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(2014) 28, 248–257 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu

REVIEW STAT factors in hematopoiesis and leukemogenesis: opportunities for therapeutic intervention

KA Dorritie1, JA McCubrey2 and DE Johnson3

Signal transducer and of transcription (STAT) comprise a family of transcription factors that are activated by , hormones and growth factors. The activation of STAT proteins plays a key role in the production of mature hematopoietic cells via effects on cellular proliferation, survival and lineage-specific differentiation. Emerging evidence also demonstrates frequent, constitutive activation of STATs in primary leukemia specimens. Moreover, roles for STATs in promoting leukemia development have been delineated in numerous preclinical studies. This review summarizes our current understanding of STAT involvement in normal hematopoiesis and leukemogenesis, as well as recent advances in the development and testing of novel STAT inhibitors.

Leukemia (2014) 28, 248–257; doi:10.1038/leu.2013.192 Keywords: STAT3; STAT5; hematopoiesis; BCR/ABL; FLT3-ITD

INTRODUCTION SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION The process of hematopoiesis is driven by cytokines and growth (STAT) PROTEINS factors and leads to the production of mature hematopoietic cells To date, seven STAT proteins have been identified: STAT1, STAT2, with varying vital functions. The engagement of cytokines and STAT3, STAT4, STAT5A, STAT5B and STAT6.1,2 encoding the growth factors with cognate receptors on the surface of bone seven family members are clustered in three chromosomal marrow-derived progenitor and differentiating hematopoietic regions: 2 (STAT 1 and 4), 12 (STATs 2 and 6) and cells results in the activation of key transcription factors, which 17 (STAT 3, 5A, 5B).1 Several STAT isoforms have also been act to promote cellular proliferation, survival or differentiation. identified, as discussed further below. Among the transcription factors that facilitate lineage commit- Despite functional differences, the STAT proteins share ment and production of lineage-specific cell types are several considerable homology.3,4 In addition, crystal structures for full- whose expression is primarily restricted to hematopoietic cells. In length proteins or portions of STATs 1, 3, 4 and 6 have been addition, several more broadly expressed transcription factors, reported and suggest similarities in structure.5,6 Full-length STAT including members of the STAT family, also have been shown to proteins range in size from 90 to 115 kDa3 and contain several have key roles in hematopoiesis. In this review, we summarize domains that are conserved among all the STATs and are critical recent experimental evidence from both in vitro and in vivo for function (Figure 1).3,7,8 The DNA-binding domain determines models that has shed on the role of different STAT proteins in the DNA-binding specificity for each STAT. The highly conserved mediating the effects of cytokines and growth factors during Src-homology-2 (SH2) domain recognizes phosphorylated normal hematopoiesis. residues and thereby mediates interactions between STAT Maintenance of cellular homeostasis in the hematopoietic proteins and specific phosphorylated proteins, including growth system also requires the ability to downmodulate STAT activation. factor receptors, other STATs and Janus (JAK) family However, in many hematological malignancies, as well as solid members (JAK1, JAK2, JAK3 and -2 (TYK2)).3,7,9 The tumors, constitutive hyperactivation of STATs is observed in a high oligomerization domain in the N-terminal regions of STATs percentage of patient specimens. As described in this review, provides sites for additional protein–protein interactions.3,9,10 overexpression or hyperactivation of STATs, particularly STAT3 and Key tyrosine residues (Tyr701 for STAT1, 690 for STAT2, 705 for STAT5, has been implicated in the development of several STAT3, 693 for STAT4, 694 for STAT5A, 699 for STAT5B and 641 for different types of . Multiple molecular mechanisms that STAT6) represent sites of phosphorylation by upstream activating are responsible for the constitutive activation of STAT signaling in (Figures 1, 2 and 3). Phosphorylation at these sites results leukemias have also been reported, and will be summarized. in recognition by the SH2 domain of another STAT protein and Lastly, the identification of STATs 3 and 5 as promising targets for leads to STAT dimerization. Following dimerization, the STAT anti- therapies has led to the development of several novel proteins translocate to the nucleus where they bind to sequence inhibitors of STAT activation and signaling. The application elements in the promoters of target genes, activating transcription of these inhibitors in leukemia models is described. of these genes (Figure 3).1–3,7,9 The C-terminal domains of STAT

