This material may be protected by Copyright law (Title 17 U.S. Code)
`
`1
`
`

`

`Clinical Cancer Research 535
`Vol. 9. 535-550, February 2003
`
`
`
`This material may be protected by Copyright law (Title 17 U5. Code)
`
`Minireview
`
`Modulation of Cellular Signaling Pathways: Prospects for Targeted
`Therapy in Hematological Malignancies
`
`Farhad Ravandi,1 Moshe Talpaz, and Zeev Estrov
`Department of Hematology/Oncology, The University of Illinois at
`Chicago, Chicago, Illinois 60612 [F. R.], and Department of
`Bioimmunotherapy, The University of Texas M. D. Anderson Cancer
`Center, Houston, Texas 77030 [M. T., Z. E.]
`
`Abstract
`
`The high remission rates observed in patients with
`chronic myelogenous leukemia who receive Imatinib mesy-
`late (Gleevec) indicate that targeted therapy for hematolog-
`ical malignancies is achievable. At the same time, progress in
`cellular biology over the past decade has resulted in a better
`understanding of the process of malignant transformation, a
`better classification of subtypes of each disease on the basis
`of molecular markers, and a better characterization of the
`molecular targets for drug development. These advances
`have already spawned the development of such effective
`agents as monoclonal antibodies and specific enzyme inhib-
`itors. This review attempts to provide a practical introduc-
`tion to the complex and growing field of targeted therapy in
`hematological malignancies.
`
`Introduction
`
`Cellular proliferation, differentiation, and death are regu-
`lated by a number of extracellular molecules such as cytokines
`and hormones, as well as intercellular interactions mediated by
`neighboring cell surface antigens. These effectors mediate gene
`transcription either directly or indirectly by activating intracel—
`lular signaling pathways, which in turn activate appropriate
`cellular machinery (1). Cell-surface receptors that convert ex—
`ternal stimuli into intracellular signals are pivotal in this signal—
`ing process. They activate intracellular pathways either through
`their inherent enzymatic function or as a result of their associ-
`ation with other catalytic proteins. Indeed, most growth factors
`and cytokines bind these receptors and exert
`their function
`through their activation, commonly by phosphorylation (1).
`Normal hematopoiesis is dependent on intricately regulated
`signaling cascades that are mediated by cytokines and their
`receptors. Orderly function of these pathways leads to the gen—
`eration of appropriate constellation of hematopoietic cells, and
`their abnormal activation results in neoplastic transformation,
`impaired apoptosis, and uncontrolled proliferation. Cytokines
`
`Received 6/25/02; revised 9/5/02; accepted 9/6/02.
`The costs of publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby marked
`advertisement in accordance with 18 U.S.C. Section 1734 solely to
`indicate this fact.
`1 To whom requests for reprints should be addressed, at University of
`Illinois—Chicago, 840 South Wood Street, MC 787 Chicago, IL 60612—
`7323. Phone: (312) 996-5982; Fax: (312) 413-4131; E-mail: ravandi@
`, uic.edu.
`
`function in a redundant and pleiotropic manner; different cyto—
`kines can exert similar effects on the same cell type, and any
`particular cytokine can have several differing biological func—
`tions (2). This complexity of function is a result of shared
`receptor subunits as well as overlapping downstream pathways
`culminating in activation of common transcription factors (3).
`The signal
`transduction cascades involve three major
`classes of proteins: kinases, adaptor or docking proteins, and
`transcription factors. Early insights into the cellular signaling
`pathways came from studies of IFN function (4—6). These
`experiments led to the identification of a family of nonreceptor
`TKs2 called Jaks and their target proteins, Stats, which mediate
`gene transcription (4, 7). The Jak—Stat pathways are commonly
`activated during cytokine signaling through phosphorylation of
`specific tyrosine residues (3). The interaction of a cytokine with
`its receptor induces its tyrosine phosphorylation and leads to
`activation of downstream protein TKs including Jaks and Stats.
`Apart from their catalytic domain, protein TKs contain several
`other characteristic motifs including the SH2, SH3, and pleck-
`strin homology domain, which enable them to interact with
`other signaling molecules and propagate the message (8, 9).
