Impact of RAS-Pathway Activation on Phenotype and Outcome in Patients with Chronic Myelomonocytic Leukemia and a TET2/SRSF2 Comutation
Article Information
Klaus Geissler1,2*, Eva Jäger3, Agnes Barna4, Michael Gurbisz3, Temeida Graf2, Elmir Graf2, Maike Stegemann2, Thomas Nösslinger5, Michael Pfeilstöcker5, Sigrid Machherndl-Spandl6, Reinhard Stauder7, Armin Zebisch8,9, Heinz Sill8, Leopold Öhler10, Rajko Kusec11, Gregor Hoermann3,12,13, and Peter Valent13,14
1Medical School, Sigmund Freud University, Vienna, Austria
2Department of Internal Medicine V with Hematology, Oncology and Palliative Medicine, Hospital Hietzing, Vienna, Austria
3Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
4Blood Transfusion Service, Blood Transfusion Service for Upper Austria, Austrian Red Cross, Linz, Austria
5Department of Internal Medicine III, Hanusch Hospital, Vienna, Austria
6Department of Internal Medicine I with Hematology with Stem Cell Transplantation, Hemostaseology and Medical Oncology, Ordensklinikum Linz Barmherzige Schwestern - Elisabethinen, Linz, Austria
7Internal Medicine V with Hematology and Oncology, Medical University of Innsbruck, Innsbruck, Austria
8Department of Internal Medicine, Division of Hematology, Medical University of Graz, Graz, Austria
9Otto-Loewi Research Center for Vascular Biology, Immunology and Inflammation, Division of Pharmacology, Medical University of Graz, Austria
10Department of Internal Medicine/Oncology, St. Josef Hospital, Vienna, Austria
11School of Medicine, University of Zagreb, University Hospital Dubrava, Zagreb, Croatia
12Central Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Innsbruck, Innsbruck, Austria
13Ludwig Boltzmann Institute for Hematology and Oncology (LBI HO), Medical University of Vienna, Vienna, Austria
14Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria
Parameter |
TET2/SRSF2 without RASopathy mutations N=50 |
TET2/SRSF2 with RASopathy Mutations N=37 |
p-Value |
Age |
74.5 (50-92) |
77 (55-93) |
0.1936 |
Sex (% males) |
29/50 (58%) |
28/37 (76%) |
0.0864 |
WBC G/L |
12.8 (2.9-121.5) |
22.3 (3.8-86.7) |
0.0151 |
Hb g/dL |
11.9 (8.1-15.1) |
10.8 (7.0-15.3) |
0.0188 |
Plt G/L |
108 (22-448) |
67 (16-309) |
0.0071 |
PB Blasts (% present) |
8/45 (17%) |
12/34 (35%) |
0.0763 |
Splenomegaly |
4/23 (17%) |
9/15 (60%) |
0.0068 |
Transformation |
2/50 (4%) |
7/37 (19%) |
0.0239 |
Table 1: The phenotype stratified by the presence or absence of RASopathy gene mutations in patients with CMML and a TET2/SRSF2 comutation.
Figure 2: Box plots showing the distribution of spontaneous myeloid colony numbers in TET2/SRSF2-mutant CMML patients with or without additional mutations in RASopathy genes including median values, minimum values, maximum values, as well as upper and lower quartiles, respectively. Cultures were plated in duplicates or triplicates, respectively, at 25-100 × 103 PBMNC/mL. Aggregates with more than 40 translucent, dispersed cells were counted as CFU-GM. CFU-GM data from patients are expressed as mean values from cultures.
4.Discussion
The RAS signaling pathway is one of the major signaling pathways which are involved into the the transduction of growth stimulatory signals from GF receptors to the nucleus where cell programs associated with cell growth are activated. The “RASopathies” are a group of genetic syndromes caused by germline mutations in genes that encode components of the RAS signaling pathway including NRAS, KRAS, NF-1, CBL, and PTPN11 [17-22]. Besides their developmental defects they share a predisposition to juvenile myelomonocytic leukemia, a hematologic malignancy of early childhood. Moreover, spontaneous molecular aberrations of components of this pathway are the most frequently mutated genes in cancers. In the preclinical mouse model myelomonocytic leukemias can be recapitulated by transplantation of mouse BM cells harboring an oncogenic mutation in the Nras locus [23]. Interestingly, alterations of the other RASopathy genes may also lead to a similar phenotype in preclinical mouse models [24-28]. Mice develop a myeloproliferative disorder with clonal expansion of the granulomonopoiesis in vivo and show spontaneous in vitro myeloid colony formation without exogenous growth factors due to aberrant GM-CSF signaling.