1Department of Medicine, University of Pittsburgh and the University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA; 2Department of Microbiology and Immunology, School of Medicine, East Carolina University, Greenville, NC, USA and 3Department of Pharmacology and Chemical Biology, University of Pittsburgh and the University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA. Correspondence: Professor DE Johnson, Division of Hematology/Oncology, University of Pittsburgh, Hillman Cancer Center, Room 2.18c, 5117 Centre Avenue, Pittsburgh, PA 15213, USA. E-mail: [email protected] Received 15 April 2013; revised 30 May 2013; accepted 13 June 2013; accepted article preview online 25 June 2013; advance online publication, 19 July 2013 STAT transcription factors in hematopoiesis and leukemogenesis KA Dorritie 249 proteins, also known as the transcriptional activation domains, are STAT3b and STAT3g.7,10 The shorter b and g isoforms are thought to involved in the transcriptional activity. Within these domains lie be generated by alternative mRNA splicing and proteolytic conserved residues (except in STATs 2 and 6), which serve processing, respectively.7 The STAT3b isoform is truncated by 55 as additional phosphorylation sites (Figures 1 and 2). Although amino acids at the C-terminal end, resulting in a frameshift and the the impact of serine phosphorylation on STATs is still poorly addition of seven new amino acids.13,14 The STAT3g isoform results understood, phosphorylation of Ser727 in STAT3 has been shown from post-translational modification via proteolysis,15,16 and while it to enhance target transcription and contribute to optimal can still be recruited to tyrosine-phosphorylated receptors via the biological activation of STAT3 during cellular transformation by SH2 domain, it is defective in signal propagation.9 STAT5A and Src.3,7,9,11 Ser727 phosphorylation also may be involved in the STAT5B are encoded by two separate genes and have at least two recruitment of coregulators such as histone acetyl transferases or isoforms each, a and b. STAT5Ab results from proteolysis between targeted translocation of STAT3 to the mitochondria. In addition, amino acids 709 and 740, whereas the STAT5B b isoform represents specific cytokines, such as (SCF), preferentially a truncation between amino acids 714 and 745.17 promote Ser727 phosphorylation.12 STAT ACTIVATION STAT ISOFORMS Activated STAT proteins mediate expression of target genes in Truncated STAT isoforms that lack the C-terminal domain have response to extracellular stimuli including cytokines, growth been described for STAT3, STAT5A and STAT5B (Figure 2), although 7 factors and hormones. In unstimulated cells, STATs reside in the their role in STAT-dependent signaling is not fully understood. cytoplasm as monomeric proteins. STAT activation involves Early reports indicated that these truncated isoforms exert a by upstream kinases, leading to STAT dominant-negative impact on the full-length STAT3/5 (STATa) dimerization. that detect phosphorylation at the isoforms, though this remains an unresolved area of controversy. tyrosine activation residues in the STAT proteins are commonly In the case of STAT3, three isoforms have been described: STAT3a, used to assess STAT activation. The phosphorylation/activation of STATs can occur via JAK-dependent or JAK-independent, - mediated pathways (depicted and described in Figure 3). In Y S addition, certain cytoplasmic nonreceptor tyrosine kinases have NH OD DBD SH2 TAD COOH 2 been shown to promote JAK-independent phosphorylation/activa- Figure 1. STAT protein structure. The STAT proteins are comprised tion of STATs.3,8 Specifically, oncogenic Src promotes constitutive of multiple functional domains. The oligomerization domain activation of STAT3, whereas the BCR-ABL fusion protein promotes (OD) mediates oligomerization of dimers to form tetramers. activation of STATs 1, 5 and, to a lesser degree, STAT3.9,18 The DNA-binding domain (DBD) determines the DNA-binding specificity for each STAT. The SH2 domain recognizes phosphory- lated tyrosine residues found in specific sequence contexts REGULATION OF STATS and facilitates interactions of STAT proteins with activated 3,19 receptors, JAKs and other STATs. The domain (TAD) In normal cells, STAT activation is typically a transient process. is important for transcription of target genes and contains key The downmodulation of STAT signaling allows for time-dependent tyrosine (Y) and serine (S) residues that are phosphorylated by attenuation of cellular and biological responses resulting from upstream activators. activated and receptors. The termination of

701 727

Y S STAT1 NH2 OD DBD SH2 TAD COOH 91 kDa

701 STAT1 NH2 OD DBD SH2 TAD COOH 84 kDa

690 STAT 2 NH2 OD DBD SH2 TAD COOH 113 kDa

705 727 STAT3  NH2 OD DBD SH2 TAD COOH 92 kDa

705 STAT3  NH2 OD DBD SH2 TAD COOH 84 kDa

705 STAT3  NH2 OD DBD SH2 TAD COOH 72 kDa

693 721 STAT4 NH2 OD DBD SH2 TAD COOH 81 kDa 725 779 NH 694 STAT5A  2 OD DBD SH2 TAD COOH 96 kDa 694 STAT5A  NH2 OD DBD SH2 TAD COOH 77 kDa 699 730 STAT5B  NH2 OD DBD SH2 TAD COOH 94 kDa 699 STAT5B  NH2 OD DBD SH2 TAD COOH 80 kDa 641 STAT6 NH2 OD DBD SH2 TAD COOH 110 kDa Figure 2. STAT proteins and their isoforms.

& 2014 Macmillan Publishers Limited Leukemia (2014) 248 – 257 STAT transcription factors in hematopoiesis and leukemogenesis KA Dorritie 250 JAK-dependent JAK-independent STAT activation STAT activation

P JAK JAK P BCR/ABL 2 112 P Y Y P P Y Y P Y P P Y Y P STAT P Y Y P STAT STAT STAT STAT

11 Src 22

Y P

P Y STAT Target Gene Transcription

Figure 3. Activation of STATs via JAK-dependent and JAK-independent pathways. During JAK-dependent activation of STATs, binding to a that lacks intrinsic kinase activity leads to the activation of JAK enzymes that are associated with the cytoplasmic region of the receptor. The activated JAKs phosphorylate tyrosine residues in the receptor cytoplasmic region (step 1), leading to recognition and binding by the SH2 domain of monomeric STAT proteins. JAKs then phosphorylate the recruited STAT proteins on their activation tyrosine residues (step 2). The phosphorylated STAT proteins undergo dimerization and translocation to the nucleus, where they induce expression of STAT target genes. In the case of JAK-dependent STAT activation, higher order multimerization of cytokine receptors (for example, dodecamer complexes) may be necessary for JAK phosphorylation of STATs.138 During JAK-independent activation, ligand binding to a receptor with intrinsic tyrosine kinase activity leads to activation/autophosphorylation of the receptor cytoplasmic region (step 1). Phosphorylated tyrosine residues on the receptor serve as recruitment sites for the SH2 domain of STATs. The recruited STATs are then directly phosphorylated on their activation tyrosine residues by the activated receptor (step 2), leading to STAT dimerization, translocation to the nucleus and induction of gene transcription. Nonreceptor, cytoplasmic tyrosine kinases including BCR/ABL and Src, can also promote JAK-independent phosphorylation/activation of STATs.