`The phosphorylation of serine and threonine residues is
`integral to the activation of numerous other intracellular proteins
`that mediate a number of other signaling pathways (10). Cyto-
`kine receptors without intrinsic kinase activity transmit their
`signals primarily through activation of Jak kinases. These re-
`ceptors as well as those with intrinsic kinase activity, the RTKs,
`were previously thought to transmit their signals independently
`of the serine/threonine kinase cascades. More recently, it has
`been established that both of these pathways interact with ser-
`ine/threonine kinase cascades such as the Ras/Raf/MEK/ERK
`(MAPK; Ref. 10). For example, after ligand binding, the [3—
`subunit of IL-3 and GM-CSF receptors are phosphorylated and,
`through recruitment of adaptor proteins such as She, Grb2, and
`
`2 The abbreviations used are: TK, tyrosine kinase; Jak, janus kinase;
`Stat, signal transducer and activator of transcription; ERK, extracellular
`signal—regulated kinase; MAPK, mitugcndiclivated protein kinase; IL,
`interleukin: GM—CSF, grunultacytc macrophage colony—stimulating fac—
`tor; JNK, c—Jun NHZ—terminal kinase; SAPK, stress—activated protein
`kinase; PKB, protein kinase B; PI3K, phosphatidylinositol 3’—kinase;
`PDGFR, platelet—derived growth factor receptor; TNF, tumor necrosis
`factor; SHP—l, SH—2 domain containing protein tyrosine phosphatase-l;
`SOCS, suppressor of cytokine signaling; PIAS, protein inhibitor of
`activated stat; RTK, receptor tyrosine kinase; FLT3R, FMS-like tyrosine
`kinase 3 receptor; Grb2, growth factor receptor binding protein 2; RSK,
`ribosomal S6 kinase; AML, acute myeloid leukemia; CML, chronic
`myulugenous leukemia; ALL, acute lymphoblastic leukemia; MEK,
`milngcwactivatcd protein/extracellular signal-regulated kinase kinase;
`ALK, anaplastic lymphoma kinase: NPM, nuclcophusmin; A'I‘RA, all-
`lrmis-reiinoic acid; AP}... acute promyclocytic leukemia; NFKB. nuclear
`factor KB; IKB, inhibitor of nuclear factor—KB; HDAC, histone deacety-
`lase; IAP, inhibitor of apoptotic pathway; CDK, cyclin—dependent ki-
`nase; CDKI, cyclin-dependent kinase inhibitor; Rb, retinoblastoma;
`RAR, retinoic acid receptor; XIAP, X—linked inhibitor of apoptosis.
`
`2
`
`

`

`536 Cellular Signaling Pathways as Therapeutic Targets
`
`Sos, activate the Ras signaling pathway (11). This in turn
`activates Raf followed by downstream activation of ERK1 and
`ERK2, and increased expression of transcription factors c—fos
`and c—jun (12—14). Other members of the MAPK family such as
`p38, and JNK/SAPK are also activated after phosphorylation of
`their serine/threonine residues as a result of cytokine/receptor
`interaction (15—18). Similarly, PI3K associates with the B chain
`of IL-3 receptor, recruits PKB/AKT by phosphorylation of its
`serine residues, and transmits cellular survival signals (19—21).
`Another downstream protein to IL-3 activation is the p7OS6
`kinase, which also interacts with the B chain and mediates
`appropriates signals (22, 23). Ultimately, these pathways influ—
`ence gene transcription through their ability to recruit transcrip—
`tion factors, regulate apoptosis through the phosphorylation of
`apoptotic proteins, and cause the cell to progress through cell-
`cycle checkpoints by activation of specific kinases.
`Of considerable interest is the description of a number of
`oncogenes with constitutive kinase activity. These molecules are
`derived from genes including c-ABL, c-FMS, FLT3, c-KIT, and
`PDGFRB, which are normally involved in the regulation of
`hematopoiesis (24). The kinase activity of the oncogene is
`constitutively activated by mutations that remove inhibitory
`domains of the molecule or induce the kinase domain to adopt
`an activated configuration (24). As a result of such constitutive
`activation a number of signaling cascades such as the Jak-Stat
`pathway, the Ras/Raf/MAPK pathway, and the PI3K pathway
`are activated.
