scholarly journals Role of SHP2 phosphatase in KIT-induced transformation: identification of SHP2 as a druggable target in diseases involving oncogenic KIT

Blood ◽  
2012 ◽  
Vol 120 (13) ◽  
pp. 2669-2678 ◽  
Author(s):  
Raghuveer Singh Mali ◽  
Peilin Ma ◽  
Li-Fan Zeng ◽  
Holly Martin ◽  
Baskar Ramdas ◽  
...  
Keyword(s):  
Systemic Mastocytosis ◽  
Myelogenous Leukemia ◽  
Myeloproliferative Disease ◽  
Growth And Survival ◽  
Intracellular Mechanism ◽  
Acute Myelogenous ◽  
Lipid Kinases

Abstract Intracellular mechanism(s) that contribute to promiscuous signaling via oncogenic KIT in systemic mastocytosis and acute myelogenous leukemia are poorly understood. We show that SHP2 phosphatase is essential for oncogenic KIT-induced growth and survival in vitro and myeloproliferative disease (MPD) in vivo. Genetic disruption of SHP2 or treatment of oncogene-bearing cells with a novel SHP2 inhibitor alone or in combination with the PI3K inhibitor corrects MPD by disrupting a protein complex involving p85α, SHP2, and Gab2. Importantly, a single tyrosine at position 719 in oncogenic KIT is sufficient to develop MPD by recruiting p85α, SHP2, and Gab2 complex to oncogenic KIT. Our results demonstrate that SHP2 phosphatase is a druggable target that cooperates with lipid kinases in inducing MPD.

scholarly journals Topoisomerase II levels and drug sensitivity in adult acute myelogenous leukemia

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 517-530 ◽  
Author(s):  
SH Kaufmann ◽  
JE Karp ◽  
RJ Jones ◽  
CB Miller ◽  
E Schneider ◽  
...  
Keyword(s):  
Acute Myelogenous Leukemia ◽  
Topoisomerase Ii ◽  
Drug Sensitivity ◽  
Myelogenous Leukemia ◽  
Clinical Samples ◽  
Immunoperoxidase Staining ◽  
Acute Myelogenous ◽  
Topo Ii

Abstract The topoisomerase (topo) II-directed agents etoposide, daunorubicin (DNR), and amsacrine (m-AMSA) are widely used in the treatment of acute myelogenous leukemia (AML). In the present study, multiple aspects of topo II-mediated drug action were examined in marrows from adult AML patients. Colony-forming assays revealed that the dose of etoposide, DNR, or m-AMSA required to diminish leukemic colony formation by 90% (LD90) varied over a greater than 20-fold range between different pretreatment marrows. Measurement of nuclear DNR accumulation in the absence and presence of quinidine revealed evidence of P-glycoprotein (Pgp) function in 8 of 82 samples at diagnosis and 5 of 36 samples at first relapse, but the largest quinidine-induced increment in DNR accumulation (< 2-fold) was too small to explain the variations in drug sensitivity. Restriction enzyme-based assays and sequencing of partial topo II alpha and topo II beta cDNAs from the most highly resistant specimens failed to demonstrate topo II gene mutations that could account for resistance. Western blotting of marrow samples containing greater than 80% blasts revealed that the content of the two topo II isoenzymes varied over a greater than 20-fold range, but did not correlate with drug sensitivity in vitro or in vivo. In addition, levels of topo II alpha and topo II beta in 46 of 47 clinical samples were lower than in human AML cell lines. Immunoperoxidase staining showed that these low topo II levels were accompanied by marked cell-to- cell heterogeneity, with topo II alpha being abundant in some blasts and diminished or absent from others. There was a trend toward increasing percentages of topo II alpha-positive cells in pretreatment marrows that contained more S-phase cells. Consistent with this observation, treatment of patients with granulocyte-macrophage colony- stimulating factor for 3 days before chemotherapy resulted in increases in topo II alpha-positive cells concomitant with increases in the number of cells traversing the cell cycle. These observations have implications for the regulation of topo II in AML, for the use of topo II-directed chemotherapy, and for future attempts to relate drug sensitivity to topo II levels in clinical material.

