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Modulation of Cellular Signaling Pathways: Prospects for Targeted

Therapy in Hematological Malignancies

Farhad Ravandi, 1 Moshe Talpaz, and Zeev EstrovDepartment of Hematology/Oncology, The University of Illinois atChicago, Chicago, Illinois 60612 [F. R.], and Department of Bioimmunotherapy, The University of Texas M. D. Anderson CancerCenter, Houston, Texas 77030 [M. T., Z. E.]

AbstractThe 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 incellular biology over the past decade has resulted in a betterunderstanding of the process of malignant transformation, abetter classification of subtypes of each disease on the basisof molecular markers, and a better characterization of themolecular targets for drug development. These advanceshave already spawned the development of such effectiveagents 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 inhematological malignancies.

Introduction

Cellular proliferation, differentiation, and death are regu-lated by a number of extracellular molecules such as cytokinesand hormones, as well as intercellular interactions mediated byneighboring cell surface antigens. These effectors mediate genetranscription either directly or indirectly by activating intracel-lular signaling pathways, which in turn activate appropriatecellular 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 throughtheir inherent enzymatic function or as a result of their associ-ation with other catalytic proteins. Indeed, most growth factorsand cytokines bind these receptors and exert their functionthrough their activation, commonly by phosphorylation (1).

Normal hematopoiesis is dependent on intricately regulatedsignaling cascades that are mediated by cytokines and theirreceptors. Orderly function of these pathways leads to the gen-eration of appropriate constellation of hematopoietic cells, andtheir abnormal activation results in neoplastic transformation,impaired apoptosis, and uncontrolled proliferation. Cytokines

function in a redundant and pleiotropic manner; different cyto-kines can exert similar effects on the same cell type, and anyparticular cytokine can have several differing biological func-tions (2). This complexity of function is a result of sharedreceptor subunits as well as overlapping downstream pathwaysculminating in activation of common transcription factors (3).

The signal transduction cascades involve three majorclasses of proteins: kinases, adaptor or docking proteins, andtranscription factors. Early insights into the cellular signalingpathways came from studies of IFN function (4 6). Theseexperiments led to the identification of a family of nonreceptorTKs 2 called Jaks and their target proteins, Stats, which mediategene transcription (4, 7). The Jak-Stat pathways are commonlyactivated during cytokine signaling through phosphorylation of specific tyrosine residues (3). The interaction of a cytokine withits receptor induces its tyrosine phosphorylation and leads toactivation of downstream protein TKs including Jaks and Stats.Apart from their catalytic domain, protein TKs contain severalother characteristic motifs including the SH2, SH3, and pleck-strin homology domain, which enable them to interact withother signaling molecules and propagate the message (8, 9).

The phosphorylation of serine and threonine residues isintegral to the activation of numerous other intracellular proteinsthat mediate a number of other signaling pathways (10). Cyto-kine receptors without intrinsic kinase activity transmit theirsignals 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 independentlyof the serine/threonine kinase cascades. More recently, it hasbeen 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 -subunit of IL-3 and GM-CSF receptors are phosphorylated and,through recruitment of adaptor proteins such as Shc, Grb2, and

Received 6/25/02; revised 9/5/02; accepted 9/6/02.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1

To whom requests for reprints should be addressed, at University of IllinoisChicago, 840 South Wood Street, MC 787 Chicago, IL 60612-7323. Phone: (312) 996-5982; Fax: (312) 413-4131; E-mail: [email protected].

2

The abbreviations used are: TK, tyrosine kinase; Jak, janus kinase;Stat, signal transducer and activator of transcription; ERK, extracellularsignal-regulated kinase; MAPK, mitogen-activated protein kinase; IL,interleukin; GM-CSF, granulocyte macrophage colony-stimulating fac-tor; JNK, c-Jun NH 2 -terminal kinase; SAPK, stress-activated proteinkinase; PKB, protein kinase B; PI3K, phosphatidylinositol 3 -kinase;PDGFR, platelet-derived growth factor receptor; TNF, tumor necrosisfactor; SHP-1, SH-2 domain containing protein tyrosine phosphatase-1;SOCS, suppressor of cytokine signaling; PIAS, protein inhibitor of activated stat; RTK, receptor tyrosine kinase; FLT3R, FMS-like tyrosinekinase 3 receptor; Grb2, growth factor receptor binding protein 2; RSK,ribosomal S6 kinase; AML, acute myeloid leukemia; CML, chronicmyelogenous leukemia; ALL, acute lymphoblastic leukemia; MEK,mitogen-activated protein/extracellular signal-regulated kinase kinase;ALK, anaplastic lymphoma kinase; NPM, nucleophosmin; ATRA, all-trans -retinoic acid; APL, acute promyelocytic leukemia; NF B, nuclear

factor B; I B, inhibitor of nuclear factor- B; 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.

