Combined Targeting of β-Catenin and FLT-3 Is Synergistically Lethal Against Cultured and Primary AML Blast Progenitor Cells Expressing FLT3 Internal Tandem Duplication (ITD)

Abstract β-catenin acts as a co-activator for the T-cell factor (TCF) 4/lymphoid enhancer factor (LEF) 1 bipartite transcription factor at the promoters of the WNT-β-catenin target genes, including cyclin D1, c-Myc and survivin. The canonical WNT-β-catenin pathway is documented to be essential for self-renewal, growth and survival of the AML stem and blast progenitor cells (BPCs), which has also been correlated with a poor prognosis in AML. In AML stem/BPCs expressing mutant FLT3-ITD, increased PI3K/AKT activity causes phosphorylation and inactivation of GSK3β, thereby preventing degradation, promoting stabilization and nuclear localization of β-catenin. Additionally, FLT3 can also directly mediate the tyrosine phosphorylation of β-catenin, thereby stabilizing and promoting the nuclear localization and binding of β-catenin to TCF4. TBL1 (transducin beta-like) is an adaptor protein, which binds to nuclear β-catenin and promotes its co-factor activity with TCF4/LEF1 in mediating transcription of the target genes, including c-Myc, cyclin D1 and survivin. Therefore, we hypothesized that targeted disruption of TBL1-β-catenin binding or depletion of TBL1 would abrogate the pro-growth and oncogenic signaling of β-catenin in AML BPCs, especially those expressing FLT3-ITD. Here, we demonstrate that treatment with 20 to 100 nM of BC2059 (β-Cat Pharmaceuticals), a small molecule, anthraquinone oxime-analog, disrupts the binding of β-catenin to TBL1 (by anti-TBL1 pull down and immunofluorescence analyses) and promotes proteasomal degradation of β-catenin, thereby attenuating the nuclear levels of β-catenin in the cultured (OCI-AML3, MOLM13 and MV4-11), as well as in primary (p) AML BPCs. Concomitantly, BC2059 treatment inhibited the mRNA and protein expression of c-Myc, cyclin D1 and survivin, while de-repressing p21 and Axin2. BC2059 also dose dependently inhibited growth and induced apoptosis of cultured and CD34+ pAML BPCs expressing FLT3-ITD (40 to 60%), but not of normal CD34+ bone marrow progenitor cells (p < 0.01). Transient knockdown of TBL1 or beta catenin (60 to 70%) by lentivirus-transduced shRNA caused loss of viability in MOLM13 cells, which was significantly enhanced by treatment with BC2059 (p < 0.01). BC2059 also induced apoptosis of MOLM13-TKIR cells that were isolated in vitro to exhibit resistance to FLT3 antagonists (approximately 50-fold). Notably, BC2059 treatment (10 mg/kg, t.i.w., by IV injection) also exerted potent in vivo anti-AML activity and significantly improved the survival of immune depleted mice engrafted with cultured and patient-derived pAML BPCs (p < 0.001). Since compared to the control OCI-AML3 cells, BC2059 demonstrated significantly greater lethality against the OCI-AML3 cells ectopically overexpressing FLT3-ITD (approximately 8-fold), we hypothesized that co-treatment with a FLT3 antagonist would further reduce the nuclear levels of β-catenin and enhance the lethal activity of FLT3-antagonist against AML BPCs expressing FLT3-ITD. Indeed, co-treatment with BC2059 (50 nM) and the FLT3-antagonist quizartinib or ponatinib (100 to 200 nM), versus each agent alone, caused more reduction in the nuclear levels and binding of β-catenin to TBL1 (by confocal immunofluorescence analysis). This was associated with greater decline in the expression of c-Myc, cyclin D1 and survivin, but increase in the levels of p21 and BIM. Compared to each agent alone, co-treatment with BC2059 and quizartinib or ponatinib also synergistically induced apoptosis of the FLT3-ITD expressing cultured (MOLM13 and MV4-11) and pAML BPCs (combination indices of < 1.0, by isobologram analyses) but not of normal CD34+ progenitor cells. Treatment with BC2059 (25 to 100 nM) also significantly increased the apoptosis observed by the shRNA mediated incomplete knockdown of TBL1 or β-catenin (approximately 70%) in MOLM13 cells (p < 0.01). Collectively, our findings support that targeted inhibition of the levels and binding of β-catenin to TBL by BC2059 and FLT3-antagonist is a promising approach to exert lethal activity against AML BPCs expressing FLT3-ITD. Further pre-clinical development of this combination therapy against FLT3-ITD expressing AML is progressing. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Anti-AML activity of a novel beta-catenin antagonist BC2059.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.10605 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 10605-10605

Author(s):

Kapil N. Bhalla ◽

Warren Fiskus ◽

Sunil Sharma ◽

Stephen Horrigan ◽

Uma Mudunuru ◽

...

