SPI-1 transforming properties depend upon specifically activated forms of the EPOR.

Friend erythroleukemia induced in mice by the spleen focus forming virus (SFFV) is a multi-step process. The pre-leukemic phase of the disease results from the abnormal activation of the Erythropoietin (Epo) receptor by the gp55 env gene product of SFFV. Later in disease progression, the emergence of leukemic clones is associated with recurrent genetic events, in particular the activation of the expression of SPI-1, an ETS family transcriptional regulator. We show here that the expression of either SPI-1 or GP55 with the mouse EPOR in avian primary erythroblasts only marginally affects their normal Epo-induced terminal differentiation. In contrast, the co-expression of GP55 and SPI-1 resulted in inhibition of Epo-induced differentiation of EPOR-expressing erythroblasts, promoting instead their proliferation. Co-expression of SPI-1 and GP55 also inhibited the apoptotic cell death program normally induced in response to Epo withdrawal. This cooperation between SPI-1 and GP55 to induce primary erythroblast transformation suggests that progression of Friend erythroleukemia critically depends upon inter-dependent interactions between the molecular events specific of the early and late phase of the disease.

A novel way to induce erythroid progenitor self renewal: cooperation of c-Kit with the erythropoietin receptor.

Red blood cells are of vital importance for oxygen transport in vertebrates. Thus, their formation during development and homeostasis requires tight control of both progenitor proliferation and terminal red cell differentiation. Self renewal (i.e. long-term proliferation without differentiation) of committed erythroid progenitors has recently been shown to contribute to this regulation. Avian erythroid progenitors expressing the EGF receptor/c-ErbB (SCF/TGFalpha progenitors) can be induced to long-term proliferation by the c-ErbB ligand transforming growth factor alpha and the steroids estradiol and dexamethasone. These progenitors have not yet been described in mammals and their factor requirements are untypical for adult erythroid progenitors. Here we describe a second, distinct type of erythroid progenitor (EpoR progenitors) which can be established from freshly isolated bone marrow and is induced to self renew by ligands relevant for erythropoiesis, i.e. erythropoietin, stem cell factor, the ligand for c-Kit and the glucocorticoid receptor ligand dexamethasone. Limiting dilution cloning indicates that these EpoR progenitors are derived from normal BFU-E/CFU-E. For a detailed study, mEpoR progenitors were generated by retroviral expression of the murine Epo receptor in bone marrow erythroblasts. These progenitors carry out the normal erythroid differentiation program in recombinant differentiation factors only. We show that mEpoR progenitors are more mature than SCF/TGFalpha progenitors and also do no longer respond to transforming growth factor alpha and estradiol. In contrast they are now highly sensitive to low levels of thyroid hormone, facilitating their terminal maturation into erythrocytes.

FLI-1 inhibits differentiation and induces proliferation of primary erythroblasts.

Friend virus-induced erythroleukemia involves two members of the ETS family of transcriptional regulators, both activated via proviral insertion in the corresponding loci. Spi-1/PU.1 is expressed in the disease induced by the original Friend virus SFFV(F-MuLV) complex in adult mice. In contrast, FLI-1 is overexpressed in about 75% of the erythroleukemias induced by the F-MuLV helper virus in newborn mice. To analyse the consequences of the enforced expression of FLI-1 on erythroblast differentiation and proliferation and to compare its activity to that of PU.1/Spi-1, we used a heterologous system of avian primary erythroblasts previously described to study the cooperation between Spi-1/PU.1 and the other molecular alterations observed in SFFV-induced disease. FLI-1 was found: (i) to inhibit the apoptotic cell death program normally activated in erythroblasts following Epo deprivation; (ii) to inhibit the terminal differentiation program induced in these cells in response to Epo and; (iii) to induce their proliferation. However, in contrast to Spi-1/PU.1, the effects of FLI-1 on erythroblast, differentiation and proliferation did not require its cooperation with an abnormally activated form of the EpoR. Enhanced survival of FLI-1 expressing erythroblasts correlated with the upregulation of bcl2 expression. FLI-1 also prevented the rapid downregulation of cyclin D2 and D3 expression normally observed during Epo-induced differentiation and delayed the downregulation of several other genes involved in cell cycle or cell proliferation control. Our results show that overexpression of FLI-1 profoundly deregulates the normal balance between differentiation and proliferation in primary erythroblasts. Thus, the activation of FLI-1 expression observed at the onset of F-MuLV-induced erythroleukemia may provide a proliferative advantage to virus infected cells that would otherwise undergo terminal differentiation or cell death.

A TEL-JAK2 fusion protein with constitutive kinase activity in human leukemia.

The Janus family of tyrosine kinases (JAK) plays an essential role in development and in coupling cytokine receptors to downstream intracellular signaling events. A t(9;12)(p24;p13) chromosomal translocation in a T cell childhood acute lymphoblastic leukemia patient was characterized and shown to fuse the 3' portion of JAK2 to the 5' region of TEL, a gene encoding a member of the ETS transcription factor family. The TEL-JAK2 fusion protein includes the catalytic domain of JAK2 and the TEL-specific oligomerization domain. TEL-induced oligomerization of TEL-JAK2 resulted in the constitutive activation of its tyrosine kinase activity and conferred cytokine-independent proliferation to the interleukin-3-dependent Ba/F3 hematopoietic cell line.

Cooperation of Spi-1/PU.1 with an activated erythropoietin receptor inhibits apoptosis and Epo-dependent differentiation in primary erythroblasts and induces their Kit ligand-dependent proliferation.