`
`With better characterization of aberrant signaling through
`the cell surface receptors and their downstream pathways in
`neoplastic cells, current research is exploring ways to reverse
`such dysregulated stimuli (25). Here, we will briefly review the
`role of cellular signaling pathways in normal cellular processes,
`neoplastic transformation, and development of hematological
`malignancies. We then explore the possible ways that their
`modulation can lead to clinically meaningful benefits.
`
`Jak-Stat Signaling Pathways
`Hematopoietic cell proliferation and differentiation is reg-
`ulated by a number of soluble polypeptides such as IFNs,
`interleukins, and colony-stimulatory factors known collectively
`as cytokines (26). Cytokines bind to their cognate receptors and
`mediate downstream effects. A cytokine receptor consists of a
`unique ligand binding subunit as well as a signal transducing
`subunit, which may be structurally similar to the other cytokine
`receptors (27—29). On the basis of their characteristic structural
`motifs in their extracellular domains a number of subfamilies of
`
`cytokine receptors have been identified (27, 29, 30). These
`include the gpl30 family, the IL—2 receptor family, the growth
`hormone receptor family, the IFN family, and the gpl40 family
`of receptors (3). A detailed description of the structure of these
`receptors is beyond the scope of this review, but in general they
`consist of two or more subunits including the OL, B, and y chains
`(3, 27). For example,
`the IL-2 receptor family includes the
`receptors for IL-2, IL-4, IL—7, IL-9, and IL—15 each consisting of
`a ligand-binding ot—subunit, and signal
`transducing B and y
`subunits. Alternatively, the gp140 family including the receptors
`for IL—3,
`IL—5, and GM—CSF have a unique ligand—binding
`(it—subunit and a common B (gpl40) signal—transducing unit (3).
`
`3
`
`Cytokine
`receptor
`
`
`
`Eli!
`Phosphate groups
`Nuciaar
`membrane
`
`Phonphnnrlallon
`by Jaks of Stats
`
`/
`
`
`
`(—)\g
` fl
`
`[+) Slat dimer
`
`
`
`
`
`
`
`Cell surface
`
`Fig. 1 The Jak-Stat pathway and its regulation.
`
`Unlike growth factor receptors (RTKs), cytokine receptors
`do not possess a cytoplasmic kinase domain, and most cytokines
`transmit their signal by recruiting other TKs (3). Dimerization of
`the cytoplasmic component of the cytokine receptor as a result
`of ligand binding is the initial step in the initiation of cellular
`signaling (31). The dimerized subunits then associate with in-
`tracellular TKs such as members of the Src or Jak families of
`
`kinases, and the signal is propagated (Fig. 1). Different Src
`family members are associated with different receptors and
`phosphorylate distinct but overlapping sets of downstream tar—
`get molecules. For example, Lck, Lyn, and Fyn can be activated
`by IL-2 (29, 32).
`The Jak family of kinases comprises four known relatively
`large proteins (Jakl, Jak2, Jak3, and Tyk2) that can bind cyto-
`kine receptor subunits, phosphorylate them, and in doing so
`creat docking sites on the receptors for binding of SH2-contain-
`ing proteins (33, 34). In general, Jaks consist of several domains
`(JHl-JH7) of which the functional significance has been char-
`acterized by mutational analysis and include a TK domain (JH1;
`Refs. 3, 33, 35, 36). The precise functions of JH2-JH7 domains
`are under current investigation (3). Jaks are able to associate
`with the cytokine receptors as well as with each other (37, 38).
`Dimerization/oligomerization of cytokine receptor subunits as a
`result of ligand binding leads to juxtaposition of Jaks (3). This
`results in transphosphorylation and activation of their kinase
`activity and the phosphorylation of downstream signaling 'pro-
`teins such as Stats, Src—kinases, and adaptors such as Shc, Grb2,
`and Cbl (Fig. 1; Refs. 39, 40).
