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LOSS OF RAF KINASE INHIBITOR PROTEIN IS INVOLVED IN MYELOMONOCYTIC LINEAGE COMMITMENT AND AGGRAVATES THE DEVELOPMENT OF CHRONIC MYELOMONOCYTIC LEUKEMIA IN A MURINE IN-VIVO MODEL
Author(s):
Veronica Caraffini
,
Veronica Caraffini
Affiliations:
Division of Hematology,Medical University of Graz,Graz,Austria
Olivia Geiger
,
Olivia Geiger
Affiliations:
Division of Hematology,Medical University of Graz,Graz,Austria
Angelika Rosenberger
,
Angelika Rosenberger
Affiliations:
Division of Hematology,Medical University of Graz,Graz,Austria
Stefan Hatzl
,
Stefan Hatzl
Affiliations:
Division of Hematology,Medical University of Graz,Graz,Austria
Bianca Perfler
,
Bianca Perfler
Affiliations:
Division of Hematology,Medical University of Graz,Graz,Austria
Johannes Lorenz Berg
,
Johannes Lorenz Berg
Affiliations:
Division of Hematology,Medical University of Graz,Graz,Austria
Clarice Lim
,
Clarice Lim
Affiliations:
Institute of Pathophysiology and Immunology,Medical University of Graz,Graz,Austria
Herbert Strobl
,
Herbert Strobl
Affiliations:
Institute of Pathophysiology and Immunology,Medical University of Graz,Graz,Austria
Karl Kashofer
,
Karl Kashofer
Affiliations:
Institute of Pathology,Medical University of Graz,Graz,Austria
Gerald Hoefler
,
Gerald Hoefler
Affiliations:
Institute of Pathology,Medical University of Graz,Graz,Austria
Klaus Geissler
,
Klaus Geissler
Affiliations:
Medical Department with Hematology, Oncology and Palliative Medicine,Hospital Hietzing,Vienna,Austria
Walter Kolch
,
Walter Kolch
Affiliations:
Systems Biology Ireland & Conway Institute,University College Dublin,Dublin,Ireland
Karen Blyth
,
Karen Blyth
Affiliations:
Cancer Research UK Beatson Institute,Glasgow,United Kingdom
Dimitris Athineos
,
Dimitris Athineos
Affiliations:
Cancer Research UK Beatson Institute,Glasgow,United Kingdom
Albert Wölfler
,
Albert Wölfler
Affiliations:
Division of Hematology,Medical University of Graz,Graz,Austria
Heinz Sill
,
Heinz Sill
Affiliations:
Division of Hematology,Medical University of Graz,Graz,Austria
Armin Zebisch
Armin Zebisch
Affiliations:
Division of Hematology,Medical University of Graz,Graz,Austria
(Abstract release date: 05/18/17) EHA Library. Caraffini V. 06/24/17; 181706; S419
Veronica Caraffini
Veronica Caraffini
Contributions
PDF Abstract
Abstract

Abstract: S419

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 12:00 - 12:15

Location: Hall C

Background

Chronic myelomonocytic leukemia (CMML) is characterized by increased proliferation and myelomonocytic lineage commitment of hematopoietic stem cells (HSCs). Mutations in the RAS-pathway occur frequently in CMML patients and lead to a CMML-like myeloproliferative disorder (CMML-MPD) in mice via causing hypersensitivity to GM-CSF. Loss of RAF kinase inhibitor protein (RKIP), a negative regulator of RAS signaling, is frequent in myelomonocytic and monocytic subtypes of acute myeloid leukemia (AML) and is often associated with RAS mutations. Moreover, RKIP loss has recently been shown to increase the proliferation of AML cell lines.

Aims
In this work, we aimed at investigating the role of RKIP in the development of CMML.

Methods
RKIP expression was measured by immunoblot and quantitative real-time PCR in 23 primary CMML patient samples as well as in CD34+ HSCs, B-lymphocytes, granulocytes and monocytes of four healthy donors. Sequence analysis of CMML samples was done with an Ion Torrent Next Generation Sequencing platform using an amplicon panel covering 39 genes recurrently mutated in myeloid neoplasms. Effects of RKIP on GM-CSF-induced myelomonocytic differentiation were studied in human CD34+ HSCs lentivirally transduced with RKIP shRNA, as well as in a genetic mouse model for RKIP deletion (RKIP-/-). Effects of RKIP on CMML development were initially studied in the same RKIP-/- model. Additionally, these mice were crossed with animals exhibiting a somatically inducible mutation in NRAS (RKIP-/-Mx1-Cre-NRASG12D) and the severity of CMML-MPD onset was studied at an age of six months.