STAT signaling is due to a number of different factors, including members, with certain members acting to bind phosphotyrosine receptor degradation via the lysosome and , activation residues on activated cytokine receptors or JAK proteins. This of cellular and expression and activation of specific results in decreased binding of STATs to receptors, inhibition of regulatory proteins. At least three different classes of regulatory JAK activity and increased proteasomal degradation of STATs and proteins have been implicated in the deactivation of STATs: JAKs.3 Induction of SOCS proteins provides a negative feedback tyrosine phosphatases, suppressors of cytokine signaling (SOCS) loop to attenuate cytokine and STAT signaling. The first SOCS proteins, and protein inhibitors of activated STATs (PIAS). family member identified, CIS1, is induced by -2 (IL-2), While certain tyrosine phosphatases have been shown to IL-3 and (EPO) and has been shown to suppress downmodulate STAT signaling via dephosphorylation and inacti- STAT5 signaling by binding to one of the STAT5-binding sites on vation of kinases upstream of STATs, other phosphatases have the EPO receptor.24 The second family member, SOCS-1, also been identified that can directly dephosphorylate STATs. The known as JAK-binding protein, may act as a tumor suppressor. tyrosine phosphatases SHP1 and SHP2 are localized primarily in Epigenetic silencing of the socs-1 gene has been associated with the the cytoplasm, and interact, via their SH2 domains with activated, development of both solid and hematological malignancies, tyrosine-phosphorylated receptors and JAK enzymes. These including leukemias.25,26 Additionally, epigenetic silencing of the interactions lead to activation of SHP1 and SHP2, with resultant gene has been shown to result in cellular proliferation in both dephosphorylation of either the receptor or JAK proteins, thereby solid and hematological malignancies.8 Qiu et al.27 demon- shutting down these upstream activators of STATs. CD45, strated a requirement for BCR-ABL-dependent phosphorylation of a transmembrane tyrosine , is present on the surface SOCS-1 and SOCS-3 in BCR-ABL-induced tumorigenesis.27 Phospho- of all nucleated hematopoietic cells and directly dephosphorylates rylation of SOCS-1 and -3 was shown to interfere with their ability to JAK enzymes.20 Genetic knockdown of CD45 leads to downregulate JAK/STAT signaling in human CML K562 cells. hyperactivation of JAK1 and JAK3 in mice.20 The T-cell protein SOCS-2 is a key negative regulator of the growth hormone/ tyrosine phosphatase (TC-PTP) is primarily found in hematopoietic -like growth factor pathway and is also induced by various cells and serves as a phosphatase for STAT1, STAT3 and likely cytokines.28 The role of SOCS-2 in leukemogenesis remains STAT5.21 Zhang et al.22 have shown that the protein tyrosine unclear. phosphatase receptor T (PTPRT), which is the most commonly Lastly, the protein inhibitors of activated STATs (PIAS) have mutated PTPR in human , directly dephosphorylates been identified as specific negative regulators of STATs. The Tyr705 in STAT3. Recent evidence also implicates PTPRD in the PIAS family contains four members: PIAS1, PIAS3, PIASx, and direct dephosphorylation of STAT3.23 PIASy.29 PIAS1 and PIASy interact with STAT1, while PIAS3 The suppressors of the cytokine signaling (SOCS) proteins, also and PIASx interact with STAT3 and STAT4, respectively.30 PIAS known as cytokine-induced SH2-containing (CIS) proteins and proteins inhibit STATs by blocking DNA-binding activity, STAT-induced STAT inhibitors, have a key role in downmodulation promoting STAT sumoylation or through the recruitment of STAT signaling. The SOCS protein family is comprised of eight of other corepressors.30–32

