Puchit Samorapoompichit

Synergistic growth-inhibitory effects of two tyrosine kinase inhibitors, dasatinib and PKC412, on neoplastic mast cells expressing the D816V-mutated oncogenic variant of KIT

Background and Objectives In a majority of all patients with systemic mastocytosis (SM) including those with mast cell leukemia (MCL), neoplastic mast cells (MC) display the D816V-mutated variant of KIT. The respective oncoprotein, KIT D816V, exhibits constitutive tyrosine kinase (TK) activity and has been implicated in malignant cell growth. Therefore, several attempts have been made to identify KIT D816V-targeting drugs. Design and Methods We examined the effects of the novel TK-inhibitor dasatinib alone and in combination with other targeted drugs on growth of neoplastic MC. Results Confirming previous studies, dasatinib was found to inhibit the TK activity of wild type (wt) KIT and KIT-D816V as well as growth and survival of neoplastic MC and of the MCL cell line, HMC-1. The growth-inhibitory effects of dasatinib in HMC-1 cells were found to be associated with a decrease in expression of CD2 and CD63. In addition, we found that dasatinib blocks KIT D816V-induced cluster-formation and viability in Ba/F3 cells. In drug combination experiments, dasatinib was found to co-operate with PKC412, AMN107, imatinib, and 2CdA in producing growth-inhibition and apoptosis in neoplastic MC. In HMC-1.1 cells lacking KIT D816V, all drug interactions were found to be synergistic in nature. By contrast, in HMC-1.2 cells exhibiting KIT D816V, only the combinations dasatinib+PKC412 and dasatinib+2CdA were found to produce synergistic effects. Interpretation and Conclusions Combinations of targeted drugs may represent an interesting pharmacologic approach for the treatment of aggressive SM or MCL.

On the way to targeted therapy of mast cell neoplasms: identification of molecular targets in neoplastic mast cells and evaluation of arising treatment concepts

Several emerging treatment concepts for myeloid neoplasms are based on novel drugs targeting cell surface antigens, signalling pathways, or critical effector molecules. Systemic mastocytosis is a haematopoietic neoplasm that behaves as an indolent myeloproliferative disease in most patients, but can also present as aggressive disease or even as an acute leukaemia. In patients with aggressive disease or mast cell leukaemia, the response to conventional therapy is poor in most cases, and the prognosis is grave. Therefore, a number of attempts have been made to define novel treatment strategies for these patients. One promising approach may be to identify novel targets and to develop targeted drug therapies. In this article, we support the notion that neoplastic mast cells indeed express a number of potential molecular targets including immunoreactive CD antigens, the microphthalmia transcription factor (MITF), and members of the Bcl‐2 family. In addition, the tyrosine kinase receptor KIT and downstream signalling pathways have been proposed as targets of a specific pharmacological intervention. A particular challenge is the disease‐related D816V‐mutated variant of KIT, which is resistant against diverse tyrosine kinase inhibitors including STI571, but may be sensitive to more recently developed targeted compounds. The therapeutic potential of target‐specific approaches in malignant mast cell disorders should be evaluated in forthcoming clinical trials in the near future.

Effects of various statins on cytokine-dependent growth and IgE-dependent release of histamine in human mast cells

Background:  Statins are inhibitors of hydroxymethylglutaryl coenzyme A (HMG CoA) reductase, a key enzyme in mevalonic acid (MVA)‐dependent signaling. Recent data suggest that statins exhibit profound inhibitory effects on growth and function of various immune cells. In the present study, we examined the in vitro effects of five different statins on primary human mast cells (MCs), MC progenitors, and the human MC line HMC‐1.

Cerivastatin and atorvastatin inhibit IL-3-dependent differentiation and IgE-mediated histamine release in human basophils and downmodulate expression of the basophil-activation antigen CD203c/E-NPP3.

Recent data suggest that the statins, apart from their lipid‐lowering activity, exhibit profound anti‐inflammatory effects. Basophils are major proinflammatory effector cells in diverse pathologic reactions. We have examined the in vitro effects of five different statins on primary human basophils, their progenitors, and the basophil cell line KU‐812. Preincubation of blood basophils with cerivastatin or atorvastatin (0.1–100 μM) for 24 h reduced their capacity to release histamine on immunoglobulin E (IgE)‐dependent stimulation in a dose‐dependent manner. These statins also inhibited IgE‐dependent up‐regulation of the basophil‐activation antigen CD203c. Moreover, both statins suppressed interleukin‐3‐induced differentiation of basophils from their progenitors as well as 3H‐thymidine uptake in KU‐812 cells. All inhibitory effects of cerivastatin and atorvastatin were reversed by mevalonic acid (200 μM). The other statins tested (lovastatin, simvastatin, pravastatin) did not show significant inhibitory effects on basophils. Together, these data identify cerivastatin and atorvastatin as novel inhibitors of growth and activation of human basophils.