Results

Loss of RKIP protein expression was observed in 6/23 (26%) CMML patient specimens and was associated with decreased mRNA levels as well (P<0.001). Patients with RKIP loss exhibited an increased percentage of myelomonocytic cells in the peripheral blood (86% vs 75%; P=0.0226). One or more mutations affecting the RAS signaling pathway were detected in all specimens with RKIP loss. In addition to the previously demonstrated induction of proliferation, we then aimed to delineate a role of RKIP loss in myeloid lineage commitment. When studying healthy donors, we observed that RKIP expression was high in HSCs and lymphoid cells, but significantly decreased in cells belonging to the myeloid lineage (monocytes, P=0.001 and granulocytes, P<0.001). In functional experiments, knockdown of RKIP increased the GM-CSF-induced myelomonocytic lineage commitment of both, human and murine HSCs (P<0.05 and P=0.0295, respectively). These results could be corroborated in-vivo, as intraperitoneal injection of GM-CSF caused a significant increase of myelomonocytic cells in the intraperitoneal cavity (P=0.006), bone marrow (P=0.007) and peripheral blood (P=0.027) in RKIP-/- mice when compared to their wildtype littermates. In a final step, we evaluated the potential of RKIP loss to cause CMML-MPD in mice. While it proved to be insufficient to cause the disease as a single event in RKIP-/- mice, it aggravated the CMML-MPD phenotype in animals carrying an additional mutation in NRAS. In this case, RKIP deletion caused worsening of leucocytosis (P=0.036) and splenomegaly (P=0.035), which was associated with increased levels of myelomonocytic cells in the bone marrow (P=0.028), peripheral blood (P=0.002) and spleen (P=0.025).

Conclusion
RKIP loss is a frequent event in CMML and is associated with mutations affecting the RAS signaling cascade. Loss of RKIP is functionally involved in myelomonocytic lineage commitment of HSCs and aggravates CMML-MPD development in mice carrying an additional mutation in NRAS.

Session topic: 15. Myeloproliferative neoplasms - Biology

Abstract: S419

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 12:00 - 12:15

Location: Hall C

Background

Chronic myelomonocytic leukemia (CMML) is characterized by increased proliferation and myelomonocytic lineage commitment of hematopoietic stem cells (HSCs). Mutations in the RAS-pathway occur frequently in CMML patients and lead to a CMML-like myeloproliferative disorder (CMML-MPD) in mice via causing hypersensitivity to GM-CSF. Loss of RAF kinase inhibitor protein (RKIP), a negative regulator of RAS signaling, is frequent in myelomonocytic and monocytic subtypes of acute myeloid leukemia (AML) and is often associated with RAS mutations. Moreover, RKIP loss has recently been shown to increase the proliferation of AML cell lines.

Aims
In this work, we aimed at investigating the role of RKIP in the development of CMML.

Methods
RKIP expression was measured by immunoblot and quantitative real-time PCR in 23 primary CMML patient samples as well as in CD34+ HSCs, B-lymphocytes, granulocytes and monocytes of four healthy donors. Sequence analysis of CMML samples was done with an Ion Torrent Next Generation Sequencing platform using an amplicon panel covering 39 genes recurrently mutated in myeloid neoplasms. Effects of RKIP on GM-CSF-induced myelomonocytic differentiation were studied in human CD34+ HSCs lentivirally transduced with RKIP shRNA, as well as in a genetic mouse model for RKIP deletion (RKIP-/-). Effects of RKIP on CMML development were initially studied in the same RKIP-/- model. Additionally, these mice were crossed with animals exhibiting a somatically inducible mutation in NRAS (RKIP-/-Mx1-Cre-NRASG12D) and the severity of CMML-MPD onset was studied at an age of six months.

Results

Loss of RKIP protein expression was observed in 6/23 (26%) CMML patient specimens and was associated with decreased mRNA levels as well (P<0.001). Patients with RKIP loss exhibited an increased percentage of myelomonocytic cells in the peripheral blood (86% vs 75%; P=0.0226). One or more mutations affecting the RAS signaling pathway were detected in all specimens with RKIP loss. In addition to the previously demonstrated induction of proliferation, we then aimed to delineate a role of RKIP loss in myeloid lineage commitment. When studying healthy donors, we observed that RKIP expression was high in HSCs and lymphoid cells, but significantly decreased in cells belonging to the myeloid lineage (monocytes, P=0.001 and granulocytes, P<0.001). In functional experiments, knockdown of RKIP increased the GM-CSF-induced myelomonocytic lineage commitment of both, human and murine HSCs (P<0.05 and P=0.0295, respectively). These results could be corroborated in-vivo, as intraperitoneal injection of GM-CSF caused a significant increase of myelomonocytic cells in the intraperitoneal cavity (P=0.006), bone marrow (P=0.007) and peripheral blood (P=0.027) in RKIP-/- mice when compared to their wildtype littermates. In a final step, we evaluated the potential of RKIP loss to cause CMML-MPD in mice. While it proved to be insufficient to cause the disease as a single event in RKIP-/- mice, it aggravated the CMML-MPD phenotype in animals carrying an additional mutation in NRAS. In this case, RKIP deletion caused worsening of leucocytosis (P=0.036) and splenomegaly (P=0.035), which was associated with increased levels of myelomonocytic cells in the bone marrow (P=0.028), peripheral blood (P=0.002) and spleen (P=0.025).

Conclusion
RKIP loss is a frequent event in CMML and is associated with mutations affecting the RAS signaling cascade. Loss of RKIP is functionally involved in myelomonocytic lineage commitment of HSCs and aggravates CMML-MPD development in mice carrying an additional mutation in NRAS.

Session topic: 15. Myeloproliferative neoplasms - Biology

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