Deepa B. Shankar

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 leukemiaoverexpress 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.

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.

CREB regulates Meis1 expression in normal and malignant hematopoietic cells

1 Borrow J, Stanton Jr VP, Andresen JM, Becher R, Behm FG, Chaganti RS et al. The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein. Nat Genet 1996; 14: 33–41. 2 Troke PJ, Kindle KB, Collins HM, Heery DM. MOZ fusion proteins in acute myeloid leukaemia. Biochem Soc Symp 2006; 73: 23–29. 3 Schmidt HH, Strehl S, Thaler D, Strunk D, Sill H, Linkesch W et al. RT-PCR and FISH analysis of acute myeloid leukemia with t(8;16)(p11;p13) and chimeric MOZ and CBP transcripts: breakpoint cluster region and clinical implications. Leukemia 2004; 18: 1115–1121. 4 Murati A, Adélaı̈de J, Mozziconacci MJ, Popovici C, Carbuccia N, Letessier A et al. Variant MYST4-CBP gene fusion in a t(10;16) acute myeloid leukaemia. Br J Haematol 2004; 125: 601–604. 5 Chaffanet M, Gressin L, Preudhomme C, Soenen-Cornu V, Birnbaum D, Pébusque MJ. MOZ is fused to p300 in an acute monocytic leukemia with t(8;22). Genes Chromosomes Cancer 2000; 28: 138–144. 6 Carapeti M, Aguiar RC, Goldman JM, Cross NC. A novel fusion between MOZ and the nuclear receptor coactivator TIF2 in acute myeloid leukemia. Blood 1998; 91: 3127–3133. 7 Murati A, Adélaı̈de J, Popovici C, Mozziconacci MJ, Arnoulet C, Lafage-Pochitaloff M et al. A further case of acute myelomonocytic leukemia with inv(8) chromosomal rearrangement and MOZNCOA2 gene fusion. Int J Mol Med 2003; 12: 423–428. 8 Xu J, Li Q. Review of the in vivo functions of the p160 steroid receptor coactivator family. Mol Endocrinol 2003; 17: 1681–1692.

Potential role of CREB as a prognostic marker in acute myeloid leukemia

The cAMP response element binding protein (CREB) is a leucine zipper transcription factor that regulates genes responsible for cell proliferation, differentiation and survival. CREB is overexpressed in the bone marrow from most patients with acute leukemia. Overexpression of CREB occurs both at the protein and at the transcript levels and is associated with gene amplification in leukemic blast cells. Higher levels of CREB correlate with a less favorable prognosis in a small cohort of adult patients with acute myeloid leukemia. In one study, patients whose bone marrow over-expresses CREB had an increased risk of relapse and decreased event-free survival. Mice that overexpress CREB in myeloid cells develop a myeloproliferative/myelodysplastic syndrome. These findings suggest that CREB plays an important role in the pathogenesis of acute leukemia and is a potential biomarker of disease.

A highly conserved regulatory element controls hematopoietic expression of GATA-2 in zebrafish

BackgroundGATA-2 is a transcription factor required for hematopoietic stem cell survival as well as for neuronal development in vertebrates. It has been shown that specific expression of GATA-2 in blood progenitor cells requires distal cis-acting regulatory elements. Identification and characterization of these elements should help elucidating transcription regulatory mechanisms of GATA-2 expression in hematopoietic lineage.ResultsBy pair-wise alignments of the zebrafish genomic sequences flanking GATA-2 to orthologous regions of fugu, mouse, rat and human genomes, we identified three highly conserved non-coding sequences in the genomic region flanking GATA-2, two upstream of GATA-2 and another downstream. Using both transposon and bacterial artificial chromosome mediated germline transgenic zebrafish analyses, one of the sequences was established as necessary and sufficient to direct hematopoietic GFP expression in a manner that recapitulates that of GATA-2. In addition, we demonstrated that this element has enhancer activity in mammalian myeloid leukemia cell lines, thus validating its functional conservation among vertebrate species. Further analysis of potential transcription factor binding sites suggested that integrity of the putative HOXA3 and LMO2 sites is required for regulating GATA-2/GFP hematopoietic expression.ConclusionRegulation of GATA-2 expression in hematopoietic cells is likely conserved among vertebrate animals. The integrated approach described here, drawing on embryological, transgenesis and computational methods, should be generally applicable to analyze tissue-specific gene regulation involving distal DNA cis-acting elements.