Keyword(s):

Transcription Factor ◽

Cyclin D1 ◽

Progenitor Cells ◽

Survivin Expression ◽

Beta Catenin ◽

Tail Vein ◽

Nsg Mice ◽

Induced Apoptosis

10605 Background: The canonical WNT-β-catenin pathway is essential for self-renewal, growth and survivalof AML stem and progenitor cells. Deregulated WNT signaling inhibits degradation of β-catenin, causing increased nuclear translocation and interaction of β-catenin with the TCF/LEF transcription factor, which up regulates cyclin D1, Myc and survivin expression in AML progenitor cells. BC2059 (β-Cat Pharmaceuticals) is a potent, small molecule, anthraquinone oxime-analog, which inhibits WNT-β catenin pathway by promoting the degradation and attenuation of β-catenin levels. Methods: We determined the in vitro anti-AML activity of BC2059 (BC) (250 to 1000 nM) against cultured and primary human AML blast progenitors, as well as evaluated the in vivo anti-AML efficacy of BC in NOD-SCID and NOD-SCID-IL2γ receptor deficient (NSG) mice. Results: BC induced cell cycle G1 phase accumulation and apoptosis (40%) of the cultured OCI-AML3, HL-60 and HEL92.1.7 (HEL) AML cells. BC dose-dependently also induced apoptosis of primary AML versus normal progenitors. Treatment with BC resulted in proteasomal degradation and decline in the nuclear levels of β-catenin, which led to decreased activity of the LEF1/TCF4 transcription factor highlighted by reduced TOP-FLASH luciferase activity in the AML cells. This was associated with reduced protein levels of cyclin D1, MYC and survivin. Co-treatment with BC and the histone deacetylase inhibitor panobinostat (PS) (10 to 20 nM) synergistically induced apoptosis of cultured and primary AML blasts. Following tail vein infusion and establishment of AML by OCI-AML3 or HEL cells in NOD-SCID mice, treatment with BC (5, 10 or 15 mg/kg b.i.w, IV) for three weeks demonstrated improved survival, as compared to the control mice (p <0. 001). Survival was further improved upon co-treatment with BC and PS (5 mg/kg IP, MWF). BC treatment (5 or 10 mg/kg IV) also dramatically improved survival of NSG mice with established human AML following tail-vein injection of primary AML blasts expressing FLT3 ITD. Mice did not experience any toxicity or weight loss. Conclusions: These findings highlight the notable pre-clinical in vitro and in vivo activity and warrant further development and in vivo testing of BC against human AML.

Download Full-text

The Carboxy-Terminal Domain of Calreticulin (CALR) Exports the Glucocorticoid Receptor (GR) from the Nucleus to the Cytoplasm of Human Erythroid Cells Resetting Their Stress Response

Blood ◽

10.1182/blood.v128.22.545.545 ◽

2016 ◽

Vol 128 (22) ◽

pp. 545-545

Author(s):

Anna Rita Migliaccio ◽

Mario Falchi ◽

Lilian Varricchio ◽

Fabrizio Martelli ◽

Francesca Masiello ◽

...

Keyword(s):

