed with antibodies against the Egr-1 protein to demonstrate the presence of the protein product in the shifted complex. Our studies suggest that the Egr-1 protein may further stimulate transcription of the egr-1 gene in response to GM-CSF as a secondary event.

The role of Fas-associated phosphatase 1 in leukemia stem cell persistence during tyrosine kinase inhibitor treatment of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by expression of Bcr-abl, a tyrosine kinase oncogene. Clinical outcomes in CML were revolutionized by development of Bcr-abl-targeted tyrosine kinase inhibitors (TKIs), but CML is not cured by these agents. CML leukemia stem cells (LSCs) are relatively TKI insensitive and persist even in remission. LSC persistence results in relapse upon TKI discontinuation, or drug resistance or blast crisis (BC) during prolonged treatment. We hypothesize that increased expression of Fas-associated phosphatase 1 (Fap1) in CML contributes to LSC persistence and BC. As Fap1 substrates include Fas and glycogen synthase kinase-3ß (Gsk3ß), increased Fap1 activity in CML is anticipated to induce Fas resistance and stabilization of ß-catenin protein. Resistance to Fas-induced apoptosis may contribute to CML LSC persistence, and ß-catenin activity increases during BC. In the current study, we directly tested the role of Fap1 in CML LSC persistence using in an in vivo murine model. In TKI-treated mice, we found that inhibiting Fap1, using a tripeptide or small molecule, prevented TKI resistance, BC and relapse after TKI discontinuation; all events observed with TKI alone. In addition, Fap1 inhibition increased Fas sensitivity and decreased ß-catenin activity in CD34(+) bone marrow cells from human subjects with CML. Therapeutic Fap1 inhibition may permit TKI discontinuation and delay in progression in CML.

Small molecule inhibition of cAMP response element binding protein in human acute myeloid leukemia cells.

The transcription factor CREB (cAMP Response-Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell-cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell-cycle and survival pathways, which may represent a novel approach for AML therapy.

HTLV-I and HTLV-II tax trans-activate the human EGR-1 promoter through different cis-acting sequences.

The immediate-early response gene, EGR-1, encodes a zinc finger-containing transcription factor that is involved in growth and differentiation of a variety of cell types. EGR-1 is induced in normal T cells following mitogenic stimulation and has recently been shown to be constitutively expressed in human T-cell leukemia virus type I (HTLV-I)- and type II (HTLV-II)-transformed T-cell lines. The trans-activating protein of HTLV-I, Tax, has been demonstrated to trans-activate promoters of a number of cellular genes, some of which may be critical in regulating T-cell proliferation. In this study, we examine the effect of Tax on expression of EGR-1 in three T-cell lines and demonstrate that both HTLV-I and -II Tax are capable of trans-activating human EGR-1 recombinant promoter constructs. Interestingly, HTLV-I and -II Tax trans-activate the human EGR-1 promoter through different promoter regions in the Jurkat cell line, suggesting that HTLV-I and -II Tax may lead to constitutive expression of EGR-1 through different signaling pathways. Deregulated expression of EGR-1 may contribute to uncontrolled cell growth and transformation during early stages of T-cell activation in HTLV-I and -II-infected cells.

5' upstream sequence and genomic structure of the human primary response gene, EGR-1/TIS8.

EGR-1/TIS8 is a primary response gene that encodes a zinc finger containing protein and is induced in a number of cell types by a variety of ligands. We have isolated and mapped the human EGR-1/TIS8 gene and sequenced the 5' upstream flanking region. A 'TATA' homology and several putative regulatory elements, including two Sp1 sites, five serum response-like elements, two cAMP response-like elements, an EGR-1 binding site (EBS), and a tetra-decanoyl phorbol acetate (TPA)-responsive element have been identified within 700 nucleotides of the upstream region. We demonstrated that a 500-base pair fragment, which includes several of these possible regulatory sequences, is functional and responsive to TPA in transient transfection assays. A further understanding of the regulation and function of human EGR-1/TIS8 gene expression may provide insight into the mechanisms that control normal and neoplastic proliferation of human cells.

