Supplementary Figure 4 from Targeting the Oligomerization Domain of ETO Interferes with RUNX1/ETO Oncogenic Activity in t(8;21)-Positive Leukemic Cells

Crucial for immune and anti-inflammatory cellular responses, signal transducer and activator of transcription 6 (STAT6) regulates transcriptional activation in response to interleukin-4 and -13 -induced tyrosine phosphorylation by direct interaction with coactivators. The interaction of STAT6 with nuclear coactivator 1 (NCoA1) is mediated by a short region of the STAT6 transactivation domain that includes the motif LXXLL and interacts with the PAS-B domain of NCoA1. Despite the availability of an X-ray structure of the PAS-B domain/Leu(794)-Gly(814)-STAT6 complex, the mechanistic details of this interaction are still poorly understood. Here, we determine the structure of the NCoA1(257-385)/STAT6(783-814) complex using Nuclear Magnetic Resonance (NMR) and X-ray crystallography. The STAT6(783-814) peptide binds with additional N-terminal amino acids to NCoA1(257-385), compared to the STAT6(794-814) peptide, explaining its higher affinity. Secondary and tertiary structures existing in the free peptide are more highly populated in the complex, suggesting binding by conformational selection.

Regulation of gene expression represents a central issue in signal-regulated cellular responses. STAT6 is a critical mediator of IL-4 stimulated gene activation. To mediate this function, STAT6 recruits co-activator complexes. We have previously shown that STAT6 binds the PAS-B domain of the co-activator NCoA-1 via an LXXLL motif in its transactivation domain. Our recent finding that the PAS-B domain of NCoA-1 is also essential for co-activator complex formation points to an additional level of regulation of the co-activator assembly. In this study, we discovered that dephosphorylation of NCoA-1 is essential for the interaction with STAT6 and for IL-4-dependent transcriptional activation. PP2A dephosphorylates NCoA-1 and facilitates the activation of STAT6 target genes. Interestingly, simultaneous inhibition of phosphatase and cyclin-dependent kinases rescues the NCoA-1/STAT6 interaction. Moreover, arrest of cells at G1/S results in enhanced NCoA-1 phosphorylation. In summary, our results indicate that the interaction of NCoA-1 and STAT6 is dynamically regulated by the phosphatase PP2A and by cyclin-dependent kinases. This provides a mechanism for integrating transcriptional regulation by STAT6 with cell cycle progression.

Classical Hodgkin lymphoma (cHL) is a malignant lymphoid disorder characterized by aberrant activation of signaling pathways. Constitutive activation of several components of the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway has been observed in Hodgkin and Reed/Sternberg cells, the tumor cells of cHL. In this study, we investigate the function of STAT6 in cHL cell lines and show that STAT6 promotes survival of these cells. Microarray expression analysis of STAT6-shRNA (short hairpin RNA)-expressing cHL cell lines was carried out to analyze the STAT6-mediated survival mechanism. Some of the identified genes with potentially important regulatory functions were also interleukin (IL)-4 dependently regulated in Ramos B cells and binding of STAT6 to the regulatory regions of several genes could be confirmed, indicating that these are direct STAT6 target genes. Importantly, STAT6 knockdown increased the expression and activation of STAT1 as well as the expression of known STAT1 target genes, indicating a cross-regulation between these signaling molecules. Forced expression of STAT1 was able to induce apoptosis in cHL cell line L1236. These findings indicate that both STAT6 and STAT1 can act as important antagonistic regulators in the pathogenesis of cHL.