Stress Response ◽

Confocal Microscopy ◽

Nuclear Localization ◽

Nuclear Export ◽

Active Sites ◽

Conflicts Of Interest ◽

Total Content ◽

Terminal Domain ◽

Nuclear Levels

Abstract Steady state erythropoiesis is regulated by interaction of EPO with its receptor, EPO-R which activates Ca2+signaling and terminal maturation [Miller, Blood 1989;73:1188]. Under stress, GR switches EPO-R signaling to a proliferation mode allowing erythroblast (Ery) amplification [Zhang, Nature 2013;499:92]. However, for recovery from anemia to occur, a mechanism, still to be identified, must counteract GR and switch EPO-R signaling back to a maturation mode. CALR chaperones other proteins to their active sites[Michalak, Biochem J 1999;344: 281]. By importing GR from the nucleus, CALR resets the stress response of murine fibroblasts. This function is regulated by the exposure of its C-terminal domain (C-CALR) sustained by Ca2+ [Holaska, Mol Cell Biol 2002;22:6286]. Whether CALR regulates nuclear import of GR in human Ery and how this regulation is modulated by Ca2+and EPO is unknown. Studies were performed to fill this gap. By WB, human Ery expanded in vitro from adult blood expressed robust levels of CALR, detectable with N-CALR- and C-CALR antibodies, and of GR. By flow cytometry, N-CALR and C-CALR were detectable on the surface of cells from the proerythroblast (proEry) to polychromatic stage. Expression of N-CALR, the epitope most exposed on the surface, was up-regulated by EPO. By confocal microscopy, both N-CALR and C-CALR were detected in non-permeabilized Ery but only C-CALR was detectable in cells permeabilized to reveal their cytoplasmic content. Intra-cytoplasmic C-CALR staining was robust in proEry, barely detectable in orthochromatic cells and enhanced by adding Ca2+ to the permeabilization buffer. Double staining of C-CALR with GR or LaminB1 (an inner nuclear membrane protein) indicated that C-CALR/GR were associated in the perinuclear area of the cytoplasm but that C-CALR remained distinct from Lamin B1, suggesting that C-CALR/GR co-localize at the cytoplasm/nuclear boundary. These results suggest that C-CALR mediates nuclear export of GR and that this function is regulated by Ca2+. This hypothesis was tested first by determining content and localization of GRα and C-CALR in nuclear and cytoplasmic fractions of Ery treated with Dex, EPO and SCF by WB. Although none of these stimulations altered the total content of these proteins, some of them affected their compartmentalization: Dex rapidly (15') induced GR S211 phosphorylation and increased the levels of GRα in nuclear fractions. These effects were antagonized by addition of the GR inhibitor RU486. The nuclear levels of GRα were modestly increased also by SCF but strongly decreased by EPO. By contrast, regardless of the stimuli, C-CALR was detected mostly in cytoplasmic fractions. The effects of these stimulations were also assessed by confocal microscopy. Exposure to Dex and EPO increased by 15' but decreased by 4h the nuclear localization of GR and significantly increased the levels of C-CALR and of C-CALR/GR co-localization detected in the cytoplasm by 4h, suggesting that C-CALR drives nuclear export of GR. In agreement with this hypothesis, multi-regression analyses comparing single C-CALR and total and cytoplasmic GR signals as independent parameters against merged signals as dependent parameter in 410 individual proEry indicated that the levels of C-CALR/GR co-localization directly correlated with the cytoplasmic levels of both C-CALR and GR but that the greatest p-values were observed for C-CALR (p=0.0000 vs 0.045). Moreover, treatment with the nuclear export inhibitor Leptomycin greatly decreased C-CALR/GR co-localization, increased nuclear localization of GR in proEry, both in untreated and EPO-treated cells, and reduced Ery proliferation in response to Dex down to levels sustained by Dex with RU486. To assess the role of Ca2+ in the nuclear export activity of C-CALR confocal microscopy and proliferation studies on Ery exposed or not to the Ca2+ chelator BAPTA in combination with Ionomycin (Io, a raiser of intracellular Ca2+) or Thapsigargin (TG, a reducer of Ca2+in endoplasmic reticulum) were performed. These treatments greatly decreased C-CALR and C-CALR/GR co-localization in the cytoplasm, increased GR signals in nuclei and reduced the proliferation of Ery in response to Dex. These results suggest that conformation changes of C-CALR induced by Ca2+signaling downstream to EPO-R sustain nuclear export of GR, resetting the stress-response of normal proEry allowing them to undergo terminal maturation. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Combined Therapy With BRD4 Antagonist and FLT3 Inhibitor Exerts Synergistic Activity Against Cultured and Primary AML Blast Progenitors Expressing FLT-ITD

Blood ◽

10.1182/blood.v122.21.3821.3821 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3821-3821

Author(s):

Melissa Rodriguez ◽

Warren Fiskus ◽

Sunil Sharma ◽

Jun Qi ◽

John A Valenta ◽

...

Keyword(s):

