Horm Mol Biol Clin Invest 2012;10(2):281–286 © 2012 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/hmbci-2012-0009
Progress towards direct inhibitors of Stat5 protein
Abbarna A. Cumaraswamy and Patrick T. Gunning* Chemical and Physical Sciences, University of Toronto at Mississauga, Mississauga, Ontario, Canada
Abstract Molecular approaches to inhibit STAT5 signaling have been hailed as a viable targeted anticancer therapy. In particular, many drugs and drug candidates have been developed to successfully inhibit upstream effectors of STAT5 by indirectly targeting cell surface receptors and protein kinases (FLT-3, JAK2, and BCR-ABL). Indirect strategies have yielded potent agents, such as imatinib, AC2207, and EXEL823 which effectively silence STAT5 activity but which suffer from offtarget effects and toxicity. This article will focus on reviewing the current literature pertaining to direct inhibitors of STAT5 protein and assess the prospects for a future STAT5-targeting therapeutic. Keywords: anticancer drugs; molecular recognition; proteinprotein interaction; Stat5.
Introduction The Jak/Stat signaling pathway (Figure 1) is of intense interest to molecular biologists, clinical oncologists and medicinal chemists, and is the subject of much investigation. In particular, the role of the Stat proteins in oncogenic signaling has been the subject of much research. The Stat family comprises seven members; Stat1, Stat2, Stat3, Stat4, Stat6, and the focus of this review, Stat5a and Stat5b proteins. The Stats perform dual roles as cytosolic signaling proteins and as nuclear transcription factors that control the expression of specific sets of genes, particularly those involved in proliferation (Bcl-xl, c-Myc, pim-1), apoptosis (JAB), cell differentiation (p21) [1], and inflammation (Osm). Stat proteins can become constitutively active through somatic mutations, as well as mutated or dysregulated expression of the upstream signaling components of this pathway and, in turn, can act as proto-oncogenes resulting in the aberrant expression of proteins they regulate. Stat5 proteins have a broad activation profile and are phosphorylated downstream of interleukin-2 (IL-2), granulocyte
*Corresponding author: Patrick T. Gunning, 3359 Mississauga Road, Mississauga, Ontario, L5L 1C6, Canada Phone: +1-905-828-5354, Fax: +1-905-828-5425, E-mail: [email protected] Received February 20, 2012; accepted April 12, 2012
macrophage colony-stimulating factor (GM-CSF), erythropoietin, IL-2, IL-5, IL-7, thrombopoietin, prolactin, and growth hormone receptor [2] by receptor-associated kinases, such as the Janus kinases (Jak). Binding of these extracellular ligands induces receptor dimerization and intracellular activation of receptor-associated Jak kinases. Jak-mediated tyrosine phosphorylation of the cytoplasmic receptor tails creates docking sites for monomeric, non-phosphorylated STAT5 proteins through their phosphotyrosine binding Src Homology 2 domain (SH2). Activated receptor-associated Jak2 kinases phosphorylate the associated STAT5 at specific tyrosine (Y) residues in the C-terminus (Y694 for STAT5a and Y699 for STAT5b), resulting in receptor dissociation. Activated STAT5 (pSTAT5) homo- or heterodimerizes [3, 4] through reciprocal SH2-phosphotyrosine interactions, producing transcriptionally active complexes. Upon nuclear translocation, STAT5-STAT5 dimers bind their respective STAT5 response elements on DNA and induce target gene transcription of cell cycle regulators, such as cyclin D1 and D2, antiapoptotic genes, and genes involved in regulation of cell differentiation. Natural deactivation of STATs can occur directly by dephosphorylation by phosphatases, including protein inhibitors of activated STAT proteins (PIAS), protein tyrosine phosphatases (SHP-2) or indirectly by downregulation of cytokine signaling via SOCS1, SOCS3, and CIS proteins which block the cytokine signal by direct inhibition of JAKs (SOCS1) and by competitive binding to tyrosine phosphorylated receptors to exclude further binding of STAT5 [2, 5]. Aberrant STAT5, along with STAT3, has been detected with high frequency in diverse human cancers, including those of the breast, prostate, liver, skin, blood, head, and neck [2]. STAT5 protein is constitutively activated by cytosolic oncogenic tyrosine kinases, TEL-Jak2 and Bcr-Abl, mutated FMS-like tyrosine kinase 3 (FLT3), as well as by overactive receptor tyrosine kinases, including SRC and EGFR [6]. Antiapoptotic genes, such as Bcl-xl, Bcl-2, Myc, and MCL, which serve to counteract other proapoptotic pathways have been found to be overactivated as a result of elevated STAT5 protein activity. Molecular approaches to inhibit STAT5 signaling have been hailed as a viable targeted anticancer therapy. In particular, many drugs and drug candidates have been developed to successfully inhibit upstream effectors of Stat5 by indirectly targeting cell surface receptors and protein kinases (FLT-3, JAK2, and BCR-ABL). Indirect strategies have yielded potent agents, such as imatinib [7], AC220 [8], and EXEL8232 [9], which effectively silence STAT5 activity but which suffer from off-target effects and toxicity. This article will focus on reviewing the current literature pertaining to direct inhibitors of STAT5 protein and assess the prospects for a future Stat5-targeting therapeutic.
