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V. Caraffini et al.
hematopoiesis. RAS-signaling mutations skew hematopoiesis into the myelomonocytic lineage and ulti- mately drive the proliferation of these cells.1 Mechanistically, they constitutively activate downstream signaling cascades, including the RAS-mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and Phosphoinositide 3-kinase/AKT path- ways.2 While this already causes myelomonocytic lineage commitment and increased proliferation of hematopoietic stem and progenitor cells (HSPC) per se,1,3,4 it also increases the sensitivity to granulocyte macrophage-colony-stimulat- ing factor (GM-CSF),5 which augments these biological effects even further. Importantly, both increased myelomonocytic lineage commitment and proliferation are considered as key steps in the pathogenesis of myelomono- cytic leukemias. Indeed, RAS-signaling mutations are essen- tial players within the development of these malignancies and cause a myeloproliferative disease (MPD) with hyper- proliferation of the monocytic and granulocytic lineages in mice.6-9 In agreement with these data, RAS-signaling muta- tions are frequently detected in myeloid neoplasias. More than 10-20% of acute myeloid leukemia (AML) cases exhibit either NRAS or KRAS mutations, respectively.10-12 A myeloid neoplasia with particular dependence on aberrant RAS-signaling is chronic myelomonocytic leukemia (CMML), an aggressive malignancy characterized by increased myelomonocytic differentiation and prolifera- tion. Indeed, more than 40% of CMML patients exhibit one or more mutations in the RAS-signaling genes.13,14 Recently, it was shown that the extent of RAS-signaling activation in myeloid neoplasias is not only determined by the presence of mutations, but also by the aberrant expression profiles of one or more of its regulators. This can also be of relevance for RAS-driven myeloid leukemogenesis, as shown for the RAS-signaling inhibitor SPRY2, which demonstrates decreased expression levels in TET2-mutated patients.15
RAF kinase inhibitor protein (RKIP) is a negative regulator of various intracellular signaling modules, including the RAS-MAPK/ERK and nuclear factor-κB pathways.16,17 A somatic loss of RKIP expression has been described in a variety of solid cancers and a metastasis-suppressor func- tion could be shown in vitro and in vivo.18,19 We have previ- ously shown that a leukemia-specific loss of RKIP occurs in patients with therapy-related AML with a predisposing germline mutation in CRAF.20 On a functional level, RKIP drives the oncogenic potential of mutant CRAF, thereby contributing to leukemogenesis in these patients. Subsequently, we could show that RKIP loss is of relevance for other subtypes of AML as well.12,21,22 It occurs in up to 20% of AML cases and contributes to leukemogenesis by increasing the proliferation of AML cells.12,21 In agreement with the data from therapy-related AML (t-AML) patients with CRAF germline mutations, RKIP loss is correlated with RAS-signaling mutations and increased the leukemogenic potential of mutant RAS in a series of in vitro assays. Interestingly, we observed that RKIP loss is also correlated with myelomonocytic and monocytic AML phe- notypes, which suggests that RKIP might play a role in myelomonocytic differentiation as well.12,20,23
In this study, we aimed to clarify a connection between RKIP and myeloid skewing of hematopoiesis in more detail and demonstrate that RKIP loss contributes to myelomonocytic lineage commitment of HSPC in vitro and in vivo. We further show relevance of RKIP for RAS-driven myelomonocytic leukemogenesis, by demon-
strating that Rkip deletion aggravates myelomonocytic MPD development in NrasG12D-mutated mice. Mechanistically, we show that RKIP loss potentiates the RAS-induced activation of the RAS-MAPK/ERK signaling cascade. Finally, we prove the clinical relevance of these findings by showing that RKIP loss is a frequent event in primary CMML patient samples and frequently co-occurs with RAS-signaling mutations. These data establish RKIP as a novel player in RAS-driven myeloid leukemogenesis.
Methods
Primary patient samples and cell lines
Chronic myelomonocytic leukemia patient samples were col- lected at the Division of Hematology, Medical University of Graz, Austria, as well as in the Austrian Biodatabase for CMML. All samples were processed and stored as described in detail in the Online Supplementary Methods. Healthy CD34+ HSPC were collect- ed from umbilical cord blood specimens (EasySep, STEMCELL Technologies) according to the manufacturer´s instructions and processed as described before.24 Peripheral blood samples from healthy donors were used to collect CD14+ monocytes (MACS, Miltenyi Biotec), B lymphocytes and granulocytes (LymphoprepTM, STEMCELL Technologies and human B Lymphocyte enrichment set, BD biosciences) according to the manufacturer’s protocol. 293T, NB4 and HL-60 cell lines were obtained from the German National Resource Center for Biological Material (DSMZ, Braunschweig, Germany). Low pas- sage stocks were frozen and cells were always passaged for less than six months after resuscitation. Additionally, cells were screened by variable number of tandem repeat profiling (VNTR) for authenticity.21 Lentiviral transduction of cell lines and primary HSPC were performed as previously described.12,21,22
Mouse experiments
All mouse experiments were performed on a C57BL/6 strain background. Survival analyses were based on groups of at least eleven animals, all other experiments comprised at least three ani- mals. Genotyping was performed using tail tips as previously described.19,25 Mice with complete deletion of Rkip (Rkip-/-) as well as their controls (Rkip+/+) were obtained from Professor John Sedivy (Brown University, Providence, RI, USA). Mx1-Cre mice were obtained from Dr. Karen Blyth (Cancer Research UK Beatson Institute, Glasgow, UK), Nras-LSLG12D (JAX stock #008304; here- after referred to as Nras) from The Jackson Laboratory (Bar Harbor, ME, USA).26 Mx1-Cre and Nras animals were kept in a heterozygous situation and crossed to Rkip-/- and Rkip+/+ mice to obtain Mx1-Cre/Nras/Rkip-/- and Mx1-Cre/Nras/Rkip+/+ genotypes, respectively. Detailed procedures of mouse analysis are presented in the Online Supplementary Methods.
Immunoblot analysis, real time quantitative polymerase chain reaction, next-generation sequenc- ing, flow cytometry and in vitro differentiation assays
These assays were extensively described previously11,21,22,27–30 and are presented in detail in the Online Supplementary Methods.
Database retrieval and statistical analyses
Microarray expression data for RKIP expression in murine hematopoietic cell compartments were downloaded via the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/), accession numbers GSE27787,31 GSE5677,32 GSE2781633 and GSE20377.34 For the statistical analysis of in vitro and in vivo experiments, paired and unpaired Student’s t-tests, respectively, were employed. For
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