The calcineurin/NFAT pathway is activated in diagnostic breast cancer cases and is essential to survival and metastasis of mammary cancer cells.

Nuclear factor of activated T cells 1 (NFAT1) expression has been associated with increased migratory/invasive properties of mammary tumor-derived cell lines in vitro. It is unknown, however, if NFAT activation actually occurs in breast cancer cases and whether the calcineurin/NFAT pathway is important to mammary tumorigenesis. Using a cohort of 321 diagnostic cases of the major subgroup of breast cancer, we found Cn/NFAT pathway activated in ER(-)PR(-)HER2(-) triple-negative breast cancer subtype, whereas its prevalence is less in other subgroups. Using a small hairpin RNA-based gene expression silencing approach in murine mammary tumor cell line (4T1), we show that not only NFAT1 but also NFAT2 and their upstream activator Cn are essential to the migratory and invasive properties of mammary tumor cells. We also demonstrate that Cn, NFAT1 and NFAT2 are essential to the tumorigenic and metastatic properties of these cells in mice, a phenotype which coincides with increased apoptosis in vivo. Finally, global gene expression analyses identified several NFAT-deregulated genes, many of them being previously associated with mammary tumorigenesis. In particular, we identified the gene encoding a disintegrin and metalloproteinase with thrombonspondin motifs 1, as being a potential direct target of NFAT1. Thus, our results show that the Cn/NFAT pathway is activated in diagnostic cases of breast cancers and is essential to the tumorigenic and metastatic potential of mammary tumor cell line. These results suggest that pharmacological inhibition of the Cn/NFAT pathway at different levels could be of therapeutical interest for breast cancer patients.

Leukemia-initiating cell activity requires calcineurin in T-cell acute lymphoblastic leukemia.

Despite their initial efficient response to induction chemotherapy, relapse remains frequent in patients with T-cell acute lymphoblastic leukemia (T-ALL), an aggressive malignancy of immature T-cell progenitors. We previously reported sustained calcineurin (Cn) activation in human lymphoid malignancies, and showed that Cn inhibitors have antileukemic effects in mouse models of T-ALL. It was unclear, however, from these studies whether these effects resulted from Cn inhibition in leukemic cells themselves or were an indirect consequence of impaired Cn function in the supportive tumor microenvironment. We thus generated a Notch (intracellular Notch 1, ICN1)-induced T-ALL mouse model, in which conditional Cn genetic deletion is restricted to leukemic cells. Ex vivo, Cn deletion altered the adhesive interactions between leukemic cells and their supportive stroma, leukemic cell survival, proliferation, migration and clonogenic potential. In vivo, Cn activation was found to be critical for leukemia initiating/propagating cell activity as demonstrated by the failure of Cn-deficient leukemic cells to transplant the disease to syngeneic recipient mice. Importantly, combination of vincristine treatment with Cre-mediated Cn ablation cooperated to induce long-term remission of ICN1-induced T-ALL. These findings indicate that Cn is a promising target in T-ALL relapse prevention, and call for clinical trials incorporating Cn inhibitors during consolidation therapy.

Functional analyses of the TEL-ARNT fusion protein underscores a role for oxygen tension in hematopoietic cellular differentiation.

The transcription factor hypoxia inducible factor 1 (HIF1), an HIF1alpha-aryl hydrocarbon receptor nuclear translocator (ARNT) dimeric factor, is essential to the cellular response to hypoxia. We described a t(1;12)(q21;p13) chromosomal translocation in human acute myeloblastic leukemia that involves the translocated Ets leukemia (TEL/ETV6) and the ARNT genes and results in the expression of a TEL-ARNT fusion protein. Functional studies show that TEL-ARNT interacts with HIF1alpha and the complex binds to consensus hypoxia response element. In low oxygen tension conditions, the HIF1alpha/TEL-ARNT complex does not activate transcription but exerts a dominant-negative effect on normal HIF1 activity. Differentiation of normal human CD34+ progenitors cells along all the erythrocytic, megakaryocytic and granulocytic pathways was accelerated in low versus high oxygen tension conditions. Murine 32Dcl3 myeloid cells also show accelerated granulocytic differentiation in low oxygen tension in response to granulocyte colony-stimulating factor. Interestingly, stable expression of the TEL-ARNT in 32Dcl3 subclones resulted in impaired HIF1-mediated transcriptional response and inhibition of differentiation enhancement in hypoxic conditions. Taken together, our results underscore the role of oxygen tension in the modulation of normal hematopoietic differentiation, whose targeting can participate in human malignancies.