Characterization of Human Prostate Mast Cells and Their Increase in Periprostatic Vein Thrombosis

Recent data suggest that mast cells (MCs) and their products are involved in the pathophysiology of thrombosis. In the present study, we analyzed the number, distribution, and phenotype of prostate MCs and periprostatic MCs in patients with unilateral periprostatic vein thrombosis (PVT) by immunohistochemical analysis and electron microscopy. MCs reacted with monoclonal antibodies to tryptase, chymase, and c-kit/CD117 and stained positively for tissue-type plasminogen activator (tPA) and urokinase receptor (uPAR/CD87) but did not express detectable urokinase (uPA) or plasminogen activator inhibitors (PAI-1, PAI-2). We found an increase in the mean +/- SEM number of MCs in PVT compared with control (PVT, 14.36 +/- 1.57 vs control, 5.23 +/- 0.57/mm2). The majority of MCs accumulated in the adventitia of thrombosed veins and showed a decrease in chymase expression. As MCs increase in number in PVT and express a profibrinolytic phenotype, we hypothesize that MC-derived molecules have a role in endogenous fibrinolysis.

Metal ion-induced toxic histamine release from human basophils and mast cells

Recent data suggest that distinct metal ions can be released from dental alloys or other biomaterials, and may cause toxic effects on various cells. In this study, the effects of 14 metal ions on histamine release from human blood basophils (n = 4), isolated tissue mast cells (lung n = 8, uterus n = 2, skin n = 1, gingiva n = 1), the basophil cell line KU-812, and the mast cell line HMC-1 were analyzed. Of the 14 metal ions, Ag+ (0.33 mM) and Hg2+ (0.33 mM) were found to induce release of histamine in blood basophils, KU-812, mast cells, and HMC-1. The effects of Ag+ and Hg2+ were dose dependent and were observed within 60 min of incubation. In primary mast cells and basophils, AU3+ (0.33 mM) also induced histamine release, whereas no effects of Au3+ on HMC-1 or KU-812 cells were seen. The other metal ions showed no effects on primary or immortal cells within 60 min. However, Pt4+ (0.33 mM) induced histamine liberation in HMC-1 and lung mast cells after 12 h. The Ag+- and Hg2+-induced rapid release of histamine from HMC-1 was associated with ultrastructural signs of necrosis, but not apoptosis. In contrast, prolonged exposure to Pt4+ (0.33 mM, 14 h) induced apoptotic cell death in HMC-1 cells, as assessed by electron microscopy and DNA analysis. Together, certain metal ions induce distinct cytopathogenic effects in mast cells and basophils. Whereas Ag+, Hg2+, and Au3+ cause direct toxicity, Pt4 causes cell death through induction of apoptosis. Whether such effects contribute to local adverse reactions to metal-containing biomaterials in vivo remains to be determined.

Targeting of mTOR is associated with decreased growth and decreased VEGF expression in acute myeloid leukaemia cells.

Background  The mammalian target of rapamycin (mTOR) has recently been implicated in leukaemic cell growth, tumour‐associated angiogenesis and expression of vascular endothelial growth factor (VEGF). We examined whether mTOR plays a role as regulator of growth and VEGF‐expression in acute myeloid leukaemia (AML). Three mTOR‐targeting drugs, rapamycin, everolimus (RAD001) and CCI‐779, were applied. The effects of these drugs on growth, survival, apoptosis and VEGF expression in primary AML cells and various AML cell lines were examined.

Dasatinib inhibits the growth and survival of neoplastic human eosinophils (EOL-1) through targeting of FIP1L1-PDGFRα

OBJECTIVE Chronic eosinophilic leukemia (CEL) is a myeloproliferative disorder characterized by molecular and/or cytogenetic evidence of clonality of eosinophils, marked eosinophilia, and organ damage. In many patients, the transforming mutation FIP1L1-PDGFRalpha and the related CHIC2 deletion are found. The respective oncoprotein, FIP1L1-PDGFRalpha, is considered to play a major role in malignant cell growth in CEL. The tyrosine kinase (TK) inhibitor imatinib (STI571) has been described to counteract the TK activity of FIP1L1-PDGFRalpha in most patients. However, not all patients with CEL show a response to imatinib. Therefore, several attempts have been made to identify other TK inhibitors that counteract growth of neoplastic eosinophils. MATERIALS AND METHODS We provide evidence that dasatinib, a multi-targeted kinase inhibitor, blocks the growth and survival of EOL-1, an eosinophil leukemia cell line carrying FIP1L1-PDGFRalpha. RESULTS The effects of dasatinib on proliferation of EOL-1 cells were dose-dependent, with an IC50 of 0.5 to 1 nM, which was found to be in the same range when compared to IC50 values produced with imatinib. Dasatinib was also found to induce apoptosis in EOL-1 cells in a dose-dependent manner (IC50: 1-10 nM). The apoptosis-inducing effects of dasatinib on EOL-1 cells were demonstrable by light microscopy, flow cytometry, and in a TUNEL assay. In Western blot experiments, dasatinib completely blocked the phosphorylation of FIP1L1-PDGFRalpha in EOL-1 cells. CONCLUSIONS Dasatinib inhibits the growth of leukemic eosinophils through targeting of the disease-related oncoprotein FIP1L1-PDGFRalpha. Based on this observation, dasatinib may be considered as a new interesting treatment option for patients with CEL.