Leukemia (2014) 248 – 257 & 2014 Macmillan Publishers Limited STAT transcription factors in hematopoiesis and leukemogenesis KA Dorritie 251 PHYSIOLOGICAL ROLE OF STATS IN HEMATOPOIESIS prevent production of mature neutrophils.50 Strikingly, mice The JAK-STAT pathway is involved in multiple processes, including lacking bone marrow STAT3 develop neutrophilia, and the bone early development, immune responses, cellular proliferation, marrow cells exhibit enhanced proliferative responses when 50 survival and differentiation. Moreover, cytokines and growth treated with G-CSF. These findings suggest that, in vivo, STAT3 factors, including , granulocyte-colony-stimulating activates a negative feedback loop that represses G-CSF-induced factor (G-CSF), EPO and (TPO) have all been granulopoiesis. demonstrated to activate the JAK-STAT pathway. Therefore, it is The influence of STAT3 on cell cycle progression and suppres- not surprising that this pathway has important roles in both sion of has become particularly apparent through the normal and dysregulated hematopoiesis. identification of STAT3 target genes. Key proteins encoded by STAT3 target genes include, among others, cyclin D1, cdc25A, The role of STAT proteins in hematopoiesis has been 3,54 investigated using both in vitro and in vivo models. In particular, c-, Bcl-XL, p21 and 27. In addition, genome-wide screening key evidence has been generated through the use of knockout using chromatin immunoprecipitation assays has identified a broad number of STAT3 target genes important for cellular mice. STAT1 knockout mice exhibit normal lymphoid and myeloid 55 development, but are defective in signaling, resulting proliferation, differentiation and oncogenesis. Clearly, more in increased susceptibility to pathogens.33,34 Additionally, À / À research is needed to determine the role of these target genes STAT1 À / À double knockouts show increased tumorigenesis in mediating the impact of STAT3 on normal hematopoiesis, compared with p53 À / À mice, suggesting a possible function of as well as leukemogenesis. STAT1 as a tumor suppressor.35 STAT4 À / À mice manifest Similar to STAT3, STAT5 is activated by a variety of cytokines defective Th1 cell differentiation, largely due to impairment and growth factors. STAT5 has a particularly important role 36,37 during fetal erythropoiesis. Nullizygous STAT5A/STAT5B of IL-12-mediated functions. STAT6 knockout mice have (STAT5A À / À 5B À / À ) mice develop fetal anemia due to decreased defects in IL-4-mediated signaling, leading to impaired T-cell responsiveness to Epo and decreased expression of Bcl-X .56 development and Th2 differentiation, as well as immunoglobulin L Adult STAT5A À / À B À / À mice exhibit markedly reduced rates class switching.38,39 As with the STAT1 and STAT4 knockouts, of erythropoiesis when hemolytic anemia is induced by STAT6 À / À mice demonstrate normal maturation within the phenylhydrazine.57 The degree of anemia in neonatal and adult myeloid lineage. / / STAT5A À À B À À mice correlates with reduced expression of the STAT3 is activated by various cytokines, including G-CSF, 57 Bcl-XL protein. Together, these findings support a critical role GM-CSF, SCF, IL-3, IL-5 and IL-6, and also has a role in mediating 58 of STAT5 in erythropoiesis. In support of this, Kirito et al. the effects of synergy between certain cytokines, such as the have shown that treatment of human megakaryoblastic leukemia synergy between SCF and G-CSF.12 Initial attempts by Takeda 40 UT-7/GM cells with EPO induced activation of STAT5 concomitant et al. to generate homozygous STAT3 knockout mice resulted in with induction of erythroid differentiation. Moreover, exogenous embryonic lethality. However, use of a Cre-loxP system allowed expression of dominant-negative STAT5A in the cytokine- successful generation of T-cell-specific, conditional STAT3 40 dependent, murine myeloid 32D cell line resulted in knockout mice. T cells from these mice demonstrate reduced impairment of IL-3-induced proliferation and G-CSF-dependent proliferative responses to IL-6 and enhanced levels of apoptosis. differentiation.59 Additional in vivo studies by others have confirmed the STAT5 also acts to mediate IL-7 signaling during early B-cell importance of STAT3 in mediating the effects of IL-6/gp130 41 development, in part, by inducing expression of anti-apoptotic signaling on hematopoiesis. The induction of c-Myc by IL-6 is myeloid cell leukemia sequence-1 (Mcl-1).60 STAT5 À / À mice also dependent on STAT3, and expression of the constitutively 42,43 exhibit arrest of B-cell development in the pre-pro-B-cell stage, active STAT3 mutant, STAT3C, leads to c-Myc upregulation. with absence of more mature cells likely due to loss of Mcl-1 Selective deletion in the B-cell lineage has revealed expression.60–62 IL-7 and STAT5 are also thought to have a role a critical role for STAT3 in B-cell development, particularly the 44 in immunoglobulin gene rearrangement, with STAT5 acting terminal differentiation of IgG B cells. Mice deficient in bone to suppress Igk rearrangement.60 Lastly, STAT5 has a role in marrow expression of STAT3 exhibit reduced levels of pro-B, pre-B, maintenance of myeloid leukemic stem cells. In CD34 þ AML cells immature B and mature B cells, and reduced bone marrow characterized by expression of constitutively activated STAT5, 45 proliferation in response to IL-7. STAT3 deletion in bone marrow knockdown of STAT5 using RNAi resulted in impaired long-term hematopoietic cells also leads to enhanced anti-tumor immunity growth.63 However, suppression of STAT5 also slowed the growth and premature death due to development of a Crohn’s-like of normal, CD34 þ cord blood progenitor cells, indicating a role for 46–48 disease. Such effects suggest that STAT3 may act normally, in STAT5 in both normal and leukemic hematopoiesis.63 some capacity, to limit certain immune cell functions and hyperactivation of immune responses. Mice deficient in STAT3 also exhibit deficiencies in mitochondrial electron transport, accompanied by overproduction of reactive oxygen species and STATS IN LEUKEMOGENESIS a rapid-aging blood .48 Additionally, bone marrow from In view of the key roles that STATs have in hematopoietic cell STAT3 À / À mice exhibit characteristics similar to myelodysplastic proliferation, survival and differentiation, their involvement in and myeloproliferative neoplasms, including erythroid dysplasia leukemogenesis is not unexpected. Constitutive activation of and shifts in the lymphoid to myeloid ratio, suggesting a role for STATs 1, 3 and 5 has been demonstrated in both acute and STAT3 in hematopoiesis.48 chronic leukemias. In many cases, this activation has been shown The precise role of STAT3 in myeloid cell differentiation and result from hyperactivation or overexpression of cellular onco- myelopoiesis remains somewhat unresolved. In vitro studies genic tyrosine kinases or leukemic fusion oncoproteins. suggest that STAT3 is important for myeloid cell survival and differentiation, while in vivo studies indicate that STAT3 negatively regulates granulopoiesis.49–51 Expression of dominant-negative STATs in v-Src-induced leukemia STAT3 in M1 murine myeloid leukemia cells has been shown to Yu et al.64 reported one of the earliest observations of constitutive block IL-6-induced monocytic differentiation, while expression of activation of STATs resulting from expression of an oncogenic the dominant-negative in murine myeloid LGM-1 cells blocked tyrosine kinase. Constitutive activation of STAT3 was observed G-CSF-induced granulocytic differentiation.51–53 By contrast, following transformation of NIH3T3 fibroblasts by v-Src.64 In in vivo studies have shown that knockout of stat3 does not hematopoietic cells (32Dcl3), expression of v-Src was shown to