Constitutive STAT5 activation specifically cooperates with the loss of p53 function in B-cell lymphomagenesis.

Signal transducers and activator of transcription 5 (STAT5) A and B are transcriptional regulators that play a central role in cytokine signaling in the hematopoietic lineage and which are frequently activated in a persistent manner in human leukemia/lymphoma, as assessed by their constitutive tyrosine phosphorylation and DNA-binding activity. To study the intrinsic oncogenic properties of persistent STAT5 activation, we generated transgenic mice in which a constitutively activated point mutant of STAT5A, STAT5A(S711F), was expressed at physiological level in their lymphoid compartment. In this model, persistent STAT5 activation is weakly oncogenic, leading to the late emergence of clonal B-cell lymphoma/leukemia at a low incidence. In contrast, STAT5(S711F) was found to cooperate with the loss of function of the p53 tumor suppressor gene to both accelerate disease onset and to skew the large tumor spectrum that normally characterize p53-deficient mice to strongly favor B-cell lymphoma/leukemia. The emergence of STAT5A(S711F)-induced B-cell tumors is associated with the activation of STAT5 tyrosine phosphorylation and DNA-binding activity, indicating that activation of STAT5 oncogenic properties in transgenic STAT5A (TgSTAT5A) (S711F) mice involves the deregulation of STAT5 phosphorylation dynamics.

The TEL-Jak2 oncoprotein induces Socs1 expression and altered cytokine response in Ba/F3 cells.

The leukemia-associated TEL-Jak2 fusion protein possesses a constitutive tyrosine kinase activity and transforming properties in hematopoietic cell lines and animal models. In the murine pro-B Ba/F3 cell line, this fusion constitutively activates the Signal Transducer and Activator of Transcription 5 (Stat5) factors and, as a consequence, induces the sustained expression of various Stat5-target genes including the Cytokine Inducible SH2-containing protein (Cis) gene, which codes for a member of the Suppressor of Cytokine Signaling (Socs) protein family. In TEL-Jak2-transformed Ba/F3 cells, we also observed the upregulation of the Socs1 gene, whose product has been reported to negatively regulate the Jak kinase activity. In transient transfection experiments, Socs1 physically interacts with TEL-Jak2 and interferes with the TEL-Jak2-induced phosphorylation and activation of Stat5 factors, probably through the Socs1-induced proteasome-mediated degradation of the fusion protein. Interestingly, TEL-Jak2-expressing Ba/F3 cells were found to be resistant to the anti-proliferative activities of gamma interferon (IFN-gamma) seemingly as a consequence of Socs1 constitutive expression. These results indicate that the Socs1-dependent cytokine feedback loop, although active, is bypassed by the TEL-Jak2 fusion, but may play a role in the leukemogenic process by altering the cytokine responses of the leukemic cells. Our results also suggest that Socs1 plays a role in shutting down the signaling from the normally activated Jak2 kinase by inducing its proteasome-dependent degradation.

Characterization of a novel ETS gene, TELB, encoding a protein structurally and functionally related to TEL.

The TEL/ETV6 gene is located at 12p13 and is frequently involved in chromosomal translocations in human malignancies usually resulting in the expression of fusion proteins between the amino terminal part of TEL, and either unrelated transcription factors or protein tyrosine kinases. We report here a novel gene named TELB which is located on human chromosomal band 6p21 and encodes a protein highly related to TEL. TELB is widely expressed in different tissues and, similarly to TEL encodes a sequence-specific transcriptional repressor.

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.

Recruitment of the nuclear receptor corepressor N-CoR by the TEL moiety of the childhood leukemia-associated TEL-AML1 oncoprotein.