Progenitor Cells ◽

Conflicts Of Interest ◽

Activation Loop ◽

Synergistic Activity ◽

Gene Promoters ◽

Histone Proteins ◽

Cellular Models ◽

Induced Apoptosis

Abstract Mutations in FLT3 are detected in approximately 30% of AML and are associated with poor overall survival. Although first (PKC412, sorafenib and CEP701) and second generation (AC220) FLT3 tyrosine kinase inhibitors (TKI) induce remissions, resistance-causing gatekeeper (F691I/L), activation loop (AL) mutations (D835V/Y/F) or compound FLT3-ITD/F691I AL mutations are known to impair the in vitro and in vivo activity of the FLT3-TKIs. The BET (bromodomain and extraterminal) protein family members including BRD4 bind to acetylated lysines on the histone proteins, help assemble transcriptional regulators at the target gene promoters and enhancers, and regulate the expression of important oncogenes, e.g., MYC and BCL-2. BRD4 antagonists JQ1 and I-BET151 disrupt the binding of the bromodomain of BRD4 to acetylated lysines on histone proteins, thereby inhibiting expressions of c-MYC and BCL-2 and inducing apoptosis of AML cells. Based on this, we evaluated the in vitro and in vivo activity of JQ1 and FLT3 antagonists AC220 and ponatinib against cultured mouse lymphoid (Ba/F3/FLT3-ITD), as well as human cultured (MOLM13 and MV4-11) and primary AML blast progenitor cells (BPCs) expressing FLT3-ITD. JQ1, but not its inactive enantiomer R-JQ1, potently induced apoptosis of not only Ba/F3/FLT3-ITD but also of Ba/F3/FLT3-ITD expressing the highly FLT3 TKI-resistant mutations F691L and D835V (IC50 values for JQ1 were 697, 1588 and 909 nM, in the three cell lines, respectively). This was associated with attenuation of c-MYC, but the induction of BIM levels. Both JQ1 and I-BET151 dose-dependently induced apoptosis of MOLM13 and MV4-11 cells, as well as of primary AML BPCs expressing FLT3-ITD. Concomitantly, JQ1 treatment attenuated c-MYC, BCL2 and CDK6, while inducing p21, p27, BIM and cleaved PARP levels. JQ1 and I-BET151 did not induce apoptosis of CD34+ normal bone marrow progenitor cells. Following engraftment of NOD/SCID mice with MOLM13 cell xenograft, treatment with JQ1 (50 mg/kg, formulated in 10% 2-hydroxypropyl-β-cyclodextrin, daily x 5 days per week x 3 weeks), versus treatment with vehicle control, significantly improved survival of the mice (p< 0.05), without causing any toxicity. This was associated with the in vivo attenuation of c-MYC and BCL-2 levels in the harvested AML cells from the mice. Co-treatment with JQ1 or I-BET151 and FLT3 antagonist AC220 or ponatinib synergistically induced apoptosis of MOLM13 and MV4-11 cells. This was associated with greater reduction in the levels of MYC, BCL2 and CDK6, but more induction of BIM, p27 and cleaved PARP levels. Knockdown of BRD4 by treatment with specific shRNA phenocopied the effects of JQ1 and sensitized MOLM13 cells to ponatinib and AC220. As compared to each agent alone, treatment with JQ1 and ponatinib or AC220 also induced more apoptosis of primary AML BPCs expressing FLT3-ITD, associated again with greater reduction of the levels of MYC, BCL2 and CDK6, but more induction of BIM, p27 and cleaved PARP levels. We also determined the effects of JQ1 against MOLM13/TKIR cells that were generated under the continuous selection pressure of FLT3 TKI, and exhibited > 10 fold resistance to ponatinib but > 50 fold resistance to AC220. Importantly, as compared to the parental MOLM13, the MOLM13/TKIR cells were markedly more sensitive to JQ1-induced apoptosis (p< 0.001). Additionally, co-treatment with JQ1 and ponatinib but not AC220 synergistically induced apoptosis of MOLM13/TKIR cells. Supporting our previous findings (Blood. 2005;105:1768) that FLT3-ITD is a heat shock protein (hsp) 90 client-oncoprotein, the non-geldanamycin hsp90 inhibitor AUY922 was equally effective in inducing apoptosis of MOLM13 versus MOLM13/TKIR cells. Collectively, these findings demonstrate that BRD4 antagonist exhibits potent activity against cultured and primary AML cells expressing FLT-3-ITD, as well as against cellular models of FLT3 with gate-keeper and activation loop mutations. These findings also highlight the novel and synergistic activity of the combination of BRD4 antagonist and AC220 or ponatinib against AML BPCs expressing FLT3-ITD, and support the rationale for testing ponatinib and BRD4 antagonist against TKI-refractory AML expressing FLT3-ITD. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Expression Profiling of a Hemopoietic Cell Survival Transcriptome Identifies a Functional Prognostic Gene Signature in Normal Karyotype AML.

Blood ◽

10.1182/blood.v114.22.1618.1618 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1618-1618

Author(s):

Mark Guthridge ◽

Daniel Thomas ◽

Emma F Barry ◽

Kok H Chung ◽

Barbara J McClure ◽

...

Keyword(s):

