Publications by authors named "Barbara Peter"

32 Publications

Phenotypic characterization of leukemia-initiating stem cells in chronic myelomonocytic leukemia.

Leukemia 2021 Mar 30. Epub 2021 Mar 30.

Department of Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria.

Chronic myelomonocytic leukemia (CMML) is a stem cell-derived neoplasm characterized by dysplasia, uncontrolled expansion of monocytes, and substantial risk to transform to secondary acute myeloid leukemia (sAML). So far, little is known about CMML-initiating cells. We found that leukemic stem cells (LSC) in CMML reside in a CD34/CD38 fraction of the malignant clone. Whereas CD34/CD38 cells engrafted NSGS mice with overt CMML, no CMML was produced by CD34/CD38 progenitors or the bulk of CD34 monocytes. CMML LSC invariably expressed CD33, CD117, CD123 and CD133. In a subset of patients, CMML LSC also displayed CD52, IL-1RAP and/or CLL-1. CMML LSC did not express CD25 or CD26. However, in sAML following CMML, the LSC also expressed CD25 and high levels of CD114, CD123 and IL-1RAP. No correlations between LSC phenotypes, CMML-variant, mutation-profiles, or clinical course were identified. Pre-incubation of CMML LSC with gemtuzumab-ozogamicin or venetoclax resulted in decreased growth and impaired engraftment in NSGS mice. Together, CMML LSC are CD34/CD38 cells that express a distinct profile of surface markers and target-antigens. During progression to sAML, LSC acquire or upregulate certain cytokine receptors, including CD25, CD114 and CD123. Characterization of CMML LSC should facilitate their enrichment and the development of LSC-eradicating therapies.
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http://dx.doi.org/10.1038/s41375-021-01227-zDOI Listing
March 2021

Agreement and repeatability of four different devices to measure non-invasive tear breakup time (NIBUT).

Cont Lens Anterior Eye 2020 10 3;43(5):507-511. Epub 2020 Mar 3.

Optometry and Vision Science, Aston University, Birmingham, UK.

Purpose: Since tear film stability can be affected by fluorescein, the Dry Eye Workshop (DEWSII) recommended non-invasive measurement of tear breakup time (NIBUT). The aim of this study was to investigate the agreement and repeatability of four different instruments in the measurement of NIBUT.

Methods: 72 participants (mean 24.2 ± 3.6 years) were recruited for this multi-centre, cross-sectional study. NIBUT was measured three times from one eye using each of the instruments in randomized order on two separate sessions during a day, separated by at least 2 h. NIBUT was performed at three sites (Switzerland, Germany and UK) using three subjective instruments, Tearscope Plus (Keeler, Windsor, UK) (TS), Polaris (bon Optic, Lübeck, Germany) (POL), EasyTear Viewplus (Easytear, Rovereto, Italy) (ET) and the objective Keratograph 5 M (Oculus Optikgeräte GmbH, Wetzlar, Germany) (KER). As the latter instrument only analyses for 24 s, all data was capped at this value.

Results: NIBUT measurements (average of both sessions) between the four instruments were not statistically significantly different: TS (median 10.4, range 2.0-24.0 s), POL (10.1, 1.0-24.0 s), ET (10.6, 1.0-24.0 s) and KER (11.1, 2.6-24.0 s) (p = 0.949). The objective KER measures were on average (1.2 s ± 9.6 s, 95 % confidence interval) greater than the subjective evaluations of NIBUT with the other instruments (mean difference 0.4 s ± 7.7 s, 95 % confidence interval), resulting in a higher limits of agreement. The slope was -0.08 to 0.11 indicating no bias in the difference between instruments with the magnitude of the NIBUT. Repeated measurements from the two sessions were not significantly different for TS (p = 0.584), POL (p = 0.549), ET (p = 0.701) or KER (p = 0.261).

Conclusions: The four instruments evaluated for their measurement of tear stability were reasonably repeatable and give similar average results.
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http://dx.doi.org/10.1016/j.clae.2020.02.018DOI Listing
October 2020

Effects of ibrutinib on proliferation and histamine release in canine neoplastic mast cells.

Vet Comp Oncol 2019 Dec 13;17(4):553-561. Epub 2019 Aug 13.

Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Vienna, Austria.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is effective in the treatment of human chronic lymphocytic leukaemia and mantle cell lymphoma. Recent data have shown that ibrutinib also blocks IgE-dependent activation and histamine release in human basophils (BAs) and mast cells (MCs). The aim of this study was to investigate whether BTK serves as a novel therapeutic target in canine mast cell tumours (MCTs). We evaluated the effects of ibrutinib on two canine MC lines, C2 and NI-1 and on primary MCs obtained from canine MCTs (n = 3). Using flow cytometry, we found that ibrutinib suppresses phosphorylation of BTK and of downstream STAT5 in both MC lines. In addition, ibrutinib decreased proliferation of neoplastic MCs, with IC values ranging between 0.1 and 1 μM in primary MCT cells and between 1 and 3 μM in C2 and NI-1 cells. In C2 cells, the combination "ibrutinib + midostaurin" produced synergistic growth-inhibitory effects. At higher concentrations, ibrutinib also induced apoptosis in both MC lines. Finally, ibrutinib was found to suppress IgE-dependent histamine release in primary MCT cells, with IC values ranging from 0.05 to 0.1 μM in NI-1 cells, and from 0.05 to 1 μM in primary MCT cells. In summary, ibrutinib exerts anti-proliferative effects in canine neoplastic MCs and counteracts IgE-dependent histamine release in these cells. Based on our data, ibrutinib may be considered as a novel therapeutic agent for the treatment of canine MCT. The value of BTK inhibition in canine MCT patients remains to be elucidated in clinical trials.
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http://dx.doi.org/10.1111/vco.12520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900099PMC
December 2019

Comparative oncology: The paradigmatic example of canine and human mast cell neoplasms.

Vet Comp Oncol 2019 Mar 24;17(1):1-10. Epub 2018 Sep 24.

Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Vienna, Austria.