`Abnormalities of Jak function have been associated with a
`
`number of disorders (34, 41). For example, chromosomal trans-
`locations resulting in TEL—JAK2 constructs lead to the con-
`stitutive activation of Stat5, IL-3-independent cellular proli-
`feration,
`and leukemogenesis
`(42, 43). The translocation
`t(9;12)(p24;p13) results in the fusion of the kinase catalytic
`
`3
`
`

`

`Clinical Cancer Research 537
`
`
`region of JAK2 with the transcription factor TEL generating the
`constitutively active TEL-IAKZ. Similarly, infection with on—
`cogenic viruses such as human T-cell lymphotrophic virus, type
`I, and Abelson murine leukemia viruses results in enhanced TK
`activity of Jaks, possibly accounting for their leukemogenic
`potential (44, 45).
`The Stat
`transcription factors are coded by six known
`mammalian genes and include 10 different Stat proteins includ—
`ing different isomers of Stats 1, 3, 4, and 5 (3, 46). Like other
`transcription factors Stats have a well-defined structure includ—
`ing a DNA—binding domain, a conserved NH2-terminal domain,
`a COOH-terminal transactivation domain, and SH2 and SH3
`domains (3). Their activation through tyrosine phosphorylation
`results in their dimerization and translocation into the nucleus
`
`where they activate specific genes (6, 47—49).
`Jak proteins activate a number of intracellular signaling
`proteins, among which Stats are the best defined (46, 50).
`Binding of a cytokine to its receptor rapidly induces tyrosine
`phosphorylation of the cytoplasmic domains of the receptor by
`activated Jak kinases, thus providing a docking site for Stat
`proteins, which are then phosphorylated. This phosphorylation
`of Stats leads to their homo- or heterodimerization and translo-
`cation to the nucleus, followed by DNA binding and gene
`activation (Fig. l; Refs. 51, 52). The specificity for Stat phos-
`phorylation is determined by the receptor docking sites and not
`the Jak kinases (53, 54). Also, different Stat proteins have
`different DNA-binding affinities, resulting in activation of spe—
`cific genes. Stats also interact with other transcription factors
`such as the p300/cyclic AMP-responsive element binding pro-
`tein farme of coactivators to activate genes (55, 56). The
`transcriptional activity of Stats may also be regulated by the
`phosphorylation of their scrine and threonine residues, although
`the implications of such regulation are not known (7, 57).
`Stats mediate diverse and sometimes opposite cellular
`events affecting growth, differentiation, and apoptosis (58, 59).
`For example, Stats can mediate both growth arrest and cellular
`proliferation. Specifically, Statl mediates the gro\\-'tli—inhibitory
`effects of lFN—y. through the induction of the CDKI p'_’l“““t
`whereas Stat5 mediates proliferative effects of IL-3 and GM-
`CSF (60, 61). Similarly, phosphorylation of Stat3 can result both
`in IL—6- and lL—lO—induced growth arrest, and in GM-CSF— and
`IL-3—induced proliferation (61—63). Stats also modulate cellular
`differentiation and apoptosis. Reconstitution of Statl in Statl—
`null U3A cells (which do not respond to TNF-OL) restores basal
`caspase expression and renders them sensitive to TNF—induced
`apoptosis (64). Conversely, Stat3 and Stat5 mediate the anti—
`apoptotic effects of IL-6 and IL-2, respectively (65, 66). Statl
`activates the caspase cascade through up—regulation of Fas and
`FasL expression in response to IFN-y (67). The exact mecha-
`nisms underlying these diverse effects are being elucidated.
`An important property of cellular signaling pathways is
`that their activation is both rapid and transient. This is because
`of effective mechanisms of inactivation. In the Jak—Stat system,
`
`proteasome—mediated degradation, tyrosine dephosphorylation,
`and inhibition by various inhibitory proteins are responsible for
`this process (4). The ubiquitin—proteasome pathway governs the
`degradation of many intracellular proteins including activated
`Stats, and effective inhibitors of this system have shown prom—
`ising early results in clinical
`trials (68, 69). The cytokine-
`
`activated Jak—Stat pathways are also inhibited by tyrosine de-
`phosphorylation mediated by cytoplasmic phosphatases such as
`SHP—l
`(70, 71). SHP-l-deficient mice demonstrate multiple
`hematopoietic abnormalities, including hyperproliferation and
`abnormal activation of granulocytes and macrophages in the
`lungs and in the skin (72). SOCS and PIAS are other important
`inhibitors of the activated Jaks and Stats (70, 73). Recent studies
`have established that SOCS are negative regulators of cytokines,
`and there is ample evidence suggesting the importance of Stats
`in the induction of SOCS expression,
`thereby constituting a
`negative feedback mechanism (74—76). The role of these inhib-
`itory proteins in the pathogenesis of neoplastic transformation is
`also becoming clearer (77).