Cell Survival ◽

Progenitor Cells ◽

Target Genes ◽

Kinase Inhibitors ◽

Conflicts Of Interest ◽

Therapeutic Potential ◽

Normal Karyotype ◽

Prognostic Indicator ◽

Hemopoietic Cell

Abstract Abstract 1618 Poster Board I-644 Deregulated cell survival programs are a classical hallmark of cancer. We have previously identified a serine residue (Ser585) in the cytoplasmic domain of the bc subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) receptors that selectively and independently promotes hemopoietic cell survival. We now show that Ser585 phosphorylation is constitutive in the majority of acute myeloid leukemia (AML) patient samples indicating that this hemopoietic “survival-only” pathway is frequently deregulated in leukemia. This constitutive Ser585 phosphorylation is refractory to tyrosine kinase inhibitors allowing the survival of a quiescent population of leukemic progenitor cells in vitro. We performed a global expression screen to identify gene targets of this survival-only pathway and report a unique bc Ser585-regulated transcriptome. Pathway analysis defines a gene network enriched for PI 3-kinase target genes as well as a cluster of genes involved in cancer and cell survival. From this panel of Ser585-regulated genes, we have validated by Q-RT-PCR a “six-gene Ser585-signature” that is deregulated in AML blasts exhibiting constitutive Ser585 phosphorylation. We show that siRNA-mediated knockdown of key components of this Ser585-signature induces apoptosis in CD34+CD38-CD123+ leukemic stem and progenitor cells. Furthermore, multivariate analysis indicates that the Ser585-signature is an independent prognostic indicator of overall patient survival in normal karyotype AML (n=56, HR=2.0, p=0.01). These results delineate a novel cytokine-regulated Ser585/PI3-kinase signalling network that is deregulated in AML and identify new prognostic targets with therapeutic potential. Disclosures No relevant conflicts of interest to declare.

Download Full-text

STAT5 and Oct-1 Form a Stable Complex That Modulates Cyclin D1 Expression

Molecular and Cellular Biology ◽

10.1128/mcb.23.24.8934-8945.2003 ◽

2003 ◽

Vol 23 (24) ◽

pp. 8934-8945 ◽

Cited By ~ 61

Author(s):

Sophie Magné ◽

Sandrine Caron ◽

Martine Charon ◽

Marie-Christine Rouyez ◽

Isabelle Dusanter-Fourt

Keyword(s):

Cyclin D1 ◽

Target Genes ◽

Promoter Sequence ◽

Stable Complex ◽

Homeodomain Protein ◽

Carboxy Terminal Region ◽

Carboxy Terminal ◽

Cell Cycle Regulator Cyclin

ABSTRACT Signal transducer and activator of transcription 5 (STAT5) is activated by numerous cytokines that control blood cell development. STAT5 was also shown to actively participate in leukemogenesis. Among the target genes involved in cell growth, STAT5 had been shown to activate cyclin D1 gene expression. We now show that thrombopoietin-dependent activation of the cyclin D1 promoter depends on the integrity of a new bipartite proximal element that specifically binds STAT5A and -B transcription factors. We demonstrate that the stable recruitment of STAT5 to this element in vitro requires the integrity of an adjacent octamer element that constitutively binds the ubiquitous POU homeodomain protein Oct-1. We observe that cytokine-activated STAT5 and Oct-1 form a unique complex with the cyclin D1 promoter sequence. We find that STAT5 interacts with Oct-1 in vivo, following activation by different cytokines in various cellular contexts. This interaction involves a small motif in the carboxy-terminal region of STAT5 which, remarkably, is similar to an Oct-1 POU-interacting motif present in two well-known partners of Oct-1, namely, OBF-1/Bob and SNAP190. Our data offer new insights into the transcriptional regulation of the key cell cycle regulator cyclin D1 and emphasize the active roles of both STAT5 and Oct-1 in this process.

Download Full-text

Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex

Development ◽

10.1242/dev.126.22.5137 ◽

1999 ◽

Vol 126 (22) ◽

pp. 5137-5148 ◽

Cited By ~ 8

Author(s):

H.D. Ryoo ◽

T. Marty ◽

F. Casares ◽

M. Affolter ◽

R.S. Mann

Keyword(s):

Nuclear Localization ◽

Target Genes ◽

Dominant Negative ◽

Homeodomain Protein ◽

Hox Proteins ◽

Trimeric Complex ◽

Binding Residue ◽

Dominant Negative Form

To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle (EXD). For EXD to bind DNA, it must be in the nucleus, and its nuclear localization requires a third homeodomain protein, Homothorax (HTH). Here we show that a conserved N-terminal domain of HTH directly binds to EXD in vitro, and is sufficient to induce the nuclear localization of EXD in vivo. However, mutating a key DNA binding residue in the HTH homeodomain abolishes many of its in vivo functions. HTH binds to DNA as part of a HTH/Hox/EXD trimeric complex, and we show that this complex is essential for the activation of a natural Hox target enhancer. Using a dominant negative form of HTH we provide evidence that similar complexes are important for several Hox- and exd-mediated functions in vivo. These data suggest that Hox proteins often function as part of a multiprotein complex, composed of HTH, Hox, and EXD proteins, bound to DNA.