In humans, advanced mast cell (MC) neoplasms are rare malignancies with a poor prognosis. Only a few preclinical models are available, and current treatment options are limited. In dogs, MC neoplasms are the most frequent malignant skin tumours. Unlike low-grade MC neoplasms, high-grade MC disorders usually have a poor prognosis with short survival. In both species, neoplastic MCs display activating KIT mutations, which are considered to contribute to disease evolution. Therefore, tyrosine kinase inhibitors against KIT have been developed. Unfortunately, clinical responses are unpredictable and often transient, which remains a clinical challenge in both species. Therefore, current efforts focus on the development of new improved treatment strategies. The field of comparative oncology may assist in these efforts and accelerate human and canine research regarding diagnosis, prognostication, and novel therapies. In this article, we review the current status of comparative oncology approaches and perspectives in the field of MC neoplasms.
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http://dx.doi.org/10.1111/vco.12440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6378619PMC
March 2019

Ludwig Boltzmann Cluster Oncology (LBC ONC): first 10 years and future perspectives.

Wien Klin Wochenschr 2018 Sep 13;130(17-18):517-529. Epub 2018 Jul 13.

Ludwig Boltzmann Cluster Oncology, Vienna, Austria.

In 2008 the Ludwig Boltzmann Cluster Oncology (LBC ONC) was established on the basis of two previous Ludwig Boltzmann Institutes working in the field of hematology and cancer research. The general aim of the LBC ONC is to improve treatment of hematopoietic neoplasms by eradicating cancer-initiating and disease-propagating cells, also known as leukemic stem cells (LSC) in the context of leukemia. In a first phase, the LBC ONC characterized the phenotype and molecular aberration profiles of LSC in various malignancies. The LSC phenotypes were established in acute and chronic myeloid leukemia, in acute lymphoblastic leukemia and in chronic lymphocytic leukemia. In addition, the concept of preleukemic (premalignant) neoplastic stem cells (pre-L-NSC) was coined by the LBC ONC and was tested in myelodysplastic syndromes and myeloproliferative neoplasms. Phenotypic characterization of LSC provided a solid basis for their purification and for the characterization of specific target expression profiles. In a second phase, molecular markers and targets were validated. This second phase is ongoing and should result in the development of new diagnostics parameters and novel, more effective, LSC-eradicating, treatment strategies; however, many issues still remain to be solved, such as sub-clonal evolution, LSC niche interactions, immunologic control of LSC, and LSC resistance. In the forthcoming years, the LBC ONC will concentrate on developing LSC-eradicating strategies, with special focus on LSC resistance, precision medicine and translation of LSC-eradicating concepts into clinical application.
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http://dx.doi.org/10.1007/s00508-018-1355-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6132878PMC
September 2018

Preclinical human models and emerging therapeutics for advanced systemic mastocytosis.

Haematologica 2018 11 5;103(11):1760-1771. Epub 2018 Jul 5.

Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Austria.

Mastocytosis is a term used to denote a group of rare diseases characterized by an abnormal accumulation of neoplastic mast cells in various tissues and organs. In most patients with systemic mastocytosis, the neoplastic cells carry activating mutations in Progress in mastocytosis research has long been hindered by the lack of suitable models, such as permanent human mast cell lines. In fact, only a few human mast cell lines are available to date: HMC-1, LAD1/2, LUVA, ROSA and MCPV-1. The HMC-1 and LAD1/2 cell lines were derived from patients with mast cell leukemia. By contrast, the more recently established LUVA, ROSA and MCPV-1 cell lines were derived from CD34 cells of non-mastocytosis donors. While some of these cell lines (LAD1/2, LUVA, ROSA and MCPV-1) do not harbor mutations, HMC-1 and ROSA cells exhibit activating mutations found in mastocytosis and have thus been used to study disease pathogenesis. In addition, these cell lines are increasingly employed to validate new therapeutic targets and to screen for effects of new targeted drugs. Recently, the ROSA subclone has been successfully used to generate a unique model of advanced mastocytosis by injection into immunocompromised mice. Such a model may allow validation of data obtained with targeted drugs directed against mastocytosis. In this review, we discuss the major characteristics of all available human mast cell lines, with particular emphasis on the use of HMC-1 and ROSA cells in preclinical therapeutic research in mastocytosis.
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http://dx.doi.org/10.3324/haematol.2018.195867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6278969PMC
November 2018

The KIT and PDGFRA switch-control inhibitor DCC-2618 blocks growth and survival of multiple neoplastic cell types in advanced mastocytosis.

Haematologica 2018 05 8;103(5):799-809. Epub 2018 Feb 8.

Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Austria

Systemic mastocytosis is a complex disease defined by abnormal growth and accumulation of neoplastic mast cells in various organs. Most patients exhibit a D816V-mutated variant of , which confers resistance against imatinib. Clinical problems in systemic mastocytosis arise from mediator-related symptoms and/or organ destruction caused by malignant expansion of neoplastic mast cells and/or other myeloid cells in various organ systems. DCC-2618 is a spectrum-selective pan KIT and PDGFRA inhibitor which blocks KIT D816V and multiple other kinase targets relevant to systemic mastocytosis. We found that DCC-2618 inhibits the proliferation and survival of various human mast cell lines (HMC-1, ROSA, MCPV-1) as well as primary neoplastic mast cells obtained from patients with advanced systemic mastocytosis (IC <1 μM). Moreover, DCC-2618 decreased growth and survival of primary neoplastic eosinophils obtained from patients with systemic mastocytosis or eosinophilic leukemia, leukemic monocytes obtained from patients with chronic myelomonocytic leukemia with or without concomitant systemic mastocytosis, and blast cells obtained from patients with acute myeloid leukemia. Furthermore, DCC-2618 was found to suppress the proliferation of endothelial cells, suggesting additional drug effects on systemic mastocytosis-related angiogenesis. Finally, DCC-2618 was found to downregulate IgE-mediated histamine release from basophils and tryptase release from mast cells. Together, DCC-2618 inhibits growth, survival and activation of multiple cell types relevant to advanced systemic mastocytosis. Whether DCC-2618 is effective in patients with advanced systemic mastocytosis is currently under investigation in clinical trials.
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http://dx.doi.org/10.3324/haematol.2017.179895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5927976PMC
May 2018

The JAK2/STAT5 signaling pathway as a potential therapeutic target in canine mastocytoma.

Vet Comp Oncol 2018 Mar 11;16(1):55-68. Epub 2017 Apr 11.

Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria.

Background: Mastocytoma are frequently diagnosed cutaneous neoplasms in dogs. In non-resectable mastocytoma patients, novel targeted drugs are often applied. The transcription factor STAT5 has been implicated in the survival of human neoplastic mast cells (MC). Our study evaluated the JAK2/STAT5 pathway as a novel target in canine mastocytoma.