INTRODUCTION:The prolactin-Janus-kinase-2-signal transducer and activator of transcription-5 (JAK2-STAT5) pathway is essential for the development and functional differentiation of the mammary gland. The pathway also has important roles in mammary tumourigenesis. Prolactin regulated target genes are not yet well defined in tumour cells, and we undertook, to the best of our knowledge, the first large genetic screen of breast cancer cells treated with or without exogenous prolactin. We hypothesise that the identification of these genes should yield insights into the mechanisms by which prolactin participates in cancer formation or progression, and possibly how it regulates normal mammary gland development.METHODS:We used subtractive hybridisation to identify a number of prolactin-regulated genes in the human mammary carcinoma cell line SKBR3. Northern blotting analysis and luciferase assays identified the gene encoding heat shock protein 90-alpha (HSP90A) as a prolactin-JAK2-STAT5 target gene, whose function was characterised using apoptosis assays.RESULTS:We identified a number of new prolactin-regulated genes in breast cancer cells. Focusing on HSP90A, we determined that prolactin increased HSP90A mRNA in cancerous human breast SKBR3 cells and that STAT5B preferentially activated the HSP90A promoter in reporter gene assays. Both prolactin and its downstream protein effector, HSP90alpha, promote survival, as shown by apoptosis assays and by the addition of the HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in both untransformed HC11 mammary epithelial cells and SKBR3 breast cancer cells. The constitutive expression of HSP90A, however, sensitised differentiated HC11 cells to starvation-induced wild-type p53-independent apoptosis. Interestingly, in SKBR3 breast cancer cells, HSP90alpha promoted survival in the presence of serum but appeared to have little effect during starvation.CONCLUSIONS:In addition to identifying new prolactin-regulated genes in breast cancer cells, we found that prolactin-JAK2-STAT5 induces expression of the HSP90A gene, which encodes the master chaperone of cancer. This identifies one mechanism by which prolactin contributes to breast cancer. Increased expression of HSP90A in breast cancer is correlated with increased cell survival and poor prognosis and HSP90alpha inhibitors are being tested in clinical trials as a breast cancer treatment. Our results also indicate that HSP90alpha promotes survival depending on the cellular conditions and state of cellular transformation.

Transcriptional activation involves the ordered recruitment of coactivators via direct interactions between distinct binding domains and recognition motifs.The p160/SRC/NCoA coactivator family comprises three members (NCoA-1, -2 and -3), which are organized in multiprotein coactivator complexes.We had identified the PAS-B domain of NCoA-1 as an LXXLL motif binding domain.Here we show that NCoA family members are able to interact with other full-length NCoA proteins via their PAS-B domain and they specifically interact with the CBPinteraction domain (CID/AD1) of NCoA-1.Peptide competition, binding experiments and mutagenesis of LXXLL motifs point at distinct binding motif specificities of the NCoA PAS-B domains.NMR studies of different NCoA-1-PAS-B/LXXLL peptide complexes revealed similar although not identical binding sites for the CID/AD1 and STAT6 transactivation domain LXXLL motifs.In mechanistic studies, we found that overexpression of the PAS-B domain is able to disturb the binding of NCoA-1 to CBP in cells and that a CID/AD1 peptide competes with STAT6 for NCoA-1 in vitro.Moreover, the expression of an endogenous androgen receptor target gene is affected by the overexpression of the NCoA-1 or NCoA-3 PAS-B domains.Our study discloses a new, complementary mechanism for the current model of coactivator recruitment to target gene promoters.

Abstract About 12% of all de novo acute myeloid leukemias are characterized by the translocation t(8;21), which generates the oncogenic fusion protein RUNX1/ETO. RUNX1/ETO has a modular structure and contains several docking sites for heterologous proteins, including transcriptional corepressors like N-CoR, SMART, and mSIN3A. RUNX1/ETO is found in high molecular weight complexes, which are crucial for the block in myeloid differentiation observed in RUNX1/ETO–transformed cells. Essential for high molecular weight complex formation is the nervy homology region 2 (NHR2) within ETO, which serves as interacting surface for oligomerization as well as association with members of the ETO protein family. Here, we show that the expression of a fusion peptide consisting of 128 amino acids (NC128), including the entire NHR2 domain of ETO, disrupts the stability of the RUNX1/ETO high molecular weight complexes, restores transcription of RUNX1/ETO target genes, and reverts the differentiation block induced by RUNX1/ETO in myeloid cells. In the presence of NC128, RUNX1/ETO–transformed cells lose their progenitor cell characteristics, are arrested in cell cycle progression, and undergo cell death. Our results indicate that selective interference with the oligomerization domain of ETO could provide a promising strategy to inhibit the oncogenic properties of the leukemia-associated fusion protein RUNX1/ETO. [Cancer Res 2007;67(5):2280–9]

The MLL gene is frequently involved in chromosomal translocations associated with high-risk acute leukaemia. Infant and therapy-related acute leukaemia patients display chromosomal breakpoints preferentially clustered in the telomeric portion of the MLL breakpoint cluster region (SCII). Here, we demonstrate that SCII colocalizes with a gene-internal promoter element in the mouse and human MLL gene, respectively. The mRNA generated encodes an N-terminally truncated version of MLL that still exhibits many functional regions, including the C-terminal SET-domain. Etoposide-induced DNA double-strand breaks colocalize with the binding site of RNA polymerase II and the transcription initiation region, but not with a nearby Topo II consensus sequence. Thus, the observed genomic instability of the human MLL gene is presumably linked to transcriptional processes. The consequences of this novel finding for the creation of chromosomal translocations, the biology of the MLL protein and for MLL-mediated acute leukaemia are discussed.