The t(12;21)(p13;q22) chromosomal translocation is the most frequent illegitimate gene recombination in a pediatric cancer and occurs in approximately 25% of common acute lymphoblastic leukemia (cALL) cases. This rearrangement results in the in frame fusion of the 5'-region of the ETS-related gene, TEL (ETV6), to almost the entire acute myeloid leukemia 1 (AML1) (also called CBFA2 or PEBP2AB1) locus and expression of the TEL-AML1 chimeric protein. Although AML1 stimulates transcription, TEL-AML1 functions as a repressor of some AML1 target genes. In contrast to the wild type AML1 protein, both TEL and TEL-AML1 interact with N-CoR, a component of the nuclear receptor corepressor complex with histone deacetylase activity. The interaction between TEL and N-CoR requires the central region of TEL, which is retained in TEL-AML1, and TEL lacking this domain is impaired in transcriptional repression. Taken together, our results suggest that TEL-AML1 may contribute to leukemogenesis by recruiting N-CoR to AML1 target genes and thus imposing an altered pattern of their expression.

The t(1;12)(q21;p13) translocation of human acute myeloblastic leukemia results in a TEL-ARNT fusion.

The TEL/ETV6 gene is located at 12p13 and encodes a member of the ETS family of transcription factors. Translocated ETS leukemia (TEL) is frequently involved in chromosomal translocations in human malignancies, usually resulting in the expression of fusion proteins between the amino-terminal part of TEL and either unrelated transcription factors or protein tyrosine kinases. We have characterized a t(1;12)(q21;p13) translocation in an acute myeloblastic leukemia (AML-M2). At the protein level, the untranslocated TEL copy and, as a result of the t(1;12) translocation, a fusion protein between TEL and essentially all of aryl hydrocarbon receptor nuclear translocator (ARNT) are expressed. The involvement of ARNT in human leukemogenesis has not been previously described. The ARNT protein belongs to a subfamily of the "basic region helix-loop-helix" (bHLH) protein that shares an additional region of similarity called the PAS (Per, ARNT, SIM) domain. ARNT is the central partner of several heterodimeric transcription factors, including those containing the aryl hydrocarbon (dioxin) receptor (AhR) and the hypoxia-inducible factor 1alpha (HIF1alpha). Our results show that the TEL-ARNT fusion protein is the crucial product of the translocation and suggest that interference with the activity of AhR or HIF1alpha can contribute to leukemogenesis.

TEL-JAK2 transgenic mice develop T-cell leukemia.

We previously reported a fusion between TEL and JAK2 in a t(9;12)(p24;p13) chromosomal translocation in childhood acute T-cell leukemia. This fusion gene encodes a TEL-JAK2 chimeric protein in which the 336 amino-terminal residues of TEL, including its specific self-association domain, are fused to the kinase domain of JAK2. TEL-JAK2 exhibits constitutive activation of its tyrosine kinase activity which, in turn, confers growth factor-independent proliferation to the interleukin-3-dependent Ba/F3 hematopoietic cell line. To elucidate the properties of TEL-JAK2 in primary cells and to create an animal model for TEL-JAK2-induced leukemia, we generated transgenic mice in which the TEL-JAK2 complementary DNA was placed under the transcriptional control of the EmuSRalpha enhancer/promoter. TEL-JAK2 founder mice and their transgenic progeny developed fatal leukemia at 4 to 22 weeks of age. Selective amplification of CD8-positive T cells was observed in blood, lymph nodes, thymus, spleen, and bone marrow. Expression of a tyrosine-phosphorylated TEL-JAK2 protein and activation of STAT1 and STAT5 (signal transducer and activator of transcription) were detected in leukemic tissues. TEL-JAK2 diseased mice also displayed invasion of nonhematopoietic organs, including liver, brain, lung, and kidney, by leukemic T cells. Leukemic organs of founder and transgenic progeny contained a monoclonal/oligoclonal T-cell population as analyzed by the rearrangement of the TCRbeta locus. Transplantation of TEL-JAK2 leukemic cells in nude mice confirmed their invasive nature. We conclude that the TEL-JAK2 fusion is an oncogene in vivo and that its expression in lymphoid cells results in the preferential expansion of CD8-positive T cells. (Blood. 2000;95:3891-3899)

Erythroid cell development and leukemic transformation: interplay between signal transduction, cell cycle control and oncogenes.

