TNF-ALPHA PROMOTES CLONAL EXPANSION OF KIT D816V+ CELLS IN SYSTEMIC MASTOCYTOSIS
Author(s): ,
Georg Greiner
Affiliations:
Department of Laboratory Medicine, Medical University of Vienna,Medical University of Vienna,Vienna,Austria;Ludwig Boltzmann Institute of Hematology and Oncology, Medical University of Vienna,Medical University of Vienna,Vienna,Austria
,
Nadine Witzeneder
Affiliations:
Department of Laboratory Medicine, Medical University of Vienna,Medical University of Vienna,Vienna,Austria;Ludwig Boltzmann Institute of Hematology and Oncology, Medical University of Vienna,Medical University of Vienna,Vienna,Austria;Department of Internal Medicine I, Division of Hematology and Hemostaseology,Medical University of Vienna,Vienna,Austria
,
Klara Klein
Affiliations:
Institute of Pharmacology and Toxicology,University of Veterinary Medicine Vienna,Vienna,Austria
,
Eva Jaeger
Affiliations:
Department of Laboratory Medicine, Medical University of Vienna,Medical University of Vienna,Vienna,Austria
,
Michael Gurbisz
Affiliations:
Department of Laboratory Medicine, Medical University of Vienna,Medical University of Vienna,Vienna,Austria
,
Marlene Gerner
Affiliations:
Department of Laboratory Medicine, Medical University of Vienna,Medical University of Vienna,Vienna,Austria
,
Sigrid Baumgartner
Affiliations:
Division of Neonatology, Pediatric Intensive Care and Neuropediatrics, Department of Pediatrics and Adolescent Medicine,Medical University of Vienna,Vienna,Austria
,
Simone Tangermann
Affiliations:
Unit of Laboratory Animal Pathology,University of Veterinary Medicine Vienna,Vienna,Austria
,
Karin Fruehwirth
Affiliations:
Department of Laboratory Medicine, Medical University of Vienna,Medical University of Vienna,Vienna,Austria;University of Applied Science,FH Campus Vienna,Vienna,Austria
,
Ilse Schwarzinger
Affiliations:
Department of Laboratory Medicine, Medical University of Vienna,Medical University of Vienna,Vienna,Austria
,
Ingrid Simonitsch-Klupp
Affiliations:
Department of Pathology,Medical University of Vienna,Vienna,Austria
,
Klaus Schmetterer
Affiliations:
Department of Laboratory Medicine, Medical University of Vienna,Medical University of Vienna,Vienna,Austria
,
Matthias Mayerhofer
Affiliations:
Ludwig Boltzmann Institute of Osteology,Hanusch Hospital,Vienna,Austria
,
Lukas Kenner
Affiliations:
Unit of Laboratory Animal Pathology,University of Veterinary Medicine Vienna,Vienna,Austria;Department of Pathology,Medical University of Vienna,Vienna,Austria;Ludwig Boltzmann Institute for Cancer Research,Ludwig Boltzmann Institute for Cancer Research,Vienna,Austria
,
Wolfgang R. Sperr
Affiliations:
Ludwig Boltzmann Institute of Hematology and Oncology, Medical University of Vienna,Medical University of Vienna,Vienna,Austria;Department of Internal Medicine I, Division of Hematology and Hemostaseology,Medical University of Vienna,Vienna,Austria
,
Veronika Sexl
Affiliations:
Institute of Pharmacology and Toxicology,University of Veterinary Medicine Vienna,Vienna,Austria
,
Michel Arock
Affiliations:
Laboratory of Hematology,Pitié-Salpêtrière Hospital,Paris,France
,
Peter Valent
Affiliations:
Ludwig Boltzmann Institute of Hematology and Oncology, Medical University of Vienna,Medical University of Vienna,Vienna,Austria;Department of Internal Medicine I, Division of Hematology and Hemostaseology,Medical University of Vienna,Vienna,Austria
Gregor Hoermann
Affiliations:
Department of Laboratory Medicine, Medical University of Vienna,Medical University of Vienna,Vienna,Austria;Ludwig Boltzmann Institute of Hematology and Oncology, Medical University of Vienna,Medical University of Vienna,Vienna,Austria;Central Institute of Medical and Chemical Laboratory Diagnostics,University Hospital Innsbruck,Innsbruck,Austria
EHA Library. Greiner G. Jun 15, 2019; 267051; S888
Georg Greiner
Georg Greiner
Contributions
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Abstract

