Insertional mutagenesis of the spi-1 gene is associated with the emergence of malignant proerythroblasts during Friend virus-induced acute erythroleukemia. To determine the role of spi-1/PU.1 in the genesis of leukemia, we generated spi-1 transgenic mice. In one founder line the transgene was overexpressed as an unexpected-size transcript in various mouse tissues. Homozygous transgenic animals gave rise to live-born offspring, but 50% of the animals developed a multistep erythroleukemia within 1.5 to 6 months of birth whereas the remainder survived without evidence of disease. At the onset of the disease, mice became severely anemic. Their hematopoietic tissues were massively invaded with nontumorigenic proerythroblasts that express a high level of Spi-1 protein. These transgenic proerythroblasts are partially blocked in differentiation and strictly dependent on erythropoietin for their proliferation both in vivo and in vitro. A complete but transient regression of the disease was observed after erythrocyte transfusion, suggesting that the constitutive expression of spi-1 is related to the block of the differentiation of erythroid precursors. At relapse, erythropoietin-independent malignant proerythroblasts arose. Growth factor autonomy could be partially explained by the autocrine secretion of erythropoietin; however, other genetic events appear to be necessary to confer the full malignant phenotype. These results reveal that overexpression of spi-1 is essential for malignant erythropoiesis and does not alter other hematopoietic lineages.

The erythroleukemia developed by spi-1/PU.1 transgenic mice is a multistage process characterized by an early arrest of the proerythroblast differentiation followed later on by malignant transformation. Herein, we report the presence of acquired mutations in the SCF receptor gene (Kit) in 86% of tumors isolated during the late stage of the disease. Kit mutations affect codon 814 or 818. Ectopic expression of Kit mutants in nonmalignant proerythroblasts confers erythropoietin independence and tumorigenicity to cells. Using PP1, PP2, and imatinib mesylate, we show that Kit mutants are responsible for the autonomous expansion of malignant cells via Erk1/2 and PI3K/Akt activations. These findings represent a proof of principle for oncogenic cooperativity between one proliferative and one differentiation blocking event for the development of an overt leukemia.

The erythroleukemia developed by spi-1/ PU.1-transgenic mice is a model of multistage oncogenic process. Isolation of tumor cells representing discrete stages of leukemic progression enables the dissection of some of the critical events required for malignant transformation. To elucidate the molecular mechanisms of multistage leukemogenesis, we developed a microarray transcriptome analysis of nontumorigenic (HS1) and tumorigenic (HS2) proerythroblasts from spi-1-transgenic mice. The data show that transcriptional up-regulation of the sphingosine kinase gene (SPHK1) is a recurrent event associated with the tumorigenic phenotype of these transgenic proerythroblasts. SPHK1 is an enzyme of the metabolism of sphingolipids, which are essential in several biologic processes, including cell proliferation and apoptosis. HS1 erythroleukemic cells engineered to overexpress the SPHK1 protein exhibited growth proliferative advantage, increased clonogenicity, and resistance to apoptosis in reduced serum level by a mechanism involving activation of the extracellular signal-related kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase ( IntroductionThe acute erythroleukemia caused by the Friend virus is a multistage neoplasm suitable to study the sequence of oncogenic events involved in the progression of erythroblastic cells toward malignancy. 1 The early step of the Friend disease is characterized by a deregulation of growth and differentiation of erythropoietin (Epo)-independent erythroblasts. This phase is induced by the viral gp55 glycoprotein that constitutively activates the Epo receptor. 2 At a later stage, a clonal population of proerythroblasts emerges that are arrested in their differentiation and tumorigenic in vivo. 3,4 In these tumor cells, 2 recurrent genetic alterations have been identified: the transcriptional dysregulation of the spi-1 gene 5 (also called PU.1) and the extinction of the p53 gene. [6][7][8] Spi-1/PU.1 is a master gene in the development of B-lymphoid, monocyte, and neutrophilic lineages. [9][10][11] Ectopic overexpression of spi-1/PU.1 in proerythroblasts leads to cell transformation as demonstrated in vitro in avian erythroid progenitors infected by spi-1-transducing retroviruses 12 or in vivo in transgenic mice overexpressing spi-1 in hematopoietic cells. 13 Spi-1-transgenic mice develop an erythroleukemia arising from the proliferation of proerythroblasts arrested in their differentiation (HS1 stage). At disease onset, survival and growth of erythroblasts are under the control of Epo. Thus, the major effect of spi-1 overexpression in transgenic mice is a blockade in the differentiation of the erythroid lineage. Later during disease progression, malignant proerythroblasts characterized by Epo-autonomous growth and tumorigenicity in vivo can be isolated (HS2 stage). This multistep process suggested that genetic lesions were required to confer full malignancy. 13 Because of its recurrent alteration during Friend erythroleukemia, 1,7,8 mutation of the tumor suppressor gene p53 was a prime cand...