Studies using genetically modified mice and ex vivo tissue culture of erythroid progenitors converge to show that generation of mature erythroid cells depends on the interplay between specific transcriptional regulators and intracellular signals controlled by cytokines and growth factors. These studies also show that terminal differentiation in the erythroid lineage is unusual since the acquisition of the phenotypic traits of mature cells occurs while the cells are still actively dividing. Furthermore, under specific stress conditions, a massive and sustained self-renewal of committed erythroid progenitors can take place to replenish the pool of terminally differentiated cells. We review here how the erythroid genetic program and its interplay with specific cytokines, growth factors and hormones controls survival, proliferation and differentiation of erythroid progenitors both in normal and stress conditions. Special emphasis is laid on our present understanding of the differences in cell cycle control, which result either in self-renewal of erythroid progenitors or in the particular cell divisions which accompany terminal differentiation. Finally, we discuss how deregulation of the various aspects of the physiological control of erythroid progenitor survival, proliferation and differentiation can lead to erythroblast transformation and erythroleukemia.

IL-2 and long-term T cell activation induce physical and functional interaction between STAT5 and ETS transcription factors in human T cells.

Activation of Stat5 by many cytokines implies that it cannot alone insure the specificity of the regulation of its target genes. We have evidenced a physical and functional interaction between members of two unrelated transcription factor families, Ets-1, Ets-2 and Stat5, which could contribute to the proliferative response to interleukin 2. Competition with GAS- and EBS-specific oligonucleotides and immunoassays with a set of anti-Stat and anti-Ets families revealed that the IL-2-induced Stat5-Ets complex recognizes several GAS motifs identified as target sites for activated Stat5 dimers. Coimmunoprecipitation experiments evidenced that a Stat5/Ets-1/2 complex is formed in vivo in absence of DNA. GST-pull down experiments demonstrated that the C-terminal domain of Ets-1 is sufficient for this interaction in vitro. Cotransfection experiments in Kit225 T cells resulted in cooperative transcriptional activity between both transcription factors in response to a combination of IL-2, PMA and ionomycin. A Stat5-Ets protein complex was the major inducible DNA-binding complex bound to the human IL-2rE GASd/EBSd motif in long-term proliferating normal human T cells activated by CD2 and CD28. These results suggest that the inducible Stat5-Ets protein interaction plays a role in the regulation of gene expression in response to IL-2 in human T lymphocytes.

Transforming properties of chimeric TEL-JAK proteins in Ba/F3 cells.

The involvement of the cytokine signaling pathway in oncogenesis has long been postulated. Recently, rearrangements of the gene encoding the tyrosine Janus kinase 2 (JAK2) have been reported in human leukemias indicating a direct JAK-signal transduction and activator of transcription (STAT)-mediated leukemic process. The leukemia-associated TEL-JAK2 fusion protein is formed by the oligomerization domain of the translocated ets leukemia (TEL) protein fused to the catalytic domain of JAK2. TEL-mediated oligomerization results in a constitutive tyrosine kinase activity that, in turn, is able to confer growth factor independence to the murine hematopoietic interleukin-3 (IL-3)-dependent Ba/F3 cell line. Results of the present study indicate that fusion proteins containing the oligomerization domain of TEL and the tyrosine kinase domains of Jak1, Jak2, JAK3, or TYK2 share similar properties and are able to efficiently substitute for the survival and mitogenic signals controlled by IL-3, without concomitant activation of the IL-3 receptor. Electrophoretic mobility shift assays demonstrated Stat5 as the only activated Stat factor in TEL-Jak2- and TEL-Jak1-expressing cells, whereas other Stats, namely Stat1 and Stat3, could be detected in TEL-JAK3-, TEL-TYK2-, and also in TEL-ABL-expressing Ba/F3 cells. High levels of expression of the Stat5-target genes pim-1, osm, and Cis were observed in all the cytokine-independent cell lines. Furthermore, the expression of a dominant negative form of Stat5A markedly interfered with the growth factor independence process mediated by TEL-Jak2 in Ba/F3 cells. Because the BCR-ABL and TEL-PDGFbetaR oncoproteins also activate Stat5, activation of this factor should be a crucial step in activated tyrosine kinase-mediated leukemogenesis. (Blood. 2000;95:2076-2083)

Proteins of the ETS family with transcriptional repressor activity.