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Sos, activate the Ras signaling pathway (11). This in turnactivates Raf followed by downstream activation of ERK1 andERK2, and increased expression of transcription factors c- fosand c- jun (12 14). Other members of the MAPK family such asp38, and JNK/SAPK are also activated after phosphorylation of their serine/threonine residues as a result of cytokine/receptorinteraction (15 18). Similarly, PI3K associates with the chainof IL-3 receptor, recruits PKB/AKT by phosphorylation of itsserine residues, and transmits cellular survival signals (19 21).Another downstream protein to IL-3 activation is the p70S6kinase, which also interacts with the chain and mediatesappropriates 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 arederived from genes including c-ABL, c-FMS , FLT3 , c-KIT , andPDGFR , which are normally involved in the regulation of hematopoiesis (24). The kinase activity of the oncogene isconstitutively activated by mutations that remove inhibitorydomains of the molecule or induce the kinase domain to adoptan activated configuration (24). As a result of such constitutiveactivation a number of signaling cascades such as the Jak-Statpathway, the Ras/Raf/MAPK pathway, and the PI3K pathwayare activated.

With better characterization of aberrant signaling throughthe cell surface receptors and their downstream pathways inneoplastic cells, current research is exploring ways to reversesuch dysregulated stimuli (25). Here, we will briefly review the

role of cellular signaling pathways in normal cellular processes,neoplastic transformation, and development of hematologicalmalignancies. We then explore the possible ways that theirmodulation can lead to clinically meaningful benefits.

Jak-Stat Signaling PathwaysHematopoietic cell proliferation and differentiation is reg-

ulated by a number of soluble polypeptides such as IFNs,interleukins, and colony-stimulatory factors known collectivelyas cytokines (26). Cytokines bind to their cognate receptors andmediate downstream effects. A cytokine receptor consists of aunique ligand binding subunit as well as a signal transducing

subunit, which may be structurally similar to the other cytokinereceptors (27 29). On the basis of their characteristic structuralmotifs in their extracellular domains a number of subfamilies of cytokine receptors have been identified (27, 29, 30). Theseinclude the gp130 family, the IL-2 receptor family, the growthhormone receptor family, the IFN family, and the gp140 familyof receptors (3). A detailed description of the structure of thesereceptors is beyond the scope of this review, but in general theyconsist of two or more subunits including the , , and chains(3, 27). For example, the IL-2 receptor family includes thereceptors for IL-2, IL-4, IL-7, IL-9, and IL-15 each consisting of a ligand-binding -subunit, and signal transducing and subunits. Alternatively, the gp140 family including the receptorsfor IL-3, IL-5, and GM-CSF have a unique ligand-binding

-subunit and a common (gp140) signal-transducing unit (3).

Unlike growth factor receptors (RTKs), cytokine receptorsdo not possess a cytoplasmic kinase domain, and most cytokinestransmit their signal by recruiting other TKs (3). Dimerization of the cytoplasmic component of the cytokine receptor as a resultof ligand binding is the initial step in the initiation of cellularsignaling (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 Srcfamily members are associated with different receptors andphosphorylate distinct but overlapping sets of downstream tar-get molecules. For example, Lck , Lyn , and Fyn can be activatedby IL-2 (29, 32).

The Jak family of kinases comprises four known relativelylarge proteins (Jak1, Jak2, Jak3, and Tyk2) that can bind cyto-kine receptor subunits, phosphorylate them, and in doing socreat docking sites on the receptors for binding of SH2-contain-ing proteins (33, 34). In general, Jaks consist of several domains(JH1-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 domainsare under current investigation (3). Jaks are able to associatewith the cytokine receptors as well as with each other (37, 38).Dimerization/oligomerization of cytokine receptor subunits as aresult of ligand binding leads to juxtaposition of Jaks (3). Thisresults in transphosphorylation and activation of their kinaseactivity 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 anumber 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 translocationt(9;12)(p24;p13) results in the fusion of the kinase catalytic

Fig. 1 The Jak-Stat pathway and its regulation.

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region of JAK2 with the transcription factor TEL generating theconstitutively active TEL-JAK2. Similarly, infection with on-cogenic viruses such as human T-cell lymphotrophic virus, typeI, and Abelson murine leukemia viruses results in enhanced TKactivity of Jaks, possibly accounting for their leukemogenicpotential (44, 45).