FIP1L1/PDGFRa Synergizes with SCF/c-Kit Signaling To Induce Systemic Mastocytosis in a Chronic Eosinophilic Leukemia Murine Model

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1540-1540
Author(s):  
Yoshiyuki Yamada ◽  
Jose A. Cancelas ◽  
Eric B. Brandt ◽  
Abel Sanchez-Aguilera ◽  
Melissa McBride ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Small Intestine ◽  
Murine Model ◽  
Fusion Gene ◽  
Systemic Mastocytosis ◽  
Wild Type ◽  
Chronic Eosinophilic Leukemia

Abstract Systemic mastocytosis (SM) associated with chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome (HES) is a result of expression of the Fip1-like1 (FIP1L1)/platelet-derived growth factor receptor alpha (PDGFRa) (F/P) fusion gene. We have previously described a murine CEL/HES model (CEL-like mice) induced by F/P fusion gene transduction and T-cell overexpression of IL-5 (Yamada Y et al., Blood 2006). We have now validated a preclinical murine model of F/P-induced SM/CEL and analyzed the pathogenesis of SM in this model. F/P+ mast cells (MC, defined as EGFP+/c-kit+/FceRI+) were significantly increased in the small intestine, bone marrow (BM) and spleen of CEL-like mice compared to wild-type mice (Table). CEL-like mice also developed cutaneous MC infiltration. In addition, mMCP-1 serum levels, which correlate well with MC expansion and activation in vivo, were significantly higher in CEL-like mice than in wild-type mice (64,000 ± 23,800 and 38 ± 41.4 pg/ml, respectively). F/P induces increased expansion of BM-derived MC in vitro (∼2,000-fold) and F/P+ BM-derived MC survive longer than wild-type MC in cytokine-deprived medium (28.0 ± 2.3% vs. 8.7 ± 3.1% 7AAD−/Annexin V− cells after 48 hours). This correlated with increased Akt phosphorylation in the F/P+ MC. Since c-kit mutations are the most frequent cause of SM, we analyzed the possible synergistic role of SCF and F/P signaling. F/P and SCF/c-kit signaling indeed synergize in the development of BM-derived MC (16-fold greater expansion than in the absence of SCF) and F/P+ BM-derived MC showed a 3.7-fold greater migratory response to SCF than wild-type BM-derived MC. In order to determine the role of SCF/c-kit signaling in F/P+ MC development, activation and tissue infiltration in vivo,these responses were evaluated in mice that were treated with a blocking anti-c-kit blocking antibody, ACK-2, or an isotype-matched control antibody. ACK-2 treatment suppressed intestinal MC infiltration and elevated plasma levels of mMCP-1 induced by F/P expression by 95 ± 6.0% and 98 ± 0.76%, respectively, whereas MC and plasma mMCP-1 were completely undetectable in wild-type mice treated with ACK2. This suggests that SCF/c-kit interactions may synergize with F/P to induce SM. In summary, mice with CEL-like disease also develop SM. F/P-induced SM is a result of increased in vivo MC proliferation, survival, activation and tissue infiltration. SCF/c-kit signaling synergizes with F/P in vivo and in vitro to promote mast cell development, activation and survival. EGFP+/c-kit+/FcεRI+ cell frequency in tissues of control and CEL-like mice (%) Control mice CEL-like mice Small intestine 1.0±0.95 47±21.4* Bone marrow 0.2±0.14 3±1.9* Spleen 0.05±0.01 3±0.8*

In vitro and in vivo activity of ATP-based kinase inhibitors AP23464 and AP23848 against activation-loop mutants of Kit

Blood ◽  
2005 ◽  
Vol 106 (1) ◽  
pp. 227-234 ◽  
Author(s):  
Amie S. Corbin ◽  
Shadmehr Demehri ◽  
Ian J. Griswold ◽  
Yihan Wang ◽  
Chester A. Metcalf ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitors ◽  
Therapeutic Potential ◽  
Systemic Mastocytosis ◽  
Myelogenous Leukemia ◽  
Therapeutic Window ◽  
Hematopoietic Progenitor Cell ◽  
Activation Loop

Oncogenic mutations of the Kit receptor tyrosine kinase occur in several types of malignancy. Juxtamembrane domain mutations are common in gastrointestinal stromal tumors, whereas mutations in the kinase activation loop, most commonly D816V, are seen in systemic mastocytosis and acute myelogenous leukemia. Kit activation-loop mutants are insensitive to imatinib mesylate and have been largely resistant to targeted inhibition. We determined the sensitivities of both Kit mutant classes to the adenosine triphosphate (ATP)–based inhibitors AP23464 and AP23848. In cell lines expressing activation-loop mutants, low-nM concentrations of AP23464 inhibited phosphorylation of Kit and its downstream targets Akt and signal transducer and activator of transcription 3 (STAT3). This was associated with cell-cycle arrest and apoptosis. Wild-type Kit–and juxtamembrane-mutant–expressing cell lines required considerably higher concentrations for equivalent inhibition, suggesting a therapeutic window in which cells harboring D816V Kit could be eliminated without interfering with normal cellular function. Additionally, AP23464 did not disrupt normal hematopoietic progenitor-cell growth at concentrations that inhibited activation-loop mutants of Kit. In a murine model, AP23848 inhibited activation-loop mutant Kit phosphorylation and tumor growth. Thus, AP23464 and AP23848 potently and selectively target activation-loop mutants of Kit in vitro and in vivo and could have therapeutic potential against D816V-expressing malignancies.