The role of p55CDC in cell cycle control and mammalian cell proliferation, differentiation, and apoptosis

The p55CDC (cell division cycle) protein is a key regulator of the cell cycle. p55CDC is related to both the CDC20 and the CDH1 proteins in yeast. p55CDC has been shown to activate the ubiquitin ligase anaphase promoting complex (APC), which is involved in degradation of proteins that control mitosis. To define the role of p55CDC during the mammalian cell cycle, we overexpressed this protein in the murine myeloid cell line 32Dcl3. 32Dcl3 cells are an ideal model system because these cells can be induced to proliferate, differentiate, or activate cellular programs leading to apoptosis. Our work suggests that p55CDC participates in cell growth, maturation, and death. Thus, p55CDC may play a more diverse role in modulating cellular functions in addition to controlling the cell cycle.

145 TARGETING BCL-2 IN ACUTE MYELOID LEUKEMIA CELLS.

Purpose of Study Proteins of the Bcl-2 family members are critical regulators of programmed cell death, and members that inhibit apoptosis, including Bcl-2 and Bcl-XL, are overexpressed in many cancers and contribute to tumor initiation, progression, and resistance to therapy. ABT-737 is a small molecule inhibitor of Bcl-2, Bcl-XL, and Bcl-w that was identified using nuclear magnetic resonance-based screen, parallel synthesis, and structure-based design. Previous studies revealed that this inhibitor does not initiate apoptosis but enhances the effects of death signals and acts synergistically with chemotherapy and radiation to induce cytotoxicity. ABT-737 as a single agent kills cells from lymphoma and small cell lung carcinoma lines in vitro and in vivo. In this study, we investigated the effects of ABT-737 on three different human acute myeloid leukemia (AML) cell lines. Methods Two human AML cell lines, KG-1 and MV-411, were treated with varying concentrations of ABT-737 ranging from 1 pM to 10 μM in complete media containing 10% fetal calf serum (FCS). Another human AML cell line, Molm-13, was treated with ABT-737 at concentrations ranging from 1 nM to 10 μM in media containing either 10% FCS or 1% FCS. Percent viability was determined using trypan blue exclusion at 24, 48, 72 hours following treatment with ABT-737. Results ABT-737 was found to inhibit KG-1 cell proliferation at an IC50 of 100 nM following treatment with ABT-737 for 96 hours. The IC50 of the MV-411 cell line was 10 nM after 24 hours of treatment. Molm-13 cells cultured in media containing 10% FCS showed an IC50 of 1 μM at 24 hours following treatment and 100 nM after 48 and 72 hours. Molm-13 cells grown in media containing 1% FCS demonstrated a decrease in percent viability with an IC50 of 100 nM after 24 hours of treatment, 10 nM after 48 hours, and 1 nM after 72 hours following treatment with ABT-737. Conclusions The AML cell lines KG-1, MV-411, and Molm-13 were susceptible to the pro-apoptotic effects of ABT-737 in vitro. Thus, further investigation into the effects of ABT-737 on additional AML cell lines in vitro and in vivo is warranted. We conclude that ABT-737 either alone or in combination with chemotherapy may provide an alternative mode of therapy for patients with AML.