The Stat transcription factors are coded by six knownmammalian genes and include 10 different Stat proteins includ-ing different isomers of Stats 1, 3, 4, and 5 (3, 46). Like othertranscription factors Stats have a well-defined structure includ-ing a DNA-binding domain, a conserved NH 2 -terminal domain,a COOH-terminal transactivation domain, and SH2 and SH3domains (3). Their activation through tyrosine phosphorylationresults in their dimerization and translocation into the nucleuswhere they activate specific genes (6, 47 49).

Jak proteins activate a number of intracellular signalingproteins, among which Stats are the best defined (46, 50).Binding of a cytokine to its receptor rapidly induces tyrosinephosphorylation of the cytoplasmic domains of the receptor byactivated Jak kinases, thus providing a docking site for Statproteins, which are then phosphorylated. This phosphorylationof Stats leads to their homo- or heterodimerization and translo-cation to the nucleus, followed by DNA binding and geneactivation (Fig. 1; Refs. 51, 52). The specificity for Stat phos-phorylation is determined by the receptor docking sites and notthe Jak kinases (53, 54). Also, different Stat proteins havedifferent DNA-binding affinities, resulting in activation of spe-cific genes. Stats also interact with other transcription factorssuch as the p300/cyclic AMP-responsive element binding pro-tein family of coactivators to activate genes (55, 56). Thetranscriptional activity of Stats may also be regulated by the

phosphorylation of their serine and threonine residues, althoughthe implications of such regulation are not known (7, 57).

Stats mediate diverse and sometimes opposite cellularevents affecting growth, differentiation, and apoptosis (58, 59).For example, Stats can mediate both growth arrest and cellularproliferation. Specifically, Stat1 mediates the growth-inhibitoryeffects of IFN- , through the induction of the CDKI p21 waf1 ,whereas Stat5 mediates proliferative effects of IL-3 and GM-CSF (60, 61). Similarly, phosphorylation of Stat3 can result bothin IL-6- and IL-10-induced growth arrest, and in GM-CSF- andIL-3-induced proliferation (61 63). Stats also modulate cellulardifferentiation and apoptosis. Reconstitution of Stat1 in Stat1-null U3A cells (which do not respond to TNF- ) restores basal

caspase expression and renders them sensitive to TNF-inducedapoptosis (64). Conversely, Stat3 and Stat5 mediate the anti-apoptotic effects of IL-6 and IL-2, respectively (65, 66). Stat1activates the caspase cascade through up-regulation of Fas andFasL expression in response to IFN- (67). The exact mecha-nisms underlying these diverse effects are being elucidated.

An important property of cellular signaling pathways isthat their activation is both rapid and transient. This is becauseof effective mechanisms of inactivation. In the Jak-Stat system,proteasome-mediated degradation, tyrosine dephosphorylation,and inhibition by various inhibitory proteins are responsible forthis process (4). The ubiquitin-proteasome pathway governs thedegradation of many intracellular proteins including activatedStats, 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 asSHP-1 (70, 71). SHP-1-deficient mice demonstrate multiplehematopoietic abnormalities, including hyperproliferation andabnormal activation of granulocytes and macrophages in thelungs and in the skin (72). SOCS and PIAS are other importantinhibitors of the activated Jaks and Stats (70, 73). Recent studieshave established that SOCS are negative regulators of cytokines,and there is ample evidence suggesting the importance of Statsin the induction of SOCS expression, thereby constituting anegative feedback mechanism (74 76). The role of these inhib-itory proteins in the pathogenesis of neoplastic transformation isalso becoming clearer (77).

RTK and Serine/Threonine Signaling PathwaysRTKs are membrane-bound enzymes with an extracellular

ligand-binding domain, a transmembrane domain, and a highlyconserved intracellular domain that mediates the activation,through tyrosine phosphorylation, of a number of downstreamsignaling proteins (78 80). These enzymes are activated byligand 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 thesereceptors serve as high-affinity docking sites for SH2-contain-ing adaptor and effector proteins (82). RTKs include diversemolecules, which are considered as members of several distinctclasses: class I including epidermal growth factor receptor; classII including insulin-like growth factor-1 receptor; class IIIincluding PDGFR, macrophage colony-stimulating factor(FMS-R or CSF-1R), 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 thepossibility of modulating them as therapeutic targets are sub- jects of intense research. The recent reports of constitutiveactivation of FLT3R resulting in stimulation of multiple signal-ing pathways and leading to malignant transformation has beenof 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-describedmolecular events. Internal tandem duplication mutations of FLT3R gene occur at exons 11 and 12 of the gene that code forthe juxtamembrane 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 in7% of patients with AML (87, 91). Inhibitors of such aberrantactivation are undergoing clinical evaluation (92, 93).