`
`RTK and Serine/Threonine Signaling Pathways
`RTKs are membrane—bound enzymes with an extracellular
`ligand—binding domain, a transmembrane domain, and a highly
`conserved intracellular domain that mediates the activation,
`
`through tyrosine phosphorylation, of a number of downstream
`signaling proteins (78—80). These enzymes are activated by
`ligand binding, by cell-cell interactions via cell adhesion mol-
`ecules, and by stimulation of G-protein coupled receptors (81).
`Phosphorylated tyrosine residues in specific domains of these
`receptors serve as high-affinity docking sites for SH2-contain-
`ing adaptor and effector proteins (82). RTKs include diverse
`molecules, which are considered as members of several distinct
`classes: class I including epidermal growth factor receptor; class
`11 including insulin-like growth factor-1 receptor; class 111
`including PDGFR, macrophage
`colony-stimulating factor
`(FMS-R or CSF—lR), stem cell factor receptor (KIT), and
`FLT3R; and class IV including FGFR (79, 83, 84). The impor-
`tance of these receptors in malignant transformation and the
`possibility of modulating them as therapeutic targets are sub—
`jects of intense research. The recent reports of constitutive
`activation of FLT3R resulting in stimulation of multiple signal—
`ing pathways and leading to malignant transformation has been
`of significant interest in leukemia research (85, 86). Such con—
`stitutive activation of this receptor has been reported in >30%
`of patients with AML and results from two well—described
`molecular events.
`Internal
`tandem duplication mutations of
`FLT3R gene occur at exons 11 and 12 of the gene that code for
`the juxtarnembrane domain of receptor (87—90). More recently,
`point mutations of codon 835 of FLT3R receptor gene, located
`in the activation loop of its TK domain, have been reported in
`7% of patients with AML (87, 91).*Inhibitors of such aberrant
`activation are undergoing clinical evaluation (92, 93).
`Many intracellular signaling proteins bind the phosphoty-
`rosine on the activated RTKs. These proteins include GTPasc
`activating protein, PI3K, Grb2, and Src—like tyrosine-kinases (1,
`10, 78). The activation of these proteins by scrine/threonine
`phosphorylation in turn activates a number of downstream sig—
`naling cascades that lead to gene transcription (10).
`Although knowledge of the Jak-Stat pathway has been
`instrumental in understanding cytokine signaling (94), the im—
`portance of signaling cascades that involve the activation of
`serine/threonine kinases is increasingly apparent (10). The ser—
`ine/threonine MAPKs, which include the Ras—Raf-MEK-ERK
`pathway,
`the p38 family of kinases, and the JNK (SAPK)
`
`4
`
`

`

`538 Cellular Signaling Pathways as Therapeutic Targets
`
`family, are activated by upstream signals and mediate effects on
`inflammation, cell growth, cell cycle progression, cell differen-
`tiation, and apoptosis (95). The Ras family of proteins belongs
`to the large superfamily of GTPases that localize to the inner
`surface of the plasma membrane (1, 96). Ras proteins play a
`pivotal role in a number of signaling pathways mediated by
`RTKs and other receptors. Ligand binding to these receptors
`initiates the autophosphorylation of specific tyrosine residues in
`their cytoplasmic domain and creates phosphotyrosyl—binding
`sites for adapter proteins such as Shc and Grb2, which in turn
`recruit guanine nucleotide exchange factors and thereby initiate
`Ras activation (97, 98).
`Once induced, Ras activates Raf serine/threonine kinase,
`which then phosphorylates MAPK kinases (otherwise known as
`MEKs; Refs. 10, 97, 99). These in turn activate MAPKs (or
`ERKs; Refs. 100, 101), which in turn move to the nucleus where
`they phosphorylate and activate nuclear transcription factors
`such as Elk-1 (102). ERKs were initially described as a novel
`family of protein kinases that, when activated, produced prolif—
`erative stimuli (103). ERKs can also activate other kinases such
`as RSKs (also known as MAPK—activated protein kinases),
`which are involved in cell-cycle regulation and apoptosis (104).