& 2014 Macmillan Publishers Limited Leukemia (2014) 248 – 257 STAT transcription factors in hematopoiesis and leukemogenesis KA Dorritie 252 induce constitutive STAT3 activation and block cytokine-induced express the STAT5B-RARa fusion protein are resistant to all-trans differentiation.65 retinoic acid. Transfection experiments have determined that STAT5B-RARa impairs transcription and arrests myeloid STATs in FLT3 signaling differentiation, in part, due to recruitment of the corepressor SMRT.78,79 FMS-related tyrosine kinase 3 (FLT3) has an important role in normal hematopoiesis via its activation of downstream mediators including STATs, MAPK and AKT/PI3K.66 Moreover, BCR/ABL and STATs activating in FLT3 result in constitutive activation of BCR/ABL results from the reciprocal translocation of chromosomes these downstream signaling pathways and dysregulated cellular 9 and 22 t(9;22)(q34;q11), commonly known as the Philadelphia proliferation. Clinically, activating mutations of FLT3 are associated . Depending on the breakpoint site of BCR, one with poor outcome in AML. Mutations in the FLT3 juxtamembrane of the three fusion proteins result, p190BCR/ABL, p210BCR/ABL or domain (FLT3 internal tandem duplications (FLT3-ITDs)) and p230BCR/ABL.80,81 The p210BCR/ABL protein is most commonly tyrosine kinase domain (FLT3-TKD) are found in roughly 30–35% associated with chronic myelogenous leukemia (CML), whereas of patients with AML.67 The FLT3 mutations observed in AML are p190BCR/ABL is typically seen in ALL. The p230BCR/ABL form is seen activating mutations that result in ligand-independent activation in chronic neutrophilic leukemia.81 These fusion proteins serve as of the FLT3 protein.67 Exogenous expression of mutant FLT3 in IL- constitutive tyrosine kinases and have been shown to promote 3-dependent 32D cells or Ba/F3 (murine lymphoid) cells results constitutive activation of STAT1, STAT5 and, to a lesser extent, in constitutive activation of both STAT5 and MAP kinase, STAT3.18,82 Carlesso et al.83 first described the constitutive whereas expression of the wild-type FLT3 protein leads activation of STAT1 and STAT5 in the Philadelphia chromosome- to activation of MAP kinase, but not STAT5.68 Injection of FLT3- positive, human cell lines K562 and BV173. Further, enforced ITD-expressing 32D cells into syngeneic mice leads to the expression of p210BCR/ABL resulted in IL-3-independent activation development of leukemia.69 FLT3-ITD activation of STAT5 also of STAT1 and STAT5 in 32D, Ba/F3 or TF-1 (human erythroleukemia appears to induce Mcl-1, promoting leukemic stem cell survival.70 cell line).83 By contrast, p210BCR/ABL expression did not increase JAK activities, demonstrating that STAT1/5 activation was 83 TEL-JAK2 and STATs independent of JAKs (Figure 3). Similar experiments by Ilaria 18 The translocation responsible for production of the ETS leukemia et al. showed activation of STAT5 and low levels of STAT1 and (TEL)-JAK2 leukemic fusion protein was initially discovered in STAT3 activation in Ba/F3 and FDC-P1 (murine myeloid cell line) cells transformed by p210BCR/ABL or p190BCR/ABL. Cells transformed a patient with T-cell childhood acute lymphoblastic leukemia (ALL) BCR/ABL and has since been reported in B-cell leukemias such as atypical with p190 also exhibited prominent activation of STAT6. chronic myeloid leukemia.71 The TEL-JAK2 fusion protein involves Expression of dominant-negative JAK mutants failed to impact STAT5 activation, again suggesting that BCR/ABL-mediated fusion of the oligomerization domain of TEL and the kinase 18 domain of JAK2.9,71,72 Stable expression of TEL-JAK2 protein in phosphorylation of STATs is JAK independent. Later studies have revealed that BCR/ABL activates hematopoietic cell kinase Ba/F3 cells results in constitutive activation of STATs 1, 3 and 5, 84 along with IL-3-independent .73 TEL-JAK2 transgenic (Hck), which in turn phosphorylates STAT5 in myeloid cells. mice manifest a fatal T-cell leukemia that arises from BCR/ABL-mediated STAT5 activation confers resistance to apoptosis, in part, by upregulating Bcl-XL. Ba/F3 cells engineered immature thymocytes and is characterized by hyperactivation of BCR/ABL 74 to express p210 or a constitutively active STAT5 mutant STAT1 and 5. 85 exhibit increased transcription of bcl-xL. An analysis of 27 CML patients revealed a correlation between increased levels of TEL-platelet derived growth factor b receptor (TEL-PDGFbR) and 86 pSTAT5 and Bcl-XL in a majority of patients in blast crisis. In STATs contrast, a majority of patients in the chronic phase of CML had Chronic myelomonocytic leukemia is often associated with the lower levels of Bcl-XL expression. Pharmacological inhibition of t(5;12) translocation that results in formation of the TEL-platelet BCR/ABL in cell lines and patient samples resulted in apoptosis 72 derived growth factor b receptor (PDGFbR) fusion protein. induction via decreased activation of STAT5 and reduced binding 87 Expression of TEL-PDGFbR in 32D and Ba/F3 cells results of STAT5 to the bcl-XL . Taken together, these studies in increased phosphorylation of STAT5 and IL-3-independent demonstrate a role for BCR/ABL in resistance to apoptosis via 75 growth. Full transformation by TEL-PDGFbR requires hyper- constitutive activation of STAT5 with resultant effects on phosphorylation of PI3K and PLCg in addition to constitutive downstream anti-apoptotic signals. STAT5 activation.75

