ERG is a megakaryocytic oncogene.

Ets-related gene (ERG) is a member of the ETS transcription factor gene family located on Hsa21. ERG is known to have a crucial role in establishing definitive hematopoiesis and is required for normal megakaryopoiesis. Truncated forms of ERG are associated with multiple cancers such as Ewing's sarcoma, prostate cancer, and leukemia as part of oncogenic fusion translocations. Increased expression of ERG is highly indicative of poor prognosis in acute myeloid leukemia and ERG is expressed in acute megakaryoblastic leukemia (AMKL); however, it is unclear if expression of ERG per se has a leukemogenic activity. We show that ectopic expression of ERG in fetal hematopoietic progenitors promotes megakaryopoiesis and that ERG alone acts as a potent oncogene in vivo leading to rapid onset of leukemia in mice. We observe that the endogenous ERG is required for the proliferation and maintenance of AMKL cell lines. ERG also strongly cooperates with the GATA1s mutated protein, found in Down syndrome AMKL, to immortalize megakaryocyte progenitors, suggesting that the additional copy of ERG in trisomy 21 may have a role in Down syndrome AMKL. These data suggest that ERG is a hematopoietic oncogene that may play a direct role in myeloid leukemia pathogenesis.

Trisomy of chromosome 21 in leukemogenesis.

Extra copies of chromosome 21 are often found in sporadic leukemias. Constitutional trisomy 21 of Down syndrome (DS) is associated with markedly increased risk for childhood leukemia. Thus the oncogenic role of trisomy 21 in the more common sporadic childhood leukemias may be revealed through the investigations of the relatively rare leukemias of DS. Recent studies of the megakaryoblastic leukemias of Down syndrome have uncovered a developmental leukemogenic mechanism characterized by a unique pre-natal collaboration between overexpressed genes from chromosome 21 and an acquired mutation in the transcription factor GATA1. The base of the markedly enhanced risk for acute lymphoblastic leukemia conferred by trisomy 21 is still unclear. Studies of the leukemias of DS are likely to contribute to the general understanding of the oncogenic mechanisms of chromosomal aneuploidies, the most common abnormalities in cancer.

The proto-oncogene ERG in megakaryoblastic leukemias.

Aneuploidy is one of the hallmarks of cancer. Acquired additions of chromosome 21 are a common finding in leukemias, suggesting a contributory role to leukemogenesis. About 10% of patients with a germ line trisomy 21 (Down syndrome) are born with transient megakaryoblastic leukemia. We and others have shown acquired mutations in the X chromosome gene GATA1 in all these cases. The gene or genes on chromosome 21 whose overexpression promote the megakaryoblastic phenotype are presently unknown. We propose that ERG, an Ets transcription factor situated on chromosome 21, is one such candidate. We show that ERG is expressed in hematopoietic stem cells, megakaryoblastic cell lines, and in primary leukemic cells from Down syndrome patients. ERG expression is induced upon megakaryocytic differentiation of the erythroleukemia cell lines K562 and UT-7, and forced expression of ERG in K562 cells induces erythroid to megakaryoblastic phenotypic switch. We also show that ERG activates the gpIb megakaryocytic promoter and binds the gpIIb promoter in vivo. Furthermore, both ERG and ETS2 bind in vivo the hematopoietic enhancer of SCL/TAL1, a key regulator of hematopoietic stem cell and megakaryocytic development. We propose that trisomy 21 facilitates the occurrence of megakaryoblastic leukemias through a shift toward the megakaryoblastic lineage caused by the excess expression of ERG, and possibly by other chromosome 21 genes, such as RUNX1 and ETS2, in hematopoietic progenitor cells, coupled with a differentiation arrest caused by the acquisition of mutations in GATA1.

The scaffolding A/Tpd3 subunit and high phosphatase activity are dispensable for Cdc55 function in the Saccharomyces cerevisiae spindle checkpoint and in cytokinesis.

Protein serine/threonine phosphatase 2A (PP2A) is a multifunctional enzyme whose trimeric form consists of a scaffolding A subunit, a catalytic C subunit, and one of several regulatory B subunits (B, B', and B''). The adenovirus E4orf4 protein associates with PP2A by directly binding the B or B' subunits. An interaction with an active PP2A containing the B subunit, or its homologue in yeast, Cdc55, is required for E4orf4-induced apoptosis in mammalian cells and for induction of growth arrest in Saccharomyces cerevisiae. In this work, Cdc55 was randomly mutagenized by low-fidelity PCR amplification, and Cdc55 mutants that lost the ability to transduce the E4orf4 toxic signal in yeast were selected. The mutations obtained by this protocol inhibited the association of Cdc55 with E4orf4, or with the PP2A-AC subunits, or both. Functional analysis revealed that a mutant that does not bind Tpd3, the yeast A subunit, as well as wild type Cdc55 in a tpd3Delta background, can form a heterodimer with the catalytic subunit. This association requires C subunit carboxyl methylation. The residual phosphatase activity associated with Cdc55 in the absence of Tpd3 is sufficient to maintain a partially active spindle checkpoint and to prevent cytokinesis defects.

Mutations in exon 2 of GATA1 are early events in megakaryocytic malignancies associated with trisomy 21.

Patients with Down syndrome (DS) frequently develop 2 kinds of clonal megakaryocytosis: a common, congenital, spontaneously resolving, transient myeloproliferative disorder (TMD) and, less commonly, childhood acute megakaryoblastic leukemia (AMKL). Recently, acquired mutations in exon 2 of GATA1, an X-linked gene encoding a transcription factor that promotes megakaryocytic differentiation, were described in 6 DS patients with AMKL. The mutations prevent the synthesis of the full-length GATA1, but allow the synthesis of a shorter GATA1 protein (GATA1s) that lacks the transactivation domain. To test whether mutated GATA1 is involved in the initiation of clonal megakaryoblastic proliferation or in the progression to AMKL, we screened 35 DS patients with either AMKL or TMD and 7 non-DS children with AMKL for mutations in exon 2 of GATA1. Mutations were identified in 16 of 18 DS patients with AMKL, in 16 of 17 DS patients with TMD, and in 2 identical twins with AMKL and acquired trisomy 21. Analysis revealed various types of mutations in GATA1, including deletion/insertions, splice mutations, and nonsense and missense point mutations, all of which prevent the generation of full-length GATA1, but preserve the translation of GATA1s. We also show that the likely mechanism of generation of GATA1 isoforms is alternative splicing of exon 2 rather than, or in addition to, alternative translation initiation, as was proposed before. These findings suggest that acquired intrauterine inactivating mutations in GATA1 and generation of GATA1s cooperate frequently with trisomy 21 in initiating megakaryoblastic proliferation, but are insufficient for progression to AMKL.