Download Full-text

Physiological and Receptor-selective Retinoids Modulate Interferon γ Signaling by Increasing the Expression, Nuclear Localization, and Functional Activity of Interferon Regulatory Factor-1

Journal of Biological Chemistry ◽

10.1074/jbc.m505749200 ◽

2005 ◽

Vol 280 (43) ◽

pp. 36228-36236 ◽

Cited By ~ 20

Author(s):

Xin M. Luo ◽

A. Catharine Ross

Keyword(s):

Retinoic Acid ◽

Nuclear Localization ◽

Target Genes ◽

Interferon Γ ◽

Epithelial Cell Line ◽

Transcriptional Level ◽

Signal Transducer ◽

Regulatory Factor

Synergistic actions between all-trans-retinoic acid (atRA) and interferon γ (IFNγ) on modulation of cellular functions have been reported both in vitro and in vivo. However, the mechanism of atRA-mediated regulation of IFNγ signaling is poorly understood. In this study, we have used the human lung epithelial cell line A549 to examine the effect of atRA on IFNγ-induced expression of IFN regulatory factor-1 (IRF-1), an important transcription factor involved in cell growth and apoptosis, differentiation, and antiviral and antibacterial immune responses. At least 4 h of pretreatment with atRA followed by suboptimal concentrations of IFNγ induced a faster, higher, and more stable expression of IRF-1 than IFNγ alone. Actinomycin D completely blocked the induction of IRF-1 by the combination, suggesting regulation at the transcriptional level. Further, we found that activation of signal transducer and activator of transcription-1 was induced more dramatically by atRA and IFNγ than by IFNγ alone. Expression of IFNγ receptor-1 on the cell surface was also increased upon atRA pretreatment. Experiments using receptor-selective retinoids revealed that ligands for retinoic acid receptor-α (RARα), including atRA, 9-cis-retinoic acid, and Am580, sequentially increased the levels of IFNγ receptor-1, activated signal transducer and activator of transcription-1, and IRF-1 and that an RARα antagonist was able to inhibit the effects of atRA and Am580. In addition, atRA pretreatment affected the transcriptional functions of IFNγ-induced IRF-1, increasing its nuclear localization and DNA binding activity as well as the transcript levels of IRF-1 target genes. These results suggest that atRA, an RARα ligand, regulates IFNγ-induced IRF-1 by affecting multiple components of the IFNγ signaling pathway, from the plasma membrane to the nuclear transcription factors.

Download Full-text

The Histone Deacetylase Inhibitor LAQ824 Maintains Normal Hematopoietic Progenitor cells (HPC) Associated with Induction of Notch Target Genes and Does Not Eliminate Leukemic HPC in Vitro.

Blood ◽

10.1182/blood.v112.11.1352.1352 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1352-1352

Author(s):

Kerstin Schwarz ◽

Oliver Ottmann ◽

Annette Romanski ◽

Anja Vogel ◽

Jeffrey W. Scott ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Progenitor Cells ◽

Gene Expression Analysis ◽

Target Genes ◽

Notch Pathway ◽

Cycle Arrest ◽

Cd34 Cells ◽

20 Nm

Abstract Introduction: Histone deacetylase inhibitors (DACi) have shown promising antileukemic activity by overcoming the differentiation block and inducing apoptosis in AML blasts. Recent data demonstrating enhanced maintenance and functional capacity of normal, but also leukemic hematopoietic progenitor cells (HPC) by the selective class I DACi valproic acid (VPA) have raised concerns about VPA in AML therapy. As more potent pan-DACi have entered clinical trials, we analysed the impact of the hydroxamic acid LAQ824 on phenotype and function of normal and leukemic CD34+ HPC and studied LAQ824- induced gene expression in the most primitive CD34+CD38- population of normal HPC. Methods: Differentiation and proliferation of CD34+ cells of bone marrow of healthy donors and peripheral blood samples of newly diagnosed AML patients were evaluated after one week of culture in presence of SCF, FLT3 ligand, TPO, IL-3 +/− LAQ824. The effect of LAQ824 on gene expression profiles in normal CD34+CD38− cells was assessed in three independent cell samples following incubation with cytokines +/− LAQ824 for 48 hours using Affymetrix GeneChip Human Genome U133 Plus 2.0 and Gene Spring Software. Serial replating of murine Sca1+Lin- HPC was performed in the presence of SCF, G-CSF, GM-CSF, IL-3, IL-6 +/− LAQ824. Results: Treatment of murine Sca1+Lin- HPC with LAQ824 (10 nM) significantly augmented colony numbers (p<0.01; n=3), and supported colony growth after four cycles of replating whereas no colonies developed in its absence beyond the second plating indicating preservation of functionally active multipotent progenitor cells. LAQ824 (10–20 nM) mediated acetylation of histone H3 in human normal and leukemic HPC. In normal HPC, LAQ824 (0–20 nM) lead to a dose-dependent increase in the proportion of CD34+ cells (20% w/o LAQ824 vs. 36% with LAQ824 20nM, p=0.07) and a significant reduction of CD14+ monocytes (18% vs. 3%, p= 0.02; n=3). The total number of CD34+ cells remained stable up to 10 nM and decreased at 20 nM. Gene expression analysis showed, that LAQ824 (20 nM) lead to an at least 3-fold up-regulation of 221 genes in all three HPC samples tested including HDAC11 and the cell cycle inhibitor p21waf1/cip1 known to be induced by most DACi in HPC. We identified several members of the notch pathway such as mastermind-like protein 2 (MAML2, a component of the active notch transcriptional complex) and notch target genes including the transcription factors HES1, HEY1 and HOXA10 and confirmed increase of protein levels by Western blotting. Reduced gene expression of mini-chromosome-maintenance (MCM) protein family members was observed which - in addition to up-regulation of p21 - has previously been associated with notch-mediated cell cycle arrest. To compare the effect of LAQ824 (20 nM) with VPA (150 ng/ml) on leukemic HPC, cells were cultured for one week with or w/o DACi. Of note, LAQ824 resulted in a 0.8-fold reduction of CD34+ leukemic HPC, while VPA expanded this population 2.2-fold compared with cytokine-treated controls (p=0.03; n=12). CFU numbers growing from CD34+ leukemic HPC in presence of LAQ824 did not differ significantly from controls (n=9). Conclusion: LAQ824 seems to diminish, but not eliminate normal as well as leukemic HPC as determined by phenotypic and functional in vitro analyses. Our gene expression analysis suggested an association with coactivator and target genes of the notch pathway and cell cycle arrest-inducing genes. In contrast to VPA, LAQ824 does not seem to support growth of leukemic HPC which may contribute to its more potent antileukemic effect.

