Ectopic production of the EVI1 transcriptional repressor zinc finger protein is seen in 4 -6% of human acute myeloid leukemias. Overexpression also transforms Rat1 fibroblasts by an unknown mechanism, which is likely to be related to its role in leukemia and which depends upon its repressor activity. We show here that mutant murine Evi-1 proteins, lacking either the N-terminal zinc finger DNA binding domain or both DNA binding zinc finger clusters, function as dominant negative mutants by reverting the transformed phenotype of Evi-1 transformed Rat1 fibroblasts. The dominant negative activity of the non-DNA binding mutants suggests sequestration of transformation-specific cofactors and that recruitment of these cellular factors might mediate Evi-1 transforming activity. C-terminal binding protein (CtBP) co-repressor family proteins bind PLDLS-like motifs. We show that the murine Evi-1 repressor domain has two such sites, PFDLT (site a, amino acids 553-559) and PLDLS (site b, amino acids 584 -590), which independently can bind CtBP family co-repressor proteins, with site b binding with higher affinity than site a. Functional analysis of specific CtBP binding mutants show site b is absolutely required to mediate both transformation of Rat1 fibroblasts and transcriptional repressor activity. This is the first demonstration that the biological activity of a mammalian cellular transcriptional repressor protein is mediated by CtBPs. Furthermore, it suggests that CtBP proteins are involved in the development of some acute leukemias and that blocking their ability to specifically interact with EVI1 might provide a target for the development of pharmacological therapeutic agents.A small number of transcription factors are frequently targets for de-regulation by recurring chromosome translocations in acute leukemias, and these events play a pivotal role in disease progression (1). The EVI-1 gene encodes one of these transcription factors, which is activated in 4 -6% of acute myeloid leukemia (AML) 1 patients with various karyotypic abnormalities of chromosome 3q26 (2), which result in the ectopic production of intact or, occasionally, C-terminal-truncated EVI1 proteins (3-6). In addition, novel EVI1 fusion proteins are sometimes produced. For example, patients with karyotypes t(3;21) (q26;q22) or t(3;12) (q26;p13) express AML1/EVI1 (7) and ETV6(TEL)/EVI1 (8) chimeras, respectively, and similar fusions with a naturally occurring MDS1/EVI1 isoform (9).The precise contribution of ectopic EVI1 and EVI1 fusion protein production in leukemia progression is unknown, but a combination of enforced transgene expression and intervention studies shows a causative role, affecting both cell differentiation and proliferation. Expression of AML1/MDS1/EVI1 induces AML in mice, resulting in the accumulation of myeloid blast cells and immature differentiated myelocytic and monocytic lineages (10). EVI1 or AML1/EVI1 expression in either 32Dcl3 cells or murine primary bone marrow cells abrogates granulocyte colony-stimulating factor and eryth...

The myeloid transforming gene Evi-1 encodes a protein with two zinc ®nger domains, designated ZF1 and ZF2, with distinct DNA binding speci®cities. For the ®rst time we demonstrate that Evi-1 has transcriptional repressor activity which is directly proportional to the amount of Evi-1 protein in cells. Repression has been observed with two distinct promoters: the minimal HSV-1 tk promoter and a VP16 inducible adenovirus E1b minimal promoter. Optimal repression is DNA binding dependent and is mediated by either ZF1 or a heterologous GAL4 DNA binding domain (GAL4DBD) but is signi®cantly less e cient through the ZF2 binding site. Both GAL4DBD/ Evi-1 fusion and non-fusion proteins have been used to map the repressor activity to a proline-rich region located within amino acids 514 ± 724 between the ZF1 and ZF2 domains. Constitutive expression of mutant proteins lacking the repressor domain are defective for transformation of Rat1 ®broblasts demonstrating that this region is required for the oncogenic activity of the Evi-1 protein. These studies show that the Evi-1 gene encodes a transcriptional repressor and has important implications for the mechanism of action of the Evi-1 protein both in development and in the progression of some myeloid leukaemias.

The Runx genes are important in development and cancer, where they can act either as oncogenes or tumour suppressors. We compared the effects of ectopic Runx expression in established fibroblasts, where all three genes produce an indistinguishable phenotype entailing epithelioid morphology and increased cell survival under stress conditions. Gene array analysis revealed a strongly overlapping transcriptional signature, with no examples of opposing regulation of the same target gene. A common set of 50 highly regulated genes was identified after further filtering on regulation by inducible RUNX1-ER. This set revealed a strong bias toward genes with annotated roles in cancer and development, and a preponderance of targets encoding extracellular or surface proteins, reflecting the marked effects of Runx on cell adhesion. Furthermore, in silico prediction of resistance to glucocorticoid growth inhibition was confirmed in fibroblasts and lymphoid cells expressing ectopic Runx. The effects of fibroblast expression of common RUNX1 fusion oncoproteins (RUNX1-ETO, TEL-RUNX1 and CBFB-MYH11) were also tested. Although two direct Runx activation target genes were repressed (Ncam1 and Rgc32), the fusion proteins appeared to disrupt the regulation of downregulated targets (Cebpd, Id2 and Rgs2) rather than impose constitutive repression. These results elucidate the oncogenic potential of the Runx family and reveal novel targets for therapeutic inhibition.