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Direct inhibitors of Stat5 STAT5 suppression through truncated STAT5 inhibitors
Truncated STAT proteins have been reported in nature, including splice variants of both STAT1 and STAT3 [10]. Both protein fragments were shown to inhibit the transcriptional activity of their respective STAT genes. N-terminal and C-terminal truncated STAT5 variants are expressed in both healthy and cancerous cell lines. In healthy cells, the low concentrations of truncated isoforms do not induce inhibitory effects against wild-type STAT5. By contrast, within various breast (TD47 and MCF7) [11] and prostate (CWR22Rv1, DU145, PC-3, LNCaP) [12] cancer cell lines, the function of truncated STAT5 is, in a dominant-negative manner, to suppress the expression of endogenous genes normally regulated by wild-type STAT5. The two most common truncated STAT5 proteins are the N-terminal and C-terminal truncated isoforms. C-terminal truncation Two naturally occurring carboxytruncated STAT5 variant proteins have been identified of molecular weights, 77 and 80 kDa. These dominant-negative C-terminal truncated Stat5 proteins are characterized by a partial or complete loss of the transactivation domain. Numerous C-terminal truncated STAT5 variants have been reported (Δ750, Δ740) [13, 14], with most variants retaining the conserved tyrosine residues, Y694 and 699, residues necessary for activation. These truncated variants are able to form heterodimers with wild-type Stat5 and bind to DNA but
have nitric oxide (NO) transcriptional activity. The tyrosine phosphorylation of the truncated Stat5 variants were found to form more stable dimers than those of wild-type and the overexpression of these proteins caused inhibition of genes (oncostatin M and Cis) normally regulated by wild-type Stat5 [15]. Thus, dominant-negative variants of C-terminal truncated STAT5 have been used to investigate the knockdown role of STAT5 in estrogen receptor (ER)-positive breast cancer cell lines [16, 17]. Evidence suggests that co-transfection experiments of wild-type STAT5 with the STAT5 aΔ740 isoform completely blocked transcriptional activity of endogenous ER in T47D (0.4 µg) and MCF7 (0.4 µg) breast cancer cells as well as ERα and ERβ in COS7 breast cancer cells. In addition, high-efficiency adenoviral delivery of these dominant-negative STAT5 isoforms into T47D breast cancer cells was found to induce apoptosis through a capsase-3-mediated pathway [11]. N-terminal truncation Whereas C-terminal truncated STAT5 proteins inhibit breast cancer cell growth, the overexpression of N-terminal truncated STAT5 proteins was shown to promote the growth of prostate cancer cells. Normally, STAT5 activation is regulated by PIAS3, which suppresses STAT activity by binding directly the N-terminal region of STAT5. In prostate cancer cells, the N-terminal truncated STAT5 protein generated at the protein level evades this transcriptional repression and continually binds to DNA leading to metastasis, tumor growth, and disease progression [12]. Recent evidence has shown that the C-terminally truncated proteins that have been reported as naturally occurring Stat5
Cumaraswamy and Gunning: Direct inhibitors of Stat5 protein
isoforms are, in fact, generated during sample preparation rather than through a post-translational in vivo modification as seen for N-terminally truncated STAT5 proteins [18–20]. This evidence brings into question where there is a physiological significance to the C-terminal truncated proteins. By contrast, N-terminal truncations were shown to exist prior to sample preparation, indicating their natural occurrence in nature [12]. One potential molecular approach would be to inhibit the proteases responsible for STAT5 N-terminal proteolytic cleavage, using small molecule inhibitors to restore the PIAS inhibitory mechanism. Suppression of STAT5 through Runx proteomimetics
Runt-related (Runx) transcription factors regulate hematopoietic cell-specific genes and play a critical role in T-lymphocyte differentiation similar to that of STAT proteins. It was discovered that interactions between Runx and STAT proteins played a negative regulatory role and served to mutually inhibit their transcriptional activity [21, 22]. Growth hormone stimulation in Saos-2 cells lead to the formation of STAT1-Runx2 and STAT3-Runx2 heterodimers. Protein complexation inhibited both Runx2 and STAT transcriptional activity while not effecting DNA binding potency. Similarly, STAT5-Runx complexes have been investigated and identified a key interaction between the Runt domain of Runx and the DNA binding domain and α-helix loop structure of STAT5. Most interestingly, this interaction prevented STAT5 nuclear translocation and inhibited transcriptional function of STAT5. Three possible mechanisms were suggested for the suppression of STAT5 transcriptional activity. First, STAT5 may block the dimerization of Runx proteins by competing for the transcriptional coactivator binding site (CBFβ) in the Runt domain which is responsible for dimerization [23]. Second, STAT5 may mask the DNA binding surface of the Runx protein, as the Runt domain is needed to bind DNA. Finally, STAT5 association with Runx proteins inhibits the mechanism of both STAT5 and Runx nuclear transport [23]. Thus, Runx transcription factors act as negative regulators of STAT5 activity and may warrant investigation. It is envisaged that mimicry of the key binding regions of Runx could yield high affinity inhibitors of STAT5 transcriptional activity. Mimicking key Runx binding epitopes could yield a new peptidomimetic/small molecule class of binders. Suppression of STAT5 through phospholipase C (PLC)-β3 proteomimetics
Stat5 activity is negatively regulated by phosphatases, such as SH2 domain-containing protein phosphatase (SHP-1) within the cytoplasm. Very recently, a study found an increased rate of Stat5 downregulation by SHP-1 through the phospholipase C (PLC)-β3 enzyme. One role of the PLC enzyme is to determine the duration, amplitude, and on-off cycling of signaling. Stat5 and SHP-1 were found to bind to PLC-β3 via its non-catalytic C-terminal domain (PLC-β3-CT) forming a multimolecular complex. As a result, SHP-1 and Stat5 are brought into
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close proximity facilitating an accelerated rate of Stat5-Tyr694 dephosphorylation [24]. Upon further investigation to understand the growth-suppressive function of PLC-β3-CT, a multitude of short peptides was generated corresponding to the regions to which Stat5 and SHP-1 bound. Two peptides (peptide b and its shorter derivative b11) substantially inhibited ( > 80% potency) in vitro proliferation and differentiation within two hematopoietic cells, Ba/F3 pro-B and TF-1 erythroleukemia cells. This is consistent with the hypothesis that reduced Stat5 activity seems to be the molecular basis for the growth-inhibitory function of PLC-β3-CT and its shorter derivatives. Interestingly, these peptides had no effect on Stat3-Tyr705 and Akt and little effect on MAPK phosphorylation, illustrating specificity for Stat5. The introduction of these peptides into target cells has been considered as a therapeutic strategy; however, there are significant drawbacks for its applications. First, there is reported evidence that there needs to be high expression levels of peptide b11 to measure any growth inhibition. Second, the stability of the peptide within the target cells against proteases needs to be accounted for. Suppression of Stat5 using oligonucleotide-based strategies