Materials And Methods: We employed inhibitors of JAK2 (R763, TG101348, AZD1480, ruxolitinib) and STAT5 (pimozide, piceatannol) and evaluated their effects on 2 mastocytoma cell lines, C2 and NI-1.

Results: Activated JAK2 and STAT5 were detected in both cell lines. The drugs applied were found to inhibit proliferation and survival in these cells with the following rank-order of potency: R763 > TG101348 > AZD1480 > pimozide > ruxolitinib > piceatannol. Moreover, synergistic anti-neoplastic effects were obtained by combining pimozide with KIT-targeting drugs (toceranib, masitinib, nilotinib, midostaurin) in NI-1 cells.

Conclusion: The JAK2/STAT5 pathway is a novel potential target of therapy in canine mastocytoma.
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http://dx.doi.org/10.1111/vco.12311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5824979PMC
March 2018

Identification of CD25 as STAT5-Dependent Growth Regulator of Leukemic Stem Cells in Ph+ CML.

Clin Cancer Res 2016 Apr 25;22(8):2051-61. Epub 2015 Nov 25.

Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria. Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Vienna, Austria.

Purpose: In chronic myelogenous leukemia (CML), leukemic stem cells (LSC) represent a critical target of therapy. However, little is known about markers and targets expressed by LSCs. The aim of this project was to identify novel relevant markers of CML LSCs.

Experimental Design: CML LSCs were examined by flow cytometry, qPCR, and various bioassays. In addition, we examined the multipotent CD25(+)CML cell line KU812.

Results: In contrast to normal hematopoietic stem cells, CD34(+)/CD38(-)CML LSCs expressed the IL-2 receptor alpha chain, IL-2RA (CD25). STAT5 was found to induce expression of CD25 in Lin(-)/Sca-1(+)/Kit(+)stem cells in C57Bl/6 mice. Correspondingly, shRNA-induced STAT5 depletion resulted in decreased CD25 expression in KU812 cells. Moreover, the BCR/ABL1 inhibitors nilotinib and ponatinib were found to decrease STAT5 activity and CD25 expression in KU812 cells and primary CML LSCs. A CD25-targeting shRNA was found to augment proliferation of KU812 cellsin vitroand their engraftmentin vivoin NOD/SCID-IL-2Rγ(-/-)mice. In drug-screening experiments, the PI3K/mTOR blocker BEZ235 promoted the expression of STAT5 and CD25 in CML cells. Finally, we found that BEZ235 produces synergistic antineoplastic effects on CML cells when applied in combination with nilotinib or ponatinib.

Conclusions: CD25 is a novel STAT5-dependent marker of CML LSCs and may be useful for LSC detection and LSC isolation in clinical practice and basic science. Moreover, CD25 serves as a growth regulator of CML LSCs, which may have biologic and clinical implications and may pave the way for the development of new more effective LSC-eradicating treatment strategies in CML.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-0767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817228PMC
April 2016

Transcriptional plasticity promotes primary and acquired resistance to BET inhibition.

Nature 2015 Sep 14;525(7570):543-547. Epub 2015 Sep 14.

Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria.

Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukaemia (AML), bromodomain and extra terminal protein (BET) inhibitors are being explored as a promising therapeutic avenue in numerous cancers. While clinical trials have reported single-agent activity in advanced haematological malignancies, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukaemia, here we perform a chromatin-focused RNAi screen in a sensitive MLL-AF9;Nras(G12D)-driven AML mouse model, and investigate dynamic transcriptional profiles in sensitive and resistant mouse and human leukaemias. Our screen shows that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET inhibitor resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodelling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukaemias regardless of their sensitivity, resistant leukaemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signalling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic chromatin immunoprecipitation sequencing and self-transcribing active regulatory region sequencing of enhancer profiles reveal that BET-resistant states are characterized by remodelled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signalling as a driver and candidate biomarker of primary and acquired BET resistance in leukaemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.
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http://dx.doi.org/10.1038/nature14898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4921058PMC
September 2015

Prominin-1 (CD133, AC133) and dipeptidyl-peptidase IV (CD26) are indicators of infinitive growth in colon cancer cells.

Am J Cancer Res 2015 15;5(2):560-74. Epub 2015 Jan 15.

Ludwig Boltzmann Cluster Oncology, Medical University of Vienna Waehringer Guertel 18-20, A-1090 Vienna, Austria ; Department of Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna Waehringer Guertel 18-20, A-1090 Vienna, Austria.

Advanced colorectal cancer is characterized by uncontrolled growth and resistance against anti-cancer agents, including ErbB inhibitors. Recent data suggest that cancer stem cells (CSC) are particularly resistant. These cells may reside within a CD133+ fraction of the malignant cells. Using HCT116 cells we explored the role of CD133 and other CSC markers in drug resistance in colon cancer cells. CD133+ cells outnumbered CD133- cells over time in long-term culture. Both populations displayed the KRAS mutation 38G > A and an almost identical target profile, including EGFR/ErbB1, ErbB2, and ErbB4. Microarray analyses and flow cytometry identified CD26 as additional CSC marker co-expressed on CD133+ cells. However, knock-down of CD133 or CD26 did not affect short-term growth of HCT116 cells, and both cell-populations were equally resistant to various targeted drugs except irreversible ErbB inhibitors, which blocked growth and ERK1/2 phosphorylation in CD133- cells more efficiently than in CD133+ cells. Moreover, the MEK inhibitor AS703026 was found to overcome resistance against ErbB blockers in CD133+ cells. Together, CD133 and CD26 are markers of long-term growth and resistance to ErbB blockers in HCT116 cells, which may be mediated by constitutive ERK activity.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396035PMC
May 2015

Chronic mast cell leukemia (MCL) with KIT S476I: a rare entity defined by leukemic expansion of mature mast cells and absence of organ damage.

Ann Hematol 2015 Feb 11;94(2):223-31. Epub 2014 Sep 11.