Download Full-text

Treatment in Vitro with a Combination of Bcl-Xl Inhibitor-ABT-737 and a JAK2 Inhibitor Selectively Eliminates JAK2V617F MPN Progenitor Cells.

Blood ◽

10.1182/blood.v114.22.752.752 ◽

2009 ◽

Vol 114 (22) ◽

pp. 752-752 ◽

Cited By ~ 1

Author(s):

Min Lu ◽

Wei Zhang ◽

Jiapeng Wang ◽

Yan Li ◽

Xiaoli Wang ◽

...

Keyword(s):

Progenitor Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Lower Percentage ◽

Normal Cells ◽

Jak2 Inhibitor ◽

Cd34 Cells ◽

Hel Cells ◽

Jak2 Inhibitors

Abstract Abstract 752 The Ph- myeloproliferative neoplasm (MPN) are associated with excessive production of red cells, platelets and granulocytes which largely determines their clinical manifestations. A mutation in the JAK2 tyrosine kinase (JAK2V617F) was identified in the majority of patients with MPNs. The JAK2V617F mutation has been shown to play a pivotal role in the pathogenesis of MPNs. We have reported that erlotinib (Tarceva), a kinase inhibitor which inhibits the epidermal growth factor induced kinase activity, is also a potent inhibitor of JAK2V617F activity. It has been shown that erythroblasts from patients with polycythemia vera (PV) express elevated levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL. In addition, we have recently documented that megakaryocytes derived from patients with primary myelofibrosis (PMF) undergo a delayed pattern of apoptosis in vitro which might be attributed to the over-expression of Bcl-xL. We hypothesize that a combination of a JAK2V617F inhibitor and a Bcl-xL inhibitor might be capable of selectively eliminating MPN cells while sparing normal cells, and therefore, providing an optimal treatment strategy for Ph− MPNs. We then evaluated the combinations of a JAK2V617 inhibitor (erlotinib or INCB018424) and a Bcl-xL inhibitor (ABT-737) for their ability to selectively eliminate MPN (JAK2V617F positive) cells while sparing normal cells in vitro using a variety of screening systems. We first tested the ability of each of the JAK2 inhibitors alone or in combination with ABT-737 to induce death of HEL cells, which harbors JAK2V617F. Among these treatments, ABT-737 (0.25 uM) plus either erlotinib (1.0 uM), or INCB018424 (1 nM) were shown to have similar capability of inducing HEL cell apoptosis (50-70%) which was significantly greater than that by each of the single agents (<5%). However, the combination of ABT-737 (0.25 uM) plus erlotinib (1.0 uM) was the least potent of inducing normal CD34+ cells to undergo apoptosis (∼5%) as compared to ABT-737 plus INCB018424 (∼20%). We next examined the effects of each of the JAK2 inhibitors alone or in combination with ABT-737 on CD34+ cells isolated from patients with PV or PMF. PV or PMF CD34+ cells were incubated in the absence or presence of agent(s) for 4 days; the percentage of apoptotic cells was then determined using Annexin V/PI staining by flow cytometry. A fraction of both untreated and treated CD34+ cells were assayed for hematopoietic colonies in the presence of SCF, IL-6, IL-3, G-CSF and EPO; individual colonies were then randomly plucked for JAK2V617F genotyping using nested allele-specific PCR. Similar to HEL cells, the percentage of MPD CD34+ cells undergoing apoptosis were similar when cells were treated with ABT-737 (0.25 uM) plus either erlotinib (1.0 uM), or INCB018424 (1.0 nM) (∼40%), but were significantly higher than that of MPN CD34+ cells treated with each of the agents alone (∼5%). Interestingly, when the percentages of JAK2V617F positive colonies within each of the treated and untreated cell populations were analyzed, MPN CD34+ cells treated with ABT-737 plus each of the JAK2 inhibitors contained a significantly lower percentage of JAK2V617F positive colonies than those untreated or treated with each of the single agents. These data indicate that the combination of ABT-737 plus a JAK2 inhibitor specifically targets on the JAK2V617F positive progenitor cells. Collectively, we identified an agent combination of ABT-737 (0.25 uM) plus a JAK2 inhibitor that is capable of selectively eliminating JAK2V617F positive MPN progenitor cells while sparing normal progenitor cells in vitro. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