PML-RARa and STATs STATS IN LEUKEMIAS Most cases of acute promyelocytic leukemia (APL) are character- AML ized by the translocation t(15;17), resulting in the expression of Analyses of primary peripheral blood and bone marrow specimens the fusion protein PML-RARa. PML is thought to act as a tumor has demonstrated constitutive activation of STATs 3 and 5 in suppressor, whereas PML-RARa acts as an oncoprotein and AML.15,67,88–94 In one such study, analysis of 50 AML patient promotes arrest of myeloid differentiation at the promyelocytic samples showed constitutive activation of STAT5 in 80% of the stage.76 Further, PML has been shown to inhibit STAT3, whereas cases and STAT3 in 74% of the cases.91 Importantly, constitutive PML-RARa enhances STAT3 activity. When expressed in Ba/F3 activation of STATs 3 and 5 has been linked with disease outcomes cells, PML inhibited STAT3-dependent cell growth, whereas in AML. Benekli et al.94 evaluated 63 pretreatment bone marrow PML-RARa augmented growth.76 This suggests a role for STAT3 samples from AML patients for expression of pSTAT3. In the 44% dysregulation in the development of PML-RARa-associated APL. of patients with constitutive activation of STAT3, disease-free While most cases of APL are characterized by PML-RARa, other survival was significantly shorter (8.7 months) than in patients fusion partners for RARa have been described, including STAT5B- without STAT3 activation (20.6 months). Zhong et al.95 performed RARa. This fusion protein results from a deletion on chromosome single-nucleotide polymorphism analyses of the stat3 gene and 17, which fuses sequences encoding the STAT5B oligomerization determined an association between stat3 genotype and domain, DNA-binding domain and a truncated SH2 domain leukemia. Additionally, in 130 AML patients undergoing standard with sequences encoding nearly full-length RARa.8,77 Patients that induction , the GG genotype in rs9909659

Leukemia (2014) 248 – 257 & 2014 Macmillan Publishers Limited STAT transcription factors in hematopoiesis and leukemogenesis KA Dorritie 253 (stat3 polymorphism) was found to be associated with BCR/ABL induces an ALL-like disease in mice with only one gene. unfavorable cytogenetics and failure to achieve complete Mice with both genes intact develop CML-like disease. This remission, whereas the GA/AA genotype was found in younger supports a key role for STAT5A/B in the induction of BCR/ABL- patients who generally exhibited better outcomes than older driven ALL and CML.103 patients.95 Expression of pSTAT5 has also been associated with poor outcome in patients with AML. Bone marrow evaluation of Large granular lymphocytic leukemia 112 newly diagnosed AML patients revealed that activation of STAT5 was significantly associated with poor overall survival and Large granular lymphocytic leukemia (LGL) is a rare chronic reduced progression-free survival.96 lymphoproliferative disorder that arises from clonal proliferation of cytotoxic T lymphocytes or natural killer cells.104,105 Nuclear extracts of peripheral blood mononuclear cells from 19 LGL Acute lymphoblastic leukemia patients revealed increased levels of activated STAT3 and/or As previously discussed, STAT5 has a key role in lymphopoiesis STAT1 compared with normal patients.106 Treatment with the JAK and aberrant activation of STAT5 has been implicated in the inhibitor AG490 resulted in decreased STAT1 and STAT3 activities pathogenesis of pre-B acute lymphoblastic leukemia (B-ALL). and induction of apoptosis in the primary leukemic cells, but not Roughly 40% of adult, and 5% of pediatric, ALL cases are in normal peripheral blood mononuclear cells.106 More recently, characterized by expression of BCR/ABL, leading to constitutive Koskela et al.104 have performed exome sequencing on 77 STAT5 activation and poorer prognosis. Additionally, studies in patients with T-cell LGL (T-LGL). Remarkably, STAT3 mutations mice have shown that transplantation of Abelson virus-trans- were found in 40% of patients and all were located in the coding formed (Ab-MuLV) STAT5A/Bnull/null cells fails to induce leukemia, region for the SH2 domain. In patients harboring the mutations, whereas transplantation of Ab-MuLV STAT5A/B þ / þ or Ab-MuLV STAT3 was activated and localized primarily in the nucleus.104 STAT5A/Bnull/ þ cells results in the development of lymphoid Patients with a STAT3 were more likely to have leukemia, underscoring the role of STAT5 in this disease.61 neutropenia and than those without Aberrant activation of STAT5 has also been observed in high- mutations.104 In another study, sequencing of the stat3 gene in risk, -like factor 2-rearranged B-ALL.97 170 cases, including both chronic lymphoproliferative disorder- natural killer