Perspective of strategic plasma therapy for prognostic improvement of patients with ovarian cancer

2013 ◽  
Vol 1598 ◽  
Author(s):  
Hiroaki Kajiyama ◽  
Fumi Utsumi ◽  
Kae Nakamura ◽  
Hiromasa Tanaka ◽  
Masaru Hori ◽  
...  
Keyword(s):  
Atmospheric Pressure ◽  
Antitumor Effect ◽  
Intraperitoneal Administration ◽  
Atmospheric Pressure Plasma ◽  
Plasma Therapy ◽  
Intracellular Mechanism ◽  
Patients Experience

ABSTRACTEpithelial ovarian carcinoma (EOC) is the leading cause of cancer-related death in women in Western countries. Once patients experience recurrence, complete cure is almost impossible. We elucidated the effect of nonequilibrium atmospheric pressure plasma on the growth of EOC, particularly in plasma-activated medium (PAM). Furthermore, we examined the role of reactive oxygen species (ROS) or their scavengers in chronic antineoplastic-resistant EOC cells. As a result, we showed PAM induced the antitumor effect of EOC cells in vitro and in vivo, even in chemoresistant cells. To apply the plasma treatment for advanced or recurrent EOC, we suggest adopting indirect plasma therapy instead of direct plasma considering intraperitoneal administration in the future. However, there are several problems under investigation, including intracellular mechanism of antitumor effect by PAM and adverse event in vivo.

scholarly journals The role of phosphoinositide 3-kinase in spreading osteoclasts induced by colony-stimulating factor-1

2001 ◽  
pp. 431-440 ◽  
Author(s):  
S Palacio ◽  
R Felix
Keyword(s):  
Bone Marrow Cells ◽  
Colony Stimulating Factor ◽  
Lipid Kinase ◽  
Growth And Survival ◽  
Phosphoinositide 3 Kinase ◽  
Stimulating Factor ◽  
K Activity

BACKGROUND: Colony-stimulating factor-1 (CSF-1), a growth and survival factor for osteoclasts, stimulates these cells to spread and migrate towards a gradient of CSF-1. This may support the translocation of osteoclasts to new sites on the bone surface to be resorbed. Phosphoinositide 3-kinase (PI 3-K) is a lipid kinase participating in various signal transduction pathways. OBJECTIVE: To investigate the role of PI 3-K in the CSF-1-induced spreading of osteoclasts. METHODS: In isolated rat osteoclasts treated with or without CSF-1, the distribution of PI 3-K and proteins phosphorylated on tyrosine were investigated using immunofluorescence. In murine osteoclast-like cells grown from bone marrow cells co-cultured with osteoblasts, the activation of the PI 3-K by CSF-1 was determined both in vivo and in vitro. In vivo, the enzyme product in the cell was determined after extraction and separation with thin layer chromatography; in vitro, PI 3-K activity was measured in the pellet immunoprecipitated from the cell lysate. RESULTS: Inhibition of PI 3-K blocked the CSF-1-induced spreading of osteoclasts. In spreading osteoclasts, a portion of PI 3-K was translocated to the periphery where proteins phosphorylated on tyrosine appeared simultaneously. In osteoclast-like cells, CSF-1 stimulated PI 3-K activity. This activity could be immunoprecipitated with antibody against phophotyrosine residues. CONCLUSION: PI 3-K participates in the CSF-1-induced spreading of osteoclasts. The activated PI 3-K may induce the reorganization of the cytoskeleton resulting in spreading and migration.

scholarly journals Acute myelogenous leukemia of the Wistar/Furth rat: establishment of a continuous tissue culture line producing lysozyme in vitro and in vivo

