Tryptase Regulates the Epigenetic Modification of Core Histones in Mast Cell Leukemia Cells.

Mast cells are immune cells that store large amounts of mast cell-restricted proteases in their secretory granules, including tryptase, chymase and carboxypeptidase A3. In mouse mast cells, it has been shown that tryptase, in addition to its canonical location in secretory granules, can be found in the nuclear compartment where it can impact on core histones. Here we asked whether tryptase can execute core histone processing in human mast cell leukemia cells, and whether tryptase thereby can affect the epigenetic modification of core histones. Our findings reveal that triggering of cell death in HMC-1 mast cell leukemia cells is associated with extensive cleavage of core histone 3 (H3) and more restricted cleavage of H2B. Tryptase inhibition caused a complete blockade of such processing. Our data also show that HMC-1 cell death was associated with a major reduction of several epigenetic histone marks, including H3 lysine-4-mono-methylation (H3K4me1), H3K9me2, H3 serine-10-phosphorylation (H3S10p) and H2B lysine-16-acetylation (H2BK16ac), and that tryptase inhibition reverses the effect of cell death on these epigenetic marks. Further, we show that tryptase is present in the nucleus of both viable and dying mast cell leukemia cells. In line with a role for tryptase in regulating nuclear events, tryptase inhibition caused increased proliferation of the mast cell leukemia cells. Altogether, the present study emphasizes a novel principle for how epigenetic modification of core histones is regulated, and provides novel insight into the biological function of human mast cell tryptase.

The upregulation of Pim kinases is essential in coordinating the survival, proliferation, and migration of KIT D816V-mutated neoplastic mast cells.

About ˜80% of mast cell neoplasm patients harbor the c-Kit activating mutation D816 V, which is associated with c-Kit inhibitor resistance and poor prognosis. However, the molecular basis for these effects is not fully known. To address this issue, in this study we screened molecules whose expression is altered by KIT D816 V mutation and found that Pim kinases were overexpressed in D816V-mutant neoplastic mast cells. This was accompanied by upregulation of signal transducer and activator of transcription (STAT) and mammalian target of rapamycin (mTOR) and downregulation of Akt and extracellular signal-regulated kinase (ERK1/2). Activated Pim kinases promoted the survival of D816 V cells by maintaining mTOR and p70S6K activation even under nutrient starvation. Conversely, cell proliferation was suppressed by inhibiting Pim kinases. The mRNA level of C-X-C chemokine receptor type 4 (CXCR4) was about 2-fold higher in D816 V cells; this was associated with a 2-fold increase in migratory capacity, which was modulated by Pim kinases. We also confirmed that upregulation of Pim kinases is a feature specific to cells with the D816 V mutation and is not observed in cells with the c-Kit activating N822 K mutation. These data suggest Pim kinases as a promising therapeutic target for the treatment of mast cell neoplasms with KIT D816 V mutation.

Acute mast cell leukemia associated with t(4;5)(q21;q33).

To the best of our knowledge, this manuscript describes clinical and pathologic findings of the first case of acute mast cell leukemia harboring t(4;5)(q21;q33), compatible with fusion of the PDGFRB gene to a rare partner, PRKG2. Translocation involving the PDGFRB gene is confirmed by fluorescence in situ hybridization study. This case presented a relatively fulminant clinical course with acute mast cell leukemia and "C" findings (cytopenia, hepatosplenomegaly, and weight loss), mast cell sarcoma, and severe basophilia. Despite aggressive presentation initially, the patient responded well to tyrosine kinase inhibitor treatment and is currently in complete remission 33 months after diagnosis. This case significantly extends the disease spectrum associated with PRKG2/PDGFRB fusion gene. Recognizing the whole spectrum of diseases associated with this fusion is critical because tyrosine kinase inhibitor treatment has been exceedingly effective in these patients.

Advanced systemic mastocytosis: the impact of KIT mutations in diagnosis, treatment, and progression.