`ERK-activated RSK kinase catalyzes the proapoptotic protein
`Bad and suppresses Bad-mediated apoptosis (105). Similarly,
`the Ras-Raf-MEK-ERK cascade modulates cellular prolifera—
`tion by regulating the activity of several proteins, including
`cell-cycle regulators (e.g., cyclin D1, p21wnfmip], p27kip], and
`cchSA) and transcription factors (e.g., c-Myc; Ref. 106).
`The Gl/S cell cycle checkpoint is a critical point determin-
`ing the commitment of cells to growth arrest or proliferation.
`During this stage cells are responsive to cytokines (107). Reg-
`ulatory proteins p21"’“f“Cipl and p27kipl are of particular impor-
`tance in this transition, which is controlled by both positive and
`negative regulators. Distinct Rb—E2F repressor complexes sup—
`press the transcription of genes required for progression of
`various phases of cell cycle. For example, Rb—E2Fl complex
`suppresses the progression through G1 (108). During this pro—
`gression from G1 to S—phase cyclin/CDKs are sequentially ac—
`tivated, which then inactivate suppressor complexes such as
`Rb—E2F1 (109). Cyclin/CDK activity results in Rb phosphoryl—
`ation and its dissociation from E2Fl leading to activation of
`genes necessary for S phase (110). Activity of a number of these
`cyclin/CDKs as well their inhibitors such as p21‘w‘fllCipl is mod-
`ulated by cytokine-mediated signals through their phosphory-
`lation.
`
`The p38 family of MAPKs is involved in various cellular
`processes such as inflammation, cell cycle progression, and cell
`death (111, 112). The four different p38 isoforms (or, B, y, and
`8) are activated by two MEK isoforms (113). Originally, the p38
`kinase pathway was reported to have a critical role in the
`generation of signals in response to stress stimuli. However, its
`role in cytokine signaling and regulation of the Jak—Stat pathway
`has been elucidated recently (114). In particular, from the stand—
`point of leukemogenesis,
`it modulates the growth—inhibitory
`effect of type I IFNs in BCR-ABL—expressing cells as well as
`normal hematopoietic progenitors (115, 116).
`The third group of MAPKs includes the JNK (otherwise
`known as SAPK; Ref. 95). The four different JNK kinases have
`a similar role to p38 kinases in cellular function and aretacti—
`
`vated by specific MAPK kinases (MEKKs) in response to
`inflammatory cytokines such as TNF—a, and other stress stimuli
`such as reactive oxygen species, heat, and withdrawal of growth
`factors (117). The MEKKl/JNK signaling increases p53 stabil—
`ity and transcriptional activation, and MEKKl/JNK potentiates
`the ability of p53 to initiate apoptosis (118).
`Normal functioning of MAPK-mediated signaling necessi—
`tates its efficient inactivation (95). A number of dual—specificity
`MAPK phosphatases serve to dephosphorylate and, hence, in-
`activate MAPKs (119—121). Similarly, protein phosphatases
`PM and PP2 dephosphorylate and inactivate a number of phos—
`phoproteins including components of the MAPK pathway
`(122, 123).
`Other signaling pathways such as those mediated by PI3K,
`AKT (also known as PKB), and protein kinase C are also
`controlled by serine/threonine phosphorylation (Fig. 2; Ref. 10).
`PI3K consists of two subunits, the p85 regulatory subunit and
`the p110 catalytic subunit (124, 125). The p85 subunit binds to
`the cytokine receptor as a consequence of ligand-receptor inter-
`action and receptor autophosphorylation (126). As a result,
`phosphatidylinositol—dependent kinases and their downstream
`substrate AKT/PKB are recruited to the membrane (127). PI3K—
`AKT pathway activates several downstream targets including
`p70 RSK, forkhead transcription factors, and NFKB (128—130).
`The serine/threonine kinase AKT is an important component of
`the cell survival machinery (10, 131—133). Its activation via the
`PI3K pathway leads to a number of events (10, 131, 134, 135).
