Leukemia (2004) 18, 189–218 & 2004 Nature Publishing Group All rights reserved 0887-6924/04 $25.00 www.nature.com/leu

REVIEW

JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis

LS Steelman1, SC Pohnert2, JG Shelton1, RA Franklin1,3, FE Bertrand1 and JA McCubrey1,3

1Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Greenville, NC, USA; 2Department of Biochemistry, Brody School of Medicine at East Carolina University, Greenville, NC, USA; and 3Leo Jenkins Cancer Center, Brody School of Medicine at East Carolina University, Greenville, NC, USA

The roles of the JAK/STAT, Raf/MEK/ERK and PI3K/Akt signal were known under different names including persisting-cell- transduction pathways and the BCR-ABL oncoprotein in stimulating-factor, Thy-1-inducing factor, multilineage hemato- leukemogenesis and their importance in the regulation of cell cycle progression and apoptosis are discussed in this review. poietic growth factor, histamine-producing cell-stimulating These pathways have evolved regulatory proteins, which serve factor, multicolony stimulating factor (CSF), CFU-stimulating to limit their proliferative and antiapoptotic effects. Small factor, eosinophil-CSF, megakaryocyte-CSF, erythroid-CSF, molecular weight cell membrane-permeable drugs that target burst-promoting activity, neutrophil–granulocyte-CSF and he- these pathways have been developed for leukemia therapy. One mopoietin-2, before it was cloned.21 Once it was cloned, it was such example is imatinib mesylate, which targets the BCR-ABL shown to have all these activities. The human IL-3 peptide kinase as well as a few structurally related kinases. This drug consists of 133 amino acids while the mouse peptide is 140 has proven to be effective in the treatment of CML patients. 21–24 However, leukemic cells have evolved mechanisms to become amino acids in length. The biological activity of the IL-3 22 resistant to this drug. A means to combat drug resistance is to molecule lies in residues 17–118, and is not dependent on the target other prominent signaling components involved in the glycosylation state of the protein.25 The half-life of the molecule pathway or to inhibit BCR-ABL by other mechanisms. Treat- is 40 min with clearance being mainly via re-absorption in renal ment of imatinib-resistant leukemia cells with drugs that target tubules.26 Ras (farnysyl transferase inhibitors) or with the protein IL-3 is primarily synthesized by T lymphocytes with a small destabilizer geldanamycin has proven to be a means to inhibit 1,2 the growth of resistant cells. This review will tie together three contribution from mast cells. IL-3 mRNA stability is regulated 27 important signal transduction pathways involved in the regula- by a rapamycin-sensitive pathway. This likely involves the tion of hematopoietic cell growth and indicate how their target of rapamycin (TOR) and its downstream target p70S6K. expression is dysregulated by the BCR-ABL oncoprotein. Frequent injections of mice with either IL-3 or inoculations of IL- Leukemia (2004) 18, 189–218. doi:10.1038/sj.leu.2403241 3-producing cells increase the number of myeloid, mast and Keywords: JAK; Ras; Raf; PI3K; BCR-ABL megakaryocytic cells.28,29 The IL-3 receptor (IL-3R) is a member of the cytokine receptor gene family and is comprised of a- and b-subunits.1 Both subunits contain extracellular domains common to the cytokine Hematopoietic cytokines and receptors 30–42 receptor family. The intracellular domains are more variable. The a subunit of the IL-3R is divided into three main Cytokines interact with cell-surface receptors initiating signaling domains: ligand-binding, transmembrane and intracellular cascades that promote cell growth and division, while inhibiting signaling domains. The ligand-binding domain has homology the pathways of apoptotic cell death.1–20 The JAK/STAT, Raf/ with other cytokine receptors (GM-CSF and IL-5); however, they MEK/ERK and PI3K/Akt signaling pathways are activated by a differ in their binding specificity. The intracellular signaling variety of cytokines that function to potentiate or inhibit domain of the a subunit lacks intrinsic kinase activity, but is hematopoiesis. These include IL-3, IL-7, SCF, G-CSF, type I S6K 30–32 required for activation of STAT5, Raf-1 and p70 . interferons and TGF-b. Signaling through IL-3/IL-3R interaction Transcriptional regulation of c-Fos and c-Jun is mediated has been widely studied as a prototype for how cytokine through the interaction of intracellular signaling domain with signaling activates JAK/STAT, Raf/MEK/ERK and PI3K/Akt path- 30 downstream targets. ways to modulate normal and abnormal hematopoiesis. The common b subunit is a member of the cytokine receptor IL-3 is a soluble highly glycosylated 26 kDa protein that superfamily, consisting of GM-CSF, IL-3 and IL-5 receptors. The belongs to the class of proteins called cytokines. The JAK/STAT, 33–36 b subunit also has no intrinsic kinase activity. The gene for Raf/MEK/ERK and PI3K/Akt signaling pathways elicited by IL-3 37 the b-subunit gene is located on mouse chromosome 15 and promote survival, growth and differentiation of pluripotent the a-chain gene is located on chromosome 14. The human b hematopoietic cells. A depiction of three pathways is presented chain is located on chromosome 22q13.1 and the a chain is diagrammatically in Figure 1. IL-3 was initially characterized by located on chromosome Xp22.3 (Table 1). The cytokine its ability to induce activity of 20-a-hydroxy-steroid-dehydro- receptor superfamily is characterized by a 200-amino-acid genase in spleen cells.21 It turns out that IL-3 was being studied extracellular domain consisting of two fibronectin b barrels. The by many different investigators, and the diverse bioactivities highly conserved consensus ‘WSXWS box’ is characteristic of the membrane proximal region of cytokine receptors.35,38 The Correspondence: JA McCubrey, Department of Microbiology and transduction of a signal by the cytokine receptor requires Immunology, Brody School of Medicine at East Carolina University, 600 Moye Blvd, Greenville, NC 27858, USA; Fax: þ 1 252 744 3104; conformational changes in the cytoplasmic domain of the b E-mail: [email protected] subunit. This conformational change promotes the binding and Received 29 August 2003; accepted 24 September 2003 activation of downstream targets including JAK, STAT and Roles of JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in leukemia LS Steelman et al 190

Figure 1 Overview of effects of IL-3 on JAK/STAT, Raf/MEK/ERK and PI3K/Akt signaling. The figure depicts some of the signal transduction pathways activated after ligation of the IL-3 receptor. Activation of the IL-3 receptor induces the activity of the JAK/STAT, Raf/MEK/ERK and PI3K/ Akt pathways, which results in cell proliferation and prevention of apoptosis. Activation of the JAK/STAT pathway can induce the expression of antiapoptotic genes such as Bcl-XL but it can also induce the expression of CIS and SOCS proteins, which serve as negative feedback regulators. IL- 3 can induce the Ras/Raf/MEK/ERK pathway, which results in phosphorylation of transcription factors that can promote cell proliferation. Akt can phosphorylate many targets (Bad, IkK, caspase-9, GSK3b, Foxo3), which have effects on apoptosis and cell cycle progression. Cytokines also induce the tyrosine phosphorylation of the Gab2 protein, which is a docking molecule. Gab2 associates with the Shp-2 phosphatase, Grb-2 and PI3PI3K. Gab2 can regulate PI3K and Ras activities. Cytokines also regulate the expression of phosphates such as Shp2 and SHIP1, which regulate these pathway. Dotted lines indicate potential interactions between the pathways. Solid lines indicate direct paths of signal transduction in the individual pathways.

Table 1 Chromosomal locations of genes involved in signal like growth factor (IGF) signaling mediated by their receptors. transduction discussed in this review Gab2 serves as a docking site for PI3K and Shp2 and also connects with the Ras/Raf/MEK/ERK pathway via Grb-2. Gab-2 Gene Chromosomal Gene Chromosomal is also a target of BCR-ABL and will be discussed later. location location The cytoplasmic portion of the IL-3bc subunit is divided into two regions, which were identified by truncation mutants: the JAK1 1p32.3 Ha-Ras 11p15.5 35 JAK2 9p24 Ki-Ras 12p12.1 proximal and distal portions. The proximal portion of the JAK3 19p13.1 Raf-1 3p25 bc subunit interacts with and activates JAKs, SOCS, CIS, STATs, STAT1 2q32.2 A-Raf Xp11.2 c-Src, PI3K and Vav.1,35 In the area spanning the distal and STAT2 12q13.13 B-Raf 7q34 proximal regions of the bc chain are the binding sites for the Shc, STAT3 17q21 MEK1 15q21 Shp1 and Shp2 proteins. The distal portion the b subunit STAT4 2q32.2 ERK1 16p11.2 c mediates transcription of c-Fos and c-Jun via its ability to STAT5 17q11.2 ERK2 22q11.2 S6K 30,31 STAT6 17q13.3–q14.1 p85a PI3K 5q12–q13 promote activation of Raf-1 and p70 . At the very end of CIS 3p21.3 p85bPI3K 19q13.2 the membrane distal domain are regions that inhibit cell growth SOCS1 16p13 p110a PI3K 3q16.3 but promote viability.39 SOCS2 12q p110b 3q22.3 SOCS3 17q25.3 p110 PI3Ka˜ 7q22 SOCS4 18q22.2 p110 PI3Ka¨ 1p36.2 Roles of hematopoietic cytokine receptors in hematopoietic SOCS5 2p21 PTEN 10q23 Gab2 11q13.4 SHIP1 2q36–q37.1, 2 disease and neoplasia Grb2 17q24–q25 SHIP2 11q23 SHC 1q21 BCR 22q11.23 The A/J strain of mice is nonresponsive to IL-3 and contains a SHP-2 12q24 ABL 9q34.1 naturally occurring mutation in the IL-3Ra-chain gene.40 Knockout mice have been created that lack either the IL-3 or IL-3R genes.41,42 Mice lacking IL-3 or IL-3R or both have a similar phenotype. These mice exhibit a normal population of phosphatidyl-inositol-3-kinase (PI3K) and other shuttling mole- hematopoietic cells, except for a reduction in eosinophils.41,42 cules such as Shc, which transduces the signal through Grb2 This is likely due to the fact that the IL-3Rb chain is also the and SOS to the Ras/Raf/MEK/ERK cascade.1,35 More recently, IL-5Rb chain and hence the mice are also deficient in another docking molecule Gab2 and the associated Shp2 IL-5-mediated responses. The observation that most other phosphatase have been shown to play important roles in hematopoietic cells appear normal suggests that the effects of cytokine signaling.38 Cytokines induce the phosphorylation of IL-3 and its receptor can be compensated for by other cytokines. Gab2, which associates with Shp-2 and Grb-2. Shp-2 has a Overexpression of the IL-3R chains was first observed in binding site on the IL-3Rb chain.1 The Gab2 docking molecule murine hematopoietic cells, which grew in either the absence of contains a pleckstrin homology (PH) and two Src homology (Src) exogenous IL-3 or in relatively low concentrations of IL-3.17 domains. The Gab2 docking protein is functionally similar to the Mutations that result in constitutive activation or overexpression insulin-regulated substrate (IRS1) involved in insulin and insulin- of the IL-3/GM-CSF/IL-5 receptors have been detected in human