Apart from indolent systemic mastocytosis (SM), which is associated with a favorable prognosis, other subtypes of SM (SM with associated clonal hematologic non-mast cell lineage disease, aggressive SM, and mast cell leukemia - collectively referred to in this review as advanced SM) can be debilitating. The complexity of SM makes both the diagnosis and design of response criteria challenging for clinical studies. The tyrosine kinase KIT has been shown to play a crucial role in the pathogenesis of SM and has been a focal point in the development of targeted therapy. Mutations within various domains of the KIT receptor that lead to constitutive activation have been identified in patients, and those involving the activation loop of the KIT receptor are the mutations most frequently detected in patients with mastocytosis. Aberrant activation of the KIT receptor results in increased production of mast cells in extracutaneous organs that may lead to organ failure or early death. This review discusses the diagnosis and management of patients with advanced SM, including the relevance of KIT in this disease, potential therapies targeting this kinase, and criteria for measuring responses to these therapies.

Immunohistochemical detection of histidine decarboxylase in neoplastic mast cells in patients with systemic mastocytosis.

Synthesis of histamine in hematopoietic progenitor cells may be one of the earliest events in mastopoiesis. We therefore asked whether the key enzyme involved in histamine production, histidine decarboxylase (HDC), can be used as an immunohistochemical marker for the detection of immature neoplastic mast cells (MC) in patients with MC-proliferative disorders. To address this question, we examined bone marrow biopsy specimens in a cohort of 102 patients with mastocytosis using an antibody against HDC. Independent of the maturation stage of MC, the anti-HDC antibody produced clear diagnostic staining results in all patients with systemic MC disease examined including those with MC leukemia and MC sarcoma, in which MCs are particularly immature. In these patients, expression of HDC was reconfirmed at the messenger RNA level by reverse transcriptase polymerase chain reaction analyses performed with RNA of highly enriched CD117(+) MC. In summary, HDC is expressed in neoplastic MC in patients with systemic mastocytosis independent of the maturation stage of cells or the variant of disease. Histidine decarboxylase should therefore be considered as a new MC marker in the screen panel of antigens used to diagnose high-grade MC malignancies.

Expression of activated M-Ras in hemopoietic stem cells initiates leukemogenic transformation, immortalization and preferential generation of mast cells.

Cultures of purified hemopoietic stem cells transduced with an activated mutant of M-Ras contained abnormal cells that, despite the presence of only low levels of growth factors, generated large, dense colonies of macrophages and blast cells. Cells from these colonies survived and grew continuously in the absence of growth factors and generated clonal cell-lines that were mainly composed of well-differentiated mast cells, with a low frequency of undifferentiated cells. When transplanted into sublethally irradiated syngeneic mice, four out of four such clones gave rise to a systemic mastocytosis and mast-cell leukemia. However, the donor clones also generated low percentages of cells with the morphological and cell-surface characteristics of erythrocytes, granulocytes, monocytes and T- and B-lymphocytes. These data indicate that signals downstream of activated M-Ras are sufficient to transform hemopoietic stem cells, and while preserving their capacity to generate other cell-lineages in vivo, result in preferential generation of mast cells.

Evaluation of neoplastic human mast cells by tryptase-immunoelectron microscopy.