Stat5 Disrupting Stat5-DNA binding Targeting nuclear traffic is another effective strategy at developing new therapeutics, specifically using oligonucleotide-based strategies to target Stat5-Stat5:DNA binding activity. There are two commonly used approaches: first, strategies that downregulate Stat5 gene expression and those that prevent Stat5 binding to DNA. Antisense oligodeoxynucleotides (asODNs) and RNA interference (RNAi) have been used to target Stat5a and Stat5b with potent downregulation of their target genes. As both isoforms share 96% sequence homology, one equivalent of either RNAi or asODN would be effective at knocking down both isoforms and downregulating gene expression [25]. This has been successful with short interference RNA (siRNA) [25]; however, siRNA suffers from specificity and efficacy resulting in off-target effects [26]. To overcome this problem, the more stable short hairpin RNA (shRNA) was introduced using viral vectors and plasmids, which were then processed into siRNA [27]. Alternatively, the delivery of asODNs to target Stat5 isoforms do not suffer from nonspecific effects, although a two-fold greater molar excess is required compared with the corresponding siRNA to achieve effective knock-down [25, 28]. Decoy ODNs have been established as an efficient approach to block gene expression by competing with the binding of the transcription factor to the DNA cis-element [28, 29]. This sequesters the transcription factor within the cytoplasm and prevents its target gene expression [30]. This approach has been successful with the downregulation of Stat5 target genes in various cancer cell lines (K562 [28], HL-60 [31]), with no observation of non-specific effects. Although decoy ODNs provide an effective approach over siRNA or asODNs for inhibition, their delivery through transfection or other
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studies in KU812 and K562 CML cells revealed that pimozide elicited a dose-dependent decrease in STAT5 tyrosine phosphorylation at concentrations of 3–10 µM. Treatment with 2 (10 µM) resulted in a decreased viability of CML cells through the induction of apoptosis with no deleterious effects on healthy cells at similar concentrations of inhibitor. Nelson and coworkers demonstrated that phospho-STAT5 knockdown was not the result of inhibiting the upstream kinases of STAT5, rather they proposed that inhibition is based on direct association of 2 with STAT5. Importantly, pimozide in combination with drugs (1, imatinib and nilotinib) targeting the BCR-ABL kinase enhanced the inhibitory effects against STAT5 phosphorylation and induced apoptosis in T3151 BCR/ABL mutant resistant CML cells [34]. Although potent knock-down of STAT5 phosphorylation was observed, the authors did not present data to show the physical association of 2 with STAT5. The authors hypothesized that 2 may be inducing a phosphatase to dephosphorylate STAT5, or 2 is altering the stability of STAT5 through interactions with chaperone proteins (HSP90). Gunning and coworkers rationally designed and developed a library of salicylic acid-containing inhibitors (∼200) for targeting the SH2 domain of STAT3, wherein the salicylic acid component was proposed to effectively mimic the key pTyr binding residue. Given that the STAT SH2 domains showed moderate sequence homology, an in vitro screen of this library for STAT5 selectivity was conducted. Several potent leads were identified with Ki < 10 µM. Most notably, 3, 4, and 5 with Ki-values of 7.9, 2.8, and 8.3 µM, respectively, exhibited a three-fold selectivity for STAT5 over STAT3 and STAT1. Of these STAT5 inhibitors, 3 exhibited the highest STAT5 specificity (i.e., STAT5 Ki = 7.9 µM cf. STAT1, > 25 µM cf. STAT3 > 25 µM). When evaluated in K562 and MV-4-11 human leukemia cells, lead agents demonstrated potent and selective suppression of STAT5 phosphorylation (via Western analysis), inhibited STAT5 target genes and induced apoptosis (IC50∼20 µM). Moreover, lead
methods limits their subcellular compartmentalization, which may restrict their ability to inhibit Stat5 in the cytoplasm. Suppression of STAT5 using small molecule inhibitors Disrupting STAT5-STAT5 dimerization Identification of small molecule inhibitors of STAT5 protein has been limited. Presently, the majority of work has focused on targeting the SH2 domain of STAT3 [32]. This can be attributed to the determination of the STAT3-STAT3:DNA bound crystal structure. The structure showed the binding conformation and residues pertinent to phosphotyrosinylated STAT3 binding another STAT3 monomer, and more particularly shed light on the key residues required to bind the SH2 domain of STAT3. To date, the analogous STAT5-STAT5:DNA bound crystal structure has not been obtained. Nonetheless, there has been recent progress made towards targeting the SH2 domain of STAT5 using small molecule inhibitors. A highthroughput screen of diverse chemical libraries of a total 17,298 compounds, using a fluorescence polarization assay developed by Berg and coworkers led to the identification of a chromone-derived acyl hydrazone of which N′-[(4-Oxo4H-chromen-3-yl)methylene]nicotinohydrazide (1) (Figure 2) demonstrated the most potent and selective inhibition of STAT5 (STAT5, IC50 = 47 µM cf. STAT3, IC50 = 180 µM and STAT1, IC50 = 130 µM). Treatment of lymphoma cells (Daudi) with 1 (100–200 µM) resulted in inhibition of STAT5-STAT5 dimerization and suppression of STAT5 DNA binding activity [33]. Similarly, a STAT5 function-based screening approach was used to show that pimozide (2) (Figure 2), an FDA approved neuroleptic drug used for the treatment of Tourette’s syndrome, potently inhibited STAT5 phosphorylation. Pimozide was found to downregulate STAT5 target gene expression without decreasing the autophosphorylation of the upstream kinases of Stat5 (BCR-ABL, MAPK, and JAK2). Cellular
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agents showed negligible cytotoxic effects against healthy bone marrow cells, which do not harbor constitutively activated STAT5 [35].
Conclusion Potent and selective inhibition of STAT5 via targeted molecule therapeutics represents a most appealing candidate for treatment of human cancers. With growing evidence implicating the prominent role of STAT5 in inflammation, interest in a STAT5 drug has extended to inflammatory diseases, such as rheumatoid arthritis [36] and inflammatory bowel disease [37]. In addition, a STAT5 drug may have application as an adjuvant therapy for the treatment of CMLs and prostate cancer. Although there has been little progress towards a clinical candidate, there is growing evidence to suggest that STAT5 protein can be targeted selectively and with moderate potency. It remains to be seen whether suitably potent inhibitors can be developed with suitable in vivo activity to enter preclinical trials. However, the limited studies do suggest that more potent agents will be uncovered with more intensive high-throughput screens and rational drug design investigations.
References 1. Nosaka T, Kawashima T, Misawa K, Ikuta K, Mui AL, Kitamura T. STAT5 as a molecular regulator of proliferation, differentiation and apoptosis in hematopoietic cells. EMBO J 1999;18:4754–65. 2. Shyh-Han T, Nevaleinen MT. Signal transducer and activator of transcription 5A/B in prostate and breast cancers. Endocr Relat Cancer 2008;15:367–90. 3. Becker S, Groner B, Muller CW. Three-dimensional structure of the Stat3beta homodimer bound to DNA. Nature 1998;394:145–51. 4. Chen X, Vinkemeier U, Zhao Y, Jeruzalmi D, Darnell JE, Jr, Kuriyan J. Crystal structure of a tyrosine phosphorylated Stat-1 dimer bound to DNA. Cell 1998;93:827–39. 5. Peltola KJ, Paukku K, Aho TL, Ruuska M, Silvennoinen O, Koskinen PJ. Pim-1 kinase inhibits Stat5-dependant transcription via its interactions with SOCS1 and SOCS3. Blood 2004;103:3744–50. 6. Yu H, Jove R. The Stats of cancer – new molecular targets come of age. Nat Rev Cancer 2004;2:97–105. 7. Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J, Lydon NB. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996;5:561–6. 8. Sato T, Yang X, Knapper S. FLT-3 ligand impedes the efficacy of FLT-3 inhibitors in vitro and in vivo. Blood 2011;117: 3286–93. 9. Wernig G, Kharas MG, Mullally A, Leeman DS, Okabe R, George T, Clary DO, Gilliland, DG. EXEL-8232, a small-molecule JAK2 inhibitor, effectively treats thrombocytosis and extramedullary hematopoiesis in a murine model of myeloproliferative neoplasm induced by MPLW515L. Leukemia 2012;26:720–7. 10. Caldenhoven E, Van Dijk TB, Solari R, Armstrong J, Raaijmakers JA, Lammers JW, Koenderman L, de Groot RP. Stat3beta, a splice variant of transcription factor Stat3, is a dominant negative regulator of transcription. J Biol Chem 1996;271:13221–7.