Leukemia Roles of JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in leukemia LS Steelman et al 191 and murine leukemias.43–47 Some naturally occurring cytokine Loss of JAK1 produces prenatal lethality due to neurological receptor mutants act as dominant negative (DN) mutations, disorders,53 while JAK2À/À results in embryonic lethality due to which may serve to dampen the response to cytokines.47 defects in erythropoiesis.54,55 JAK3 expression is limited to Experimentally we have demonstrated that overexpression of the hematopoietic cells, and JAK3 knockout mice have develop- IL-3Ra or IL-3Rb chains will lower the concentrations of mental defects in lymphoid cells.64–66 cytokines necessary to promote proliferation of cytokine- Aggregation of cytokine receptors following activation allows dependent cells.5,17 formation of receptor homodimers and heterodimers.67 Recep- tor aggregation allows transphosphorylation of receptors and activation of the associated JAKs and STATs. The best evidence of transphosphorylation is JAK1 mutant cell lines, which cannot JAK/STAT pathway activate Tyk2 after stimulation with interferon a/b.52 Another example of this transphosphorylation is that IL-2 cannot activate The JAK/STAT pathway consists of three families of genes: the JAK1 in the absence of JAK3.53,68 Together these data indicate JAK, or Janus family of tyrosine kinases, the STAT (signal that receptor aggregation and transphosphorylation are impor- transducers and activators of transcription) family and the tant in activation of the JAKs. CIS/SOCS family, which serves to downregulate the activity The STAT gene family consists of seven proteins (STAT1, of the JAK/STAT pathway.48–67 The JAK/STAT pathway involves STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6) ranging in signaling from the cytokine receptor to the nucleus. JAKs are molecular weights from 75 to 95 kDa (748–851 aa). The stimulated by activation of a cytokine receptor. Stimulation of structure of the STAT family proteins consists of an amino- JAKs results in STAT transcription factor activity. terminal oligomerization domain, a DNA-binding domain in the JAKs are a family of large tyrosine kinases, having molecular central part of the protein, an Src homology 2 (SH2) domain and weights in the range of 120–140 kDa (1130–1142 aa). Four JAKs a transactivation domain near the carboxyl terminus (Figure 2). (JAK1, JAK2, JAK3 and Tyk2) have been identified in mam- The transactivation domain is the most divergent in size and mals.49 JAK proteins consist of seven different conserved sequence and is responsible for activation of transcription. The domains (JH1–JH7). The JH1 constitutes a kinase domain, while oligomerization domain contains a tyrosine that is rapidly JH2 is a pseudokinase domain. Many possible roles have been phosphorylated by JAKs, allowing the phosphotyrosine product proposed for the different domains of the JAK proteins:54–61 (1) to interact with the SH2 domains of other STAT proteins. JH2 inhibits JH1; (2) JH2 promotes STAT binding; (3) JH2 is Formation of STAT dimers promotes movement to the nucleus, required for kinase activity of JH1; and (4) JH6 and JH7 are DNA binding and activation of transcription, as well as necessary for association of JAKs with cytokine receptors. A increased protein stability. Threonine (T) phosphorylation has diagram illustrating the key features of the JAK, STAT and SOCS been proposed to play a further role in the regulation of STAT proteins is presented in Figure 2. activity.69,70 This may be mediated by ERK,70 indicating a point

Figure 2 Structure of the JAK, STAT and SOCS proteins. The JAK family of proteins is centrally involved in cytokine-mediated signal transduction (see Kisseleva et al49 and Krebs and Hilton50 and references therein). The JAK family consists of JAK1, JAK2, JAK3 and TYK2. The JAK family of kinases contains seven JAK homology (JH) regions. Shown in this diagram is the JAK3 protein with the amino-acid residues indicated in the JH regions. The JH1 region is the kinase domain that contains a regulatory tyrosine residue. The STAT family of proteins consists of at least seven STAT proteins, which all have a STAT dimerization domain, a coiled-coil domain, a DNA-binding domain, an SH2 domain for interaction with tyrosine-phosphorylated proteins and finally a transactivation domain. The SOCS (suppressor of cytokine signaling proteins) consists of at least six SOCS proteins and a CIS (cytokine-inducible SH2-containing) protein. These proteins contain an amino-terminal domain which inhibits JAK activity, an SH2 domain which binds tyrosine-phosphorylated proteins and a SOCS box which promotes protein degradation via interaction with the proteosome.

Leukemia Roles of JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in leukemia LS Steelman et al 192 of interaction between the Raf/MEK/ERK and JAK/STAT path- ways. The roles of the STAT and Jak proteins in hematopoietic growth have been investigated by the creation of knockout strains of mice.71–73 STAT3À/À mice have severe developmental problems resulting in fetal death.73 Cytokine signaling abnorm- alities are associated with other STAT knockout models, but all mice are viable. Constitutive STAT activity is associated with viral infections, but only STAT3 is known to have oncogenic properties.74–78 v- Abl and BCR-ABL induce constitutive STAT4 activity.74,75 STAT transcription factors can induce antiapoptotic gene expression including Bcl-XL. The JAK/STAT pathway is negatively regulated by the suppressors of cytokine signaling (SOCS) and cytokine-induced SH2-containing (CIS) family of proteins. The more accepted name for this family is SOCS. This protein family consists of SOCS1–SOCS5 and CIS1.79–89 This family of genes has a conserved SH2 domain and SOCS box79–89 (Figure 2). The SOCS box, the carboxy-terminal 40 amino acids, is implicated in stability and degradation of the protein by targeting it to the proteosome.82–84 CIS inhibits STAT5 activation by competing for its receptor-binding site.85,86 SOCS1 directly binds and inhibits the kinase domain (JH1) of JAK2.86–89 Gene ablation studies have indicated that the SOCS proteins have important roles in regulating the effects of interferon-g, growth hormone and erythropoietin. SOCS1 and SOCS3 knockout mice are lethal, Figure 3 Structure of TEL-JAK proteins. (a) Breakpoints in chimeric whereas SOCS2 knockout mice are 30% larger than their wild- TEL-JAK proteins and (b) chimeric TEL-JAK proteins that have been type counterparts.89–92 observed in different types of leukemias. Note that the chimeric TEL- There are other mechanisms to downregulate JAK/STAT JAK proteins contain the TEL-encoded oligomerization domain and the JAK-encoded kinase. The TEL oligomerization domain makes the JAK signaling. Protein phosphatases, including CD45 and PTPeC, kinase domain constitutively active. This diagram was derived in part are also implicated in the negative regulation of JAK–STAT from the work published by Drs Bernard and Gilliland and others.95–99 signaling.93,94

determination of the ability of STAT5 to interact with other Roles of JAK/STAT pathway in neoplasia oncoproteins to transform primary hematopoietic cells. Interest- ingly, one activated STAT5 retrovirus contains the necessary A chromosomal translocation forming the TEL-JAK2 fusion activating mutation in the SH2 domain (N642H) and the other protein that results in constitutive kinase activity has been activated STAT5a construct requires two mutations in different observed in a limited number of patients with ALL and CML.95,96 domains (the coiled-coil) and the transactivation effector This chimeric protein has been shown to abrogate the cytokine domains to be able to abrogate the cytokine dependence of dependence of certain hematopoietic cell lines.95–98 On the hematopoietic cell lines. other hand, the partner TEL gene (translocated ETS in leukemia) is often rearranged in human leukemia.99 TEL rearrangement partners include Abl, PDGF-R and JAK, all of which encode Ras/Raf/MEK/ERK pathway tyrosine kinases. The fusion products encode constitutively active tyrosine kinases involved in human leukemia. Activation The Ras/Raf/MEK/ERK pathway is a central signal transduction of JAK in TEL-JAK is due to the oligomerization domain provided pathway, which transmits signals from multiple cell surface by the TEL transcription factor, which results in the constitutive, receptors to transcription factors in the nucleus.119–121 This ligand-independent activation of the JAK kinase domain.95–98 A pathway is frequently referred to as the MAP kinase pathway as diagram illustrating the breakpoints and resulting TEL-JAK fusion MAPK stands for mitogen-activated protein kinase indicating protein is presented in Figure 3.95–99 Activated JAK proteins that this pathway can be stimulated by mitogens, cytokines and have been made from the TEL-JAK rearranged chromosomal growth factors. The pathway can be activated by Ras stimulating translocation. the membrane translocation of Raf. This pathway also interacts STAT overexpression is frequently observed in human with many different signal transduction pathways including cancers.74–78,100–115 The STAT3 protein can function as an PI3K/Akt and JAK/STAT (see below). oncogene,78 and other STAT proteins may function in oncogenic Ras is a small GTP-binding protein, which is the common transformation.105–115 The STAT molecules provide novel upstream molecule of several signaling pathways including Raf/ therapeutic targets for oncogenic transformation.114,115 Activat- MEK/ERK, PI3K/Akt and RalEGF/Ral.119–121 The switch regions ing mutants of STAT5a have been made, which will abrogate the of the Ras proteins are in part responsible for the switch between cytokine dependence of hematopoietic cells.116–118 the inactive and active states of the protein. Switching between Some STAT mutants were isolated based on their ability to these states has been associated with conformational changes in abrogate the cytokine dependence of hematopoietic cell the switch regions, which allows the binding of GTPase- lines.95–99,116,118 A diagram of these retroviral constructs is activating proteins (GAPs, of which the tumor suppressor gene presented in Figure 4. Use of these constructs has allowed NF-1 is a member) and guanine nucleotide exchange factors

Leukemia Roles of JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in leukemia LS Steelman et al 193

Figure 4 TEL-JAK and mutated STAT5 retroviruses. The cDNAs encoding the TEL-JAK oncoprotein and activated STAT5a mutants have been inserted into retroviruses encoding neor.95–99,116,118 These retroviruses allow the relatively easy transfer of the genes into hematopoietic cells. Thus the effects of activated TEL-JAK and STAT5a proteins on other signal transduction pathways as well as the prevention of apoptosis can be determined. The amino-acid substitutions that result in STAT5 activation are indicated above the different STAT5 mutants. These constructs were made in the laboratories of Drs Bernard and Kitamura.96,97,116,117