AIMS: To analyse and characterize the ultrastructural morphology of normal tissue mast cells (MC) and neoplastic bone marrow MC. METHODS: We have examined the ultrastructure and cytomorphological features of MC derived from cord blood cells, neoplastic bone marrow MC in patients with systemic mastocytosis (SM, n = 4), myelomastocytic leukaemia (MML, n = 2), mast cell leukaemia (MCL, n = 2) and tryptase-positive acute myeloid leukaemia (AML, n = 4). RESULTS: Based on their ultrastructure and morphology, four distinct cell types could be delineated: (i) mature well-granulated tissue MC exhibiting a round central nucleus; (ii) atypical MC type I with oval nuclei, hypogranulated cytoplasm, and prominent surface projections; (iii) immature atypical MC with bi- or polylobed nuclei (atypical MC type II = promastocytes); and (iv) metachromatic blasts. Type I atypical MC were detected in a patient with indolent SM, whereas type II MC and metachromatic blasts were primarily found in MML, MCL and tryptase-positive AML. In all samples examined, the identity of MC could be reconfirmed by immunoelectron microscopy, irrespective of the stage of cell maturation or the disease variant, all types of MC contained tryptase in their cytoplasmic granules. CONCLUSION: Immunoelectron microscopy may be a helpful approach in confirming the identity of neoplastic MC in myeloid neoplasms.

Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930.

OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3' kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non-receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell leukemia.

Monomeric IgE stimulates NFAT translocation into the nucleus, a rise in cytosol Ca2+, degranulation, and membrane ruffling in the cultured rat basophilic leukemia-2H3 mast cell line.

Mast cells are key regulators in allergy and inflammation, and release histamine, cytokines, and other proinflammatory mediators. In the classical view, IgE acts merely to prime mast cells, attaching to FcepsilonRs but not evoking any cell signaling response until cross-linked by the presence of a multivalent allergen. However, several recent studies have reported that IgE alone can promote cell survival and cytokine production in the absence of cross-linking by allergen. In this study we demonstrate that acute addition of monomeric IgE elicits a wide spectrum of responses in the rat basophilic leukemia-2H3 mast cell line, including activation of phospholipases Cgamma and D, a rise in cytosol Ca(2+), NFAT translocation, degranulation, and membrane ruffling within minutes. Calcium transients persist for hours as long as IgE is present resulting in the maintained translocation of the transcription factor NFAT to the nucleus. Removal of IgE reverses the signaling processes. Our results indicate that, far from simply preparing the cells for a response to allergen, monomeric IgE can stimulate signaling pathways that lead to degranulation, membrane ruffling, and NFAT translocation. The mechanism of activation is likely to be via aggregation of the FcepsilonR1 because activation by IgE can be inhibited with monovalent hapten.

Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukemia; activation following aggregation of FcepsilonRI or FcgammaRI.

Two novel stem cell factor (SCF) dependent human mast cell lines, designated LAD 1 and 2, were established from bone marrow aspirates from a patient with mast cell sarcoma/leukemia. LAD 1 and 2 cells have the ultrastructural features of human mast cells, and express FcepsilonRI, CD4, 9, 13, 14, 22, 31, 32, 45, 64, 71, 103, 117, 132, CXCR4 (CD184), CCR5 (CD195); and intracytoplasmic histamine, tryptase and chymase. LAD 1 and 2 do not exhibit activating mutations at codon 816 of c-kit. Both LAD 1 and 2 release beta-hexosaminidase following FcepsilonRI or FcgammaRI aggregation. The availability of these cell lines offers an unparalleled circumstance to examine the biology of human mast cells.

Inhibitory effect of retinoic acid on proliferation, maturation and tryptase level in human leukemic mast cells (HMC-1).

Mast cells play important role in allergic inflammation by releasing histamine, tryptase and several inflammatory cytokines. Human leukemic mast cells (HMC-1) have been used to study mast cell mediator and their role in inflammatory mechanisms. HMC-1 contain and release several inflammatory mediators, of which the proteolytic enzyme tryptase is most characteristic. Retinoids, including retinoic acid, are naturally occurring and synthetic derivatives of vitamin A. All-trans-retinoic (ATRA) acid had been previously reported to inhibit cell proliferation, differentiation and apoptosis. In the present study, we investigated the effect of ATRA on the proliferation and secretion of tryptase in HMC-1. HMC-1 were treated with ATRA at 10(-4M), 10(-5M) or 10(-6M) for 3, 4 or 5 days in culture. Control HMC-1 were treated with equal amount of culture medium only. ATRA decreased the number of HMC-1 as compared to the control group. The same treatment for 3, 4 or 5 days also decreased intracellular tryptase levels. These results indicate that ATRA significantly inhibits both proliferation and growth as shown by the decreased intracellular tryptase levels in HMC-1. ATRA may be a useful agent in the treatment of mast cell proliferative disorders.

