Abstract
In human hematopoietic malignancies, RAS mutations are frequently observed. Yet, little is known about signal transduction pathways that mediate KRAS-induced phenotypes in human CD34(+) stem/progenitor cells. When cultured on bone marrow stroma, we observed that KRAS(G12V)-transduced cord blood (CB) CD34(+) cells displayed a strong proliferative advantage over control cells, which coincided with increased early cobblestone (CAFC) formation and induction of myelomonocytic differentiation. However, the KRAS(G12V)-induced proliferative advantage was transient. By week three no progenitors remained in KRAS(G12V)-transduced cultures and cells were all terminally differentiated into monocytes/macrophages. In line with these results, LTC-IC frequencies were strongly reduced. Both the ERK and p38 MAPK pathways, but not JNK, were activated by KRAS(G12V) and we observed that proliferation and CAFC formation were mediated via ERK, while differentiation was predominantly mediated via p38. Interestingly, we observed that KRAS(G12V)-induced proliferation and CAFC formation, but not differentiation, were largely mediated via secreted factors, since these phenotypes could be recapitulated by treating non-transduced cells with conditioned medium harvested from KRAS(G12V)-transduced cultures. Multiplex cytokine arrays and genome-wide gene expression profiling were performed to gain further insight into the mechanisms by which oncogenic KRAS(G12V) can contribute to the process of leukemic transformation. Thus, angiopoietin-like 6 (ANGPTL6) was identified as an important factor in the KRAS(G12V) secretome that enhanced proliferation of human CB CD34(+) cells.
Highlights
(2008), and KWF (RUG 2009-4275). □S The on-line version of this article contains supplemental Table S1. 1 To whom correspondence should be addressed: Dept. of Hematology, myeloid leukemias, activating mutations have been found in NRAS and KRAS genes, in particular in acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), and juvenile myelomonocytic leukemia (JMML) [11,12,13]
Sabnis et al investigated the effect of KRasG12D on murine hematopoietic stem and progenitor cells and they found that KRasG12D induced a strong proliferative advantage, increased the fraction of proliferating HSCs, and initiated T-lineage leukemia/lymphoma which was associated with secondary Notch1 mutations
Transduced and sorted MiNR1 control and KRASG12V cells were used to initiate MS5 cocultures and expansion and differentiation were analyzed weekly. These experiments revealed that KRAS overexpression in cord blood (CB) CD34ϩ cells induced a massive proliferation within the first weeks but after the second week KRASG12V-transduced cells stopped proliferating
Summary
Cell Cultures and Cell Lines—Neonatal CB was collected from healthy full-term pregnancies after informed consent from the obstetrics departments of the Martini Hospital Groningen and the University Medical Center Groningen (UMCG) in The Netherlands. Conditioned medium was collected from MS5 cocultures initiated with either MiNR1 control or KRAS-G12V-transduced CD34ϩ CB cells. For CFC assays CD34ϩ cells or suspension cells from cocultures (1–5 ϫ 103 cells) were plated in duplicate in 35-mm tissue culture dishes containing 1 ml assay medium consisting of methylcellulose (StemCell Technologies, Grenoble, France) supplemented with IMDM (PAA Laboratories, Coble, Germany), 20 ng/ml IL-3, 20 ng/ml IL-6 (both of them from GistBrocades, Delft, The Netherlands), 20 ng/ml G-CSF (RhonePoulenc Rorer, Amstelveen, The Netherlands), 20 ng/ml c-kit ligand (Amgen, Thousand Oaks), and 6 units/ml erythropoietin (Janssen-Cilag B.V., Tilburg, The Netherlands). Viral particles for retroviral transduction were collected after 8 –12 h of culturing virus producers in hematopoietic progenitor cell growth medium (HPGM) (Lonza, Leusden, The Netherlands). For Western blot analysis we used control and KRAS G12V-transduced CB CD34ϩ cells, which were cultured in MS5 cocultures for 1 week. Differences were considered statistically significant at p Ͻ 0.05
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