Acetylation of signaling molecules can lead to apoptosis or differentiation of carcinoma cells. The molecular mechanisms underlying these processes and the biological role of enzymes mediating the transfer or removal of an acetyl-group are currently under intense investigation. Our study shows that Stat1 is an acetylated protein. Stat1 acetylation depends on the balance between Stat1-associated histone deacetylases (HDACs) and histone acetyltransferases (HATs) such as CBP. Remarkably both inhibitors of HDACs and the cytokine interferon alpha alter this equilibrium and induce Stat1 acetylation. The analysis of Stat1 mutants reveals Lys 410 and Lys 413 as acetylation sites. Experiments with Stat1 mutants mimicking either constitutively acetylated or nonacetylated states show that only acetylated Stat1 is able to interact with NF-kappaB p65. As a consequence, p65 DNA binding, nuclear localization, and expression of anti-apoptotic NF-kappaB target genes decrease. These findings show how the acetylation of Stat1 regulates NF-kappaB activity and thus ultimately apoptosis.

While chemo- and radiotherapy is far developed and successfully employed by default for cancer treatment, severe side effects point to the urgent need for more specific therapies based on the molecular mechanisms of this disease. Strategies to specifically inhibit signaling pathways that are known to force proliferation, prevent apoptosis or promote angiogenesis are expected to have a substantial impact on the future direction taken in cancer therapy. The Janus Kinase (JAK) / Signal transducer and activator of transcription (STAT) pathway is one major signaling pathway converting the signal of cytokines, growth factors and hormones into gene expression programs regulating essential cellular functions like proliferation, differentiation and survival. The suppressors of cytokine signaling (SOCS) as well as phosphatases normally tightly regulate the JAK/STAT pathway. Frequently, however this pathway is constitutively activated in a wide variety of human malignancies and substantially contributes to carcinogenesis. Consequently, new strategies for targeting the JAK/STAT pathway have been developed. This review discusses the biology of the JAK/STAT signaling pathway, which offers several molecular strategies for therapeutic interruption.

STAT proteins have the function of signaling from the cell membrane into the nucleus, where they regulate gene transcription. Latent mammalian STAT proteins can form dimers in the cytoplasm even before receptor-mediated activation by specific tyrosine phosphorylation. Here we describe the 3.21-angstrom crystal structure of an unphosphorylated STAT5a homodimer lacking the N-terminal domain as well as the C-terminal transactivation domain. The overall structure of this fragment is very similar to phosphorylated STATs. However, important differences exist in the dimerization mode. Although the interface between phosphorylated STATs is mediated by their Src-homology 2 domains, the unphosphorylated STAT5a fragment dimerizes in a completely different manner via interactions between their beta-barrel and four-helix bundle domains. The STAT4 N-terminal domain dimer can be docked onto this STAT5a core fragment dimer based on shape and charge complementarities. The separation of the dimeric arrangement, taking place upon activation and nuclear translocation of STAT5a, is demonstrated by fluorescence resonance energy transfer experiments in living cells.

Extravillous trophoblast cells resemble cancer cells with regard to their intrinsic invasiveness. They invade decidual tissue, but, unlike tumor cells, shut down their invasive properties, when they become inappropriate. Stimuli involved in the modulation of invasion, as well as their underlying signaling mechanisms require further clarification. We were especially interested in discovering signals capable of stimulating invasion in otherwise low-invasive cells involved in reproduction. Using the choriocarcinoma cell line Jeg-3 as a model, we have addressed the potential role of cytokine/growth factor-driven activation of signal transducer and activator of transcription 3 (STAT3) in this process. Jeg-3 cells were treated with various factors known to induce trophoblast proliferation, differentiation, migration, or invasiveness (insulin-like-growth-factor-II (IGF-II), hepatocyte growth factor (HGF), interleukin-6 (IL-6), and leukemia inhibitory factor (LIF)). Only LIF elicited strong tyrosine phosphorylation and specific DNA-binding activity of STAT3. It induced a significant acceleration of cell proliferation and promoted the capability of Jeg-3 cells to invade into an artificial extracellular matrix. Moreover, LIF influenced the expression pattern of proteases and protease inhibitors with potential relevance for invasiveness (downregulation of mRNA for tissue inhibitor of metalloproteinase 1 (TIMP-1) and upregulation of mRNA for caspase-4). In conjunction with earlier work, in which we found that STAT3 DNA-binding activity was increased in invasive cells (choriocarcinoma, first trimester trophoblasts) and absent in non-invasive cells (term trophoblasts), these findings suggest a connection between LIF-driven STAT3 activity and invasiveness of choriocarcinoma and trophoblast cells.