Abstract: S888

Type: Oral Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 16:45 - 17:00

Location: Elicium 1

Background
Systemic mastocytosis (SM) is characterized by clonal mast cell (MC) infiltration in the bone marrow (BM). Most patients harbor the activating KIT mutation D816V. Cytokines are substantially involved in the interactions between neoplastic cells and the BM microenvironment in SM and in classical myeloproliferative neoplasms (MPN) and have been recognized as a major trigger of disease evolution. The pro-apoptotic/anti-proliferative and pro-inflammatory cytokine tumor necrosis factor alpha (TNFa) has been identified as an important driver for the expansion of JAK2 V617F+ clones and the establishment of MPN. 

Aims
Investigation of the potential role of TNFa in disease evolution of SM patients. 

Methods
In vitro and in vivo experiments with MC lines and primary material of SM patients.

Results
In a first step, lentivirally-driven expression of the KIT D816V mutant gene in the human Mo7e cells suggested an oncogene-dependent regulation of the production of TNFa. Correspondingly, the human neoplastic KIT D816V+ MC lines HMC-1.2 and ROSAKIT D816V expressed and secreted substantial amounts of TNFa and inhibition of KIT D816V either by the recently approved multi-kinase inhibitor midostaurin or by RNAi-mediated knockdown of KIT significantly reduced expression of this cytokine. Mechanistically, we studied auto-/paracrine effects of TNFa on the clonal selection of neoplastic MC using KIT D816V negative (ROSAKIT WT) and positive (ROSAKIT D816V) subclones of ROSA cells. TNFa reduced proliferation of ROSAKIT WT cells (84.9%±4.6% of control, p<0.01) whereas ROSAKIT D816V cells were resistant to TNFa (102.7%±3.7% of control, n.s.). Preliminary data also indicate a relative resistance of primary neoplastic cells obtained from KIT D816V+ SM patients against TNFa in colony-forming unit (CFU) assay. To determine the mechanism of TNFa resistance in KIT D816V+ cells, we screened for key regulators of apoptosis and identified the anti-apoptotic protein survivin to be highly upregulated in KIT D816V+ ROSA cells upon TNFa treatment but not in KIT D816V- ROSA cells. We hypothesize that KIT D816V+ MC produce substantial amounts of TNFa that induces apoptosis and suppresses proliferation of normal BM cells while MC themselves are relatively resistant via upregulation of survivin. In a next step, we studied the effect of TNFa on growth of neoplastic MC in vivo in a xenotransplant model using ROSAKIT D816V cells with and without knockout of TNFa in NOD-SCID IL-2Rγ-null (NSG) mice. CRISPR/Cas9-mediated knockout completely abolished expression and secretion of TNFa in ROSAKIT D816V cells and resulted in a significantly longer survival of mice compared to mice injected with TNFa expressing controls (median 49 vs. 58 days, p<0.05). Finally, the clinical relevance of TNFa expression was studied in a SM patient’s cohort (n=45). TNFa levels in sera of patients were significantly higher (median 1.95, range 0.72-16.64 pg/ml, p=0.002) compared to age- and sex-matched controls (median 1.49, range 0.51-5.63 pg/ml). Importantly, when we stratified SM patients into those with clearly elevated TNFa levels and those with TNFa levels within the reference range of controls (≤3.63 pg/ml) a significantly lower overall survival was observed for SM patients with high TNFa serum levels (median 6.3 years vs. not reached, p<0.005).

Conclusion
In summary, we have identified TNFa as a critical, KIT D816V-dependent, cytokine-mediator that promotes clonal expansion of KIT D816V+ MC and may thereby be involved in clonal evolution and disease progression in SM.

Session topic: 15. Myeloproliferative neoplasms - Biology & Translational Research

Keyword(s): Cytokine, Tumor necrosis factor alpha (TNFa)

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