Spi-1/PU.1 is a myeloid- and B-cell specific transcription factor which is also involved in Friend virus-induced murine erythroleukemia. The pre-leukemic phase of Friend erythroleukemia results from activation of the erythropoietin receptor (EpoR) by the spleen focus forming virus (SFFV) envelope glycoprotein, followed by the emergence of leukemic clones characterized by overexpression of Spi-1 and mutation of the p53 tumor suppressor gene. We developed a heterologous system to analyze the contribution of these alterations to the induction of primary erythroblast transformation. Avian erythroblasts expressing the activated mouse EpoR(R129C) differentiated into erythrocytes in response to hEpo. Expression of Spi-1 in these cells inhibited this ability to differentiate and rescued the cells from the apoptotic cell death program normally induced upon hEpo withdrawal. Although devoid of any effect by itself, a mutant p53 cooperated with Spi-1 and EpoR(R129C) to reinforce both phenotypes. Analysis of erythroblasts co-expressing Spi-1 and the wild-type mouse EpoR showed that differentiation arrest and inhibition of apoptosis depended on specific cooperation between Spi-1 and EpoR(R129C). This cooperation was also required to induce the sustained proliferation of differentiation-blocked erythroblasts in response to ligand activation of the endogenous tyrosine kinase receptor c-Kit. These results show that Spi-1/PU.1 requires signals emanating from specific cytokine and growth factor receptors to affect the survival, proliferation and differentiation control of primary erythroblasts. They also suggest that the function of Spi-1/PU.1 in the late phase of Friend leukemia requires specific signaling from the gp55-modified EpoR generated during the early phase of the disease.

A domain of TEL conserved in a subset of ETS proteins defines a specific oligomerization interface essential to the mitogenic properties of the TEL-PDGFR beta oncoprotein.

TEL is a novel member of the ETS family of transcriptional regulators which is frequently involved in human leukemias as the result of specific chromosomal translocations. We show here by co-immunoprecipitation and GST chromatography analyses that TEL and TEL-derived fusion proteins form homotypic oligomers in vitro and in vivo. Deletion mutagenesis identifies the TEL oligomerization domain as a 65 amino acid region which is conserved in a subset of the ETS proteins including ETS-1, ETS-2, FLI-1, ERG-2 and GABP alpha in vertebrates and PNTP2, YAN and ELG in Drosophila. TEL-induced oligomerization is shown to be essential for the constitutive activation of the protein kinase activity and mitogenic properties of TEL-platelet derived growth factor receptor beta (PDGFR beta), a fusion oncoprotein characteristic of the leukemic cells of chronic myelomonocytic leukemia harboring a t(5;12) chromosomal translocation. Swapping experiments in which the TEL oligomerization domain was exchanged by the homologous domains of representative vertebrate ETS proteins including ETS-1, ERG-2 and GABP alpha show that oligomerization is a specific property of the TEL amino-terminal conserved domain. These results indicate that the amino-terminal domain conserved in a subset of the ETS proteins has evolved to generate a specialized protein-protein interaction interface which is likely to be an important determinant of their specificity as transcriptional regulators.

Phosphorylation of Ets1 regulates the complementation of a CSF-1 receptor impaired in mitogenesis.

Ets1, the founder member of the Ets transcription factor family, is involved in a variety of developmental and cellular processes. Previous studies have shown that serine phosphorylation of Ets1 inhibits its DNA binding activity, suggesting that phosphorylation is important in the regulation of Ets1 function. To further examine Ets1 phosphorylation, we ectopically expressed Ets1 in fibroblasts and stimulated these cells with serum. Using two-dimensional tryptic phosphopeptide analysis and site-directed mutagenesis, we found that Ets1 was phosphorylated on threonine 38, a residue conserved in several Ets proteins. Substitution of this residue with alanine enhanced CSF-1-dependent colony formation in semi-solid medium of NIH3T3 cells expressing a mitogenically defective CSF-1 receptor [Y809F]. Threonine 38 is part of a consensus amino-acid sequence frequently recognized and targeted by members of the MAP kinase family. Moreover, this residue is phosphorylated in vitro by recombinant ERK2, which suggests that the kinase which phosphorylates threonine 38 in vivo is a member of the MAP kinase family. In addition, phosphorylation on threonine 38 seems to negatively regulate Ets1 activity in response to growth-factor stimulation.

Spi-1 and mutant p53 regulate different aspects of the proliferation and differentiation control of primary erythroid progenitors.

The emergence of leukemic cells in Friend virus complex-induced erythroleukemia is associated with two recurrent genetic alterations, namely the inactivation of the p53 tumor suppressor gene and the overexpression of Spi-1, a member of the Ets family of transcriptional regulators. In order to determine the role of these genetic alterations on the proliferation and differentiation control of erythroblasts, we expressed Spi-1 and the temperature sensitive mutant p53(V135A) in avian primary erythroid progenitors. We show that enforced expression of Spi-1 in erythroblasts obtained from bone marrow cells by expression of the ts-Sea tyrosine kinase inhibits the execution of the differentiation program normally induced in these cells in response to Epo and insulin and following inactivation of ts-Sea function. In contrast, overexpression of p53(V135A) is without effect on the ability of these cells to differentiate into erythrocytes. However, expression of p53(V135A) in erythroid progenitors obtained from bone marrow cells in the presence of SCF, TGF alpha and estradiol, was found to relieve these cells from their absolute TGF alpha requirement for long term proliferation. This phenotype is dependent upon the expression of the mutant form of p53(V135A) as it is not observed at a temperature at which p53(V135A) regains wild type p53 function. Our results show that each of the genetic alterations which characterize Friend erythroleukemic cells affect in a distinct manner the proliferation and differentiation control of primary erythroid progenitors.