The HMC-1 human mast cell line expresses the hepatocyte growth factor receptor c-met.

Hepatocyte growth factor (HGF) was originally characterized as a strong inducer of liver regeneration. However, it is now clear that HGF and its receptor, the proto-oncogene c-met, can be expressed in many other tissues, and that HGF can mediate diverse biological activities. We investigated the expression and function of c-met in a human mast cell line (HMC-1). We found that HMC-1 cells express c-met and that c-met expression can be upregulated by treatment of the cells with phorbol 12-myristate 13-acetate (PMA). Although HGF did not detectably influence the proliferation or morphology of HMC-1 cells, HGF inhibited the cells' ability to release tumor necrosis factor-alpha (TNF-alpha) in response to stimulation with PMA and the calcium ionophore, A23187. These results add the inhibition of TNF-alpha production to the other recognized effects of HGF/c-met on cellular function.

Brefeldin A inhibits antigen- or calcium ionophore-mediated but not PMA-induced phospholipase D activation in rat basophilic leukemia (RBL-2H3) cells.

Recent reports have indicated that ADP-ribosylation factor (ARF) plays a role in the regulation of phospholipase D (PLD) activity in the in vitro assay system. Since a fungal metabolite brefeldin A (BFA) is known to interfere with ARF function, the effect of BFA on antigen-induced PLD activation was examined in rat basophilic leukemia (RBL-2H3) cells. BFA inhibited the antigen-induced formation of phosphatidylbutanol (PBut), a specific and stable metabolite produced by PLD activity in a concentration-dependent manner. The maximal inhibition obtained at 10 micrograms/ml of the drug was nearly 70% and further inhibition was not observed at higher concentrations. Ca(2+)-ionophore A23187-mediated PLD activation was also prevented by BFA. In contrast, BFA failed to inhibit PLD activation in response to 4 beta-phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC). This indicates that there are BFA-sensitive and BFA-insensitive pathways leading to PLD activation in RBL-2H3 cells and also that the PKC-mediated pathway may be insensitive to BFA treatment, suggesting the existence of PLD isozymes. BFA inhibited Ag-induced serotonin release at a concentration 20-fold lower than that needed for the inhibition of PLD. Moreover, PMA caused a marked production of PBut, but it failed to elicit secretory response. This implies that PLD may be not a crucial element for secretory responses.

Purification of tryptase from a human mast cell line.

The neutral protease tryptase has been isolated from a human mast cell line, HMC-1. The HMC-1 line was established from the peripheral blood of a patient with mast cell leukemia and maintained as continuously proliferating clones in vitro and as solid mast cell tumors in nude mice. HMC-1-derived tryptase was purified by sequential chromatography on Dowex 1, DEAE 5 PW, and heparin-agarose. Purified tryptase has an apparent molecular weight of 150,000, as determined by molecular sieve HPLC, but migrates as a doublet of bands of 32/35,000 on SDS-PAGE gels. Maximal enzymatic activity was observed at pH 8.5. Cleavage of tosyl-L-arginine methyl ester by purified tryptase was inhibited by dansyl-L-glutamyl-glycyl-L-arginine chloromethyl ketone 2 HCl, HgCl2, tosyl-L-lysine chloromethyl ketone, leupeptin, and PMSF but not by benzamidine, aprotinin, tosyl-L-phenyl-alanine chloromethyl ketone, soybean trypsin inhibitor, human plasma, ovomucoid inhibitor, or lima bean trypsin inhibitor. Microsequencing of purified tryptase yielded an amino terminal sequence that was identical to that previously reported for human pituitary-derived tryptase.