Over-expression of p55Cdc inhibits granulocyte differentiation and accelerates apoptosis in myeloid cells.

p55Cdc is a protein identified in cycling mammalian cells. It is highly expressed in proliferating but not in differentiated or growth-arrested cells. Structurally, p55Cdc is similar to the Cdc4 and Cdc20 proteins, which have been proposed to regulate DNA synthesis and mitosis in Saccharomyces cerevisiae. To define the role of p55Cdc during myelopoiesis, we studied the expression and regulation of this protein in response to the hematopoietic growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte-colony stimulating factor (G-CSF). We analysed the time course of expression of p55Cdc in response to GM-CSF and G-CSF stimulation in the murine factor-dependent myeloid leukemic cell line, 32Dc13, and demonstrated differential regulation of p55Cdc in response to these two growth factors. Over-expression of p55Cdc resulted in acceleration of apoptosis in growth factor- and serum-free conditions, although no difference was observed in the rate of cell proliferation. Decreases in p55Cdc protein levels correlated with cells undergoing apoptosis. p55Cdc over-expression also inhibited granulocyte differentiation of 32Dc13 cells treated with G-CSF. Our studies suggest that p55Cdc regulation is critical for normal cell cycle control during myeloid cell proliferation and differentiation.

p55CDC/hCDC20 is associated with BUBR1 and may be a downstream target of the spindle checkpoint kinase.

Eukaryotic cells have evolved a mechanism that delays the progression of mitosis until condensed chromosomes are properly positioned on the mitotic spindle. We have been studying genes that regulated the spindle checkpoint in human cells. Enforced expression of human BUBR1, but not a BUBR1 mutant allele, enhances accumulation of mitotic cells. Yeast two-hybrid system and GST-pulldown analyses show that p55CDC/hCdc20, a protein known to link spindle checkpoint components such as MAD2 to anaphase promoting complex (APC), interacts with BUBR1. In addition, p55CDC is capable of pulling down BUBR1 in sf-9 cells infected with both p55CDC and His6-BUBR1 recombinant baculoviruses but not in the cells infected with p55CDC baculoviruses or with the baculoviral vector alone. Moreover, immunoprecipitation followed by Western blot analyses confirmed that native p55CDC is associated with BUBR1 in HeLa cells. Spindle checkpoint activation by nocodazole treatment enhances the association between p55CDC and His6-BUBR1. In nocodazole-arrested mitotic cells, both CDC16 and hyperphosphorylated CDC27, two APC components, preferentially associate with His6-BUBR1 resins, but not the control resins. Furthermore, BUBR1 phosphorylates p55CDC in vitro, and the phosphorylation of p55CDC by BUBR1 appears to be correlated with spindle checkpoint activation. Together, our studies strongly suggest that BUBR1 may target APC via p55CDC.

Transcription factors and translocations in lymphoid and myeloid leukemia.

Chromosomal translocations involving transcription factors and aberrant expression of transcription factors are frequently associated with leukemogenesis. Transcription factors are essential in maintaining the regulation of cell growth, development, and differentiation in the hematopoietic system. Alterations in the mechanisms that normally control these functions can lead to hematological malignancies. Further characterization of the molecular biology of leukemia will enhance our ability to develop disease-specific treatment strategies, and to develop effective methods of diagnosis and prognosis.

Analysis of cell death in myeloid cells inducibly expressing the cell cycle protein p55Cdc.

p55Cdc, a cell cycle protein is expressed in cycling mammalian cells and is required for normal cell division. Expression of this protein is regulated during the cell cycle, peaking in late G1 and S. We have previously shown that constitutive expression of p55Cdc results in inhibition of granulocyte differentiation. Degradation of p55Cdc is also required for apoptosis in growth factor and serum starved cells. In the present study we prepared stably transfected cells conditionally expressing p55Cdc in response to zinc stimulation to investigate the role of inducible p55Cdc expression in apoptosis of myeloid cells. We report that inducible expression of p55Cdc in the myeloid leukemic cell line 32Dc13 resulted in increased cell death. p55Cdc overexpression led to a statistically significant decrease in the viability of 32Dc13 cells compared with that of control cells. Furthermore, cell staining and flow cytometry analysis revealed that p55Cdc-overexpressing 32Dc13 cells progressed to apoptosis much earlier than uninduced cells. These results suggest that inducible expression of p55Cdc leads to earlier increases in cell death in the absence of growth factor and serum in myeloid leukemic cells.

