Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Caraffini, V.
Analysis of the role of RAF kinase inhibitor protein in the development of myeloid neoplasias
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Graz Medical University; 2019. pp. 136
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Höfler Gerald
- Sill Heinz
- Wölfler Albert
- Zebisch Armin
- Altmetrics:
- Abstract:
- RAF kinase inhibitor protein (RKIP) is a negative regulator of the RAS-MAPK/ERK pathway. It is a well-known metastasis-suppressor in solid cancers and a somatic loss of RKIP has been described frequently in these entities. Around 20% of acute myeloid leukemia (AML) cases show a functionally relevant loss of RKIP, which correlates with RAS mutations and myelomonocytic AML phenotypes. In this thesis, we aimed at extending the knowledge about RKIP in hematologic neoplasias even further. First, we clarified the role of RKIP in myeloid sarcoma (MS). MS is a subtype of AML, in which leukemic blasts form solid tumor masses in non-hematopoietic tissues. As this process resembles the development of metastases in solid tumors, we hypothesized that RKIP loss drives the tissue infiltration of leukemic cells. We could show that knockdown of RKIP promotes the invasion and migration of AML cells in-vitro. This could be confirmed in-vivo, where AML cells with RKIP knockdown invaded into, and formed tumor masses in the chorioallantoic membrane of chicken embryos. Mechanistically, we could show that RKIP loss induces the tissue infiltration of leukemic blasts via RAS-MAPK/ERK independent mechanisms and identified candidate effector genes via microarray analyses in RKIP knockdown cells. Finally, we could prove the clinical relevance of these findings by demonstrating that RKIP loss correlates with the presence of MS in primary AML patient specimens. We could additionally show that RKIP loss correlated with RAS-signaling mutations in MS using Next-Generation Sequencing (NGS). This suggests a functional synergism between these events in myeloid leukemogenesis. Secondly, we studied the role of RKIP in myelomonocytic differentiation. Since the RAS-MAPK/ERK pathway plays a role in the myeloid lineage commitment of hematopoietic stem and progenitor cells (HSPCs), and as RKIP loss correlates with myelomonocytic AML phenotypes, we hypothesized that RKIP loss plays a role in myelomonocytic differentiation and in the development of myelomonocytic neoplasias. In physiologic hematopoiesis, we observed low RKIP levels in myeloid lineage cells. Then, we could prove a functional involvement of RKIP loss in myelomonocytic differentiation by modulating RKIP in CD34+ HSPCs and HL-60 AML cells. These results could be confirmed in mice with a complete deletion of Rkip (Rkip-/-): GM-CSF induced formation of myelomonocytic cells was significantly increased in Rkip-/- animals. We then studied the role of RKIP in chronic myelomonocytic leukemia (CMML), which is characterized by increased myelomonocytic proliferation and frequent occurrence of RAS-signaling mutations. Therefore, it is an ideal model to study RKIP in myelomonocytic leukemogenesis and to investigate a potential interaction between RKIP loss and RAS mutations. We crossed Rkip-/- mice with animals carrying an inducible NrasG12D mutation. These mice develop a myeloproliferative disease resembling CMML (CMML-MPD). Additional deletion of Rkip caused a significant aggravation of CMML-MPD, proving the functional synergism between RKIP loss and RAS-signaling mutations in myeloid leukemogenesis. Mechanistically, we could show that loss of RKIP expression increases the activity of the RAS-MAPK/ERK signaling cascade within these leukemia models. Finally, we studied a cohort of primary CMML patient specimens. RKIP loss occurred in more than 30% of cases and again co-existed with RAS-signaling mutations. Altogether, we show that loss of RKIP expression is a relevant event for myeloid leukemogenesis. Firstly, we demonstrate that it drives the tissue infiltration of myeloid blasts and thereby contributes to the development of MS. Then, we show that it contributes to myelomonocytic differentiation and the development of CMML. Finally, we corroborate the association between RKIP loss and RAS-signaling mutations and demonstrate a functional synergism between these events in myeloid leukemogenesis.