Self-renewal, maturation, and differentiation of the rat myelomonocytic hematopoietic stem cell

Hematopoiesis is viewed as a differentiating system emanating from a pluripotent hematopoietic stem cell capable of both self‐renewal and differentiation. By identifying and characterizing a novel and highly specific in vitro mitogenic response to the N‐acetyl glucosamyl/sialic acid specific, stem cell‐binding lectin wheat germ agglutinin (WGA), we demonstrate the existance of a rare (0.1%), plastic adherent precursor in rat bone marrow capable of proliferation (two to seven divisions) in response to WGA. Stimulated cells possess a lineage (lin)low/− immunophenotype and immature blastoid morphology (WGA blasts). A subsequent proliferative response to stem cell factor (SCF), the ligand for the proto‐oncogene receptor tyrosine kinase c‐kit, is characterized by an initial maturation in immunophenotype and subsequent self‐renewal of cells (SCF blasts) without differentiation for at least 50 generations. Although granulocyte colony‐stimulating factor (G‐CSF), interleukin (IL) ‐6, IL‐7, and IL‐11 synergize with SCF to increase blast colony formation, cytokines such as granulocyte‐macrophage CSF or IL‐3 are without significant effect. At all time points in culture, however, cells rapidly differentiate to mature neutrophils with dexamethasone or to mainly monocytes/macrophages in the presence of 1α,25‐dihydroxyvitamin D3, characterized by cell morphology and cytochemistry. Removal of SCF during blast maturation, self‐renewal, or induction of differentiation phases results in apoptotic cell death. Data indicate a pivotal role for SCF/c‐kit interaction during antigenic maturation, self‐renewal, and apoptotic protection of these lineage‐restricted progenitors during non‐CSF‐mediated induction of differentiation. This approach provides a source of many normal, proliferating myelomonocytic precursor cells, and introduces possible clinical applications of ex vivo expanded myeloid stem cells.—Lucas, T., Krugluger, W., Samorapoompichit, P., Gamperl, R., Beug, H., Förster, O. Self‐renewal, maturation, and differentiation of the rat myelomonocytic hematopoietic stem cell. FASEB J. 13, 263–272 (1999)

Targeting of heat-shock protein 32/heme oxygenase-1 in canine mastocytoma cells is associated with reduced growth and induction of apoptosis

OBJECTIVE Advanced mast cell (MC) neoplasms are usually resistant to conventional therapy. Therefore, current research focuses on new targets in neoplastic MC and development of respective targeted drugs. Mastocytomas in dogs often behave as aggressive tumors. We report that heat-shock protein 32 (Hsp32), also known as heme oxygenase-1, is a survival-enhancing molecule and new target in canine mastocytoma cells. MATERIALS AND METHODS As assessed by reverse transcriptase polymerase chain reaction, Northern blotting, immunocytochemistry, and Western blotting, primary neoplastic dog MC, and the canine mastocytoma-derived cell line C2 expressed Hsp32 mRNA and the Hsp32 protein in a constitutive manner. RESULTS The KIT-targeting drug midostaurin inhibited expression of Hsp32, as well as survival in C2 cells. Confirming the functional role of Hsp32, the inhibitory effect of midostaurin on C2 cells was markedly reduced by the Hsp32-inductor hemin. Two pharmacologic Hsp32-inhibitors, styrene maleic-acid micelle-encapsulated ZnPP (SMA-ZnPP) and pegylated zinc-protoporphyrin (PEG-ZnPP) were applied. Both drugs were found to inhibit proliferation of C2 cells as well as growth of primary neoplastic canine MC. The growth-inhibitory effects of SMA-ZnPP and PEG-ZnPP were dose- and time-dependent (IC(50): 1-10 muM) and found to be associated with induction of apoptosis. CONCLUSIONS Hsp32 is an important survival factor and interesting new target in neoplastic canine MC. Trials with Hsp32-targeted drugs are now warranted to define the clinical efficacy of these drugs.