285
11. Yamashita H, Iwase H, Toyama T, Fujii Y. Naturally occurring dominant-negative Stat5 suppresses transcriptional activity of estrogen receptors and induces apoptosis in T47D breast cancer cells. Oncogene 2003;22:1638–52. 12. Dagvadorj A, Shyh-Han T, Liao Z, Xie J, Nurmi M, Alanen K, Rui H, Mirtti T, Nevalainen MT. N-terminal truncation of Stat5a/b circumvents PIAS3-mediated transcriptional inhibition of Stat5 in prostate cancer cells. Int J Biochem Cell Biol 2010;42:2037–46. 13. Moriggl R, Gouilleux-Gruart V, Jahne R, Berchtold S, Gartmann C, Liu X, Hennighausen L, Sotiropoulos A, Groner B, Gouilleux F. Deletion of the carboxy-terminal transactivation domain of MGF-Stat5 results in sustained DNA binding and a dominant negative phenotype. Mol Cell Biol 1996;10:5691–700. 14. Kazansky AV, Raught B, Lindsey SM, Wang Y, Rosen JM. Regulation of mammary gland factor/stat5a during mammary gland development. Mol Endocrinol 1995;9:1598–609. 15. Wang D, Stravopodis D, Teglund S, Kitazawa J, Ihle JN. Naturally occurring dominant negative variants of Stat5. Mol Cell Biol 1996;16:6141–8. 16. DaSilva L, Rui H, Erwin RA, Howard OM, Kirken RA, Malabarba MG, Hackett RH, Larner AC, Farrar WL. Prolactin recruits Stat1, Stat3 and Stat5 independent of conserved receptor tyrosines TYR402, TYR479, TYR515 and TYR580. Mol Cell Endocrinol 1996;117:131–40. 17. Schaber JD, Fang H, Xu J, Grimley PM, Rui H. Prolactin activates Stat1 but does not antagonize Stat1 activation and growth inhibition by type 1 interferons in human breast cancer cells. Cancer Res 1998;58:1914–9. 18. Azam M, Lee C, Strehlow I, Schindler C. Functionally distinct isoforms of STAT5 are generated by protein processing. Immunity 1997;6:691–701. 19. Garimorth K, Welte T, Doppler W. Generation of carboxy-terminally deleted forms of STAT5 during preparation of cell extracts. Exp Cell Res 1999;246:148–51. 20. Schuster B, Hendry L, Byers H, Lynham SF, Ward MA, John S. Purification and identification of the STAT5 protease in myeloid cells. Biochem J 2007;404:81–7. 21. Kim S, Koga T, Isobe M, Kern BE, Yokochi T, Chin YE, Karsenty G, Taniguchi T, Takayanagi H. Stat1 functions as a cytoplasmic attenuator of Runx2 in the transcriptional program of osteoblast differentiation. Genes Dev 2003;17:1979–91. 22. Ziros PG, Georgakopoulos T, Habeos I, Basdra EK, Papavassiliou AG. Growth hormone attenuates the transcriptional activity of Runx2 by facilitating its physical association with Stat3β. J Bone Miner Res 2004;19:1892–904. 23. Ogawa S, Satake M, Ikuta K. Physical and functional interactions between Stat5 and Runx transcription factors. J Biochem 2008;143:695–709. 24. Yasudo H, Ando T, Xiao W, Kawakami Y, Kawakami T. Short stat5-interacting peptide derived from phospholipase C-β3 inhibits hematopoietic cell proliferation and myeloid differentiation. PLoS ONE 2011;6:e24995. 25. Liu X, Robinson GW, Gouilleux F, Groner B, Hennighausen L. Cloning and expression of Stat5 and an additional homologue (Stat5b) involved in prolactin signal transduction in mouse mammary tissue. Proc Natl Acad Sci USA 1995;92:8831–5. 26. Sledz CA, Williams BR. RNA interference and double-stranded-RNA-activated pathways. Biochem Soc Trans 2004;32: 952–6. 27. Klosek SK, Nakashiro K, Hara S, Goda H, Hamakawa H. Stat3 as a molecular target in RNA interference-based treatment of oral squamous cell carcinoma. Oncol Rep 2008;20:873–8. 28. Wang X, Zeng J, Shi M, Zhao S, Bai W, Cao W, Tu Z, Huang Z, Feng W. Targeted blockage of signal transducer and activator of
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transcription 5 signaling pathway with decoy oligodeoxynucleotides suppresses leukemic K562 cell growth. DNA Cell Biol 2011;30:71–8. 29. Ahn JD, Morishita R, Kaneda Y, Lee S, Kwon K, Choi S, Lee K, Park J, Moon I, Park JG, Yoshizuma M, Ouchi Y, Lee I. Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo. Circ Res 2002;90:1325–32. 30. Cutroneo KR, Ehrlich H. Silencing or knocking out eukaryotic gene expression by oligodeoxynucleotide decoys. Crit Rev Eukaryot Gene Expr 2006;16:23–30. 31. Cao S, Wang C, Zheng Q, Qiao Y, Xu K, Jiang T, Wu A. Stat5 regulates glioma cell invasion by pathways dependant and independent of Stat5 DNA binding. Neurosci Lett 2011;487:228–33. 32. Avadisian M, Haftchenary S, Gunning PT. Inhibiting aberrant Stat3 function with molecular therapeutics: a progress report. Anticancer Drugs 2011;22:115–27.
33. Muller J, Sperl B, Reindl W, Kiessling A, Berg T. Discovery of chromone-based inhibitors of the transcription factor Stat5. ChemBioChem 2008;9:723–7. 34. Nelson EA, Walker SR, Weisberg E, Bar-Natan M, Barrett R, Gashin LB, Terrell S, Klitgaard JL, Santo L, Addorio MR, Ebert BL, Griffin JD, Frank DA. The Stat5 inhibitor pimozide decreases survival of chronic myelogenous leukemia cells resistant to kinase inhibitors. Blood 2011;117:3421–9. 35. Page BD, Khoury H, Laister RC, Fletcher S, Vellozo M, Manzoli A, Yue P, Turkson J, Minden MD, Gunning PT. Small molecule STAT5-SH2 domain inhibitors exhibit potent antileukemia activity. J Med Chem 2012;55:1047–55. 36. Naka T, Kishimoto T. Joint diseases caused by defective gp130-mediated STAT signaling. Arthritis Res 2002;4: 154–8. 37. O’Shea JJ, Murray PJ. Cytokine signaling modules in inflammatory responses. Immunity 2008;28:477–87.