(GEFs). When Ras is active GTP is bound, whereas when Ras is The Raf proteins have three distinct functional domains: CR1, inactive GDP is bound. GTPases inactivate the Ras proteins CR2 and CR31 (Figure 6). The CR1 domain is necessary for Ras while GEFs activate the Ras proteins by either stimulating the binding and subsequent activation. The CR2 domain is a removal of phosphate from GTP or addition of GTP respectively. regulatory domain. CR3 is the kinase domain. Deletion of the A diagram of the activation/inactivation of the Ras protein is CR1 and CR2 domains produces a constitutively active Raf presented in Figure 5. protein due in part to the removal of phosphorylation sites that Different mutation frequencies have been observed between serve to negatively regulate the kinase in the CR2 domain. An Ras genes in human cancer.122,123 An excellent concise overview of the Raf proteins is presented in Figure 7. summary of the modes of regulation, activities and frequency The A-Raf gene is located on X chromosome in humans137 of Ras mutations is presented in the website of Drs Watzinger and produces a 68 kDa protein. The highest level of A-Raf and Lion.123 The structural domains of the Ras proteins, the sites expression in the adult is in the urogenital tract. A-Raf deletion of the Ras protein that are mutated, the structures of the naturally results in postnatal lethality, attributed to neurological and occurring Ras retrovirus and a genetically engineered Ras gastrointestinal defects.138 The role of A-Raf has remained retrovirus124 are presented in Figure 6. elusive. A-Raf is the weakest Raf kinase in terms of activation of There are three different Ras family members: Ha-Ras, Ki-Ras ERK activity. ERK activation occurs in A-Raf knockout mice, and N-Ras. The Ras proteins show varying abilities to activate indicating that other Raf isoforms can compensate for this the Raf/MEK/ERK and PI3K/Akt cascades, as Ki-Ras has been deficiency.139 Some studies have indicated that A-Raf has an associated with Raf/MEK/ERK while Ha-Ras is associated with important role in stimulating the growth of hematopoietic PI3K/Akt activation.125 Amplification of ras proto-oncogenes cells.140 Moreover, we have observed that overexpression of and activating mutations that lead to the expression of activated A-Raf will abrogate the cytokine dependence of constitutively active Ras proteins are observed in approximately hematopoietic cells (Table 2). 30% of all human cancers.123,126,127 The effects of Ras on The B-Raf gene is located on chromosome 7q34.141,142 B-Raf proliferation and tumorigenesis have been documented in encodes a 94 kDa protein. Highest expression of B-Raf is in the immortal cell lines.128–136 However, antiproliferative responses testes and neuronal tissue. The B-Raf gene produces splice of oncogenic Ras have also been observed in nontransformed variants, the physiological roles of which are not yet eluci- fibroblasts, primary rat Schwann cells and primary fibroblast dated.141 Loss of B-Raf expression in mice results in intrauterine 133,134 142 cells of human and murine origin. This premature G1 death between days 10.5 and 12.5. The B-Raf knockout arrest and subsequent senescence is dependent on the Raf/MEK/ mouse embryo displays enlarged blood vessels and increased ERK pathway and was shown to be mediated by many cell cycle apoptosis of differentiated endothelial cells. This is an indication inhibitory molecules including p15Ink4b/p16Ink4a and p21Cip1.135 that Raf kinases can regulate apoptosis. B-Raf and Raf-1 activity The Raf protein family consists of A-Raf, B-Raf and Raf-1, are negatively regulated by Akt phosphorylation.143,144 Akt also which are involved in the regulation of proliferation, differentia- has other effects on the presentation of apoptosis, discussed in tion and apoptosis induced after cytokine stimulation.1,136–139 more detail later. In contrast, other studies have shown that

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Figure 5 Ras activation and recruitment of Raf. The activity of the Ras protein is regulated by its conformational state. There are switch regions in the Ras protein, which effect whether Ras is in an active or inactive conformation (see Figure 6). Switching between these states has been associated with conformational changes in the switch regions, which allows the binding of guanine nucleotide exchange factors (GEFs) or GTPase- activating proteins (GAPs, of which the tumor suppressor gene NF-1 is a member). When Ras is active GTP is bound, whereas when Ras is inactive GDP is bound. GTPases inactivate the Ras proteins while GEFs activate the Ras proteins by either stimulating the removal of phosphate from GTP or addition of GTP respectively. Active Ras can then bind Raf on the Ras-binding domain (BD) present in Raf. This results in the translocation of Raf to the cell membrane. Raf is then activated by phosphorylation and dephosphorylation as shown in Figure 8.

overexpression of B-Raf in Rat-1 cells results in decreased (CR3) ligated to the hormone-binding (hb) domain of the apoptosis due to inhibition of caspase activity.145 Historically, estrogen receptor, rendering their activity dependent on the B-Raf is the strongest Raf in terms of induction of MEK activity as presence of b-estradiol or the estrogen receptor antagonist 4 determined by in vitro kinase assays. B-Raf activation is different hydroxyl tamoxifen (4HT) (Figure 7). Dr McMahon and Dr from either Raf-1 or A-Raf, as its activation requires phosphor- Hartmund Land (ICRF, London, UK)162 have also made ylation of one regulatory residue while activation of Raf-1 and conditional Raf-1 kinases which contain the hb domain of the A-Raf requires two phosphorylation events.146,147 B-Raf is also androgen receptor linked to the DRaf-1, which makes Raf-1 regulated by Akt and the serum glucocorticoid-regulated kinase activity dependent on the presence of testosterone. The ability of (SGK).148,149 Recently, it has been shown that B-Raf may be Raf proteins to phosphorylate MEK1 varies from B-Raf4Raf- required in the activation of Raf-1 and that B-Raf and Raf-1 may 14A-Raf.159,161 The ability of Raf to abrogate cytokine be induced at different times following stimulation.149,150 dependency is inversely proportional to their MEK1 activity, Moreover, the three different Raf proteins may have different with A-Raf4Raf-14B-Raf.160,161 Stimulation of Raf activates subcellular pools, and Raf-1 and A-Raf may be localized in MEK1 and ERK resulting in phosphorylation of transcription some cases to the mitochondrion.151 The roles of B-Raf in factors, proliferation, and inhibition of apoptosis. human neoplasia will be discussed later. The activation of the three Raf isoforms is complex and not The Raf-1 gene is located on chromosome 3p25 and produces totally understood. Ras is known to activate B-Raf independently a 74 kDa protein.137 The Raf-1 proto-oncogene was the first of Src activity, while Raf-1 and A-Raf require Src for complete cloned Raf gene as it is the cellular homolog to v-Raf contained activation.146,147 Src phosphorylates Y340, Y341 of Raf-1 and in the naturally occurring acute retrovirus 3611-MSV.152 Raf-1 is Y301, Y302 on A-Raf, which are not present in B-Raf.163 B-Raf ubiquitously expressed in adult tissues, with highest expression contains aspartic acid (D) at the corresponding residues. in muscle, cerebellum and fetal brain.137 It is the most studied Aspartic acid residues are negatively charged and are believed Raf isoform. A DN version of Raf-1 inhibits Ha-Ras-induced to confer a constitutively active configuration at this site. B-Raf is transformation and tumor formation.153 Raf-1 has important constitutively phosphorylated on S445, a site equivalent to S338 roles in apoptosis as it phosphorylates and inactivates Bad.154 in Raf-1, which is phosphorylated as a result of Ras activa- Raf-1 phosphorylates and co-immunoprecipitates with Bcl- tion.163 The Ras-binding domains of B-Raf and Raf-1 have a 2155,156 as well as regulates Bag and Bad expression, in BCR/ greater affinity for Ras than A-Raf.164 This suggests that A-Raf ABL-expressing cells.155 Recently, Raf-1 has been proposed to may have a primary activator other than Ras. Phosphorylation of have roles independent of MEK/ERK that are often involved in the activation loop of Raf-1 and B-Raf is necessary but not the regulation of cell cycle progression and apoptosis.156,157 sufficient for activation.164 Deletion or mutation of inhibitory These new roles for Raf-1 will be discussed below. phosphorylation sites present at certain residues by site-directed Conditionally active Raf proteins have been made by Dr mutagenesis activates Raf proteins.165 Akt (PKB) also phosphor- Martin McMahon’s laboratory first at DNAX (Palo Alto, CA, ylates Raf-1 at S259, which has been associated with inhibition USA) and then at the University of California (San Francisco, of Raf-1 activity.142,143 Akt and SGK phosphorylate B-Raf at the CA, USA).158–161 These constructs contain the kinase domain equivalent residue.148,149

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Figure 6 Structure of wild-type and activated ras proteins. (a) Structures of the wild-type Ras proteins containing the phosphate-binding motifs, the switch regions, the guanine base-binding domains and the CAAX motifs are presented. The switch regions of the Ras proteins are in part responsible for the switch between the inactive and active states of the protein. The Ras proteins have been divided into four regions: the amino- terminal region which is identical in Ha-Ras, Ki-Ras and N-Ras, which is followed by a 70– 80% conserved region which contains the sequences necessary for binding the guanine bases, which is followed by a hypervariable domain, and finally a CAAX domain which is necessary for interaction with the cell membrane. The amino-acid residues in the CAAX domain are C ¼ cysteine, A ¼ any aliphatic residue and X ¼ any uncharged amino acid. (b) Sites of mutations in the Ras proteins. The sites of mutations detected in Ras are indicated. These mutations occur in codons that are associated with guanine nucleotide-binding sites. These mutations result in reduced GTPase activity (12, 13, 59, 61, 63) or decreased nucleotide affinity (residues 116, 117, 119, 146, 165). These mutations result in locking the Ras protein in an active state, which cannot be inactivated by the GTPase. Deletion of the NF-1 tumor suppressor gene also results in deregulated Ras expression and has been observed in certain childhood leukemias. Much of the information presented in (a,b) was derived from the Watzinger and Lion Ras family website.123 (c) Structures of the naturally occurring HaSV and engineered v-Ha-Ras-encoding retroviruses. The engineered v-Ha-Ras retrovirus allows the introduction of the activated v-Ha-Ras gene into diverse cells. The SV-X-Ha-Ras is described in detail in the work of Dr Witte and colleagues.124