`For example, the phosphorylation of the cytosolic protein IKB
`by AKT releases NFKB from its association with IKB. N_FKB
`then moves into the nucleus, where it induces a number of genes
`involved in cell survival (131). Meanwhile, the inhibitory pro—
`tein IKB is degraded by the proteasome (136). AKT also phos-
`phorylates the proapoptotic protein Bad, which leads to higher
`levels of free antiapoptotic Bcl-xL and thereby inhibits the
`cell-death protease caspase-9 (134). The tumor suppressor gene
`PTEN codes for a phosphatase that acts by removing a phos—
`phate group from the 3 position of the inositol ring of the
`PIP3’4_5 phospholipids located at the cellular membrane. This
`prevents the proximation of AKT and phosphatidylinositol-
`dependent kinases, and prevents AKT activation (137—140).
`Several lines of evidence including studies of PTEN knockout
`mice support the role of PTEN as a tumor suppressor gene (141).
`Serine/threonine kinases, in general, also influence the activity
`of other antiapoptotic proteins of the Bcl—2 family (10, 135,
`142). In the normal cell cycle, Bel-2 is phosphorylated on its
`serine/threonine residues at several points during the G2 to M
`phase transition (10, 143).
`PKC, another important signaling enzyme, phosphorylates
`specific serine or threonine residues on target proteins in differ—
`ent ways (Fig. 2). For example, PKC is a potent activator of
`Raf—1, which activates the MAPK cascade. This leads to phos—
`phorylation of IKB, release of NFKB, translocation of NFKB into
`the nucleus, and gene transcription as described above (144—
`146). PKC also regulates cytokine signals through its effects on
`the Jak—Stat pathway in some myeloid progenitor cell
`lines
`(147). The significant role of PKC in phosphorylation and
`activation of Raf has led to its targeting for inhibition of the
`Raf—Ras-MEK—ERK pathway (95, 148). For example, stauros—
`
`5
`
`

`

`Clinical Cancer Research 539
`
`
`Neighboring cell
`
` Cytokine
`receptor
`
`
`
`Fig. 2
`ways.
`
`Interactions of diverse signaling path—
`
`
`30172 4'" """“' " "
`
`
`
`
`
`Cailular membrane
`
`porine analogs UCN—Ol and CGP 41251 are being examined as
`inhibitors of PKC and MAPK signaling (149, 150).
`Various signaling pathways interact resulting in their mod-
`ulation at several levels. For example, PI3K interacts with and
`enhances Raf-Ras-MEK—ERK pathway (151—153). Other ser-
`ine/threonine phosphorylation pathways modulate cytokine sig-
`nals through the Jak—Stat pathway (10, 154). In addition to being
`tyrosine phosphorylated, several Stats (StatIoL, Stat3, and Stat4)
`are serine phosphorylated by ERKs at conserved serine residues
`(155). In fact, during cytokine signaling, Jak and Raf kinases
`carry on an intricate cross-talk (10).
`
`Therapeutic Implications
`Signaling pathways may be particularly attractive targets in
`cancer therapy because they may be inappropriately activated in
`malignant cells. However, several factors must be carefully
`considered~while developing agents that modulate these path-
`ways. One is the possible toxicity of such therapy. Because
`these pathways are activated to a significantly greater degree in
`malignant cells than normal cells, their partial inhibition may be
`sufficient to interfere with malignant cell growth without caus—
`ing significant toxicity (25). Therefore, despite the pivotal role
`of these pathways in normal cellular function, their inhibition
`may not be toxic to normal cells.
`Another point of consideration in designing inhibitors is
`the specificity of the target pathway and the selectivity of its
`inhibitors. Adding to this challenge is the fact that these path—
`ways are part of a complex network of interconnecting cascades
`resulting in a certain degree of redundancy and overlap.