Many intracellular signaling proteins bind the phosphoty-rosine on the activated RTKs. These proteins include GTPaseactivating protein, PI3K, Grb2, and Src -like tyrosine-kinases (1,10, 78). The activation of these proteins by serine/threoninephosphorylation in turn activates a number of downstream sig-naling cascades that lead to gene transcription (10).

Although knowledge of the Jak-Stat pathway has beeninstrumental 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-ERKpathway, the p38 family of kinases, and the JNK (SAPK)

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family, are activated by upstream signals and mediate effects oninflammation, cell growth, cell cycle progression, cell differen-tiation, and apoptosis (95). The Ras family of proteins belongsto the large superfamily of GTPases that localize to the innersurface of the plasma membrane (1, 96). Ras proteins play apivotal role in a number of signaling pathways mediated byRTKs and other receptors. Ligand binding to these receptorsinitiates the autophosphorylation of specific tyrosine residues intheir cytoplasmic domain and creates phosphotyrosyl-bindingsites for adapter proteins such as Shc and Grb2, which in turnrecruit guanine nucleotide exchange factors and thereby initiateRas activation (97, 98).

Once induced, Ras activates Raf serine/threonine kinase,which then phosphorylates MAPK kinases (otherwise known asMEKs; Refs. 10, 97, 99). These in turn activate MAPKs (orERKs; Refs. 100, 101), which in turn move to the nucleus wherethey phosphorylate and activate nuclear transcription factorssuch as Elk-1 (102). ERKs were initially described as a novelfamily of protein kinases that, when activated, produced prolif-erative stimuli (103). ERKs can also activate other kinases suchas 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 proteinBad and suppresses Bad-mediated apoptosis (105). Similarly,the Ras-Raf-MEK-ERK cascade modulates cellular prolifera-tion by regulating the activity of several proteins, includingcell-cycle regulators ( e.g. , cyclin D1, p21 waf1/cip1 , p27 kip1 , andcdc25A) and transcription factors ( e.g. , c-Myc; Ref. 106).

The G 1 /S cell cycle checkpoint is a crit ical point determin-ing the commitment of cells to growth arrest or proliferation.During this stage cells are responsive to cytokines (107). Reg-

ulatory proteins p21waf1/cip1

and p27kip1

are of particular impor-tance in this transition, which is controlled by both positive andnegative regulators. Distinct Rb-E2F repressor complexes sup-press the transcription of genes required for progression of various phases of cell cycle. For example, Rb-E2F1 complexsuppresses the progression through G 1 (108). During this pro -gression from G 1 to S-phase cyclin/CDKs are sequentially ac-tivated, which then inactivate suppressor complexes such asRb-E2F1 (109). Cyclin/CDK activity results in Rb phosphoryl-ation and its dissociation from E2F1 leading to activation of genes necessary for S phase (110). Activity of a number of thesecyclin/CDKs as well their inhibitors such as p21 waf1/cip1 is mod-ulated by cytokine-mediated signals through their phosphory-

lation.The p38 family of MAPKs is involved in various cellularprocesses such as inflammation, cell cycle progression, and celldeath (111, 112). The four different p38 isoforms ( , , , and

) are activated by two MEK isoforms (113). Originally, the p38kinase pathway was reported to have a critical role in thegeneration of signals in response to stress stimuli. However, itsrole in cytokine signaling and regulation of the Jak-Stat pathwayhas been elucidated recently (114). In particular, from the stand-point of leukemogenesis, it modulates the growth-inhibitoryeffect of type I IFNs in BCR-ABL-expressing cells as well asnormal hematopoietic progenitors (115, 116).

The third group of MAPKs includes the JNK (otherwiseknown as SAPK; Ref. 95). The four different JNK kinases havea similar role to p38 kinases in cellular function and are acti-

vated by specific MAPK kinases (MEKKs) in response toinflammatory cytokines such as TNF- , and other stress stimulisuch as reactive oxygen species, heat, and withdrawal of growthfactors (117). The MEKK1/JNK signaling increases p53 stabil-ity and transcriptional activation, and MEKK1/JNK potentiatesthe ability of p53 to initiate apoptosis (118).