The Runx genes play paradoxical roles in cancer where they can function either as dominant oncogenes or tumor suppressors according to context. We now show that the ability to induce premature senescence in primary murine embryonic fibroblasts (MEF) is a common feature of all three Runx genes. However, ectopic Runx-induced senescence contrasts with Ras oncogene-induced senescence, as it occurs directly and lacks the hallmarks of proliferative stress. Moreover, a fundamental role for Runx function in the senescence program is indicated by the effects of Runx2 disruption, which renders MEFs prone to spontaneous immortalization and confers an early growth advantage that is resistant to stress-induced growth arrest.

The mammalian Runx gene family (Runx1-3) are transcription factors that play essential, lineage-specific roles in development. A growing body of evidence implicates these genes as mutational targets in cancer where, in different contexts, individual family members have been reported to act as tumour suppressors, dominant oncogenes or mediators of metastasis. We are exploring these paradoxical observations by ectopic expression of RUNX genes in primary murine embryonic fibroblasts where, in common with a number of other dominant oncogenes, RUNX1 induces senescence-like growth arrest in the presence of an intact p19 ARF -p53 pathway. We now report that, in MEFs lacking functional p53, RUNX1 has apparently prooncogenic effects on cell growth that include cytoskeletal reorganization, reduced contact inhibition at confluence and accelerated tumour expansion in vivo. On the other hand, RUNX1 conferred no obvious growth advantage at low cell density and actually delayed entry of primary MEFs into S phase. We also found that ectopic RUNX1 interferes with the morphological and growth responses of p53-null MEFs to TGFb indicating that these effects are mediated by overlapping pathways. These observations help to elucidate the context-dependent consequences of loss and gain of Runx activity.

The Evi-1 gene encodes a zinc ®nger transcriptional repressor protein that normally plays a role in development and is frequently activated in myeloid leukaemias. Evi-1 has two distinct DNA binding domains, ZF1 and ZF2, and a de®ned repressor domain but the function of the remainder of the molecule is unknown. The ZF2 and repressor domains have been shown to be required for transformation and we show here that ZF1 is also required. An alternative splice variant of Evi-1, designated D324, encodes a protein which lacks a portion of the ZF1 DNA binding domain and the intervening amino acids 239 ± 514 (designated IR) located between ZF1 and the repressor domain. We show that D324 can neither bind ZF1, repress transcription through this site nor transform Rat1 ®broblasts. Reconstitution studies demonstrate that the defect in D324 is partially complemented by recreating the ZF1 DNA binding activity. However, full function also requires the IR region which has transcriptional repressor activity. This study shows therefore, that ZF1, ZF2 and repressor domains and the IR region all contribute to the transformation e ciency of the Evi-1 protein.

A role for the RUNX genes in cancer failsafe processes has been suggested by their induction of senescence-like growth arrest in primary murine fibroblasts and the failure of RAS-induced senescence in Runx2 deficient cells. We now show that RUNX1 induces senescence in human primary fibroblasts. High affinity DNA binding is necessary but not sufficient, as shown by the functional attenuation of the truncated RUNX1/AML1a isoform and the TEL-RUNX1 fusion oncoprotein. However, a similar phenotype was potently induced by the RUNX1-ETO (AML1-ETO) oncoprotein, despite its dominant negative potential. Detailed comparison of H-RASV12, RUNX1 and RUNX1-ETO senescent phenotypes showed that the RUNX effectors induce earlier growth stasis with only low levels of DNA damage signalling and a lack of chromatin condensation, a marker of irreversible growth arrest. In human fibroblasts, all effectors induced p53 in the absence of detectable p14ARF, while only RUNX1-ETO induced senescence in p16INK4a null cells. Correlation was noted between induction of p53, reactive oxygen species and phospho-p38, while p38MAPK inhibition rescued cell growth markedly. These findings reveal a role for replication-independent pathways in RUNX and RUNX1-ETO senescence, and show that the context-specific oncogenic activity of RUNX1 fusion proteins are mirrored in their distinctive interactions with failsafe responses.

The Runx genes (Runx1, 2, and 3) regulate cell fate in development and can operate as either oncogenes or tumor suppressors in cancer. The oncogenic potential of ectopic Runx expression has been shown in transgenic mice that develop lymphoma in potent synergy with overexpressed Myc, and in established fibroblasts that display altered morphology and increased tumorigenicity. Candidate oncogenic functions of overexpressed Runx genes include resistance to apoptosis in response to intrinsic and extrinsic stresses. In a search for gene targets responsible for this aspect of Runx phenotype, we have identified three key enzymes in sphingolipid metabolism (Sgpp1, Ugcg, and St3gal5/Siat9) as direct targets for Runx transcriptional regulation in a manner consistent with survival and apoptosis resistance. Consistent with these changes in gene expression, mass spectrometric analysis showed that ectopic Runx reduces intracellular long-chain ceramides in NIH3T3 fibroblasts and elevated extracellular sphingosine 1 phosphate. Runx expression also opposed the activation of c-Jun-NH 2 -kinase and p38MAPK , key mediators of ceramide-induced death, and suppressed the onset of apoptosis in response to exogenous tumor necrosis factor α. The survival advantage conferred by ectopic Runx could be partially recapitulated by exogenous sphingosine 1 phosphate and was accompanied by reduced phosphorylation of p38 MAPK . These results reveal a novel link between transcription factor oncogenes and lipid signaling pathways involved in cancer cell survival and chemoresistance.