Department of Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria,

Mast cell leukemia (MCL) is a rare, life-threatening malignancy defined by a substantial increase in neoplastic mast cells (MCs) in bone marrow (BM) smears, drug-resistance, and a poor prognosis. In most patients, the survival time is less than 1 year. However, exceptional cases may present with a less malignant course. We report on a 49-year-old female patient with MCL diagnosed in 2013. In February 2013, first symptoms, including flushing, headache, and diarrhea, were recorded. In addition, mild anemia was detected. The disease was characterized by a massive increase in well-granulated, mature, and often spindle-shaped MCs (80 %) in BM smears. The serum tryptase level amounted to 332 ng/mL. Like in most other MCL patients, no skin lesions were detected. However, unlike in other patients, tryptase levels remained stable, and no other signs or symptoms of MCL-induced organ damage were found. Sequencing studies revealed an isolated S476I point mutation in KIT but no mutation in codon 816. The patient received histamine receptor blockers but refused cytoreductive therapy. After 9 months, still no progression or organ damage was detected. However, progression with transformation to acute MCL occurred after 12 months. We propose that the chronic type of MCL with stable conditions, absence of organ damage, and a mature MC morphology is recognized as a distinct entity that should be distinguished from the acute variant of MCL.
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http://dx.doi.org/10.1007/s00277-014-2207-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4896380PMC
February 2015

Identification of heat shock protein 32 (Hsp32) as a novel target in acute lymphoblastic leukemia.

Oncotarget 2014 Mar;5(5):1198-211

Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Austria.

Heat shock proteins (Hsp) are increasingly employed as therapeutic targets in oncology. We have shown that Hsp32, also known as heme oxygenase-1 (HO-1), serves as survival factor and potential target in Ph+ chronic myeloid leukemia. We here report that primary cells and cell lines derived from patients with acute lymphoblastic leukemia (ALL) express Hsp32 mRNA and the Hsp32 protein in a constitutive manner. Highly enriched CD34+/CD38- ALL stem cells also expressed Hsp32. Two Hsp32-targeting drugs, pegylated zinc protoporphyrine (PEG-ZnPP) and styrene maleic acid-micelle-encapsulated ZnPP (SMA-ZnPP), induced apoptosis and growth arrest in the BCR/ABL1+ cell lines, in Ph- lymphoblastic cell lines and in primary Ph+ and Ph- ALL cells. The effects of PEG-ZnPP and SMA-ZnPP on growth of leukemic cells were dose-dependent. In Ph+ ALL, major growth-inhibitory effects of the Hsp32-targeting drugs were observed in imatinib-sensitive and imatinib-resistant cells. Hsp32-targeting drugs were found to synergize with imatinib, nilotinib, and bendamustine in producing growth inhibition and apoptosis in Ph+ ALL cells. A siRNA against Hsp32 was found to inhibit growth and survival of ALL cells and to synergize with imatinib in suppressing the growth of ALL cells. In conclusion, Hsp32 is an essential survival factor and potential new target in ALL.
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http://dx.doi.org/10.18632/oncotarget.1805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4012724PMC
March 2014

Identification of Ponatinib as a potent inhibitor of growth, migration, and activation of neoplastic eosinophils carrying FIP1L1-PDGFRA.

Exp Hematol 2014 Apr 6;42(4):282-293.e4. Epub 2014 Jan 6.

Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Austria; Ludwig Boltzmann Cluster Oncology, Vienna, Austria. Electronic address:

In chronic eosinophilic leukemia, the transforming oncoprotein FIP1L1-PDGFRA is a major target of therapy. In most patients, the tyrosine kinase inhibitor (TKI) imatinib induces complete remission. For patients who are intolerant or resistant, novel TKIs have been proposed. We examined the in vitro effects of 14 kinase blockers on growth and function of EOL-1 cells, a FIP1L1-PDGFRA(+) eosinophil cell line. Major growth-inhibitory effects were seen with all PDGFR-blocking agents, with IC50 values in the low nanomolar range: ponatinib, 0.1-0.2 nmol/L; sorafenib, 0.1-0.2 nmol/L; masitinib, 0.2-0.5 nmol/L; nilotinib, 0.2-1.0 nmol/L; dasatinib, 0.5-2.0 nmol/L; sunitinib, 1-2 nmol/L; midostaurin, 5-10 nmol/L. These drugs were also found to block activation of PDGFR-downstream signaling molecules, including Akt, S6, and STAT5 in EOL-1 cells. All effective TKIs produced apoptosis in EOL-1 cells as determined by microscopy, Annexin-V/PI, and caspase-3 staining. In addition, PDGFR-targeting TKIs were found to inhibit cytokine-induced migration of EOL-1 cells. In all bioassays used, ponatinib was found to be the most potent compound in EOL-1 cells. In addition, ponatinib was found to downregulate expression of the activation-linked surface antigen CD63 on EOL-1 cells and to suppress the growth of primary neoplastic eosinophils. We also examined drug effects on Ba/F3 cells expressing two clinically relevant, imatinib-resistant, mutant forms of FIP1L1-PDGFRA, namely T674I and D842V. Strong inhibitory effects on both mutants were seen only with ponatinib. In summary, novel PDGFR-targeting TKIs may be alternative agents for the treatment of patients with imatinib-resistant chronic eosinophilic leukemia. Although several different PDGFR-targeting agents are effective, the most potent drug appears to be ponatinib.
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http://dx.doi.org/10.1016/j.exphem.2013.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338611PMC
April 2014

The pan-Bcl-2 blocker obatoclax promotes the expression of Puma, Noxa, and Bim mRNA and induces apoptosis in neoplastic mast cells.

J Leukoc Biol 2014 Jan 19;95(1):95-104. Epub 2013 Sep 19.

1.Division of Hematology and Hemostaseology and Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, A-1090 Vienna, Austria.

Advanced SM is an incurable neoplasm with short survival time. So far, no effective therapy is available for these patients. We and others have shown recently that neoplastic MC in ASM and MCL express antiapoptotic Mcl-1, Bcl-2, and Bcl-xL. In this study, we examined the effects of the pan-Bcl-2 family blocker obatoclax (GX015-070) on primary neoplastic MC, the human MC leukemia cell line HMC-1, and the canine mastocytoma cell line C2. Obatoclax was found to inhibit proliferation in primary human neoplastic MC (IC₅₀: 0.057 μM), in HMC-1.2 cells expressing KIT D816V (IC₅₀: 0.72 μM), and in HMC-1.1 cells lacking KIT D816V (IC₅₀: 0.09 μM), as well as in C2 cells (IC₅₀: 0.74 μM). The growth-inhibitory effects of obatoclax in HMC-1 cells were accompanied by an increase in expression of Puma, Noxa, and Bim mRNA, as well as by apoptosis, as evidenced by microscopy, TUNEL assay, and caspase cleavage. Viral-mediated overexpression of Mcl-1, Bcl-xL, or Bcl-2 in HMC-1 cells was found to introduce partial resistance against apoptosis-inducing effects of obatoclax. We were also able to show that obatoclax synergizes with several other antineoplastic drugs, including dasatinib, midostaurin, and bortezomib, in producing apoptosis and/or growth arrest in neoplastic MC. Together, obatoclax exerts major growth-inhibitory effects on neoplastic MC and potentiates the antineoplastic activity of other targeted drugs. Whether these drug effects can be translated to application in patients with advanced SM remains to be determined.
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http://dx.doi.org/10.1189/jlb.1112609DOI Listing
January 2014

Synergistic growth-inhibitory effects of ponatinib and midostaurin (PKC412) on neoplastic mast cells carrying KIT D816V.