Several protein kinases are known to regulate Raf-1 activity. activated by a Raf-1 mutant, which no longer binds or CAMP-dependent protein kinase (PKA) inhibits Raf-1. CAMP phosphorylates MEK1.186 This suggests that Raf-1 has physiolo- activates PKA resulting in phosphorylation of Raf-1 on S43 and gical substrates other than MEK1. S621, inhibiting Raf-1 activity.166–168 This contrasts with B-Raf Recently, the roles of Raf-1 in the Raf/MEK/ERK signal activation in response to PKA.168–170 Raf-1 is activated through transduction pathway have become controversial due to the p21-associated protein (Pak).171 Activation of conventional discovery that B-Raf was the more potent activator of MEK and protein kinase C isoforms (a, b and g) stimulates Raf-1 activity that many of the ‘functions’ of Raf-1 still persist in Raf-1 but not in a baculovirus system.172 Protein kinase Ce is associated with B-Raf knockout mice. Raf-1 has been postulated to have c-N-Ras and Raf-1 and is necessary for activation of Raf-1 by important roles in cell cycle progression, activation of the p53 phorbol 12 myristate 13 acetate in fibroblasts.173 RKIP, a Raf-1- and NF-kB transcription factors and the prevention of apoptosis. interacting protein, inhibits Raf-1 activation of MEK1.174 A Raf-1 has been postulated to have nonenzymatic functions and model for activation of Raf-1 is presented in Figure 8a. serve as a docking protein. An excellent summary of these non- 14-3-3 proteins are known to bind Raf. Interaction of MEK/ERK-related activities of Raf-1 is presented in the review by 14-3-3 proteins with phosphorylated S259 and S621 inhibits Hindley and Kolch.157 Raf-1 has been proposed to have Raf-1.175–178 Protein phosphorylase 2A can remove the important functions at the mitochondrial membrane. Interest- phosphate on S259 allowing Raf to become disassociated from ingly, it has been shown that BCR-ABL may interact with Raf-1 14-3-3. This allows Raf-1 to assume a conformation in which it to alter the distribution of Bag at the mitochondrial membrane can be phosphorylated and activated by other kinases including and hence regulate (prevent) apoptosis in hematopoietic PAK and Src family kinases. cells.155,157 A diagram depicting some of the non-MEK1/ERK Raf-1 was originally characterized as phosphorylating MEK1, postulated effects of Raf-1 is presented in Figure 9. but B-Raf is the primary MEK1 activator in bovine brain extracts, Recently, the mechanism of B-Raf regulation has been more NIH 3T3 cells, PC12 cells and other cells.179–185 Raf-dependent intensively investigated.136,179–185 A comparison of Raf-1 and B- activation of p90 ribosomal S6 kinase (p90Rsk) and NF-kBis Raf activation is presented in Figure 8a and b. B-Raf activation

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Figure 7 Structures of Raf proteins. Depicted are the structures of A-Raf, B-Raf and Raf-1 proteins. These proteins consist of three domains: CR1, CR2 and CR3. CR1 contains the Ras-binding domain (RBD) and the cysteine-rich domain (CRD). CR2 contains the regulatory domain which has sites for B-Raf and Raf-1 phosphorylation by Akt and SGK. CR3 is the kinase domain which also has many serine (S), threonine (T) and tyrosine (Y) residues which are involved in activation/inactivation. More regulatory sites have been identified on the Raf-1 protein than either the B-Raf or A-Raf proteins. This is because Raf-1 has been more intensively studied. The protein sizes, chromosomal locations and estimates of the gene sizes are indicated. Interestingly, each Raf gene is comprised of at least 15 exons and the genes range in size from 11 to 80 kb. Different alternative splicing variants of B-Raf have been detected. In the bottom half of this panel are retroviral constructs encoding activated versions of Raf. 3611- MSV is a naturally occurring Raf retrovirus. Retroviruses encoding activated versions of the Raf proteins have been made by Dr Martin McMahon and colleagues at DNAX (Palo Alto, CA, USA) and the University of California, San Francisco (San Francisco, CA, USA)158–161 and Dr Hartmund Land (Imperial Cancer Research Fund, London, UK).162 These engineered Raf constructs have the Ras-binding and -regulatory domains deleted.15 The hormone-binding (hb) domains of the estrogen receptor (ER) or the androgen receptor (AR) were inserted to make the constructs conditionally active in that they require either b-estradiol or testosterone for activation, respectively. The GFPDRaf-1:ER and GFPDRaf-1:AR have the green fluorescent protein (GFP) moiety fused to the amino terminus making them fluorescent upon activation by light of the appropriate wave length.15 Neo ¼ gene encoding G418 (geneticin, G418) resistance, Puro ¼ gene encoding puromycin resistance, Blast ¼ gene encoding blasticidin resistance.

may occur through the GTP-binding protein Rap-1, which is MEK in turn is activated by RA-GEF-1 and Src. PKA may activate Src in some cells, which results in Rap-1 activation (Figure 10). MEK proteins are the primary downstream targets of Raf. The There are three regulatory residues on B-Raf: S444, T598 MEK family of genes consists of five genes: MEK1, MEK2, MEK3, and S601. The S/T kinases which phosphorylate these MEK4 and MEK5. This family of dual-specificity kinases has both residues are not known. Activated B-Raf can interact with S/T and Y kinase activity. The structure of MEK consists of an Ras, which in turn results in the activation of Raf-1.184 B-Raf amino-terminal negative regulatory domain and a carboxy- can activate MEK, which results in ERK and downstream terminal MAP kinase-binding domain, which is necessary for kinases and transcription factors. Rheb (Ras-enriched homolog binding and activation of ERKs.187–189 Deletion of the regulatory found in brain) and 14-3-3 proteins can bind and inhibit B-Raf MEK1 domain results in constitutive MEK1 and ERK activation. activity.183 B-Raf can also be negatively regulated by phosphor- MEK1 is a 393-amino-acid protein with a molecular weight of ylation by either SGK or Akt. Akt has been shown to 44 kDa.189 MEK1 is modestly expressed in embryonic develop- phosphorylate B-Raf on three residues: S364, S428 and T439 ment and is elevated in adult tissue with the highest levels (see below). However, Akt may have greater affinity for other detected in brain tissue.189 Knockout of functional MEK1 results substrates besides B-Raf. SGK has been proposed to be a more in lethality due to placental vascularization problems.190 Mice relevant kinase for regulating B-Raf through phosphorylation of with DN MEK1 have also been generated; these mice were S364.185 Potential mechanisms for activation of B-Raf are viable although defects in T-cell development occurred.191 presented in Figure 10. MEK1 requires phosphorylation of S218 and S222 for activation, B-Raf may also activate Raf-1 via Ras-GTP.184 While Rap-1 and substitution of these residues with D or glutamic acid (E) led activates B-Raf, it may also serve to inactivate Raf-1.186 Thus the to an increase in activity and foci formation in NIH3T3 pathways for activation and inactivation of B-Raf and Raf-1 are cells.187,192–194 Mutated MEK1 constructs demonstrate that complex and may appear contradictory. activation of ERK does not require a functional MEK1 kinase

Leukemia Roles of JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in leukemia LS Steelman et al 197 Table 2 Abrogation of cytokine dependence of hematopoietic cells kinases.189 This dual phosphorylation activates ERK; however, by activated oncogenes ERK activity is downregulated by S/T phosphatases, Y phospha- tases or dual-specificity (Y and S/T) phosphatases.206–211 ERK Oncogene Hematopoietic cell line dimerization occurs subsequent to phosphorylation. This dimerization maintains the activation of ERK and promotes TF-1 FDC-P1 FL5.12 nuclear localization of the protein.212 Activated ERK preferen- 213 BCR-ABL ++ +++ ++ tially phosphorylates S/T residues preceding a proline. v-Ha-Ras ++ +++ À Mice lacking ERK1 have defective T-cell development similar DRaf-1 +++ + À to transgenic mice containing a DN MEK1.214 Recently, DRaf-1:ER +++ + À different roles have been suggested for ERK1 and ERK2. While DA-Raf:ER +++ ++ À these two proteins are often detected at similar levels, their DB-Raf:ER + + À DMEK-1:ER ++ + À expression patterns may change. ERK1 has been postulated to PI3K (act) ÀÀ Àinhibit ERK2 expression. Increased ERK2 activity is associated 215 DAkt (act) ÀÀ Àwith cell proliferation. Interestingly, ERK1 has been asso- DAkt:ER (Myr+) ÀÀ Àciated with cognitive brain functions including learning.216 DAkt:ER (MyrÀ) ÀÀ À Tel-JAK ++ ++ + STAT5 (act) ++ ++ + Rsk DRaf-1:ER+DAkt:ER (Myr+)NDND+ p90 and downstream transcription factors DRaf-1:ER+DAkt:ER (MyrÀ)NDND À DA-Raf:ER+DAkt:ER (Myr+)NDND+ Downstream targets of ERK include the p90Rsk kinase and the DB-Raf:ER+DAkt:ER (Myr+)NDND+ CREB, c-Myc and other transcription factors. ERK and p90Rsk DRaf-1:ER+PI3K (Act) ND ND + can enter the nucleus to phosphorylate transcription factors DA-Raf:ER+PI3K (Act) ND ND + which can lead to their activation. The effects of activation of DB-Raf:ER+PI3K (Act) ND ND + the Raf/MEK/ERK cascade on downstream transcription factors ND ¼ not determined, À¼inability of oncogene to abrogate cytokine have been recently reviewed.121 dependence, + ¼ low efficiency of abrogation of cytokine dependence, ++ ¼ readily abrogates cytokine dependence. Roles of the Ras/Raf/MEK/ERK pathway in neoplasia domain.194 Replacement of the amino terminus of MEK1 with the hormone-binding domain of the estrogen receptor produces Mutations at three different Ras codons (12, 13 and 61) convert a construct with kinase activity responsive to the presence of Ras into a constitutively active protein.217–227 These point estrogen analogs.195–197 This construct is an invaluable tool in mutations can be induced by environmental mutagens.224 research into MEK1 signaling. Studies with this construct Given the high level of mutations that have been detected at demonstrated that activated MEK1 could abrogate cytokine Ras, this pathway has always been considered a key target for dependency of certain hematopoietic cells.195–199 Constitutive therapeutic intervention. Approximately 30% of human cancers activity of MEK1 in primary cell culture promotes senescence have Ras mutations. Ras mutations are frequently observed in and induces p53 and p16INK4a, and the opposite was observed certain hematopoietic malignancies including myelodysplastic in immortalized cells or cells lacking either p53 or p16INK4a.200 syndromes, juvenile myelomonocytic leukemia and acute Constitutive activity of MEK1 inhibits NF-kB transcription by myeloid leukemia.122,225–227 Ras mutations are often one step negatively regulating p38MAPK activity.201 in tumor progression, and mutations at other genes (chromoso- Pharmaceutical companies have developed inhibitors of mal translocations, gene amplification and tumor suppressor MEK.202–204 The two most widely used are U0126 and gene inactivation) have to occur for a complete malignant PD98059, as they are commercially available. These two phenotype to be manifested. Pharmaceutical companies have 204 inhibitors have IC50 values of 70 nM and 2 mM, respectively. developed many farnesyl transferase (FT) inhibitors, which PD98059 inhibits activation, while U0126 inhibits activity. Both suppress the farnesylation of Ras, preventing it from localizing inhibitors have a noncompetitive mechanism of inhibiting ERK to the cell membrane.228–247 The sites of action of FT and other activity.202–204 small molecular weight membrane-permeable signal transduc- tion pathway inhibitors are presented in Figure 11. As stated previously, there are three different Ras genes: Ki- ERK Ras, Ha-Ras and N-Ras. The biochemical differences between these Ras proteins have remained elusive. Ki-Ras mutations have The main physiological substrates of MEK are the members of been more frequently detected in human neoplasia than Ha-Ras the ERK (extracellular signal-regulated kinase) or MAPK (mito- mutations. Ras has been shown to activate both the Raf/MEK/ gen activated protein kinase) family of genes. The ERK family ERK and the PI3K/Akt pathways. Thus, mutations at Ras should consists of four distinct groups of kinases: ERK, Jun amino- theoretically activate both pathways simultaneously. Ras muta- terminal kinases (JNK1/2/3), p38MAPK (p38 a/b/g/d) and ERK5. In tions have a key role in malignant transformation, as both of addition, there are ERK3, ERK4, ERK6, ERK7 and ERK8 kinases, these pathways can prevent apoptosis as well as regulate cell which while related to ERK1 and ERK2 have different modes of cycle progression. Recently, it was shown that there is activation, and their biochemical roles are not as well specificity in terms of the ability of Ki-Ras and Ha-Ras to induce characterized. The ERK1 and ERK2 proteins are the most studied the Raf/MEK/ERK and PI3K/Akt pathways.125,248,249 Ki-Ras with regard to Raf signaling in hematopoietic cells. preferentially activates the Raf/MEK/ERK pathway while Ha- ERK1 and ERK2 encode 42 and 44 kDa proteins. These Ras preferentially activates the PI3K/Akt pathway. Therefore, if proteins were originally isolated by their ability to phosphorylate Ras inhibitors could be developed which would specifically microtubule-associated protein 2.205 ERKs are activated through inhibit one particular Ras protein, it might be possible to inhibit dual phosphorylation of T182 and Y184 residues by MEK1 one of the downstream pathways. This might under certain