Normal functioning of MAPK-mediated signaling necessi-tates its efficient inactivation (95). A number of dual-specificityMAPK phosphatases serve to dephosphorylate and, hence, in-activate MAPKs (119 121). Similarly, protein phosphatasesPP1 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 alsocontrolled by serine/threonine phosphorylation (Fig. 2; Ref. 10).PI3K consists of two subunits, the p85 regulatory subunit andthe p110 catalytic subunit (124, 125). The p85 subunit binds tothe cytokine receptor as a consequence of ligand-receptor inter-action and receptor autophosphorylation (126). As a result,phosphatidylinositol-dependent kinases and their downstreamsubstrate AKT/PKB are recruited to the membrane (127). PI3K-AKT pathway activates several downstream targets includingp70 RSK, forkhead transcription factors, and NF B (128 130).The serine/threonine kinase AKT is an important component of the cell survival machinery (10, 131 133). Its activation via thePI3K pathway leads to a number of events (10, 131, 134, 135).For example, the phosphorylation of the cytosolic protein I Bby AKT releases NF B from its association with I B. NF Bthen moves into the nucleus, where it induces a number of genesinvolved in cell survival (131). Meanwhile, the inhibitory pro-tein I B is degraded by the proteasome (136). AKT also phos-phorylates the proapoptotic protein Bad, which leads to higherlevels of free antiapoptotic Bcl-x L and thereby inhibits thecell-death protease caspase-9 (134). The tumor suppressor genePTEN codes for a phosphatase that acts by removing a phos-phate group from the 3 position of the inositol ring of thePIP 3,4,5 phospholipids located at the cellular membrane. Thisprevents the proximation of AKT and phosphatidylinositol-dependent kinases, and prevents AKT activation (137 140).Several lines of evidence including studies of PTEN knockoutmice 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, Bcl-2 is phosphorylated on itsserine/threonine residues at several points during the G 2 to Mphase transition (10, 143).

PKC, another important signaling enzyme, phosphorylatesspecific 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 I B, release of NF B, translocation of NF B intothe nucleus, and gene transcription as described above (144 146). PKC also regulates cytokine signals through its effects onthe Jak-Stat pathway in some myeloid progenitor cell lines(147). The significant role of PKC in phosphorylation andactivation of Raf has led to its targeting for inhibition of theRaf-Ras-MEK-ERK pathway (95, 148). For example, stauros-



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porine analogs UCN-O1 and CGP 41251 are being examined asinhibitors of PKC and MAPK signaling (149, 150).

Various signaling pathways interact resulting in their mod-ulation at several levels. For example, PI3K interacts with andenhances 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 (Stat1 , Stat3, and Stat4)are serine phosphorylated by ERKs at conserved serine residues(155). In fact, during cytokine signaling, Jak and Raf kinasescarry on an intricate cross-talk (10).

Therapeutic ImplicationsSignaling pathways may be particularly attractive targets in

cancer therapy because they may be inappropriately activated inmalignant cells. However, several factors must be carefullyconsidered while developing agents that modulate these path-ways. One is the possible toxicity of such therapy. Becausethese pathways are activated to a significantly greater degree inmalignant 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 roleof these pathways in normal cellular function, their inhibitionmay not be toxic to normal cells.

Another point of consideration in designing inhibitors isthe specificity of the target pathway and the selectivity of itsinhibitors. Adding to this challenge is the fact that these path-ways are part of a complex network of interconnecting cascadesresulting in a certain degree of redundancy and overlap.

Diseases induced by specific oncogenic alterations leadingto constitutive activation of pivotal molecules in the pathwaysmay provide us with such specificity and selectivity. A numberof translocations occurring in hematological malignancies areknown 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. Itstransforming activity is the product of activation of a number of signaling molecules such as Ras, Raf, PI3K, JNK/SAPK, Crkl,and Stat5 (156 160). As a result of their activation, a number of pathways, which inhibit apoptosis and promote cell survival, areinduced (24). Bcr-Abl is constitutively phosphorylated on anumber of tyrosine residues, allowing the docking of adaptor

proteins such as Cbl, Crkl, Shc, and Grb2, which recruit the Rassignaling pathway, the p85 regulatory subunit of PI3K, the focaladhesion proteins paxillin and talin, and a number of othersignaling 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 andALL (164 170).

Although the platelet-derived growth factors, PDGFRand PDGFR have significant homology, only the PDGFR hasbeen implicated in hematological cancers where a significantnumber of patients with chronic myelomonocytic leukemia havet(5;12)(q33;p13) generating the fusion protein TEL-PDGFR(171, 172). As a result of ligand-independent dimerization and

autophosphorylation of the PDGF

-subunit, the TK is consti-tutively active. PDGF is able to stimulate the growth of primi-tive hematopoietic, erythroid, and megakaryocytic precursors,and TEL-PDGFR can confer cytokine-independent growth toBa/F3cells (173). Imatinib mesylate inhibits the kinasePDGFR and has been shown anecdotally to be effective inpatients with this translocation (174).

Similarly, the chimeric gene NPM-ALK is produced as aresult of translocation t(2;5)(p23;q35; Refs. 175, 176). Fusion of NH 2 -terminal domain of NPM to the cytoplasmic region of theALK 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 theactivation of the downstream signaling proteins such as PI3K,AKT, and Stat5 (179 182). Therefore, design of specific inhib-

Fig. 2 Interactions of diverse signaling path-ways.