Replication factor C3 is a CREB target gene that regulates cell cycle progression through the modulation of chromatin loading of PCNA.

CREB (cyclic AMP response element-binding protein) is a transcription factor overexpressed in normal and neoplastic myelopoiesis and regulates cell cycle progression, although its oncogenic mechanism has not been well characterized. Replication factor C3 (RFC3) is required for chromatin loading of proliferating cell nuclear antigen (PCNA) which is a sliding clamp platform for recruiting numerous proteins in the DNA metabolism. CREB1 expression, which was activated by E2F, was coupled with RFC3 expression during the G1/S progression in the KG-1 acute myeloid leukemia (AML) cell line. There was also a direct correlation between the expression of RFC3 and CREB1 in human AML cell lines as well as in the AML cells from the patients. CREB interacted directly with the CRE site in RFC3 promoter region. CREB-knockdown inhibited primarily G1/S cell cycle transition by decreasing the expression of RFC3 as well as PCNA loading onto the chromatin. Exogenous expression of RFC3 was sufficient to rescue the impaired G1/S progression and PCNA chromatin loading caused by CREB knockdown. These studies suggest that RFC3 may have a role in neoplastic myelopoiesis by promoting the G1/S progression and its expression is regulated by CREB.

Regulation of egr-1 gene expression by retinoic acid in a human growth factor-dependent cell line.

Retinoic acid (RA) has profound suppressive effects on growth and survival of human growth factor-dependent cell line, M07e. Treatment of M07e cells by RA reduced expression of egr-1 gene, while the levels of c-myc gene expression remained similar. Suppression of egr-1 gene expression by RA was dosage-dependent and reached maximum at 4 h after RA addition. The decay of egr-1 mRNA was similar in M07e cells treated with or without RA. The transcriptional activity of the promoter region up to -600 or -480 bp upstream of the egr-1 gene was greatly reduced by RA treatment. These data suggest that biological effects of RA on hematopoietic cells may, in part, be mediated by transcriptional suppression of egr-1 gene through its promoter region within -480 bp.

Combined radiation and cytochrome CYP4B1/4-ipomeanol gene therapy using the EGR1 promoter.

BACKGROUND: Cytochrome p450 isozyme CYP4B1 converts the inert prodrug 4-ipomeanol (4-IM) into toxic alkylating metabolites. Induction of cytotoxicity by 4-IM combined with ionizing radiation (IR) in cells transfected with a fusion protein of rabbit cytochrome CYP4B1 under control of the radiation inducible EGR1 promoter was investigated. The capability of activated 4-IM to sensitize cells to IR was also assessed. MATERIALS AND METHODS: Survival fractions of cells, determined by MTT assays, stably transfected with EGR1-CYP4B1 were compared with that of cells transfected with a control plasmid after IR followed by 4-IM. Radiosensitization was tested by comparing clonogenic survival curves of cells transfected with the CYP4B1 cassette under a CMV promoter instead of EGR-1, irradiated with or without 4-IM. RESULTS: MTT assays for cytotoxicity indicated a decrease in relative survival fractions (survival with 4-IM/survival without 4-IM) of the EGR1-CYP4B1 transfected cells with increasing radiation dosage, but not of control cells. Clonogenic assays revealed decreased survival fractions with increasing radiation doses (CYP4B1 transfected and control cells) and 4-IM concentrations (CYP4B1 transfected cells), but showed no significant differences in slope of survival curves with 4-IM. CONCLUSION: The results indicate IR potentiates the cytotoxic activity of the EGR1-CYP4B1/4-IM transgene system, but activated 4-IM does not sensitize cells to IR. Thus, the EGR1-CYP4B1/4-IM system is a viable radiation-gene therapy system that may allow for improved spatial and temporal control of cytotoxicity by therapeutic radiation fields.