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Figure 8 Models for Raf activation. (a) Raf-1, (b) B-Raf. These models are adapted from the publications of Dr Walter Kolch174 and Dr Catrin Pritchard.136

circumstances be advantageous. Furthermore, decreases in ERK MEK or ERK were thought to be relatively rare in human expression may affect differentiation responses. Thus in certain neoplasia. For many years, numerous scientists felt that the tumors, one might desire to inhibit the effects Ras has on the activation of the Raf/MEK/ERK cascade was mainly due to PI3K/Akt pathway as opposed to the effects Ras has on Raf. mutations at Ras and hence studies aimed at elucidating the Targeting of Ha-Ras as opposed to Ki-Ras might inhibit apoptosis mechanisms of Ras activation were promulgated. suppression by Ha-Ras but not the effects Ki-Ras has on cell While mutations at the Raf gene in human neoplasia have cycle progression and differentiation. A diagram illustrating the been detected, they have not until recently gained the interactions between the Raf/MEK/ERK and PI3K/Akt cascades is clinical importance that Ras mutations readily achieved. Due presented in Figure 11. to more innovative, high-throughput DNA sequencing, Overexpression of the Raf/MEK/ERK cascade has been scientists have recently discovered that the B-Raf gene is frequently observed in human neoplasia.250–270 A prime frequently mutated in certain cancers including hematopoietic consequence of this activation may be the increased expression tumors.136,251–253,255,259–261,265 Approximately 60% of the of growth factors that can potentially further activate this melanomas surveyed in one study were observed to have cascade by an autocrine loop. Many cytokine and growth factor mutations at B-Raf.265 This result provides relevance to genes contain transcription factor-binding sites, which are investigating signal transduction pathways, as by understanding bound by transcription factors (Ets, Elk, Jun, Fos, CREB) whose how B-Raf is activated, one Ras-dependent and one Ras- activities are often activated by the Raf/MEK/ERK cascade. independent event, scientists could predict why a single Identification of the mechanisms responsible for activation of missense mutation in B-Raf permitted ligand-independent this cascade has remained elusive. Genetic mutations at Raf, activation, whereas similar mutation events would not be

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Figure 9 Novel Raf-1 pathways that are independent of MEK1/ERK. Many non-MEK1/ERK-mediated functions of Raf-1 have been postulated. Some of the purported roles (eg, cell cycle regulatory, antiapoptotic effects on NF-kB activation) may be more likely than others (eg, effects of Raf-1 on p53 transcription). BCR-ABL can activate Raf-1 activity and thus have antiapoptotic and cell cycle effects. Raf can activate the Cdk25 phosphatase, which results in CDK activation. This diagram was adapted from the information presented in the excellent reviews on non-MEK1/ ERK-mediated effects on Raf-1 by Hindley and Kolch.157 predicted to result either in Raf-1 or A-Raf activation as they other Raf genes under certain circumstances might prove required multiple activation events. Interestingly, in 22 mela- detrimental. Thus the development of unique and broad- nomas, colorectal and NSCLC tumors examined, there were 10 spectrum Raf inhibitors may prove useful in human cancer with mutations at B-Raf and 10 with mutations at Ras. Two therapy. tumors had mutations at B-Raf and Ras and two did not have Activation of Raf is complex and requires numerous mutations at either gene. Thus, B-Raf transformation does not phosphorylation and dephosphorylation events.119–121 Preven- appear to require Ras, and many tumors had mutations at one or tion of Raf activation by targeting kinases (eg, Src, PKC, PKA, the other but not both genes. A diagram of sites of mutation PAK or Akt) and phosphatases (eg, PP2A) involved in Raf detected in the B-Raf protein is presented in Figure 12. Recently, activation may be a mechanism to inhibit/regulate Raf it has been suggested that B-Raf mutations occur during the activity.282–310 It is worth noting that some of these kinases process of tumor progression as opposed to establishment.263 normally inhibit Raf activation (Akt, PKA). A major limitation of This was suggested by analysis of B-Raf mutations in different this approach would be the specificities of these kinases and developmental stages of melanomas. phosphatases. Inhibiting these kinases/phosphatases could result Raf inhibitors have been developed and some are being used in activation or inactivation of other proteins and would have in clinical trials.271–282 Raf-1 has at least 13 regulatory other effects on cell physiology. phosphorylation residues. Therefore, inhibition of Raf activity Dimerization of Raf proteins is critical for their activity. We is difficult as certain phosphorylation events stimulate Raf often think of a single Raf protein carrying out its biochemical activity while others inhibit Raf activity and promote Raf activity. However, Raf proteins dimerize with themselves and association with 14-3-3 proteins, which render it inactive and other Raf isoforms to become active. Drugs such as coumermy- present in the cytoplasm.279–282 Certain Raf inhibitors were cin, which inhibit Raf dimerization, and others such as developed that inhibit the Raf kinase activity as determined by geldanamycin, which prevent interaction of Raf with 14-3-3 assays with purified Raf proteins and substrates (MEK). Some Raf proteins, suppress Raf activity.279–282 Various Raf isoforms may inhibitors may affect a single Raf isoform (eg, Raf-1), others may dimerize and result in chimeric molecules, which may have affect Raf proteins, which are more similar (Raf-1 and A-Raf),278 different biochemical activities. Little is known about the while other Raf inhibitors may affect all three Raf proteins (Raf- heterodimerization of Raf proteins. 1, A-Raf and B-Raf). We have observed that the L-779,450 Downstream of Raf lies MEK. Currently, it is believed that inhibitor suppresses the effects of A-Raf and Raf-1 more than the MEK1 is not frequently mutated in human cancer. However, effects of B-Raf.278 Knowledge of the particular Raf gene aberrant expression of MEK1 has been detected in many mutated or overexpressed in certain tumors may provide critical different types of cancer, and mutated forms of MEK1 will information regarding how to treat the patient. Inhibition of transform fibroblast, hematopoietic and other cell types. Useful certain Raf genes might prove beneficial while inhibition of inhibitors to MEK have been developed that display high

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Figure 10 Potential mechanisms of activation of B-Raf. B-Raf can be activated by interactions with Rap-1. Akt and SGK can also phosphorylate B-Raf ,which results in its inactivation. Rheb and 14-3-3 can negatively affect Raf activity. B-Raf may also activate Raf-1 through interaction with Ras. The S/T kinases which activate B-Raf remain controversial.

degrees of specificity. The successful development of MEK The MAP kinase phosphatase-1 (MKP-1) removes the phos- inhibitors may be due to the relatively few phosphorylation sites phates from ERK.314 MKP-1 is mutated in certain tumors and on MEK involved in activation/inactivation. MEK inhibitors are could be considered a tumor suppressor gene.314,315 An in clinical trials.119 inhibitor to this phosphatase has been developed (Ro-31- Downstream of MEK lies ERK. To our knowledge no small 8220).315 molecular weight ERK inhibitors have been developed yet; however, inhibitors to ERK could prove very useful as ERK can phosphorylate many targets (Rsk, c-Myc, Elk, etc) that have PI3K/Akt pathway growth-promoting effects. There are at least two ERK molecules regulated by the Raf/MEK/ERK cascade: ERK1 and ERK2. Little is Activation of the IL-3 receptor also stimulates the PI3K/Akt known about the different in vivo targets of ERK1 and ERK2. pathway. PI3K is a family of proteins that catalyze transfer of the However, ERK2 has been postulated to have pro-proliferative g-phosphate of ATP to the D3 position of phosphoinositides. effects while ERK1 has antiproliferative effects.311–313 Develop- This protein family can be divided into three classes. Class I ment of specific inhibitors to ERK1 and ERK2 might eventually PI3K is a multisubunit protein consisting of an approximately prove useful in the treatment of certain diseases. 110 kDa catalytic subunit and a regulatory subunit of 50–

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Figure 11 Interactions between the Raf/MEK/ERK and PI3K/Akt pathways and sites of action of small molecular weight signal transduction pathway inhibitors. Interactions between PI3K/Akt and Raf/MEK/ERK pathways are indicated. Established paths of signal transduction pathways are represented by solid lines. Potential sites of interaction between these two pathways are indicated by dotted lines. These pathways interact and crossregulate each other by both direct and indirect mechanisms. These pathways regulate gene expression by transcriptional, post-transcriptional and post-translational mechanisms. Black boxes indicate sites of inhibition of small molecular weight signal transduction inhibitors. Round black ovals represent proteins that serve to inhibit gene expression, induce apoptosis or inactivate proteins.