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itors of the NPM-ALK may be of considerable interest intreating patients with anaplastic large cell lymphoma where upto 50% of patients express the chromosomal translocation (183).

The TK modulated pathways such as the Jak-stat cascadecan be targeted at several steps along the way (Fig. 3). Becausea number of cytokines and growth hormones play an importantrole in the suppression of apoptosis in the malignant clone inhematological cancers ( e.g. , IL-6 in myeloma; IL-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 aplausible therapeutic strategy (185, 186). Furthermore, wherethere is constitutive activation of the receptor leading to neo-plastic change, selective inhibition of kinase activity of thereceptor is likely to be of benefit. For example, inhibition of FLT3-R TK activity is selectively cytotoxic to AML blastsharboring 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 canbe targeted by selective inhibitors (92, 188, 189). Clinical trialsexamining the efficacy and safety of such inhibitors are cur-

rently under way. Intracellular activators of Stats such as Jaksand Src are also likely targets (164 166). For example, the Jak2inhibitor AG490 inhibited the growth of ALL cells in a mousemodel without affecting normal hematopoiesis (190). AG490also acts by inhibiting Stat function, and induces apoptosis inSezary cells and myeloma cells (191, 192). Furthermore, nu-merous other cellular TKs have been identified that likely haveroles in neoplastic cell survival and proliferation, and a numberof TK inhibitors are already being explored for their effects onsignaling cascades. In general, two classes of TK inhibitors arebeing developed: inhibitors of the ATP binding site and inhib-itors of the substrate binding sites (164, 193, 194).

There are other ways to interrupt Stat signaling. One is thespecific inhibition of the Stat SH2 domain, because it is essentialfor the recruitment of Stats to the receptor subunit and for Stat

dimerization (25). Antisense oligonucleotides directed at Stats(195), modulation of the activity of natural Stat inhibitors suchas SOCS and PIAS, and inhibition of the binding of activatedStat dimers to their DNA targets (25, 196, 197) are othermechanisms currently under investigation.

A better understanding of the effects of current antineo-plastic agents on these pathways may also elucidate their mech-anism of action and lead to the development of more effectiveand less toxic agents. For example, fludarabine causes a pro-found and specific loss of Stat1 (198), which may be at leastpartly responsible for its antineoplastic properties as well as itsimmunosuppressive properties, which is similar to Stat1 knock-out mice.

Multiprotein complexes, which can modify the structure of chromatin and influence gene regulation, are often aberrant inhematological malignancies. Although their interactions with var-ious signaling pathways have not been clearly defined, they meritspecific mention as they are subject of intense research. Suchcomplexes include proteins such as HDAC, histone acetyltrans-ferase, and DNA methyltransferases. In a significant proportion of patients with AML-M2, the translocation t(8;21)(q22;q22) resultsin the fusion of the DNA-binding domain of AML1 with ETO,which interacts with corepressor complexes such HDAC and re-press cellular differentiation (199, 200). Similarly, t(12;21)(p13;q22) in childhood ALL results in recruitment of the repressorN-CoR by the fusion protein TEL-AML1 (201, 202). Inhibitors of HDACs and DNA methyltransferase such as butyrates, trichostatinA, 5-azacytidine, and 2 -deoxy-azacytidine are currently underinvestigation in clinical trials (203, 204).

The most common translocation in APL, t(15;17)(q22;q11.2), results in the fusion of RAR with the PML genes, with

the resulting fusion protein retaining the functional domains of both (205, 206). The fusion protein recruits nuclear receptorcorepressors SMRT and N-CoR, which together with HDACskeep the chromatin in the closed configuration and suppress/ inactivate retinoid receptor target genes in myeloid development(207, 208). In patients with PML-RAR , pharmacological dosesof ATRA can convert the fusion protein from a dominant-negative inhibitor of retinoid-regulated transcription to anactivator (209). Signaling pathways that interact with retinoid-mediated transcription may be modulated to potentiate ATRA-induced differentiation (210). ATRA may also exert effects onupstream signaling pathways. For example, ATRA up-regulatesStat1 and Stat2 activity in the IFN- signaling pathway, thereby

potentiating the growth-inhibitory effects of IFN- (211 213).Another novel therapeutic agent in APL, arsenic trioxide, affectsa number of cellular signaling pathways, leading to induction of apoptosis and differentiation (214 217). Therapeutic effectsof arsenic trioxide in APL are partly related to its degradation of PML-RAR fusion protein through targeting PML to proteaso-mal degradation by Sumolation (218). However, arsenic trioxidehas a number of other effects such as activation of JNK kinases;translocation of PKC from cytosol to plasma membrane tomediate downstream signaling; enhancement of CDKIs,p21 waf1/cip1 , and p27 kip1 ; and inhibition of I B kinase and NF B(214).