Signal transducer and activator of transcription 5 (STAT5) is a member of the STAT family of transcription factors that relay the effect of diverse cytokines, hormones, and growth factors by regulating the transcription of distinct target genes. This function is emphasized by its crucial role in the development of the mammary gland and the hematopoietic system. Cytokine receptor-associated Janus kinases (JAKs) induce dimerization, nuclear translocation, and DNA binding through tyrosine phosphorylation of STAT5. STAT5 regulates the expression of cytokine target genes by binding to gamma interferon-activated sequence (GAS) motifs. Transcriptional activation requires the contact of STAT5 to coactivators and components of the transcription machinery. Another important point in transcriptional activation is the cooperation with other transcription factors that bind in close vicinity to the target gene promoters and enhancers. Their concerted action can result in an enhanced binding to the promoters or in cooperative recruitment of coactivators. In addition, cross-talk with other signaling pathways as well as secondary modifications of STAT5 have been described to affect transactivation function.

The essential regulators in the pathogenesis of classical Hodgkin lymphoma (cHL) are still largely unknown. The malignant Hodgkin/Reed-Sternberg (HRS) cells of cHL secrete various cytokines leading to the activation of signaling pathways such as the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. In this study, we investigate the role of distinct JAK/STAT pathway components in the regulation of proliferation and survival of cHL cell lines. Electrophoretic mobility shift assay and western blot analysis revealed that the activation status of STAT family members varies in different cHL cell lines. Tyrosine kinase inhibitors of the JAK/STAT pathway blocked the activation of most of the STAT family members. This was accompanied with a strong antiproliferative effect and enhanced death of the treated cHL cell lines. Specific downregulation of STAT3 by siRNA expression decreased cell proliferation and induced apoptosis. Overexpression of SOCS1 and SOCS3 resulted in a proliferation arrest of cells with limited endogenous amount of these negative regulators, but not in cells that already express high amounts of SOCS1 and SOCS3. Our findings highlight the importance of STAT3 in cHL transformation and suggest SOCS1 and SOCS3 as potential targets for therapeutic intervention in distinct forms of cHL. (c) 2005 Wiley-Liss, Inc.

Colorectal carcinoma (CRC) is a major cause of morbidity and mortality in Western countries. It has so far been molecularly defined mainly by alterations of the Wnt pathway. We show here for the first time that aberrant activities of the signal transducer and activator of transcription STAT3 actively contribute to this malignancy and, thus, are a potential therapeutic target for CRC. Constitutive STAT3 activity was found to be abundant in dedifferentiated cancer cells and infiltrating lymphocytes of CRC samples, but not in non-neoplastic colon epithelium. Cell lines derived from malignant colorectal tumors lost persistent STAT3 activity in culture. However, implantation of colon carcinoma cells into nude mice resulted in restoration of STAT3 activity, suggesting a role of an extracellular stimulus within the tumor microenvironment as a trigger for STAT activation. STAT3 activity in CRC cells triggered through interleukin-6 or through a constitutively active STAT3 mutant promoted cancer cell multiplication, whereas STAT3 inhibition through a dominant-negative variant impaired IL-6-driven proliferation. Blockade of STAT3 activation in CRC-derived xenograft tumors slowed down their development, arguing for a contribution of STAT3 to colorectal tumor growth.

Signal transducer and activator of transcription 6 (STAT6) regulates transcriptional activation in response to interleukin-4 (IL-4) by direct interaction with coactivators. Among them, NCoA-1, a member of the p160/steroid receptor coactivator (SRC) family, has been found to bind to STAT6 with the region B of its putative Per-Arnt-Sim (PAS) domain. STAT6 interacts specifically with NCoA-1 via an LXXLL motif in its transactivation domain. Crystals of the NCoA-1(257-385) domain in complex with the STAT6(794-814) LXXLL motif were obtained in two hexagonal space groups. The crystals in space group P6(1), with unit-cell parameters a = 61.7, b = 61.7, c = 146.5 A, alpha = beta = 90, gamma = 120 degrees, diffract to 2.8 A at a home source. Crystals belonging to space group P6(2), with unit-cell parameters a = 62.0, b = 62.0, c = 73.6 A, alpha = beta = 90, gamma = 120 degrees, diffract to 1.8 A at a synchrotron source.