100 kDa. Class I PI3K is regulated by growth factor receptor activation. The preferred in vivo substrate of class I PI3K is phosphoinositide-(4,5) diphosphate.316,317 Class II PI3K is a single peptide of 170–210 kDa with a carboxy-terminal C2 domain, necessary for phospholipid binding and kinase activity.318,319 Class III PI3K are related to Saccharomyces cerevisiae vacuolar protein sorting mutant.320 Class I PI3K is the most studied class of PI3K and the most interesting with regard to signaling in hematopoietic cells. PI3K activity is associated with cytoskeletal organization, cell division, inhibition of apoptosis and glucose uptake.321–323 Deletion of the PI3K p85 regulatory subunit, or the p110 catalytic subunit, results in Figure 12 Sites of mutation in B-Raf genes. The sites of mutations embryonic lethality.324,325 The deletion of the carboxyl terminus detected in the B-Raf protein are presented. In all, 90% of B-Raf 326,327 mutations in melanoma involve V599.136,260 V599E is the most of p85 produces a constitutively active oncogenic PI3K. 252 Moreover, a constitutively active mutant was made by mutating common genetic change in thyroid papillary cancers. The residues 323 that are important in B-Raf regulation by AKT are indicated. Mutations lysine to glutamic acid (K227E) in the Ras-binding domain. A at S364, S428 and T439 interfere with Akt phosphorylation of B-Raf, depiction of the structures of the class I PI3K proteins and their which may negatively affect the induction of apoptosis.260 retroviral counterparts used to investigate the oncogenic effects of PI3K is presented in Figure 13. The prototype regulatory subunit p85 consists of an amino- terminal SH3 domain, a break point-cluster-region homology interactions between the p85 and p110 subunits occur by the domain (BCR), a proline-rich region, an interSH2 (iSH2) region iSH2 region.340–344 Phosphorylation of S608 present in p85 and an amino-terminal SH2 domain.328–330 The BCR domain is results in a three- to seven-fold decrease in PI3K activity. The highly homologous to the GTPase-activating domain of the catalytic subunit has been implicated as the kinase phosphor- break-point-cluster gene product331 and specifically interacts ylating S608.342–347 PI3K is inhibited by the interaction of the with Cdc42 and Rac1, although no GTPase activity has been proline-rich domain of the adaptor protein Ruk (SH3-domain found.332,333 The proline-rich regions associate with the SH3 kinase-binding protein 1) with an SH3 domain of p85. The regions of v-Src, Lyn, Fyn, GRB2, Abl and Lck.334–339 The inhibition of PI3K by Ruk overexpression promotes apoptosis,

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Figure 13 Structure of PI3K. Sh2 ¼ Src homology 2 region responsible for binding tyrosine-phosphorylated residues on proteins, Sh3 ¼ Src homology region 3 region, which is a proline-rich region. BCR – region homologous to the break point cluster region. The structures of the PI3K p85 and p110 subunits are indicated as well as the sizes of the proteins. An illustration of the PI3K-containing constructs made by Dr Jullian Downward and colleagues at ICRF (London, UK), which have been used to investigate the PI3K pathways, is presented in the bottom of the figure.327

which is inhibited by expression of a constitutively active of the PDK family – PDK1 and PDK2 – although the identity of p110.348 PDK2 remains elusive. PDK1 consists of two domains: a The p110 catalytic subunit of PI3K has both lipid and S/T carboxy-terminal PH domain and an amino-terminal kinase protein kinase activity. The p110 PI3K is a kinase domain that domain. The PH domain has a greater affinity for binding has homology to many protein kinases.349,350 L802 present in phosphoinositides than the PH domain of Akt.368 PDK1 is the kinase domain is involved in the coordination of the b- ubiquitously expressed in human tissues and is exclusively phosphate of the ATP, and is covalently modified to a Schiff base located in the cytosol.367,369–371 by the PI3K inhibitor wortmannin.351,352 LY294002, a querce- In vitro, PDK1 phosphorylates T308 of Akt, but not S473, tin-derived PI3K inhibitor, inhibits ATP binding.353–355 The p85 which is required for full activation of Akt.372 In vivo, activation subunit inhibits and stabilizes the catalytic subunit.356 of PDK1 and phosphorylation of T308 of Akt is independent of Activation of class I PI3K requires translocation to the plasma PH-dependent lipid association. Association of Akt with membrane and association with an activated receptor tyrosine phosphoinositides produces a conformational change allowing kinase or its substrates.357,358 The localization of PI3K to the S473 to be phosphorylated by PDK1.367,373 Alternatively, membrane is activated by the IL-3 receptor, Ras or the proline- interaction of PDK1 with the carboxy-terminal PDK1-interacting rich regions of Shc, Lyn, Fyn, Grb2, v-Src, Abl, Lck, Cbl, or fragment of PRK2 (protein kinase C-like 2) allows PDK1 to dynamin.336–348,359–362 These interactions activate PI3K produ- phosphorylate T308 and S473 activating Akt.374 The precise cing phosphoinositide-3-phosphate and/or phosphoinositide- mechanism of Akt S473 phosphorylation remains controversial. 3,4,5-triphosphate. The phosphoinositide-3-phosphate product It may be phosphorylated by the integrin-linked kinase (ILK).375– is degraded by phosphatases, including the PTEN tumor 380 Translocation of PDK1 is inhibited by increased cAMP levels suppressor. The kinase activity of PI3K is inhibited by cAMP.363 in the cell.381 The phospholipid products of PI3K activate downstream targets, including PDK, Akt and PKC.364–366 PI3K inhibitors that show specificity have been developed. These and their derivative Akt inhibitors show promise in suppressing this pathway in human cancer. The downstream target of PDK1 and likely PDK2 is Akt or protein kinase B (PKB). Activated Akt was originally isolated from cells of the leukemia and lymphoma prone AKR strain of PDK mice.382,383 c-Akt was cloned by several different groups using degenerate PCR with primers for the kinase domains,384–387 low Phosphoinositide-dependent kinase (PDK) requires the phos- stringency screening for protein kinase A386 and sequencing of pholipid product of PI3K for activation. Originally isolated from human DNA hybridizing to v-Akt.387 Multiple clonings of the skeletal muscle using a PKB glutathione-S-affinity column, PDK gene resulted in several different names for the Akt family of is a lipid-requiring kinase.367 It is a 63 kDa S/T kinase and part of genes, including Akt, PKB, RAC-PK (related to A and C protein the PDK family of genes. There are believed to be two members kinase). The Akt family of genes in mammals consists of three

Leukemia Roles of JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in leukemia LS Steelman et al 203 genes: Akt1 (PKBa), Akt2 (PKBb) and Akt3 (PKBg). A diagram of A model of Akt activation requires translocation of activated these three proteins and certain retroviral constructs encoding PI3K to the membrane385 as a result of PIP3 binding to the Akt Akt counterparts is presented in Figure 14. Akt1 has been PH domain. Next, PI3K phosphorylates phosphoinositides mapped to chromosome 14q32, a region frequently involved in resulting in the translocation and activation of PDKs. PDK1 translocations in leukemia and lymphoma.382,385,388 Akt2 has phosphorylates and activates Akt first on Thr (Akt1 T308, Akt2 been mapped to 19q13.1–q13.2, a chromosome region known T309 and Akt3 T305). PDK2 may then phosphorylate Ser (Akt1 to be amplified in ovarian and pancreatic cancers.389,390 Akt2 is S473, Akt2 S474 and Akt3 S472).385 However, the precise amplified in 12.1% of ovarian and 2.8% breast cancers.391 Akt1 identity of PDK2 and mechanism(s) regulating serine phosphor- and Akt2 are expressed in all tissues, but the highest expression ylation of Akt are not fully defined. is in brain, thymus, heart, and lung.385–387 Akt3 expression is The most studied biological activities of Akt are the regulation high in brain and testes.391 of glucose transport/metabolism and apoptosis. PI3K is required Akt proteins contain three domains: PH, S/T kinase and for glucose transport. Moreover, inhibition of PI3K decreases regulatory. The PH interacts with lipids, and targets the protein glucose transport.396–398 Elevated Akt activity increases glucose to the membrane after activation.392,393 The oncogenic ability of transport.399,400 Akt phosphorylates glycogen synthase kinase-3 Akt was first observed in the original isolation of the v-Akt- (GSK-3) and 6-phosphofructose-2-kinase (PFK2) in vitro,401,402 containing retrovirus which contained a GAG-Akt fusion thus regulating glucose flux to glycogen and glycolysis. protein. This retroviral-GAG protein contains a myristylation Activated Akt increases protein synthesis in the presence of site, which alters the normal localization from the cytosol (90%) PI3K inhibitors,403–407 presumably through the increased to the plasma membrane (40%), nucleus (30%) and cytosol phosphorylation of the inhibitory binding protein PHAS-1. (30%).394 Similar targeting has been achieved with c-Akt The antiapoptotic effects of Akt occur through its phosphor- constructs used by adding a v-src-encoded myristylation domain ylation of a wide variety of targets. The first antiapoptotic target in place of the normal PH domain. In addition, various activating identified was Bad, a member of the Bcl-2 family. Phosphoryla- point mutations were introduced into the modified Akt gene by tion of Bad at S136 by Akt allows phosphorylated Bad to interact site-directed mutagenesis. This resulted in a constitutively with 14-3-3 proteins, promoting cell survival.408,409 Interaction activated Akt construct (DAkt(Myr þ )). Furthermore, an addi- of Bad with 14-3-3 proteins inhibits the ability of Bad to interact tional construct was made conditional by the addition of a 4- with Bcl-2 and Bcl-XL. This allows Bcl-XL to bind to pro- hydroxy-tamoxifen-responsive ERn domain (DAkt:ERn (Myr þ )) apoptotic Bax molecules and prevent the formation of pro- (Figure 14). As a control, a construct lacking the v-Src apoptotic Bax homodimers (Figure 15). However, as the model myristylation domain (DAkt:ERn(MyrÀ)) was also made.395 presented in Figure 15 indicates, Bad is also phosphorylated on

Figure 14 Structure of Akt genes. Depicted is the structure of the Akt proteins and some modified proteins used to investigate signal transduction and apoptotic pathways in hematopoietic cells. Akt consists of three domains: the pleckstrin homology domain (PH), the kinase domain and the regulatory domain. The three Akt isoforms depicted in the top part of this diagram were adapted in part from the publication of Nicholson and Anderson.385 Various naturally occurring and recombinant retroviral Akt constructs are presented in the bottom portion of the figure. The AKT8 retrovirus contains part of Akt fused with the myristylation site of Mo-MuLV retrovirus GAG protein, which targets the protein to the membrane.382,383 DAkt(Myr þ ) is a modified Akt gene with the PH domain deleted and a v-Src-encoded myristylation site in its place making it constitutively active.399 DAkt:ERn (MyrÀ) and DAkt:ERn (Myr þ ) have the PH domain deleted and either lack or contain a v-Src-encoded myristylation site and a mutated hormone-binding domain (n) of the estrogen receptor, making the kinase activity regulated by 4HT.395 The mutated ERn domain binds 4HT 100-fold better than b-estradiol.395