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Progress towards direct inhibitors of Stat5 protein
Abbarna A. Cumaraswamy and Patrick T. Gunning* Chemical and Physical Sciences, University of Toronto at Mississauga, Mississauga, Ontario, Canada
Abstract Molecular approaches to inhibit STAT5 signaling have been hailed as a viable targeted anticancer therapy. In particular, many drugs and drug candidates have been developed to successfully inhibit upstream effectors of STAT5 by indirectly targeting cell surface receptors and protein kinases (FLT-3, JAK2, and BCR-ABL). Indirect strategies have yielded potent agents, such as imatinib, AC2207, and EXEL823 which effectively silence STAT5 activity but which suffer from offtarget effects and toxicity. This article will focus on reviewing the current literature pertaining to direct inhibitors of STAT5 protein and assess the prospects for a future STAT5-targeting therapeutic. Keywords: anticancer drugs; molecular recognition; proteinprotein interaction; Stat5.
Introduction The Jak/Stat signaling pathway (Figure 1) is of intense interest to molecular biologists, clinical oncologists and medicinal chemists, and is the subject of much investigation. In particular, the role of the Stat proteins in oncogenic signaling has been the subject of much research. The Stat family comprises seven members; Stat1, Stat2, Stat3, Stat4, Stat6, and the focus of this review, Stat5a and Stat5b proteins. The Stats perform dual roles as cytosolic signaling proteins and as nuclear transcription factors that control the expression of specific sets of genes, particularly those involved in proliferation (Bcl-xl, c-Myc, pim-1), apoptosis (JAB), cell differentiation (p21) [1], and inflammation (Osm). Stat proteins can become constitutively active through somatic mutations, as well as mutated or dysregulated expression of the upstream signaling components of this pathway and, in turn, can act as proto-oncogenes resulting in the aberrant expression of proteins they regulate. Stat5 proteins have a broad activation profile and are phosphorylated downstream of interleukin-2 (IL-2), granulocyte
*Corresponding author: Patrick T. Gunning, 3359 Mississauga Road, Mississauga, Ontario, L5L 1C6, Canada Phone: +1-905-828-5354, Fax: +1-905-828-5425, E-mail: [email protected] Received February 20, 2012; accepted April 12, 2012
macrophage colony-stimulating factor (GM-CSF), erythropoietin, IL-2, IL-5, IL-7, thrombopoietin, prolactin, and growth hormone receptor [2] by receptor-associated kinases, such as the Janus kinases (Jak). Binding of these extracellular ligands induces receptor dimerization and intracellular activation of receptor-associated Jak kinases. Jak-mediated tyrosine phosphorylation of the cytoplasmic receptor tails creates docking sites for monomeric, non-phosphorylated STAT5 proteins through their phosphotyrosine binding Src Homology 2 domain (SH2). Activated receptor-associated Jak2 kinases phosphorylate the associated STAT5 at specific tyrosine (Y) residues in the C-terminus (Y694 for STAT5a and Y699 for STAT5b), resulting in receptor dissociation. Activated STAT5 (pSTAT5) homo- or heterodimerizes [3, 4] through reciprocal SH2-phosphotyrosine interactions, producing transcriptionally active complexes. Upon nuclear translocation, STAT5-STAT5 dimers bind their respective STAT5 response elements on DNA and induce target gene transcription of cell cycle regulators, such as cyclin D1 and D2, antiapoptotic genes, and genes involved in regulation of cell differentiation. Natural deactivation of STATs can occur directly by dephosphorylation by phosphatases, including protein inhibitors of activated STAT proteins (PIAS), protein tyrosine phosphatases (SHP-2) or indirectly by downregulation of cytokine signaling via SOCS1, SOCS3, and CIS proteins which block the cytokine signal by direct inhibition of JAKs (SOCS1) and by competitive binding to tyrosine phosphorylated receptors to exclude further binding of STAT5 [2, 5]. Aberrant STAT5, along with STAT3, has been detected with high frequency in diverse human cancers, including those of the breast, prostate, liver, skin, blood, head, and neck [2]. STAT5 protein is constitutively activated by cytosolic oncogenic tyrosine kinases, TEL-Jak2 and Bcr-Abl, mutated FMS-like tyrosine kinase 3 (FLT3), as well as by overactive receptor tyrosine kinases, including SRC and EGFR [6]. Antiapoptotic genes, such as Bcl-xl, Bcl-2, Myc, and MCL, which serve to counteract other proapoptotic pathways have been found to be overactivated as a result of elevated STAT5 protein activity. Molecular approaches to inhibit STAT5 signaling have been hailed as a viable targeted anticancer therapy. In particular, many drugs and drug candidates have been developed to successfully inhibit upstream effectors of Stat5 by indirectly targeting cell surface receptors and protein kinases (FLT-3, JAK2, and BCR-ABL). Indirect strategies have yielded potent agents, such as imatinib [7], AC220 [8], and EXEL8232 [9], which effectively silence STAT5 activity but which suffer from off-target effects and toxicity. This article will focus on reviewing the current literature pertaining to direct inhibitors of STAT5 protein and assess the prospects for a future Stat5-targeting therapeutic.
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Cumaraswamy and Gunning: Direct inhibitors of Stat5 protein
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Jak2/Stat5 signaling pathway.
