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V. Caraffini et al.
Figure 8. RAS-signaling mutations are frequent in chronic myelomonocytic leukemia (CMML) patients with RAF kinase inhibitor protein (RKIP) loss. Thirty-nine genes with recurrent mutations in myeloid neoplasias were screened for mutations by the means of next-generation sequencing.11 The heatmap shows the distribu- tion and number of mutations observed in each CMML patient (n=37). Every column describes one CMML patient specimen, defined as either “RKIP loss” or “RKIP normal” according to the immunoblot results. Light purple indicates the presence of one mutation; dark purple is used to demonstrate the presence of multiple mutations. Genes without sequence variations in any of the patients are not shown.
percentage of myelomonocytic cells (MMC; monocytes and granulocytes) in the peripheral blood (86% vs. 76%, P=0.030) (Online Supplementary Table S4). This is in agree- ment with our functional data and with previous findings from AML.12 To investigate the molecular landscape of CMML patients with RKIP loss, we performed next-gen- eration sequencing (NGS) covering 39 genes with recur- rent mutations in myeloid neoplasms (Figure 8). In total, these analyses could be performed in 37 of 41 patients. All together, we discovered 186 mutations in 37 of 37 (100%) of the patients. Thirty-three of 37 had more than one mutation with a median of four variants per sample (range 1-32). In agreement with previous studies,38 the most frequently affected genes were TET2 (75.7%), SRSF2 (46.0%), CBL (24.3%), and ASXL1 (24.3%). Mutations in NRAS and KRAS affected 13 of 37 (35.1%) of the patients (Online Supplementary Table S5). This high frequency was even increased, when mutations affecting RAS-signaling were grouped (including NRAS, KRAS, CBL, PTPN11, FLT3, CSF3R, KIT, JAK2, and NF1). Twenty-nine of 37 (78.4%) of CMML patients' specimens showed one or more mutations within these genes. Most interestingly, however, almost all patients with RKIP loss (11 of 12, 91.7%) exhibited one or more mutations in RAS-signaling genes. Together with the previously pub- lished data from AML, this indicates that RKIP loss and RAS-signaling mutations co-occur in myeloid neoplasms, which proves the clinical relevance of the functional in vivo data mentioned above. Of note, RAS-signaling muta- tions were also frequent in patients with normal RKIP expression (18 of 25, 72%; comparison to patients with RKIP loss, P=0.232), which suggests that RKIP loss is not the only second genetic hit that interacts with RAS-signal- ing mutations in myeloid leukemogenesis. Indeed, such
interactions have previously been shown for a variety of genetic aberrations, including aberrant expression of members of the dual specificity phosphatase (DUSP) and SPROUTY (Spry) families, as well as for mutations in ASXL1 and TET2.15,39-41 Finally, overall survival (OS) was similar between patients with and without RKIP loss (P=0.913) (Online Supplementary Figure S9). It must be noted, however, that these analyses are limited by the fact that these patients were managed with different treatment modalities, which ranged from best supportive care to high-dose chemotherapy. Subgroup analyses focusing on uniformly treated patients only could not be carried out due to the small sample size of this cohort.
Discussion
Hematopoietic stem and progenitor cells have the potential to differentiate into both myeloid and lymphoid hematopoietic cells. Although tight control and regula- tion programs are in place to maintain this system in homeostasis, skewing of hematopoiesis into the myeloid lineage can be achieved by a multitude of genetic aberra- tions. This includes RAS-signaling mutations,1 which drive the myelomonocytic lineage commitment by increasing the sensitivity of intracellular signaling cas- cades to extracellular growth factors, such as GM-CSF.5,42 In this study, we hypothesized that a loss of the RAS-sig- naling inhibitor RKIP plays a role in myelomonocytic dif- ferentiation as well. Therefore, we initially analyzed RKIP expression during human and murine hematopoiet- ic differentiation and observed that it is high in HSPC and lymphoid cells but decreases in cells belonging to the myeloid lineage. By modulating RKIP expression in
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haematologica | 2020; 105(2)