Leukemia Roles of JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in leukemia LS Steelman et al 204

Figure 15 Interaction of Akt with bad. Bad in the unpho- sphorylated state associates with Bcl-2 or Bcl-XL, promoting apoptosis. Akt phosphorylates Bad on S136. ERK phosphorylate Bad on S112 and PKA phosphorylates Bad on S155. These phosphorylation events cause Figure 16 Structures of the phosphatases PTEN and SHIP. In the Bad to associate with 14-3-3 proteins, thus inhibiting apoptosis. top portion of the figure, the domain structure of the PTEN protein is presented. The PEST domains are involved in the regulation of protein stabilization. In the bottom of the figure, the structure of the SHIP-1 phosphatase is depicted. In the top panel, the domains present in the PTEN phosphatase are presented. In the bottom panel, the domains different sites by members of the Raf/MEK/ERK (S112) and PKA present in the SHIP1 protein are presented. The bottom panel was (S112, S155) pathways. Thus, signal transduction pathways are adapted from Rohrschneider et al.428 The two H above the inositol complicated and intricately control the activity of apoptotic 50phosphatase domain represent homology regions with other regulatory molecules such as Bad. As stated previously, Raf-1 phosphatases. When the two NPXX (asparagine, proline, X ¼ any has many effects on the regulation of apoptosis. Some of these amino acid) motifs are tyrosine phosphorylated, they have the effects occur at the mitochondrial membrane and are indepen- potential to bind protein tyrosine-binding domain (PTB) or SH2 domains contained on proteins. The PxxP polyproline motifs can bind dent of MEK and ERK activity. SH3 domains present in proteins. A rough scale of the amino acid Bad and Bcl-XL have been shown to be expressed in normal present in the PTEN and SHIP1 protein is presented underneath the and leukemic hematopoietic precursor cells.409 Immature proteins. hematopoietic cells do not express Bcl-2 but do express þ Bcl-XL. CD34 cells express Bcl-2, Bcl-XL and Bad. Bcl-2 expression is higher on CD34 þ cells than on AML cells. Phosphorylated Bad was expressed in AML.409 some 10) also known as MMAC1, mutated in multiple advanced In human cells, Akt phosphorylates and inactivates caspase- cancers) is considered a tumor suppressor gene.423–428 PTEN is a 9.410 Overexpression of Akt inhibits cytochrome c-induced dual-specificity lipid and protein phosphatase that removes the 410 activation of caspase-9. Phosphorylation of the forkhead 3-phosphate from the PI3K lipid product PtdIns(3,4,5)P3 to (FOXO) family of transcription factors is also attributed to produce PtdIns(4,5)P2, which prevents Akt activation. PTEN is Akt.411–414 This phosphorylation results in forkhead transcrip- often mutated in human cancer. Recently, two other phospha- tion factors translocation to the cytoplasm, thus inhibiting tases, SHIP-1 and SHIP-2, have been shown to remove the 5- 412 429,430 transcription of pro-apoptotic genes such as FasL. Akt phosphate from PtdIns(3,4,5)P3 to produce PtdIns(3,4)P2. activates transcription of antiapoptotic genes through phosphor- DN SHIP1 mutations have been detected in human leuke- ylation of IKK and regulation of NF-kB.415 Akt also promotes cell mia.431 This pathway provides proliferative and antiapoptotic survival and cell cycle progression by its ability to phosphorylate signals, and its dysregulation has often been linked with MDM2 and GSK-3.416,417 Once phosphorylated by Akt, MDM2 malignant transformation.432–441 A diagram of the PTEN and translocates to the nucleus and interacts with p300.417 p300 SHIP phosphatases is presented in Figure 16. dissociates from p19ARF, resulting in the degradation of p53 and cell cycle progression.417 Akt phosphorylates GSK-3, inhibiting its activity.418,419 The decreased GSK-3 activity increases stability of b-catenin and enhances its association with Roles of the PI3K/Akt/PTEN pathway in neoplasia lymphoid enhancer factor–T cell factor (LEF/TCF).416 The b- catenin–LEF/TCF complex increases transcription of proteins Ras can activate PI3K, and some Ras mutations result in such as cyclin D1 and c-myc, promoting cell cycle progres- deregulated PI3K and downstream Akt activation.428 PTEN sion.416 Akt also phosphorylates the tumor suppressor BRCA1, negatively regulates Akt activity; hence mutations that result in but the implications are not yet identified.420–422 It appears that PTEN loss may lead to persistently increased levels of Akt Akt has many targets, whether all these targets are directly activity.424–427 Mutations and hemizygous deletions of PTEN phosphorylated by Akt remains unclear. Clearly, Akt can affect have been detected in some primary acute leukemias and non- both cell cycle progression and apoptosis. Akt inhibitors have Hodgkin’s lymphomas, and many hematopoietic cell lines lack been developed; however, those that are currently commer- or have low PTEN protein expression.424–427 Increased Akt cially available do not show the effectiveness and specificity expression has also been linked with tumor progression; the Akt- that PI3K inhibitors do.119–121,278 related Akt-2 gene is amplified in some cervical, ovarian and pancreatic cancers as well as non-Hodgkin’s lymphomas.441–443 In cancers where there are mutations in the PI3K/Akt/PTEN Regulation of the PI3K pathway by phosphatases pathway, there are usually mutations at PTEN or Akt and not both. A diagram of the effects of the PTEN and SHIP The PI3K pathway is negatively regulated by phosphatases. phosphatases on the regulation of PI3K/Akt pathway is presented PTEN ((phosphatase and tensin homolog deleted on chromo- in Figure 17.

Leukemia Roles of JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in leukemia LS Steelman et al 205

Figure 17 Regulation of apoptosis and leukemia by BCR-ABL, v-Ha-Ras, PI3K, PTEN and SHIP. The effects of activating mutations on gene expression on the regulation of apoptosis and the induction of leukemia are illustrated. Different mechanisms of activation of this pathway by genetic mutations are indicated. Some of the oncogenes may induce or repress these pathways. For example, BCR-ABL suppresses SHIP1 expression, which results in elevated levels of phosphatidylinositol (3,4,5)-trisphosphate, while BCR-ABL also induces Gap2, which interacts with SHIP1.

Crosstalk between the Raf/MEK/ERK and PI3K/Akt signal Effects of activated JAK/STAT, Ras/Raf/MEK/ERK and PI3K/ transduction pathways Akt oncogenes on the cytokine dependence of hematopoietic cells Interactions between the Raf and PI3K/Akt pathways, or crosstalk, is an area of intense research. The first implication The effects of activated forms of the JAK/STAT, Ras/Raf/MEK/ERK of crosstalk between these pathways was observed after and PI3K/Akt pathways on the cytokine dependence of treatment of cells with pharmaceutical inhibitors of PI3K, which hematopoietic cells have been investigated extensively in our decreased ERK activity.444–447 Secondly, PI3K activated through as well as other laboratories. Abrogation of cytokine depen- the fibronectin receptor results in increased Raf activity.448 dence is a key step in leukemogenesis. Results that we have Thirdly, a DN mutant of the p85 subunit of PI3K decreased obtained regarding the abilities of these oncoproteins to MEK1 and ERK activities.449 A decrease in proliferation of cells abrogate the cytokine dependence of human (TF-1) and murine transfected with DN p85 was also observed.449 PI3K/Akt can (FDC-P1 and FL5.12) hematopoietic cells are summarized in affect the Raf pathway, through the interaction of Akt with Table 2. In order to investigate the comparative ability of these the Raf family of proteins. Zimmerman and Moelling demon- oncoproteins on cytokine dependence, it is important to strated that Raf-1 activity is decreased by Akt-mediated investigate the effects of these oncogenes in a single laboratory phosphorylation of S259.143,144 An increase in Akt activity where culture conditions are relatively constant. While activated decreased Raf-1 activity, which was differentiation specific in TEL-JAK, STAT5A and BCR-ABL can efficiently abrogate the skeletal muscle culture models.143 Another report examined the cytokine dependence of these cells, activated forms of Ras, Raf, ability of Akt activity to regulate B-Raf activity by phosphoryla- MEK, PI3K and Akt show significant differences in the ability to tion on S364 and S428, residues not present in Raf-1 or A- abrogate cytokine dependence. Activated Ras and Raf will Raf.450 The addition of active Akt decreased B-Raf activity in abrogate the cytokine dependence of FDC-P1 and TF-1 cells but HEK293 cells. Inhibition of Akt by LY294002 increased B-Raf not FL.12 cells. In contrast, activated PI3K or Akt did not activity. Many interactions between the Raf pathway and PI3K/ abrogate the cytokine dependence of any of the cell lines Akt pathway have been demonstrated.143,144,445–449 Recently, it although they did prolong the survival of the cells. However, on was demonstrated that it is more effective to inhibit the growth addition of an activated Raf or MEK1 oncoprotein, cytokine- of Raf- and MEK1-transformed hematopoietic cells with independent cells were isolated from PI3K- or Akt-infected cells inhibitors that target both the Raf/MEK/ERK and PI3K/Akt indicating that activation of both the Raf/MEK/ERK and PI3K/Akt pathways.423 pathways could synergize and lead to abrogation of cytokine