Despite a number of studies investigating the role of the RAS signaling pathway in CMML remains controversial. There are studies showing that mutations RAS components are associated with inferior survival but there are also studies which failed to show this. The presence or absence of a RAS point mutation was not a significant prognostic parameter in a univariate analysis from 66 CMML patients reported by Onida in 2002 [5]. In univariate analysis OS rates decreased in 312 molecularly characterized CMML patients with mutations in CBL and there was a trend towards shorter survival in NRAS mutated patients in a study by Itzykson [6]. Since NRAS mutations significantly affected survival in univariate analysis and retained its prognostic value in multivariable Cox regression mutations in NRAS were integrated in the risk assessment of 214 patients with CMML by Elena [7]. An international dataset analysed by Padron revealed in addition to ASXL1 mutations mutations in CBL but not in NRAS or KRAS as a new molecular independent prognostic parameter [8]. Restricting analysis to molecularly defined subgroups may be more appropriate to study the effects of RAS pathway mutations on the phenotype and clinical outcome. In the preclinical mouse model it is possible to work with clearly defined molecular categories. Im mice with a TET2-mutant background, the transfection of NRAS mutation convincingly aggravated the disease [29, 30]. Here we show in CMML cells from patients with TET2/SRSF2 comutation that the presence of mutations in RASopathy genes clearly changed the phenotype of disease and shortened survival.
The basis for all RAS pathway-oriented treatment concepts is the identification of RAS pathway hyperactivation in patients. Due to the fact that in CMML more than one molecular aberration can be detected in the majority of patients, functional tests may be important to better estimate the contribution of a particular molecular aberration in the pathogenesis of the malignancy. We recently were able to show that spontaneous CFU-GM formation may be a functional surrogate of RAS pathway activation [12, 13]. Hyperactivation of the RAS signaling pathway in this study was not only demonstrated at the molecular level but also at a functional level by comparing the number of growth factor independent myeloid colony formation in CMML patients with the TET2/SRSF2 comutation and the presence or absence of additional mutations in RASopathy genes. Spontaneous colony formation was much higher in patients with molecular aberrations of the RAS signaling pathway as compared to patients without these aberrations. In one patient who had serial determinations of the mutational landscape and in vitro colony formation we can directly demonstrate that the evolution of an NRAS clone was paralleled by increasing myeloid colony growth.
The clinical implication of our findings is supported by the recent development of novel RAS pathway inhibitors [31]. Since RAS is the most frequently mutated gene family in cancers, investigators have sought an effective RAS inhibitor for more than three decades. RAS inhibitors, however, were so elusive that RAS was termed ‘undruggable’. With the recent success of allele- specific covalent inhibitors we have now the opportunity to evaluate the best therapeutic strategies to treat RAS-driven cancers. Inhibition of the RAS pathway with or without other agents etsablished in the treatment of myeloid malignancies may be attractive approaches to treat RAS- mutant CMML.
Authors Contributions
K.G. directed the research, collected, analyzed and interpreted the data and wrote the manuscript; E.J. performed colony assays; A.B., M.G. performed NGS analyses; T.G. and E.G. performed the administration of data; G.H. interpreted molecular data; T.N., M.P., S.M.,R.S., A.Z,. H.S., L.Ö., R.K., P.V, provided patient samples and clinical information; All authors had the opportunity to review the manuscript.
Funding
This study was supported by the “Gesellschaft zur Erforschung der Biologie und Therapie von Tumorkrankheiten” – ABCMML-112015” and the “Austrian Science Fund (FWF) – grant F4704-B20”.
Conflict of Interest Disclosure
The authors declare no conflict of interest.
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