Direct inhibitors of Stat5 STAT5 suppression through truncated STAT5 inhibitors
Truncated STAT proteins have been reported in nature, including splice variants of both STAT1 and STAT3 [10]. Both protein fragments were shown to inhibit the transcriptional activity of their respective STAT genes. N-terminal and C-terminal truncated STAT5 variants are expressed in both healthy and cancerous cell lines. In healthy cells, the low concentrations of truncated isoforms do not induce inhibitory effects against wild-type STAT5. By contrast, within various breast (TD47 and MCF7) [11] and prostate (CWR22Rv1, DU145, PC-3, LNCaP) [12] cancer cell lines, the function of truncated STAT5 is, in a dominant-negative manner, to suppress the expression of endogenous genes normally regulated by wild-type STAT5. The two most common truncated STAT5 proteins are the N-terminal and C-terminal truncated isoforms. C-terminal truncation Two naturally occurring carboxytruncated STAT5 variant proteins have been identified of molecular weights, 77 and 80 kDa. These dominant-negative C-terminal truncated Stat5 proteins are characterized by a partial or complete loss of the transactivation domain. Numerous C-terminal truncated STAT5 variants have been reported (Δ750, Δ740) [13, 14], with most variants retaining the conserved tyrosine residues, Y694 and 699, residues necessary for activation. These truncated variants are able to form heterodimers with wild-type Stat5 and bind to DNA but
have nitric oxide (NO) transcriptional activity. The tyrosine phosphorylation of the truncated Stat5 variants were found to form more stable dimers than those of wild-type and the overexpression of these proteins caused inhibition of genes (oncostatin M and Cis) normally regulated by wild-type Stat5 [15]. Thus, dominant-negative variants of C-terminal truncated STAT5 have been used to investigate the knockdown role of STAT5 in estrogen receptor (ER)-positive breast cancer cell lines [16, 17]. Evidence suggests that co-transfection experiments of wild-type STAT5 with the STAT5 aΔ740 isoform completely blocked transcriptional activity of endogenous ER in T47D (0.4 µg) and MCF7 (0.4 µg) breast cancer cells as well as ERα and ERβ in COS7 breast cancer cells. In addition, high-efficiency adenoviral delivery of these dominant-negative STAT5 isoforms into T47D breast cancer cells was found to induce apoptosis through a capsase-3-mediated pathway [11]. N-terminal truncation Whereas C-terminal truncated STAT5 proteins inhibit breast cancer cell growth, the overexpression of N-terminal truncated STAT5 proteins was shown to promote the growth of prostate cancer cells. Normally, STAT5 activation is regulated by PIAS3, which suppresses STAT activity by binding directly the N-terminal region of STAT5. In prostate cancer cells, the N-terminal truncated STAT5 protein generated at the protein level evades this transcriptional repression and continually binds to DNA leading to metastasis, tumor growth, and disease progression [12]. Recent evidence has shown that the C-terminally truncated proteins that have been reported as naturally occurring Stat5
Cumaraswamy and Gunning: Direct inhibitors of Stat5 protein
isoforms are, in fact, generated during sample preparation rather than through a post-translational in vivo modification as seen for N-terminally truncated STAT5 proteins [18–20]. This evidence brings into question where there is a physiological significance to the C-terminal truncated proteins. By contrast, N-terminal truncations were shown to exist prior to sample preparation, indicating their natural occurrence in nature [12]. One potential molecular approach would be to inhibit the proteases responsible for STAT5 N-terminal proteolytic cleavage, using small molecule inhibitors to restore the PIAS inhibitory mechanism. Suppression of STAT5 through Runx proteomimetics
Runt-related (Runx) transcription factors regulate hematopoietic cell-specific genes and play a critical role in T-lymphocyte differentiation similar to that of STAT proteins. It was discovered that interactions between Runx and STAT proteins played a negative regulatory role and served to mutually inhibit their transcriptional activity [21, 22]. Growth hormone stimulation in Saos-2 cells lead to the formation of STAT1-Runx2 and STAT3-Runx2 heterodimers. Protein complexation inhibited both Runx2 and STAT transcriptional activity while not effecting DNA binding potency. Similarly, STAT5-Runx complexes have been investigated and identified a key interaction between the Runt domain of Runx and the DNA binding domain and α-helix loop structure of STAT5. Most interestingly, this interaction prevented STAT5 nuclear translocation and inhibited transcriptional function of STAT5. Three possible mechanisms were suggested for the suppression of STAT5 transcriptional activity. First, STAT5 may block the dimerization of Runx proteins by competing for the transcriptional coactivator binding site (CBFβ) in the Runt domain which is responsible for dimerization [23]. Second, STAT5 may mask the DNA binding surface of the Runx protein, as the Runt domain is needed to bind DNA. Finally, STAT5 association with Runx proteins inhibits the mechanism of both STAT5 and Runx nuclear transport [23]. Thus, Runx transcription factors act as negative regulators of STAT5 activity and may warrant investigation. It is envisaged that mimicry of the key binding regions of Runx could yield high affinity inhibitors of STAT5 transcriptional activity. Mimicking key Runx binding epitopes could yield a new peptidomimetic/small molecule class of binders. Suppression of STAT5 through phospholipase C (PLC)-β3 proteomimetics
Stat5 activity is negatively regulated by phosphatases, such as SH2 domain-containing protein phosphatase (SHP-1) within the cytoplasm. Very recently, a study found an increased rate of Stat5 downregulation by SHP-1 through the phospholipase C (PLC)-β3 enzyme. One role of the PLC enzyme is to determine the duration, amplitude, and on-off cycling of signaling. Stat5 and SHP-1 were found to bind to PLC-β3 via its non-catalytic C-terminal domain (PLC-β3-CT) forming a multimolecular complex. As a result, SHP-1 and Stat5 are brought into
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close proximity facilitating an accelerated rate of Stat5-Tyr694 dephosphorylation [24]. Upon further investigation to understand the growth-suppressive function of PLC-β3-CT, a multitude of short peptides was generated corresponding to the regions to which Stat5 and SHP-1 bound. Two peptides (peptide b and its shorter derivative b11) substantially inhibited ( > 80% potency) in vitro proliferation and differentiation within two hematopoietic cells, Ba/F3 pro-B and TF-1 erythroleukemia cells. This is consistent with the hypothesis that reduced Stat5 activity seems to be the molecular basis for the growth-inhibitory function of PLC-β3-CT and its shorter derivatives. Interestingly, these peptides had no effect on Stat3-Tyr705 and Akt and little effect on MAPK phosphorylation, illustrating specificity for Stat5. The introduction of these peptides into target cells has been considered as a therapeutic strategy; however, there are significant drawbacks for its applications. First, there is reported evidence that there needs to be high expression levels of peptide b11 to measure any growth inhibition. Second, the stability of the peptide within the target cells against proteases needs to be accounted for. Suppression of Stat5 using oligonucleotide-based strategies