Haematologica 2013 Sep 28;98(9):1450-7. Epub 2013 Mar 28.

Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Austria.

Patients with advanced systemic mastocytosis, including mast cell leukemia, have a poor prognosis. In these patients, neoplastic mast cells usually harbor the KIT mutant D816V that confers resistance against tyrosine kinase inhibitors. We examined the effects of the multi-kinase blocker ponatinib on neoplastic mast cells and investigated whether ponatinib acts synergistically with other antineoplastic drugs. Ponatinib was found to inhibit the kinase activity of KIT G560V and KIT D816V in the human mast cell leukemia cell line HMC-1. In addition, ponatinib was found to block Lyn- and STAT5 activity in neoplastic mast cells. Ponatinib induced growth inhibition and apoptosis in HMC-1.1 cells (KIT G560V(+)) and HMC-1.2 cells (KIT G560V(+)/KIT D816V(+)) as well as in primary neoplastic mast cells. The effects of ponatinib were dose-dependent, but higher IC50-values were obtained in HMC-1 cells harboring KIT D816V than in those lacking KIT D816V. In drug combination experiments, ponatinib was found to synergize with midostaurin in producing growth inhibition and apoptosis in HMC-1 cells and primary neoplastic mast cells. The ponatinib+midostaurin combination induced substantial inhibition of KIT-, Lyn-, and STAT5 activity, but did not suppress Btk. We then applied a Btk short interfering RNA and found that Btk knockdown sensitizes HMC-1 cells against ponatinib. Finally, we were able to show that ponatinib synergizes with the Btk-targeting drug dasatinib to produce growth inhibition in HMC-1 cells. In conclusion, ponatinib exerts major growth-inhibitory effects on neoplastic mast cells in advanced systemic mastocytosis and synergizes with midostaurin and dasatinib in inducing growth arrest in neoplastic mast cells.
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http://dx.doi.org/10.3324/haematol.2012.079202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3762103PMC
September 2013

5-azacytidine and decitabine exert proapoptotic effects on neoplastic mast cells: role of FAS-demethylation and FAS re-expression, and synergism with FAS-ligand.

Blood 2012 May 21;119(18):4242-52. Epub 2012 Mar 21.

Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Waehringer Guertel 18-20, Vienna, Austria

Aggressive systemic mastocytosis (ASM) and mast cell leukemia (MCL) are advanced hematopoietic neoplasms with poor prognosis. In these patients, neoplastic mast cells (MCs) are resistant against various drugs. We examined the effects of 2 demethylating agents, 5-azacytidine and decitabine on growth and survival of neoplastic MCs and the MC line HMC-1. Two HMC-1 subclones were used, HMC-1.1 lacking KIT D816V and HMC-1.2 exhibiting KIT D816V. Both agents induced apoptosis in HMC-1.1 and HMC-1.2 cells. Decitabine, but not 5-azacytidine, also produced a G(2)/M cell-cycle arrest in HMC-1 cells. Drug-induced apoptosis was accompanied by cleavage of caspase-8 and caspase-3 as well as FAS-demethylation and FAS-re-expression in neoplastic MCs. Furthermore, both demethylating agents were found to synergize with the FAS-ligand in inducing apoptosis in neoplastic MCs. Correspondingly, siRNA against FAS was found to block drug-induced expression of FAS and drug-induced apoptosis in HMC-1 cells. Neither 5-azacytidine nor decitabine induced substantial apoptosis or growth arrest in normal MCs or normal bone marrow cells. Together, 5-azacytidine and decitabine exert growth-inhibitory and proapoptotic effects in neoplastic MCs. These effects are mediated through "FAS-re-expression" and are augmented by the FAS-ligand. Whether epigenetic drugs produce antineoplastic effects in vivo in patients with ASM and MCL remains to be determined.
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http://dx.doi.org/10.1182/blood-2011-09-382770DOI Listing
May 2012

The PI3-kinase/mTOR-targeting drug NVP-BEZ235 inhibits growth and IgE-dependent activation of human mast cells and basophils.

PLoS One 2012 27;7(1):e29925. Epub 2012 Jan 27.

Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria.

The phosphoinositide 3-kinase (PI3-kinase) and the mammalian target of rapamycin (mTOR) are two major signaling molecules involved in growth and activation of mast cells (MC) and basophils (BA). We examined the effects of the dual PI3-kinase/mTOR blocker NVP-BEZ235 on growth of normal and neoplastic BA and MC as well as immunoglobulin E (IgE)-dependent cell activation. Growth of MC and BA were determined by measuring (3)H-thymidine uptake and apoptosis. Cell activation was determined in histamine release experiments and by measuring upregulation of CD63 and CD203c after challenging with IgE plus anti-IgE or allergen. We found that NVP-BEZ235 exerts profound inhibitory effects on growth of primary and cloned neoplastic MC. In the MC leukemia cell line HMC-1, NVP-BEZ235 showed similar IC(50) values in the HMC-1.1 subclone lacking KIT D816V (0.025 µM) and the HMC-1.2 subclone expressing KIT D816V (0.005 µM). Moreover, NVP-BEZ235 was found to exert strong growth-inhibitory effects on neoplastic MC in a xenotransplant-mouse model employing NMR1-Foxn1(nu) mice. NVP-BEZ235 also exerted inhibitory effects on cytokine-dependent differentiation of normal BA and MC, but did not induce growth inhibition or apoptosis in mature MC or normal bone marrow cells. Finally, NVP-BEZ235 was found to inhibit IgE-dependent histamine release in BA and MC (IC(50) 0.5-1 µM) as well as anti-IgE-induced upregulation of CD203c in BA and IgE-dependent upregulation of CD63 in MC. In summary, NVP-BEZ235 produces growth-inhibitory effects in immature neoplastic MC and inhibits IgE-dependent activation of mature BA and MC. Whether these potentially beneficial drug effects have clinical implications is currently under investigation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029925PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3267720PMC
July 2012

Polo-like kinase-1 as a novel target in neoplastic mast cells: demonstration of growth-inhibitory effects of small interfering RNA and the Polo-like kinase-1 targeting drug BI 2536.