ETS proteins form one of the largest families of signal-dependent transcriptional regulators, mediating cellular proliferation, differentiation and tumorigenesis. Most of the known ETS proteins have been shown to activate transcription. However, four ETS proteins (YAN, ERF, NET and TEL) can act as transcriptional repressors. In three cases (ERF, NET and TEL) distinct repression domains have been identified and there are indications that NET and TEL may mediate transcription via Histone Deacetylase recruitment. All four proteins appear to be regulated by MAPKs, though for YAN and ERF this regulation seems to be restricted to ERKs. YAN, ERF and TEL have been implicated in cellular proliferation although there are indications suggesting a possible involvement of YAN and TEL in differentiation as well. Other ETS-domain proteins have been shown to repress transcription in a context specific manner, and there are suggestions that the ETS DNA-binding domain may act as a transcriptional repressor. Transcriptional repression by ETS domain proteins adds an other level in the orchestrated regulation by this diverse family of transcription factors that often recognize similar if not identical binding sites on DNA and are believed to regulate critical genes in a variety of biological processes. Definitive assessment of the importance of this novel regulatory level will require the identification of ETS proteins target genes and the further analysis of transcriptional control and biological function of these proteins in defined pathways.

Characterization of the chronic myelomonocytic leukemia associated TEL-PDGF beta R fusion protein.

The t(5;12) translocation, associated with chronic myelomonocytic leukemia, generates a novel gene encoding a protein, TEL-PDGF beta R, composed of the 154 amino-terminal amino acids of the transcription factor TEL and the transmembrane and intracellular part of the PDGF beta-receptor (PDGF beta R). TEL also occurs as a tumor-associated fusion partner for the tyrosine kinases c-ABL, JAK2 and TRK-C. Previous studies have demonstrated growth promoting activity of TEL-PDGF beta R and also indicated that the TEL moiety activates the tyrosine kinase of the PDGF beta R through the formation of TEL-PDGF beta R oligomers. We demonstrate that tyrosine phosphorylation of the fusion protein can be attenuated through overexpression of the TEL part of TEL-PDGF beta R, suggesting a strategy for antagonizing the signaling of TEL-PDGF beta R, and other TEL-fusion proteins containing tyrosine kinase domains. Comparison of BaF/3 cell lines expressing TEL-PDGF beta R and ligand-stimulated PDGF beta R revealed that only TEL-PDGF beta R expression conferred IL-3-independent growth, suggesting differences in signaling capacity of the two proteins. Finally, tyrosine residues 17 and 27 in TEL-PDGF beta R was identified as autophosphorylation sites in TEL-PDGF beta R.

TEL is a sequence-specific transcriptional repressor.

TEL is a gene frequently involved in specific chromosomal translocations in human leukemia and sarcoma that encodes a member of the ETS family of transcriptional regulators. TEL is unusual among other ETS proteins by its ability to self-associate in vivo, a property that is essential to the oncogenic activation of TEL-derived fusion proteins. We show here that TEL is a sequence-specific transcriptional repressor of ETS-binding site-driven transcription of model and natural promoters. Deletion of the oligomerization domain of TEL or its substitution by the homologous region of monomeric ETS1 impaired the ability of TEL to repress. In contrast, substitution of the oligomerization domain of TEL by unrelated oligomerization domains resulted in an active repressor, showing that the ability of TEL to repress depends on its ability to self-associate. The study of the properties of TEL fusions to the heterologous DNA binding domain of Gal4 identified two autonomous repression domains in TEL, distinct from its oligomerization domain, that are essential to the ability of TEL to repress ETS-binding site-containing promoters. These results have implications for the normal function of TEL, its relation to other ETS proteins, and its role in leukemogenesis.