Stat5 Disrupting Stat5-DNA binding Targeting nuclear traffic is another effective strategy at developing new therapeutics, specifically using oligonucleotide-based strategies to target Stat5-Stat5:DNA binding activity. There are two commonly used approaches: first, strategies that downregulate Stat5 gene expression and those that prevent Stat5 binding to DNA. Antisense oligodeoxynucleotides (asODNs) and RNA interference (RNAi) have been used to target Stat5a and Stat5b with potent downregulation of their target genes. As both isoforms share 96% sequence homology, one equivalent of either RNAi or asODN would be effective at knocking down both isoforms and downregulating gene expression [25]. This has been successful with short interference RNA (siRNA) [25]; however, siRNA suffers from specificity and efficacy resulting in off-target effects [26]. To overcome this problem, the more stable short hairpin RNA (shRNA) was introduced using viral vectors and plasmids, which were then processed into siRNA [27]. Alternatively, the delivery of asODNs to target Stat5 isoforms do not suffer from nonspecific effects, although a two-fold greater molar excess is required compared with the corresponding siRNA to achieve effective knock-down [25, 28]. Decoy ODNs have been established as an efficient approach to block gene expression by competing with the binding of the transcription factor to the DNA cis-element [28, 29]. This sequesters the transcription factor within the cytoplasm and prevents its target gene expression [30]. This approach has been successful with the downregulation of Stat5 target genes in various cancer cell lines (K562 [28], HL-60 [31]), with no observation of non-specific effects. Although decoy ODNs provide an effective approach over siRNA or asODNs for inhibition, their delivery through transfection or other
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studies in KU812 and K562 CML cells revealed that pimozide elicited a dose-dependent decrease in STAT5 tyrosine phosphorylation at concentrations of 3–10 µM. Treatment with 2 (10 µM) resulted in a decreased viability of CML cells through the induction of apoptosis with no deleterious effects on healthy cells at similar concentrations of inhibitor. Nelson and coworkers demonstrated that phospho-STAT5 knockdown was not the result of inhibiting the upstream kinases of STAT5, rather they proposed that inhibition is based on direct association of 2 with STAT5. Importantly, pimozide in combination with drugs (1, imatinib and nilotinib) targeting the BCR-ABL kinase enhanced the inhibitory effects against STAT5 phosphorylation and induced apoptosis in T3151 BCR/ABL mutant resistant CML cells [34]. Although potent knock-down of STAT5 phosphorylation was observed, the authors did not present data to show the physical association of 2 with STAT5. The authors hypothesized that 2 may be inducing a phosphatase to dephosphorylate STAT5, or 2 is altering the stability of STAT5 through interactions with chaperone proteins (HSP90). Gunning and coworkers rationally designed and developed a library of salicylic acid-containing inhibitors (∼200) for targeting the SH2 domain of STAT3, wherein the salicylic acid component was proposed to effectively mimic the key pTyr binding residue. Given that the STAT SH2 domains showed moderate sequence homology, an in vitro screen of this library for STAT5 selectivity was conducted. Several potent leads were identified with Ki < 10 µM. Most notably, 3, 4, and 5 with Ki-values of 7.9, 2.8, and 8.3 µM, respectively, exhibited a three-fold selectivity for STAT5 over STAT3 and STAT1. Of these STAT5 inhibitors, 3 exhibited the highest STAT5 specificity (i.e., STAT5 Ki = 7.9 µM cf. STAT1, > 25 µM cf. STAT3 > 25 µM). When evaluated in K562 and MV-4-11 human leukemia cells, lead agents demonstrated potent and selective suppression of STAT5 phosphorylation (via Western analysis), inhibited STAT5 target genes and induced apoptosis (IC50∼20 µM). Moreover, lead
methods limits their subcellular compartmentalization, which may restrict their ability to inhibit Stat5 in the cytoplasm. Suppression of STAT5 using small molecule inhibitors Disrupting STAT5-STAT5 dimerization Identification of small molecule inhibitors of STAT5 protein has been limited. Presently, the majority of work has focused on targeting the SH2 domain of STAT3 [32]. This can be attributed to the determination of the STAT3-STAT3:DNA bound crystal structure. The structure showed the binding conformation and residues pertinent to phosphotyrosinylated STAT3 binding another STAT3 monomer, and more particularly shed light on the key residues required to bind the SH2 domain of STAT3. To date, the analogous STAT5-STAT5:DNA bound crystal structure has not been obtained. Nonetheless, there has been recent progress made towards targeting the SH2 domain of STAT5 using small molecule inhibitors. A highthroughput screen of diverse chemical libraries of a total 17,298 compounds, using a fluorescence polarization assay developed by Berg and coworkers led to the identification of a chromone-derived acyl hydrazone of which N′-[(4-Oxo4H-chromen-3-yl)methylene]nicotinohydrazide (1) (Figure 2) demonstrated the most potent and selective inhibition of STAT5 (STAT5, IC50 = 47 µM cf. STAT3, IC50 = 180 µM and STAT1, IC50 = 130 µM). Treatment of lymphoma cells (Daudi) with 1 (100–200 µM) resulted in inhibition of STAT5-STAT5 dimerization and suppression of STAT5 DNA binding activity [33]. Similarly, a STAT5 function-based screening approach was used to show that pimozide (2) (Figure 2), an FDA approved neuroleptic drug used for the treatment of Tourette’s syndrome, potently inhibited STAT5 phosphorylation. Pimozide was found to downregulate STAT5 target gene expression without decreasing the autophosphorylation of the upstream kinases of Stat5 (BCR-ABL, MAPK, and JAK2). Cellular
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Cumaraswamy and Gunning: Direct inhibitors of Stat5 protein
agents showed negligible cytotoxic effects against healthy bone marrow cells, which do not harbor constitutively activated STAT5 [35].
Conclusion Potent and selective inhibition of STAT5 via targeted molecule therapeutics represents a most appealing candidate for treatment of human cancers. With growing evidence implicating the prominent role of STAT5 in inflammation, interest in a STAT5 drug has extended to inflammatory diseases, such as rheumatoid arthritis [36] and inflammatory bowel disease [37]. In addition, a STAT5 drug may have application as an adjuvant therapy for the treatment of CMLs and prostate cancer. Although there has been little progress towards a clinical candidate, there is growing evidence to suggest that STAT5 protein can be targeted selectively and with moderate potency. It remains to be seen whether suitably potent inhibitors can be developed with suitable in vivo activity to enter preclinical trials. However, the limited studies do suggest that more potent agents will be uncovered with more intensive high-throughput screens and rational drug design investigations.
References 1. Nosaka T, Kawashima T, Misawa K, Ikuta K, Mui AL, Kitamura T. STAT5 as a molecular regulator of proliferation, differentiation and apoptosis in hematopoietic cells. EMBO J 1999;18:4754–65. 2. Shyh-Han T, Nevaleinen MT. Signal transducer and activator of transcription 5A/B in prostate and breast cancers. Endocr Relat Cancer 2008;15:367–90. 3. Becker S, Groner B, Muller CW. Three-dimensional structure of the Stat3beta homodimer bound to DNA. Nature 1998;394:145–51. 4. Chen X, Vinkemeier U, Zhao Y, Jeruzalmi D, Darnell JE, Jr, Kuriyan J. Crystal structure of a tyrosine phosphorylated Stat-1 dimer bound to DNA. Cell 1998;93:827–39. 5. Peltola KJ, Paukku K, Aho TL, Ruuska M, Silvennoinen O, Koskinen PJ. Pim-1 kinase inhibits Stat5-dependant transcription via its interactions with SOCS1 and SOCS3. Blood 2004;103:3744–50. 6. Yu H, Jove R. The Stats of cancer – new molecular targets come of age. Nat Rev Cancer 2004;2:97–105. 7. Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J, Lydon NB. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996;5:561–6. 8. Sato T, Yang X, Knapper S. FLT-3 ligand impedes the efficacy of FLT-3 inhibitors in vitro and in vivo. Blood 2011;117: 3286–93. 9. Wernig G, Kharas MG, Mullally A, Leeman DS, Okabe R, George T, Clary DO, Gilliland, DG. EXEL-8232, a small-molecule JAK2 inhibitor, effectively treats thrombocytosis and extramedullary hematopoiesis in a murine model of myeloproliferative neoplasm induced by MPLW515L. Leukemia 2012;26:720–7. 10. Caldenhoven E, Van Dijk TB, Solari R, Armstrong J, Raaijmakers JA, Lammers JW, Koenderman L, de Groot RP. Stat3beta, a splice variant of transcription factor Stat3, is a dominant negative regulator of transcription. J Biol Chem 1996;271:13221–7.