Haematologica 2011 May 17;96(5):672-80. Epub 2011 Jan 17.

Department of Internal Medicine I, Division of Hematology & Hemostaseology, Medical University of Vienna, Austria.

Background: In advanced systemic mastocytosis the response of neoplastic mast cells to conventional drugs is poor and the prognosis is bad. Current research is, therefore, attempting to identify novel drug targets in neoplastic mast cells. Polo-like kinase-1 is a serine/threonine kinase that plays an essential role in mitosis and has recently been introduced as a new target in myeloid leukemias and solid tumors.

Design And Methods: In the present study, we analyzed the expression and function of Polo-like kinase-1 in neoplastic mast cells in systemic mastocytosis.

Results: As determined by immunostaining, primary neoplastic mast cells as well as the human mast cell leukemia cell line HMC-1 displayed phosphorylated Polo-like kinase-1. In addition, neoplastic mast cells expressed Polo-like kinase-1 mRNA. Polo-like kinase-1-specific small interfering RNA induced apoptosis in neoplastic mast cells, whereas no effect was seen with a control small interfering RNA. BI 2536, a drug targeting Polo-like kinase-1, was found to inhibit proliferation in HMC-1 cells in a dose-dependent manner. BI 2536 also inhibited the growth of primary neoplastic mast cells and cells of the canine mastocytoma cell line C2. The growth-inhibitory effects of BI 2536 on neoplastic mast cells were found to be associated with mitotic arrest and subsequent apoptosis. Finally, BI 2536 was found to synergize with the KIT-targeting kinase inhibitor midostaurin (PKC412) in inhibiting the growth of neoplastic mast cells. In control experiments, BI 2536 did not induce apoptosis in normal cultured mast cells.

Conclusions: Collectively, our data show that Polo-like kinase-1 is a potential therapeutic target in neoplastic mast cells. Targeting Polo-like kinase-1 may be an attractive pharmacological concept in the management of advanced systemic mastocytosis.
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http://dx.doi.org/10.3324/haematol.2010.031328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084913PMC
May 2011

KIT polymorphisms and mutations determine responses of neoplastic mast cells to bafetinib (INNO-406).

Exp Hematol 2010 Sep 26;38(9):782-91. Epub 2010 May 26.

Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria.

Objective: Advanced systemic mastocytosis (SM) is characterized by uncontrolled growth of neoplastic mast cells (MC) and drug resistance. The tyrosine kinase receptor KIT is often mutated and activated and thus contributes to malignant growth of MC. Therefore, KIT-targeting drugs are currently tested for their ability to block growth of malignant MC.

Materials And Methods: We determined the effects of the multikinase inhibitor INNO-406 (bafetinib) on primary neoplastic MC, the canine mastocytoma cell line C2, the human MC leukemia cell line HMC-1.1 bearing the KIT mutant V560G, and HMC-1.2 cells harboring KIT V560G and KIT D816V.

Results: INNO-406 was found to inhibit proliferation in HMC-1.1 cells (IC(50): 30-40 nM), but not in HMC-1.2 cells or primary neoplastic cells in patients with KIT D816V-positive SM. In canines, growth-inhibitory effects of INNO-406 were seen in C2 cells (IC(50): 50-100 nM) exhibiting a KIT exon 11 internal tandem-duplication and in primary neoplastic MC harboring wild-type exon 11, whereas no effects were seen in MC exhibiting a polymorphism at amino acid 581 in exon 11. INNO-406 was found to block KIT phosphorylation and expression in HMC-1.1 cells and C2 cells, but not in HMC-1.2 cells, whereas Lyn-phosphorylation was blocked by INNO-406 in all types of MC.

Conclusions: In neoplastic MC, the major target of INNO-406 appears to be KIT. Drug responses may depend on the presence and type of KIT mutation. In human MC, the KIT D816V mutant introduces resistance, and in canine mastocytomas, an exon 11 polymorphism may be indicative of resistance against INNO-406.
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http://dx.doi.org/10.1016/j.exphem.2010.05.004DOI Listing
September 2010

H1-receptor antagonists terfenadine and loratadine inhibit spontaneous growth of neoplastic mast cells.

Exp Hematol 2010 Oct 1;38(10):896-907. Epub 2010 Jun 1.

Department for Companion Animals and Horses, Clinic for Internal Medicine and Infectious Diseases, University of Veterinary Medicine Vienna, Vienna, Austria.

Objective: In mast cell (MC) neoplasms, clinical problems requiring therapy include local aggressive and sometimes devastating growth of MCs and mediator-related symptoms. A key mediator of MCs responsible for clinical symptoms is histamine. Therefore, use of histamine receptor (HR) antagonists is an established approach to block histamine effects in these patients.

Materials And Methods: We screened for additional beneficial effects of HR antagonists and asked whether any of these agents would also exert growth-inhibitory effects on primary neoplastic MCs, the human MC line HMC-1, and on two canine MC lines, C2 and NI-1.

Results: We found that the HR1 antagonists terfenadine and loratadine suppress spontaneous growth of HMC-1, C2, and NI-1 cells, as well as growth of primary neoplastic MCs in all donors tested (human patients, n = 5; canine patients, n = 8). The effects of both drugs were found to be dose-dependent (IC(50): terfenadine, 1-20 μM; loratadine, 10-50 μM). Both agents also produced apoptosis in neoplastic MCs and augmented apoptosis-inducing effects of two KIT-targeting drugs, PKC412 and dasatinib. The other HR1 antagonists (fexofenadine, diphenhydramine) and HR2 antagonists (famotidine, cimetidine, ranitidine) tested did not exert substantial growth-inhibitory effects on neoplastic MCs. None of the histamine receptor blockers were found to modulate cell-cycle progression in neoplastic MCs.

Conclusions: The HR1 antagonists terfenadine and loratadine, in addition to their antimediator activity, exert in vitro growth-inhibitory effects on neoplastic MCs. Whether these drugs (terfenadine) alone, or in combination with KIT inhibitors, can also affect in vivo neoplastic MC growth remains to be determined.
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http://dx.doi.org/10.1016/j.exphem.2010.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4337971PMC
October 2010

In vitro and in vivo growth-inhibitory effects of cladribine on neoplastic mast cells exhibiting the imatinib-resistant KIT mutation D816V.