156 ANALYSIS OF A TARGETED RECEPTOR TYROSINE KINASE INHIBITOR IN THE TREATMENT OF ACUTE MYELOGENOUS LEUKEMIA

Receptor Tyrosine kinases (RTK) play an important role in cell signaling, proliferation, and differentiation. Increased activation or levels of RTKs have been reported in approximately 90% of acute myelogenous leukemias (AML). Therefore, a drug designed to specifically target and inhibit RTKs is a rational approach to treat AML. We studied the effects of the RTKI, ABT869, on proliferation of AML cells and normal bone marrow in vitro. Methods Several myeloid leukemia cell lines were analyzed, including K562, NB-4, HL-60, KG-1, M-NFS-60, 32DCl.3, and TF-1. Cells were plated at a density of 2×10e5 cells/ml in growth media with ABT869 at concentrations between 100 μM to 1 pM. Cell viability was determined using Trypan Blue exclusion and MTT assays. Normal human bone marrow was plated in methylcellulose containing cytokines with or without ABT869. Results ABT869 rapidly induced cell death in the AML cell lines tested at concentrations above 10μM. At drug concentrations of 1, 10 and 100 nM, greater than 70% inhibition of cell growth was observed compared to the DMSO control treated cells within 48 hours after treatment. We performed methylcellulose assays to test for sensitivity of normal human bone marrow cells to ABT869. After 14 days, little difference in colony number or morphology was observed up to 10 nM concentration of ABT869. We are testing the effects of ABT869 on primary AML cells in colony forming assays and in NOD/SCID mice to further establish the compounds efficacy in vitro and in vivo. Conclusions Both growth factor-dependent cell lines (TF-1, 32D Cl.3, and NFS-60) and growth factor independent cell lines (KG-1, HL-60, and NB-4) were equally susceptible to the targeted receptor tyrosine kinase inhibitor, ABT869. In addition, physiologic concentrations of ABT869 did not affect the growth of normal human bone marrow progenitor cells in methylcellulose. Our results demonstrate that ABT869 may be a potentially effective drug in the treatment of patients with AML.

155 THE CYCLIC ADENOSINE MONOPHOSPHATE RESPONSE ELEMENT BINDING PROTEIN REGULATES HEMATOPOIETIC PROGENITOR CELL PROLIFERATION AND MYELOID ENGRAFTMENT

Purpose The cAMP response element binding protein (CREB) is a transcription factor that regulates gene expression in a variety of cell types and promotes cell proliferation and survival. We previously reported that more than 60% of AML patients overexpressed CREB in the bone marrow. To understand the role of CREB in myelopoiesis, we generated transgenic mice in which CREB is overexpressed in myeloid cells. We analyzed the hematopoietic progenitor cells from CREB transgenic mice in methylcellulose colony and bone marrow transplantation assays. Methods Bone marrow cells obtained from hMRP8-CREB transgenic mice were plated in methylcellulose containing IL-3, IL-6, and SCF. After 14 days the colonies were counted and analyzed using FACs and cytospin preparations. Bone marrow cells (4 ×106) from CREB transgenic mice were transplanted into lethally irradiated wild type C57/BL6 recipient mice. Peripheral blood counts were obtained every 4 weeks and FACs analysis was performed. Results CREB transgenic mice showed evidence of monocytosis, compared to age-matched littermate controls. Bone marrow cells from CREB transgenic mice formed robust colonies earlier and had increased numbers of colony forming units (CFU-GM). Bone marrow from CREB transgenic mice also had evidence of more immature myeloid cells compared to controls. We observed a 10-fold increase in the numbers of bone marrow progenitor cells from CREB transgenic mice (two different founder lines) compared to controls when cultured in the absence of cytokines. In bone marrow transplant experiments, mice transplanted with CREB transgenic mouse bone marrow had signs of earlier myeloid engraftment at 6 weeks following transplantation compared to controls. Recipients of CREB transgenic bone marrow showed increased monocytes and neutrophils in the peripheral blood with a corresponding increase in Mac-1+, Gr-1+ cell populations. The lymphocyte count was significantly lower in mice transplanted with CREB transgenic bone marrow compared to controls. Conclusions Our results suggest that CREB plays a critical role in the regulation of normal myelopoiesis and hematopoietic progenitor cell proliferation.