and T-LGL, identified mutations in 28% of cases.105 105 Chronic lymphocytic leukemia Again, all mutations were located in the SH2 domain. Inhibition of STAT3 with STA-21 resulted in apoptosis of LGL cells.105 STAT3 Hyperactivation of STAT3 has been described in chronic lympho- mutations in T-LGL, as well as CD30 þ T-cell lymphomas, have also cytic leukemia (CLL). Curiously, in contrast to the constitutive been reported by Ohgami et al.107 Tyr705 phosphorylation found in many solid and hematological Recently, mutations in the SH2 domain of STAT5B were malignancies, Hazan-Halevy et al. observed that STAT3 was 108 98 discovered in a small number of patients with LGL. These primarily phosphorylated on Ser727 in primary CLL specimens. mutations result in enhanced STAT5B activation and upregulation Treatment of CLL cells with IL-6 resulted in transient induction of of STAT5B target genes. Patients harboring one of the newly phospho-Tyr705 phosphorylation, followed by a rapid decline in identified STAT5B mutations exhibited a particularly aggressive levels. Conversely, phospho-Ser727 STAT3 levels remained disease compared with LGL patients lacking the mutation.108 elevated for extended periods.98 Similar to phospho-Tyr705 STAT3, phospho-Ser727 STAT3 was found to translocate to the nucleus, bind to DNA and induce gene transcription.98 As with THERAPEUTIC TARGETING OF STATS STAT3, STAT1 appears to be constitutively phosphorylated on Ser727 in B lymphocytes from CLL patients.99 However, the precise On the basis of studies investigating STAT expression and role of STAT1 in the pathogenesis of CLL remains unclear. function, and correlation with clinical outcomes, the STAT An interesting interaction between STAT3 and NF-kB has been proteins, particularly STAT3 and STAT5, can be considered well- identified in CLL. NF-kB is known to have an important role validated targets in certain leukemias. Thus, there is considerable in B-cell survival and proliferation, and is hyperactivated in CLL.100 interest in developing novel agents and strategies for inhibiting In CLL patient samples, Liu et al.101 demonstrated binding of STAT signaling. Currently, however, there are very few highly STAT3 to the activated NF-kB protein. The STAT3/NF-kB complex specific, direct inhibitors of STAT proteins. Most agents that are was shown to translocate to the nucleus and induce expression of described as STAT3 inhibitors actually target upstream activators NF-kB target genes. Moreover, infection of CLL cells with STAT3- such as JAKs, Src, BCR/ABL or receptor tyrosine kinases. shRNA resulted in downregulation of mRNA levels for NF-kB target Fortunately, targeting of these upstream kinases represents a genes, indicating a role for STAT3 in NF-kB activation in CLL therapeutically viable approach for attenuating STAT activation/ cells.101 signaling. Inhibition of STAT signaling in Philadelphia chromosome- positive leukemias can be achieved through the use of inhibitors Chronic myelogenous leukemia targeting BCR/ABL. treatment of BCR/ABL-expressing cell As previously discussed, BCR/ABL promotes constitutive phos- lines and CD34 þ cells from CML patients results in apoptosis 87 phorylation/activation of STATs 1, 5 and, to a lesser degree, induction via interference with the STAT5/Bcl-XL pathway. STAT3.49 This has been demonstrated in BCR/ABL-positive cell Imatinib has also been to shown to successfully target lines, cell lines engineered to express BCR/ABL, and in primary TEL-PDGFR.109 The development of resistance to TKIs in BCR/ patient samples.18,83,86 Specimens from CML patients in blast crisis ABL-expressing leukemias is a significant clinical problem, and is have increased levels of Bcl-XL and phosphorylated STAT5 due, in part, to point mutations such as T315I in the ABL kinase compared with those in the chronic phase.86 Blocking BCR/ABL domain.110,111 Recent studies have suggested a role for STAT3 activity with imatinib in CML, megakaryocytic and pre- lines and STAT5 in TKI resistance in CML.112,113 In this regard, the results in growth inhibition and induction of apoptosis associated STAT5 selective inhibitor pimozide has been shown to promote 102 with decreased levels of pSTAT5 and Bcl-XL. reduction in pSTAT5 levels, decreased expression of STAT5 target The use of a BCR/ABL retroviral mouse transplantation model genes and induction of apoptosis in CML cell lines.114 Pimozide has helped to clarify the roles of the STAT5A and STAT5B in inhibition of STAT5 also inhibited colony formation of primary BCR/ABL-driven leukemogenesis. Deletion of both stat5A/B genes CML blasts and induced apoptosis in 32D cells engineered to abrogates BCR/ABL-induced leukemia.103 On the other hand, express the T3151 BCR/ABL mutation.114