`Diseases induced by specific oncogenic alterations leading
`to constitutive activation of pivotal molecules in the pathways
`may provide us with such specificity and selectivity. A number
`of translocations occurring in hematological malignancies are
`known to result in fusion genes with enhanced kinase activity
`(24). The oncoprotein Bcr—Abl results from a translocation be-
`
`tween chromosomes 9 and 22, and occurs in CML and ALL. Its
`transforming activity is the product of activation of a number of
`signaling molecules such as Ras, Raf, PI3K, INK/SAPK, Crkl,
`and Stat5 (156—160). As a result of their activation, a number of
`pathways, which inhibit apoptosis and promote cell survival, are
`induced (24). Bcr-Abl is constitutively phosphorylated on a
`number of tyrosine residues, allowing the docking of adaptor
`proteins such as Cbl, Crkl, Shc, and Grb2, which recruit the Ras
`signaling pathway, the p85 regulatory subunit of PI3K, the focal
`adhesion proteins paxillin and talin, and a number of other
`signaling pathways (159, 161—163). The specific Bcr-Abl inhib-
`itor Imatinib mesylate has proven to be very effective in sup-
`pressing these pathways in vitro and in patients with CML and
`ALL (164—170).
`Although the platelet—derived growth factors, PDGFROI.
`and PDGFRB have significant homology, only the PDGFRB has
`been implicated in hematological cancers where a significant
`number of patients with chronic myelomonocytic leukemia have
`t(5;12)(q33;p13) generating the fusion protein TEL—PDGFRB
`(171, 172). As a result of ligand-independent dimerization and
`autophosphorylation of the PDGF B—subunit, the TK is consti-
`tutively active. PDGF is able to stimulate the growth of primi-
`tive hematopoietic, erythroid, and megakaryocytic precursors,
`and TEL-PDGFRB can confer cytokine-independent growth to
`Ba/F3cells
`(173).
`Imatinib mesylate
`inhibits
`the kinase
`PDGFRB and has been shown anecdotally to be effective in
`patients with this translocation (174).
`Similarly, the chimeric gene NPM-ALK is produced as a
`result of translocation t(2;5)(p23;q35; Refs. 175, 176). Fusion of
`NHz—terminal domain of NPM to the cytoplasmic region of the
`ALK TK receptor results in constitutive activation of its cata-
`lytic domain (177, 178). NPM-ALK associates with a number of
`adaptor proteins such as Grb2 and Crkl, and results in the
`activation of the downstream signaling proteins such as PI3K,
`AKT, and Stat5 (179—182). Therefore, design of specific inhib-
`
`6
`
`

`

`540 Cellular Signaling Pathways as Therapeutic Targets
`
` "scri t'on
`
`pl
`
`Fig. 3 Targets for inhibition in Jak-Stat signaling pathway.
`
`Gene 1:33
`
`Cell Membrane
`
`in
`itors of the NPM-ALK may be of considerable interest
`treating patients with anaplastic large cell lymphoma where up
`to 50% of patients express the chromosomal translocation (183).
`The TK modulated pathways such as the Jak-stat cascade
`can be targeted at several steps along the way (Fig. 3). Because
`a number of cytokines and growth hormones play an important
`role in the suppression of apoptosis in the malignant clone in
`hematological cancers (e.g.,
`IL—6 in myeloma; lL—2 in some
`T-cell lymphomas; and IL-1, IL—3, and GM-CSF in AML; Refs.
`25, 184), inhibition of the cytokines and their receptors is a
`plausible therapeutic strategy (185, 186). Furthermore, where
`there is constitutive activation of the receptor leading to neo—
`plastic change, selective inhibition of kinase activity of the
`receptor is likely to be of benefit. For example, inhibition of
`FLT3-R TK activity is selectively cytotoxic to AML blasts
`harboring the appropriate activating mutations (92, 187). Acti—
`vating mutations of FLT3—R including the internal tandem du—
`plication mutation and mutation of the TK domain occur in
`>30% of patients with AML, confer adverse prognosis, and can
`be targeted by selective inhibitors (92, 188, 189). Clinical trials
`examining the efficacy and safety of such inhibitors are cur-
`rently under way. Intracellular activators of Stats such as Jaks
`and Src are also likely targets (164—166). For example, the Jak2
`inhibitor AG490 inhibited the growth of ALL cells in a mouse
`model without affecting normal hematopoiesis (190). AG490
`also acts by inhibiting Stat function, and induces apoptosis in
`Sezary cells and myeloma cells (191, 192). Furthermore, nu-
`merous other cellular TKs have been ident