285
11. Yamashita H, Iwase H, Toyama T, Fujii Y. Naturally occurring dominant-negative Stat5 suppresses transcriptional activity of estrogen receptors and induces apoptosis in T47D breast cancer cells. Oncogene 2003;22:1638–52. 12. Dagvadorj A, Shyh-Han T, Liao Z, Xie J, Nurmi M, Alanen K, Rui H, Mirtti T, Nevalainen MT. N-terminal truncation of Stat5a/b circumvents PIAS3-mediated transcriptional inhibition of Stat5 in prostate cancer cells. Int J Biochem Cell Biol 2010;42:2037–46. 13. Moriggl R, Gouilleux-Gruart V, Jahne R, Berchtold S, Gartmann C, Liu X, Hennighausen L, Sotiropoulos A, Groner B, Gouilleux F. Deletion of the carboxy-terminal transactivation domain of MGF-Stat5 results in sustained DNA binding and a dominant negative phenotype. Mol Cell Biol 1996;10:5691–700. 14. Kazansky AV, Raught B, Lindsey SM, Wang Y, Rosen JM. Regulation of mammary gland factor/stat5a during mammary gland development. Mol Endocrinol 1995;9:1598–609. 15. Wang D, Stravopodis D, Teglund S, Kitazawa J, Ihle JN. Naturally occurring dominant negative variants of Stat5. Mol Cell Biol 1996;16:6141–8. 16. DaSilva L, Rui H, Erwin RA, Howard OM, Kirken RA, Malabarba MG, Hackett RH, Larner AC, Farrar WL. Prolactin recruits Stat1, Stat3 and Stat5 independent of conserved receptor tyrosines TYR402, TYR479, TYR515 and TYR580. Mol Cell Endocrinol 1996;117:131–40. 17. Schaber JD, Fang H, Xu J, Grimley PM, Rui H. Prolactin activates Stat1 but does not antagonize Stat1 activation and growth inhibition by type 1 interferons in human breast cancer cells. Cancer Res 1998;58:1914–9. 18. Azam M, Lee C, Strehlow I, Schindler C. Functionally distinct isoforms of STAT5 are generated by protein processing. Immunity 1997;6:691–701. 19. Garimorth K, Welte T, Doppler W. Generation of carboxy-terminally deleted forms of STAT5 during preparation of cell extracts. Exp Cell Res 1999;246:148–51. 20. Schuster B, Hendry L, Byers H, Lynham SF, Ward MA, John S. Purification and identification of the STAT5 protease in myeloid cells. Biochem J 2007;404:81–7. 21. Kim S, Koga T, Isobe M, Kern BE, Yokochi T, Chin YE, Karsenty G, Taniguchi T, Takayanagi H. Stat1 functions as a cytoplasmic attenuator of Runx2 in the transcriptional program of osteoblast differentiation. Genes Dev 2003;17:1979–91. 22. Ziros PG, Georgakopoulos T, Habeos I, Basdra EK, Papavassiliou AG. Growth hormone attenuates the transcriptional activity of Runx2 by facilitating its physical association with Stat3β. J Bone Miner Res 2004;19:1892–904. 23. Ogawa S, Satake M, Ikuta K. Physical and functional interactions between Stat5 and Runx transcription factors. J Biochem 2008;143:695–709. 24. Yasudo H, Ando T, Xiao W, Kawakami Y, Kawakami T. Short stat5-interacting peptide derived from phospholipase C-β3 inhibits hematopoietic cell proliferation and myeloid differentiation. PLoS ONE 2011;6:e24995. 25. Liu X, Robinson GW, Gouilleux F, Groner B, Hennighausen L. Cloning and expression of Stat5 and an additional homologue (Stat5b) involved in prolactin signal transduction in mouse mammary tissue. Proc Natl Acad Sci USA 1995;92:8831–5. 26. Sledz CA, Williams BR. RNA interference and double-stranded-RNA-activated pathways. Biochem Soc Trans 2004;32: 952–6. 27. Klosek SK, Nakashiro K, Hara S, Goda H, Hamakawa H. Stat3 as a molecular target in RNA interference-based treatment of oral squamous cell carcinoma. Oncol Rep 2008;20:873–8. 28. Wang X, Zeng J, Shi M, Zhao S, Bai W, Cao W, Tu Z, Huang Z, Feng W. Targeted blockage of signal transducer and activator of
286
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transcription 5 signaling pathway with decoy oligodeoxynucleotides suppresses leukemic K562 cell growth. DNA Cell Biol 2011;30:71–8. 29. Ahn JD, Morishita R, Kaneda Y, Lee S, Kwon K, Choi S, Lee K, Park J, Moon I, Park JG, Yoshizuma M, Ouchi Y, Lee I. Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo. Circ Res 2002;90:1325–32. 30. Cutroneo KR, Ehrlich H. Silencing or knocking out eukaryotic gene expression by oligodeoxynucleotide decoys. Crit Rev Eukaryot Gene Expr 2006;16:23–30. 31. Cao S, Wang C, Zheng Q, Qiao Y, Xu K, Jiang T, Wu A. Stat5 regulates glioma cell invasion by pathways dependant and independent of Stat5 DNA binding. Neurosci Lett 2011;487:228–33. 32. Avadisian M, Haftchenary S, Gunning PT. Inhibiting aberrant Stat3 function with molecular therapeutics: a progress report. Anticancer Drugs 2011;22:115–27.
33. Muller J, Sperl B, Reindl W, Kiessling A, Berg T. Discovery of chromone-based inhibitors of the transcription factor Stat5. ChemBioChem 2008;9:723–7. 34. Nelson EA, Walker SR, Weisberg E, Bar-Natan M, Barrett R, Gashin LB, Terrell S, Klitgaard JL, Santo L, Addorio MR, Ebert BL, Griffin JD, Frank DA. The Stat5 inhibitor pimozide decreases survival of chronic myelogenous leukemia cells resistant to kinase inhibitors. Blood 2011;117:3421–9. 35. Page BD, Khoury H, Laister RC, Fletcher S, Vellozo M, Manzoli A, Yue P, Turkson J, Minden MD, Gunning PT. Small molecule STAT5-SH2 domain inhibitors exhibit potent antileukemia activity. J Med Chem 2012;55:1047–55. 36. Naka T, Kishimoto T. Joint diseases caused by defective gp130-mediated STAT signaling. Arthritis Res 2002;4: 154–8. 37. O’Shea JJ, Murray PJ. Cytokine signaling modules in inflammatory responses. Immunity 2008;28:477–87.
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