& 2014 Macmillan Publishers Limited Leukemia (2014) 248 – 257 STAT transcription factors in hematopoiesis and leukemogenesis KA Dorritie 254 As expression of FLT3-ITD results in activation of the STAT An alternative method for achieving highly selective inhibition pathway, targeting of FLT3 activity can be used to inhibit of activated STATs involves the use of decoy oligonucleotides. STAT signaling in AML. Among the agents currently being Double-stranded decoy oligonucleotides mimic DNA-response evaluated are the multi-kinase inhibitors , elements and act by competitively inhibiting the binding of and (CEP701), as well as agents more specific for FLT3, transcription factors to the corresponding response elements including (AC220) and tandutinib (MLN518).115 in target genes. A STAT3 decoy oligonucleotide has been A number of these inhibitors have shown promise in vitro, but successfully tested in preclinical models of , mixed results have been observed in early clinical trials.115 and was recently evaluated in a phase 0 trial of this disease, but Treatment with other tyrosine kinase inhibitors, such as , has not been applied against hematological malignancies.132,133 and , can also be used to achieve inhibition of However, treatment with a STAT5 decoy oligonucleotide has been c-Kit-, Src- or BCR/ABL-mediated STAT activation. shown to downregulate STAT5 target genes and induce apoptosis Targeting of STAT5 using pimozide has been tested in vitro and in K562 cells.134 Further research is needed to ascertain the in a mouse model of FLT3-ITD-positive AML, where pimozide possible therapeutic benefit of STAT decoy oligonucleotides in the treatment resulted in decreased viability of AML cell lines and treatment of leukemias. decreased tumor burden in mice.116 The combination of pimozide Another approach toward inhibiting STATs involves selective with the multi-kinase inhibitor resulted in further activation of negative regulators, including PTPs, and SOCS and inhibition of STAT5 phosphorylation, suggesting the potential PIAS proteins. While the development of agents that induce or utility of this combination.116 activate SOCS and PIAS is largely unexplored, the potential for There has also been considerable interest in targeting JAK targeting PTPs is now emerging.9 For example, BCR/ABL is known enzymes as a means of inhibiting STAT signaling. Inhibition to functionally inactivate the phosphatase PP2A by enhancing of AML cell lines with the JAK2 inhibitor results in expression of the PP2A inhibitor SET protein.135 Restoration of decreased phosphorylation of STAT3 and STAT5 with induction of PP2A activity by activators such as FTY720 and forskolin results in apoptosis.117 WP1066 and its parent compound AG490, inhibit the dephosphorylation and activation of SHP1 phosphatase.136,137 JAK2 activation, thereby blocking downstream STAT activation Activated SHP1 dephosphorylates BCR/ABL, making it sus- and promoting apoptosis in human AML OCIM2 and K562 cells.118 ceptible to proteasome-dependent degradation. This results in WP-1034, a derivative of AG490, was found to block STAT3 and downmodulation of BCR/ABL-mediated STAT5 activation, leading 5 activation and induce cell cycle arrest and apoptosis in human to growth inhibition in BCR/ABL-expressing cells.136,137 AML OCIM2 cells, as well as primary AML cells.119 The JAK1/JAK2 inhibitor demonstrated efficacy in the treatment of post-myeloproliferative neoplasm AML in a recent phase SUMMARY/PERSPECTIVES II study.120 Interestingly, ex vivo treatment of primary AML cells The STAT proteins have distinct and critical roles in mediating the with the JAK2 inhibitor AZ960 resulted in unexpected induction effects of cytokines and growth factors on mature hematopoietic of cell cycling and downregulation of p21waf1 in the normally cells and during the process of hematopoiesis. In general, findings quiescent CD34 þ /CD38 À leukemic stem cell population, from in vitro and in vivo studies of STATs have been consistent, suggesting a unique opportunity to target these cells.121 although this is not always the case. As most in vitro studies have Recently, C188-9, a novel small-molecule inhibitor of STAT3 utilized leukemic cell line models, the delineation of normal STAT signaling has been reported. C188-9 inhibits G-CSF-induced STAT3 function using these models may be flawed. Further development phosphorylation and target , and suppresses blast of tissue- and lineage-specific knockout models is needed to fully colony formation in leukemia cell lines and primary AML cells.90 clarify the role of the different STATs during hematopoiesis. There Other small molecules that disrupt STAT function or signaling is also a need to more clearly define the function and regulation of include S3l-201, STA-21, Stattic and S3I-1757, although these the shortened STAT3 and STAT5 isoforms, as most studies have compounds have not been rigorously evaluated in hematological focused exclusively on the full-length proteins. malignancies.122–125 Findings from preclinical in vivo models and analyses of primary A number of naturally occurring compounds have also been patient specimens have also revealed unique roles for STATs in the identified that inhibit STAT phosphorylation/activation, including development of several forms of leukemia. While STAT1 likely acts guggulsterone, curcumin, indirubin, icariside II, capsaicin, cucurbi- as a tumor suppressor, aberrant activation of STAT3 and STAT5 tacin, ursolic acid and galiellalactone. Although several of these clearly promotes leukemia development and correlates with poor compounds are well tolerated and may hold promise in the clinical prognosis. Multiple mechanisms have been identified that treatment of leukemias, in general, they lack specificity for STATs can lead to STAT3 and STAT5 hyperactivation, including aberrant and potentially exert their effects through inhibition of multiple activation of upstream receptor tyrosine kinases, inactivation or signaling pathways. downregulation of negative regulators and mutation of STAT Another means of directed STAT targeting is through the use of proteins. STATs also are constitutively activated in cells harboring antisense oligonucleotides and small interfering RNAs (siRNAs).126 fusion oncoproteins with kinase activities, and act to mediate at Downregulation of STAT3 and STAT5 has been achieved in least some of the oncogenic effects of these proteins. solid tumors, with more limited success in hematological Collectively, findings from preclinical leukemia models and malignancies.127–129 Treatment of K562 cells with antisense primary leukemia cells have served to validate STAT3 and STAT5 oligonucleotides or siRNA has been used to achieve down- as valuable targets for anti-cancer therapies. A great deal of effort regulation of STAT3, STAT5A and STAT5B with accompanying is currently being invested to develop STAT inhibitors. It will be increased apoptosis.126 Knockdown of STAT3 with siRNA in the important in these ventures to develop inhibitors that are specific human cutaneous T-cell lymphoma line Hut78 downregulated for either STAT3 or STAT5, but do not inhibit structurally related STAT3 target gene expression, inhibited cell growth and induced STAT1, as STAT1 has tumor suppressive properties. Although a apoptosis.130 More recently, Zhang et al.131 have utilized CpG(A)- number of small-molecule and naturally occurring STAT inhibitors coupled siRNAs to target cells expressing toll-like receptor 9. have been reported, for many, questions remain regarding their Intratumoral injection of CpG(A)-coupled siRNA directed against specificities, mechanisms of action, potencies and bioavailabilities. STAT3 or Bcl-XL mRNAs inhibited the growth of xenotransplanted Lastly, while a high degree of specificity may be achieved through AML and myeloma tumors.131 Currently, there is a need for more the use of siRNAs/shRNAs or decoy oligonucleotides, the delivery research to improve the delivery of antisense and RNAi molecules of these agents to leukemic cells remains a significant obstacle to to normal and malignant hematopoietic cells. clinical development. Thus, further identification of novel STAT

Leukemia (2014) 248 – 257 & 2014 Macmillan Publishers Limited STAT transcription factors in hematopoiesis and leukemogenesis KA Dorritie 255 inhibitors and refinement of existing agents is likely to continue as binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors. an area of active research in the coming years. EMBO J 1995; 14: 2816–2826. 25 Chen CY, Tsay W, Tang JL, Shen HL, Lin SW, Huang SY et al. SOCS1 methylation in patients with newly diagnosed . Genes Chromosomes CONFLICT OF INTEREST Cancer 2003; 37: 300–305. The authors declare no conflict of interest. 26 Chim CS, Wong KY, Loong F, Srivastava G. SOCS1 and SHP1 hypermethylation in mantle cell lymphoma and : implications for epigenetic activation of the Jak/STAT pathway. Leukemia 2004; 18: 356–358. 27 Qiu X, Guo G, Chen K, Kashiwada M, Druker B, Rothman P et al. A requirement for ACKNOWLEDGEMENTS SOCS-1 and SOCS-3 phosphorylation in Bcr-Abl-induced tumorigenesis. 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