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RKIP loss aggravates RAS-driven leukemogenesis
healthy HSPC and undifferentiated AML cell lines, we could further show that loss of RKIP expression is an important driver of myelomonocytic lineage commit- ment. This could be corroborated in subsequent in vivo studies, where we did show that RKIP loss increases the activation of RAS-MAPK/ERK signaling, and consequent- ly, the GM-CSF-induced myelomonocytic differentiation of HSPC. Of note, we observed that RKIP exerts its role in myelomonocytic lineage commitment of HSPC by act- ing as an amplifier of GM-CSF signaling rather than inducing the differentiation process on its own. This has previously been shown for other alterations affecting RAS-signaling as well43-45 and further highlights the impor- tance of physiological and pathological GM-CSF/RAS-sig- naling regulation in hematopoiesis.
Increased myelomonocytic lineage commitment has also been proposed to be an essential pre-phase of myeloid neoplasms.46 Indeed, a role of RKIP in myeloid leukemogenesis has been suggested previously, as its somatic loss of expression was described as a frequent event in AML.12,20-22 In line with our functional data pre- sented above, it thereby correlated with myelomonocytic AML phenotypes.12 In the current study, we further strengthen these data by demonstrating that RKIP loss is indeed of functional relevance for the development of myelomonocytic leukemias. Again, it acted as an amplifi- er of pathologic RAS-signaling, as it aggravated the activ- ity of the RAS-MAPK/ERK pathway as well as the devel- opment of a myelomonocytic MPD in mice that carry a somatically inducible mutation in Nras within the hematopoietic system. These data are further strength- ened by our analysis of 41 primary CMML patients' spec- imens, where we observed that RKIP loss occurs in almost 30% of cases on the one hand, and that it co- occurs with RAS-signaling mutations on the other. The data are, therefore, in agreement with previous studies of our group, where we did observe a clinical correlation and a functional synergism between RAS-signaling muta- tions and RKIP loss in different subtypes of AML.12,20,22 They are also in agreement with previous observations, where RAS-driven leukemogenesis could be significantly aggravated by additional inactivation of RAS-MAPK/ERK signaling inhibitors belonging to the dual specificity phos- phatase (DUSP) and SPROUTY (Spry) families.15,39,41 Together with the previously shown aggravation of RAS-induced myeloid leukemogenesis by mutations in ASXL1 and TET2,15,40 respectively, these data indicate that activated RAS-signaling in human leukemias is far more complex than initially believed and cannot be explained by the occurrence of RAS-signaling mutations alone.
Finally, our data might also be of relevance for the future development of targeted treatment approaches in myeloid neoplasias, particularly for those aiming to inhibit specific signal transduction cascades. This is based on our observation that both the signaling and leuke- mogenic effects of RAS mutations can be influenced by aberrant expression of RAS-signaling regulator proteins. So far, development of these agents has often been hin- dered by the fact they showed disappointing efficacy in
clinical trials, even though the results from pre-clinical models had been promising. An example for such a histo- ry of drug development are MEK-inhibitors, which effi- ciently attenuate Ras-driven MPD in mice, but show dis- appointing results in clinical trials of myeloid malignan- cies.47 Among others, one reason for this is the fact that the monogenic pre-clinical model does not adequately reflect the situation in myeloid neoplasia patients, who usually exhibit a complex network of co-occurring and interacting genetic aberrations within their neoplastic clone. Therefore, more detailed knowledge of the co- occurrence of mutational and non-mutational aberrations in patients' specimens, as well as the functional conse- quences thereof, might not only help to extend our knowledge about the pathogenesis of this aggressive malignancy, but also to more specifically select patients that might profit from targeted therapies directed at cellu- lar signaling. One successful example of this approach is the recent observation that sensitivity to MEK inhibitors in Nras-mutated mice can be increased by the co-occur- rence of Tet2 deletion and decreased Spry2 expression lev- els.15 With these data, the authors identified a specific sub- group of RAS-mutated patients that will be the best candi- dates for MEK-directed therapy. The fact that simultane- ous occurrence of RAS mutations and RKIP loss potentiat- ed RAS-MAPK/ERK signaling as well, might identify another group of patients with particular sensitivity to this therapeutic approach. Future studies will, therefore, be warranted to specifically test this hypothesis.
In conclusion, we show that the RAS-signaling regula- tor RKIP plays a central role in myelomonocytic lineage commitment of HSPC. We further show its relevance for myelomonocytic leukemogenesis by demonstrating that Rkip deletion enhances RAS-MAPK/ERK signaling and aggravates the development of a myelomonocytic MPD in Nras-mutated mice. Finally, we prove the clinical rele- vance of these findings by showing that RKIP loss is a fre- quent event in primary CMML patients' samples and fre- quently co-occurs with RAS-signaling mutations. These data establish RKIP as a novel player in RAS-driven myeloid leukemogenesis.
Acknowledgments
The authors would like to thank Prof. John Sedivy for provid- ing Rkip-/- mice.
Funding
This study was supported by research funding from the Austrian Science Fund (grant P26619-B19 to A. Zebisch) and from the Science Foundation Ireland (grant 14/IA/2395 to W. Kolch). Work in the laboratories of A. Zebisch, A. Wölfler, and H. Sill is further supported by Leukämiehilfe Steiermark. PhD candidate V. Caraffini received funding from the Austrian Science Fund (grant P26619-B19 to A. Zebisch) and was trained within the frame of the PhD Program Molecular Medicine of the Medical University of Graz. PhD candidate J.L. Berg received funding from the Medical University of Graz within the PhD Program Molecular Medicine.
This work was supported by Biobank Graz.
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