Leukemic transformation of normal murine erythroid progenitors: v- and c-ErbB act through signaling pathways activated by the EpoR and c-Kit in stress erythropoiesis Marieke von Lindern 1,5 , Eva Maria Deiner 2,5 , Helmut Dolznig 3 , Martine Parren-van Amelsvoort 1 , Michael J Hayman 4 , Ernst W Mullner 3 and Hartmut Beug* ,2 1 Institute of Hematology, Erasmus Medical Centre Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands; 2 Institute of Molecular Pathology, Dr. Bohrgasse 7, A-1030 Vienna, Austria; 3 Institute of Medical Biochemistry, Vienna Biocenter (VBC), Dr. Bohr-Gasse 9, A-1030 Vienna, Austria; 4 Department of Molecular Genetics & Microbiology, Life Sciences Bldg, State University of New York at Stony Brook, Stony Brook, New York, NY 11794, USA Primary erythroid progenitors can be expanded by the synergistic action of erythropoietin (Epo), stem cell factor (SCF) and glucocorticoids. While Epo is required for erythropoiesis in general, glucocorticoids and SCF mainly contribute to stress erythropoiesis in hypoxic mice. This ability of normal erythroid progenitors to undergo expansion under stress conditions is targeted by the avian erythroblastosis virus (AEV), harboring the oncogenes v- ErbB and v-ErbA. We investigated the signaling path- ways required for progenitor expansion under stress conditions and in leukemic transformation. Immortal strains of erythroid progenitors, able to undergo normal, terminal dierentiation under appropriate conditions, were established from fetal livers of p537/7 mice. Expression and activation of the EGF-receptor (HER-1/ c-ErbB) or its mutated oncogenic version (v-ErbB) in these cells abrogated the requirement for Epo and SCF in expansion of these progenitors and blocked terminal dierentiation. Upon inhibition of ErbB function, dier- entiation into erythrocytes occurred. Signal transducing molecules important for renewal induction, i.e. Stat5- and phosphoinositide 3-kinase (PI3K), are utilized by both EpoR/c-Kit and v/c-ErbB. However, while v-ErbB transformed cells and normal progenitors depended on PI3K signaling for renewal, c-ErbB also induces progenitor expansion by PI3K-independent mechanisms. Oncogene (2001) 20, 3651 ± 3664. Keywords: erythropoiesis; signal transduction; cellular transformation Introduction In hematopoiesis, the pluripotent stem cell has to decide between self-renewal (long-term proliferation without lineage commitment) and entering commit- ment/dierentiation pathways. During commitment, the cells undergo a limited number of cell divisions, followed by terminal dierentiation. General concepts state that the stem cell's ability for self-renewal is irreversibly lost during lineage commitment (for review see Keller, 1992; Till and McCulloch, 1980). However, in leukemia, progenitors derived from both pluripotent stem cells and multi- or unipotent progenitors can undergo long-term proliferation without entering terminal dierentiation. The recent observation, that multipotent (Nutt et al., 1999), B-lymphoid (Rolink et al., 1991) and erythroid progenitors (Steinlein et al., 1995) can be continuously propagated as undieren- tiated cells in vitro strongly suggests that committed progenitors may have a large, or even unlimited, renewal potential under particular physiological condi- tions. However, the mechanisms responsible for this `renewal' of normal or leukemic committed progenitors are unknown. Transformation by the retrovirus AEV (avian ery- throblastosis virus) enables erythroid progenitors to undergo prolonged renewal. AEV encodes v-ErbB, a truncated and mutated version of the avian EGF- receptor, EGFR/c-ErbB (Downward et al., 1984), which is sucient for in vitro transformation and leukemogen- esis in chicks (Beug et al., 1985). Subsequently, nontransformed committed erythroid progenitors cap- able of extended renewal were identi®ed (Pain et al., 1991), which express endogenous c-ErbB (Hayman et al., 1993). These cells undergo renewal in response to ligands for c-ErbB (TGFa) and estrogen- and glucocorticoid receptors (ER, GR, Wessely et al., 1997a,b). A second, apparently distinct avian erythroid progenitor can be expanded in the presence of Erythropoietin (Epo) and stem cell factor (SCF), again in cooperation with ligands for the GR and ER (Wessely et al., 1999). This type of progenitor has also been identi®ed in human bone marrow, cord blood (von Lindern et al., 1999) and mouse fetal liver (Reichardt et al., 1998). Interestingly, expression of the v-ErbB oncogene or the avian EGFR in Epo- dependent avian progenitors was found to substitute for the combined action of the EpoR and c-Kit to Oncogene (2001) 20, 3651 ± 3664 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc *Correspondence: H Beug, E-mail: beug@nt.imp.univie.ac.gt 5 The ®rst two authors contributed equally to this work Received 25 January 2001; revised 21 March 2001; accepted 2 April 2001