HoxA10 Regulates Myeloid Progenitor Proliferation by Activating Transcription of the Gene Encoding Transforming Growth Factor Beta 2.

Blood ◽

10.1182/blood.v116.21.1572.1572 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1572-1572

Author(s):

Chirag Shah ◽

Hao Wang ◽

Elizabeth A. Eklund

Keyword(s):

Progenitor Cells ◽

Transforming Growth Factor Beta ◽

Target Genes ◽

Transforming Growth Factor ◽

Hematopoietic Stem ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells ◽

Growth Factor Beta ◽

Differentiation Block

Abstract Abstract 1572 HoxA10 is a homeodomain transcription factor which functions as a myeloid leukemia promoter. Correlative clinical studies found that increased expression of a group of HoxA proteins, including HoxA10, in acute myeloid leukemia (AML) was associated with poor prognosis. In murine models, overexpression of HoxA10 in the bone marrow was associated with development of a myeloproliferative disease which progressed to AML with time. These results suggested that HoxA10-overexpression dysregulated cell proliferation and/or survival, and predisposed to acquisition of additional mutations which led to differentiation block and AML. Additional investigations, we and others demonstrated that HoxA10 overexpression in murine hematopoietic stem cells (HSC) expanded the granulocyte/monocyte progenitor (GMP) population in vitro and in vivo. Despite this information about the impact of HoxA10 overexpression on myeloid leukemogenesis, the mechanisms by which HoxA10 exerts this effect are largely unknown. To investigate such mechanisms, we have been identifying HoxA10 target genes. In previous studies, we identified a number of HoxA10 target genes that encode phagocyte effector proteins. HoxA10 represses transcription of these gene in myeloid progenitors, and decreased HoxA10 repression activity contributes to phenotypic differentiation as myelopoiesis proceeds. This provided a potential mechanism for HoxA10 involvement in differentiation block, but not progenitor survival or expansion. We used a chromatin immuno-precipitation based approach to identify additional HoxA10 target genes involved in these activities. Previously, we reported that HoxA10 activated the DUSP4 gene in myeloid progenitor cells. This gene encodes Mitogen Activated Protein Kinase Phosphatase 2 (Mkp2) which inhibits Jnk-induced apoptosis in myeloid progenitor cells. This provided a mechanism for increased cell survival in HoxA10-overexpressing cells. In the current studies, we identified TGFB2 as a HoxA10 target gene. This gene encodes Transforming Growth Factor Beta 2 (TgfB2) a member of the TgfB super family of cytokines. Similar to TgfB1 and 3, TgfB2 interacts with TgfB-receptors I and II. However, unlike these more classical family members, TgfB2 induces proliferation of hematopoietic stem and progenitor cells. We found that HoxA10 activated the TGFB2 promoter via tandem cis elements in the proximal promoter. This resulted in autocrine stimulation of proliferation in HoxA10-overexpressing GMP and leukemia cells in vitro. Increased proliferation in HoxA10-overexpressing cells involved activation of the MAP kinase pathway in a TgfB2 dependent manner. These studies identify autocrine production of pro-proliferative cytokines as a novel mechanism for the function of Hox proteins. These findings have implications for ex vivo expansion of HSC and myeloid progenitors for tissue engineering. These result also have implications for therapeutic approaches to poor prognosis AML characterized by increased Hox expression. Disclosures: No relevant conflicts of interest to declare.

Download Full-text