Blood ◽  
1975 ◽  
Vol 46 (1) ◽  
pp. 27-38 ◽  
Author(s):  
JS Greenberger ◽  
DS Rosenthal ◽  
SA Aaronson ◽  
WC Moloney
Keyword(s):  
Tissue Culture ◽  
Acute Myelogenous Leukemia ◽  
Fluorescent Antibody ◽  
Virus Production ◽  
Histochemical Staining ◽  
Myelogenous Leukemia ◽  
Acute Myelogenous ◽  
Inbred Rat

Abstract A transplantable myelogenous leukemia of an inbred Wistar/Furth rat has been established in tissue culture and cloned. The resulting transplantable leukemia line demonstrates in vitro doubling time of 20 hr, colony-forming efficiency of 5% in liquid and methylcellulos- containing medium, and a saturation density of 3.0 x 106 cells/sq cm in liquid medium. Following intraperitoneal inoculation, newborn rats developed solid tumors, ascities, and leukemia with ld50 of5 x 103 cells and mean latency of 60 days. The tumor cell morphology was consistent with that of acute myelogenous leukemia. Histochemical staining for myeloid enzymes revealed no evidence of myeloperoxidase, esterase, or leukocyte alkaline phosphatase; however, fluorescent antibody staining for lysozyme was markedly positive. Serum, urine, and ascitic fluid from rats with transplanted leukemia also contained elevated levels of lysozyme. There was no detectable type-CRNA virus production by this cell line after as long as 100 days in vitro. This inbred rat myelogenous leukemia should provide a useful model for studies of chemotherapy and immunoltherapy of human acute myelogenous leukemia.

A-Type Proanthocyanidins Prevent Engraftment Of Primary Acute Myelogenous Leukemia Cells In Mice and Exhibit Potentially Novel Anti-Leukemia Mechanisms

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3962-3962
Author(s):  
Laura M Bystrom ◽  
Hongliang Zong ◽  
Hsiao-Ting Hsu ◽  
Neng Yang ◽  
Noa Greenberg ◽  
...  
Keyword(s):  
Cell Lines ◽  
Iron Metabolism ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Survival Pathways ◽  
Leukemia Cell Lines ◽  
Acute Myelogenous

Abstract Acute myelogenous leukemia (AML) is often a fatal disease where after strong induction therapy most patients relapse and die. AML originates and is maintained by leukemia stem cells (LSCs). Failure to eliminate LSCs by chemotherapy is likely to result in disease relapse. Therefore, it is a priority to identify new therapies that eliminate blasts while ablating LSCs and preventing a relapse. We have found that a unique class of compounds in cranberries (Vaccinium macrocarponAit.), known as A-type proanthocyanidins (A-PACs), were effective against several leukemia cell lines and primary AML samples in vitro. A-PACs consist of monomeric epicatechin units attached to one another by a carbon-carbon bond and a distinctive ether bond that differentiates these compounds from other proanthocyanidins found in nature. Moreover, A-PACs possess ortho-hydroxyl phenolic groups that have the potential to bind to iron and alter redox status. Preliminary work showed that pre-treatment with antioxidants or holo-transferrin (iron-saturated transferrin) partially protected AML cells from A-PAC induced cell death (p<0.01). A-PACs were also found to selectively ablate leukemia stem and progenitor cells, with minimal effects on normal hematopoetic stem cells. Furthermore, AML engraftment of cells treated ex vivo with 62.5 µg/ml A-PACs was decreased (90.6%, n=3, p<0.001), while normal CD34+ cells retained engraftment capability in immunodeficient mice. It was also found that a fraction of A-PACs of up to 7 degree of polymerization was more effective than individual A-PACs. This information prompted us to investigate the in vivo anti-leukemia effects of A-PACs in xenotransplanted mice with primary AML samples, and to further investigate the mechanisms associated with these compounds. Primary AML cells were injected in sub-lethally irradiated NOD/SCID mice. Four weeks after injections, when human leukemia cells have engrafted, intraperitoneal injections of cytarabine (AraC) at 60 mg/kg were given to the mice for 1 week everyday or A-PACs (100 mg/kg dose every 3 days for A-PACs) and vehicle control (1% DMSO in PBS every 3 days) were injected for 2.5 weeks. Mice were sacrificed and leukemia engraftment evaluated using anti-human CD45 and CD33. Moreover, primary cells treated with A-PACs were assessed for effects on iron metabolism, ROS, and survival pathways either by gene expression analysis, flow cytometry or mass spectrometry. Administration of A-PACs to NOD-SCID mice bearing AML tumors reduced tumor burden. Mice that were treated with the vehicle control had engraftment of AML primary cells equivalent to 16.1% (95% CI: -6.0, 38.37; n=4), whereas the mice treated with the A-PACs and AraC showed a level of engraftment of 4.9% (95% CI: 2, 8; n=5) and 5.8% (95% CI: -1.1, 12.7; n=5), respectively. No significant changes in hemoglobin or weight were found between the different treatment groups. Moreover, qPCR analysis of sensitive leukemia cell lines treated with A-PACs showed changes in gene expression of several iron metabolism genes in sensitive leukemia cell lines (up-regulation of ferritin and transferrin receptors 1 and down-regulation of ferroportin) and several ROS-relevant genes (down-regulation of nuclear factor erythroid-2-related factor 2 and glutamate-cysteine ligase regulatory subunit). Mass spectrometry also confirmed that A-PACs bind iron. The results indicate that A-PACs not only target primary AML cells in vitro but are also effective in vivo. Secondary transplants are also being performed to determine the effects on LSC activity. Some of the anti-leukemia mechanisms under investigation include effects related to iron metabolism, ROS or inhibition of survival pathways. Understanding the unique structure and biological effects of A-PACs may provide novel information about pathways involved in the survival of LSCs and provide crucial information in preparation for clinical trials and/or optimal combination drug therapies. Disclosures: Rivella: Novartis: Consultancy; Bayer: Consultancy; Isis: Consultancy, Research Funding; Merganser: Equity Ownership, Research Funding; Biomarin: Consultancy; Alexion: Consultancy; Imago: Consultancy.