Monoclonal antibodies such as rituximab (anti-CD20) andalemtuzumab (anti-CD52) are increasingly used in treating he-matological malignancies (219 221). Their exact mechanism of

Fig. 3 Targets for inhibition in Jak-Stat signaling pathway.



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action is largely unknown. However, understanding their effectson the signaling pathways downstream of the target receptor islikely to point the way to more effective agents. The ligation of CD20 by rituximab in the presence of FcR-expressing cells mayinitiate signal transduction events that induce an elevation of intracellular Ca2 and lead to apoptosis of the target malignantB cells (222). Rituximab has also been shown in vivo to activate

caspases in blood leukemia cells immediately after its infusionsuggesting a mechanism of action independent of complement-mediated cell lysis or antibody-dependent cellular cytotoxicity(223, 224).

Inhibition of serine/threonine kinases involved in RTKsignaling is another major area of research and development. Asnoted previously, Ras proteins play a significant role in humancarcinogenesis. Therefore, inhibition of Ras signal transductionhas been the subject of intense research. Ras-mediated signalingcan be inhibited by prevention of its membrane localization, byinhibition of Ras protein expression using antisense nucleotides,and by inhibition of its downstream targets (95, 225). Pharma-cological inhibitors of farnesyltransferase, the enzyme respon-

sible for the COOH-terminal prenylation of Ras and, hence, itsmembrane association and transforming activity, have alreadybeen developed, and clinical trials of their efficacy in leukemiaare under way (96, 226 229). Four such farnesyl transferaseinhibitors are available at various stages of testing: R115777,SCH66336, L778123, and BMS214662 (95, 230, 231). FTIsmay also have Ras-independent effects on other cellular signal-ing components that contribute to their antineoplastic action(232). Other steps in the serine/threonine cascades, from the cellsurface receptor to gene transcription, are also being examinedas possible targets for therapy (Ref. 95; Fig. 4). Compoundssuch as geldanamycin and derivatives of radicicol are known todestabilize Raf protein and interfere with Raf signaling (233 235). Several staurosporine derivatives including UCN-O1,CGP41251, and PKC412 are able to inhibit PKC signaling, and

have been examined in cell lines and clinical studies (149, 150,236). Aberrant MEK and ERK activity has been demonstratedin AML and CML (237 240), and MEK inhibitors such asPD098059, PD184352, and UO126 are able to modulate cellu-lar proliferation, differentiation, and apoptosis (241 243).PD184352 has been examined in Phase I trials in patients (244).Pharmacological inhibitors of PI3K, wortmannin, andLY294002 have shown significant potency in preclinical studies(245, 246).

Downstream targets of these signaling pathways involvedin cell-cycle regulation, cell survival, and apoptosis can also bemodulated (Table 1). For examples, agents capable of promot-ing apoptosis are being developed (247). These may function byactivating the caspase cascade through blockade of the IAPs,inhibition of the NF B pathway (using I B kinase inhibitors orproteasome inhibitors), and inhibition of the PI3K pathway(using kinase inhibitors; Refs. 69, 134, 248). IAPs including theXIAP can block the process of programmed cell death throughtheir interaction with members of the caspase family of cysteineproteases (249). The human family of caspases includes 11members involved in processing of inflammatory cytokines aswell as execution of apoptosis (250). Dysregulation of caspasecascade has been implicated in human neoplasia, and IAPs, thenatural inhibitors of caspases, are pivotal in this regulatoryprocess (250, 251). XIAP, the most potent member of the IAPfamily blocks both the initiator caspase-9, and the effectorcaspases-3 and -7 (252). An association between XIAP levelsand survival has been reported in AML (253). Down-regulationof XIAP protein by adenoviral antisense vector results in sen-sitization to radiation-induced cell death (254).

The ubiquitin-proteasome pathway is the central pathway

for degradation of intracellular proteins (255, 256). Targetingof proteins to proteasome is through covalent attachment of a poly-ubiquitin chain. This system is specific allowing up-regulation of degradation of certain proteins without affectingthe proteolysis of other substrates; this enables the proteasometo function as a regulator of metabolic pathways (256). There-fore, different classes of proteasome inhibitors can be used todifferentially affect cellular levels of oncogenic proteins (256).Important substrates for proteasome degradation include cyclinsand CDKIs; transcription factors such as p53, NF B, c-Myc,c-fos, and c-Jun; a number of apoptosis family of proteins; IAPs;and some caspases (256 262). PS-341 is a specific and potentinhibitor of proteasome (263). It induces apoptosis and over-

comes resistance in a number of cell lines as well as primarycells from patients with chronic lymphocytic leukemia (264 268). Its in vitro activity in myeloma has led to its ongoingexamination in clinical trials (269 271).

