PRO-NECROPTOTIC RIPK3 AS A STAGE-DEPENDENT MARKER IN THE BONE MARROW OF PATIENTS WITH MDS AND CMML
Author(s): ,
Veronika Dill
Affiliations:
III. Medizinische Klinik für Hämatologie und internistische Onkologie,Klinikum rechts der Isar,München,Germany
,
Stefanie Jilg
Affiliations:
III. Medizinische Klinik für Hämatologie und internistische Onkologie,Klinikum rechts der Isar,München,Germany
,
Celina Wagner
Affiliations:
III. Medizinische Klinik für Hämatologie und internistische Onkologie,Klinikum rechts der Isar,München,Germany
,
Richard Hauch
Affiliations:
III. Medizinische Klinik für Hämatologie und internistische Onkologie,Klinikum rechts der Isar,München,Germany
,
Lars Buschhorn
Affiliations:
III. Medizinische Klinik für Hämatologie und internistische Onkologie,Klinikum rechts der Isar,München,Germany
,
Timo Odinius
Affiliations:
III. Medizinische Klinik für Hämatologie und internistische Onkologie,Klinikum rechts der Isar,München,Germany
,
Johanna Kauschinger
Affiliations:
III. Medizinische Klinik für Hämatologie und internistische Onkologie,Klinikum rechts der Isar,München,Germany
,
Adam Wahida
Affiliations:
III. Medizinische Klinik für Hämatologie und internistische Onkologie,Klinikum rechts der Isar,München,Germany
,
Ulrike Höckendorf
Affiliations:
III. Medizinische Klinik für Hämatologie und internistische Onkologie,Klinikum rechts der Isar,München,Germany
,
Julia Slotta-Huspenina
Affiliations:
Institut für Pathologie,Klinikum rechts der Isar,München,Germany
,
Peter Prodinger
Affiliations:
Klinik und Poliklinik für Orthopädie und Sportorthopädie,Klinikum rechts der Isar,München,Germany
,
Burkhard Schmidt
Affiliations:
Onkologie Pasing,München,Germany
,
Catharina Müller-Thomas
Affiliations:
Klinik für Hämatologie, Onkologie, Palliativmedizin, Immunologie und Innere Medizin,Klinikum Schwabing,München,Germany
,
Torsten Haferlach
Affiliations:
MLL,München,Germany
,
Wolfgang Kern
Affiliations:
MLL,München,Germany
,
Katharina Götze
Affiliations:
III. Medizinische Klinik für Hämatologie und internistische Onkologie,Klinikum rechts der Isar,München,Germany
,
Christian Peschel
Affiliations:
III. Medizinische Klinik für Hämatologie und internistische Onkologie,Klinikum rechts der Isar,München,Germany
,
Florian Bassermann
Affiliations:
III. Medizinische Klinik für Hämatologie und internistische Onkologie,Klinikum rechts der Isar,München,Germany
Philipp Jost
Affiliations:
III. Medizinische Klinik für Hämatologie und internistische Onkologie,Klinikum rechts der Isar,München,Germany
EHA Library. Dill V. Jun 15, 2019; 266944; PS1327
Veronika Dill
Veronika Dill
Contributions
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Abstract

Abstract: PS1327

Type: Poster Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 17:30 - 19:00

Location: Poster area

Background
Chronic inflammation plays a central role in the development of hematological neoplasms. In Myelodysplastic syndromes (MDS) pro-inflammatory microenvironment is suspected to drive disease development. Several studies have shown increased rates of programmed cell death in marrow cells of patients with early stage MDS. In these patients improvement of cytopenia is a major therapeutic approach. Overcoming resistance to programmed cell death is a central therapeutic aim in patients with high-risk MDS/sAML. RIPK3 is a crucial player of regulated necrosis (necroptosis). Necroptosis has a strong inflammatory capacity and pro-inflammatory processes trigger necroptosis. 

Aims
The aim of this project is to identify pro-necroptotic proteins in the bone marrow of patients with MDS, CMML and sAML and to correlate the necroptotic capacity with the stage of the disease. By pharmacological modulation of necroptosis we would like to evaluate the effects of necroptotic signaling on inflammation, cell death and cell differentiation. 

Methods
We therefore analyzed the impact of pro-necroptotic signaling on bone marrow bulk and the subset of CD34+stem/progenitor cells of patients with MDS and chronic myelomonocytic leukemia (CMML) in vitro. Bone marrow mononuclear cells (BMMNCs) were isolated from BM aspirates of 5 patients with early MDS (MDS-MLD, MDS-RS-SLD and MDS-RS-MLD), 5 patients with late MDS (MDS-EB1 and MDS-EB2), 5 patients with CMML-1, 7 patients with CMML-2 and 4 patients with sAML after a history of MDS and 4 patients with sAML after a history of CMML. Bulk of BMMNCs and purified CD34+stem/progenitor cells were stained intracellular for RIPK3 and analyzed by flow cytometry. The patient samples were compared to 12 age-matched BM samples obtained from hip replacement surgery from otherwise healthy individuals. Furthermore, immunohistochemistry was performed in an enlarged cohort of patients. Therefore paraffin embedded bone marrow biopsies of individuals with MDS or CMML-1/2 were analyzed over time and disease progression. Additionally BMMNCs of patients with MDS were treated with specific RIPK1 (Nec1s) and RIPK3 (GSK 843A) inhibitors for 72h. Short term viability analysis by flow cytometry were performed and long-term analysis using growth factor enriched methylcellulose.

Results
Expression of  RIPK3 was significantly increased in sAML after a history of MDS compared to late MDS and compared to healthy controls. Furthermore patients with sAML after a history of CMML showed a significantly higher expression of RIPK3 than patients with CMML-1, CMML-2 and healthy controls. These findings were confirmed by immunohistochemistry in an enlarged cohort of patients. Here paraffin embedded bone marrow biopsies of individuals with MDS or CMML-1/2 were analyzed over time and disease progression.

Additionally BMMNCs of patients with MDS or CMML were treated with specific RIPK1 (Nec1s) and RIPK3 (GSK 843A) inhibitorsfor 72h. Short term viability analysis by flow cytometry showed no impact of inhibitor treatment. However after culture in growth factor enriched methylcellulose for 10-14 days colony forming capacity was significantly increased in low-risk MDS patients after inhibition of pro-necroptotic signaling. Here increase in colony numbers was not restricted to a specific lineage.

Conclusion
We conclude that RIPK3 plays a critical stage-dependent role in patients with MDS and CMML. Disease progression to sAML is associated with an increased RIPK3-Expression. We further hypothesize that pro-inflammatory processes like necroptosis have an impact on aberrant myeloid homeostasis.

Session topic: 9. Myelodysplastic syndromes - Biology & Translational Research

Keyword(s): Bone Marrow, Inflammation, MDS, Stem and progenitor cell

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