Exp Hematol 2010 Sep 27;38(9):744-55. Epub 2010 May 27.

Department of Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria.

Objective: In most patients with systemic mastocytosis (SM), including aggressive SM (ASM) and mast cell (MC) leukemia (MCL), neoplastic cells express the oncogenic KIT mutation D816V, which confers resistance to imatinib. Cladribine (2CdA) is a nucleoside analog that has been introduced as a promising agent for treatment of advanced SM.

Materials And Methods: We examined the in vitro effects of 2CdA on growth of neoplastic MC, and the in vivo effects of 2CdA (0.13 mg/kg/day intravenously, days 1-5; three to eight cycles) in seven patients with advanced SM.

Results: Cladribine was found to inhibit growth of primary MC and the MC line HMC-1 in a dose-dependent manner, with lower IC(50) values recorded in HMC-1.2 cells harboring KIT D816V (IC(50): 10 ng/mL) compared to HMC-1.1 cells lacking KIT D816V (IC(50): 300 ng/mL). In two patients with progressive smoldering SM, 2CdA produced a long-lasting response with a sustained decrease in serum tryptase levels, whereas in patients with progressive ASM or MCL, 2CdA showed little if any effects. The drug was well-tolerated in most cases. However, one patient developed a massive generalized purulent long-lasting skin rash. The antiproliferative effects of 2CdA on MC were found to be associated with morphologic signs of apoptosis and caspase cleavage. Cladribine did not counteract the kinase activity of KIT D816V or KIT-downstream signaling molecules.

Conclusions: Cladribine may be a promising agent for treatment of progressive smoldering KIT D816V(+) SM. In rapidly progressing ASM or MCL, additional or alternative drugs are required to induce long-lasting antineoplastic effects.
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http://dx.doi.org/10.1016/j.exphem.2010.05.006DOI Listing
September 2010

Polo-like kinase 1 (Plk1) as a novel drug target in chronic myeloid leukemia: overriding imatinib resistance with the Plk1 inhibitor BI 2536.

Cancer Res 2010 Feb 9;70(4):1513-23. Epub 2010 Feb 9.

Department of Internal Medicine I, Division of Hematology and Hemostaseology, Institute of Immunology, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, A-1090 Vienna, Austria.

In most patients with chronic myeloid leukemia (CML), the disease can be kept under control using the BCR/ABL kinase inhibitor imatinib. Nevertheless, resistance or intolerance to imatinib and other BCR/ABL inhibitors may occur during therapy. Therefore, CML research is focusing on novel targets and targeted drugs. Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays an essential role in mitosis. In this study, we examined the expression of Plk1 in CML cells and its potential role as a therapeutic target. Plk1 was found to be expressed in phosphorylated form in the CML cell line K562 as well as in primary CML cells in all patients tested. Inhibition of BCR/ABL by imatinib or nilotinib (AMN107) led to decreased expression of the Plk1 protein in CML cells, suggesting that BCR/ABL promotes Plk1 generation. Silencing of Plk1 in CML cells by a small interfering RNA approach was followed by cell cycle arrest and apoptosis. Furthermore, the Plk1-targeting drug BI 2536 was found to inhibit proliferation of imatinib-sensitive and imatinib-resistant CML cells, including leukemic cells, carrying the T315 mutation of BCR/ABL with reasonable IC(50) values (1-50 nmol/L). The growth-inhibitory effects of BI 2536 on CML cells were found to be associated with cell cycle arrest and apoptosis. Moreover, BI 2536 was found to synergize with imatinib and nilotinib in producing growth inhibition in CML cells. In conclusion, Plk1 is expressed in CML cells and may represent a novel, interesting target in imatinib-sensitive and imatinib-resistant CML.
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http://dx.doi.org/10.1158/0008-5472.CAN-09-2181DOI Listing
February 2010

Identification of proapoptotic Bim as a tumor suppressor in neoplastic mast cells: role of KIT D816V and effects of various targeted drugs.

Blood 2009 Dec 22;114(26):5342-51. Epub 2009 Oct 22.

Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria.

Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In most cases, neoplastic cells display the D816V-mutated variant of KIT. KIT D816V exhibits constitutive tyrosine kinase (TK) activity and has been implicated in increased survival and growth of neoplastic MCs. Recent data suggest that the proapoptotic BH3-only death regulator Bim plays a role as a tumor suppressor in various myeloid neoplasms. We found that KIT D816V suppresses expression of Bim in Ba/F3 cells. The KIT D816-induced down-regulation of Bim was rescued by the KIT-targeting drug PKC412/midostaurin. Both PKC412 and the proteasome-inhibitor bortezomib were found to decrease growth and promote expression of Bim in MC leukemia cell lines HMC-1.1 (D816V negative) and HMC-1.2 (D816V positive). Both drugs were also found to counteract growth of primary neoplastic MCs. Furthermore, midostaurin was found to cooperate with bortezomib and with the BH3-mimetic obatoclax in producing growth inhibition in both HMC-1 subclones. Finally, a Bim-specific siRNA was found to rescue HMC-1 cells from PKC412-induced cell death. Our data show that KIT D816V suppresses expression of proapoptotic Bim in neoplastic MCs. Targeting of Bcl-2 family members by drugs promoting Bim (re)-expression, or by BH3-mimetics such as obatoclax, may be an attractive therapy concept in SM.
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http://dx.doi.org/10.1182/blood-2008-08-175190DOI Listing
December 2009

Growth-inhibitory effects of four tyrosine kinase inhibitors on neoplastic feline mast cells exhibiting a Kit exon 8 ITD mutation.

Vet Immunol Immunopathol 2009 Dec 18;132(2-4):243-50. Epub 2009 May 18.

Department of Companion Animals and Horses, Clinic for Internal Medicine and Infectious Diseases, University of Veterinary Medicine Vienna, Austria.