Cell-cycle proteins such as the CDKs and their inhibitors(CDKIs), which modulate cell-cycle progression in response toappropriate signals, are also possible targets. Orderly cell divi-sion requires a tight control of cyclins and CDKs, particularly atthe G 1 /S checkpoint (272). Complexes of Rb family of tumorsuppressors with E2F transcription factors inhibit the transcrip-tion of several genes involved in the S phase of cell cycle bothby active repression as well as through the recruitment of HDACs (273, 274). Phosphorylation of Rb by cyclin/CDKcomplexes leads to release of E2Fs and transactivation of targetgenes by binding of E2Fs to E2F response elements (275). The

Fig. 4 Targets for inhibition in RTK signaling pathways.



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activity of cyclin D:CDK4/6 and cyclin E:CDK2 complexes isregulated by CDKIs of the INK4 and KIP families such asp16 ink4a , p15 ink4b , p18 ink4c , p19 ink4d , p21 waf1/cip1 , p27 kip1 , andp57 kip2 , which act as inhibitors of G 1 /S transition. Disruptedactivity of a number of G 1 checkpoint genes/proteins has beenimplicated in carcinogenesis (275). Decreased Rb expression,

and a negative correlation between Rb expression and survivalhave been reported in ALL (276). Deletions and mutations of Rbhave been reported in a number of hematological malignancies(276). Genes for p16 ink4a and p15 ink4b are suppressed in varioushematological cancers mainly by deletion and by hypermethyl-ation of their promoters (277, 278). The role of inactivation of these genes as a prognostic indicator has been controversial(275). Alternatively, oncogenic amplification, and overexpres-sion of cyclins and CDKs have been reported in a numberof hematological cancers (279). Of note are overexpressionof cyclin D1 in mantle cell lymphoma and hyperactivation of cdk4/cdk6 in human T-cell leukemia virus type 1 leukemias(279). Inhibitors of cyclins and CDKs such as flavopiridol, andthe staurosporine derivative UCN-01 are under active develop-ment (248, 280). Early phase clinical trials of these agents havebeen reported recently (279, 281). However, it is likely that themajority these drugs will be used in combination with standardcytotoxic chemotherapy agents or for therapy of minimal resid-ual disease.

Concluding RemarksOver the past decade extensive research has elucidated the

role of cellular signaling pathways in cellular growth, differen-tiation, and apoptosis. The disruption of these pathways, on theother hand, has been shown to promote the neoplastic transfor-mation of cells. This knowledge has led to the development of a number of molecules that in preclinical and clinical studieshave been shown to be capable of reversing the neoplastic

process. Our current challenge is to further understand thecomplexity of these processes and to devise specific agents thattarget the neoplastic cells whereas sparing normal tissues. Thiswould truly realize the concept of targeted therapy in treatinghematological malignancies and solid tumors alike.

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Table 1 Targets for drug development

Target Prototype drug Mode of action

Tyrosine and serine/threonine kinases Rituxan, Campath-1H Ligand-binding inhibitionCEP-701, AG1295 FLT3R kinase inhibitionGenistein Non-specific kinaseLimonene, perillyl alcohol, R115777, SCH66336,

L778123, BMS214662Ras FT inhibition a

Geldanamycin, radicicol Raf inhibitionWortmannin, LY294002 PI3K inhibitionRapamycin P70S6K inhibitionPD098059, PD184352, UO126 MEK inhibitionAG490 JAK inhibitionImatinib mesylate ABL, PDGFR, KIT

Cell-cycle proteins Flavopiridol, Olomoucine, UCN-01 CDK inhibitionPurvalanol Cdc2 inhibition

Apoptosis-re lated proteins Staurosporine, UCN-O1, CGP41251, PKC412 Bcl -2 inhibit ion (PKC)Bryostatin PKC activation P53-MDM2 inhibitionPS341 Proteasome inhibition

Angiogenesis-related proteins Suramin, SU5416 VEGFR and bFGFRCell life span targets Anthraquinones Telomerase inhibitionTranscription factors (Myc, Ets, Fos,

Jun, Rel, Myb)a Ras FT, Ras farnesyl transferase; MDM2, mouse double minute-2; PKC, protein kinase C; VEGFR, vascular endothelial growth factor receptor;

bFGFR, basic fibroblast growth factor receptor.



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rvandi 02, jak-targeting in leukemia

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