Leukemia Roles of JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in leukemia LS Steelman et al 206 dependence in cells that were normally refractory to abrogation autophosphorylation site (Y177) present on BCR-ABL binds the of cytokine dependence. Grb2 adaptor protein and is partly responsible for the lineage and severity of experimentally induced disease.464 Mutation of Y177 to Y117F dramatically impaired myeloid leukemogenesis Involvement and crosstalk of BCR-ABL between the Ras/Raf/ but had less of an effect on lymphoid leukemogenesis. These MEK/ERK and PI3K/Akt and JAK/STAT pathways investigators also determined that Y177 recruits the scaffolding adaptor protein Gab2 via a Grb2/Gab2 complex. In mutant For almost 20 years now, it has been known that the BCR-ABL Y177F cells, Gab2 phosphorylation was reduced. Gab2 is chromosomal translocation plays a key role in the pathogenesis important for activation of both the PI3K/Akt and Raf/MEK/ERK of CML.451–464 The BCR-ABL oncoprotein induces a prolifera- pathways. Furthermore, these authors demonstrated that mye- tive effect, while full transformation requires additional genetic loid progenitors from Gab2 knockout mice are resistant to events (translocation of other oncogenes, for example, AML1/ transformation by BCR-ABL as well as activation of the PI3K/Akt EVI1, AML/ETO, NuP98/HOXA) that block differentiation.451 and Ras/Raf/MEK/ERK pathways. Thus these studies indicate the Over the past few years, it has been discovered that BCR-ABL role that BCR-ABL oncoprotein has on the PI3K/Akt and Ras/Raf/ also has effects on pre-existing cellular signaling circuits. BCR- MEK/ERK pathways and the critical importance of Gab2 in ABL activates the Ras/Raf/MEK/ERK, JAK/STAT and PI3K/Akt transformation mediated by BCR-ABL. Furthermore, as dis- signal transduction pathways, which results in a proliferative cussed previously, STAT5 is constitutively phosphorylated and 451–464 75 effect. BCR-ABL-transformed cells display hyperactivity Bcl-XL is overexpressed in BCR-ABL cells. An overview of the of Ras, Raf and JAK/STAT. This could occur by multiple effects of BCR-ABL on the JAK/STAT, PI3K/Akt and Ras/Raf/MEK/ mechanisms, by BCR-ABL activating these pathways directly, ERK pathways is presented in Figure 18. or by the induction of autocrine cytokines, which in turn There has been great success in targeting the BCR-ABL activate these pathways. BCR-ABL lies at a pinnacle position in tyrosine kinase with a signal transduction inhibitor developed by signal transduction pathways. It can interact with Grb-2, which Novartis, imatinib (also known as gleevec, STI571). However, couples its response to Gab2 and Shc. Moreover, BCR-ABL has upon treatment of some patients with this drug, resistance negative effects on the SHIP1 phosphatase, which results in an occurs.465–468 This has also been observed in in vitro studies antiapoptotic effect.463 Recently, it was shown that the with cells transformed to grow in response to BCR-ABL. There

Figure 18 BCR-ABL activation of cytokine signal transduction and sites of inhibition. BCR-ABL is a potent oncogene that can affect many downstream signaling pathways including JAK/STAT, PI3K/Akt and Raf/MEK/ERK. Furthermore, BCR-ABL can affect adaptor proteins such as Gab2 and phosphatases such as SHIP1, which further fine-tune these pathways. Many sites of inhibition of cell growth have been discussed in this review and some of them are indicated in the figure. As chemotherapeutic drugs such as imatinib are used more frequently to treat CML, more imatinib resistance will be observed. Potential methods to circumvent this drug resistance include treatment of the cells with inhibitors that target other molecules in the pathway dysregulated by BCR-ABL (eg, Ras, Raf, MEK, PI3K, BCL-2) or affect BCR-ABL function by different mechanism (eg, geldanamycin).

Leukemia Roles of JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in leukemia LS Steelman et al 207 are different mechanisms of resistance to imatinib, for example, References point mutations in the BCR-ABL kinase domain or gene amplification. Therefore, scientists have searched for additional 1 Blalock WL, Weinstein-Oppenheimer C, Chang F, Hoyle PE, methods to suppress the growth of these BCR-ABL-transformed, Wang XY, Algate PA et al. Signal transduction, cell cycle imatinib-resistant cells. One method that is showing success in regulatory, and anti-apoptotic pathways regulated by IL-3 in suppressing the growth of the imatinib-resistant cells is to treat hematopoietic cells: possible sites for intervention with anti- 469 neoplastic drugs. Leukemia 1999; 13: 1109–1166. them with inhibitors that target either Ras or PI3K. It may also 2 Wang XY, McCubrey JA. Regulation of interleukin-3 expression in be effective to treat the cells with inhibitors that target the JAK normal and autocrine transformed hematopoietic cells. Int J kinases or a cocktail of inhibitors. An additional method to treat Oncol 1997; 10: 989–1001. imatinib-resistant cells is geldanamycin treatment.470–474 Gel- 3 McCubrey JA, May WS, Duronio V, Mufson A. Serine/threonine danamycin can target molecules that bind Hsp-90. Geldana- phosphorylation in cytokine signal transduction. Leukemia 2000; mycin will destabilize the BCR-ABL protein and inhibit the 14: 9–21. 4 Lee Jr JT, McCubrey JA. The Raf/MEK/ERK signal transduction growth of imatinib-resistant CML cells. cascade as a target for chemotherapeutic intervention in leukemia. Leukemia 2002; 16: 486–507. 5 Steelman LS, Algate PA, Blalock WL, Wang XY, Prevost KD, Hoyle PE et al. Oncogenic effects of overexpression of the Summary interleukin-3 receptor on hematopoietic cells. Leukemia 1996; 10: 528–542. 6 Wang XY, Steelman LS, McCubrey JA. Abnormal activation of In this review, the effects of three different signal transduction cytokine gene expression by intracisternal type A particle pathways and the BCR-ABL chimeric oncoprotein on normal transposition: effects of mutations, which result in autocrine and malignant hematopoietic cell growth have been summar- growth stimulation and malignant transformation. Cytokines Cell ized. The three pathways are normally activated after cytokine Mol Ther 1997; 3: 3–19. receptor ligation and they can induce growth, cell cycle 7 McKearn JP, McCubrey JA, Fagg B. Enrichment of hematopoietic progression and prevent apoptosis. Their effects on normal cell precursor cells and cloning of multipotential B lymphocyte precursors. Proc Natl Acad Sci USA 1985; 82: –7418. growth are often kept in check by naturally occurring inhibitors 8 McCubrey JA, Holland G, McKearn J, Risser R. Abrogation of or tumor suppressor proteins. For example, the JAK/STAT factor-dependence in two IL-3-dependent cell lines can occur by pathway has the SOCS/CIS family of proteins, which serve to two distinct mechanisms. Oncogene Res 1989; 4: 97–109. limit its effects by a negative feedback pathway. Deletion of the 9 Algate PA, McCubrey JA. Autocrine transformation of hemopoie- SOCS genes can lead to lethality by altering the responses to tic cells resulting from cytokine message stabilization after interferon-g, erythropoietin and other cytokines. Moreover, intracisternal A particle transposition. Oncogene 1993; 8: deletion of the SOCS gene can result in large mice due to 1221–1232. 10 Mayo MW, Wang X-Y, Algate PA, Arana GF, Hoyle PE, Steelman defects in limiting the effects of certain cytokines that serve to LS et al. Synergy between AUUUA motif disruption and enhancer limit cell growth. The abnormal TEL-JAK chimeric proteins can insertion results in autocrine transformation of interleukin-3- contribute to the neoplastic transformation of hematopoietic dependent hematopoietic cells. Blood 1995; 86: 3139–3150. cells. This occurs in part due to the constitutive, cytokine- 11 Wang X-Y, McCubrey JA. Malignant transformation induced by independent activation of the JAK kinase activity. cytokine genes: a comparison of the abilities of germline and Experimentally induced deletion of Raf and MEK genes can mutated interleukin 3 genes to transform hematopoietic cells by transcriptional and posttranscriptional mechanisms. Cell Growth result in embryonic lethality indicating the critical roles that Differ 1996; 7: 487–500. these genes play in life. Mutations in Ras have been known for 12 Wang XY, McCubrey JA. Differential effects of retroviral long many years and shown to be important in myelodysplastic terminal repeats on interleukin-3 gene expression and autocrine syndromes. Recently, mutations at Raf genes, particularly B-Raf, transformation. Leukemia 1997; 11: 1711–1725. 13 Wang X-Y, McCubrey JA. Characterization of proteins binding have been shown to play roles in human cancer, especially 0 melanomas. The Raf/MEK/ERK pathway can be negatively specifically to the interleukin 3 (IL-3) mRNA 3 untranslated region in IL-3-dependent and autocrine transformed cells. regulated by the PI3K/Akt cascade as well as the MKP1 Leukemia 12; 1998: 520–531. phosphatase, which inactivates phosphorylated ERK. 14 McCubrey JA, Lee JT, Steelman LS, Blalock WL, Moye PW, Chang The PI3K/Akt pathway has the PTEN and SHIP phosphatases, F et al. Interactions between the PI3K and Raf signaling pathways which serve to fine-tune its antiapoptotic effects. Mutations at can result in the transformation of hematopoietic cells. Cancer the PI3K/Akt/PTEN pathway can contribute to leukemogenesis Detect Prevent 2001; 25: 375–393. by elevating the expression of the pleiotropic antiapoptotic Akt 15 Moye PW, Blalock WL, Hoyle PE, Chang F, Franklin RA, Weinstein-Oppenheimer C et al. Synergy between Raf and protein, which can both stimulate antiapoptotic and inhibit pro- BCL2 in abrogating the cytokine-dependency of hematopoietic apoptotic proteins. There are many different mechanisms by cells. Leukemia 2000; 14: 1060–1079. which mutations at the PI3K/PTEN/Akt pathway can contribute 16 Franklin RA, McCubrey JA. Kinases: positive and negative to neoplastic transformation, including point mutation (PTEN, regulators of apoptosis. Leukemia 2000; 14: 2019–2034. SHIP), amplification (Akt) or deletion (PTEN). 17 Algate PA, Steelman LS, Mayo MW, Miyajima A, McCubrey JA. As our knowledge of the particular genes mutated in cancers Regulation of the interleukin (IL-3) receptor by IL-3 in the fetal liver-derived FL5.12 cell line. Blood 1994; 83: 2459–2468. improves, our ability to treat patients afflicted with certain 18 Lee Jr JT, McCubrey JA. Targeting the Raf kinase cascade in diseases will increase substantially. Hopefully we will be able cancer therapy – novel molecular targets and therapeutic to target the particular gene(s) in a pathway(s) affected. The strategies. Expert Opin Targeted Ther 2002; 6: 659–678. genetic mutation may affect multiple signal transduction 19 McCubrey JA, Wang XY, Algate PA, Blalock WL, Steelman LS. pathways. This may require development of specific treatments Autocrine transformation: cytokine model. In: Blagosklonny M that target more than one pathway. Moreover, targeting multiple (ed). Cell Cycle Checkpoints and Cancer. Austin, TX: Landes Bioscience Press, 2001, pp 1–16. pathways may be more efficacious as this approach may 20 McCubrey JA, Blalock WL, Chang F, Steelman LS, Pohnert SC, suppress or eliminate tumor growth at lower concentrations of Navolanic PM et al. Signal transduction pathways: cytokine the drugs than that required to inhibit growth by targeting a model. In: Blagosklonny M (ed). Cell Cycle Checkpoints and single pathway. Cancer. Austin, TX: Landes Bioscience Press, 2001, pp 17–51.

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