Semaxanib (SUGEN).

SUGEN (owned by Pharmacia) is developing semaxanib (SU-5416), the lead in a series of small molecule inhibitors of the flk-1 tyrosine kinase receptor (flk-1 RTK), for the potential treatment of solid tumors (via suppression of metastasis and angiogenesis) [191353], [264484]. In July 1999, phase III trials for colorectal and lung cancer were initiated [326969]. In March 2001, phase III trials were initiated for the compound as an addition to a standard chemotherapy regimen in colorectal cancer [402241]; at this time, Pharmacia, as well as the NCI, was conducting clinical studies for numerous other solid and hematological cancers [402241]. By October 2000, oral forms of the compound were also being evaluated [385699]. In July 2000, Pharmacia anticipated US and international filing in 2001 [374505]. Taiho and SUGEN have agreed a joint development program for SUGEN's angiogenesis inhibitors [293021]. In August 1998, the USPTO issued US-05792783 to SUGEN, covering a family of compounds, including semaxanib. The patent claims cover the compounds and composition, as well as methods of use in a variety of diseases, including cancer [294467]. In August 1998, the USPTO issued US-05792783 to SUGEN, covering a family of compounds, including semaxanib. The patent claims cover the compounds and composition, as well as methods of use in a variety of diseases, including cancer [294467].

Onogenomics: dissecting cancer through genome research.

The Oncogenomics meeting focused on bioinformatics, molecular pathways and global gene expression profiles relating to cancer. Several sessions were devoted to updating the audience on the latest status of the human genome project. Future directions will focus on mining the genome for new information about the genetic code in humans. Proteomics is becoming a useful tool for helping to understand the structure and function of proteins and their partners, which will, in turn, enable us to more rationally use proteins as targets for therapy.

Targeting steroid hormone receptors for ubiquitination and degradation in breast and prostate cancer.

Proteolysis targeting chimeric molecules (Protacs) target proteins for destruction by exploiting the ubiquitin-dependent proteolytic system of eukaryotic cells. We designed two Protacs that contain the peptide 'degron' from hypoxia-inducible factor-1alpha, which binds to the Von-Hippel-Lindau (VHL) E3 ubiquitin ligase complex, linked to either dihydroxytestosterone that targets the androgen receptor (AR; Protac-A), or linked to estradiol (E2) that targets the estrogen receptor-alpha (ERalpha; Protac-B). We hypothesized that these Protacs would recruit hormone receptors to the VHL E3 ligase complex, resulting in the degradation of receptors, and decreased proliferation of hormone-dependent cell lines. Treatment of estrogen-dependent breast cancer cells with Protac-B induced the degradation of ERalpha in a proteasome-dependent manner. Protac-B inhibited the proliferation of ERalpha-dependent breast cancer cells by inducing G(1) arrest, inhibition of retinoblastoma phosphorylation and decreasing expression of cyclin D1, progesterone receptors A and B. Protac-B treatment did not affect the proliferation of estrogen-independent breast cancer cells that lacked ERalpha expression. Similarly, Protac-A treatment of androgen-dependent prostate cancer cells induced G(1) arrest but did not affect cells that do not express AR. Our results suggest that Protacs specifically inhibit the proliferation of hormone-dependent breast and prostate cancer cells through degradation of the ERalpha and AR, respectively.

Molecular targets and the treatment of myeloid leukemia.

Leukemia is a multistep process involving accumulation of genetic alterations over time. These genetic mutations destroy the delicate balance between cell proliferation, differentiation, and apoptosis. Traditional approaches to treatment of leukemia involve chemotherapy, radiation, and bone marrow transplantation. In recent years, specific targeted therapies have been developed for the treatment of leukemia. The success of treatment of acute promyelocytic leukemia with All Trans Retinoic Acid (ATRA) and CML with imatinib have lead to increased efforts to identify targets that can be inhibited by small molecules for treatment of hematological malignancies. In this review, we describe the current advances in the development of targeted therapy in acute myeloid leukemia.