Systemic mastocytosis (SM) in felines is a rare neoplasm defined by increased growth and accumulation of immature mast cells (MC) in various organs including the spleen. Although in many cases splenectomy is an effective approach, relapses may occur. In these patients, treatment options are limited. Recent data suggest that various Kit tyrosine kinase inhibitors (TKI) interfere with growth of neoplastic MC in humans. In the current study, we examined the effects of four TKI, imatinib, midostaurin, nilotinib, and dasatinib, on growth of spleen-derived feline neoplastic MC in three SM patients. Expression of Kit in neoplastic MC was confirmed by flow cytometry and/or Western blotting. In all three cases, a 12-bp internal tandem duplication in exon 8, resulting in a four amino acid-insertion between residues Thr418 and His419 in Kit, was detectable. As assessed by (3)H-thymidine incorporation experiments, all four TKI were found to inhibit the growth of feline neoplastic MC in a dose-dependent manner. The growth-inhibitory TKI effects were found to be associated with morphologic signs of apoptosis in MC. In conclusion, various Kit-targeting TKI can inhibit the in vitro growth and survival of feline neoplastic MC in SM.
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http://dx.doi.org/10.1016/j.vetimm.2009.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763335PMC
December 2009

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

Exp Hematol 2008 Nov 23;36(11):1461-70. Epub 2008 Aug 23.

Department of Companion Animals and Horses, Clinic for Internal Medicine and Infectious Diseases, University of Veterinary Medicine Vienna, Austria.

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.
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http://dx.doi.org/10.1016/j.exphem.2008.06.002DOI Listing
November 2008

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

Exp Hematol 2008 Oct 10;36(10):1244-53. Epub 2008 Jul 10.

Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria.

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.
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http://dx.doi.org/10.1016/j.exphem.2008.04.017DOI Listing
October 2008

Fiberoptic intubation and laryngeal morbidity: a randomized controlled trial.

Anesthesiology 2007 Oct;107(4):585-90

Department of Anaesthesia, Spitalregion Rheintal Werdenberg Sarganserland, Walenstadt, Switzerland.

Background: Tracheal intubation with neuromuscular blocking agents is associated with a low incidence of minor vocal cord sequelae (8%). The aim of this noninferiority trial was to demonstrate that the frequency of vocal cord sequelae after fiberoptic intubation with a flexible silicone tube without neuromuscular blocking agents was less than 25% (maximum tolerable inferiority).

Methods: Two-hundred seventy patients were prospectively randomized to two groups. All intubations were performed by anesthesiologists with extensive experience in fiberoptic and conventional techniques. Fiberoptic nasotracheal intubation consisted of a bolus dose of 2 microg/kg fentanyl; 0.25 ml cocaine instillation, 10%, into nasal canals; cricothyroid injection of 2 ml lidocaine, 1%; bronchoscopy; administration of 0.3 mg/kg etomidate; and advancing a flexible silicone tube after loss of consciousness. Orotracheal intubation was performed with a polyvinyl chloride tube after induction with 2 microg/kg fentanyl, 2 mg/kg propofol, and 0.6 mg/kg rocuronium. Patients were examined by laryngoscopy before surgery, 24 h after surgery, and daily until complete restitution. Postoperative hoarseness was assessed by a standardized interview.

Results: The incidence of vocal cord sequelae was 11 out of 130 (8.5%) in the fiberoptic group versus 12 out of 129 (9.3%) in the control group (chi-square = 0.057, df = 1, P = 0.81; upper limit of the one-sided 95% confidence interval for the difference: +5.1%). There were no persistent injuries. The incidence of postoperative hoarseness was 4% in both groups.

Conclusions: Because fiberoptic intubation without neuromuscular blocking agents is safe regarding vocal cord sequelae, routine use is justified for anesthesiologists experienced in this technique.
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http://dx.doi.org/10.1097/01.anes.0000281925.61143.b5DOI Listing
October 2007

Expression of activins C and E induces apoptosis in human and rat hepatoma cells.

Carcinogenesis 2003 Nov 29;24(11):1801-9. Epub 2003 Aug 29.

Institute of Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria.

Activins C and E (homodimers of the betaC and betaE subunits), which are almost exclusively expressed in the liver, are members of the transforming growth factor beta (TGFbeta) superfamily of growth factors. We examined their expression in three different hepatoma cell lines and found that, compared with normal liver or primary hepatocytes, human hepatoblastoma (HepG2), human hepatocellular carcinoma (Hep3B) and rat hepatoma (H4IIEC3) cells have either completely lost or drastically reduced the expression of activins C and E. In order to elucidate the biological function of these proteins we transiently transfected HepG2, Hep3B and H4IIEC3 cell lines with rat activin betaC or betaE cDNA to study the consequences of restoring activin expression in hepatoma cells. Transfection with activin betaA, a known inhibitor of hepatic DNA synthesis and inducer of apoptosis, served as a positive control. We found that transfection of the three cell lines with activin betaC or betaE, as well as with activin betaA, reduced the increase in cell number by up to 40% compared with cells transfected with a control plasmid. Co-culture with a CHO cell clone secreting activin C also inhibited HepG2 cell multiplication. Furthermore, the three hepatoma cell lines studied showed an enhanced rate of apoptosis and elevated levels of active caspases in response to activin transfection. These results indicate that activins C and E share the potential to induce apoptosis in liver derived cell lines with activin A and TGFbeta1.
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http://dx.doi.org/10.1093/carcin/bgg154DOI Listing
November 2003

Follistatin overexpression in rodent liver tumors: a possible mechanism to overcome activin growth control.

Mol Carcinog 2002 Sep;35(1):1-5

Institute for Cancer Research, University of Vienna, Austria.

The activin-follistatin system is a potent growth regulatory system of liver tissue homeostasis. Activin A inhibits hepatocellular DNA synthesis and induces cell death. Follistatin binds activin and sequesters it from the signaling pathway. Consistently, follistatin has been reported to act as an inducer of DNA synthesis in the liver. Using RNase protection analysis, we studied the expression of follistatin in rat and mouse liver tumors as a possible mechanism to overcome activin growth control. Approximately 40% of the tumors (nine of 24 each), most of them hepatocellular carcinomas, displayed increased levels of follistatin mRNA when compared to tumor-surrounding liver tissue. The degree of overexpression was highly variable but independent of the carcinogen treatment that animals had received. It was also independent from the histological stage of malignancy and further found in rat liver adenomas. Follistatin expression was also observed in cell lines derived from human hepatocellular carcinomas. Overexpression of follistatin may represent a unique strategy of hepatic tumors to overcome the inhibitory action of a growth factor, activin, by decreasing its local bioavailability.
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http://dx.doi.org/10.1002/mc.10068DOI Listing
September 2002
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