scholarly journals Transformation by Tribbles homolog 2 (Trib2) requires both the Trib2 kinase domain and COP1 binding

Blood ◽  
2010 ◽  
Vol 116 (23) ◽  
pp. 4948-4957 ◽  
Author(s):  
Karen Keeshan ◽  
Will Bailis ◽  
Priya H. Dedhia ◽  
Maria E. Vega ◽  
Olga Shestova ◽  
...  
Keyword(s):  
Function Analysis ◽  
Kinase Domain ◽  
Myelogenous Leukemia ◽  
Granulocytic Differentiation ◽  
Functional Regions ◽  
N Terminus ◽  
Catalytic Loop ◽  
Acute Myelogenous

Abstract Tribbles homolog 2 (Trib2) is a pseudokinase that induces acute myelogenous leukemia (AML) in mice and is highly expressed in a subset of human AML. Trib2 has 3 distinct regions, a proline-rich N-terminus, a serine/threonine kinase homology domain, and a C-terminal constitutive photomorphogenesis 1 (COP1)–binding domain. We performed a structure-function analysis of Trib2 using in vitro and in vivo assays. The N-terminus was not required for Trib2-induced AML. Deletion or mutation of the COP1-binding site abrogated the ability of Trib2 to degrade CCAAT/enhancer-binding protein-α (C/EBP-α), block granulocytic differentiation, and to induce AML in vivo. Furthermore, COP1 knockdown inhibited the ability of Trib2 to degrade C/EBP-α, showing that it is important for mediating Trib2 activity. We also show that the Trib2 kinase domain is essential for its function. Trib2 contains variant catalytic loop sequences, compared with conventional kinases, that we show are necessary for Trib2 activity. The kinase domain mutants bind, but cannot efficiently degrade, C/EBP-α. Together, our data demonstrate that Trib2 can bind both COP1 and C/EBP-α, leading to degradation of C/EBP-α. Identification of the functional regions of Trib2 that are essential to its oncogenic role provides the basis for developing inhibitors that will block Trib functions in cancer.

scholarly journals A bi-faceted role of estrogen receptor β in breast cancer

2013 ◽  
Vol 20 (3) ◽  
pp. R127-R139 ◽  
Author(s):  
Etienne Leygue ◽  
Leigh C Murphy
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Large Body ◽  
Estrogen Receptor Β ◽  
Cancer Tissue ◽  
Culture Studies ◽  
Growth And Survival

Despite over 15 years of research, the exact role, if any, played by estrogen receptor β (ERβ) in human breast cancer remains elusive. A large body of data bothin vitroandin vivosupports its role as an antiproliferative, pro-apoptotic factor especially when co-expressed with ERα. However, there is a smaller body of data associating ERβ with growth and survival in breast cancer. In clinical studies and most often in cell culture studies, the pro-growth and pro-survival activity of ERβ occurs in ERα-negative breast cancer tissue and cells. This bi-faceted role of ERβ is discussed in this review.

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